Immunology, Endocrine & Metabolic Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry - Immunology, Endocrine and Metabolic Agents) - Volume 8, Issue 1, 2008

When I was a graduate student in the late 1970s, much of the study that was conducted on male reproductive biology which formed the basis of male contraceptive research focused on three main areas: (i) endocrine regulation at the level of the hypothalamic-pituitary-testicular axis, (ii) morphological studies to delineate the events of sperm maturation in the testis and epididymis, and (iii) the biology of spermatozoa (e.g., sperm-specific antigens for vaccine development) and sperm-egg interactions. Some efforts were also made to use extracts of natural products (e.g., gossypol from cotton seeds), chemicals (e.g., GnRH antagonists and/or agonists, lonidamine) and several others for male contraception. At that time, I was studying sperm function in the human. However, developments in biochemistry, medicinal chemistry, and cell and molecular biology in the next two decades have drastically transformed the approaches used to study male reproductive biology and contraceptive research. With these powerful research techniques in hand, investigators have tackled many basic questions which would not have been possible just a decade ago. In this Special Issue entitled: “Male Contraception” published in Immunology, Endocrine & Metabolic Agents in Medicinal Chemistry, I have attempted to bring forth a few selected topics written by investigators in the field. These articles have introduced some thought-provoking concepts, which I felt might interest both young and senior investigators. Due to space limitations, I was not able to cover all areas of research interest in the field, such as recent developments in the use of testosterone or testosterone and progestin combinations to induce reversible infertility in men. Nonetheless, I have attempted to provide a balanced treatment on the topic of “Male Contraception” by recruiting a diverse group of investigators to contribute to this issue, making it a timely collection of review articles for reproductive biologists and investigators in male contraceptive research. While we have seen a significant drop in funding in contraceptive research in recent years, enthusiasm among scientists has not diminished. I hope that this Special Issue will further spark interest among investigators in the field. I wish to thank my colleagues who have taken their time to write an article for this Special Issue. I am also grateful to Bentham Science who has helped me to assemble this issue from its inception to its publication.

Androgens play a key role in the control of spermatogenesis and interference with their intratesticular secretion and action is a critical element in many contraceptive strategies. Nonetheless, the cellular and molecular mechanisms by which androgens control germ cell development remain poorly understood. Recent transgenic models in which the androgen receptor (AR) is selectively ablated in Sertoli cells show unambiguously that the Sertoli cell is the main target for androgen action in the control of spermatogenesis. A number of additional mouse models have been developed mimicking human diseases in which mutations of the AR cause disturbed fertility without affecting male development. Transcriptional profiling studies in mice with Sertoli cell-selective AR ablation and in some other experimental paradigms have tried to identify androgen-regulated genes relevant to the control of spermatogenesis. The overlap in genes identified in different studies is poor but this may be due mainly to dissimilarities in experimental setup. In all studies, relatively large numbers of genes rather than a few key genes seem to be affected by androgen action. Genes related to tubular restructuring, cell junction dynamics, cytoskeleton, solute transportation and vitamin A metabolism are prominently present. Although further work is obviously needed, it may be anticipated that these studies will result in the identification of subsets of genes that can be used as diagnostic tools as well as in the identification of targets for the development of novel contraceptives.

The post-translational modification of proteins by covalent attachment of ubiquitin molecules targets protein for degradation by 26S proteasome composed of a 20S core unit capped with one 19S regulatory complex on each side. Such event plays a key role in numerous cellular functions including spermatogenesis. It is conceivable that ubiquitination is involved in all phases of spermatogenesis and its destinated functions control various cellular activities in the testis, ranging from the mitotic spindle formation in germ cells to the structural formation of the spermatid flagella. In this review, we aim to summarize the recent findings in the study of ubiquitination in the testis. It also highlights specific areas for future research toward a better understanding of the precise physiological relationship between ubiquitin-mediated proteolysis and spermatogenesis. Furthermore, a thorough understanding of this system that can lead to developing new approaches for male contraception is discussed.

