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Male fertility is dependent on spermatogonial stem cells (SSCs) that self-renew and produce differentiating germ cells. Growth factors produced within the testis are essential for SSC maintenance but intrinsic factors that dictate the SSC response to these stimuli are poorly characterised. Here, we have studied the role of GILZ, a TSC22D family protein and spermatogenesis regulator, in spermatogonial function and signalling. Although broadly expressed in the germline, GILZ was prominent in undifferentiated spermatogonia and Gilz deletion in adults resulted in exhaustion of the GFRalpha1+ SSC-containing population and germline degeneration. GILZ loss was associated with mTORC1 activation, suggesting enhanced growth factor signalling. Expression of deubiquitylase USP9X, an mTORC1 modulator required for spermatogenesis, was disrupted in Gilz mutants. Treatment with an mTOR inhibitor rescued GFRalpha1+ spermatogonial failure, indicating that GILZ-dependent mTORC1 inhibition is crucial for SSC maintenance. Analysis of cultured undifferentiated spermatogonia lacking GILZ confirmed aberrant activation of ERK MAPK upstream mTORC1 plus USP9X downregulation and interaction of GILZ with TSC22D proteins. Our data indicate an essential role for GILZ-TSC22D complexes in ensuring the appropriate response of undifferentiated spermatogonia to growth factors via distinct inputs to mTORC1.Copyright © 2018. Published by The Company of Biologists Ltd.

The aim of this study was to prove the effects of dexamethasone on immune responses in rats with testicular torsion. This was an experimental study with post test only control group design. In this study, 27 rats were randomly divided into 3 groups. In normal or sham group (CN) orchidectomy was performed on the right testis on day 14. The positive control group (CP) had testicular torsion for 10 h, then manual detorsion was performed after 4 h, and on day 14 orchidectomy was performed on ipsilateral and contralateral testes. The treatment group (N) had testicular torsion for 10 h and received 10 mg/kgBW dexamethasone subcutaneously 30 minutes before manual detorsion 4 hours later, and on day 14 orchidectomy was performed to ipsilateral and contralateral testes. The spermatogonium count was calculated based on the average number of intratubular seminiferous tubule section observed in contralateral testicular incision with Haematoxiliyn Eosin (HE) staining. IgG count was calculated based on the average number of extratubular seminiferous tubules observed at 5 contralateral testicular incision with immunohistochemical staining (IHC). The number of spermatogonia was significantly different between CN and CP, CN and N, and CP and N (p<0.05). IgG production also showed significant differences between CN and CP, CN and N, as well as CP and N (p<0.05). In conclusion, administration of dexamethasone provides a protective effect on spermatogonium count and IgG production on a 10-hour testicular torsion.

Male fertility is dependent on spermatogonial stem cells (SSCs) that self-renew and produce differentiating germ cells. Growth factors produced within the testis are essential for SSC maintenance but intrinsic factors that dictate the SSC response to these stimuli are poorly characterised. Here, we have studied the role of GILZ, a TSC22D family protein and spermatogenesis regulator, in spermatogonial function and signalling. Although broadly expressed in the germline, GILZ was prominent in undifferentiated spermatogonia and Gilz deletion in adults resulted in exhaustion of the GFRalpha1+ SSC-containing population and germline degeneration. GILZ loss was associated with mTORC1 activation, suggesting enhanced growth factor signalling. Expression of deubiquitylase USP9X, an mTORC1 modulator required for spermatogenesis, was disrupted in Gilz mutants. Treatment with an mTOR inhibitor rescued GFRalpha1+ spermatogonial failure, indicating that GILZ-dependent mTORC1 inhibition is crucial for SSC maintenance. Analysis of cultured undifferentiated spermatogonia lacking GILZ confirmed aberrant activation of ERK MAPK upstream mTORC1 plus USP9X downregulation and interaction of GILZ with TSC22D proteins. Our data indicate an essential role for GILZ-TSC22D complexes in ensuring the appropriate response of undifferentiated spermatogonia to growth factors via distinct inputs to mTORC1.Copyright © 2018. Published by The Company of Biologists Ltd.

The aim of this study was to prove the effects of dexamethasone on immune responses in rats with testicular torsion. This was an experimental study with post test only control group design. In this study, 27 rats were randomly divided into 3 groups. In normal or sham group (CN) orchidectomy was performed on the right testis on day 14. The positive control group (CP) had testicular torsion for 10 h, then manual detorsion was performed after 4 h, and on day 14 orchidectomy was performed on ipsilateral and contralateral testes. The treatment group (N) had testicular torsion for 10 h and received 10 mg/kgBW dexamethasone subcutaneously 30 minutes before manual detorsion 4 hours later, and on day 14 orchidectomy was performed to ipsilateral and contralateral testes. The spermatogonium count was calculated based on the average number of intratubular seminiferous tubule section observed in contralateral testicular incision with Haematoxiliyn Eosin (HE) staining. IgG count was calculated based on the average number of extratubular seminiferous tubules observed at 5 contralateral testicular incision with immunohistochemical staining (IHC). The number of spermatogonia was significantly different between CN and CP, CN and N, and CP and N (p<0.05). IgG production also showed significant differences between CN and CP, CN and N, as well as CP and N (p<0.05). In conclusion, administration of dexamethasone provides a protective effect on spermatogonium count and IgG production on a 10-hour testicular torsion.

The aim of this study was to prove the effects of dexamethasone on immune responses in rats with testicular torsion. This was an experimental study with post test only control group design. In this study, 27 rats were randomly divided into 3 groups. In normal or sham group (CN) orchidectomy was performed on the right testis on day 14. The positive control group (CP) had testicular torsion for 10 h, then manual detorsion was performed after 4 h, and on day 14 orchidectomy was performed on ipsilateral and contralateral testes. The treatment group (N) had testicular torsion for 10 h and received 10 mg/kgBW dexamethasone subcutaneously 30 minutes before manual detorsion 4 hours later, and on day 14 orchidectomy was performed to ipsilateral and contralateral testes. The spermatogonium count was calculated based on the average number of intratubular seminiferous tubule section observed in contralateral testicular incision with Haematoxiliyn Eosin (HE) staining. IgG count was calculated based on the average number of extratubular seminiferous tubules observed at 5 contralateral testicular incision with immunohistochemical staining (IHC). The number of spermatogonia was significantly different between CN and CP, CN and N, and CP and N (p<0.05). IgG production also showed significant differences between CN and CP, CN and N, as well as CP and N (p<0.05). In conclusion, administration of dexamethasone provides a protective effect on spermatogonium count and IgG production on a 10-hour testicular torsion.

