Gonadotropin suppression in men leads to a reduction in claudin-11 at the Sertoli cell tight junction.

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 5α-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), ß-catenin (adherens junction), connexin-43 (gap junction), vinculin (ectoplasmic specialization) and ß-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 control men. Regardless of gonadotropin suppression treatment, claudin-11 localization was markedly disrupted and was broadly associated with the extent of meiotic/post-meiotic germ cell suppression; claudin-11 staining was (i) punctate (i.e. 'spotty' appearance) at the basal aspect of tubules when the average numbers of adluminal germ cells were <15% of control, (ii) presented as short fragments with cytoplasmic extensions when numbers were 15-25% of control or (iii) remained continuous when numbers were >40% of control. Changes in localization of connexin-43 and vinculin were also observed (smaller effects than for claudin-11) but ZO-1, ß-catenin and ß-actin did not differ, compared with control. LIMITATIONS, REASONS FOR CAUTION: Claudin-11 was the only Sertoli cell TJ protein investigated, but it is considered to be the most pivotal of constituent proteins given its known implication in infertility and BTB function. We were limited to testis samples which had been gonadotropin-suppressed for 8 weeks, shorter than the 74-day spermatogenic wave, which may account for the heterogeneity in claudin-11 and germ cell response observed among the men. Longer suppression (12-24 weeks) is known to suppress germ cells further and claudin-11 disruption may be more uniform, although we could not access such samples. WIDER IMPLICATIONS OF THE FINDINGS: These findings are important for our understanding of the sites of action of male hormonal contraception, because they suggest that BTB function could be ablated following long-term hormone suppression treatment. STUDY FUNDING/COMPETING INTERESTS: National Health and Medical Research Council (Australia) Program Grants 241000 and 494802; Research Fellowship 1022327 (to R.I.M.) and the Victorian Government's Operational Infrastructure Support Program. None of the authors have any conflicts to disclose. TRIAL REGISTRATION NUMBER: Not applicable.

Inhibin B in male reproduction: pathophysiology and clinical relevance.

The recent availability of specific inhibin assays has demonstrated that inhibin B is the relevant circulating inhibin form in the human male. Inhibin B is a dimer of an alpha and a betaB subunit. It is produced exclusively by the testis, predominantly by the Sertoli cells in the prepubertal testis, while the site of production in the adult is still controversial. Inhibin B controls FSH secretion via a negative feedback mechanism. In the adult, inhibin B production depends both on FSH and on spermatogenic status, but it is not known in which way germ cells contribute to inhibin B production. The regulation of inhibin B production changes during life. There is an inhibin B peak in serum shortly after birth only partly correlated with an increase in serum FSH, probably reflecting the proliferating activity of the Sertoli cells during this phase of life. Afterwards, inhibin B levels decrease and remain low until puberty, when they rise again, first as a consequence of FSH stimulation and then as a result of the combined regulation by FSH and the ongoing spermatogenesis. In the adult, serum inhibin B shows a clear diurnal variation closely related to that of testosterone. The administration of FSH increases the secretion of inhibin B in normal men, but is much more pronounced in males with secondary hypogonadism. The treatment of infertile men with FSH, however, does not result in an unequivocal inhibin B increase. There is a clear inverse relationship between serum inhibin B and FSH in the adult. Serum inhibin B levels are strongly positively correlated with testicular volume and sperm counts. In infertile patients, inhibin B decreases and FSH increases. In general, there is very good correlation with the degree of spermatogenetic damage, with the arrest at the earlier stages having the lowest inhibin B levels. However, for unknown reasons, there are cases of Sertoli-cell-only syndrome with normal inhibin B levels. Inhibin B and FSH together are a more sensitive and specific marker for spermatogenesis than either one alone. However, the inhibin B concentrations are not a reliable predictor of the presence of sperm in biopsy samples for testicular sperm extraction. Suppression of spermatogenesis with testosterone and gestagens leads to a partial reduction of inhibin B in serum but it is never completely suppressed. In contrast, testicular irradiation in monkeys or humans leads to a rapid and dramatic decrease of inhibin B, which becomes undetectable when germ cells are completely absent. In summary, although inhibin B is a valuable index of spermatogenesis, the measurement of serum inhibin B levels is still of limited clinical relevance for individual patients.

