Liza O'Donnell

Spermiation: The process of sperm release.

Spermiation is the process by which mature spermatids are released from Sertoli cells into the seminiferous tubule lumen prior to their passage to the epididymis. It takes place over several days at the apical edge of the seminiferous epithelium, and involves several discrete steps including remodelling of the spermatid head and cytoplasm, removal of specialized adhesion structures and the final disengagement of the spermatid from the Sertoli cell. Spermiation is accomplished by the co-ordinated interactions of various structures, cellular processes and adhesion complexes which make up the “spermiation machinery”. This review addresses the morphological, ultrastructural and functional aspects of mammalian spermiation. The molecular composition of the spermiation machinery, its dynamic changes and regulatory factors are examined. The causes of spermiation failure and their impact on sperm morphology and function are assessed in an effort to understand how this process may contribute to sperm count suppression during contraception and to phenotypes of male infertility.

CHAPTER 21 – Endocrine Regulation of Spermatogenesis

Spermatogenesis occurs within the seminiferous tubules of the testis, in close association with the somatic cells of the seminiferous epithelium, the Sertoli cells. At the completion of spermatogenesis, mature spermatids are released from the Sertoli cells into the seminiferous tubule lumen, and proceed through the excurrent duct system, known as the rete testis, until they enter the epididymis via the efferent ducts. The duration of the proliferative period and the number of Sertoli cells produced, together with the subsequent maturation period, determines the spermatogenic potential of the testis, with each Sertoli cell capable of supporting a finite number of germ cells. The endocrine regulation of spermatogenesis is accomplished via a classic negative feedback loop involving interactions between the hypothalamus, pituitary, and testis (the hypothalamic–pituitary–testis, or HPT, axis). The production of spermatozoa is dependent on stimulation by the pituitary gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), which are secreted in response to hypothalamic gonadotropinreleasing hormone (GnRH).

Characterization of Normal Spermiation and Spermiation Failure Induced by Hormone Suppression in Adult Rats

Abstract At the end of spermatogenesis, elongated spermatids are released from supporting Sertoli cells via the process termed spermiation. Previous studies have shown that spermiation failure occurs after hormone suppression, in which spermatids are retained instead of releasing. However, the molecular mechanisms involved in spermiation and spermiation failure are largely unknown. The aims of the present study were, first, to characterize the ultrastructural events associated with normal spermiation and spermiation failure using light and electron microscopy and, second, to investigate the localization of cell adhesion-associated (β1-integrin and cadherins) and junction-associated molecules (integrin-associated kinase [ILK], β-catenin, and espin) during these processes. Four adult Sprague-Dawley rats received testosterone and estradiol implants and FSH antibody (2 mg kg−1 day−1) for 7 days to suppress testicular testosterone and FSH and to induce spermiation failure. Four rats treated with saline were used as controls. After testosterone and FSH suppression, spermiation at the ultrastructural level appeared to be normal until the final disengagement of the spermatids from Sertoli cells (stage VIII), at which stage a large number of retained spermatids were noted. Immunohistochemical localization of espin showed that during spermiation, removal of the ectoplasmic specialization (ES) occurred 30 h before spermatid disengagement, suggesting that non-ES junctions mediate the spermatid-Sertoli cell interaction before and during disengagement. β1-Integrin and β-catenin remained associated with spermatids after ES removal and until disengagement; however, ILK was removed along with the ES. Though detectable, N-cadherin was not associated with the spermatid-Sertoli cell junction. After testosterone and FSH suppression, β1-integrin, but not N-cadherin or β-catenin, remained associated with spermatids that failed to spermiate. In conclusion, hormone suppression-induced spermiation failure is caused by defects in the disengagement of spermatids from the Sertoli cell, and this process likely is mediated by β1-integrin in an ILK-independent mechanism.

Sertoli Cell Ectoplasmic Specializations in the Seminiferous Epithelium of the Testosterone-Suppressed Adult Rat

Abstract The Sertoli cell ectoplasmic specialization is a unique junctional structure involved in the interaction between elongating spermatids and Sertoli cells. We have previously shown that suppression of testicular testosterone in adult rats by low-dose testosterone and estradiol (TE) treatment causes the premature detachment of step 8 round spermatids from the Sertoli cell. Because these detaching round spermatids would normally associate with the Sertoli cell via the ectoplasmic specialization, we hypothesized that ectoplasmic specializations would be absent in the seminiferous epithelium of TE-treated rats, and the lack of this junction would cause round spermatids to detach. In this study, we investigated Sertoli cell ectoplasmic specializations in normal and TE-treated rat testis using electron microscopy and localization of known ectoplasmic specialization-associated proteins (espin, actin, and vinculin) by immunocytochemistry and confocal microscopy. In TE-treated rats where round spermatid detachment was occurring, ectoplasmic specializations of normal morphology were observed opposite the remaining step 8 spermatids in the epithelium and, importantly, in the adluminal Sertoli cell cytoplasm during and after round spermatid detachment. When higher doses of testosterone were administered to promote the reattachment of all step 8 round spermatids, newly elongating spermatids associated with ectoplasmic specialization proteins within 2 days. We concluded that the Sertoli cell ectoplasmic specialization structure is qualitatively normal in TE-treated rats, and thus the absence of this structure is unlikely to be the cause of round spermatid detachment. We suggest that defects in adhesion molecules between round spermatids and Sertoli cells are likely to be involved in the testosterone-dependent detachment of round spermatids from the seminiferous epithelium.

