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Deficient spermiogenesis in mice lacking Rlim

  1. Feng Wang
  2. Maria Gracia Gervasi
  3. Ana Bošković
  4. Fengyun Sun
  5. Vera D Rinaldi
  6. Jun Yu
  7. Mary C Wallingford
  8. Darya A Tourzani
  9. Jesse Mager
  10. Lihua Julie Zhu
  11. Oliver J Rando
  12. Pablo E Visconti
  13. Lara Strittmatter
  14. Ingolf Bach  Is a corresponding author
  1. Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, United States
  2. Department of Veterinary & Animal Sciences, University of Massachusetts Amherst, United States
  3. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, United States
  4. Program in Molecular Medicine, University of Massachusetts Medical School, United States
  5. Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, United States
  6. Electron Microscopy Core, University of Massachusetts Medical School, United States
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Cite this article as: eLife 2021;10:e63556 doi: 10.7554/eLife.63556

Abstract

The X-linked gene Rlim plays major roles in female mouse development and reproduction, where it is crucial for the maintenance of imprinted X chromosome inactivation in extraembryonic tissues of embryos. However, while females carrying a systemic Rlim knockout (KO) die around implantation, male Rlim KO mice appear healthy and are fertile. Here, we report an important role for Rlim in testis where it is highly expressed in post-meiotic round spermatids as well as in Sertoli cells. Systemic deletion of the Rlim gene results in lower numbers of mature sperm that contains excess cytoplasm, leading to decreased sperm motility and in vitro fertilization rates. Targeting the conditional Rlim cKO specifically to the spermatogenic cell lineage largely recapitulates this phenotype. These results reveal functions of Rlim in male reproduction specifically in round spermatids during spermiogenesis.

Introduction

In testes of adult animals, the differentiation of spermatogonial stem cells during spermatogenesis occurs within seminiferous tubules. Spermiogenesis represents a late stage during spermatogenesis, in which post-meiotic round spermatids differentiate into mature spermatozoa by condensation of the spermatid DNA and formation of the sperm head and tail (O'Donnell, 2014; O'Donnell et al., 2011). Spermatozoa are then released into the lumen of seminiferous tubules, in a process called spermiation, which involves the remodeling and reduction of cytoplasm (França et al., 2016). Even though these processes are crucial for male reproduction, they are poorly understood due to lack of knowledge on spermiation molecular mechanisms.

The ubiquitin proteasome system (UPS) plays important roles in male reproduction (Richburg et al., 2014) with a testis-specific version of the proteasome complex (Kniepert and Groettrup, 2014). Indeed, the ubiquitination of proteins in cells of the testis is required for functional spermatogenesis including spermiogenesis, and multiple steps during the progression of spermatogonial stem cells to mature spermatozoa critically depend on the UPS (Richburg et al., 2014). The UPS pathway is critically dependent upon E3 ubiquitin ligases, which provide substrate specificity by selecting target proteins for ubiquitination (Metzger et al., 2014; Pickart, 2001).

The X-linked gene Rlim (also known as Rnf12) encodes a RING H2 type E3 ligase (Metzger et al., 2014; Joazeiro and Weissman, 2000; Bach et al., 1999). While Rlim mRNA is widely expressed in many organs and cell types, RLIM protein is more selectively detected (Bach et al., 1999; Ostendorff et al., 2006). In cells RLIM shuttles between the cytoplasm and nucleus. Nuclear translocation is regulated by phosphorylation, and in many cell types RLIM is primarily detected in the nucleus (Jiao et al., 2013), where it controls not only levels and dynamics of various proteins and protein complexes involved in transcriptional regulation (Bach et al., 1999; Ostendorff et al., 2002; Krämer et al., 2003; Güngör et al., 2007; Gontan et al., 2012; Johnsen et al., 2009; Her and Chung, 2009; Huang et al., 2011; Wang et al., 2019), but also its own expression via autoubiquitination (Ostendorff et al., 2002). In female mice, Rlim functions as a major epigenetic regulator of nurturing tissues. RLIM promotes the survival of milk-producing alveolar cells in mammary glands of pregnant and lactating females (Jiao et al., 2012). Moreover, RLIM is crucial for imprinted X chromosome inactivation (iXCI) (Shin et al., 2010; Wang et al., 2016; Gontan et al., 2018), the epigenetic silencing of one X chromosome in placental trophoblast cells early during female embryogenesis to achieve X dosage compensation (Payer, 2016). Indeed, due to inhibited placental trophoblast development, the deletion of a maternally inherited Rlim allele results in peri-implantation lethality specifically of females (Shin et al., 2010; Wang et al., 2016). In contrast, males systemically lacking Rlim appear to develop normally, are born at Mendelian ratios and are fertile as adults (Shin et al., 2010).

Here, we report high and dynamic Rlim mRNA and protein expression in the testis of male mice, where expression of RLIM protein is highly detected in round spermatids during spermiogenesis as well as in Sertoli cells. Indeed, we show that Rlim is required for the generation of normal sperm numbers with normal sperm cytoplasmic volume. Even though Sertoli cells are known to regulate cytoplasmic reduction in spermatozoa (O'Donnell, 2014; O'Donnell et al., 2011), our genetic analyses reveal that this activity is mediated by Rlim expressed in the spermatogenic cell lineage. These results assign important functions of Rlim during spermiogenesis.

Results

Rlim expression in testis is highly regulated

To investigate potential functions of Rlim in mice in addition to XCI, we examined mRNA expression in various tissues isolated from adult mice via Northern blots. Rlim mRNA was detected in many tissues with highest levels in testis (Figure 1A), consistent with published results (Bach et al., 1999; Ostendorff et al., 2000). However, while the Rlim-encoding mRNA in most tissues migrated around 7.5 kb, a variant band at 2.4 kb was detected in testis. Based on published RNA-seq data sets on mouse testes isolated at various post-partum stages (Margolin et al., 2014), mapping of reads to the Rlim locus revealed relatively homogenously distribution over all exons in 6–20 days post-partum (dpp) animals. However, most of the reads in sexually mature (38 dpp) animals mapped in exonic regions upstream of the TGA Stop codon, encompassing the 5’ non-coding region and the entire open-reading frame (ORF), while most of the 3’ noncoding region was underrepresented (Figure 1B). Indeed, consistent with the length of the observed variant Rlim mRNA, closer examination the Rlim cDNA sequence revealed a consensus alternative polyadenylation site (Proudfoot, 2011) starting 69 bp downstream of the TGA Stop codon (Figure 1C). Thus, in mature mouse testes a short, variant Rlim RNA is generated by alternative polyadenylation (Tian and Manley, 2017).

A short Rlim mRNA variant highly expressed in testis is generated via alternative polyadenylation in mature male mice.

(A) A Northern blot containing RNA extracts from various adult mouse tissues (WT for Rlim) was hybridized with an Rlim probe (upper panel) and a probe recognizing β-actin as loading control (lower panel). (B) Modified from the UCSC Genome Browser: Cumulative raw reads from RNA-seq datasets of testes RNA isolated from post-natal mice at 6, 12, 16, 20, and 38 days post-partum (dpp) (Margolin et al., 2014) were mapped on the Rlim locus (variable scales). Structure of the Rlim gene is shown below in blue with boxed exon regions. Protein coding regions are indicated in thicker stroke. Blue arrow indicates direction of transcription. ATG start codon, TGA stop codon and site of alternative polyadenylation sequence (red dotted line) is indicated. Note low relative read density 3’ of the alternative polyadenylation site specifically in 38 dpp animals. (C) Nucleotide sequence containing an alternative polyadenylation site downstream of the TGA stop codon. Conserved motifs including a T-rich sequence, A/TATAAA, and CA motifs are boxed. The cleavage position is indicated (green arrow).

Because expression of Rlim mRNA and protein can be strikingly different (Ostendorff et al., 2006), we examined RLIM protein in testes using an established RLIM antibody (Ostendorff et al., 2006; Ostendorff et al., 2002). Consistent with our mRNA analyses (Figure 1), western blots on protein extracts of testis, brain, and spleen confirmed high expression of full-length RLIM protein in testis (Figure 2—figure supplement 1A). Using immunohistochemistry (IHC) on testes sections, we detected strong immunoreactivity in specific regions of some seminiferous tubules, representing differentiating spermatogenic cells (Figure 2A). Moreover, we detected single RLIM-positive cells at the periphery of all tubules. Consistent with published results (Wang et al., 2016), little to no RLIM staining was detected in males carrying a Sox2-Cre (Hayashi et al., 2002) – mediated conditional knockout of the Rlim gene (cKO/YSox2-Cre), as these animals lack RLIM in somatic tissues as well as the germline (Wang et al., 2016; Shin et al., 2014). Lack of RLIM protein in testes of cKO/YSox2-Cre animals was confirmed by western blot (Figure 2—figure supplement 1B). Because high RLIM levels appeared to be expressed at specific stages during spermatogenesis (Figure 2A), we performed co-staining using an antibody against peanut agglutinin (PNA) that stains acrosomal structures allowing staging of spermatogenic cells within seminiferous tubules (Kotaja et al., 2004; Oakberg, 1956a; Oakberg, 1956b). Around seminiferous stage II / III, RLIM levels are detectable but low in all spermatogenic cells, including post-meiotic step 2/3 spermatids (Figure 2B). However, indicated by RLIM/PNA co-staining, RLIM levels are high in step 6–8 round spermatids (stages VI–VIII), a mid-timepoint in spermiogenesis before spermatids begin to elongate (O'Donnell, 2014; Qian et al., 2014). Thus, RLIM protein levels are low in step 1–5 spermatids, dramatically upregulated in step 6–8 spermatids, and then downregulated in elongating spermatids at step 10 prior to their release (O'Donnell et al., 2011; Figure 2B). We noted that the nuclei of RLIM-positive cells located at the periphery of seminiferous tubules displayed a triangular shape characteristic for Sertoli cells (Figure 2—figure supplement 1C). To identify this RLIM-positive cell type (Figure 2A), we performed IHC, co-staining with antibodies against RLIM and the Sertoli cell marker GATA1 (Yomogida et al., 1994). Positive co-staining identified these cells as Sertoli cells (Figure 2C). This dynamic and regulated expression of Rlim suggests roles during male reproduction.

Figure 2 with 1 supplement see all
High RLIM protein expression specifically in round spermatids and in Sertoli cells.

Tissue sections of mouse testes were stained using IHC with indicated antibodies. Boxed areas are shown in higher magnification. (A) DAB staining of testes sections generated from fl/Y and, as negative control, Rlim cKO/YSox2-Cre males littermates using antibodies against RLIM. Left panel: fl/Y male. Red arrows (box 1) and yellow arrows (box 2) point at spermatogenic cells and cells located in the periphery of seminiferous tubules that exhibit high RLIM staining, respectively. Right panels: cKO/Y male. Scale bars = 150 μm. (B) IHC on fl/Y testis co-staining with antibodies against RLIM (green) and PNA (red) to determine differentiation stages of spermatogenic cells within seminiferous tubules (indicated). Scale bar = 60 μm. (C) IHC on fl/Y testis co-staining with antibodies against RLIM (green) and GATA1 (red), a Sertoli cell marker. Scale bar = 25 μm.