Background: Our understanding of the molecular mechanisms implementing pubertal maturation of the testis in vertebrates is incomplete. This topic is relevant in Atlantic salmon aquaculture, since precocious male puberty negatively impacts animal welfare and growth. We hypothesize that certain miRNAs modulate mRNAs relevant for the initiation of puberty. To explore which miRNAs regulate mRNAs during initiation of puberty in salmon, we performed an integrated transcriptome analysis (miRNA and mRNA-seq) of salmon testis at three stages of development: an immature, long-term quiescent stage, a prepubertal stage just before, and a pubertal stage just after the onset of single cell proliferation activity in the testis. Results: Differentially expressed miRNAs clustered into 5 distinct expression profiles related to the immature, prepubertal and pubertal salmon testis. Potential mRNA targets of these miRNAs were predicted with miRmap and filtered for mRNAs displaying negatively correlated expression patterns. In summary, this analysis revealed miRNAs previously known to be regulated in immature vertebrate testis (miR-101, miR-137, miR-92b, miR-18a, miR-20a), but also miRNAs first reported here as regulated in the testis (miR-new289, miR-30c, miR-724, miR-26b, miR-new271, miR-217, miR-216a, miR-135a, miR-new194 and the novel predicted n268). By KEGG enrichment analysis, progesterone signaling and cell cycle pathway genes were found regulated by these differentially expressed miRNAs. During the transition into puberty we found differential expression of miRNAs previously associated (let7a/b/c), or newly associated (miR-15c, miR-2184, miR-145 and the novel predicted n7a and b) with this stage. KEGG enrichment analysis revealed that mRNAs of the Wnt, Hedgehog and Apelin signaling pathways were potential regulated targets during the transition into puberty. Likewise, several regulated miRNAs in the pubertal stage had earlier been associated (miR-20a, miR-25, miR-181a, miR-202

STUDY QUESTION Are Sertoli cell tight junctions (TJs) disrupted in men undergoing hormonal contraception? SUMMARY ANSWER Localization of the key Sertoli cell TJ protein, claudin-11, was markedly disrupted by 8 weeks of gonadotropin suppression, the degree of which was related to the extent of adluminal germ cell suppression. WHAT IS KNOWN ALREADY Sertoli cell TJs are vital components of the blood-testis barrier (BTB) that sequester developing adluminal meiotic germ cells and spermatids from the vascular compartment. Claudin-11 knockout mice are infertile; additionally claudin-11 is spatially disrupted in chronically gonadotropin-suppressed rats coincident with a loss of BTB function, and claudin-11 is disorganized in various human testicular disorders. These data support the Sertoli cell TJ as a potential site of hormonal contraceptive action. STUDY DESIGN, SIZE, DURATION BTB proteins were assessed by immunohistochemistry (n = 16 samples) and mRNA (n = 18 samples) expression levels in available archived testis tissue from a previous study of 22 men who had undergone 8 weeks of gonadotropin suppression and for whom meiotic and post-meiotic germ cell numbers were available. The gonadotropin suppression regimens were (i) testosterone enanthate (TE) plus the GnRH antagonist, acyline (A); (ii) TE + the progestin, levonorgestrel, (LNG); (iii) TE + LNG + A or (iv) TE + LNG + the 5alpha-reductase inhibitor, dutasteride (D). A control group consisted of seven additional men, with three archived samples available for this study. PARTICIPANTS/MATERIALS, SETTINGS, METHODS Immunohistochemical localization of claudin-11 (TJ) and other junctional type markers [ZO-1 (cytoplasmic plaque), beta-catenin (adherens junction), connexin-43 (gap junction), vinculin (ectoplasmic specialization) and beta-actin (cytoskeleton)] and quantitative PCR was conducted using matched frozen testis tissue. MAIN RESULTS AND THE ROLE OF CHANCE Claudin-11 formed a continuous staining pattern at the BTB in

STUDY QUESTION Are Sertoli cell tight junctions (TJs) disrupted in men undergoing hormonal contraception? SUMMARY ANSWER Localization of the key Sertoli cell TJ protein, claudin-11, was markedly disrupted by 8 weeks of gonadotropin suppression, the degree of which was related to the extent of adluminal germ cell suppression. WHAT IS KNOWN ALREADY Sertoli cell TJs are vital components of the blood-testis barrier (BTB) that sequester developing adluminal meiotic germ cells and spermatids from the vascular compartment. Claudin-11 knockout mice are infertile; additionally claudin-11 is spatially disrupted in chronically gonadotropin-suppressed rats coincident with a loss of BTB function, and claudin-11 is disorganized in various human testicular disorders. These data support the Sertoli cell TJ as a potential site of hormonal contraceptive action. STUDY DESIGN, SIZE, DURATION BTB proteins were assessed by immunohistochemistry (n = 16 samples) and mRNA (n = 18 samples) expression levels in available archived testis tissue from a previous study of 22 men who had undergone 8 weeks of gonadotropin suppression and for whom meiotic and post-meiotic germ cell numbers were available. The gonadotropin suppression regimens were (i) testosterone enanthate (TE) plus the GnRH antagonist, acyline (A); (ii) TE + the progestin, levonorgestrel, (LNG); (iii) TE + LNG + A or (iv) TE + LNG + the 5alpha-reductase inhibitor, dutasteride (D). A control group consisted of seven additional men, with three archived samples available for this study. PARTICIPANTS/MATERIALS, SETTINGS, METHODS Immunohistochemical localization of claudin-11 (TJ) and other junctional type markers [ZO-1 (cytoplasmic plaque), beta-catenin (adherens junction), connexin-43 (gap junction), vinculin (ectoplasmic specialization) and beta-actin (cytoskeleton)] and quantitative PCR was conducted using matched frozen testis tissue. MAIN RESULTS AND THE ROLE OF CHANCE Claudin-11 formed a continuous staining pattern at the BTB in

Spermatogonia stem cell (SSC) self-renewal and differentiation are tightly regulated processes that ensure a continued production of mature sperm throughout male adulthood. In the present study, we investigated the role of glucocorticoid-induced leucine zipper (GILZ) in maintenance of the male germline and spermatogenesis. GILZ was detectable in germ cells of wild type mice on the day of birth, suggesting a role for GILZ in prospermatogonia and SSC pool formation. Gilz KO mice were generated and adult males were azoospermic and sterile. During the first wave of spermatogenesis in Gilz KO mice, spermatogenesis arrested part way through pachytene of meiosis I. Subsequent waves resulted in a progressive depletion of germ cells through apoptosis to ultimately produce a Sertoli cell-only phenotype. Further, in contrast to wild type littermates, PLZF+ cells were detected in the peri-luminal region of Gilz KO mice at day 6 post-natal, suggesting a defect in prospermatogonia migration in the absence of GILZ. At age 30 days, transient accumulation of PLZF+ cells in a subset of tubules and severely compromised spermatogenesis were observed in Gilz KO mice, consistent with defective SSC differentiation. GILZ deficiency was associated with an increase in FOXO1 transcriptional activity, which leads to activation of a selective set of FOXO1 target genes, including a pro-apoptotic protein, BIM. On the other hand, no evidence of a heightened immune response was observed. Together, these results suggest that GILZ suppresses FOXO1 nuclear translocation, promotes SSC differentiation over self-renewal, and favours germ cell survival through inhibition of BIM-dependent pro-apoptotic signals. These findings provide a mechanism for the effects of GILZ on spermatogenesis and strengthen the case for GILZ being a critical molecule in the regulation of male fertility. © 2013 Ngo et al.