Effect of chorionic gonadotropin and flutamide on Leydig cell and macrophage populations in the testosterone-estradiol-implanted adult rat.

The objective of this study was to investigate the role of androgens and nonandrogenic Leydig cell products in maintaining Leydig cell and macrophage numbers in the testis of the adult rat. Adult male Sprague-Dawley rats received Silastic implants containing testosterone and estradiol (T-E) in order to suppress endogenous luteinizing hormone (LH) for 9 weeks. After T-E treatment, Leydig cell and macrophage numbers, quantified using the optical disector approach, were reduced by 40 and 60%, respectively, compared with controls. Administration of human chorionic gonadotropin (hCG) for a period of 10 days restored Leydig cell numbers to control levels, and macrophage numbers were partially restored. Administration of the antiandrogen, flutamide, in combination with hCG treatment in T-E implanted animals prevented the restoration of Leydig cell numbers but did not prevent the recovery of macrophage numbers. In the T-E-implanted animals, there was a decrease in testicular macrophage nuclear size, which was not restored by either hCG or hCG plus flutamide treatment. The results of this study support the hypothesis that LH is the main pituitary regulator of both Leydig cell and macrophage number in the adult rat testis and further indicate that androgens are responsible for maintaining Leydig cell numbers and/or differentiation, but nonandrogenic Leydig cell factors are primarily responsible for controlling macrophage numbers. Testicular macrophage function, as indicated by nuclear size, does not appear to be influenced by LH or testosterone in the adult rat.

Follicle-stimulating hormone is required for the initial phase of spermatogenic restoration in adult rats following gonadotropin suppression.

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 < 0.05). T24+FSHAb treatment increased the number of pachytene spermatocyte and round spermatids (56 and 22% of normal, respectively; P < 0.05), whereas T24+ConAb treatment increased these cells forms to 79 and 31% of normal, respectively. In conclusion, T alone is unable to restore spermatogenic cell populations in the setting of chronic FSH withdrawal. Although acute FSH withdrawal markedly impairs the restoration process, higher doses of T can partially compensate for the lack of FSH. These data suggest that FSH is important for the initial phase of spermatogenic restoration.

FSH immunoneutralization acutely impairs spermatogonial development in normal adult rats.

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 (approximately 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 (approximately 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 (approximately 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. Effects at other sites in spermatogenesis are suggested by the changes in spermatocyte and spermatid populations. However, to clarify these effects, selective FSH withdrawal would need to be prolonged until steady state had been achieved.

Redd1 is a novel marker of testis development but is not required for normal male reproduction.

In an effort to identify novel candidate genes involved in testis determination, we previously used suppression subtraction hybridisation PCR on male and female whole embryonic (12.0-12.5 days post coitum) mouse gonads. One gene to emerge from our screen was Redd1. In the current study, we demonstrate by whole-mount in situ hybridisation that Redd1 is differentially expressed in the developing mouse gonad at the time of sex determination, with higher expression in testis than ovary. Furthermore, Redd1 expression was first detected as Sry expression peaks, immediately prior to morphological sex determination, suggesting a potential role for Redd1 during testis development. To determine the functional importance of this gene during testis development, we generated Redd1-deficient mice. Morphologically, Redd1-deficient mice were indistinguishable from control littermates and showed normal fertility. Our results show that Redd1 alone is not required for testis development or fertility in mice. The lack of a male reproductive phenotype in Redd1 mice may be due to functional compensation by the related gene Redd2.

