C. elegans Hedgehog-related proteins are tissue- and substructure-specific components of the cuticle and pre-cuticle.

In C. elegans, expanded families of divergent Hedgehog-related and Patched-related proteins promote numerous processes ranging from epithelial and sense organ development to pathogen responses to cuticle shedding during the molt cycle. The molecular functions of these proteins have been mysterious since nematodes lack a canonical Hedgehog signaling pathway. Here we show that Hedgehog-related proteins are components of the cuticle and pre-cuticle apical extracellular matrices that coat, shape, and protect external epithelia. Of four Hedgehog-related proteins imaged, two (GRL-2 and GRL-18) stably associated with the cuticles of specific tubes and two (GRL-7 and WRT-10) labelled pre-cuticle substructures such as furrows or alae. We found that wrt-10 mutations disrupt cuticle alae ridges, consistent with a structural role in matrix organization. We hypothesize that most nematode Hedgehog-related proteins are apical extracellular matrix components, a model that could explain many of the reported functions for this family. These results highlight ancient connections between Hedgehog proteins and the extracellular matrix and suggest that any signaling roles of C. elegans Hedgehog-related proteins will be intimately related to their matrix association.

C. elegans Hedgehog-related proteins are tissue- and substructure-specific components of the cuticle and pre-cuticle.

In C. elegans, divergent Hedgehog-related (Hh-r) and Patched-related (PTR) proteins promote numerous processes ranging from epithelial and sense organ development to pathogen responses to cuticle shedding during the molt cycle. Here we show that Hh-r proteins are actual components of the cuticle and pre-cuticle apical extracellular matrices (aECMs) that coat, shape, and protect external epithelia. Different Hh-r proteins stably associate with the aECMs of specific tissues and with specific substructures such as furrows and alae. Hh-r mutations can disrupt matrix structure. These results provide a unifying model for the function of nematode Hh-r proteins and highlight ancient connections between Hh proteins and the extracellular matrix.

Noncanonical necrosis in 2 different cell types in a Caenorhabditis elegans NAD+ salvage pathway mutant.

Necrosis was once described as a chaotic unregulated response to cellular insult. We now know that necrosis is controlled by multiple pathways in response to many different cellular conditions. In our pnc-1 NAD+ salvage deficient Caenorhabditis elegans model excess nicotinamide induces excitotoxic death in uterine-vulval uv1 cells and OLQ mechanosensory neurons. We sought to characterize necrosis in our pnc-1 model in the context of well-characterized necrosis, apoptosis, and autophagy pathways in C. elegans. We confirmed that calpain and aspartic proteases were required for uv1 necrosis, but changes in intracellular calcium levels and autophagy were not, suggesting that uv1 necrosis occurs by a pathway that diverges from mec-4d-induced touch cell necrosis downstream of effector aspartic proteases. OLQ necrosis does not require changes in intracellular calcium, the function of calpain or aspartic proteases, or autophagy. Instead, OLQ survival requires the function of calreticulin and calnexin, pro-apoptotic ced-4 (Apaf1), and genes involved in both autophagy and axon guidance. In addition, the partially OLQ-dependent gentle nose touch response decreased significantly in pnc-1 animals on poor quality food, further suggesting that uv1 and OLQ necrosis differ downstream of their common trigger. Together these results show that, although phenotypically very similar, uv1, OLQ, and touch cell necrosis are very different at the molecular level.

The Caenorhabditis elegans Patched domain protein PTR-4 is required for proper organization of the precuticular apical extracellular matrix.

The Patched-related superfamily of transmembrane proteins can transport lipids or other hydrophobic molecules across cell membranes. While the Hedgehog receptor Patched has been intensively studied, much less is known about the biological roles of other Patched-related family members. Caenorhabditis elegans has a large number of Patched-related proteins, despite lacking a canonical Hedgehog pathway. Here, we show that PTR-4 promotes the assembly of the precuticle apical extracellular matrix, a transient and molecularly distinct matrix that precedes and patterns the later collagenous cuticle or exoskeleton. ptr-4 mutants share many phenotypes with precuticle mutants, including defects in eggshell dissolution, tube shaping, alae (cuticle ridge) structure, molting, and cuticle barrier function. PTR-4 localizes to the apical side of a subset of outward-facing epithelia, in a cyclical manner that peaks when precuticle matrix is present. Finally, PTR-4 is required to limit the accumulation of the lipocalin LPR-3 and to properly localize the Zona Pellucida domain protein LET-653 within the precuticle. We propose that PTR-4 transports lipids or other hydrophobic components that help to organize the precuticle and that the cuticle and molting defects seen in ptr-4 mutants result at least in part from earlier disorganization of the precuticle.

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.

Transcytosis in the development and morphogenesis of epithelial tissues.

Transcytosis is a form of specialized transport through which an extracellular cargo is endocytosed, shuttled across the cytoplasm in membrane-bound vesicles, and secreted at a different plasma membrane surface. This important process allows membrane-impermeable macromolecules to pass through a cell and become accessible to adjacent cells and tissue compartments. Transcytosis also promotes redistribution of plasma membrane proteins and lipids to different regions of the cell surface. Here we review transcytosis and highlight in vivo studies showing how developing epithelial cells use it to change shape, to migrate, and to relocalize signaling molecules.

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.

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.

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.

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.

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.

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.