The function of protease during male meiosis has not been well studied. We previously cloned and characterized four testis-specific serine proteases in the mouse testis. One of the proteases, Prss41/Tessp-1, was expressed in the germ and Sertoli cell. This time, to examine the involvement of Prss41/Tessp-1 in spermatogenesis, we conducted the organ culture of testis fragments in the presence of the anti-Prss41/Tessp-1 antibody. Because in the Sertoli cell, the Prss41/Tessp-1 protein was mostly associated with the membrane of intracellular organelles by glycosylphosphatidylinositol, the antibody was expected to affect Prss41/Tessp-1 at the plasma membrane of spermatogonia. By adding the antibody, the number of germ cells was decreased in some seminiferous tubules. The marker genes expression strongly suggested that meiosis was arrested at spermatogonia, and the number of apoptotic germ cells increased by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. These data indicated that Prss41/Tessp-1 was necessary for the progression of meiosis at the stage of spermatogonia during in vitro spermatogenesis. Together with our previous study, the current results suggest that the Prss/Tessp proteases are important for the progression of meiosis at each stage.

The function of protease during male meiosis has not been well studied. We previously cloned and characterized four testis-specific serine proteases in the mouse testis. One of the proteases, Prss41/Tessp-1, was expressed in the germ and Sertoli cell. This time, to examine the involvement of Prss41/Tessp-1 in spermatogenesis, we conducted the organ culture of testis fragments in the presence of the anti-Prss41/Tessp-1 antibody. Because in the Sertoli cell, the Prss41/Tessp-1 protein was mostly associated with the membrane of intracellular organelles by glycosylphosphatidylinositol, the antibody was expected to affect Prss41/Tessp-1 at the plasma membrane of spermatogonia. By adding the antibody, the number of germ cells was decreased in some seminiferous tubules. The marker genes expression strongly suggested that meiosis was arrested at spermatogonia, and the number of apoptotic germ cells increased by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. These data indicated that Prss41/Tessp-1 was necessary for the progression of meiosis at the stage of spermatogonia during in vitro spermatogenesis. Together with our previous study, the current results suggest that the Prss/Tessp proteases are important for the progression of meiosis at each stage.

Spermatogonial proliferation is a critical but poorly understood determinant of the spermatogenic process. In rats, exogenous testosterone plus oestradiol (TE) markedly suppresses serum LH and testicular testosterone levels. In the TE-treated rat, spermatogonial number declines to 65% of control levels while round to elongated spermatid maturation is interrupted. The partial restoration of testicular testosterone levels by exogenous testosterone administration restores spermatid maturation but neither testicular weight nor spermatogonial number are normalized. This study aimed to determine the role of testosterone and/or non-androgenic Leydig cell factors in the restoration of spermatogonial number in the testosterone-treated rat. Germ cell numbers were assessed using stereological methods and expressed as germ cell number per testis. Adult Sprague Dawley rats initially received 3 cm testosterone plus 0.4 cm oestradiol Silastic implants for 9 weeks to suppress spermatogenesis, followed by 10 days of either (i) exogenous testosterone using implants (T24 cm) or testosterone esters (5 or 25 mg sc every third day), or (ii) human chorionic gonadotrophin (hCG; 0.5, 1.25, 2.5 or 10 IU/kg sc daily) as an LH substitute to restore Leydig cell function. Following TE treatment, testicular weights and testicular testosterone levels were reduced to 31%, and 1.3% of control levels, respectively. In response to exogenous testosterone administration, testicular weight was restored to 52-58% of controls while testicular testosterone levels increased to only 3.5-17.3% of controls despite serum testosterone levels 4.5-24-fold above control. In response to hCG treatments, a graded increase in testicular testosterone levels was achieved (2.6-205% of control) anti testicular weights increased to 38-56% of control. TE suppression reduced (p < 0.05) type A spermatogonia and type B spermatogonia/preleptotene spermatocyte numbers per testis to 61% and 77% of control, respectively; however, ne

While the early studies of the inhibins, activins and follistatins concentrated on their role as endocrine regulators of FSH secretion, recent data has emphasized the local actions of the activins and follistatin. Inhibin, through its capacity to suppress FSH secretion can modulate numerous processes within the testis. However, to date, evidence to support a local role for inhibin is limited. In contrast, activin and its binding protein follistatin are produced by a large number of cell-types within the testis raising the possibility of a range of paracrine and autocrine actions. These include the modulation of androgen production, influence on the proliferation of Sertoli cells and germ cells as well as the capacity to influence the structural and functional features of mitochondria within germ cells. Some of these actions are carefully controlled in a temporal relationship during the development of testicular function in the rat in which there is no separation in time between birth and the onset of spermatogenesis. Given the range of actions of activin in different cell-types, recognition of systems that are designed to modulate its actions are crucial in enhancing our understanding of how these many roles can be compartmentalized. Copyright © 2001 Elsevier Science Ireland Ltd.

Follicle-stimulating hormone (FSH) plays an important part in testicular development. Its role in the regulation of spermatogenesis in the adult, however, remains controversial. This study aimed to explore the role of FSH in the maintenance of adult rat spermatogenesis by using immunoneutralization to selectively withdraw FSH action for periods of up to 8.5 days and then assessing the outcome by quantification of germ cell number. Adult rats received either an ovine polyclonal rat FSH antibody (FSHAb, 2 mg/kg subcutaneous daily - a dosage known to neutralize >90% of FSH in serum) for 2, 4, 7, or 8.5 days or a control sheep immunoglobulin (ConAb, 2 mg/kg) for 8.5 days. Testes were perfusion fixed, and germ cell numbers per testis were quantified using the optical disector (sic) stereological method. The percentage of seminiferous tubules displaying apoptotic cells was determined by the in situ end labeling method (TUNEL). FSHAb treatment for 4, 7, or 8.5 days significantly reduced the number of type A/intermediate spermatogonia (~74% of control values) associated with stages I-IV. Similar reductions were seen in type B spermatogonial and preleptotene spermatocyte numbers after 8.5 days of FSHAb treatment (~69% of control values; P < 0.05). Decreases (P < 0.05) in the numbers of pachytene spermatocytes in stages I- III and VIII, round spermatids in stages I-III, VII, and VIII (~70% of control values), and step 19 elongated spermatids in stage VII (51% of control values) were achieved after 8.5 days of FSHAb treatment. Compared with control, FSHAb treatment increased the percentage of stage XIV-III tubules containing TUNEL-positive cells by about twofold after 7 days of FSHAb treatment (P < 0.05). This study supports a role for FSH in the maintenance of quantitatively normal adult rat spermatogenesis, specifically by regulating A3 and A4 spermatogonial subtypes. FSH may act on these spermatogonia by enhancing the stage-dependent survival of type A spermatogonia. Effect