Androgen action on the restoration of spermatogenesis in adult rats: effects of human chorionic gonadotrophin, testosterone and flutamide administration on germ cell number.

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 approximately 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 treatment, a graded increase in testicular testosterone levels was achieved (2.6-20.5% of control) and 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, neither subsequent testosterone nor hCG treatments significantly increased either germ cell number. In a second study, hCG (1.25 IU/kg) was administered alone or in combination with the androgen receptor antagonist, flutamide (100 mg/kg sc daily), to withdraw androgenic effects at all stages of spermatogenesis. The TE-induced suppression of type A spermatogonia (59% control) and type B spermatogonia/preleptotene spermatocytes (68% control) was not affected by hCG +/- flutamide. On the other hand, as expected, hCG increased the number of elongated spermatids (p < 0.05). The co-administration of flutamide reduced (p < 0.05) the numbers of all pachytene spermatocyte forms, round and elongated spermatids below those of TE-treated animals. We conclude that neither exogenous testosterone nor hCG is capable of restoring spermatogonial number in the TE-treated rat within 10 days despite the partial or full restoration of testicular testosterone levels. No evidence was found for the involvement of non-androgenic Leydig factors in the control of spermatogonial numbers. The data from flutamide-treated animals demonstrates that residual androgen effects are present in the TE model as, even in the presence of testicular testosterone levels below that needed for spermatid maturation, further inhibition of spermatocyte development and meiosis is apparent.

Effects of testosterone on spermatogenic cell populations in the adult rat.

The aim of this study was to investigate the progression of germ cell populations through the rat spermatogenic cycle when spermatogenesis was suppressed by LH withdrawal through the use of a combination of testosterone (T) and estradiol (E) and then reinitiated by the administration of high doses of T. Adult Sprague-Dawley rats received 3-cm T and 0.4-cm E silastic implants for 6, 8, or 12 wk to suppress spermatogenesis followed by high-dose T (24-cm implants) for up to 12 wk to reinitiate spermatogenesis. The number of spermatogonia, primary spermatocytes, and round spermatids per testis was established by stereological techniques, and the elongated spermatid number was determined by the testicular content of nuclei resistant to homogenization in Triton X-100. Suppression for 6-12 wk resulted in moderate and significant (p < 0.05) reductions in the numbers of type A and type B spermatogonia (to 44-59% of control levels), preleptotene (68-72% of control), and leptotene/zygotene spermatocytes (62-79% of control) as well as in the numbers of stage I-VII (56-69% of control) and stage VIII-XIV (35-43% of control) pachytene spermatocytes. Round spermatids were suppressed to 29-45% of control levels (p < 0.05) while elongated spermatids were undetectable. The hourly production rates of germ cells (calculated using published time divisors) were used to study the cellular conversions through spermatogenesis (based on the ratios of the hourly production rates) and revealed that T withdrawal consistently abolished the conversion of round to elongated spermatids. The duration of suppression (6, 8, or 12 wk) had no effect on the degree to which germ cell populations or conversions were reduced. In response to high-dose T administration, spermatogonial and spermatocyte numbers and production rates (up to stage I-VII pachytene) remained suppressed, while stage VIII-XIV pachytene spermatocytes showed an increase of borderline significance. On the other hand, round and elongated spermatid numbers and production rates increased significantly (to 81% and 78% of control, respectively) and their conversion was normalized, i.e., the spermiogenic process was restored to a level consistent with the numbers of earlier germ cells proceeding through the cycle. These data suggest that, in the presence of low T levels, spermatogenesis proceeds at approximately 65% of normal levels between the spermatogonial and round spermatid stages, irrespective of the duration of T-induced suppression. This is followed by a precipitous decline in elongated spermatid number that is attributed to the disappearance of round spermatids.(ABSTRACT TRUNCATED AT 400 WORDS)

Neonatal exposure of rats to recombinant follicle stimulating hormone increases adult Sertoli and spermatogenic cell numbers.