Diminished production and functionality of Rlim KO sperm

Next, we investigated functions of the Rlim gene in males using our conditional knockout (cKO) mouse model (Shin et al., 2010). Results on male mice systemically lacking Rlim induced by Cre recombinase transgenes (Shin et al., 2010; Wang et al., 2016; Shin et al., 2014) or by germline KO (Figure 3—figure supplement 1A) reveals that Rlim has no essential functions during male embryogenesis and post-natal development. The germline KO/Y animals were generated by crossing heterozygous Rlim KO females (flm/KOp) with WT/Y males (Shin et al., 2010; Shin et al., 2014). To explore potential roles of the Rlim gene during male reproduction, we compared testes of 8-week-old animals systemically lacking Rlim either by a germline Rlim KO or with a Sox2-Cre-mediated Rlim cKO (cKO/YSox2-Cre) with fl/Y littermate controls. Indeed, in cKO/YSox2-Cre and KO/Y mice the size and weight of the testis was significantly decreased when compared to their respective fl/Y littermates, (Figure 3A,B; Figure 3—figure supplement 1B). This was accompanied by lower numbers of mature sperm isolated in Caudal swim-out experiments (Figure 3C; Figure 3—figure supplement 1C). Even though these numbers are biased towards motile sperm, these data suggest diminished sperm production. To examine plausible causes for these phenotypes, we analyzed PAS-stained testis sections. However, we did not detect obvious abnormalities of specific testicular cell types during spermiogenesis in animals lacking Rlim, and sperm release into tubules appeared normal (Figure 3—figure supplement 1D, not shown). Moreover, in IHC staining testes sections with antibodies against cleaved caspase 3, there were no signs of increased apoptosis in the spermatogenic cells lacking Rlim (not shown). Examining epididymal epithelia, which also express RLIM (Figure 3—figure supplement 2A), revealed no significant differences in weight between fl/Y and cKO/YSox2-Cre animals (Figure 3—figure supplement 2B). Visualizing tubules that form the inner epididymal layers via de-lipidation (Sylwestrak et al., 2016; Tomer et al., 2014), we did not detect major differences between genotypes (Figure 3—figure supplement 2C). Our results indicate testicular phenotypes in mice lacking Rlim.

Figure 3 with 2 supplements see all
Lack of Rlim affects sperm production.

Males systemically lacking Rlim were generated via Sox2-Cre mediated Rlim deletion (cKO/YSox2-Cre) and directly compared to fl/Y male littermates at 8 weeks of age. (A) Deletion of Rlim results in smaller testes. Representative testes isolated from adult male fl/Y control animals (#1,2) and cKO/YSox2-Cre littermates (#3,4) are shown. (B) Significantly decreased weight of testes isolated from cKO/YSox2-Cre animals (n = 9) when compared to fl/Y littermates (n = 7). Values were normalized against total body weight and represent the mean ± s.e.m. p Values are shown (students t-test). (C) Significantly decreased numbers of sperm in animals lacking Rlim. Cauda epididymal sperm were collected via swim-out in HTF medium. After 10 min of swim-out, total sperm numbers were determined (n = 7 fl/Y; n = 9 cKO/YSox2-Cre). s.e.m. and p Values are indicated. (D, E) Differentially expressed genes in testes of fl/Y and cKO/YSox2-Cre mice as determined by RNA-seq experiments on biological replicates. Genes significantly (p<0.05) down-regulated and up-regulated upon the Rlim deletion in each experiment are shown in (C) and (D), respectively. Arrow indicates Rlim. (F) Differentially expressed genes distribute in eight functional categories that include metabolism, signaling, transcription, cell organization, transport, UPS, ncRNA and other.

As Rlim is a transcriptional regulator and the structural integrity of testes appeared to be generally intact in cKO/YSox2-Cre males (Figure 2A), we next examined effects of Rlim on genome wide gene expression. Thus, we performed RNA-seq experiments on RNA isolated from total testis of 8-week-old animals, comparing global gene expression in cKO/YSox2-Cre and fl/Y male littermates. These experiments including library construction, sequencing, and data processing were performed as previously described (Wang et al., 2017). Consistent with findings that attribute both positive and negative functions of RLIM for gene transcription (Bach et al., 1999; Gontan et al., 2012), statistical analyses revealed 118 down-regulated and 83 up-regulated genes (threshold p<0.05) in cKO/YSox2-Cre animals (Figure 3C,D). Gene ontology analyses revealed that functions of these genes fell mostly in eight categories with around half of all differentially expressed genes involved in signaling (27.5%) and regulation of metabolism (22.5%), in particular regulatory functions on lipid metabolism (Figure 3E). Other genes are involved in transcription/chromatin (14%), cell organization (6%), transport (6%) or the UPS (3%), while 9.5% of gene functions occupy multiple other cellular pathways. Around 10.5% of transcripts constituted non-coding (nc) RNAs. No major differences in functions between up- and down-regulated genes were detected in cKO/YSox2-Cre testes. Combined, these results suggest functions of Rlim during spermatogenesis.

Next, we investigated the characteristics and functionality of sperm isolated from the Cauda of 8-week-old males via swim-out. Indeed, cKO/YSox2-Cre sperm displayed elevated rates of morphological abnormalities, including coiled midpieces and head malformations (Figure 4A,B). However, the sperm capacitation-induced phosphorylation pathways, acrosomal status and induced acrosome reaction in Rlim KO sperm were similar to controls as judged by western blot and PNA staining, respectively (Figure 4—figure supplement 1A–E), indicating that the deletion of Rlim did not affect general signaling. To examine sperm motility, we used CEROS computer-assisted semen analysis (CASA) in swim-out experiments at T0 and after 60 min (T60) under conditions that allow capacitation comparing cKO/YSox2-Cre males with fl/Y littermates. The results revealed significant motility deficiencies of cKO/YSox2-Cre sperm. Indeed, cKO/YSox2-Cre sperm displayed decreased total motility, and out of the total motile sperm the percentages of sperm with progressive motility was lower, while the percentages of slow and weakly motile sperm populations were higher (Figure 4C–E). Thus, cKO/YSox2-Cre sperm is less motile. Next, we tested for possible functional consequences of these deficiencies during in vitro fertilization (IVF) (Sharma et al., 2016), using oocytes originating from WT females and sperm isolated from either fl/Y or cKO/YSox2-Cre littermates. In these experiments, the numbers of oocytes and sperm cells were adjusted to 100–150 and 100,000, respectively, to achieve fertilization rates of around 80% for the control samples as judged by the number of embryos reaching cleavage stage 24 hr after adding sperm (Figure 4F,G). The development of IVF embryos was monitored up to (96 hr) at which point blastocyst stage was reached by the majority of embryos generated by control sperm. Indeed, sperm isolated from fl/Y control animals yielded in 84.3% cleavage stage and 77% blastocyst stage embryos, compared to 67.0% and 46.8% for cKO/YSox2-Cre sperm, respectively (Figure 4F,G). Moreover, the rate of blastocysts per cleavage staged embryos was 92% of for fl/Y sperm and only 70% for cKO/YSox2-Cre (Figure 4—figure supplement 1F), suggesting that lack of Rlim negatively affects embryonic development under in vitro conditions. Thus, sperm isolated from cKO/YSox2-Cre animals yielded in significantly less embryos reaching the appropriate developmental stage when compared to fl/Y littermate controls, both for cleavage and blastocyst stages (Figure 4F,G). Combined, these data indicate that deletion of Rlim in male mice results in reduced sperm functionality.

Figure 4 with 1 supplement see all
Increased abnormalities and decreased functionality in sperm lacking Rlim.

Cauda epididymal sperm were collected via swim-out from 8-week-old males (n = 7 fl/Y ▪; n = 9 cKO/YSox2-Cre). (A) Sperm morphology was assessed by light microscopy. Representative images of difference interference contrast (DIC) display the morphological patterns found indicated by arrows: 1, Head malformation; 2, Coiled midpiece; 3, Flipped head. Scale bar = 25 µm. (B) Quantification of the morphology patterns. Percentages of normal sperm, coiled midpiece, head malformation, and flipped head out of the sperm population. At least 100 sperm per sample were counted, total sperm counted 1146 for fl/Y and 1077 for cKO/YSox2-Cre. Values represent the mean ± s.e.m. (C) Sperm motility was evaluated in the swim-out (T0) and after 60 min of incubation in conditions that support capacitation (T60). Sperm motility was examined using the CEROS computer-assisted semen analysis (CASA) system. Percentage of total motile sperm in the population. n = 12, values represent the mean ± s.e.m. (D) Classification of type of motility (progressive, intermediate, hyperactivated, slow, and weakly motile; in percentage) out of the total motile population at T0. n = 12, values represent the mean ± s.e.m. (E) Classification of type of motility (in percentage) out of the total motile population after 60 min of incubation in capacitation conditions (T60). n = 12, values represent the mean ± s.e.m. (F) Representative images for cleavage and blastocyst stages at 24 hr and 96 hr, respectively. Asterisks indicate embryos not reaching anticipated embryonic stage. Scale bar = 100 μm. (G) Summary of IVF results. n = 142 and 313 presumed oocytes for fl/Y and cKO/Y sperm, 7 and 8 animals, respectively. Values represent the mean ± s.e.m.

Increased size of cytoplasmic droplets in Rlim cKO sperm

Because of the decreased motility of Rlim cKO sperm (Figure 4C–E), we examined the energetic status including amino acids, glycolysis, TCA cycle, pentose phosphate pathway, and nucleotide biosynthesis via metabolomic profiling of polar metabolites. We isolated 40–45 Mio cauda swim-out sperm each of 3 fl/Y and 5 cKO/YSox2-Cre animals, and polar metabolites were measured using liquid chromatography coupled with mass spectrometry (LC-MS). Results showed that, unexpectedly, 66 metabolites were significantly more abundant in the cKO/YSox2-Cre sperm, in particular levels of many amino acids, in contrast to only five metabolites that displayed lower levels (Figure 5A). Analyses of these diverse metabolites did not yield in the identification of a specific energy production pathway but rather revealed that metabolite intermediates of many cellular pathways are increased in the Rlim cKO sperm, suggesting a more general problem. Thus, we included electron microscopy (EM) in our analyses. Interrogating sperm via scanning EM (SEM) revealed significantly more Rlim cKO/YSox2-Cre sperm displaying cytoplasmic droplets in the midpiece, and these droplets were also increased in size as measured using ImageJ software (Figure 5B–D). Because Cauda sperm has matured a considerable time in the epididymis that also expresses Rlim (Figure 3—figure supplement 2A), to distinguish testicular versus epididymal functions of Rlim in droplet formation we extended these studies to testicular sperm. Indeed, isolating testicular sperm from 30 fl/Y and cKO/YSox2-Cre animals each, the numbers of isolated sperm per Rlim cKO testis were lower but this was no longer significant when compared to controls (Figure 6A). We noted that the midpieces from cKO sperm were highly vulnerable to rupturing, while the prevalence of coiled midpieces appeared similar between cKO and control sperm (Figure 6B). Moreover, we detected a low number of sperm that exhibited duplicated axonemes specifically in Rlim cKO/YSox2-Cre but not in fl/Y sperm (Figure 6B; Figure 6—figure supplement 1A). Again, the sizes of cytoplasmic droplets in testicular sperm were significantly increased (Figure 6C,D). Transmission EM (TEM) on testes sections confirmed the occurrence of duplicated axonemes in cKO/YSox2-Cre sperm as well as head malformations, while the overall structural integrity of the sperm tail appeared normal (Figure 6—figure supplement 1A–D), and no signs of decreased chromatin packaging in sperm heads was detected as judged by sperm head density. Interestingly, the cytoplasmic pockets in the Rlim cKO sperm heads appeared more pronounced in sperm of the epididymal Caput region (Figure 6E,F). These data reveal excessive cytoplasm in sperm heads and midpieces in males lacking Rlim and are consistent with testicular as opposed to epididymal functions of Rlim.

Increased size of cytoplasmic droplet in caudal sperm of males lacking Rlim.

(A) Cauda epididymal sperm were collected from 8-week-old fl/Y or cKO/YSox2-Cre mice and polar metabolites were determined via LC-MS. Samples were run and data analyzed by the Metabolite Profiling Core Facility at the Whitehead Institute. Note general increased content of metabolites in cKO/YSox2-Cre sperm. (B) Cauda sperm was collected from 8-week-old fl/Y or cKO/YSox2-Cre mice and after 10 min separation, immediately fixed in 2.5% glutaraldehyde followed by SEM analysis. Sperm with or without cytoplasmic droplets were counted. n = 250, each. (C) Increased size of cytoplasmic droplets in cKO/YSox2-Cre sperm. Representative images are shown. Droplets are indicated by arrows. (D) Increased size of cytoplasmic droplets in cKO/YSox2-Cre sperm. Droplet surface size was determined via ImageJ. n = 100, each.

Figure 6 with 1 supplement see all
Rlim plays important roles for cytoplasmic reduction.