This study examined the effect of GnRH-antagonist (GnRH-A)-induced gonadotrophin withdrawal on numbers of germ cells in adult cynomolgus monkeys and aimed to identify the site of the earliest spermatogenic lesion(s) produced. Animals received either GnRH-A (Cetrorelix; 450 mug kg-1 day-1 s.c.; n = 5) or vehicle (control, n = 4) for 25 days. One testis was removed on day 16 and the other testis on day 25. The optical disector stereological method was used to estimate germ and Sertoli cell numbers per testis. After GnRH-A treatment for 16 days, the number of type A spermatogonia was unchanged; however, type B spermatogonia (15% of control), preleptotene + leptotene + zygotene (15% control) and pachytene (55% control) spermatocytes were all reduced (P < 0.05). By day 25, these cells were further reduced together with step 1-6 spermatids (38% control; P < 0.05). More mature germ cells were unaffected. The proportion of type A pale spermatogonia at stages VII-XII was reduced (P < 0.05) in GnRH-A-treated groups (52% on day 16, 43% on day 25) compared with control (67%). After 25 days of GnRH-A treatment, the number of Sertoli cells was unaltered but nuclear volume was reduced (66% control, P < 0.05). Tubule length was unchanged but volume (50% control), diameter (62% control) and epithelial thickness (59% control) were reduced (P < 0.05). GnRH-A treatment suppressed serum testosterone concentrations into the castrate range, and testicular testosterone concentrations to 21-36% of control values. Serum inhibin (as an index of FSH action) was suppressed in GnRH-A-treated animals by day 16, declining to 38% of control concentrations at day 25. Therefore, the primary lesion produced by GnRH-A induced gonadotrophin withdrawal is the rapid and profound reduction in the number of type B spermatogonia. The time course of germ cell loss suggests the inhibition of type A pale spermatogonial mitosis as the primary mechanism. Later germ cell maturation, specifically meiosis and spermi

A detailed understanding of the hormonal regulation of spermatogenesis is required for the informed assessment and management of male fertility and, conversely, for the development of safe and reversible male hormonal contraception. An approach to the study of these issues is outlined based on the use of well-defined in vivo models of gonadotropin/androgen deprivation and replacement, the quantitative assessment of germ cell number using stereological techniques, and the directed study of specific steps in spermatogenesis shown to be hormone dependent. Drawing together data from rat, monkey, and human models, we identify differences between species and formulate an overview of the hormonal regulation of spermatogenesis. There is good evidence for both separate and synergistic roles for both testosterone and follicle-stimulating hormone (FSH) in achieving quantitatively normal spermatogenesis. Based on relatively selective withdrawal and replacement studies, FSH has key roles in the progression of type A to B spermatogonia and, in synergy with testosterone, in regulating germ cell viability. Testosterone is an absolute requirement for spermatogenesis. In rats, it has been shown to promote the adhesion of round spermatids to Sertoli cells, without which they are sloughed from the epithelium and spermatid elongation fails. The release of mature elongated spermatids from the testis (spermiation) is also under FSH/testosterone control in rats. Data from monkeys and men treated with steroidal contraceptives indicate that impairment of spermiation is a key to achieving azoospermia. The contribution of 5alpha-reduced androgens in the testis to the regulation of spermatogenesis is also relevant, as 5alpha-reduced androgens are maintained during gonadotropin suppression and may act to maintain low levels of germ cell development. These concepts are also discussed in the context of male hormonal contraceptive development.

The role of follicle-stimulating hormone (FSH) in adult rat spermatogenesis is unclear. Although exogenous testosterone (T) restores spermatogenesis following gonadotropin-releasing hormone (GnRH) immunization or T plus estradiol (TE) treatments, an assessment of the independent action of T and FSH was not possible, as exogenous T treatment maintains serum FSH levels. We have used passive immunization against FSH to determine whether T alone is capable of reinitiating spermatogenesis after chronic and acute FSH withdrawal. Adult rats received T-filled Silastic implants 6 cm (T6) or 8 cm (T24) in length for 7 days in combination with either a polyclonal sheep antisera raised against rat FSH (FSHAb, 2 mg/kg SC daily) or control sheep immunoglobulin (ConAb) after either GnRH immunization (12 weeks) or TE treatment (9 weeks). The neutralizing capacity of the FSHAb was determined using a FSH in vitro bioassay; this analysis demonstrated that administration of FSHAb in vivo reduced FSH levels by >90%. Testes were fixed and germ cell number per testis quantified using the optical dissector. GnRH immunization reduced spermatogonia, pachytene spermatocytes, and round spermatids to 50, 13, and <1% of normal, respectively. T6 and T24 Silastic implants with the inclusion of the FSHAb did not increase the number of spermatogonia, pachytene spermatocytes, and round spermatids (50, 15, and 1% of normal, respectively). T6+ConAb treatment increased spermatogonial, pachytene spermatocyte, and round spermatid numbers to 74, 30, and 3% of normaL, respectively (P < 0.05). No further increases were seen with T24 implants. TE treatment suppressed pachytene spermatocytes and round spermatids to 33 and 1% of normal, respectively (P < 0.05). T6+FSHAb treatment did not increase the number of pachytene spermatocytes and round spermatids (36 and 8%, respectively), whereas T6+ConAb treatment increased pachytene spermatocyte and round spermatid number to 50 and 28% of normal, respectively (P < 0.

Fibroblast growth factors (FGFs) are essential for embryonic development and have been implicated in testis development and function. The effects of FGFs are mediated through four high-affinity receptors (FGFRs), which have different binding affinities for each of the ligands. We have used indirect avidin-biotin-horseradish peroxidase-enhanced immunohistochemistry to localize FGFR-1, -2, -3, and -4 in fetal, immature, and adult rat testes. In the fetal testis, immunoreactivity for FGFR-1 was seen in gonocytes, Sertoli cells, Leydig cells, and mesenchyme, and FGFR-3 was localized in gonocytes. In the immature testis, FGFR-1 was localized to spermatogonia, and all four FGFRs were localized in pachytene spermatocytes, immature adultlike Leydig cells, and peritubular cells. In the adult testis epithelium, Sertoli cells were immunoreactive for FGFR-4, and germ cells were immunoreactive for all four FGFRs, with specific receptors localized to specific stages of germ cell development. In the adult testis interstitium, FGFR-1, -2, and -4 were localized in Leydig cells, and FGFR-1 and -4 were also localized in peritubular cells. The discrete cell- and stage-specific localization of FGFRs in the fetal, immature, and adult rat testis suggests that FGFs exert specific roles through these receptors in spermatogenesis, Leydig cell function, and testicular development.

