Androgens responsible for male sexual differentiation in utero are produced by Leydig cells in the fetal testicular interstitium. Leydig cells rarely proliferate and, hence, rely on constant differentiation of interstitial progenitors to increase their number during fetal development. The cellular origins of fetal Leydig progenitors and how they are maintained remain largely unknown. Here we show that Notch-active, Nestin-positive perivascular cells in the fetal testis are a multipotent progenitor population, giving rise to Leydig cells, pericytes, and smooth muscle cells. When vasculature is disrupted, perivascular progenitor cells fail to be maintained and excessive Leydig cell differentiation occurs, demonstrating that blood vessels are a critical component of the niche that maintains interstitial progenitor cells. Additionally, our data strongly supports a model in which fetal Leydig cell differentiation occurs by at least two different means, with each having unique progenitor origins and distinct requirements for Notch signaling to maintain the progenitor population.

Sexual reproduction is dependent on the activity of androgenic steroid hormones to promote gonadal development and gametogenesis. Leydig cells of the testis and theca cells of the ovary are critical cell types in the gonadal interstitium that carry out steroidogenesis and provide key androgens for reproductive organ function. In this chapter, we will discuss important aspects of interstitial androgenic cell development in the gonad, including: the potential cellular origins of interstitial steroidogenic cells and their progenitors; the molecular mechanisms involved in Leydig cell specification and differentiation (including Sertoli-cell-derived signaling pathways and Leydig-cell-related transcription factors and nuclear receptors); the interactions of Leydig cells with other cell types in the adult testis, such as Sertoli cells, germ cells, peritubular myoid cells, macrophages, and vascular endothelial cells; the process of steroidogenesis and its systemic regulation; and a brief discussion of the development of theca cells in the ovary relative to Leydig cells in the testis. Finally, we will describe the dynamics of steroidogenic cells in seasonal breeders and highlight unique aspects of steroidogenesis in diverse vertebrate species. Understanding the cellular origins of interstitial steroidogenic cells and the pathways directing their specification and differentiation has implications for the study of multiple aspects of development and will help us gain insights into the etiology of reproductive system birth defects and infertility.

The α4 Na,K-ATPase is a sperm-specific protein essential for sperm motility and fertility yet little is known about the mechanisms that regulate its expression in germ cells. Here, the potential involvement of DNA methylation in regulating the expression of this sperm-specific protein is explored. A single, intragenic CpG island (Mα4-CGI) was identified in the gene encoding the mouse α4 Na,K-ATPase (Atp1a4), which displayed reduced methylation in mouse sperm (cells that contain α4) compared to mouse kidney (tissue that lacks α4 expression). Unlike the intragenic CGI, the putative promoter (the −700 to +200 region relative to the transcriptional start site) of Atp1a4 did not show differential methylation between kidney and sperm nevertheless it did drive methylation-dependent reporter gene expression in the male germ cell line GC-1spg. Furthermore, treatment of GC-1spg cells with 5-Aza2-Deoxycytidine led to upregulation of the α4 transcript and decreased methylation of both the Atp1a4 promoter and the Mα4-CGI. In addition, Atp1a4 expression in mouse embryonic stem cells deficient in DNA methytransferases suggests that both maintenance and de novo methylation are involved in regulating its expression. In an attempt to define the regulatory function of the Mα4-CGI, possible roles of the Mα4-CGI in regulating Atp1a4 expression via methylation-dependent transcriptional elongation inhibition in somatic cells and via its ability to repress promoter activity in germ cells were uncovered. In all, our data suggests that both the promoter and the intragenic CGI could combine to provide multiple modes of regulation for optimizing the Atp1a4 expression level in a cell type-specific manner.

Specification of mouse primordial germ cells (PGCs), the precursors of sperm and eggs, involves three major molecular events: repression of the somatic program; re-acquisition of pluripotency; and reprogramming to a unique epigenetic ground state. Gene knockout studies in mouse models, along with global transcriptome analyses, have revealed the key signaling pathways and transcription factors essential for PGC specification. Knowledge obtained from these studies has been utilized extensively to develop robust in vitro PGC induction models not only in mice but also in humans. These models have, in turn, formed the basis for a detailed understanding of the signaling pathways and epigenetic dynamics during in vivo PGC specification and development. Recapitulation of human PGC specification in culture is of tremendous significance for understanding the mechanisms of human germ cell development in normal and disease states and has implications for addressing germ-cell-based causes of infertility.

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