The Roles of Luteinizing Hormone, Follicle-Stimulating Hormone and Testosterone in Spermatogenesis and Folliculogenesis Revisited.

Spermatogenesis and folliculogenesis involve cell-cell interactions and gene expression orchestrated by luteinizing hormone (LH) and follicle-stimulating hormone (FSH). FSH regulates the proliferation and maturation of germ cells independently and in combination with LH. In humans, the requirement for high intratesticular testosterone (T) concentration in spermatogenesis remains both a dogma and an enigma, as it greatly exceeds the requirement for androgen receptor (AR) activation. Several data have challenged this dogma. Here we report our findings on a man with mutant LH beta subunit (LHß) that markedly reduced T production to 1-2% of normal., but despite this minimal LH stimulation, T production by scarce mature Leydig cells was sufficient to initiate and maintain complete spermatogenesis. Also, in the LH receptor (LHR) knockout (LuRKO) mice, low-dose T supplementation was able to maintain spermatogenesis. In addition, in antiandrogen-treated LuRKO mice, devoid of T action, the transgenic expression of a constitutively activating follicle stimulating hormone receptor (FSHR) mutant was able to rescue spermatogenesis and fertility. Based on rodent models, it is believed that gonadotropin-dependent follicular growth begins at the antral stage, but models of FSHR inactivation in women contradict this claim. The complete loss of FSHR function results in the complete early blockage of folliculogenesis at the primary stage, with a high density of follicles of the prepubertal type. These results should prompt the reassessment of the role of gonadotropins in spermatogenesis, folliculogenesis and therapeutic applications in human hypogonadism and infertility.

Follicle-stimulating hormone promotes growth of human prostate cancer cell line-derived tumor xenografts.

Chemical castration in prostate cancer can be achieved with gonadotropin-releasing hormone (GnRH) agonists or antagonists. Their effects differ by the initial flare of gonadotropin and testosterone secretion with agonists and the immediate pituitary-testicular suppression by antagonists. While both suppress luteinizing hormone (LH) and follicle-stimulating hormone (FSH) initially, a rebound in FSH levels occurs during agonist treatment. This rebound is potentially harmful, taken the expression of FSH receptors (R) in prostate cancer tissue. We herein assessed the role of FSH in promoting the growth of androgen-independent (PC-3, DU145) and androgen-dependent (VCaP) human prostate cancer cell line xenografts in nude mice. Gonadotropins were suppressed with the GnRH antagonist degarelix, and effects of add-back human recombinant FSH were assessed on tumor growth. All tumors expressed GnRHR and FSHR, and degarelix treatment suppressed their growth. FSH supplementation reversed the degarelix-evoked suppression of PC-3 tumors, both in preventive (degarelix and FSH treatment started upon cell inoculation) and therapeutic (treatments initiated 3 weeks after cell inoculation) setting. A less marked, though significant FSH effect occurred in DU145, but not in VCaP xenografts. FSHR expression in the xenografts supports direct FSH stimulation of tumor growth. Testosterone supplementation, to maintain the VCaP xenografts, apparently masked the FSH effect on their growth. Treatment with the LH analogue hCG did not affect PC-3 tumor growth despite their expression of luteinizing hormone/choriongonadotropin receptor. In conclusion, FSH, but not LH, may directly stimulate the growth of androgen-independent prostate cancer, suggesting that persistent FSH suppression upon GnRH antagonist treatment offers a therapeutic advantage over agonist.

Constitutively active follicle-stimulating hormone receptor enables androgen-independent spermatogenesis.