Cytokines are known to regulate an array of physiological functions in the testis, including cell differentiation, apoptosis, steroidogenesis, and cell division. Recent studies have illustrated that cytokines also take a crucial role in the regulation of junction dynamics. These include the regulation of cell-cell adhesion and tight junction permeability barriers in multiple epithelia and endothelia, such as those found in the small intestine, kidney, skin, and testis. In this review, we summarize recent findings in this field with an emphasis on the role of cytokines in junction restructuring events during spermatogenesis in the seminiferous epithelium of testes. This review also identifies several areas of research that functional studies can be designed to unravel the physiological significance of cytokines in junction restructuring at the Sertoli-Sertoli or Sertoli-germ cell interface in the seminiferous epithelium. It is expected that multiple cytokines, such as TGF-β3 and TNFα, are working in concert with other yet-to-be identified molecules to coordinate the intriguing events of junction restructuring during different stages of the seminiferous epithelial cycle in adult testes in mammals.

During spermatogenesis, development of spermatogonia into elongated spermatids takes place in the seminiferous epithelium of the adult mammalian testis. Specifically, post-meiotic germ cell maturation occurs in a unique microenvironment sequestered from the systemic circulation by the blood-testis barrier (BTB), which is formed by adjacent Sertoli cells. Therefore, an intact BTB, as well as stable Sertoli-germ cell adhesion, are important criteria for successful spermatogenesis. To date, numerous factors have been shown to influence spermatogenesis, and among them is the well-studied nitric oxide (NO)/guanosine 3',5'-cyclic monophosphate (cGMP) signaling cascade. The enzymes of this pathway, namely nitric oxide synthase, soluble guanylate cyclase and cGMP-dependent protein kinase, have all been shown to regulate cell junctions in the testis. Likewise, recent findings have shown that this signaling cascade also plays a critical role in the regulation of Sertoli-germ cell adhesion. In this mini-review, we briefly discuss the regulatory role of each protein component of the NO/cGMP pathway in the context of testicular junction dynamics, as well as their importance in fertility and male contraception.

The acrosome is a secretory vesicle located in the mammalian sperm head. Its main function is to transport hydrolytic enzymes. These enzymes will dissolve the zona pellucid, assisting the sperm in fertilizing the egg. The acrosome is assembled in the haploid spermatid during spermiogenesis, but many of its enzymes are already synthesized in pachytene spermatocytes during the early phase of spermatogenesis. Haploid spermatids have developed unique mechanism(s) to assure the proper localization/orientation of the acrosome, attachment near the nucleus, and the targeting of acrosomal proteins towards this vesicle. During spermiogenesis, these germ cells undergo dramatic transformations in shape and intracellular distribution of organelles, and the configuration of microtubules seems to be involved with each specific step. In this context, it seems microtubules are essential for the assembly and formation of the acrosome during spermiogenesis. Microtubules are also involved in guiding proteins to the acrosome, since genetic mouse models with abnormal spermatozoa as their only phenotype were shown to have alterations of the microtubule configuration in round and elongating spermatids, while their spermatozoa were having misplacement of acrosomal proteins. A few genetic mouse models have been designed to study vesicular trafficking or acrosome assembly, and some of these models closely resemble specific human fertility pathologies such as oligoasthenospermia or acrosomeless round-headed sperm. Thus, the study of acrosome biogenesis could aid the development of new infertility tests or the discovery of new regulation targets of sperm production.

Muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors, consisting of five subtypes (M1 to M5). These receptors play different roles in autocrine and neuronal systems and are important in the physiology and pathophysiology of various organs. Activation of mAChRs may affect cell proliferation, differentiation, growth and other functions in the male reproductive tract. This review focuses on the identification and function of mAChR subtypes in the male reproductive tract with emphasis on the signaling transduction pathways activated by the agonist-mAChR complex in rat Sertoli cells. The unique localization of mAChR subtypes in specific compartments of the efferent ductules, epididymis, vas deferens, seminal vesicle and prostate found in various species have suggested a role for these receptors in the modulation of luminal fluid composition and smooth muscle contraction. In Sertoli cells, the activation of mAChRs induces transactivation of the epidermal growth factor receptor (EGFR) through βγ-subunits of G proteins that promote Src-mediated metalloprotease-dependent cleavage and the release of EGFR ligands from the cell surface, to be followed by the binding of these ligands to EGFR and activation of extracellular signal-regulated kinases (ERK)1/2. Furthermore, an increase on ERK1/2 phosphorylation via phospholipase Cβ (PLCβ) and intracellular Ca++ mobilization dependent mechanisms is also involved, but no involvement of protein kinase C (PKC) is observed. The transactivation of EGFR by the agonist-mAChR complex is also involved in Sertoli cell proliferation. Since receptors are targets for pharmacological manipulation of physiological processes, these studies may be important for the development of selective therapeutic agents and new approaches in fertility/infertility and/or male contraception.

Dynamins are large GTPases of ∼100 kDa known to participate in endocytosis and interact with the actin-based cytoskeletal network in multiple tissues. Recent studies have shown that dynamins play a critical role in the internalization of integral membrane proteins via either clathrin-mediated or clathrin-independent endocytosis. Furthermore, recent studies have shown that dynamin II interacts with junctional complex adaptors, namely ZO-1 and β-catenin, at the blood-testis barrier in the seminiferous epithelium of adult rat testes. This interaction may be responsible for pulling away tight junction- and adherens junction-based protein complexes, thereby facilitating blood-testis barrier opening to permit preleptotene and leptotene spermatocyte migration, which is a critical event in spermatogenesis occurring at stage VIII of the seminiferous epithelial cycle. In this short review, we highlight some of the latest findings on dynamins in the field, and discuss how this information can be used to further expand the functional studies to tackle the role of dynamins in spermatogenesis. It is likely that dynamins per se or their interacting protein partners can become a target for male contraceptive research to compromise spermatogenesis, leading to transient male infertility without perturbing the hypothalamic-pituitary-testicular axis.

In the mammalian testis, the irreversible conversion of androgens into estrogens is catalyzed by the cytochrome P450 aromatase, a microsomal enzymatic complex encoded by a unique gene (Cyp19) in humans which contained 18 exons, 9 of them being translated. In addition that gene includes 9 non-coding exons I, located in the 5' end and controlled by tissue-specific promoters that are spliced alternatively onto a common site in exon II. However, a unique protein of 55 kDa is produced. In most of mammals studied so far, at least in rodents, all testicular cells except peritubular cells contain aromatase which is therefore constitutively expressed and very precisely controlled (in terms of expression and enzyme activity) according to somatic and germ cell origin. In man, our data obtained from both ejaculated spermatozoa and immature gem cells demonstrate the presence of a biologically active aromatase ; moreover estrogen receptors (ER alpha and ER beta) are also present, especially ERβ in seminiferous tubules. All together according to the widespread localisation of aromatase and estrogen receptors in testicular cells, our review clearly shows that besides gonadotrophins and androgens, estrogens produced locally, should be considered as physiologically relevant hormones involved in the regulation of spermatogenesis and spermiogenesis. Therefore it is suggested that aromatase could be a helpful marker which might reflect the gene expression during spermatogenesis as well as the sperm quality and consequently, that parameter may be used as a diagnostic tool to assess the fertility in man.