Analyses of testicular sperm isolated from 8 weeks-old fl/Y or cKO/YSox2-Cre mice. (A) Quantification of sperm yield normalized against testis weight. n = 30, each genotype. (B) Quantification of sperm morphology scoring ruptured, coiled sperm and sperm with two axonemes. n = 250. (C) Larger cytoplasmic droplets in cKO/YSox2-Cre sperm. Representative images are shown. Droplets are indicated by arrows. (D) Increased size of cytoplasmic droplets in cKO/YSox2-Cre sperm. Droplet surface size was determined via ImageJ. n = 100, each. (E) Sperm head malformations within the epididymal Caput region as determined via TEM. Representative images are shown. Arrows point at cytoplasmic pocket. (F) Quantification of sperm exhibiting excessive head cytoplasmic pocket.

Functions of Rlim during spermiogenesis specifically in the spermatogenic cell lineage

Next, we addressed the question as to the cell type of Rlim action during spermiogenesis as RLIM protein is highly detectable both in the spermatogenic cell lineage specifically in round spermatids and in Sertoli cells (Figure 2). Because Sertoli cells play major roles in the regulation of spermiogenesis/spermiation (O'Donnell, 2014; O'Donnell et al., 2011; França et al., 2016) and cell numbers are correlated with sperm production capacity (Griswold, 1995), we compared number of cells positive for Sertoli cell marker GATA4, as in adult mice GATA4 expression does not vary with the cycle of the seminiferous epithelium (Yomogida et al., 1994). Counting GATA4-positive cells within seminiferous tubules, IHC revealed similar numbers of Sertoli cells in testes with or without Rlim (Figure 7—figure supplement 1A,B), indicating that Rlim is not required for Sertoli cell development and differentiation. Moreover, analyzing specific Sertoli cell structures involved in spermiation in testis sections via TEM, we did not detect major structural deficiencies in cells lacking Rlim including the formation of apical ectoplasmic specialization (ES) or the apical tubulobulbar complex (TBC), and no obvious signs of defective cytoplasmic reduction (Figure 7—figure supplement 1C,D).

In order to genetically elucidate the cell identity of Rlim function, we targeted the Rlim cKO via Ngn3-Cre (also known as Neurog3-Cre) (Schonhoff et al., 2004) to the spermatogenic cell lineage (Yoshida et al., 2004) and via Sf1-Cre (Dhillon et al., 2006) to Sertoli cells (Kim et al., 2007). IHC on testis sections using RLIM antibodies confirmed the correct, specific and penetrant targeting of both Cre drivers (Figure 7A). Because of a mixed background of these Cre-driver mouse lines, cKO males were compared to their respective littermate controls. Indeed, Rlim cKO/YNgn3-Cre mice displayed decreased testis weights and numbers of mature sperm isolated in caudal swim-out experiments (Figure 7B,C, respectively). In contrast, no significant effects on testes weights and sperm numbers were measured in cKO/YSf1-Cre animals. Consistent with these findings, SEM analyses of Caudal swim-out sperm revealed significantly increased numbers of sperm containing cytoplasmic droplets in cKO/YNgn3-Cre animals when compared with cKO/YSf1-Cre animals and their respective littermate controls (Figure 7D). Moreover, focusing on sperm with cytoplasmic droplets, the droplet sizes in cKO/YNgn3-Cre sperm were increased (Figure 7E,F), similar to those of cKO/YSox2-Cre sperm (Figure 5C,D). IVF experiments using swim-out sperm yielded in 76% cleavage stage and 58% blastocyst stage embryos for control, compared to 62% and 33% for cKO/YNgn3-Cre sperm, respectively (Figure 7G), confirming the functional deficiency of sperm. Thus, the phenotypes in males systemically lacking Rlim are largely recapitulated by cKO/YNgn3-Cre but not cKO/YSf1-Cre mice, demonstrating functions of Rlim in the spermatogenic cell lineage. Our combined results provide strong evidence that the upregulated expression of Rlim in round spermatids plays important functions for the reduction of cytoplasmic volume in sperm.

Figure 7 with 1 supplement see all
Functions of Rlim predominantly in the spermatogenic cell lineage.

Animals with an Rlim cKO in the spermatogenic cell lineage or in Sertoli cells were generated via Ngn3-Cre and SF1-Cre (cKO/YNgn3-Cre and cKO/YSF1-Cre), respectively. (A) IHC on testis sections of cKO/YNgn3-Cre and cKO/YSF1-Cre animals using RLIM antibodies. Correct targeting is indicated by lack of RLIM specifically in spermatogenic cells but not Sertoli cells (yellow arrows) in cKO/YNgn3-Cre animals, and in Sertoli cells but not round spermatids (red arrows) in cKO/YSF1-Cre males. Scale bars = 75 μm. (B) Significantly decreased weight of testes isolated from cKO/YNgn3-Cre males but not from cKO/YSf1-Cre animals (n = 18 fl/Y; 14 cKO/YNgn3-Cre) (n = 16 fl/Y; 10 cKO/YSf1-Cre). cKO animals were directly compared to their respective fl/Y male littermates at 8 weeks of age. Values were normalized against total body weight and represent the mean ± s.e.m. p Values are shown (students t-test). (C) Significantly decreased numbers of sperm isolated from cKO/YNgn3-Cre males but not from cKO/YSf1-Cre animals. Cauda epididymal sperm were collected via swim-out in HTF medium. After 10 min of swim-out, total sperm numbers were determined (n = 7 fl/Y; n = 9 cKO/YNgn3-Cre; n = 9 fl/Y; n = 11 cKO/YSf1-Cre). s.e.m. and p values are indicated. (D) Cauda sperm was collected from 8 weeks-old mice and visualized via SEM (n = 3 per genotype). Sperm with or without cytoplasmic droplets were counted. n = 250, per animal. (E) Increased size of cytoplasmic droplets in cKO/YNgn3-Cre sperm. Representative SEM images are shown. Upper panels: cKO/YNgn3-Cre and fl/Y control. Lower panel: cKO/YSf1-Cre and fl/Y control. Droplets are indicated by arrows. (F) Summary of cytoplasmic droplets in cKO/YNgn3-Cre sperm. Droplet surface size of SEM images was determined via ImageJ. n = 100, each. (G) Summary of IVF using sperm isolated from cKO/YNgn3-Cre and littermate control males. n = 262 and 324 presumed oocytes for fl/Y and cKO/Y sperm, respectively, seven animals, each. Values represent the mean ± s.e.m.

Discussion

Our results reveal robust expression of Rlim in male reproductive organs, particularly in testis, where the expression pattern was highly dynamic both at the mRNA and protein levels (Figures 1 and 2). The appearance of a variant mRNA in males coincides with sexual maturation, suggesting that this form is predominantly expressed in differentiating round spermatids, which express high levels of RLIM protein (Figure 2A), and, because Rlim acts in the spermatogenic cell lineage (Figure 7), also coincides with the exertion of its in vivo function in males. Indeed, alternative mRNAs display different time of synthesis, mRNA stability and/or translational efficiency (Tian and Manley, 2017). At the protein level, we find RLIM expression in spermatogenic cells is post-meiotically upregulated from low levels in step three spermatids to high levels peaking in round spermatids at step 6–8 and then downregulated again in elongating step nine spermatids and thereafter (Figure 2). Thus, Rlim joins many X-linked genes that are reactivated after meiotic sex chromosome inactivation (MSCI) (Ernst et al., 2019) and it is tempting to speculate that this reactivation is connected with the occurrence of the alternative Rlim mRNA. Moreover, the downregulation of RLIM in elongating spermatocytes coincides with a change in transcriptional and chromatin dynamics at this stage (Ernst et al., 2019). Even though Rlim is X-linked, the finding that the RLIM protein is detected in most/all round spermatids, including those that presumably harbor a Y chromosome, is explained by the fact that cytoplasmic bridges exist between spermatids and that RLIM efficiently shuttles between nuclei in heterokaryon cells (Jiao et al., 2013).

Our RNA-seq analyses indicate that many genes are affected in testes lacking Rlim influencing various cellular functions including signaling and metabolism (Figure 3). As Rlim regulates transcriptional factors (Bach et al., 1999; Ostendorff et al., 2002; Krämer et al., 2003; Güngör et al., 2007; Gontan et al., 2012; Johnsen et al., 2009; Her and Chung, 2009; Huang et al., 2011; Wang et al., 2019), it is thus likely that many of the differentially expressed genes (Figure 3D,E) might be affected indirectly. Because the vast majority of cells in the testis reflect spermatogenic cells, where Rlim exerts its functions (Figure 7), many differentially expressed genes reflect those expressed in this cell lineage and therefore may collectively contribute to the observed Rlim KO phenotypes in testes. However, we cannot exclude the possibility that subtle and hence undetected changes in testicular cell types in mice lacking Rlim may contribute to the observed differences in gene expression as well as testes weight (Figure 3).

Sperm produced by males lacking Rlim is dysfunctional with decreased motility and increased cytoplasm and head abnormalities. Because excess cytoplasm affects sperm motility, morphology including head morphology as well as fertilization potential (Cooper, 2011; Rengan et al., 2012), and the cytoplasmic volume is regulated during spermiogenesis just after RLIM protein is highly detected in spermatids, it is likely that the increased cytoplasmic volume is responsible for much of the defects detected in sperm lacking Rlim. Moreover, as the midpiece cytoplasm is particularly important for sperm osmoregulation (Cooper, 2011; Rengan et al., 2012), an increased size of cytoplasmic droplets is predicted to render sperm more vulnerable to osmotic challenges, which may ultimately lead to midpiece rupturing (Figure 6). Thus, while the finding of increased metabolite content in cKO sperm (Figure 5A) suggests defective cytoplasmic reduction during spermiation even though major defects were not observed (Figure 7—figure supplement 1), it is likely that the some of the increase in cytoplasmic size might have occurred in released sperm after spermiation. In this context, an increased sperm cytoplasm has been associated with higher activities of specific enzymes of the energy pathway including G6PDH (Aitken et al., 1994; Yuan et al., 2013), which diverts glucose metabolism away from glycolysis toward the pentose pathway. It is thus tempting to speculate that increased G6PDH activity might be partially responsible for the decreased Acetyl-CoA levels measured in sperm lacking Rlim (Figure 5A). Our combined data suggests that inefficient cytoplasmic reduction during spermiogenesis/spermiation renders Rlim KO sperm vulnerable to adequately adjust to the changing environment that occurs during transit through the epididymis (Gervasi and Visconti, 2017; Sullivan and Saez, 2013) leading to functional defects.

Concerning the cell type of Rlim action, RLIM protein is detected in spermatogenic cells specifically in round spermatids, in Sertoli cells and in epididymal epithelial cells. Our combined data reveals that lack of RLIM specifically in round spermatids is responsible for much of the observed sperm phenotype. This is demonstrated by targeting the Rlim cKO via Ngn3-Cre in the spermatogenic cell lineage, resulting in defective spermiogenesis, and similar sperm phenotypes when compared to the systemic Rlim cKO (Figures 3 and 7). In contrast, targeting the Rlim cKO via Sf1-Cre to Sertoli cells failed to induce a testis/sperm phenotype and providing strong evidence that Rlim in Sertoli cells is not involved in the regulation of spermiogenesis provided by this cell type. Thus, while we cannot exclude minor functions of Rlim in Sertoli cells and possibly also epididymal epithelial cells, our results provide strong evidence that Rlim in round spermatids is required for normal spermiogenesis. Therefore, Rlim adds to a very limited number of genes in the spermatogenic cell lineage that regulate the cytoplasmic reduction/droplet size (Zheng et al., 2007; Mikolcevic et al., 2012).

Considering early lethality of female mouse embryos with a maternally transmitted Rlim mutation (Shin et al., 2010), mathematical modeling of this exclusively female phenotype indicates that a deleterious mutation in the Rlim gene would shift selective evolutionary pressure entirely on females leading to a gender bias toward males in a mouse population over time (Jiao et al., 2012). Because gender biases in mouse populations represent an unfavorable strategy for reproduction (Hamilton, 1967), it is likely that the observed functions of Rlim during male reproduction will contribute to counter-act gender biases induced by its female function.