Spermatogenesis is regulated by the 2 pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). This process is considered impossible without the absolute requirement of LH-stimulated testicular testosterone (T) production. The role of FSH remains unclear because men and mice with inactivating FSH receptor (FSHR) mutations are fertile. We revisited the role of FSH in spermatogenesis using transgenic mice expressing a constitutively strongly active FSHR mutant in a LH receptor-null (LHR-null) background. The mutant FSHR reversed the azoospermia and partially restored fertility of Lhr-/- mice. The finding was initially ascribed to the residual Leydig cell T production. However, when T action was completely blocked with the potent antiandrogen flutamide, spermatogenesis persisted. Hence, completely T-independent spermatogenesis is possible through strong FSHR activation, and the dogma of T being a sine qua non for spermatogenesis may need modification. The mechanism for the finding appeared to be that FSHR activation maintained the expression of Sertoli cell genes considered androgen dependent. The translational message of our findings is the possibility of developing a new strategy of high-dose FSH treatment for spermatogenic failure. Our findings also provide an explanation of molecular pathogenesis for Pasqualini syndrome (fertile eunuchs; LH/T deficiency with persistent spermatogenesis) and explain how the hormonal regulation of spermatogenesis has shifted from FSH to T dominance during evolution.

Role of Follicle-Stimulating Hormone in Spermatogenesis.

Spermatogenesis is a concerted sequence of events during maturation of spermatogonia into spermatozoa. The process involves differential gene-expression and cell-cell interplay regulated by the key endocrine stimuli, i.e., follicle-stimulating hormone (FSH) and luteinizing hormone (LH)-stimulated testosterone. FSH affects independently and in concert with testosterone, the proliferation, maturation and function of the supporting Sertoli cells that produce regulatory signals and nutrients for the maintenance of developing germ cells. Rodents are able to complete spermatogenesis without FSH stimulus, but its deficiency significantly decreases sperm quantity. Men carrying loss-of-function mutation in the gene encoding the ligand (FSHB) or its receptor (FSHR) present, respectively, with azoospermia or suppressed spermatogenesis. Recently, the importance of high intratesticular testosterone concentration for spermatogenesis has been questioned. It was established that it can be completed at minimal intratesticular concentration of the hormone. Furthermore, we recently demonstrated that very robust constitutive FSHR action can rescue spermatogenesis and fertility of mice even when the testosterone stimulus is completely blocked. The clinical relevance of these findings concerns a new strategy of high-dose FSH in treatment of spermatogenic failure.

Mouse models of altered gonadotrophin action: insight into male reproductive disorders.

The advent of technologies to genetically manipulate the mouse genome has revolutionised research approaches, providing a unique platform to study the causality of reproductive disorders in vivo. With the relative ease of generating genetically modified (GM) mouse models, the last two decades have yielded multiple loss-of-function and gain-of-function mutation mouse models to explore the role of gonadotrophins and their receptors in reproductive pathologies. This work has provided key insights into the molecular mechanisms underlying reproductive disorders with altered gonadotrophin action, revealing the fundamental roles of these pituitary hormones and their receptors in the hypothalamic-pituitary-gonadal axis. This review will describe GM mouse models of gonadotrophins and their receptors with enhanced or diminished actions, specifically focusing on the male. We will discuss the mechanistic insights gained from these models into male reproductive disorders, and the relationship and understanding provided into male human reproductive disorders originating from altered gonadotrophin action.

Overlapping dose responses of spermatogenic and extragonadal testosterone actions jeopardize the principle of hormonal male contraception.

Testosterone (T), alone or in combination with progestin, provides a promising approach to hormonal male contraception. Its principle relies on enhanced negative feedback of exogenous T to suppress gonadotropins, thereby blocking the testicular T production needed for spermatogenesis, while simultaneously maintaining the extragonadal androgen actions, such as potency and libido, to avoid hypogonadism. A serious drawback of the treatment is that a significant proportion of men do not reach azoospermia or severe oligozoospermia, commensurate with contraceptive efficacy. We tested here, using hypogonadal luteinizing hormone/choriongonadotropin receptor (LHCGR) knockout (LHR(-/-)) mice, the basic principle of the T-based male contraceptive method, that a specific T dose could maintain extragonadal androgen actions without simultaneously activating spermatogenesis. LHR(-/-) mice were treated with increasing T doses, and the responses of their spermatogenesis and extragonadal androgen actions (including gonadotropin suppression and sexual behavior) were assessed. Conspicuously, all dose responses to T were practically superimposable, and no dose of T could be defined that would maintain sexual function and suppress gonadotropins without simultaneously activating spermatogenesis. This finding, never addressed in clinical contraceptive trials, is not unexpected in light of the same androgen receptor mediating androgen actions in all organs. When extrapolated to humans, our findings may jeopardize the current approach to hormonal male contraception and call for more effective means of inhibiting intratesticular T production or action, to achieve consistent spermatogenic suppression.