Context:. Male hormonal contraception via gonadotropin and intratesticular androgen withdrawal disrupts spermatogenesis at two principal sites: 1) spermatogonial maturation, and 2) spermiation. Objective(s): The objective of this study was to explore the relative dependence of each stage of germ cell development on FSH and LH/intratesticular androgen action. Design, Setting, and Participant(s): Eighteen men enrolled in this prospective, randomized 14-wk study at Prince Henrys Institute. Intervention(s): Subjects (n = 6/group) were assigned to 6 wk of 1) testosterone (T) implant (4 x 200 mg sc once)+depot medroxy progesterone acetate (DMPA; 150 mg im once); 2) T implant+DMPA+FSH (300 IU sc twice weekly); and 3) T implant+DMPA+human chorionic gonadotropin (hCG; 1000 IU sc twice weekly as an LH substitute). Men then underwent a vasectomy and testicular biopsy with previously reported control data used for comparison. Main Outcome Measure(s): Germ cell number (assessed by the optical disector stereological approach) and intratesticular androgen levels were determined. Result(s): T+DMPA alone significantly suppressed type B spermatogonia, preleptotene through to pachytene spermatocytes, and round spermatids from control (P < 0.05). All germ cell subtypes were maintained at control levels by either FSH or LH activity, except pachytene spermatocytes, which were found to be lower in the hCG vs. FSH (P < 0.01) and control groups (P < 0.05). Conclusion(s): FSH and LH maintained spermatogenesis independently in this gonadotropin-suppressed model. Compared with LH, FSH showed better maintenance of pachytene spermatocyte number, whereas improved conversion to round spermatids was suggested with hCG treatment. Future contraceptive treatment strategies must consider independent regulation of spermatogenesis by both FSH and LH/intratesticular androgens for maximum efficacy. Copyright © 2006 by The Endocrine Society.

Oestrogen is a metabolite of testosterone, but its role in spermatogenesis is ill-defined. Oestrogen may exert its effects on spermatogenesis, as oestrogen receptor (ER)-beta has been localised to both germ and somatic cells. This study sought to establish whether the restoration of early germ cell numbers in spermatogenesis by high-dose exogenous testosterone was influenced by its metabolite, oestrogen. The ER antagonist (ICI 182780) was administered, at a dose known to impair oestrogen action in the male reproductive tract, during testosterone treatment of gonadotrophin-releasing hormone (GnRH)-immunised rats, and germ cell numbers were determined. GnRH-immunised adult Sprague-Dawley rats (n=7-8 per group) received two doses of testosterone, either as a Silastic implant (24 cm (T24 cm)) or an injectable ester for 10 days alone or in combination with ICI 182780 (2 mg/kg, s.c. injection daily). Control rats received vehicle alone. Testes were perfusion-fixed and germ cells were quantified by the optical disector technique. GnRH-immunisation reduced (P<0.001) both type A/intermediate spermatogonial and type B spermatogonial/preleptotene spermatocyte number (56% of control) and leptotene/zygotene spermatocyte number (63% of control). Pachytene spermatocyte and round spermatids were reduced to 12% and 1% (P<0.01) of control respectively. Testosterone treatment did not increase type A/intermediate spermatogonial number compared with GnRH-immunised controls over the 10-day study period. Treatment with testosterone-esters increased type B spermatogonial/preleptotene spermatocytes and leptotene/zygotene spermatocyte numbers (both being ~83% of control, P<0.05), while T24 cm treatment did not significantly increase their numbers (~73% of control) compared with GnRH-immunised controls. Both treatments increased pachytene spermatocyte and round spermatid numbers to 55% and 8% of control respectively. Co-administration of ICI 182780 had no effect on any of these germ cell numb

The hormones that regulate spermatogonial development are ill defined, in part due to lack of appropriate experimental models. The photoinhibited hamster model provides a rich source of spermatogonia, thus making it an ideal model to study their control. This study aimed to assess the effects of FSH, in the absence of testosterone, on the reinitiation of Sertoli cell and spermatogonial development in the photosensitive adult Djungarian hamster. Hamsters raised under long photoperiods (LD, 16L:8D) were exposed to short photoperiods (SD, 8L:16D) for 11 wk, leading to suppression of gonadotropins and regression of testicular function. Groups of 10 animals then received FSH alone or in combination with the antiandrogen, flutamide, for 7 days. Two control groups maintained either under long or short photoperiods were treated with vehicle. Sertoli and germ cell number were then determined using the optical disector (sic) stereological technique. The number of Sertoli cells, type A spermatogonia, type B spermatogonia/preleptotene spermatocytes, and leptotene/zygotene spermatocytes were suppressed in SD controls to 66%, 34%, 19%, and 10% (all P < 0.01) of long-day control values, respectively. Later germ cell types were not detected. FSH treatment, with or without flutamide, increased Sertoli cell number (P < 0.01) to normal long-day values. Similarly, FSH treatment in the absence/presence of flutamide increased type A spermatogonia, type B spermatogonia/preleptotene spermatocytes, and leptotene/zygotene spermatocytes to ~85%, 69%, and 80% (all P < 0.01) of long-day controls, respectively. Our data demonstrate that the reinitiation of spermatogonial maturation in this model is dependent on FSH in the presence of an antiandrogen. Surprisingly, the adult Sertoli cell population in this model is also hormone dependent. This naturally occurring model provides a unique opportunity to understand the mechanisms (apoptotic and/or proliferative) by which FSH regulates Sertoli and ge

The BH3-only proteins of the Bcl-2 family initiate apoptosis through the activation of Bax-like relatives. Loss of individual BH3-only proteins can lead either to no phenotype, as in mice lacking Bik, or to marked cell excess, as in the hematopoietic compartment of animals lacking Bim. To investigate whether functional redundancy with Bim might obscure a significant role for Bik, we generated mice lacking both genes. The hematopoietic compartments of bik -/-bim-/- and bim-/- mice were indistinguishable. However, although testes develop normally in mice lacking either Bik or Bim, adult bik-/-bim-/- males were infertile, with reduced testicular cellularity and no spermatozoa. The testis of young bik-/-bim-/- males, like those lacking Bax, exhibited increased numbers of spermatogonia and spermatocytes, although loss of Bik plus Bim blocked spermatogenesis somewhat later than Bax deficiency. The initial excess of early germ cells suggests that spermatogenesis fails because supporting Sertoli cells are overwhelmed. Thus, Bik and Bim share, upstream of Bax, the role of eliminating supernumerary germ cells during the first wave of spermatogenesis, a process vital for normal testicular development. ©2005 European Molecular Biology Organization.