In summary, our data provide first evidence that in addition to crucial epigenetic functions in female embryogenesis and reproduction, the E3 ubiquitin ligase RLIM also occupies important roles during the reproduction of male mice. These results have major implications for epigenetic regulation and emphasize the importance of the UPS in male reproduction.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (M. musculus)RlimfloxPMID:20962847MGI:1342291
Genetic reagent (M. musculus)Sox2-CreJackson LaboratoryJAX #008454PMID:14516668
Genetic reagent (M. musculus)Neurog3-Cre
(Ngn3-Cre)
PMID:15183725JAX #005667Dr. Andrew Leiter (UMMS)
Genetic reagent (M. musculus)Sf1-CreJackson LaboratoryJAX #012462PMID:16423694
AntibodyRabbit anti-Rlim (rabbit polyclonal)PMID:11882901IHC (1:250)
WB (1:1000)
AntibodyRat anti-GATA1 (rat monoclonal)Santa Cruzsc265IHC (1:100)
AntibodyRabbit anti-GATA4 (rabbit polyclonal)Abcamab84593IHC (1:500)
AntibodyRabbit anti-phosphoPKA (rabbit monoclonal)Cell Signaling clone 100G7E9624WB (1:10000)
AntibodyMouse anti-PY (mouse monoclonal)Millipore clone 4G1005–321WB (1:10000)
AntibodyMouse anti- β-actin (mouse monoclonal)Santa Cruzsc47778WB (1:250)
AntibodyRabbit anti-cleaved caspase3 (rabbit monoclonal)Cell Signalingab9664IHC (1:300)

RNA-seq and data analyses

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RNA-seq on RNA isolated from testes of fl/Y and cKO/Y males including library construction and sequencing on a NextSeq 500 was carried out essentially as described (Wang et al., 2016; Wang et al., 2017). Reads (paired end 35 bp) were aligned to the mouse genome (mm10) using TopHat (version 2.0.12) (Trapnell et al., 2009), with default setting except set parameter read-mismatches was set to 2, followed by running HTSeq (version 0.6.1p1) (Anders et al., 2015), Bioconductor packages edgeR (version 3.10.0) (Robinson et al., 2010; Robinson and Smyth, 2007) and ChIPpeakAnno (version 3.2.0) (Zhu, 2013; Zhu et al., 2010) for transcriptome quantification, differential gene expression analysis, and annotation. For edgeR, we followed the workflow as described in Anders et al., 2013.

De-lipidation of epididymis

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De-lipidation of epididymis was performed based on previously described protocols (Sylwestrak et al., 2016; Tomer et al., 2014). Briefly, isolated organs were fixed with 4% paraformaldehyde (PFA) in PBS for 32 hr at room temperature (RT), then rinsed with PBS for three times of at least 2 hr. Tissues were kept at 4°C in PBS with 0.02% sodium azide until the time of tissue processing. In order to visualize tubules that form the inner layers of the epididymis, a de-lipidation step was performed. De-lipidation was done passively by incubating the organ with 4% SDS/PBS at RT in an orbital shaker for 2 weeks. The 4% SDS/PBS solution was changed every other day. At the end of the second week, the organ was rinsed with PBS for three times of least 4 hr and finally placed on a refractive index matching solution (RIMS: 0.17M iodixanol; 0.4M diatrizoic acid, 1M n-methyl-d-glucamine, 0.01% sodium azide), 24 hr prior to imaging.

Collection of sperm

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Epididymal Cauda sperm collected via swim-out and testicular sperm which were analyzed in this study was collected from 8 weeks-old fl/Y or cKO/Y mice. Briefly, Cauda epididymides were dissected and placed in 1 ml of modified Krebs-Ringer medium (m-TYH; 100 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5.5 mM Glucose, 0.8 mM Pyruvic Acid, 1.7 mM Calcium Chloride, 20 mM HEPES). Sperm was allowed to swim-out for 10 min at 37°C and then the epididymides were removed. Concentration of all sperm was calculated using a Neubauer hemocytometer. For mature testicular spermatozoa isolation, testes from one 8-week-old mouse were minced in a 35 mm Petri dish containing 1 ml 150 mM NaCl. Finely minced tissue slurry was then transferred to a 15 ml conical tube and set aside for 3–5 min to allow tissue pieces to settle down. Next, the cell suspension was loaded onto 10.5 ml of 52% isotonic percoll (Sigma). The tubes were then centrifuged at 15000 x g for 10 min at 10°C. The pellet was resuspended in 10 ml of 150 mM NaCl and spun at 900 x g at 4°C for 10 min followed by three washes with 150 mM NaCl at 4000 x g at 4°C for 5 min.

Sperm analyses

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Concerning the assessment of sperm morphology, after swim-out, sperm suspensions (50 µl) were fixed with paraformaldehyde 4% (w/v; EMS, Hatfield, MA) in phosphate buffered saline (PBS) for 10 min at room temperature. Then samples were centrifuged at 800 x g for 5 min, washed with PBS, and let air-dry on poly-L-lysine-coated glass slides. Samples were mounted using VectaShield mounting media (H-1000, Vector Laboratories, Burlingame, CA) and differential interference contrast (DIC) images were taken using a 60X objective (Nikon, PlanApo, NA 1.49) in an inverted microscope (Nikon Eclipse TE300). Images were analyzed using the freeware ImageJ v1.52e (http://imagej.nih.gov/ij/download.html), and sperm morphology was classified into the following categories: normal, coiled midpiece, head malformation, flipped head, and residual cytoplasmic droplet. Results are shown as percentages, at least 200 sperm per sample were counted in single-blinded experiments. Concerning the analysis of sperm acrosomal status, sperm from the swim-out suspension were loaded into glass slides and let air-dry for 15 min. After that samples were fixed, and the acrosomes were stained as described below in the sperm acrosome reaction section. Fluorescence and phase contrast images were taken in a Nikon Eclipse TE300 fluorescence microscope using a 40x objective (Nikon, Phase 2 DL, NA 0.55). Images were analyzed using the freeware ImageJ v1.52e, and at least 200 sperm per sample were counted in single-blinded experiments. Results are shown as percentage of acrosome intact sperm.

Concerning sperm capacitation and analysis of swim-out cauda sperm motility, sperm were incubated at 37°C for 60 min in m-TYH (Non-Cap) or in m-TYH supplemented with 15 mM NaHCO3 and 5 mg/ml BSA (Cap). Sperm motility was evaluated in the swim-out (T = 0) and after 60 min of incubation in capacitating conditions (T = 60). Briefly, sperm suspensions (30 µl) were loaded into pre-warmed chamber slides (Leja slides, Spectrum Technologies, Healdsburg, CA) and placed on a warmed microscope stage at 37°C. Sperm motility was examined using the CEROS computer-assisted semen analysis (CASA) system (Hamilton Thorne Research, Beverly, MA). Acquisition parameters were set as follows: frames acquired: 90; frame rate: 60 Hz; minimum cell size: four pixels; static head size: 0.13–2.43; static head intensity: 0.10–1.52; and static head elongation: 5–100. At least five microscopy fields corresponding to a minimum of 200 sperm were analyzed in each experiment. Data were analyzed using the CASAnova software (Goodson et al., 2011).

Concerning SDS-PAGE and western blotting, swim-out cauda sperm samples were centrifuged at 12,000 x g for 2 min, washed in 1 ml of PBS, and then centrifuged at 12,100 x g for 3 min. Sperm proteins were extracted by resuspending the remaining pellets in Laemmli, 1970 sample buffer, boiled for 5 min and centrifuged once more at 12,100 x g for 5 min. Protein extracts (supernatant) were then supplemented with β-mercaptoethanol 5% (v/v), and boiled again for 4 min. Protein extracts equivalent to 2.5 × 105 sperm/lane were subjected to SDS–PAGE, and electro-transferred to PVDF membranes (Bio-Rad, Waltham, MA). PVDF membranes were blocked with 5% (w/v) fat-free milk in tris buffered saline containing 0.1% (v/v) Tween 20 (T-TBS) and immunoblotted with anti-pPKAs antibody (clone 100G7E, 1:10,000) overnight at 4°C to detect phosphorylated PKA substrates. Then, membranes were incubated with HRP-conjugated anti-rabbit secondary antibody diluted in T-TBS (1:10,000) for 60 min at room temperature. Detection was done with an enhanced chemiluminescence ECL plus kit (GE Healthcare) as per manufacturer instructions. After developing of pPKAs, membranes were stripped at 55°C for 20 min in 2% (w/v) SDS, 0.74% (v/v) β-mercaptoethanol, 62.5 mM Tris (pH 6.5), blocked with fish gelatin 20% (v/v; Sigma cat # G7765, St. Louis, MO) in T-TBS for 60 min at room temperature and re-blotted with anti-PY antibody (clone 4G10, 1:10,000) to detect proteins phosphorylated in tyrosine residues. Membranes were then incubated with HRP-conjugated anti-mouse secondary antibody diluted in T-TBS (1:10,000) for 60 min at room temperature. Detection was done with an enhanced chemiluminescence ECL plus kit (GE Healthcare) as per manufacturer instructions.

Concerning swim-out cauda sperm acrosome reaction, after 60 min of capacitation in m-TYH Cap medium, sperm samples were incubated with progesterone (10 µM) or with DMSO (vehicle) in m-TYH Cap at 37°C for 30 min. Then, sperm were loaded into glass slides and let air-dry for 15 min. Sperm were fixed by incubation with paraformaldehyde 4% (w/v) in PBS at room temperature for 15 min, washed three times (5 min each) with PBS and permeabilized with 0.1% (v/v) Triton X-100 for 3 min. After permeabilization, samples were washed three times with PBS, and then incubated with Alexa Fluor 488-conjugated lectin peanut agglutinin (PNA) in PBS at room temperature for 30 min. Before mounting with Vectashield (Vector Laboratories, Burlingame, CA), samples were washed three times with PBS for 5 min each time. Epifluorescence images were taken in a Nikon Eclipse TE300 fluorescence microscope using a 40x objective (Nikon). Phase contrast images were taken in parallel. Images were analyzed using the freeware ImageJ v1.52e, and at least 200 sperm per sample were counted in single-blinded experiments. Results are shown as percentage of acrosome reacted sperm.

In vitro fertilization (IVF)

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IVF experiments were performed essentially as described (Sharma et al., 2016) with some modifications. Briefly, female C57BL/6J mice (age 6–8 weeks) were superovulated by intraperitoneal injection of pregnant mare's serum gonadotropin (PMSG; 5 U; Calbiochem) followed by human chorionic gonadotropin (hCG; 5 U; Sigma-Aldrich) 48 hr later (Yamashita et al., 2008). Metaphase II-arrested oocytes tightly packed with cumulus cells were collected from the oviductal ampulla 14 hr after hCG injection and placed in a 100 μl drop of human tubal fluid (HTF; Millipore) medium covered with mineral oil. Fresh cauda epididymal sperm of fl/Y and cKO/Y littermates (>6 animals per genotype both for Sox2-Cre and Ngn3-Cre, aged 2–4 mo) were swum-up in 1 ml HTF medium for 10 min and capacitated by incubation for another 30 min at 37°C under 5% CO2. An aliquot (1.0 × 105 cells) of the capacitated sperm suspension was added to 100 μl drop of HTF medium containing the oocytes. After incubation at 37°C under 5% CO2 for 4 hr, the presumed zygotes were washed with KSOM medium to remove cumulus cells, sperm and debris, and then incubated in a 50 μl drop of KSOM medium. In vitro fertilized embryos were analyzed at cleavage (24 hr) and blastocyst stages (96 hr).

Metabolomic profiling

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Polar metabolite measurements were performed by the Metabolite Profiling Core Facility at the Whitehead Institute for Biomedical Research (Cambridge, MA). For LC-MS analyses, 45 Mio epididymal cauda swim-out sperm were collected each from 5 cKO/YSox2-Cre and three fl/y animals in extraction mix containing 80% methanol and isotopically labeled amino acids. Peak area ratios of each metabolite were determined and log2 ratio relative to the mean of control (fl/Y) sperm determined.

Electron microscopy

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SEM was performed on caudal epididymis sperm and sperm isolated from testes. Caudal sperm was let to dissociate in HTF medium and then fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1M Na Cacodylate buffer. The fixed sperm was then placed on poly Lysine covered cover slips and left to adhere for 10 min. The samples were rinsed three times in the same fixation buffer, post fixed in aqueous 1% (w/v) OsO4 for 1 hr at RT, dehydrated through a graded ethanol series to ethanol 100% (x3), and then they were critically point dried. The cover slips were then mounted on double sided carbon tape onto aluminum SEM stubs and grounded with colloidal silver paint, sputter coated with 12 nm of gold-palladium and were imaged using secondary electron (SEI) mode with a FEI Quanta 200 MKII FEG SEM.