Feasibility of male hormonal contraception: lessons from clinical trials and animal experiments.

The general interest in the availability of male contraceptives is on the increase across different cultures and ethnic backgrounds, due in part to the fact that men are now willing more than ever, to share the responsibility of family planning. Despite the expression of interest and tremendous advances in research however, a modern male hormonal contraceptive method has remained an elusive goal. Testosterone (T) alone, or in combination with a progestin currently provides the most promising lead to male hormonal contraception. The principle relies on enhanced negative feedback of exogenous T to suppress gonadotropins, thereby blocking the endocrine stimulus for the process of spermatogenesis. A serious drawback is the inconsistent suppression among men of different ethnic backgrounds. This has increased the quest for development to include other nonhormonal methods. In reality many obstacles still have to be overcome before an acceptable method is available. In this review, we highlight recent developments in male hormonal contraceptives methods. Based on our recent findings from animal experiment, we shed light on why the method is not achieving the intended results, and suggest possible ways forward.

17beta-hydroxysteroid dehydrogenase type 1 is an independent prognostic marker in breast cancer.

Estrogens have an important role in the development and progression of breast cancer. 17beta-Hydroxysteroid dehydrogenase type 1 (17HSD1), type 2 (17HSD2), and type 5 (17HSD5) are associated with sex steroid metabolism in normal and cancerous breast tissue. The mRNA expressions of the 17HSD1, 17HSD2, and 17HSD5 enzymes were analyzed in 794 breast carcinoma specimens by using tissue microarrays and normal histologic sections. The results were correlated with the estrogen receptor alpha (ER-alpha) and beta (ER-beta), progesterone receptor, Ki67, and c-erbB-2 expressions analyzed by immunohistochemical techniques and with the Tumor-Node-Metastasis classification, tumor grade, disease-free interval, and survival of the patients. Signals for 17HSD1 mRNA were detected in 16%, 17HSD2 in 25%, and 17HSD5 in 65% of the breast cancer specimens. No association between the 17HSD1, 17HSD2, and 17HSD5 expressions was detected. A significant association was observed between ER-alpha and ER-beta (P = 0.02; odds ratio, 1.96) expressions. There was also a significant inverse association between ER-alpha and 17HSD1 (P = 0.04; odds ratio, 0.53), as well as ER-alpha and 17HSD5 (P = 0.001; odds ratio, 0.35). Patients with tumors expressing 17HSD1 mRNA or protein had significantly shorter overall and disease-free survival than the other patients (P = 0.0010 and 0.0134, log rank). The expression of 17HSD5 was significantly higher in breast tumor specimens than in normal tissue (P = 0.033; odds ratio, 5.56). The group with 17HSD5 overexpression had a worse prognosis than the other patients (P = 0.0146). ER-alpha also associated with survival (P = 0.045). Cox multivariate analyses showed that 17HSD1 mRNA, tumor size, and ER-alpha had independent prognostic significance.

17Beta-hydroxysteroid dehydrogenase type 2: independent prognostic significance and evidence of estrogen protection in female patients with colon cancer.

The mRNA expression of 17beta-hydroxysteroid dehydrogenase (17HSD) types 1 and 2 enzymes catalyzing opposite reaction of estrogen metabolism was investigated in colon cancer. Further, the significance of the 17HSD type 2 enzyme as a possible marker of colorectal cancer (CRC) prognosis was studied. In the normal mucosa, 17HSD type 2 mRNA was predominantly expressed in the surface epithelium and in the upper parts of the crypts. In the lamina propria expression was seen in endothelial cells and mononuclear phagocytes. In colorectal tumors, 17HSD type 2 expression was in most cases downregulated. Female patients had significantly more cancers with high 17HSD type 2 mRNA expression (n=11/35; 31%) than male patients (n=3/39; 8%) (P=0.02). We observed a significant impact of 17HSD type 2 mRNA expression on survival in female patients with distal colorectal cancer (n=24), with an overall cumulative 5-year survival rate of 54% in those with low 17HSD type 2 mRNA expression. None of the female patients with high 17HSD type 2 mRNA expression survived (n=11; P=0.0068; log rank 7.32). In male patients, no significant association with survival was observed. Our data provide evidence suggesting that low 17HSD type 2 mRNA expression is an independent marker of favorable prognosis in females with distal colorectal cancer, supporting the presence of gender- and location-related differences in the pathogenesis of colon cancer.

