Peter G. Stanton

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.

Claudin-11 expression and localisation is regulated by androgens in rat Sertoli cells in vitro

Claudin-11 and occludin are protein components in tight junctions (TJs) between Sertoli cells which are important for the maintenance of the blood-testis barrier. Barrier formation occurs during puberty, with evidence suggesting hormonal regulation of both claudin-11 and occludin. This study aimed to investigate the regulation of claudin-11 and occludin mRNA expression by testosterone (T) and FSH and their immunolocalisation at rat Sertoli cell TJs in vitro, and to correlate any steroid regulation with the functional capacity of TJs. Sertoli cells formed functional TJs within 3 days as assessed by transepithelial electrical resistance (TER). Both T and dihydrotestosterone significantly (P < 0.01) increased TER twofold and claudin-11 mRNA two- to threefold within 3 days. FSH partially stimulated TER and claudin-11 mRNA, but estradiol had no effect. T also promoted claudin-11 localisation into extensive intercellular contacts. In contrast to claudin-11, Tand FSH did not change occludin mRNA expression, however, T promoted localisation of occludin at cell contacts in a similar manner to claudin-11. Addition of flutamide to T-stimulated cells caused a twofold decrease in both TER and claudin-11 mRNA expression, and resulted in the loss of both proteins from cell contacts. This effect was reversible following flutamide removal. It is concluded that androgens i) co-regulate claudin-11 mRNA expression and TER, implicating claudin-11 in TJ formation and ii) promote the localisation of claudin-11 and occludin at Sertoli cell contacts. Hence, the ability of androgens to maintain spermatogenesis in vivo is partly via their effects on TJ proteins and regulation of the blood-testis barrier.

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).

Adult Sertoli Cells Are Not Terminally Differentiated in the Djungarian Hamster: Effect of FSH on Proliferation and Junction Protein Organization

Abstract Sertoli cell number is considered to be stable and unmodifiable by hormones after puberty in mammals, although recent data using the seasonal breeding adult Djungarian hamster (Phodopus sungorus) model challenged this assertion by demonstrating a decrease in Sertoli cell number after gonadotropin depletion and a return to control levels following 7 days of FSH replacement. The present study aimed to determine whether adult Sertoli cells are terminally differentiated using known characteristics of cellular differentiation, including proliferation, junction protein localization, and expression of particular maturational markers, in the Djungarian hamster model. Adult long-day (LD) photoperiod (16L:8D) hamsters were exposed to short-day (SD) photoperiod (8L:16D) for 11 wk to suppress gonadotropins and then received exogenous FSH for up to 10 days. Sertoli cell proliferation was assessed by immunofluorescence by the colocalization of GATA4 and proliferating cell nuclear antigen and quantified by stereology. Markers of Sertoli cell maturation (immature, cytokeratin 18 [KRT18]; mature, GATA1) and junction proteins (actin, espin, claudin 11 [CLDN11], and tight junction protein 1 [TJP1, also known as ZO-1]) also were localized using confocal immunofluorescence. In response to FSH treatment, proliferation was upregulated within 2 days compared with SD controls (90% vs. 0.2%, P < 0.001) and declined gradually thereafter. In LD hamsters, junction proteins colocalized at the basal aspect of Sertoli cells, consistent with inter-Sertoli cell junctions, and were disordered within the Sertoli cell cytoplasm in SD animals. Exogenous FSH treatment promptly restored localization of these junction markers to the LD phenotype. Protein markers of maturity remain consistent with those of adult Sertoli cells. It is concluded that adult Sertoli cells are not terminally differentiated in the Djungarian hamster and that FSH plays an important role in governing the differentiation process. It is proposed that Sertoli cells can enter a transitional state, exhibiting features common to both undifferentiated and differentiated Sertoli cells.

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.