Spermatogenesis is a dynamic process in which stem spermatogonia through a series of events become mature spermatozoa. Not all germ cells achieve maturity and cell death by apoptosis appears to be a constant feature of normal spermatogenesis in a variety of mammalian species to maintain proper germ cell numbers. It is because of the obvious importance of the germ cells in the context of species propagation, the testis has very active prosurvival and proapoptotic systems that together regulate the extent of germ cell apoptosis. A growing body of evidence demonstrates that both spontaneous and increased germ cell death are triggered by various regulatory stimuli, including deprivation of gonadotropins and intratesticular testosterone by gonadotropin-releasing hormone antagonist or by estradiol or testosterone treatment, exposure to local testicular heating, toxicants, and chemotherapeutic agents. This review focuses on the cell death events that occur in testis under various conditions and provide an assessment of the knowledgebase existing on the role of pro and anti-apoptotic proteins and involvement of the cell death receptor and the mitochondrial pathway during germ cell apoptosis. Various knockout or mutant models of pro and anti-apoptotic proteins are discussed with an aim to provide an overview of requirement of various genes in germ cell apoptosis.

In recent years, the impact of various environmental toxicants on male reproductive function has received considerable attention, partly fuelled by the reports of falling sperm count and rising reproductive disorders in human population. Environmental toxicants disturb the pro-oxidant/ anti-oxidant balance of the testis resulting in impairment of testicular function. The reactive oxygen species (ROS) and associated oxidative stress has grown up to the point of molecular mechanism responsible for male infertility. Therefore, there is a greater need to look into the reason behind the vulnerability of male reproductive system to ROS. Under physiological conditions, the testicular spermatogenesis and steroidogenesis are associated with production of ROS that makes them more susceptible to oxidative stress. In addition, exposure to toxicants aggravates the production of ROS to greater extent. ROS are involved in regulation of testicular functions within critical levels. The present review outlines the source of ROS, its role in testis and summarizes recent studies on ROSmediated effects of environmental toxicants on testicular function. Testicular spermatogenesis and steroidogenesis are a source of ROS and also a target for their disruptive actions. The role of environmental toxicants in changing testicular ROS levels merits further investigation.

Besides hormonal-based male contraceptives, such as testosterone undecanoate (a long-chain ester of testosterone) which can be administered either orally as contraceptive pill or by injection, some efforts have been made in the field to develop non-hormonal contraceptives to suppress spermatogenesis. One of the major goals for non-hormonal contraceptives is to avoid a disruption of the hypothalamic- pituitary-testicular axis, without affecting the systemic testosterone, LH and FSH levels, hoping to minimize the side-effects since testosterone has multiple target organs besides the testis. However, these non-hormonal male contraceptives are often met with poor absorption at the gastrointestinal tract and if they are peptide/protein based, they are subjected to proteolytic cleavage following oral administration. Thus, other non-oral routes are being considered. In this short review, we highlighted some of the latest development in the field regarding the administration of other therapeutic drugs via non-parenteral and non-oral routes. This information as briefly reviewed herein should provide some insights for delivery of male contraceptives in the future.

In adult mammalian testes, cell junctions not only maintain the integrity of the testis, their dynamic turnover also plays different crucial roles in spermatogenesis during the seminiferous epithelial cycle, such as cell-cell communication to coordinate meiosis, spermatid orientation and germ cell movement. In the testis, disassembly and reassembly of cell junctions at the Sertoli-Sertoli and Sertoli- germ cell interface allow the translocation of germ cells towards the adluminal compartment, but at the same time, germ cells remain attach to Sertoli cells for structural and nutritional supports. Temporal and spatial expressions of junction proteins in Sertoli cells and germ cells therefore become one of the major events to maintain such process. A precise control of such event could be in part achieved by transcriptional and post-transcriptional modifications of target genes pertinent to the components of the junction complexes. In this review, we aim to summarize the recent findings in the study of transcriptional and post-transcriptional regulations of junction proteins in epithelial cells and in the testis. It also highlights specific areas for future studies. A comprehensive study on this topic definitely leads to a thorough understanding of spermatogenesis and the development of new approaches for male contraception.