Differential susceptibility of male and female germ cells to glucocorticoid-mediated signaling

Reviewer #1 (Public Review):

Summary:
Cincotta et al set out to investigate the presence of glucocorticoid receptors in the male and female embryonic germline. They further investigate the impact of tissue-specific genetically induced receptor absence and/or systemic receptor activation on fertility and RNA regulation. They are motivated by several lines of research that report inter and transgenerational effects of stress and or glucocorticoid receptor activation and suggest that their findings provide an explanatory mechanism to mechanistically back parental stress hormone exposure-induced phenotypes in the offspring.

Strengths:
- A chronological immunofluorescent assessment of GR in fetal and early life oocyte and sperm development.
- RNA seq data that reveal novel cell type specific isoforms validated by q-RT PCR E15.5 in the oocyte.
- 2 alternative approaches to knock out GR to study transcriptional outcomes. Oocytes: systemic GR KO (E17.5) with low input 3-tag seq and germline-specific GR KO (E15.5) on fetal oocyte expression via 10X single cell seq and 3-cap sequencing on sorted KO versus WT oocytes - both indicating little impact on polyadenylated RNAs
- 2 alternative approaches to assess the effect of GR activation in vivo (systemic) and ex vivo (ovary culture): here the RNA seq did show again some changes in germ cells and many in the soma.
- They exclude oocyte-specific GR signaling inhibition via beta isoforms.
- Perinatal male germline shows differential splicing regulation in response to systemic Dex administration, results were backed up with q-PCR analysis of splicing factors.

Weaknesses:
- The presence of a protein cannot be entirely excluded based on IF data (staining of spermatids is referred to but not shown).
- The authors do not consider post-transcriptional level a) modifications also trigged by GR activation b) non-coding RNAs (not assessed by seq).
- Sequencing techniques used are not total RNA but either are focused on all polyA transcripts (10x) or only assess the 3' prime end and hence are not ideal to study splicing, The number of replicates in the low input seq is very low and hence this might be underpowered. Since Dex treatment showed some (modest) changes in oocyte RNA - effects of GR depletion might only become apparent upon Dex treatment as an interaction.
- Effects in oocytes following systemic Dex might be indirect due to GR activation in the soma.
- Even though ex vivo culture of ovaries shows GR translocation to the nucleus it is not sure whether the in vivo systemic administration does the same.

The conclusion that fetal oocytes are "intrinsically buffered to GR signalling" is very strong, given that "only" poly A sequencing and few replicates of 3-prime sequencing have been analyzed and information is lacking on whether GR is activated in germ cells in the systemically dex-injected animals.

This work is a good reference point for researchers interested in glucocorticoid hormone signaling fertility and RNA splicing. It might spark further studies on germline-specific GR functions and the impact of GR activation on alternative splicing.

While the study provides a characterization of GR and some aspects of GR perturbation, and the negative findings in this study do help to rule out a range of specific roles of GR in the germline, there is still a range of other potential unexplored options. The introduction of the study eludes to implications for intergenerational effects via epigenetic modifications in the germline, however, it does not mention that the indirect effects of reproductive tissue GR signaling on the germline have indeed already been described in the context of intergenerational effects of stress. Also, the study does not assess epigenetic modifications.

The conclusion that the persistence of a phenotype for up to three generations suggests that stress can induce lasting epigenetic changes in the germline is misleading. For the reader who is unfamiliar with the field, it is important to define much more precisely what is referred to as "a phenotype". Furthermore, this statement evokes the impression that the very same epigenetic changes in the germline have been observed across multiple generations.

The evidence of the presence of GR in the germline is also somewhat limited - since other studies using sequencing have detected GR in the mature oocyte and sperm.

The discussion ends again on the implications of sex-specific differences of GR signaling in the context of stress-induced epigenetic inheritance. It states that the observed differences might relate to the fact that there is more evidence for paternal lineage findings, without considering that maternal lineage studies in epigenetic inheritance are generally less prevalent due to some practical factors - such as more laborious study design making use of cross-fostering or embryo transfer. Since the authors comment on RNA-mediated inheritance it seems inevitable to again consider indirect effects.

Reviewer #2 (Public Review):

Summary: There is increasing evidence in the literature that rodent models of stress can produce phenotypes that persist through multiple generations. Nevertheless, the mechanism(s) by which stress exposure produces phenotypes are unknown in the directly affected individual as well as in subsequent offspring that did not directly experience stress. Moreover, it has also been shown that glucocorticoid stress hormones can recapitulate the effects of programmed stress. In this manuscript, the authors test the compelling hypothesis that glucocorticoid receptor (GR)-signaling is responsible for the transmission of phenotypes across generations. As a first step, the investigators test for a role of GR in the male and female germline. Using knockouts and GR agonists, they show that although germ cells in male and female mice have GR that appears to localize to the nucleus when stimulated, oocytes are resistant to changes in GR levels. In contrast, the male germline exhibits changes in splicing but no overt changes in fertility.

Strengths: Although many of the results in this manuscript are negative, this is a careful and timely study that informs additional work to address mechanisms of transmission of stress phenotypes across generations and suggests a sexually dimorphic response to glucocorticoids in the germline. The work presented here is well-done and rigorous and the discussion of the data is thoughtful. Overall, this is an important contribution to the literature.