Despite significant advances in contraceptive options for women over the last 50 yr, world population continues to grow rapidly. Scientists and activists alike point to the devastating environmental impacts that population pressures have caused, including global warming from the developed world and hunger and disease in less developed areas. Moreover, almost half of all pregnancies are still unwanted or unplanned. Clearly, there is a need for expanded, reversible, contraceptive options. Multicultural surveys demonstrate the willingness of men to participate in contraception and their female partners to trust them to do so. Notwithstanding their paucity of options, male methods including vasectomy and condoms account for almost one third of contraceptive use in the United States and other countries. Recent international clinical research efforts have demonstrated high efficacy rates (90-95%) for hormonally based male contraceptives. Current barriers to expanded use include limited delivery methods and perceived regulatory obstacles, which stymie introduction to the marketplace. However, advances in oral and injectable androgen delivery are cause for optimism that these hurdles may be overcome. Nonhormonal methods, such as compounds that target sperm motility, are attractive in their theoretical promise of specificity for the reproductive tract. Gene and protein array technologies continue to identify potential targets for this approach. Such nonhormonal agents will likely reach clinical trials in the near future. Great strides have been made in understanding male reproductive physiology; the combined efforts of scientists, clinicians, industry and governmental funding agencies could make an effective, reversible, male contraceptive an option for family planning over the next decade.

Advances in male contraception.

The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in the mammalian body. It divides the seminiferous epithelium into the basal and the apical (adluminal) compartments. Meiosis I and II, spermiogenesis, and spermiation all take place in a specialized microenvironment behind the BTB in the apical compartment, but spermatogonial renewal and differentiation and cell cycle progression up to the preleptotene spermatocyte stage take place outside of the BTB in the basal compartment of the epithelium. However, the BTB is not a static ultrastructure. Instead, it undergoes extensive restructuring during the seminiferous epithelial cycle of spermatogenesis at stage VIII to allow the transit of preleptotene spermatocytes at the BTB. Yet the immunological barrier conferred by the BTB cannot be compromised, even transiently, during the epithelial cycle to avoid the production of antibodies against meiotic and postmeiotic germ cells. Studies have demonstrated that some unlikely partners, namely adhesion protein complexes (e.g., occludin-ZO-1, N-cadherin-β-catenin, claudin-5-ZO-1), steroids (e.g., testosterone, estradiol-17β), nonreceptor protein kinases (e.g., focal adhesion kinase, c-Src, c-Yes), polarity proteins (e.g., PAR6, Cdc42, 14-3-3), endocytic vesicle proteins (e.g., clathrin, caveolin, dynamin 2), and actin regulatory proteins (e.g., Eps8, Arp2/3 complex), are working together, apparently under the overall influence of cytokines (e.g., transforming growth factor-β3, tumor necrosis factor-α, interleukin-1α). In short, a “new” BTB is created behind spermatocytes in transit while the “old” BTB above transiting cells undergoes timely degeneration, so that the immunological barrier can be maintained while spermatocytes are traversing the BTB. We also discuss recent findings regarding the molecular mechanisms by which environmental toxicants (e.g., cadmium, bisphenol A) induce testicular injury via their initial actions at the BTB to elicit subsequent damage to germ-cell adhesion, thereby leading to germ-cell loss, reduced sperm count, and male infertility or subfertility. Moreover, we also critically evaluate findings in the field regarding studies on drug transporters in the testis and discuss how these influx and efflux pumps regulate the entry of potential nonhormonal male contraceptives to the apical compartment to exert their effects. Collectively, these findings illustrate multiple potential targets are present at the BTB for innovative contraceptive development and for better delivery of drugs to alleviate toxicant-induced reproductive dysfunction in men.

The Blood-Testis Barrier and Its Implications for Male Contraception

The regulation of spermatogenesis by androgens.

Androgens are critical steroid hormones that determine the expression of the male phenotype, including the outward development of secondary sex characteristics as well as the initiation and maintenance of spermatogenesis. Their actions are mediated by the androgen receptor (AR), a member of the nuclear receptor superfamily. AR functions as a ligand-dependent transcription factor, regulating expression of an array of androgen-responsive genes. Androgen and the AR play important roles in male spermatogenesis and fertility. The recent generation and characterization of male total and conditional AR knockout mice from different laboratories demonstrated the necessity of AR signaling for both external and internal male phenotype development. As expected, the male total AR knockout mice exhibited female-typical external appearance (including a vagina with a blind end and a clitoris-like phallus), the testis was located abdominally, and germ cell development was severely disrupted, which was similar to a human complete androgen insensitivity syndrome or testicular feminization mouse. However, the process of spermatogenesis is highly dependent on autocrine and paracrine communication among testicular cell types, and the disruption of AR throughout an experimental animal cannot answer the question about how AR in each type of testicular cell can play roles in the process of spermatogenesis. In this review, we provide new insights by comparing the results of cell-specific AR knockout in germ cells, peritubular myoid cells, Leydig cells, and Sertoli cells mouse models that were generated by different laboratories to see the consequent defects in spermatogenesis due to AR loss in different testicular cell types in spermatogenesis. Briefly, this review summarizes these results as follows: 1) the impact of lacking AR in Sertoli cells mainly affects Sertoli cell functions to support and nurture germ cells, leading to spermatogenesis arrest at the diplotene primary spermatocyte stage prior to the accomplishment of first meiotic division; 2) the impact of lacking AR in Leydig cells mainly affects steroidogenic functions leading to arrest of spermatogenesis at the round spermatid stage; 3) the impact of lacking AR in the smooth muscle cells and peritubular myoid cells in mice results in similar fertility despite decreased sperm output as compared to wild-type controls; and 4) the deletion of AR gene in mouse germ cells does not affect spermatogenesis and male fertility. This review tries to clarify the useful information regarding how androgen/AR functions in individual cells of the testis. The future studies of detailed molecular mechanisms in these in vivo animals with cell-specific AR knockout could possibly lead to useful insights for improvements in the treatment of male infertility, hypogonadism, and testicular dysgenesis syndrome, and in attempts to create safe as well as effective male contraceptive methods.