Ovarian steroid receptors and their role in ovarian function

The steroidogenic pathway within the ovary gives rise to progestins, androgens and oestrogens, all of which act via specific nuclear receptors to regulate reproductive function and maintain fertility. The precise role of oestrogen in the ovary remains to be elucidated, hence the data presented here which arises from studies designed to resolve this issue. Oestrogens signal via two receptor subtypes ERalpha and ERbeta, both of which are present in the ovary. ERbeta, the most abundant mRNA, is primarily expressed by GC where it transduces signals from ovarian-derived and exogenous oestrogens. Specific roles for each of the ERs in the ovary have yet to be established, despite ER knockout studies indicating both are required for normal function. The ArKO mouse is a model of oestrogen insufficiency. These mice are infertile as a result of arrested folliculogenesis (at the antral stage) and a failure to ovulate. Trans/re-differentiation of somatic cells in the ovary gives rise to Sertoli cell-like and Leydig cell-like cells within abnormal follicular structures. Disruption to the balance of sex steroids in the ovary is likely to facilitate this phenotype. Future studies will focus on the regulation of somatic cell differentiation, assigning roles to individual ERs and establishing definitive targets of oestrogen action in the ovary.

The road to ovulation: the role of oestrogens

Oestrogens have been known for many years to have a direct influence on folliculogenesis. Oestradiol-17beta (E2) and its analogues have both proliferative and differentiative effects on somatic cells of follicles. Nevertheless, definitive proof of an obligatory role for oestrogen in folliculogenesis and elucidation of the mechanisms subserving its different actions in follicular cells remains elusive. Several recent developments permit a re-examination of the roles and actions of E2 in the follicle. They are: (i) the discovery of a second form of the oestrogen receptor, ERbeta; (ii) the advent of genetically modified mice with deletions in the ERalpha (alphaERKO) ERbeta (BERKO) and the double ER deletions (alphabetaERKO); and (iii) a mouse model of oestrogen deficiency (ArKO) by targeted disruption of the cyp 19 gene encoding the aromatase enzyme. Recent information derived from these models is reviewed to re-assess the roles and actions of oestrogens in follicular dynamics and the phenotypic differentiation of ovarian somatic cells in the ovary. The data demonstrate that oestrogen is obligatory for normal folliculogenesis and that the phenotype of the ovarian somatic cells depends on the steroid milieu. The ArKO mouse provides a model to test the roles of the respective ERs in proliferation and differentiation using specific agonists and antagonists, and to study regulation of the differentiation of ovarian and testicular somatic cells.

An essential role for katanin p80 and microtubule severing in male gamete production.

Katanin is an evolutionarily conserved microtubule-severing complex implicated in multiple aspects of microtubule dynamics. Katanin consists of a p60 severing enzyme and a p80 regulatory subunit. The p80 subunit is thought to regulate complex targeting and severing activity, but its precise role remains elusive. In lower-order species, the katanin complex has been shown to modulate mitotic and female meiotic spindle dynamics and flagella development. The in vivo function of katanin p80 in mammals is unknown. Here we show that katanin p80 is essential for male fertility. Specifically, through an analysis of a mouse loss-of-function allele (the Taily line), we demonstrate that katanin p80, most likely in association with p60, has an essential role in male meiotic spindle assembly and dissolution and the removal of midbody microtubules and, thus, cytokinesis. Katanin p80 also controls the formation, function, and dissolution of a microtubule structure intimately involved in defining sperm head shaping and sperm tail formation, the manchette, and plays a role in the formation of axoneme microtubules. Perturbed katanin p80 function, as evidenced in the Taily mouse, results in male sterility characterized by decreased sperm production, sperm with abnormal head shape, and a virtual absence of progressive motility. Collectively these data demonstrate that katanin p80 serves an essential and evolutionarily conserved role in several aspects of male germ cell development.