Structural and functional characterisation of hFSH and hLH isoforms

Human follicle-stimulating hormone (hFSH) and luteinizing hormone (hLH) are gonadotropins which are secreted as multiple forms by the pituitary. Evidence supporting the structural and functional heterogeneity of 15 purified hFSH isoforms and 20 purified hLH isoforms from pituitary extracts will be presented. Gonadotropin isoforms were purified by a combination of preparative isoelectric focusing and ion-exchange chromatography. The protein mass of each isoform was determined by amino acid analysis, which also correlated (data for hLH) (r = 0.999, P < 0.001, n = 15) with the UV area under the curve at 280 nm of the isoforms following gel-filtration HPLC. The alpha and beta subunits of FSH and LH were shown to be intact by SDS-PAGE under reducing condition, with no evidence of proteolytic nicking or presence of contaminating proteins. hFSH radioreceptor activity varied over a seven-fold range, and a positive correlation (r = 0.85, P < 0.001, n = 9) was observed between FSH receptor activity and the sialic acid (SA) content (1.5-13.7 mol SA/mol hFSH) of the isoforms, as determined by an HPLC-based microfluorometric assay. FSH in vitro activities varied over a similar range with a high correlation (r = 0.82, n = 15) with receptor activities, suggesting that the initial association of the hormone with the receptor is the key interaction with less differences attributed to subsequent effects in the signaling pathway. A similar result was seen with the hLH isoforms. To explore FSH/LH in vivo, the circulating half-life (LH/FSH) and the in vivo bioactivity (LH) using an acute in vivo assay was investigated. The clearance of hLH and hFSH showed a bi-exponential pattern for all isoform preparations with the proportion of the slower dissociating component (t 1/2 50-60 min) increasing three-fold with increasing sialic acid content of the isoform. The more rapidly cleared component (t 1/2 approx 10 min) is attributed to hepatically cleared gonadotropin, rather than gonadotropin equilibration between body compartments. The in vivo assay procedure for LH was based on the 24 h integrated plasma testosterone levels in rats following administration of graded doses of hLH isoform or standard. A 16-fold range in vivo activities between LH isoforms (n = 14) was observed. A comparison between hLH in vitro and in vivo activities showed a good correlation (r = 0.75) with the slope of the regression line (1.39) not significantly different from unity. These results suggest that in this acute in vivo assay method, the differences in circulating half-lives between hLH isoforms although large is not a key factor in their in vivo activity. However, in chronic in vivo assay systems the differences in clearance rates between isoforms may be important in their subsequent biological response. It is concluded that structural heterogeneity of FSH and LH contributes to functional differences, with a key interaction occurring at the receptor level. The contribution of sialic acid to these activities was also investigated.

Growth Differentiation Factor 9 Is a Germ Cell Regulator of Sertoli Cell Function

Oocyte-secreted growth differentiation factor (GDF) 9 and bone morphogenetic protein (BMP) 15 are critical regulatory factors in female reproduction. Together, they promote granulosa cell proliferation and stimulate the maturation of preovulatory follicles. Despite their importance in female fertility, GDF9 and BMP15 expression patterns and function during spermatogenesis have not been investigated. In this study we show that the expression and stage-specific localization of both factors are limited to the germ cells of the rat seminiferous epithelium, with GDF9 being principally localized in round spermatids and BMP15 in gonocytes and pachytene spermatocytes. To identify potential cellular targets for GDF9 actions, cells of the seminiferous tubule were isolated and screened for the expression of signaling receptors [activin-like kinase (ALK) 5, ALK6, and BMP receptor, type II)]. Individual receptor types were expressed throughout the seminiferous epithelium, but coexpression of ALK5 and BMP receptor, type II was limited to Sertoli cells and round spermatids. Based on the reproductive actions of related TGFbeta ligands in the ovary and testis, GDF9 was assessed for its ability to regulate tight junction function and inhibin B production in rat Sertoli cell cultures. When recombinant mouse GDF9 was added to immature Sertoli cell cultures, it inhibited membrane localization of the junctional proteins claudin-11, occludin, and zonula occludens-1, thereby disrupting tight junction integrity. Concomitantly, GDF9 up-regulated inhibin subunit expression and significantly stimulated dimeric inhibin B protein production. Together, these results demonstrate that GDF9 and BMP15 are germ cell-specific factors in the rat testis, and that GDF9 can modulate key Sertoli cell functions.

