Deficient spermiogenesis in mice lacking Rlim
- Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, United States
- Department of Veterinary & Animal Sciences, University of Massachusetts Amherst, United States
- Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, United States
- Program in Molecular Medicine, University of Massachusetts Medical School, United States
- Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, United States
- Electron Microscopy Core, University of Massachusetts Medical School, United States
Figures
Figure 1
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 …
Figure 2 with 1 supplement
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 …
Figure 2—figure supplement 1
RLIM protein in testis.
(A) Comparison of RLIM protein expression in mouse spleen, brain, and testis using western blot analysis. Note high levels of full-length RLIM (arrow) in testis. As loading control, the same blot …
Figure 3 with 2 supplements
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 …
Figure 3—figure supplement 1
Decreased sperm production in males lacking Rlim.
(A) Rlim has no essential functions during male embryogenesis and post-natal development. Using Rlimfl/KO as dames, a similar number of males out of 9 litters receiving a germline KO or the fl Rlim …
Figure 3—figure supplement 2
Normal epididymal appearance in males lacking Rlim.
(A) Robust staining of most/all nuclei of epithelial cells lining epididymal tubules in IHC on a cross-section through the Cauda region of fl/Y males using Rlim antibodies. cKO/YSox2-Cre sections …
Figure 4 with 1 supplement
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 …
Figure 4—figure supplement 1
Evaluation of molecular and physiological events related to sperm capacitation.
Sperm were incubated at 37°C for 60 min in m-TYH (non-capacitating conditions, NC) or in m-TYH supplemented with 15 mM NaHCO3 and 5 mg/ml BSA (capacitating conditions, (C)). (A) After 60 min of …
Figure 5
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 …
Figure 6 with 1 supplement
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 …
Figure 6—figure supplement 1
Evaluation of sperm structure within testes of cKO/YSox2-Cre animals.
(A) Examples of cKO/YSox2-Cre sperm exhibiting two axonemes (SEM). Left panel: Sperm with two axonemes. Boxed area is shown in higher magnification. Middle panel: Sperm with two axonemes and …
Figure 7 with 1 supplement
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 …
Figure 7—figure supplement 1
Normal numbers of Sertoli cells in testes lacking Rlim.
(A) DAB staining of paraffin-embedded testes sections of cKO/YSox2-Cre and fl/Y animals using antibodies against Sertoli cell marker GATA4. Scale bars = 150 μm. (B) Quantification of GATA4-positive …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (M. musculus) | Rlimflox | PMID:20962847 | MGI:1342291 | |
| Genetic reagent (M. musculus) | Sox2-Cre | Jackson Laboratory | JAX #008454 | PMID:14516668 |
| Genetic reagent (M. musculus) | Neurog3-Cre (Ngn3-Cre) | PMID:15183725 | JAX #005667 | Dr. Andrew Leiter (UMMS) |
| Genetic reagent (M. musculus) | Sf1-Cre | Jackson Laboratory | JAX #012462 | PMID:16423694 |
| Antibody | Rabbit anti-Rlim (rabbit polyclonal) | PMID:11882901 | IHC (1:250) WB (1:1000) | |
| Antibody | Rat anti-GATA1 (rat monoclonal) | Santa Cruz | sc265 | IHC (1:100) |
| Antibody | Rabbit anti-GATA4 (rabbit polyclonal) | Abcam | ab84593 | IHC (1:500) |
| Antibody | Rabbit anti-phosphoPKA (rabbit monoclonal) | Cell Signaling clone 100G7E | 9624 | WB (1:10000) |
| Antibody | Mouse anti-PY (mouse monoclonal) | Millipore clone 4G10 | 05–321 | WB (1:10000) |
| Antibody | Mouse anti- β-actin (mouse monoclonal) | Santa Cruz | sc47778 | WB (1:250) |
| Antibody | Rabbit anti-cleaved caspase3 (rabbit monoclonal) | Cell Signaling | ab9664 | IHC (1:300) |