TEM was performed on ultrathin sections on Caput epididymides and testes. Tissues were dissected and immediately immersed in 2.5% glutaraldehyde in 0.1 M Na Cacodylate buffer, pH 7.2 for 60 min at RT. The samples were rinsed three times in the same fixation buffer and post-fixed with 1% osmium tetroxide for 1 hr at room temperature. Samples were then washed three times with ddH2O for 10 min, and in block stained with a 1% Uranyl Acetate aqueous solution (w/v) at 6°C overnight. After three rinses in ddH2O the samples were dehydrated through a graded ethanol series of 20% increments, before two changes in 100% ethanol. Samples were then infiltrated first with two changes of 100% Propylene Oxide and then with a 50%/50% propylene oxide / SPI-Pon 812 resin mixture. The following day 5 changes of fresh 100% SPI-Pon 812 resin were done before the samples were polymerized at 68°C in flat embedding molds. The samples were then reoriented, and thin sections (approximately 70 nm) were placed on copper support grids and contrasted with Lead citrate and Uranyl acetate. Sections were examined using the a CM10 TEM with 100Kv accelerating voltage, and images were captured using a Gatan TEM CCD camera.

Data availability

RNAseq data have been deposited in GEO under accession code GSE114593.

The following data sets were generated
    1. Wang F
    2. Bach I
    (2020) NCBI Gene Expression Omnibus
    ID GSE114593. Analysis of functions of Rlim during reproduction in male mice.
The following previously published data sets were used
    1. Margolin G
    2. Khil PP
    3. Bellani MA
    4. Camerini-Otero RD
    (2014) NCBI Gene Expression Omnibus
    ID GSE44346. RNA-Seq and RNA Polymerase II ChIP-Seq of mouse spermatogenesis.

References

    1. Yomogida K
    2. Ohtani H
    3. Harigae H
    4. Ito E
    5. Nishimune Y
    6. Engel JD
    7. Yamamoto M
    (1994)
    Developmental stage- and spermatogenic cycle-specific expression of transcription factor GATA-1 in mouse sertoli cells
    Development 120:1759–1766.

Decision letter

  1. Patricia J Wittkopp
    Senior Editor; University of Michigan, United States
  2. Jeannie T Lee
    Reviewing Editor; Massachusetts General Hospital, United States
  3. Julie Cocquet
    Reviewer; Institut Cochin, Inserm U1016, Paris Descartes University, France

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

The new insights into Rlim and the unexpected role that it plays in male spermatogenesis will be of interest to readers of eLife.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "Deficient spermatogenesis and sperm maturation in mice lacking Rlim" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous. Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we cannot proceed with the current form of the manuscript.

Your manuscript revisits the effect of RLIM deficiency on male fertility. Previously, your work showed that Rlim deletion has no obvious effect on male fertility (Shin et al., 2010), but the current study shows expression of a testis-specific Rlim transcript variant with a shorter 3' UTR and more detailed study shows that KO males have smaller testes, epididymides, fewer sperm, more frequent morphological abnormalities, lower motility, and aberrant gene expression. Furthermore, embryos generated from in vitro fertilisation using Rlim KO sperm progress to blasts at a lower rate.

These are interesting observations, but all reviewers find the study to be on the preliminary side and all agree that a characterization of mechanism would be required for publication in eLife. The reviewers pointed out a number of additional technical issues that would need to be addressed. We recognize that you might be able to perform additional work to shed light on mechanism and address technical concerns. We would ordinarily only proceed to a revised manuscript if the amount of work required for revision can be managed in 1-2 months. In this case, we feel that much more than 2 months would be needed. Although we reject the current version of the paper, we invite you to resubmit the manuscript should you be able to perform additional experiments to elucidate how Rlim defects can lead to defective spermatogenesis. We do not know what the reviewers specifically have in mind, but we imagine that something like an RNA-seq analysis that identifies differentially expressed genes involved in KO versus control sperm at the level of metabolomics and/or lipidomics. Alternatively, a mass spec analysis for differentially ubiquinated proteins or differences in protein profiles might be helpful.

Reviewer #1:

In previous work, the Bach lab showed that Rlim deletion has no obvious effect on male fertility (Shin et al., 2010). In this follow-up manuscript, they show that RLIM protein is expressed in pachytene spermatocytes, Sertoli cells (testis) and epithelial cells (epididymis). Northern blot and RNA-seq analyses reveal expression of a testis-specific Rlim transcript variant with a shorter 3' UTR. Loss of function of Rlim using Sox2-Cre-induced whole embryonic knockout (KO) shows that Rlim KO males have smaller testes, with dysregulated gene expression. Rlim KO males also have smaller epididymides, which contain fewer sperm, more frequent morphological abnormalities and lower motility. Accordingly, embryos generated from in vitro fertilisation using Rlim KO sperm develop to blastocysts at a lower rate compared to control.

My comments are as follows:

1) In its current form the study doesn't provide mechanistic insight. The analysis is incremental and would not contribute to the significant advance of reproductive biology. Therefore, whether the findings are of the highest scientific importance which eLife aims to publish is unclear.

2) Figure 2A: western blot data of RLIM should be added. If the short variant has a full-length ORF, protein size would be the same in testis and other tissues.

3) Figure 3A, B: please indicate age of the mice used for analysis. What does "testis 1/2" mean in B? If just single testis of each mouse was weighed, the data might be biased and mean weight of both testes should have been used. Is apoptosis in Rlim KO testis observed as it could explain smaller testis size?

4) Figure 3C-E: it is unclear whether gene dysregulation in the KO occurs in germ cells or other cell types. The authors could sort different stages of spermatogenic cells in testis (example shown in Bastos et al., Cytometry Part A, 2005) and perform RNA-seq analysis.

5) Figure 3—figure supplement 1: the macro-H2A1 data is not convincing, and looks like background staining. I would suggest performing immunofluorescence staining of meiocyte nuclear spreads. Also, marker protein expression doesn't reveal whether XY silencing is affected. The authors' RNA-seq data could be used to ask whether sex-genes are de-repressed or not. Again, cell-type specific analysis suggested in comment 3 is important as the authors can focus the analysis on pachytene cell population, in which sex chromosome silencing occurs.

6) Figure 4-6: the relationship between RLIM expression and the spermiogenesis phenotype is not clear. This mechanistic deficiency could be addressed using Cre lines that deplete Rlim in specific cell types (e.g. pachytene, Sertoli cells).

Reviewer #2:

This study reported a role of Rlim in male reproduction. RLIM is a RING finger ubiquitin E3 ligase. Previous studies have shown that Rlim knockout females are embryonic lethal due to a failure in the maintenance of X-inactivation in extraembryonic tissues but KO males are fertile. In the current study, Rlim expression was examined in both testis and epididymis at both protein and RNA levels. It was concluded that RLIM protein level peaks in pachytene spermatocytes. The Rlim KO males show reduced sperm count and decreased testis weight. The KO sperm display reduced motility and reduced fertilization rate in vitro. RNA-seq on adult KO and control testes revealed altered transcriptome. While sperm and fertilization defects are well characterized and interesting, the overall study is very preliminary. It is unclear how inactivation of Rlim causes such sperm defects. No attempt was made to identify the RLIM target proteins in testis. The fact that the Rlim KO males are still fertile diminishes its significance. There is a major problem with data interpretation. Overall, I don't feel that this preliminary study represents a significant contribution.

1) Expression of RLIM protein in testis: The expression data interpretation (Figure 1) is not correct. It was concluded that RLIM expression peaks in pachytene spermatocytes at stages VI/VII (Figure 1B). RLIM-positive cells are also PNA-positive (acrosomal caps) and thus are round spermatids instead of pachytene spermatocytes. RLIM-positive round spermatids are obvious in the Figure 1A-1 tubule. Based on the data presented, the correct interpretation is that Rlim is expressed in round spermatids and Sertoli cells in the testis. While this mistake can be easily corrected, it has impacted the entire manuscript in a wrong way, which needs to be re-interpreted and re-written.

2) Figure 2 shows the generation of a short testis-specific transcript due to alternative polyadenylation. This is quite common in testis and is not really meaningful.

3) Impact on MSCI. First, Rlim is not expressed in pachytene spermatocytes (see point #1), thus discussion of MSCI is not relevant. Second, gammaH2AX and macroH2A1 are very abundant, their presence may not necessarily indicate lack of effect on MSCI. It is better to look at the expression of sex-linked (X-linked and Y-linked) genes from their RNA-seq data. If MSCI is not affected, sex-linked genes should not be preferentially affected.

4) The RNA-seq was performed on adult whole testis instead of enriched germ cell types. Given that the cKO testis is smaller and the sperm count is lower, differential expression of genes (DEGs) identified could be due to the reduced proportion of germ cells in the KO testis. This needs to be taken into consideration.

Reviewer #3:

Wang et al. identified the localization of RLIM in mouse testes and epididymis. They also made RlimcKO/Y mice and found decreased sperm production with high rates of morphological abnormalities as well as decrease in motility and fertilization rates in vitro. This study uncovered functions of RLIM in male reproduction, which may indicate potential evolutionary pressure for this gene. However, the study did not well illustrate the mechanism of the gene's functions in spermatogenesis, and some results they showed are not well analyzed. Thus, I suggest this paper to be rejected.

1) Though the authors showed abnormal sperm morphology and decreased sperm motility in RlimcKO/Y mice, they did not identify the exact stage for the appearance of the phenotype, e.g. meiosis, post meiosis or during fertilization. Thus, they did not know the functional stage of this gene, which made it impossible for mechanism studies.

2) The authors suggested that RLIM has "high and dynamic expression in specific cell types including Sertoli cells as well as differentiating spermatocytes at the pachytene stage". However, according to the staining pattern of RLIM in Figure 1B, RLIM is exclusively localized in germ cells expressing PNA, which is only expressed in post meiotic cells. Thus, RLIM is not expressed in "differentiating spermatocytes at the pachytene stage".

3) Most of the major conclusions raised in this study are supported by a single experiment, which are not very convincing.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your article "Deficient spermiogenesis in mice lacking Rlim" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Patricia Wittkopp as the Senior and Reviewing Editor. The following individual involved in review of your submission has agreed to reveal their identity: Julie Cocquet (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Summary:

This paper examines the role of Rlim E3 ubiquitin ligase during mouse spermatogenesis. The authors show that spermatozoa from Rlim knockout (KO) males are malformed, less motile and present with a defect in cytoplasmic reduction leading to a small decrease in their fertilizing abilities. They looked at Rlim testicular expression and identified a smaller transcript isoform which is highly expressed in postmeiotic cells. Expression in epididymis epithelium was also observed but its role remains unclear. Finally by producing a male germ cell specific KO, the authors conclude that Rlim postmeiotic isoform is at the basis of the observed sperm defects in the KO.

This is a resubmission of work rejected previously because it failed to elucidate how Rlim defects can lead to defective spermatogenesis. While all three current reviewers (two of which were not involved in the initial evaluation) applauded the addition of new data (i.e., additional data of metabolite and detailed morphology analyses of Sox2-Cre Rlim KO sperm, as well as conditional Rlim KO analyses using Cre lines specific to testicular germ cells or Sertoli cells), they also felt that the issues raised in the first round of revision (e.g., the molecular function of Rlim in spermatids) had not been fully addressed. They were optimistic, however, that they could be addressed with another round of revision addressing the points below.

Essential revisions:

1) Reviewer 1, point 1) The authors showed that germline Rlim KO phenocopies the Sox2-Cre Rlim KO phenotype in sperm (Figure 7). In this experiment, quantification of RLIM protein decrease in the target cell types (germ cells in Ngn3-Cre KO and Sertoli cells in Sf1-Cre KO) should be shown. Also, is the IVF phenotype (Figure 4 F-G) also observed in the Ngn3-Cre Rlim KO? This will provide more important and direct evidence than the morphology analyses in Figure 7 to show sperm defects in the Ngn3-Cre Rlim KO.

2) Reviewer 1, point 2) Figure 2A: The data requested was RLIM western blot using wildtype tissues to compare the RLIM protein size of the testis-specific short variant and the long variant expressed in other tissues. Because ORF is shared in these transcripts, their protein size would be same. This data should be added in the revision.

