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.

The germ cell-specific RNA binding protein RBM46 is essential for spermatogonial differentiation in mice.

Control over gene expression is exerted, in multiple stages of spermatogenesis, at the post-transcriptional level by RNA binding proteins (RBPs). We identify here an essential role in mammalian spermatogenesis and male fertility for 'RNA binding protein 46' (RBM46). A highly evolutionarily conserved gene, Rbm46 is also essential for fertility in both flies and fish. We found Rbm46 expression was restricted to the mouse germline, detectable in males in the cytoplasm of premeiotic spermatogonia and meiotic spermatocytes. To define its requirement for spermatogenesis, we generated Rbm46 knockout (KO, Rbm46-/-) mice; although male Rbm46-/- mice were viable and appeared grossly normal, they were infertile. Testes from adult Rbm46-/- mice were small, with seminiferous tubules containing only Sertoli cells and few undifferentiated spermatogonia. Using genome-wide unbiased high throughput assays RNA-seq and 'enhanced crosslinking immunoprecipitation' coupled with RNA-seq (eCLIP-seq), we discovered RBM46 could bind, via a U-rich conserved consensus sequence, to a cohort of mRNAs encoding proteins required for completion of differentiation and subsequent meiotic initiation. In summary, our studies support an essential role for RBM46 in regulating target mRNAs during spermatogonia differentiation prior to the commitment to meiosis in mice.

Actin-related protein ACTL7B ablation leads to OAT with multiple morphological abnormalities of the flagellum and male infertility in mice†.

The formation of fertilisation-competent sperm requires spermatid morphogenesis (spermiogenesis), a poorly understood program that involves complex coordinated restructuring and specialised cytoskeletal structures. A major class of cytoskeletal regulators are the actin-related proteins (ARPs), which include conventional actin variants, and related proteins that play essential roles in complexes regulating actin dynamics, intracellular transport, and chromatin remodeling. Multiple testis-specific ARPs are well conserved among mammals, but their functional roles are unknown. One of these is actin-like 7b (Actl7b) that encodes an orphan ARP highly similar to the ubiquitously expressed beta actin (ACTB). Here we report ACTL7B is expressed in human and mouse spermatids through the elongation phase of spermatid development. In mice, ACTL7B specifically localises to the developing acrosome, within the nucleus of early spermatids, and to the flagellum connecting region. Based on this localisation pattern and high level of sequence conservation in mice, humans, and other mammals, we examined the requirement for ACTL7B in spermiogenesis by generating and characterising the reproductive phenotype of male Actl7b KO mice. KO mice were infertile, with severe and variable oligoteratozoospermia (OAT) and multiple morphological abnormalities of the flagellum (MMAF) and sperm head. These defects phenocopy human OAT and MMAF, which are leading causes of idiopathic male infertility. In conclusion, this work identifies ACTL7B as a key regulator of spermiogenesis that is required for male fertility.

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.

Differential RA responsiveness directs formation of functionally distinct spermatogonial populations at the initiation of spermatogenesis in the mouse.

In the mammalian testis, sustained spermatogenesis relies on spermatogonial stem cells (SSCs); their progeny either remain as stem cells (self-renewal) or proliferate and differentiate to enter meiosis in response to retinoic acid (RA). Here, we sought to uncover elusive mechanisms regulating a key switch fundamental to spermatogonial fate: the capacity of spermatogonia to respond to RA. Using the developing mouse testis as a model, we found that spermatogonia and precursor prospermatogonia exhibit a heterogeneous capacity to respond to RA with at least two underlying causes. First, progenitor spermatogonia are prevented from responding to RA by catabolic activity of cytochrome P450 family 26 enzymes. Second, a smaller subset of undifferentiated spermatogonia enriched for SSCs exhibit catabolism-independent RA insensitivity. Moreover, for the first time, we observed that precursor prospermatogonia are heterogeneous and comprise subpopulations that exhibit the same differential RA responsiveness found in neonatal spermatogonia. We propose a novel model by which mammalian prospermatogonial and spermatogonial fates are regulated by their intrinsic capacity to respond (or not) to the differentiation signal provided by RA before, and concurrent with, the initiation of spermatogenesis.

Dynamic cytoplasmic projections connect mammalian spermatogonia in vivo.

Throughout the male reproductive lifespan, spermatogonial stem cells (SSCs) produce committed progenitors that proliferate and then remain physically connected in growing clones via short cylindrical intercellular bridges (ICBs). These ICBs, which enlarge in meiotic spermatocytes, have been demonstrated to provide a conduit for postmeiotic haploid spermatids to share sex chromosome-derived gene products. In addition to ICBs, spermatogonia exhibit multiple thin cytoplasmic projections. Here, we have explored the nature of these projections in mice and find that they are dynamic, span considerable distances from their cell body (≥25 µm), either terminate or physically connect multiple adjacent spermatogonia, and allow for sharing of macromolecules. Our results extend the current model that subsets of spermatogonia exist as isolated cells or clones, and support a model in which spermatogonia of similar developmental fates are functionally connected through a shared dynamic cytoplasm mediated by thin cytoplasmic projections.