Sex steroid metabolism in human gastric mucosa: 17 beta-hydroxysteroid dehydrogenase type 2 in normal, inflamed and neoplastic gastric tissues.

The 17 beta-hydroxysteroid dehydrogenase (17HSD) enzymes are involved in the regulation of the biological activity of sex steroids. We analyzed the expression of 17HSD type 1 and 2 enzymes which catalyze opposite reactions of estrogen metabolism, in normal gastric mucosa and various gastric diseases of 81 tissue specimens. No expression of 17HSD type 1 mRNA was observed in any of the specimens. 17HSD type 2 mRNA expression was observed in the surface and foveolar epithelium of normal gastric mucosa and in the duodenum, while expression was weak or absent in glandular epithelium. Gender did not have an effect on epithelial expression, but 17HSD type 2 mRNA expression decreased with increasing age in both normal and inflamed gastric mucosa (c = 0.6; p = 0.02, Spearman rank correlation). In children, the expression in glandular epithelium was significantly higher than in adults (p = 0.03). Chronic gastritis was associated with decreased expression in surface epithelium (p = 0.023). Regenerating epithelium close to ulcers and active gastritis showed up-regulation (p < 0.03). Type I intestinal metaplasia showed an up-regulation (p = 0.005) comparable to that seen in the duodenum, while type III metaplasia showed decreased expression in comparison with type I metaplasia (p = 0.003). Expression was further down-regulated in cancer (p < 0.003). 17HSD type 2 mRNA expression in gastric and duodenal epithelium suggests that estrogen and androgen inactivation and active progesterone production are physiological features of gastroduodenal mucosa. The higher 17HSD type 2 mRNA expression in normal gastric mucosa of children compared to adults may be associated with increased need for protection against the mucosal load of foreign substances. The down-regulation associated with aging may have relevance in the pathogenesis of gastric cancer.

Downregulation of estrogen-metabolizing 17 beta-hydroxysteroid dehydrogenase type 2 expression correlates inversely with Ki67 proliferation marker in colon-cancer development.

The 17HSDs are a group of isozymes that catalyze the interconversion between high-activity 17 beta-hydroxysteroids and low-activity 17-ketosteroids. In the present study, we characterized the expression of 17HSD types 1 and 2 in normal and malignant gastrointestinal tissues and cells. Using the colon as a model for cancer of the gastrointestinal tract, expression of the 17HSD enzymes in cancer development was studied and correlated with proliferation and differentiation markers as assessed by Ki67 and mucin staining, respectively. In normal colon and small intestine, 17HSD type 2 mRNA was expressed in the surface epithelial cells and, to a lesser extent, in the cryptal epithelial cells. In colon-cancer specimens, 17HSD type 2 expression was downregulated both in the tissues and in the cell lines and correlated inversely with the proliferation marker. No expression for the 17HSD type 1 enzyme was observed in normal or cancerous gastrointestinal tract tissues. In line with the expression studies, 17HSD activity measurements with colon cells showed that only the oxidative conversion of E2 to E1 was present, and Northern blot analysis showed the signal only for 17HSD type 2. Localization of the ERs alpha and beta, assessed by immunohistochemistry and in situ hybridization, showed the presence of ER beta in the lamina propria of the colon. Our study shows that 17HSD type 2 expression is associated with the functional integrity of the gastrointestinal tract. The decrease in expression of the type 2 enzyme may increase estrogen influence in colon cancer.