3) Reviewer 1, point 9) It is stated that "the Cauda region of many cKO/YSox2-Cre animals contained thinner tubules when compared to control males". The requested data was quantification of this thinner tubule phenotype. This data should be added in the revision.

Reviewer 1, point 10) Figure 4—figure supplement 1F suggests that Rlim deletion in sperm affects embryonic development after fertilisation. This point should be discussed more.

4) Throughout the Introduction including on the first line there is in appropriate reference to adult spermatogenesis involving primordial germ cells. These are not present in adult testes – should be undifferentiated spermatogonia or spermatogonial stem cells instead e.g. the progression of PGCs to mature spermatozoa should be progression of spermatogonia; stem cells to mature spermatozoa

5) First paragraph of Introduction – midpiece is a section of the sperm tail so should read “formation of the sperm head and tail” or to be more precise “the formation of the sperm head, HTCA and tail” not “formation of the sperm head, midpiece and tail”

6) First paragraph of Introduction – “the number of genes involved remain limited.” Should be “the number of genes identified to be involved remains limited.”

7) When describing which spermatid population RLIM is expressed in should use spermatid step not just seminiferous tubule stage (as some tubules have two types of spermatids in them so it is better practice) e.g. the spermatids that it is expressing in stage VI/VII – are step 6-7 spermatids. Also, the meaning would be clearer especially to non-specialists if you change the following sentence:

RLIM levels are dramatically upregulated specifically in round spermatids that have undergone meiosis at stages VI / VII, an early timepoint in spermiogenesis (O'Donnell, 2014; Qian et al., 2014).”

to

RLIM levels are dramatically upregulated specifically in post-meiotic step 6-7 round spermatids (stages VI / VII), an early timepoint in spermiogenesis (O'Donnell, 2014; Qian et al., 2014).”

As previous wording could be read to mean that round spermatids undergo meiosis at stage VI / VII which is of course incorrect. Also step 6-7 isn't super early in spermiogenesis it's about 1/3-1/2 to half the way through, what is notable is it is immediately before spermatids begin to elongate in step 8. While you make reference to the fact that its expression is no longer detected in spermatozoa that are released during spermiation – it would also be good to make reference in the results text to the fact that its expression in other spermatid steps is low e.g. once they begin to elongate it goes down and beforehand in step 3 it is low. More precise definition of the all the spermatids steps it is upregulated in would be beneficial

8) Figure 1B bottom panel is incorrectly staged. Based on acrosome morphology that is likely stage XI not IX, as the acrosome morphology shows that the apical hook/falciform shape has been acquired

9) More care needs to be taken with basic fertility phenotyping and characterisation of spermatogenesis. For example:

a) The use of swim out from the epididymis is not a quantitative method of sperm numbers as it is dependent on the cuts made as to how many sperm vacate, in addition non-motile sperm will be less likely to vacate the epididymis. Accurate sperm counts require homogenisation of the epididymis, particularly given the KO mice have lower motility the intrinsic bias of the sperm swim out towards motile sperm may results in KO sperm counts appearing to be lower than they really are

b) Basic PAS analysis of testis histology of KO testes should be conducted, with particular attention to stage 8 and 9 to see if there are defects in spermiation i.e. are all sperm properly released or do you see spermatid retention at stage 9, do residual bodies form normally, normal size etc (this would help define whether the origin of the cytoplasmic droplet is due to defects during spermiation or if it is related to defects in the normal changes that occur to it during transit through the epididymis). In addition of the steps within spermatids undergo head shaping to identify the origin of these defects.

c) Despite what the previous reviewer 3 said, testis and epididymis weight should not be expressed relative to body weight

d) The double axoneme phenotype is not commonly seen in KO models and the origins of this might reveal important insight about sperm tail formation. Have the authors characterized the head to tail coupling apparatus of these sperm and investigated if this is due to supernumerary basal bodies?

10) The authors need to include data showing validation of their KO models at an mRNA and protein level

11) How do the authors think the gene transcription changes in the RLIM KO mice relates to the phenotypes observed? Some interpretation of this is needed in the Discussion.

12) Result section Rlim expression in testis is highly regulated.

a) It is not clearly presented when exactly RLIM is highly expressed in postmeiotic cells. Is it restricted to stages VI to VIII? Since at stage IX on Figure 2B the signal is no longer strong (but see my comment below regarding staging). On Figure 2A, it seems as if many tubules display strong round spermatid signal. A precise description of RLIM protein dynamic at various stages of spermatogenesis is needed. Recapitulating IF/IHC observations using a scheme showing the different stages of spermatogenesis would be useful.

b) Figure 2

A – IHC panel:

Higher magnification insets from negative CTL would be useful to confirm that the signal observed in Sertoli cells is specific (i.e. not seen in neg control)

B - To me (but the figure is small) the image labelled "stage IX" is not a stage IX. I do not recognize the lectin PNA signal to be a stage IX: At this stage round spermatids have just started to elongate and condensed step 16 spermatids have already undergone spermiation (so are no longer present).

NB. Higher magnification of the DAPI images (in the merged figure for instance) would be helpful.

c) It seems odd to present Rlim expression in epididymal cells in the part entitled "Rlim expression in testis is highly regulated"

13) Result section Diminished production and functionality of Rlim KO sperm

a) Overall I find this part a bit confusing. At first, the testicular impairment seems to be minimized because of the observation that Rlim is expressed in epididymis epithelium. Yet it clearly appears from the last result section that sperm defects originate from a testicular (germ cell) impairment. Maybe this result section could be slightly re-organized?

specific comments/questions:

b) The observation of a small but significant reduction in testis weight indicates a (mild) spermatogenesis defect; so do testicular sperm morphological abnormalities.

Can the authors further investigate testicular cell composition – to see if one particular (likely postmeiotic) cell stage is affected? In the Discussion, it is said that increased cell death and chromatin packaging defects were investigated – even if the results are negative it would be worth presenting (or evoking) them in this section.

Since spermatogenesis is mildly impaired, RNAseq on whole testis should be interpreted with caution, as it could reflect a mild change in cell population. This emphasizes the need for a more detailed presentation of the testicular defects (or absence of visible defects).

c) Sentence: "…revealed that the Cauda region of many cKO/YSox2-Cre animals contained thinner tubules when compared to control males (Figure 3—figure supplement 1E), consistent with decreased Caudal sperm (Figure 3C)."

This sentence suggests that decreased caudal sperm is due to caudal region malformation. But to conclude, one needs to show quantification of testicular spermatozoa (or caput spermatozoa). Figure 6A data could be presented at that point.

But then I am confused: what is the authors' conclusion? is the decreased sperm count a consequence of testicular or epididymis defects? Since epididymis defects appear to be ruled out by the analysis of Ngn3-Cre KO sperm, it would increase clarity if epididymis analyses were presented separately.

14) Discussion

a) The first part about Rlim short isoform could be reduced. While it is an interesting fact, I feel it does not require so much emphasis since testis specific isoforms are quite common.

b) The fact that Rlim is X-encoded and expressed in spermatids does not mean it escapes MSCI (as MSCI occurs in pachytene primary spermatocytes). Instead, it means that Rlim short isoform is one of the many X-linked genes that are expressed after meiosis, when the X is re-activated (see for instance Ernst et al., 2019)

c) Considering the fact that the KO impact on male reproductive abilities (at least for the lab mouse) is minor, I find the part about the evolutionary consequences of a spermatogenesis role for Rlim a bit far-fetched. The paternal effect of Rlim in milk-producing alveolar cells would likely be more predominant (Jiao et al., 2012). I would recommend to put less emphasis on that aspect, especially in the Abstract.

https://doi.org/10.7554/eLife.63556.sa1

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Reviewer #1:

In previous work, the Bach lab showed that Rlim deletion has no obvious effect on male fertility (Shin et al., 2010). In this follow-up manuscript, they show that RLIM protein is expressed in pachytene spermatocytes, Sertoli cells (testis) and epithelial cells (epididymis). Northern blot and RNA-seq analyses reveal expression of a testis-specific Rlim transcript variant with a shorter 3' UTR. Loss of function of Rlim using Sox2-Cre-induced whole embryonic knockout (KO) shows that Rlim KO males have smaller testes, with dysregulated gene expression. Rlim KO males also have smaller epididymides, which contain fewer sperm, more frequent morphological abnormalities and lower motility. Accordingly, embryos generated from in vitro fertilisation using Rlim KO sperm develop to blastocysts at a lower rate compared to control.

My comments are as follows:

1) In its current form the study doesn't provide mechanistic insight. The analysis is incremental and would not contribute to the significant advance of reproductive biology. Therefore, whether the findings are of the highest scientific importance which eLife aims to publish is unclear.

We believe that the revised manuscript provides much more insight into the mechanisms underlying the reproduction phenotype in male mice lacking Rlim. Because Rlim KO sperm displays decreased motility we have investigated the energy status of sperm polar metabolites and surprisingly found increased metabolite content (Figure 5). Detailed EM analyses reveals that cytoplasmic reduction is inhibited in Rlim KO sperm (Figures 5, 6) and using specific Cre drivers to target the Rlim cKO to Sertoli cells or the spermatogenic cell lineage we continue to show that Rlim in round spermatids is required for normal spermiogenesis (Figure 7). Using germline KO we show that the testis phenotype occurs independently of the expression of Cre-recombinase (Figure 3—figure supplement 1B, C). Combined these results identify Rlim as a novel and important regulator of cytoplasmic reduction in sperm, a process that is not well studied despite its major impact on male reproduction, thereby illuminating the evolution of the Rlim gene.

2) Figure 2A: western blot data of RLIM should be added. If the short variant has a full-length ORF, protein size would be the same in testis and other tissues.

Unfortunately, in our experience Western blot data do not allow distinguishing small differences in MW of Rlim protein, e.g. Rlim deletion mutants in which the NES or NLS is deleted (lacking 13 or 20 amino acids) migrate at a very similar position than the WT protein (see also Jiao et al., 2013; Figure 6B).

3) Figure 3A, B: please indicate age of the mice used for analysis. What does "testis 1/2" mean in B? If just single testis of each mouse was weighed, the data might be biased and mean weight of both testes should have been used. Is apoptosis in Rlim KO testis observed as it could explain smaller testis size?

All analyses on sperm have been carried out from sperm originating from 8 weeks old males. Even though we have included both testes halves from all animals in the previous version of the manuscript we agree and have changed the figure to reflect the entire testes (Figure 3A, B). We did not observe apoptosis in RlimKO testes as measured via IHC using act. caspase 3 antibodies and mentioned this in the text (Discussion).

4) Figure 3C-E: it is unclear whether gene dysregulation in the KO occurs in germ cells or other cell types. The authors could sort different stages of spermatogenic cells in testis (example shown in Bastos et al., Cytometry Part A, 2005) and perform RNA-seq analysis.

We have targeted the cKO of Rlim to the early spermatogenic cell lineage or to Sertoli (and Leydig) cells using Ngn3-Cre and Sf1-Cre, respectively. While Ngn3-Cre Rlim cKO mice largely recapitulate defects of the systemic Rlim KO, SF1-Cre cKO mice do not (see also comments to point 6, below). Thus, much of the gene dysregulation in the KO likely occurs in the spermatogenic cell lineage.

5) Figure 3—figure supplement 1: the macro-H2A1 data is not convincing, and looks like background staining. I would suggest performing immunofluorescence staining of meiocyte nuclear spreads. Also, marker protein expression doesn't reveal whether XY silencing is affected. The authors' RNA-seq data could be used to ask whether sex-genes are de-repressed or not. Again, cell-type specific analysis suggested in comment 3 is important as the authors can focus the analysis on pachytene cell population, in which sex chromosome silencing occurs.

We agree with this criticism. Because Rlim exerts its function in the spermatogenic cell lineage, where it is highly expressed in round spermatids only after MSCI and meiosis has already occurred, it likely is not relevant for these processes and we have therefore removed this figure (see also point 3, reviewer 2).

6) Figure 4-6: the relationship between RLIM expression and the spermiogenesis phenotype is not clear. This mechanistic deficiency could be addressed using Cre lines that deplete Rlim in specific cell types (e.g. pachytene, Sertoli cells).