Aurora A Kinase (AURKA) is required for male germline maintenance and regulates sperm motility in the mouse.

Aurora A kinase (AURKA) is an important regulator of cell division and is required for assembly of the mitotic spindle. We recently reported the unusual finding that this mitotic kinase is also found on the sperm flagellum. To determine its requirement in spermatogenesis, we generated conditional knockout animals with deletion of the Aurka gene in either spermatogonia or spermatocytes to assess its role in mitotic and postmitotic cells, respectively. Deletion of Aurka in spermatogonia resulted in disappearance of all developing germ cells in the testis, as expected, given its vital role in mitotic cell division. Deletion of Aurka in spermatocytes reduced testis size, sperm count, and fertility, indicating disruption of meiosis or an effect on spermiogenesis in developing mice. Interestingly, deletion of Aurka in spermatocytes increased apoptosis in spermatocytes along with an increase in the percentage of sperm with abnormal morphology. Despite the increase in abnormal sperm, sperm from spermatocyte Aurka knockout mice displayed increased progressive motility. In addition, sperm lysate prepared from Aurka knockout animals had decreased protein phosphatase 1 (PP1) activity. Together, our results show that AURKA plays multiple roles in spermatogenesis, from mitotic divisions of spermatogonia to sperm morphology and motility.

Acyl-CoA synthetase 6 enriches seminiferous tubules with the ω-3 fatty acid docosahexaenoic acid and is required for male fertility in the mouse.

Docosahexaenoic acid (DHA) is an ω-3 dietary-derived polyunsaturated fatty acid of marine origin enriched in testes and necessary for normal fertility, yet the mechanisms regulating the enrichment of DHA in the testes remain unclear. Long-chain ACSL6 (acyl-CoA synthetase isoform 6) activates fatty acids for cellular anabolic and catabolic metabolism by ligating a CoA to a fatty acid, is highly expressed in testes, and has high preference for DHA. Here, we investigated the role of ACSL6 for DHA enrichment in the testes and its requirement for male fertility. Acsl6-/- males were severely subfertile with smaller testes, reduced cauda epididymal sperm counts, germ cell loss, and disorganization of the seminiferous epithelium. Total fatty acid profiling of Acsl6-/- testes revealed reduced DHA and increased ω-6 arachidonic acid, a fatty acid profile also reflected in phospholipid composition. Strikingly, lipid imaging demonstrated spatial redistribution of phospholipids in Acsl6-/- testes. Arachidonic acid-containing phospholipids were predominantly interstitial in control testes but diffusely localized across Acsl6-/- testes. In control testes, DHA-containing phospholipids were predominantly within seminiferous tubules, which contain Sertoli cells and spermatogenic cells but relocalized to the interstitium in Acsl6-/- testes. Taken together, these data demonstrate that ACSL6 is an initial driving force for germ cell DHA enrichment and is required for normal spermatogenesis and male fertility.

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.

The mTORC1 component RPTOR is required for maintenance of the foundational spermatogonial stem cell pool in mice†.

The self-renewal, proliferation, and differentiation of the spermatogonial populations must be finely coordinated in the mammalian testis, as dysregulation of these processes can lead to subfertility, infertility, or the formation of tumors. There are wide gaps in our understanding of how these spermatogonial populations are formed and maintained, and our laboratory has focused on identifying the molecular and cellular pathways that direct their development. Others and we have shown, using a combination of pharmacologic inhibitors and genetic models, that activation of mTOR complex 1 (mTORC1) is important for spermatogonial differentiation in vivo. Here, we extend those studies to directly test the germ cell-autonomous requirement for mTORC1 in spermatogonial differentiation. We created germ cell conditional knockout mice for "regulatory associated protein of MTOR, complex 1" (Rptor), which encodes an essential component of mTORC1. While germ cell KO mice were viable and healthy, they had smaller testes than littermate controls, and no sperm were present in their cauda epididymides. We found that an initial cohort of Rptor KO spermatogonia proliferated, differentiated, and entered meiosis (which they were unable to complete). However, no self-renewing spermatogonia were formed, and thus the entire germline was lost by adulthood, resulting in Sertoli cell-only testes. These results reveal the cell autonomous requirement for RPTOR in the formation or maintenance of the foundational self-renewing spermatogonial stem cell pool in the mouse testis and underscore complex roles for mTORC1 and its constituent proteins in male germ cell development.

