Bryan A. Niedenberger

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.

Marker expression reveals heterogeneity of spermatogonia in the neonatal mouse testis

Prospermatogonia transition to type A spermatogonia, which provide the source for the spermatogonial stem cell (SSC) pool. A percentage of these type A spermatogonia then differentiate to enter meiosis as spermatocytes by ∼P10. It is currently unclear as to when these distinct populations are initially formed in the neonatal testis, and when the expression of markers both characteristic of and required for the adult undifferentiated and differentiating states is established. In this study, we compared expression of known spermatogonial cell fate markers during normal development and in response to the differentiation signal provided by retinoic acid (RA). We found that some markers for the undifferentiated state (ZBTB16/PLZF and CDH1) were expressed in nearly all spermatogonia from P1 through P7. In contrast, differentiation markers (STRA8 and KIT) appeared in a subset of spermatogonia at P4, coincident with the onset of RA signaling. GFRA1, which was present in nearly all prospermatogonia at P1, was only retained in STRA8/KIT- spermatogonia. From P4 through P10, there was a great deal of heterogeneity in the male germ cell population in terms of expression of markers, as markers characteristic of the undifferentiated (except GFRA1) and differentiating states were co-expressed through this interval. After P10, these fate markers diverged to mark distinct populations of undifferentiated and differentiating spermatogonia, and this pattern was maintained in juvenile (P18) and adult (P>60) testes. Taken together, these results reveal that the spermatogonia population is heterogeneous during the first wave of spermatogenesis, and indicate that neonatal spermatogonia may not serve as an ideal substitute for studying the function of adult spermatogonia.

Nuclear localization of the actin regulatory protein Palladin in sertoli cells.

In the testis, F‐actin structures are involved in spermatid nuclear remodeling and cytoplasm reduction, maintenance of the blood–testis barrier, support of the spermatogonial stem cell niche, and release of spermatids into the tubular lumen. To gain a better understanding of actin regulation in Sertoli–germ cell interactions, we investigated the expression of the Palladin (Palld) gene, which encodes a widely expressed phosphoprotein that localizes to actin‐rich cytoplasmic structures, including focal adhesions, cell–cell junctions, podosomes, and stress fibers, and serves as a molecular scaffold to bundle actin fibers. In germ cells, PALLD was concentrated along the tubulin‐ and F‐actin‐containing cytoplasmic manchette that forms adjacent to the elongating spermatid nucleus during spermiogenesis. To our surprise, PALLD relocated from the cytoplasm to the nucleus of Sertoli cells in the juvenile testis, coincident with the onset of puberty, and this localization was maintained in the adult. We provide evidence that the 140 kDa isoform of PALLD predominates in Sertoli cells, and that it is apparently cleaved, with the C‐terminus localizing to the nucleus while the N‐terminus remains cytoplasmic. We investigated the nuclear localization of the C‐terminus of PALLD and found that it is regulated by a putative nuclear export signal. These results provide the foundation for future work employing Sertoli cell‐ and spermatid‐specific Palld‐knockout mice to study diverse roles of PALLD as both a nuclear‐actin regulatory protein and as a potential regulator of manchette formation during spermatogenesis. Mol. Reprod. Dev. 80: 403–413, 2013.

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

Abstract 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. Summary Sentence Germ cell specific Mtor knockout mice exhibit a phenotype in which spermatogonia self-renew in adult mice, but fail to proliferate, differentiate, and complete spermatogenesis.

Actin dynamics regulate subcellular localization of the F-actin-binding protein PALLD in mouse Sertoli cells

Sertoli cells undergo terminal differentiation at puberty to support all phases of germ cell development, which occurs in the mouse beginning in the second week of life. By ∼18 days postpartum (dpp), nearly all Sertoli cells have ceased proliferation. This terminal differentiation is accompanied by the development of unique and regionally concentrated filamentous actin (F-actin) structures at the basal and apical aspects of the seminiferous epithelium, and this reorganization is likely to involve the action of actin-binding proteins. Palladin (PALLD) is a widely expressed F-actin-binding and bundling protein recently shown to regulate these structures, yet it is predominantly nuclear in Sertoli cells at puberty. We found that PALLD localized within nuclei of primary Sertoli cells grown in serum-free media but relocalized to the cytoplasm upon serum stimulation. We utilized this system with in vivo relevance to Sertoli cell development to investigate mechanisms regulating nuclear localization of this F-actin-binding protein. Our results indicate that PALLD can be shuttled from the nucleus to the cytoplasm, and that this relocalization occurred following depolymerization of the F-actin cytoskeleton in response to cAMP signaling. Nuclear localization was reduced in Hpg-mutant testes, suggesting the involvement of gonadotropin signaling. We found that PALLD nuclear localization was unaffected in testis tissues from LH receptor and androgen receptor-mutant mice. However, PALLD nuclear localization was reduced in the testes of FSH receptor-mutant mice, suggesting that FSH signaling during Sertoli cell maturation regulates this subcellular localization.

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.

Advanced immunostaining approaches to study early male germ cell development

Mammalian male germ cell development takes place in the testis under the influence of a variety of somatic cells and an incompletely defined paracrine and endocrine influences. Since it is not recapitulated well in vitro, researchers studying spermatogenesis often manipulate the germline by creating transgenic or knockout mice or by administering pharmaceutical agonists/antagonists or inhibitors. The effects of these types of manipulations on germline development can often be determined following microscopic imaging, both of stained and immunostained testis sections. Here, we describe approaches for microscopic analysis of the developing male germline, provide detailed protocols for a variety of immunostaining approaches, and discuss transgenic fluorescent reporter lines for studying the early stages of spermatogenesis.

Dynamic cytoplasmic projections connect mammalian spermatogonia in vivo

ABSTRACT 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. Summary: Dynamic cytoplasmic projections connect mammalian spermatogonia and provide patent connections for the sharing of macromolecules during testis development.

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

ABSTRACT 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. Summary Sentence SSC maintenance requires RPTOR