We agree with this criticism and have therefore targeted the cKO of Rlim to the spermatogenic cell lineage or to Sertoli (and Leydig) cells using Ngn3-Cre and Sf1-Cre, respectively. While Ngn3-Cre Rlim cKO mice largely recapitulate defects of the systemic Rlim KO, SF1-Cre cKO mice do not (Figure 7; see also comments to 4). Because Rlim is highly upregulated in round spermatids (Figure 2), this suggests major functions of Rlim during spermiogenesis. Together with the finding of defective cytoplasmic reduction (Figures 5-7), we believe the results shown in the revised manuscript clarify the relation between Rlim expression and the spermiogenesis phenotype.

Reviewer #2:

This study reported a role of Rlim in male reproduction. RLIM is a RING finger ubiquitin E3 ligase. Previous studies have shown that Rlim knockout females are embryonic lethal due to a failure in the maintenance of X-inactivation in extraembryonic tissues but KO males are fertile. In the current study, Rlim expression was examined in both testis and epididymis at both protein and RNA levels. It was concluded that RLIM protein level peaks in pachytene spermatocytes. The Rlim KO males show reduced sperm count and decreased testis weight. The KO sperm display reduced motility and reduced fertilization rate in vitro. RNA-seq on adult KO and control testes revealed altered transcriptome. While sperm and fertilization defects are well characterized and interesting, the overall study is very preliminary. It is unclear how inactivation of Rlim causes such sperm defects. No attempt was made to identify the RLIM target proteins in testis. The fact that the Rlim KO males are still fertile diminishes its significance. There is a major problem with data interpretation. Overall, I don't feel that this preliminary study represents a significant contribution.

1) Expression of RLIM protein in testis: The expression data interpretation (Figure 1) is not correct. It was concluded that RLIM expression peaks in pachytene spermatocytes at stages VI/VII (Figure 1B). RLIM-positive cells are also PNA-positive (acrosomal caps) and thus are round spermatids instead of pachytene spermatocytes. RLIM-positive round spermatids are obvious in the Figure 1A-1 tubule. Based on the data presented, the correct interpretation is that Rlim is expressed in round spermatids and Sertoli cells in the testis. While this mistake can be easily corrected, it has impacted the entire manuscript in a wrong way, which needs to be re-interpreted and re-written.

We agree with this reviewer and have addressed this issue, including re-interpretation and rewriting. Thank you for pointing this out.

2) Figure 2 shows the generation of a short testis-specific transcript due to alternative polyadenylation. This is quite common in testis and is not really meaningful.

While we agree that testis-specific transcripts via alternative polyadenylation are common in testis, we disagree that this is not meaningful, as it might help in the observed upregulation of Rlim protein observed in round spermatids (Figure 2, see also Discussion section), where Rlim actively regulates spermiogenesis. Moreover, the use of this polyadenylation signal is also observed during mouse preimplantation development, when Rlim exerts its crucial role in promoting X chromosome inactivation in females (see Wang et al., 2016; Figure 1E). Finally, our results identify a new and functional polyadenylation signal in the Rlim gene.

3) Impact on MSCI. First, Rlim is not expressed in pachytene spermatocytes (see point #1), thus discussion of MSCI is not relevant. Second, gammaH2AX and macroH2A1 are very abundant, their presence may not necessarily indicate lack of effect on MSCI. It is better to look at the expression of sex-linked (X-linked and Y-linked) genes from their RNA-seq data. If MSCI is not affected, sex-linked genes should not be preferentially affected.

Thank you for pointing this out. Based on this comment we have removed this figure (see also point 5, reviewer 1).

4) The RNA-seq was performed on adult whole testis instead of enriched germ cell types. Given that the cKO testis is smaller and the sperm count is lower, differential expression of genes (DEGs) identified could be due to the reduced proportion of germ cells in the KO testis. This needs to be taken into consideration.

Because the vast majority of cells in the testis reflect spermatogenic cells, where Rlim exerts its functions (Figure 7), we believe that differentially expressed genes as detected via RNA-seq (Figures 3D-F), likely reflect those expressed in this cell lineage, even though in the KO testis there is a slightly lower proportion of this cell type. We have addressed this point in the Discussion section.

Reviewer #3:

Wang et al. identified the localization of RLIM in mouse testes and epididymis. They also made RlimcKO/Y mice and found decreased sperm production with high rates of morphological abnormalities as well as decrease in motility and fertilization rates in vitro. This study uncovered functions of RLIM in male reproduction, which may indicate potential evolutionary pressure for this gene. However, the study did not well illustrate the mechanism of the gene's functions in spermatogenesis, and some results they showed are not well analyzed. Thus, I suggest this paper to be rejected.

1) Though the authors showed abnormal sperm morphology and decreased sperm motility in RlimcKO/Y mice, they did not identify the exact stage for the appearance of the phenotype, e.g. meiosis, post meiosis or during fertilization. Thus, they did not know the functional stage of this gene, which made it impossible for mechanism studies.

We have now shown that deletion of Rlim in the spermatogenic cell lineage is largely responsible for the sperm phenotype (Figure 7). Taken together with Rlim expression pattern in this lineage (Figure 2), our results provide strong evidence that Rlim in round spermatids is required for normal spermiogenesis (see also point 6, reviewer 1).

2) The authors suggested that RLIM has "high and dynamic expression in specific cell types including Sertoli cells as well as differentiating spermatocytes at the pachytene stage". However, according to the staining pattern of RLIM in Figure 1B, RLIM is exclusively localized in germ cells expressing PNA, which is only expressed in post meiotic cells. Thus, RLIM is not expressed in "differentiating spermatocytes at the pachytene stage".

Thank you for pointing this out (see also point 1, reviewer 2). We have corrected this error.

3) Most of the major conclusions raised in this study are supported by a single experiment, which are not very convincing.

It is unclear what the reviewer means with this comment. All experiments have been carried out several times on multiple biological replicates to obtain significant results. The testis phenotype is observed in mice with three different genetic settings: Germline Rlim KO, cKO/YSox2-Cre and cKO/YNgn3-Cre but not in cKO/YSf1-Cre males (Figure 3—figure supplement 1, Figure 3 and Figure 7, respectively).

[Editors’ note: what follows is the authors’ response to the second round of review.]

Essential revisions:

1) Reviewer 1, point 1) The authors showed that germline Rlim KO phenocopies the Sox2-Cre Rlim KO phenotype in sperm (Figure 7). In this experiment, quantification of RLIM protein decrease in the target cell types (germ cells in Ngn3-Cre KO and Sertoli cells in Sf1-Cre KO) should be shown.

Based on this comment, we now show that RLIM protein is undetectable in testes of Sox2-Cre cKO/Y mice via Western blotting (new Figure 2—figure supplement 1B), corroborating IHC results in Figure 2A. We have also performed IHC on testes sections of Ngn3-Cre cKO/Y and Sf1-Cre cKO/Y mice following RLIM expression. While Ngn3-Cre cKO mice lack immunoreactivity in the spermatogenic cell lineage, the staining of Sertoli cells appears normal. For Sf1-Cre cKO it is the inverse: immunoreactivity in Sertoli cells is low/not detectable while round spermatids stain normally. These results show correct targeting with high specificity and penetrance (new Figure 7A). See also point 10, below.

Also, is the IVF phenotype (Figure 4 F-G) also observed in the Ngn3-Cre Rlim KO? This will provide more important and direct evidence than the morphology analyses in Figure 7 to show sperm defects in the Ngn3-Cre Rlim KO.

Based on this comment we now show a similar IVF phenotype in the Ngn3-Cre cKO (new Figure 7G), thereby corroborating functions of Rlim in the spermatogenic cell lineage.

2) Reviewer 1, point 2) Figure 2A: The data requested was RLIM western blot using wildtype tissues to compare the RLIM protein size of the testis-specific short variant and the long variant expressed in other tissues. Because ORF is shared in these transcripts, their protein size would be same. This data should be added in the revision.

We have performed the requested Western blot (new Figure 2—figure supplement 1A) demonstrating expression of full length protein in testes.

3) Reviewer 1, point 9) It is stated that "the Cauda region of many cKO/YSox2-Cre animals contained thinner tubules when compared to control males". The requested data was quantification of this thinner tubule phenotype. This data should be added in the revision.

Based on this comment we have tried to quantify the thinner tubule phenotype using ImageJ. However, our results did not reach significance levels (P=0.14). Because 1. data on epididymis caused confusion (see points 12c, 13a), and 2. the Rlim KO phenotype is testicular, the epididymis is not of high relevance for the paper, we have removed this statement. All data on epididymis are now shown in a single Figure (Figure 3—figure supplement 2; see also points 12c and 13a).

Reviewer 1, point 10) Figure 4—figure supplement 1F suggests that Rlim deletion in sperm affects embryonic development after fertilisation. This point should be discussed more.

Because male mice lacking Rlim develop normally during preimplantation development (see Shin et al., 2010; 2014; see also Figure 3 —figure supplement 1A), our results in IVF (Figure 4—figure supplement 1F) indicates that the lack of Rlim appears to affect early embryo development specifically under in vitro conditions. We have added this in the text.

4) Throughout the Introduction including on the first line there is in appropriate reference to adult spermatogenesis involving primordial germ cells. These are not present in adult testes – should be undifferentiated spermatogonia or spermatogonial stem cells instead e.g. the progression of PGCs to mature spermatozoa should be progression of spermatogonia; stem cells to mature spermatozoa

Thank you for pointing this out. We have corrected this.

5) First paragraph of Introduction – midpiece is a section of the sperm tail so should read “formation of the sperm head and tail” or to be more precise “the formation of the sperm head, HTCA and tail” not “formation of the sperm head, midpiece and tail”

We have corrected this.

6) First paragraph of Introduction – “the number of genes involved remain limited.” Should be “the number of genes identified to be involved remains limited.”

Corrected.

7) When describing which spermatid population RLIM is expressed in should use spermatid step not just seminiferous tubule stage (as some tubules have two types of spermatids in them so it is better practice) e.g. the spermatids that it is expressing in stage VI/VII – are step 6-7 spermatids. Also, the meaning would be clearer especially to non-specialists if you change the following sentence:

“RLIM levels are dramatically upregulated specifically in round spermatids that have undergone meiosis at stages VI / VII, an early timepoint in spermiogenesis (O'Donnell, 2014; Qian et al., 2014).”

to

“RLIM levels are dramatically upregulated specifically in post-meiotic step 6-7 round spermatids (stages VI / VII), an early timepoint in spermiogenesis (O'Donnell, 2014; Qian et al., 2014).”

As previous wording could be read to mean that round spermatids undergo meiosis at stage VI / VII which is of course incorrect. Also step 6-7 isn't super early in spermiogenesis it's about 1/3-1/2 to half the way through, what is notable is it is immediately before spermatids begin to elongate in step 8. While you make reference to the fact that its expression is no longer detected in spermatozoa that are released during spermiation – it would also be good to make reference in the results text to the fact that its expression in other spermatid steps is low e.g. once they begin to elongate it goes down and beforehand in step 3 it is low. More precise definition of the all the spermatids steps it is upregulated in would be beneficial

Thank you for pointing this out. In response, we have rewritten this paragraph, addressing these points in the revised manuscript. We have also added a panel in Figure 2B that allows more precise staging of RLIM expression in spermatids. See also our response points 8 and 12b below.

8) Figure 1B bottom panel is incorrectly staged. Based on acrosome morphology that is likely stage XI not IX, as the acrosome morphology shows that the apical hook/falciform shape has been acquired

We agree and have adjusted the staging in Figure 2B (see also point 12).

9) More care needs to be taken with basic fertility phenotyping and characterisation of spermatogenesis. For example:

a) The use of swim out from the epididymis is not a quantitative method of sperm numbers as it is dependent on the cuts made as to how many sperm vacate, in addition non-motile sperm will be less likely to vacate the epididymis. Accurate sperm counts require homogenisation of the epididymis, particularly given the KO mice have lower motility the intrinsic bias of the sperm swim out towards motile sperm may results in KO sperm counts appearing to be lower than they really are

We agree with this statement. We have changed the text to reflect for the intrinsic bias in swim out sperm numbers towards motile sperm. All sperm preps have been made by the same person (F.W.) ensuring similar cuts and the yields between animals of the same genotype were relatively reproducible (see error margins in Figure 3C).

b) Basic PAS analysis of testis histology of KO testes should be conducted, with particular attention to stage 8 and 9 to see if there are defects in spermiation i.e. are all sperm properly released or do you see spermatid retention at stage 9, do residual bodies form normally, normal size etc (this would help define whether the origin of the cytoplasmic droplet is due to defects during spermiation or if it is related to defects in the normal changes that occur to it during transit through the epididymis). In addition of the steps within spermatids undergo head shaping to identify the origin of these defects.