Androgen Receptor Roles in Spermatogenesis and Fertility: Lessons from Testicular Cell-Specific Androgen Receptor Knockout Mice

This chapter discusses the modulatory influences regulating the timing of puberty in rats. The developmental process that leads to puberty in the female rat is based on an extraordinarily complex series of interrelated events. The central nervous system (CNS) plays a critical role by controlling both anterior pituitary function, through the secretion of hypothalamic factors, and the ovary via pituitary hormones and direct neural inputs. The central neuroendocrine regulation of luteinizing hormone-releasing hormone (LHRH) production and gonadotropin secretion initiates the onset of puberty in rats through LH actions on the Leydig cells to increase androgen production and follicle-stimulating hormone (FSH) actions on the Sertoli cell to enhance Leydig cell function and initiate Sertoli cell differentiation needed for the induction of spermatogenesis. The negative feedback of androgens and inhibin regulate gonadotropin production during the progression of puberty to the adult stage. The coordinated control of this hypothalamic–pituitary axis with the testis involves molecular events at the LHRH neuron and pituitary levels and at the testis somatic cell level. The resulting endocrine events control the onset and progression of puberty in the rat.

CHAPTER 38 – Puberty in the Rat

Androgens and the androgen receptor (AR) play important roles in male fertility, although the detailed mechanisms, particularly how androgen/AR influences spermatogenesis in particular cell types, remain unclear. Using a Cre-Lox conditional knockout strategy, we generated a tissue-specific knockout mouse with the AR gene deleted only in Sertoli cells (S-AR-/y). Phenotype analyses show the S-AR-/y mice were indistinguishable from WT AR mice (B6 AR+/y) with the exception of testes, which were significantly atrophied. S-AR-/y mice were infertile, with spermatogenic arrest predominately at the diplotene premeiotic stage and almost no sperm detected in the epididymides. S-AR-/y mice also have lower serum testosterone concentrations and higher serum leuteinizing hormone concentrations than B6 AR+/y mice. Further mechanistic studies demonstrated that S-AR-/y mice have defects in the expression of anti-Mullerian hormone, androgen-binding protein, cyclin A1, and sperm-1, which play important roles in the control of spermatogenesis and/or steroidogenesis. Together, our Sertoli cell-specific AR knockout mice provide in vivo evidence of the need for functional AR in Sertoli cells to maintain normal spermatogenesis and testosterone production, and ensure normal male fertility.

https://www.pnas.org/content/pnas/101/18/6876.full.pdf

Infertility with defective spermatogenesis and hypotestosteronemia in male mice lacking the androgen receptor in Sertoli cells

Androgen and the androgen receptor (AR) have been shown to play critical roles in male fertility. Our previous data demonstrated that mice lacking AR (AR(-/y)) revealed incomplete germ cell development and lowered serum testosterone levels, which resulted in azoospermia and infertility. However, the consequences of AR loss in Leydig cells remain largely unknown. Using a Cre-LoxP conditional knockout strategy, we generated a tissue-specific knockout mouse (L-AR(-/y)) with the AR gene deleted by the anti-Mullerian hormone receptor-2 (Amhr2) promoter driven Cre expressed in Leydig cells. Phenotype analyses show that the outside appearance of L-AR(-/y) mice was indistinguishable from wild type mice (AR(+/y)), but with atrophied testes and epididymis. L-AR(-/y) mice were infertile, with spermatogenic arrest predominately at the round spermatid stage and no sperm could be detected in the epididymis. L-AR(-/y) mice also have lower serum testosterone concentrations and higher serum leuteinizing hormone and follicle-stimulating hormone concentrations than AR(+/y) mice. Further mechanistic studies demonstrated that hypotestosteronemia in L-AR(-/y) mice is not caused by reducing numbers of Leydig cells, but instead by the alterations of several key steroidogenic enzymes, including 17beta-HSD3, 3beta-HSD6, and P450c17. Together, L-AR(-/y) mice provide in vivo evidence that functional AR in Leydig cells is essential to maintain normal spermatogenesis, testosterone production, and required for normal male fertility.

Shauh-Der Yeh

Papers

Infertility with defective spermatogenesis and hypotestosteronemia in male mice lacking the androgen receptor in Sertoli cells

Infertility with defective spermatogenesis and steroidogenesis in male mice lacking androgen receptor in Leydig cells