Is the Adult Sertoli Cell Terminally Differentiated

ABSTRACT New data have challenged the convention that the adult Sertoli cell population is fixed and unmodifiable. The Sertoli cell has two distinct functions: 1) formation of the seminiferous cords and 2) provision of nutritional and structural support to developing germ cells. For these to occur successfully, Sertoli cells must undergo many maturational changes between fetal and adult life, the main switches occurring around puberty, including the loss of proliferative activity and the formation of the blood-testis barrier. Follicle-stimulating hormone plays a key role in promoting Sertoli cell proliferation, while thyroid hormone inhibits proliferative activity in early postnatal life. Together these regulate the Sertoli-germ cell complement and sperm output in adulthood. By puberty, the Sertoli cell population is considered to be stable and unmodifiable by hormones. But there is mounting evidence that the size of the adult Sertoli cell population and its maturational status is modifiable by hormones and that Sertoli cells can gain proliferative ability in the spermatogenically disrupted hamster and human model. This new information demonstrates that the adult Sertoli cell population, at least in the settings of testicular regression in the hamster and impaired fertility in humans in vivo and from mice and men in vitro, is not a terminally differentiated population. Data from the hamster now show that the adult Sertoli cell population size is regulated by hormones. This creates exciting prospects for basic and clinical research in testis biology. The potential to replenish an adult Sertoli-germ cell complement to normal in a setting of infertility may now be realized.

Post-translational modifications and protein-specific isoforms in endometriosis revealed by 2D DIGE.

Endometriosis is a chronic disorder affecting approximately 10% of women in whom endometrial tissue forms painful lesions outside the uterus. It has a major impact on their physical, mental and social well-being but has no known cure, and there is no nonsurgical means of diagnosis. We have used a proteomic approach to identify proteins with altered abundance in the eutopic endometrium of endometriosis patients in the midsecretory phase of the menstrual cycle. 2D-differential in gel electrophoresis (DIGE) and mass spectrometry identified 20 proteins that were present at different levels in endometriosis patients (p < 0.05), many of which have not previously been associated with endometriosis. Protein abundance changes did not correlate well with published gene array data, emphasizing the extensive post-translational modification that occurs in this tissue. Abundance or localization changes in endometrial tissue were validated by immunohistochemistry and Western blotting for three proteins, vimentin (VIM), peroxiredoxin 6 (PRDX6), and ribonuclease/angiogenin inhibitor 1 (RNH1), while observed changes could not be confirmed for coronin 1A (CORO1A) or transgelin (TAGLN2). In addition, multiple charge and size isoforms were observed for PDRX6 and vimentin (VIM), and an additional PDRX6 isoform was observed in endometriosis patients that was below the level of detection in healthy women. Biological pathway analysis identified that cytoskeletal remodeling via keratin intermediate filaments, processing of the cystic fibrosis transmembrane receptor (CFTR), the glucocorticoid receptor subunit alpha (GCR), and heat shock factor 1 (HSF1) were all significantly over-represented features in endometriosis patients. This study highlights the highly dynamic nature of endometrial tissue and suggests that considerable post-translational modification of proteins is a key factor in the pathology of endometriosis.

Regulation of testicular tight junctions by gonadotrophins in the adult Djungarian hamster in vivo.

This study aimed to assess the effect of gonadotrophin suppression and FSH replacement on testicular tight junction dynamics and blood-testis barrier (BTB) organisation in vivo, utilising the seasonal breeding Djungarian hamster. Confocal immunohistology was used to assess the cellular organisation of tight junction proteins and real-time PCR to quantify tight junction mRNA. The effect of tight junction protein organisation on the BTB permeability was also investigated using a biotin-linked tracer. Tight junction protein (claudin-3, junctional adhesion molecule (JAM)-A and occludin) localisation was present but disorganised after gonadotrophin suppression, while mRNA levels (claudin-11, claudin-3 and occludin) were significantly (two- to threefold) increased. By contrast, both protein localisation and mRNA levels for the adaptor protein zona occludens-1 decreased after gonadotrophin suppression. FSH replacement induced a rapid reorganisation of tight junction protein localisation. The functionality of the BTB (as inferred by biotin tracer permeation) was found to be strongly associated with the organisation and localisation of claudin-11. Surprisingly, JAM-A was also recognised on spermatogonia, suggesting an additional novel role for this protein in trans-epithelial migration of germ cells across the BTB. It is concluded that gonadotrophin regulation of tight junction proteins forming the BTB occurs primarily at the level of protein organisation and not gene transcription in this species, and that immunolocalisation of the organised tight junction protein claudin-11 correlates with BTB functionality.