Retinoic acid is dispensable for meiotic initiation but required for spermiogenesis in the mammalian testis.

Retinoic acid (RA) is the proposed mammalian 'meiosis inducing substance'. However, evidence for this role comes from studies in the fetal ovary, where germ cell differentiation and meiotic initiation are temporally inseparable. In the postnatal testis, these events are separated by more than 1 week. Exploiting this difference, we discovered that, although RA is required for spermatogonial differentiation, it is dispensable for the subsequent initiation, progression and completion of meiosis. Indeed, in the absence of RA, the meiotic transcriptome program in both differentiating spermatogonia and spermatocytes entering meiosis was largely unaffected. Instead, transcripts encoding factors required during spermiogenesis were aberrant during preleptonema, and the subsequent spermatid morphogenesis program was disrupted such that no sperm were produced. Taken together, these data reveal a RA-independent model for male meiotic initiation.

Modeling mammalian spermatogonial differentiation and meiotic initiation in vitro.

In mammalian testes, premeiotic spermatogonia respond to retinoic acid by completing an essential lengthy differentiation program before initiating meiosis. The molecular and cellular changes directing these developmental processes remain largely undefined. This wide gap in knowledge is due to two unresolved technical challenges: (1) lack of robust and reliable in vitro models to study differentiation and meiotic initiation; and (2) lack of methods to isolate large and pure populations of male germ cells at each stage of differentiation and at meiotic initiation. Here, we report a facile in vitro differentiation and meiotic initiation system that can be readily manipulated, including the use of chemical agents that cannot be safely administered to live animals. In addition, we present a transgenic mouse model enabling fluorescence-activated cell sorting-based isolation of millions of spermatogonia at specific developmental stages as well as meiotic spermatocytes.

Differential responsiveness of spermatogonia to retinoic acid dictates precocious differentiation but not meiotic entry during steady-state spermatogenesis†.

The foundation of mammalian spermatogenesis is provided by undifferentiated spermatogonia, which comprise of spermatogonial stem cells (SSCs) and transit-amplifying progenitors that differentiate in response to retinoic acid (RA) and are committed to enter meiosis. Our laboratory recently reported that the foundational populations of SSCs, undifferentiated progenitors, and differentiating spermatogonia are formed in the neonatal testis in part based on their differential responsiveness to RA. Here, we expand on those findings to define the extent to which RA responsiveness during steady-state spermatogenesis in the adult testis regulates the spermatogonial fate. Our results reveal that both progenitor and differentiating spermatogonia throughout the testis are capable of responding to exogenous RA, but their resulting fates were quite distinct-undifferentiated progenitors precociously differentiated and proceeded into meiosis on a normal timeline, while differentiating spermatogonia were unable to hasten their entry into meiosis. This reveals that the spermatogonia responding to RA must still complete the 8.6 day differentiation program prior to their entry into meiosis. Addition of exogenous RA enriched testes with preleptotene and pachytene spermatocytes one and two seminiferous cycles later, respectively, supporting recent clinical studies reporting increased sperm production and enhanced fertility in subfertile men on long-term RA analog treatment. Collectively, our results reveal that a well-buffered system exists within mammalian testes to regulate spermatogonial RA exposure, that exposed undifferentiated progenitors can precociously differentiate, but must complete a normal-length differentiation program prior to entering meiosis, and that daily RA treatments increased the numbers of advanced germ cells by directing undifferentiated progenitors to continuously differentiate.

The rapamycin analog Everolimus reversibly impairs male germ cell differentiation and fertility in the mouse†.

Sirolimus, also known as rapamycin, and its closely related rapamycin analog (rapalog) Everolimus inhibit "mammalian target of rapamycin complex 1" (mTORC1), whose activity is required for spermatogenesis. Everolimus is Food and Drug Administration approved for treating human patients to slow growth of aggressive cancers and preventing organ transplant rejection. Here, we test the hypothesis that rapalog inhibition of mTORC1 activity has a negative, but reversible, impact upon spermatogenesis. Juvenile (P20) or adult (P>60) mice received daily injections of sirolimus or Everolimus for 30 days, and tissues were examined at completion of treatment or following a recovery period. Rapalog treatments reduced body and testis weights, testis weight/body weight ratios, cauda epididymal sperm counts, and seminal vesicle weights in animals of both ages. Following rapalog treatment, numbers of differentiating spermatogonia were reduced, with concomitant increases in the ratio of undifferentiated spermatogonia to total number of remaining germ cells. To determine if even low doses of Everolimus can inhibit spermatogenesis, an additional group of adult mice received a dose of Everolimus ∼6-fold lower than a human clinical dose used to treat cancer. In these animals, only testis weights, testis weight/body weight ratios, and tubule diameters were reduced. Return to control values following a recovery period was variable for each of the measured parameters and was duration and dose dependent. Together, these data indicate rapalogs exerted a dose-dependent restriction on overall growth of juvenile and adult mice and negative impact upon spermatogenesis that were largely reversed; following treatment cessation, males from all treatment groups were able to sire offspring.