It is believed that Sertoli cells support a finite number of germ cells and that Sertoli cell number may help determine the spermatogenic capacity of the adult. In the rat, Sertoli cells divide during fetal and early postnatal life, a process controlled in part by FSH. This study examined the effect of recombinant human FSH on postnatal testicular development and the impact of neonatal FSH exposure on the adult animal. Sprague-Dawley rats received FSH (200 IU/kg s.c.) daily from birth (Day 1) until Day 5, 10, 15, and 20. In a second experiment, animals received FSH for the first 10 or 15 days of life and were killed at Day 90. Sertoli and spermatogenic cell numbers were determined by stereological methods (the optical disector technique). FSH treatment significantly (p < 0.001) increased testicular weights (135%, 193%, and 173% of controls at Days 10, 15, and 20, respectively). The absolute volume of the epithelium and interstitium increased significantly as a result of increases in tubule diameter and length. In response to FSH treatment, the number of Sertoli cells increased significantly (p < 0.01) to 168%, 139%, and 151% of control numbers at Days 10, 15, and 20, respectively. After 15 days of FSH treatment, the numbers of spermatogonia and spermatocytes also increased (169% and 220% of control, p < 0.01, p < 0.001, respectively). The labeling index of Sertoli cell nuclei, as determined by bromodeoxyuridine incorporation, was unaffected by FSH treatment, with cessation of Sertoli cell division occurring as normal at Day 15. FSH treatment for the first 10 or 15 days of neonatal life resulted in testicular hypertrophy in adulthood (testis weight 118% and 124% of control, respectively). Similar increases were seen in the absolute volume of seminiferous epithelium and lumen, which were attributed to an increase in tubule length. Sertoli cell numbers increased to 113% and 149% of control after 10 and 15 days, respectively, of FSH exposure, with similar increases in round and elongated spermatid numbers. We conclude that exposure of the neonatal rat to recombinant FSH results in testicular hypertrophy with increases in seminiferous tubule volume and length and proportionate increases in Sertoli and germ cell numbers. This trophic effect of increased perinatal FSH exposure persists into adulthood to augment the spermatogenic potential of the rat.

Identification of specific sites of hormonal regulation in spermatogenesis in rats, monkeys, and man.

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.

Impairment of spermatogenesis in mice lacking a functional aromatase (cyp 19) gene.

It is well established that spermatogenesis is controlled by gonadotrophins and testosterone. However, a role for estrogens in male reproduction recently was suggested in adult mice deficient in estrogen receptor alpha. These mice became infertile primarily because of an interruption of fluid reabsorption by the efferent ductules of the epididymis, thus leading to a disruption of the seminiferous epithelium [Hess, R. A., Bunick, D., Lee, K. H., Bahr, J., Taylor, J. A., Korach, K. S., and Lubahn, D. B. (1997) Nature (London) 390, 509-512]. Despite the demonstration of the aromatase enzyme, which converts androgens to estrogens, and estrogen receptors within the rodent seminiferous epithelium, the role of aromatase and estrogen in germ cell development is unknown. We have investigated spermatogenesis in mice that lack aromatase because of the targeted disruption of the cyp19 gene (ArKO). Male mice deficient in aromatase were initially fertile but developed progressive infertility, until their ability to sire pups was severely impaired. The mice deficient in aromatase developed disruptions to spermatogenesis between 4.5 months and 1 year, despite no decreases in gonadotrophins or androgens. Spermatogenesis primarily was arrested at early spermiogenic stages, as characterized by an increase in apoptosis and the appearance of multinucleated cells, and there was a significant reduction in round and elongated spermatids, but no changes in Sertoli cells and earlier germ cells. In addition, Leydig cell hyperplasia/hypertrophy was evident, presumably as a consequence of increased circulating luteinizing hormone. Our findings indicate that local expression of aromatase is essential for spermatogenesis and provide evidence for a direct action of estrogen on male germ cell development and thus fertility.