A new translation and reader's guide to Victor von Ebner's classical description of spermatogenesis.

This is a translation and modern interpretation of one of the most important manuscripts on spermatogenesis, published by Victor von Ebner, in 1871. It was originally written in Italian, and to the best of my knowledge has never been translated to English or examined by modern scientists.

Cell-autonomous requirement for mammalian target of rapamycin (Mtor) in spermatogonial proliferation and differentiation in the mouse†.

Spermatogonial stem cells must balance self-renewal with production of transit-amplifying progenitors that differentiate in response to retinoic acid (RA) before entering meiosis. This self-renewal vs. differentiation fate decision is critical for maintaining tissue homeostasis, as imbalances cause defects that can lead to human testicular cancer or infertility. Little is currently known about the program of differentiation initiated by RA, and the pathways and proteins involved are poorly defined. We recently found that RA stimulation of the Phosphatidylinositol 3-kinase (PI3K)/AKT/Mammalian target of rapamycin (mTOR) kinase signaling pathway is required for differentiation, and that short-term inhibition of mTOR complex 1 (mTORC1) by rapamycin blocked spermatogonial differentiation in vivo and prevented RA-induced translational activation. Since this phenotype resulted from global inhibition of mTORC1, we created conditional germ cell knockout mice to investigate the germ cell-autonomous role of MTOR in spermatogonial differentiation. MTOR germ cell KO mice were viable and healthy, but testes from neonatal (postnatal day (P)8), juvenile (P18), and adult (P > 60) KO mice were smaller than littermate controls, and no sperm were produced in adult testes. Histological and immunostaining analyses revealed that spermatogonial differentiation was blocked, and no spermatocytes were formed at any of the ages examined. Although spermatogonial proliferation was reduced in the neonatal testis, it was blocked altogether in the juvenile and adult testis. Importantly, a small population of self-renewing undifferentiated spermatogonia remained in adult testes. Taken together, these results reveal that MTOR is dispensable for the maintenance of undifferentiated spermatogonia, but is cell autonomously required for their proliferation and differentiation.

Mammalian target of rapamycin complex 1 (mTORC1) Is required for mouse spermatogonial differentiation in vivo.

Spermatogonial stem cells (SSCs) must balance self-renewal with production of transit-amplifying progenitors that differentiate in response to retinoic acid (RA) before entering meiosis. This self-renewal vs. differentiation spermatogonial fate decision is critical for maintaining tissue homeostasis, as imbalances cause spermatogenesis defects that can lead to human testicular cancer or infertility. A great deal of effort has been exerted to understand how the SSC population is maintained. In contrast, little is known about the essential program of differentiation initiated by retinoic acid (RA) that precedes meiosis, and the pathways and proteins involved are poorly defined. We recently reported a novel role for RA in stimulating the PI3/AKT/mTOR kinase signaling pathway to activate translation of repressed mRNAs such as Kit. Here, we examined the requirement for mTOR complex 1 (mTORC1) in mediating the RA signal to direct spermatogonial differentiation in the neonatal testis. We found that in vivo inhibition of mTORC1 by rapamycin blocked spermatogonial differentiation, which led to an accumulation of undifferentiated spermatogonia. In addition, rapamycin also blocked the RA-induced translational activation of mRNAs encoding KIT, SOHLH1, and SOHLH2 without affecting expression of STRA8. These findings highlight dual roles for RA in germ cell development - transcriptional activation of genes, and kinase signaling to stimulate translation of repressed messages required for spermatogonial differentiation.

Retinoic acid regulates Kit translation during spermatogonial differentiation in the mouse.

In the testis, a subset of spermatogonia retains stem cell potential, while others differentiate to eventually become spermatozoa. This delicate balance must be maintained, as defects can result in testicular cancer or infertility. Currently, little is known about the gene products and signaling pathways directing these critical cell fate decisions. Retinoic acid (RA) is a requisite driver of spermatogonial differentiation and entry into meiosis, yet the mechanisms activated downstream are undefined. Here, we determined a requirement for RA in the expression of KIT, a receptor tyrosine kinase essential for spermatogonial differentiation. We found that RA signaling utilized the PI3K/AKT/mTOR signaling pathway to induce the efficient translation of mRNAs for Kit, which are present but not translated in undifferentiated spermatogonia. Our findings provide an important molecular link between a morphogen (RA) and the expression of KIT protein, which together direct the differentiation of spermatogonia throughout the male reproductive lifespan.

The Role of Retinoic Acid (RA) in Spermatogonial Differentiation.