Pharmacological Treatment of Neonatal and Juvenile Mice to Study Spermatogenesis.

The delivery, to newborn and juvenile mice, of drugs and other compounds that manipulate the physiology or cellular/molecular state -e.g., by activating or inhibiting signaling pathways) is a powerful, yet underutilized approach to studying spermatogenesis. Here, we provide detailed protocols we have optimized in our laboratory for safely and effectively feeding and injecting mice and discuss troubleshooting approaches.

Cnot3 is required for male germ cell development and spermatogonial stem cell maintenance.

The foundation of spermatogenesis and lifelong fertility is provided by spermatogonial stem cells (SSCs). SSCs divide asymmetrically to either replenish their numbers (self-renewal) or produce undifferentiated progenitors that proliferate before committing to differentiation. However, regulatory mechanisms governing SSC maintenance are poorly understood. Here, we show that the CCR4-NOT mRNA deadenylase complex subunit CNOT3 plays a critical role in maintaining spermatogonial populations in mice. Cnot3 is highly expressed in undifferentiated spermatogonia, and its deletion in spermatogonia resulted in germ cell loss and infertility. Single cell analyses revealed that Cnot3 deletion led to the de-repression of transcripts encoding factors involved in spermatogonial differentiation, including those in the glutathione redox pathway that are critical for SSC maintenance. Together, our study reveals that CNOT3 - likely via the CCR4-NOT complex - actively degrades transcripts encoding differentiation factors to sustain the spermatogonial pool and ensure the progression of spermatogenesis, highlighting the importance of CCR4-NOT-mediated post-transcriptional gene regulation during male germ cell development.

WNK1 is required during male pachynema to sustain fertility.

WNK1 is an important regulator in many physiological functions, yet its role in male reproduction is unexplored. In the male germline, WNK1 is upregulated in preleptotene spermatocytes indicating possible function(s) in spermatogenic meiosis. Indeed, deletion of Wnk1 in mid-pachytene spermatocytes using the Wnt7a-Cre mouse led to male sterility which resembled non-obstructive azoospermia in humans, where germ cells failed to complete spermatogenesis and produced no sperm. Mechanistically, we found elevated MTOR expression and signaling in the Wnk1-depleted spermatocytes. As MTOR is a central mediator of translation, we speculated that translation may be accelerated in these spermatocytes. Supporting this, we found the acrosome protein, ACRBP to be prematurely expressed in the spermatocytes with Wnk1 deletion. Our study uncovered an MTOR-regulating factor in the male germline with potential implications in translation, and future studies will aim to understand how WNK1 regulates MTOR activity and impact translation on a broader spectrum.

Mammalian SWI/SNF chromatin remodeler is essential for reductional meiosis in males.

The mammalian SWI/SNF nucleosome remodeler is essential for spermatogenesis. Here, we identify a role for ARID2, a PBAF (Polybromo - Brg1 Associated Factor)-specific subunit, in meiotic division. Arid2cKO spermatocytes arrest at metaphase-I and are deficient in spindle assembly, kinetochore-associated Polo-like kinase1 (PLK1), and centromeric targeting of Histone H3 threonine3 phosphorylation (H3T3P) and Histone H2A threonine120 phosphorylation (H2AT120P). By determining ARID2 and BRG1 genomic associations, we show that PBAF localizes to centromeres and promoters of genes known to govern spindle assembly and nuclear division in spermatocytes. Consistent with gene ontology of target genes, we also identify a role for ARID2 in centrosome stability. Additionally, misexpression of genes such as Aurkc and Ppp1cc (Pp1γ), known to govern chromosome segregation, potentially compromises the function of the chromosome passenger complex (CPC) and deposition of H3T3P, respectively. Our data support a model where-in PBAF activates genes essential for meiotic cell division.