Follicle-stimulating hormone (FSH) plays an important part in testicular development. Its role in the regulation of spermatogenesis in the adult, however, remains controversial. This study aimed to explore the role of FSH in the maintenance of adult rat spermatogenesis by using immunoneutralization to selectively withdraw FSH action for periods of up to 8.5 days and then assessing the outcome by quantification of germ cell number. Adult rats received either an ovine polyclonal rat FSH antibody (FSHAb, 2 mg/kg subcutaneous daily - a dosage known to neutralize >90% of FSH in serum) for 2, 4, 7, or 8.5 days or a control sheep immunoglobulin (ConAb, 2 mg/kg) for 8.5 days. Testes were perfusion fixed, and germ cell numbers per testis were quantified using the optical disector (sic) stereological method. The percentage of seminiferous tubules displaying apoptotic cells was determined by the in situ end labeling method (TUNEL). FSHAb treatment for 4, 7, or 8.5 days significantly reduced the number of type A/intermediate spermatogonia (~74% of control values) associated with stages I-IV. Similar reductions were seen in type B spermatogonial and preleptotene spermatocyte numbers after 8.5 days of FSHAb treatment (~69% of control values; P < 0.05). Decreases (P < 0.05) in the numbers of pachytene spermatocytes in stages I- III and VIII, round spermatids in stages I-III, VII, and VIII (~70% of control values), and step 19 elongated spermatids in stage VII (51% of control values) were achieved after 8.5 days of FSHAb treatment. Compared with control, FSHAb treatment increased the percentage of stage XIV-III tubules containing TUNEL-positive cells by about twofold after 7 days of FSHAb treatment (P < 0.05). This study supports a role for FSH in the maintenance of quantitatively normal adult rat spermatogenesis, specifically by regulating A3 and A4 spermatogonial subtypes. FSH may act on these spermatogonia by enhancing the stage-dependent survival of type A spermatogonia. Effect

This study examined the effect of GnRH-antagonist (GnRH-A)-induced gonadotrophin withdrawal on numbers of germ cells in adult cynomolgus monkeys and aimed to identify the site of the earliest spermatogenic lesion(s) produced. Animals received either GnRH-A (Cetrorelix; 450 mug kg-1 day-1 s.c.; n = 5) or vehicle (control, n = 4) for 25 days. One testis was removed on day 16 and the other testis on day 25. The optical disector stereological method was used to estimate germ and Sertoli cell numbers per testis. After GnRH-A treatment for 16 days, the number of type A spermatogonia was unchanged; however, type B spermatogonia (15% of control), preleptotene + leptotene + zygotene (15% control) and pachytene (55% control) spermatocytes were all reduced (P < 0.05). By day 25, these cells were further reduced together with step 1-6 spermatids (38% control; P < 0.05). More mature germ cells were unaffected. The proportion of type A pale spermatogonia at stages VII-XII was reduced (P < 0.05) in GnRH-A-treated groups (52% on day 16, 43% on day 25) compared with control (67%). After 25 days of GnRH-A treatment, the number of Sertoli cells was unaltered but nuclear volume was reduced (66% control, P < 0.05). Tubule length was unchanged but volume (50% control), diameter (62% control) and epithelial thickness (59% control) were reduced (P < 0.05). GnRH-A treatment suppressed serum testosterone concentrations into the castrate range, and testicular testosterone concentrations to 21-36% of control values. Serum inhibin (as an index of FSH action) was suppressed in GnRH-A-treated animals by day 16, declining to 38% of control concentrations at day 25. Therefore, the primary lesion produced by GnRH-A induced gonadotrophin withdrawal is the rapid and profound reduction in the number of type B spermatogonia. The time course of germ cell loss suggests the inhibition of type A pale spermatogonial mitosis as the primary mechanism. Later germ cell maturation, specifically meiosis and spermi

A detailed understanding of the hormonal regulation of spermatogenesis is required for the informed assessment and management of male fertility and, conversely, for the development of safe and reversible male hormonal contraception. An approach to the study of these issues is outlined based on the use of well-defined in vivo models of gonadotropin/androgen deprivation and replacement, the quantitative assessment of germ cell number using stereological techniques, and the directed study of specific steps in spermatogenesis shown to be hormone dependent. Drawing together data from rat, monkey, and human models, we identify differences between species and formulate an overview of the hormonal regulation of spermatogenesis. There is good evidence for both separate and synergistic roles for both testosterone and follicle-stimulating hormone (FSH) in achieving quantitatively normal spermatogenesis. Based on relatively selective withdrawal and replacement studies, FSH has key roles in the progression of type A to B spermatogonia and, in synergy with testosterone, in regulating germ cell viability. Testosterone is an absolute requirement for spermatogenesis. In rats, it has been shown to promote the adhesion of round spermatids to Sertoli cells, without which they are sloughed from the epithelium and spermatid elongation fails. The release of mature elongated spermatids from the testis (spermiation) is also under FSH/testosterone control in rats. Data from monkeys and men treated with steroidal contraceptives indicate that impairment of spermiation is a key to achieving azoospermia. The contribution of 5alpha-reduced androgens in the testis to the regulation of spermatogenesis is also relevant, as 5alpha-reduced androgens are maintained during gonadotropin suppression and may act to maintain low levels of germ cell development. These concepts are also discussed in the context of male hormonal contraceptive development.

While the early studies of the inhibins, activins and follistatins concentrated on their role as endocrine regulators of FSH secretion, recent data has emphasized the local actions of the activins and follistatin. Inhibin, through its capacity to suppress FSH secretion can modulate numerous processes within the testis. However, to date, evidence to support a local role for inhibin is limited. In contrast, activin and its binding protein follistatin are produced by a large number of cell-types within the testis raising the possibility of a range of paracrine and autocrine actions. These include the modulation of androgen production, influence on the proliferation of Sertoli cells and germ cells as well as the capacity to influence the structural and functional features of mitochondria within germ cells. Some of these actions are carefully controlled in a temporal relationship during the development of testicular function in the rat in which there is no separation in time between birth and the onset of spermatogenesis. Given the range of actions of activin in different cell-types, recognition of systems that are designed to modulate its actions are crucial in enhancing our understanding of how these many roles can be compartmentalized. Copyright © 2001 Elsevier Science Ireland Ltd.

The role of follicle-stimulating hormone (FSH) in adult rat spermatogenesis is unclear. Although exogenous testosterone (T) restores spermatogenesis following gonadotropin-releasing hormone (GnRH) immunization or T plus estradiol (TE) treatments, an assessment of the independent action of T and FSH was not possible, as exogenous T treatment maintains serum FSH levels. We have used passive immunization against FSH to determine whether T alone is capable of reinitiating spermatogenesis after chronic and acute FSH withdrawal. Adult rats received T-filled Silastic implants 6 cm (T6) or 8 cm (T24) in length for 7 days in combination with either a polyclonal sheep antisera raised against rat FSH (FSHAb, 2 mg/kg SC daily) or control sheep immunoglobulin (ConAb) after either GnRH immunization (12 weeks) or TE treatment (9 weeks). The neutralizing capacity of the FSHAb was determined using a FSH in vitro bioassay; this analysis demonstrated that administration of FSHAb in vivo reduced FSH levels by >90%. Testes were fixed and germ cell number per testis quantified using the optical dissector. GnRH immunization reduced spermatogonia, pachytene spermatocytes, and round spermatids to 50, 13, and <1% of normal, respectively. T6 and T24 Silastic implants with the inclusion of the FSHAb did not increase the number of spermatogonia, pachytene spermatocytes, and round spermatids (50, 15, and 1% of normal, respectively). T6+ConAb treatment increased spermatogonial, pachytene spermatocyte, and round spermatid numbers to 74, 30, and 3% of normaL, respectively (P < 0.05). No further increases were seen with T24 implants. TE treatment suppressed pachytene spermatocytes and round spermatids to 33 and 1% of normal, respectively (P < 0.05). T6+FSHAb treatment did not increase the number of pachytene spermatocytes and round spermatids (36 and 8%, respectively), whereas T6+ConAb treatment increased pachytene spermatocyte and round spermatid number to 50 and 28% of normal, respectively (P < 0.