KATNAL1 regulation of sertoli cell microtubule dynamics is essential for spermiogenesis and male fertility

Spermatogenesis is a complex process reliant upon interactions between germ cells (GC) and supporting somatic cells. Testicular Sertoli cells (SC) support GCs during maturation through physical attachment, the provision of nutrients, and protection from immunological attack. This role is facilitated by an active cytoskeleton of parallel microtubule arrays that permit transport of nutrients to GCs, as well as translocation of spermatids through the seminiferous epithelium during maturation. It is well established that chemical perturbation of SC microtubule remodelling leads to premature GC exfoliation demonstrating that microtubule remodelling is an essential component of male fertility, yet the genes responsible for this process remain unknown. Using a random ENU mutagenesis approach, we have identified a novel mouse line displaying male-specific infertility, due to a point mutation in the highly conserved ATPase domain of the novel KATANIN p60-related microtubule severing protein Katanin p60 subunit A-like1 (KATNAL1). We demonstrate that Katnal1 is expressed in testicular Sertoli cells (SC) from 15.5 days post-coitum (dpc) and that, consistent with chemical disruption models, loss of function of KATNAL1 leads to male-specific infertility through disruption of SC microtubule dynamics and premature exfoliation of spermatids from the seminiferous epithelium. The identification of KATNAL1 as an essential regulator of male fertility provides a significant novel entry point into advancing our understanding of how SC microtubule dynamics promotes male fertility. Such information will have resonance both for future treatment of male fertility and the development of non-hormonal male contraceptives.

Stage-Specific Expression of Genes Associated with Rat Spermatogenesis: Characterization by Laser-Capture Microdissection and Real-Time Polymerase Chain Reaction

Abstract Spermatogenesis in the rat consists of 14 unique morphologic cellular associations between Sertoli cells and developing germ cells within the seminiferous epithelium. The complexity of the cellular associations leads to difficulty in the isolation of individual cells at a defined stage of development for the study of their unique patterns of gene or protein expression. Thus, laser-capture microdissection is an ideal technique to permit such analysis. This study used laser-capture microdissection and real-time reverse transcription-polymerase chain reaction (RT-PCR) to quantitate the stage-specific expression of a series of genes of functional significance in hormonal regulation and cell-cell interactions in spermatogenesis, including cathepsin-L, CREM-τ, transition protein-1, androgen receptor, β1-integrin, N-cadherin, and hypoxanthine phosphoribosyltransferase (HPRT). Frozen sections (10 μm) were obtained from normal adult rat testes. Laser-capture microdissection (LCM) was used to capture all cells in cross-sections of seminiferous tubules that were grouped into stages I–V, VII–VIII, and IX–XIII. Transition protein-1 expression was lowest during stages I–V and increased 5.9-fold during stages VII–VIII and IX–XIII (P < 0.01). Cathepsin-L expression was highest during stages I–V and VII–VIII, falling 4.9-fold during stages IX–XIII (P < 0.05). Similarly, CREM-τ expression was highest during stages I–V and VII–VIII, falling 1.6-fold during stages IX–XIII (P < 0.05). A novel CREM-τ isoform lacking the phosphorylation domain was also characterized but was not stage-specific. β1-Integrin, N-cadherin, and androgen receptor expression did not change between the spermatogenic stages examined. HPRT housekeeper expression was lowest during stages I–V but increased 1.5-fold during stages VII–VIII and IX–XIII (P < 0.05). This study is the first to apply LCM and real-time RT-PCR analysis to quantitate stage-specific changes in the expression of multiple genes in the seminiferous epithelium.

5α-Reductase Isoenzymes 1 and 2 in the Rat Testis During Postnatal Development

Abstract The pubertal initiation of spermatogenesis is reliant on androgens, and during this time, 5α-reduced androgens such as dihydrotestosterone (DHT) are the predominant androgens in the testis. Two 5α-reductase (5αR) isoenzymes (5αR1 and 5αR2) have been identified, which catalyze the conversion of testosterone to the more potent androgen DHT. The present study aimed to investigate the developmental pattern of 5αR isoenzymes and their relationship to the production of 5α-reduced androgens in the postnatal rat testis. Both 5αR1 and 5αR2 isoenzyme mRNAs were measured by real-time polymerase chain reaction, isoenzyme activity levels by specific assays, and testicular androgens by radioimmunoassay after high-performance liquid chromatographic separation. Both 5αR1 and 5αR2 mRNAs and activity levels were low in the 10-day-old (prepubertal) testis, peaked between Days 20 and 40 during puberty, and then declined to low levels at 60–160 days of age. The developmental pattern of both 5αR isoenzyme activity levels was mirrored by the testicular production of 5α-reduced metabolites. Although 5αR1 was greater than 5αR2 at all ages, it is likely, given the substrate preferences of the two, that both isoenzymes contribute to the pubertal peak of 5α-reduced androgen biosynthesis. The peak in 5αR isoenzymes and 5α-reduced metabolite production coincided with the first wave of spermatogenesis in the rat, suggesting a role for 5α-reduced metabolites in the initiation of spermatogenesis. This was explored by acute administration of a 5αR inhibitor (L685,273) to immature rats. The L685,273 markedly suppressed testicular 5αR activity during puberty by 75%–86%. However, a marked increase was observed in testicular testosterone levels (in the absence of changes in LH), and no decrease was observed in the absolute levels of 5α-reduced metabolites. Therefore, whether the formation of DHT in the presence of low testosterone levels in the pubertal testis is required for the initiation of spermatogenesis cannot be tested using 5αR inhibitors. We conclude that both 5αR1 and 5αR2 isoenzymes are involved in the peak of 5α-reduced androgen biosynthesis in the testis during the pubertal initiation of spermatogenesis.