Retinoic acid (RA) directs the sequential, but distinct, programs of spermatogonial differentiation and meiotic differentiation that are both essential for the generation of functional spermatozoa. These processes are functionally and temporally decoupled, as they occur in distinct cell types that arise over a week apart, both in the neonatal and adult testis. However, our understanding is limited in terms of what cellular and molecular changes occur downstream of RA exposure that prepare differentiating spermatogonia for meiotic initiation. In this review, we describe the process of spermatogonial differentiation and summarize the current state of knowledge regarding RA signaling in spermatogonia.

TSPAN8 Expression Distinguishes Spermatogonial Stem Cells in the Prepubertal Mouse Testis.

Precise separation of spermatogonial stem cells (SSCs) from progenitor spermatogonia that lack stem cell activity and are committed to differentiation remains a challenge. To distinguish between these spermatogonial subtypes, we identified genes that exhibited bimodal mRNA levels at the single-cell level among undifferentiated spermatogonia from Postnatal Day 6 mouse testes, including Tspan8, Epha2, and Pvr, each of which encode cell surface proteins useful for cell selection. Transplantation studies provided definitive evidence that a TSPAN8-high subpopulation is enriched for SSCs. RNA-seq analyses identified genes differentially expressed between TSPAN8-high and -low subpopulations that clustered into multiple biological pathways potentially involved in SSC renewal or differentiation, respectively. Methyl-seq analysis identified hypomethylated domains in the promoters of these genes in both subpopulations that colocalized with peaks of histone modifications defined by ChIP-seq analysis. Taken together, these results demonstrate functional heterogeneity among mouse undifferentiated spermatogonia and point to key biological characteristics that distinguish SSCs from progenitor spermatogonia.

Rhox13 is required for a quantitatively normal first wave of spermatogenesis in mice.

We previously described a novel germ cell-specific X-linked reproductive homeobox gene (Rhox13) that is upregulated at the level of translation in response to retinoic acid (RA) in differentiating spermatogonia and preleptotene spermatocytes. We hypothesize that RHOX13 plays an essential role in male germ cell differentiation, and have tested this by creating a Rhox13 gene knockout (KO) mouse. Rhox13 KO mice are born in expected Mendelian ratios, and adults have slightly reduced testis weights, yet a full complement of spermatogenic cell types. Young KO mice (at ~7-8 weeks of age) have a ≈50% reduction in epididymal sperm counts, but numbers increased to WT levels as the mice reach ~17 weeks of age. Histological analysis of testes from juvenile KO mice reveals a number of defects during the first wave of spermatogenesis. These include increased apoptosis, delayed appearance of round spermatids and disruption of the precise stage-specific association of germ cells within the seminiferous tubules. Breeding studies reveal that both young and aged KO males produce normal-sized litters. Taken together, our results indicate that RHOX13 is not essential for mouse fertility in a controlled laboratory setting, but that it is required for optimal development of differentiating germ cells and progression of the first wave of spermatogenesis.

Transcriptional and translational heterogeneity among neonatal mouse spermatogonia.

Spermatogonial stem cells (SSCs) are a subset of undifferentiated spermatogonia responsible for ongoing spermatogenesis in mammalian testes. Spermatogonial stem cells arise from morphologically homogeneous prospermatogonia, but growing evidence suggests that only a subset of prospermatogonia develops into the foundational SSC pool. This predicts that subtypes of undifferentiated spermatogonia with discrete mRNA and protein signatures should be distinguishable in neonatal testes. We used single-cell quantitative RT-PCR to examine mRNA levels of 172 genes in individual spermatogonia from 6-day postnatal (P6) mouse testes. Cells enriched from P6 testes using the StaPut or THY1(+) magnetic cell sorting methods exhibited considerable heterogeneity in the abundance of specific germ cell and stem cell mRNAs, segregating into one somatic and three distinct spermatogonial clusters. However, P6 Id4-eGFP(+) transgenic spermatogonia, which are known to be enriched for SSCs, were more homogeneous in their mRNA levels, exhibiting uniform levels for the majority of genes examined (122 of 172). Interestingly, these cells displayed nonuniform (50 of 172) expression of a smaller cohort of these genes, suggesting there is substantial heterogeneity even within the Id4-eGFP(+) population. Further, although immunofluorescence staining largely demonstrated conformity between mRNA and protein levels, some proteins were observed in patterns that were disparate from those detected for the corresponding mRNAs in Id4-eGFP(+) spermatogonia (e.g., Kit, Sohlh2, Stra8), suggesting additional heterogeneity is introduced at the posttranscriptional level. Taken together, these data demonstrate the existence of multiple spermatogonial subtypes in P6 mouse testes and raise the intriguing possibility that these subpopulations may correlate with the development of functionally distinct spermatogenic cell types.