Fibroblast growth factors (FGFs) are essential for embryonic development and have been implicated in testis development and function. The effects of FGFs are mediated through four high-affinity receptors (FGFRs), which have different binding affinities for each of the ligands. We have used indirect avidin-biotin-horseradish peroxidase-enhanced immunohistochemistry to localize FGFR-1, -2, -3, and -4 in fetal, immature, and adult rat testes. In the fetal testis, immunoreactivity for FGFR-1 was seen in gonocytes, Sertoli cells, Leydig cells, and mesenchyme, and FGFR-3 was localized in gonocytes. In the immature testis, FGFR-1 was localized to spermatogonia, and all four FGFRs were localized in pachytene spermatocytes, immature adultlike Leydig cells, and peritubular cells. In the adult testis epithelium, Sertoli cells were immunoreactive for FGFR-4, and germ cells were immunoreactive for all four FGFRs, with specific receptors localized to specific stages of germ cell development. In the adult testis interstitium, FGFR-1, -2, and -4 were localized in Leydig cells, and FGFR-1 and -4 were also localized in peritubular cells. The discrete cell- and stage-specific localization of FGFRs in the fetal, immature, and adult rat testis suggests that FGFs exert specific roles through these receptors in spermatogenesis, Leydig cell function, and testicular development.

Context:. Male hormonal contraception via gonadotropin and intratesticular androgen withdrawal disrupts spermatogenesis at two principal sites: 1) spermatogonial maturation, and 2) spermiation. Objective(s): The objective of this study was to explore the relative dependence of each stage of germ cell development on FSH and LH/intratesticular androgen action. Design, Setting, and Participant(s): Eighteen men enrolled in this prospective, randomized 14-wk study at Prince Henrys Institute. Intervention(s): Subjects (n = 6/group) were assigned to 6 wk of 1) testosterone (T) implant (4 x 200 mg sc once)+depot medroxy progesterone acetate (DMPA; 150 mg im once); 2) T implant+DMPA+FSH (300 IU sc twice weekly); and 3) T implant+DMPA+human chorionic gonadotropin (hCG; 1000 IU sc twice weekly as an LH substitute). Men then underwent a vasectomy and testicular biopsy with previously reported control data used for comparison. Main Outcome Measure(s): Germ cell number (assessed by the optical disector stereological approach) and intratesticular androgen levels were determined. Result(s): T+DMPA alone significantly suppressed type B spermatogonia, preleptotene through to pachytene spermatocytes, and round spermatids from control (P < 0.05). All germ cell subtypes were maintained at control levels by either FSH or LH activity, except pachytene spermatocytes, which were found to be lower in the hCG vs. FSH (P < 0.01) and control groups (P < 0.05). Conclusion(s): FSH and LH maintained spermatogenesis independently in this gonadotropin-suppressed model. Compared with LH, FSH showed better maintenance of pachytene spermatocyte number, whereas improved conversion to round spermatids was suggested with hCG treatment. Future contraceptive treatment strategies must consider independent regulation of spermatogenesis by both FSH and LH/intratesticular androgens for maximum efficacy. Copyright © 2006 by The Endocrine Society.

Oestrogen is a metabolite of testosterone, but its role in spermatogenesis is ill-defined. Oestrogen may exert its effects on spermatogenesis, as oestrogen receptor (ER)-beta has been localised to both germ and somatic cells. This study sought to establish whether the restoration of early germ cell numbers in spermatogenesis by high-dose exogenous testosterone was influenced by its metabolite, oestrogen. The ER antagonist (ICI 182780) was administered, at a dose known to impair oestrogen action in the male reproductive tract, during testosterone treatment of gonadotrophin-releasing hormone (GnRH)-immunised rats, and germ cell numbers were determined. GnRH-immunised adult Sprague-Dawley rats (n=7-8 per group) received two doses of testosterone, either as a Silastic implant (24 cm (T24 cm)) or an injectable ester for 10 days alone or in combination with ICI 182780 (2 mg/kg, s.c. injection daily). Control rats received vehicle alone. Testes were perfusion-fixed and germ cells were quantified by the optical disector technique. GnRH-immunisation reduced (P<0.001) both type A/intermediate spermatogonial and type B spermatogonial/preleptotene spermatocyte number (56% of control) and leptotene/zygotene spermatocyte number (63% of control). Pachytene spermatocyte and round spermatids were reduced to 12% and 1% (P<0.01) of control respectively. Testosterone treatment did not increase type A/intermediate spermatogonial number compared with GnRH-immunised controls over the 10-day study period. Treatment with testosterone-esters increased type B spermatogonial/preleptotene spermatocytes and leptotene/zygotene spermatocyte numbers (both being ~83% of control, P<0.05), while T24 cm treatment did not significantly increase their numbers (~73% of control) compared with GnRH-immunised controls. Both treatments increased pachytene spermatocyte and round spermatid numbers to 55% and 8% of control respectively. Co-administration of ICI 182780 had no effect on any of these germ cell numb

The hormones that regulate spermatogonial development are ill defined, in part due to lack of appropriate experimental models. The photoinhibited hamster model provides a rich source of spermatogonia, thus making it an ideal model to study their control. This study aimed to assess the effects of FSH, in the absence of testosterone, on the reinitiation of Sertoli cell and spermatogonial development in the photosensitive adult Djungarian hamster. Hamsters raised under long photoperiods (LD, 16L:8D) were exposed to short photoperiods (SD, 8L:16D) for 11 wk, leading to suppression of gonadotropins and regression of testicular function. Groups of 10 animals then received FSH alone or in combination with the antiandrogen, flutamide, for 7 days. Two control groups maintained either under long or short photoperiods were treated with vehicle. Sertoli and germ cell number were then determined using the optical disector (sic) stereological technique. The number of Sertoli cells, type A spermatogonia, type B spermatogonia/preleptotene spermatocytes, and leptotene/zygotene spermatocytes were suppressed in SD controls to 66%, 34%, 19%, and 10% (all P < 0.01) of long-day control values, respectively. Later germ cell types were not detected. FSH treatment, with or without flutamide, increased Sertoli cell number (P < 0.01) to normal long-day values. Similarly, FSH treatment in the absence/presence of flutamide increased type A spermatogonia, type B spermatogonia/preleptotene spermatocytes, and leptotene/zygotene spermatocytes to ~85%, 69%, and 80% (all P < 0.01) of long-day controls, respectively. Our data demonstrate that the reinitiation of spermatogonial maturation in this model is dependent on FSH in the presence of an antiandrogen. Surprisingly, the adult Sertoli cell population in this model is also hormone dependent. This naturally occurring model provides a unique opportunity to understand the mechanisms (apoptotic and/or proliferative) by which FSH regulates Sertoli and ge

The BH3-only proteins of the Bcl-2 family initiate apoptosis through the activation of Bax-like relatives. Loss of individual BH3-only proteins can lead either to no phenotype, as in mice lacking Bik, or to marked cell excess, as in the hematopoietic compartment of animals lacking Bim. To investigate whether functional redundancy with Bim might obscure a significant role for Bik, we generated mice lacking both genes. The hematopoietic compartments of bik -/-bim-/- and bim-/- mice were indistinguishable. However, although testes develop normally in mice lacking either Bik or Bim, adult bik-/-bim-/- males were infertile, with reduced testicular cellularity and no spermatozoa. The testis of young bik-/-bim-/- males, like those lacking Bax, exhibited increased numbers of spermatogonia and spermatocytes, although loss of Bik plus Bim blocked spermatogenesis somewhat later than Bax deficiency. The initial excess of early germ cells suggests that spermatogenesis fails because supporting Sertoli cells are overwhelmed. Thus, Bik and Bim share, upstream of Bax, the role of eliminating supernumerary germ cells during the first wave of spermatogenesis, a process vital for normal testicular development. ©2005 European Molecular Biology Organization.