Based on this comment we have performed PAS analyses. Focusing on stage 9 tubules, we did not see signs of improper sperm release (new Figure 3—figure supplement 1D). Because low contrast did not allow for a reliable identification and interrogation of small vesicles including residual bodies, we examined signs of cytoplasmic reduction during spermiation in TEM on testis sections. We did not observe obvious/major defects during cytoplasmic reduction in TEM (new Figure 7—figure supplement 1D). However, as we were unable to quantify droplet/lobe size on spermatids in TEM because of differing section angles combined with an uneven distribution of the droplet over the midpiece, these results do not exclude mild defects. Incorporating these data, because of increased metabolite content (Figure 4A-C), our combined results suggest some defects in cytoplasmic reduction in RlimKO spermatids during spermiation that might become exacerbated after sperm release due to difficulties in adjusting to a changing environment. This is discussed in the text.

c) Despite what the previous reviewer 3 said, testis and epididymis weight should not be expressed relative to body weight

Testis weight is commonly expressed relative to body weight. In this paper all three Cre drivers used are in a different genetic background: While Sox2-Cre mice are in a congenic C57Bl/6 background, the Sf1-Cre and the Ngn3-Cre mice are in different mixed backgrounds and animals display different sizes and weights and swim-out sperm numbers in controls differ (see Figure 7). We feel that it is important under these circumstances to normalize organ weights against the body weight to render data better comparable.

d) The double axoneme phenotype is not commonly seen in KO models and the origins of this might reveal important insight about sperm tail formation. Have the authors characterized the head to tail coupling apparatus of these sperm and investigated if this is due to supernumerary basal bodies?

The occurrence of sperm with 2 axonemes in RlimKO animals is relatively rare (Figure 6B) and thus represents a minor phenotype. Staining swim-out sperm with antibodies against Centrin2 did not reveal frequent occurrence of supernumerary basal bodies. Therefore, we believe that the proposed investigations are beyond the scope of our study.

10) The authors need to include data showing validation of their KO models at an mRNA and protein level

See also our response to point 1. Using Sox2-Cre to target the Rlim cKO, we have previously demonstrated correct targeting of the floxed region at the mRNA level via single embryo RNA-seq (see Wang et al., 2016; Figure 1E; the parental Rlim has been targeted). Correct and efficient targeting of RLIM protein in testis by Sox2-Cre is now shown via IHC (Figure 2A) and Western blot (Figure 2—figure supplement 1B). Concerning Ngn3-Cre cKO and Sf1-Cre cKO mice, we have performed IHC on testes sections showing correct targeting with high specificity and penetrance (new Figure 7A).

11) How do the authors think the gene transcription changes in the RLIM KO mice relates to the phenotypes observed? Some interpretation of this is needed in the Discussion

Based on this comment we have added a paragraph on the interpretation of our RNA-seq results in the Discussion section, as requested.

12) Result section Rlim expression in testis is highly regulated

a) It is not clearly presented when exactly RLIM is highly expressed in postmeiotic cells. Is it restricted to stages VI to VIII? Since at stage IX on Figure 2B the signal is no longer strong (but see my comment below regarding staging). On Figure 2A, it seems as if many tubules display strong round spermatid signal. A precise description of RLIM protein dynamic at various stages of spermatogenesis is needed. Recapitulating IF/IHC observations using a scheme showing the different stages of spermatogenesis would be useful.

We have added a new panel in Figure 2B that shows high RLIM expression in step 7-8 spermatids. Thus, RLIM expression is high in spermatids from step 6-8, but low at spermatid stages before and after that. We have made this clear in the text. See also our response to point 7.

b) Figure 2

A – IHC panel:

Higher magnification insets from negative CTL would be useful to confirm that the signal observed in Sertoli cells is specific (i.e. not seen in neg control)

Based on this comment we have added a higher magnification inset in Figure 2A, which we believe makes it clear that RLIM is no longer present in Sertoli cells in the Sox2-Cre cKO/Y.

B – To me (but the figure is small) the image labelled "stage IX" is not a stage IX. I do not recognize the lectin PNA signal to be a stage IX: At this stage round spermatids have just started to elongate and condensed step 16 spermatids have already undergone spermiation (so are no longer present).

NB. Higher magnification of the DAPI images (in the merged figure for instance) would be helpful.

We agree and have changed the Figure accordingly and added a higher magnification inset of DAPI images, as requested. See also our response to points 7 and 12 above.

c) It seems odd to present Rlim expression in epididymal cells in the part entitled "Rlim expression in testis is highly regulated"

In response to this comment and points 3 and 13a, to avoid confusion, we have summarized all data on epididymis in a single paragraph and figure (Figure 3—figure supplement 2), as Rlim does not play a major role in epididymis.

13) Result section Diminished production and functionality of Rlim KO sperm

a) Overall I find this part a bit confusing. At first, the testicular impairment seems to be minimized because of the observation that Rlim is expressed in epididymis epithelium. Yet it clearly appears from the last result section that sperm defects originate from a testicular (germ cell) impairment. Maybe this result section could be slightly re-organized?

specific comments/questions:

In response to this comment and points 3 and 12c, to avoid confusion, we have summarized all data on epididymis in a single paragraph and figure (Figure 3—figure supplement 2), as Rlim does not play a major role in epididymis

b) The observation of a small but significant reduction in testis weight indicates a (mild) spermatogenesis defect; so do testicular sperm morphological abnormalities.

Can the authors further investigate testicular cell composition – to see if one particular (likely postmeiotic) cell stage is affected? In the Discussion, it is said that increased cell death and chromatin packaging defects were investigated – even if the results are negative it would be worth presenting (or evoking) them in this section.

Our PAS analyses (Figure 3—figure supplement 1D; data not shown) did not reveal major visible changes at the level of testicular cell composition. We have mentioned in the Results section that no cell death was observed. No obvious chromatin packaging defect was observed as judged by sperm head density in TEM and is mentioned in the TEM section.

Since spermatogenesis is mildly impaired, RNAseq on whole testis should be interpreted with caution, as it could reflect a mild change in cell population. This emphasizes the need for a more detailed presentation of the testicular defects (or absence of visible defects).

We agree with the reviewer. We have added in the Discussion section the sentence: “However, we cannot exclude the possibility that subtle and hence undetected changes in testicular cell types in mice lacking Rlim (Figure 3—figure supplement 1D) may contribute to the observed differences in gene expression as well as testes weight (Figure 3).”

c) Sentence: "…revealed that the Cauda region of many cKO/YSox2-Cre animals contained thinner tubules when compared to control males (Figure 3—figure supplement 1E), consistent with decreased Caudal sperm (Figure 3C)."

This sentence suggests that decreased caudal sperm is due to caudal region malformation. But to conclude, one needs to show quantification of testicular spermatozoa (or caput spermatozoa). Figure 6A data could be presented at that point.

But then I am confused: what is the authors' conclusion? is the decreased sperm count a consequence of testicular or epididymis defects? Since epididymis defects appear to be ruled out by the analysis of Ngn3-Cre KO sperm, it would increase clarity if epididymis analyses were presented separately.

See also our response to points 3 and 12c. Based on this comment we have we have summarized all data on epididymis in a single paragraph and figure (Figure 3—figure supplement 2), as requested.

14) Discussion

a) The first part about Rlim short isoform could be reduced. While it is an interesting fact, I feel it does not require so much emphasis since testis specific isoforms are quite common.

We agree and have condensed this section in the Discussion.

b) The fact that Rlim is X-encoded and expressed in spermatids does not mean it escapes MSCI (as MSCI occurs in pachytene primary spermatocytes). Instead, it means that Rlim short isoform is one of the many X-linked genes that are expressed after meiosis, when the X is re-activated (see for instance Ernst et al., 2019)

Thank you for pointing this out. This has been corrected in the revised manuscript.

c) Considering the fact that the KO impact on male reproductive abilities (at least for the lab mouse) is minor, I find the part about the evolutionary consequences of a spermatogenesis role for Rlim a bit far-fetched. The paternal effect of Rlim in milk-producing alveolar cells would likely be more predominant (Jiao et al., 2012). I would recommend to put less emphasis on that aspect, especially in the Abstract.

We agree and have removed this aspect in the Abstract and shortened the corresponding paragraph in the Discussion.

https://doi.org/10.7554/eLife.63556.sa2

Article and author information

Author details

  1. Feng Wang

    Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, United States
    Contribution
    Conceptualization, Formal analysis, Investigation, Methodology, Writing - original draft
    Competing interests
    No competing interests declared
  2. Maria Gracia Gervasi

    Department of Veterinary & Animal Sciences, University of Massachusetts Amherst, Amherst, United States
    Contribution
    Formal analysis, Investigation, Methodology, Writing - original draft
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5468-2700
  3. Ana Bošković

    Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  4. Fengyun Sun

    Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  5. Vera D Rinaldi

    Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0051-1754
  6. Jun Yu

    Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  7. Mary C Wallingford

    Department of Veterinary & Animal Sciences, University of Massachusetts Amherst, Amherst, United States
    Present address
    Mother Infant Research Institute, Tufts Medical Center, Boston, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  8. Darya A Tourzani

    Department of Veterinary & Animal Sciences, University of Massachusetts Amherst, Amherst, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  9. Jesse Mager

    Department of Veterinary & Animal Sciences, University of Massachusetts Amherst, Amherst, United States
    Contribution
    Supervision
    Competing interests
    No competing interests declared
  10. Lihua Julie Zhu

    1. Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, United States
    2. Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, United States
    3. Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, United States
    Contribution
    Supervision
    Competing interests
    No competing interests declared
  11. Oliver J Rando

    Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
    Contribution
    Supervision
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1516-9397
  12. Pablo E Visconti

    Department of Veterinary & Animal Sciences, University of Massachusetts Amherst, Amherst, United States
    Contribution
    Supervision
    Competing interests
    No competing interests declared
  13. Lara Strittmatter

    Electron Microscopy Core, University of Massachusetts Medical School, Worcester, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  14. Ingolf Bach

    1. Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, United States
    2. Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, United States
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Writing - original draft, Writing - review and editing
    For correspondence
    ingolf.bach@umassmed.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4505-8946

Funding

National Institutes of Health (GM128168)

  • Ingolf Bach

National Institutes of Health (HD080224)

  • Oliver J Rando

National Institutes of Health (HD038082)

  • Pablo E Visconti

National Institutes of Health (DP1ES025458)

  • Oliver J Rando

University of Massachusetts (DK32520)

  • Ingolf Bach

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We are grateful to M Krykbaeva, L Huang, A Schlueter, G Pazour, J Shin and E Torres for advice and/or experimental help. We thank A Leiter for providing Ngn3-Cre mice and the Metabolite Profiling Core Facility at the Whitehead Institute for running metabolomics samples and for data analysis. IB is a member of the University of Massachusetts DERC (DK32520). This work was supported from NIH grants R01GM128168 to IB, R01HD080224 and DP1ES025458 to OJR, and R01HD38082 to PEV.

Ethics

Animal experimentation: All mice were housed in the animal facility of UMMS and utilized according to NIH guidelines and those established by the UMMS Institute of Animal Care and Usage Committee (IACUC; protocol #201900344).

Senior Editor

  1. Patricia J Wittkopp, University of Michigan, United States

Reviewing Editor

  1. Jeannie T Lee, Massachusetts General Hospital, United States

Reviewer

  1. Julie Cocquet, Institut Cochin, Inserm U1016, Paris Descartes University, France

Publication history

  1. Received: September 29, 2020
  2. Accepted: February 22, 2021
  3. Accepted Manuscript published: February 23, 2021 (version 1)
  4. Accepted Manuscript updated: February 24, 2021 (version 2)
  5. Version of Record published: March 5, 2021 (version 3)

Copyright

© 2021, Wang et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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