Spermatogonial proliferation is a critical but poorly understood determinant of the spermatogenic process. In rats, exogenous testosterone plus oestradiol (TE) markedly suppresses serum LH and testicular testosterone levels. In the TE-treated rat, spermatogonial number declines to 65% of control levels while round to elongated spermatid maturation is interrupted. The partial restoration of testicular testosterone levels by exogenous testosterone administration restores spermatid maturation but neither testicular weight nor spermatogonial number are normalized. This study aimed to determine the role of testosterone and/or non-androgenic Leydig cell factors in the restoration of spermatogonial number in the testosterone-treated rat. Germ cell numbers were assessed using stereological methods and expressed as germ cell number per testis. Adult Sprague Dawley rats initially received 3 cm testosterone plus 0.4 cm oestradiol Silastic implants for 9 weeks to suppress spermatogenesis, followed by 10 days of either (i) exogenous testosterone using implants (T24 cm) or testosterone esters (5 or 25 mg sc every third day), or (ii) human chorionic gonadotrophin (hCG; 0.5, 1.25, 2.5 or 10 IU/kg sc daily) as an LH substitute to restore Leydig cell function. Following TE treatment, testicular weights and testicular testosterone levels were reduced to 31%, and 1.3% of control levels, respectively. In response to exogenous testosterone administration, testicular weight was restored to 52-58% of controls while testicular testosterone levels increased to only 3.5-17.3% of controls despite serum testosterone levels 4.5-24-fold above control. In response to hCG treatments, a graded increase in testicular testosterone levels was achieved (2.6-205% of control) anti testicular weights increased to 38-56% of control. TE suppression reduced (p < 0.05) type A spermatogonia and type B spermatogonia/preleptotene spermatocyte numbers per testis to 61% and 77% of control, respectively; however, ne

We have demonstrated a role for activin A, follistatin, and FSH in male germ cell differentiation at the time when spermatogonial stem cells and committed spermatogonia first appear in the developing testis. Testis fragments from 3-day-old rats were cultured for 1 or 3 days with various combinations of these factors, incubated with bromodeoxyuridine (BrdU) to label proliferating cells, and then processed for stereological analysis and detection of BrdU incorporation. Gonocyte numbers were significantly elevated in cultures treated with activin, while the combination of FSH and the activin antagonist, follistatin, increased the proportion of spermatogonia in the germ cell population after 3 days. All fragment groups treated with FSH contained a significantly higher proportion of proliferating Sertoli cells, while activin and follistatin each reduced Sertoli cell division. In situ hybridization and immunohistochemistry on normal rat testes demonstrated that gonocytes, but not spermatogonia, contain the activin beta(A) subunit mRNA and protein. In contrast, gonocytes first expressed follistatin mRNA and protein at 3 days after birth, concordant with the transition of gonocytes to spermatogonia. Collectively, these data demonstrate that germ cells have the potential to regulate their own maturation through production of endogenous activin A and follistatin. Sertoli cells were observed to produce the activin/inhibin beta(A) subunit, the inhibin alpha subunit, and follistatin, demonstrating that these cells have the potential to regulate germ cell maturation as well as their own development. These findings indicate that local regulation of activin bioactivity may underpin the coordinated development of germ cells and somatic cells at the onset of spermatogenesis. (C) 2000 Academic Press.

We have demonstrated a role for activin A, follistatin, and FSH in male germ cell differentiation at the time when spermatogonial stem cells and committed spermatogonia first appear in the developing testis. Testis fragments from 3-day-old rats were cultured for 1 or 3 days with various combinations of these factors, incubated with bromodeoxyuridine (BrdU) to label proliferating cells, and then processed for stereological analysis and detection of BrdU incorporation. Gonocyte numbers were significantly elevated in cultures treated with activin, while the combination of FSH and the activin antagonist, follistatin, increased the proportion of spermatogonia in the germ cell population after 3 days. All fragment groups treated with FSH contained a significantly higher proportion of proliferating Sertoli cells, while activin and follistatin each reduced Sertoli cell division. In situ hybridization and immunohistochemistry on normal rat testes demonstrated that gonocytes, but not spermatogonia, contain the activin beta(A) subunit mRNA and protein. In contrast, gonocytes first expressed follistatin mRNA and protein at 3 days after birth, concordant with the transition of gonocytes to spermatogonia. Collectively, these data demonstrate that germ cells have the potential to regulate their own maturation through production of endogenous activin A and follistatin. Sertoli cells were observed to produce the activin/inhibin beta(A) subunit, the inhibin alpha subunit, and follistatin, demonstrating that these cells have the potential to regulate germ cell maturation as well as their own development. These findings indicate that local regulation of activin bioactivity may underpin the coordinated development of germ cells and somatic cells at the onset of spermatogenesis. (C) 2000 Academic Press.

We examined 32 DNA sequences of mouse and human major histocompatibility complex (MHC) genes believed to have been subjected to gene conversion events. All regions of the mouse H2 genes as well as the human HLA genes which have been implied to be involved in gene conversion events had elevated levels of CpG dinucleotides, whereas the rest of the genes showed extensive CpG suppression. Mouse MHC genes which have been suspected but not directly implied to be involved in gene conversion events also showed elevated levels of CpG dinucleotides. Moreover, both mouse and human MHC genes which have never been suspected of undergoing gene conversion had low levels of CpG throughout the genes. These results indicate that high CpG levels are correlated with gene conversion rather than with polymorphism, as non-polymorphic genes that have been implicated as gene conversion donors also have elevated levels of CpG dimers in the involved regions whereas polymorphic genes which have never been considered to undergo gene conversion events have a low level of CpG dinucleotides. We also studied the methylation pattern of CpG dimers in the Abk gene by restriction enzyme digestion of mouse testis DNA followed by Southern blot and hybridization to an Abk-specific probe. The examined CpG dimers in prepubescent mice, where the latest germline stages are spermatogonia, leptene, or pachytene, are respectively non-methylated. Accordingly, the CpG dimers appear to be non-methylated in germline DNA from the testis of prepubescent mice, where gene conversions have been reported to occur.