Hypogonadal Mice Research Articles

Genetic hypogonadal (Gnrh1hpg) mouse model uncovers influence of reproductive axis on maturation of the gut microbiome during puberty.

The gut microbiome plays a key role in human health and gut dysbiosis is linked to many sex-specific diseases including autoimmune, metabolic, and neurological disorders. Activation of the hypothalamic-pituitary-gonadal (HPG) axis during puberty leads to sexual maturation and development of sex differences through the action of gonadal sex steroids. While the gut microbiome also undergoes sex differentiation, the mechanisms involved remain poorly understood. Using a genetic hypogonadal (hpg) mouse model, we sampled the fecal microbiome of male and female wild-type and hpg mutant mice before and after puberty to determine how microbial taxonomy and function are influenced by age, sex, and the HPG axis. We showed that HPG axis activation during puberty is required for sexual maturation of the gut microbiota composition, community structure, and metabolic functions. We also demonstrated that some sex differences in taxonomic composition and amine metabolism developed independently of the HPG axis, indicating that sex chromosomes are sufficient for certain sex differences in the gut microbiome. In addition, we showed that age, independent of HPG axis activation, led to some aspects of pubertal maturation of the gut microbiota community composition and putative functions. These results have implications for microbiome-based treatments, indicating that sex, hormonal status, and age should be considered when designing microbiome-based therapeutics.

Genetic hypogonadal mouse model reveals niche-specific influence of reproductive axis and sex on intestinal microbial communities

BackgroundThe gut microbiome has been linked to many diseases with sex bias including autoimmune, metabolic, neurological, and reproductive disorders. While numerous studies report sex differences in fecal microbial communities, the role of the reproductive axis in this differentiation is unclear and it is unknown how sex differentiation affects microbial diversity in specific regions of the small and large intestine.MethodsWe used a genetic hypogonadal mouse model that does not produce sex steroids or go through puberty to investigate how sex and the reproductive axis impact bacterial diversity within the intestine. Using 16S rRNA gene sequencing, we analyzed alpha and beta diversity and taxonomic composition of fecal and intestinal communities from the lumen and mucosa of the duodenum, ileum, and cecum from adult female (n = 20) and male (n = 20) wild-type mice and female (n = 17) and male (n = 20) hypogonadal mice.ResultsBoth sex and reproductive axis inactivation altered bacterial composition in an intestinal section and niche-specific manner. Hypogonadism was significantly associated with bacteria from the Bacteroidaceae,Eggerthellaceae,Muribaculaceae, and Rikenellaceae families, which have genes for bile acid metabolism and mucin degradation. Microbial balances between males and females and between hypogonadal and wild-type mice were also intestinal section-specific. In addition, we identified 3 bacterial genera (EscherichiaShigella, Lachnoclostridium, and Eggerthellaceaegenus) with higher abundance in wild-type female mice throughout the intestinal tract compared to both wild-type male and hypogonadal female mice, indicating that activation of the reproductive axis leads to female-specific differentiation of the gut microbiome. Our results also implicated factors independent of the reproductive axis (i.e., sex chromosomes) in shaping sex differences in intestinal communities. Additionally, our detailed profile of intestinal communities showed that fecal samples do not reflect bacterial diversity in the small intestine.ConclusionsOur results indicate that sex differences in the gut microbiome are intestinal niche-specific and that sampling feces or the large intestine may miss significant sex effects in the small intestine. These results strongly support the need to consider both sex and reproductive status when studying the gut microbiome and while developing microbial-based therapies.

Revisiting the gonadotropic regulation of mammalian spermatogenesis: evolving lessons during the past decade.

Spermatogenesis is a multi-step process of male germ cell (Gc) division and differentiation which occurs in the seminiferous tubules of the testes under the regulation of gonadotropins - Follicle Stimulating Hormone (FSH) and Luteinising hormone (LH). It is a highly coordinated event regulated by the surrounding somatic testicular cells such as the Sertoli cells (Sc), Leydig cells (Lc), and Peritubular myoid cells (PTc). FSH targets Sc and supports the expansion and differentiation of pre-meiotic Gc, whereas, LH operates via Lc to produce Testosterone (T), the testicular androgen. T acts on all somatic cells e.g.- Lc, PTc and Sc, and promotes the blood-testis barrier (BTB) formation, completion of Gc meiosis, and spermiation. Studies with hypophysectomised or chemically ablated animal models and hypogonadal (hpg) mice supplemented with gonadotropins to genetically manipulated mouse models have revealed the selective and synergistic role(s) of hormones in regulating male fertility. We here have briefly summarized the present concept of hormonal control of spermatogenesis in rodents and primates. We also have highlighted some of the key critical questions yet to be answered in the field of male reproductive health which might have potential implications for infertility and contraceptive research in the future.

The role of gonadotrophins in gonocyte transformation during minipuberty.

Postnatal surge of gonadotrophins, Luteinizing hormone (LH) and Follicle-Stimulating hormone (FSH) known as minipuberty, is critical for gonocyte maturation into spermatogonial stem cells (SSC) in the testis. Gonadotrophins are essential for optimum fertility in men, but very little is known how they regulate germ cells during minipuberty. This study examined whether gonadotrophins play a role on gonocyte transformation in vivo. Testes from hypogonadal (hpg) mice and their wild type (WT) littermates (n = 6/group) were weighed, and processed in paraffin at postnatal days (D) 0, 3, 6 and 9. Mouse VASA homologue (germ cell marker), anti-Müllerian hormone (Sertoli cell marker) antibodies and DAPI (nuclei marker) were used for immunofluorescence followed by confocal imaging. Germ cells on or off basement membrane (BM) and Sertoli cells/tubule were counted using Image J and analyzed with GraphPad. Comparing to WT littermates, there were significantly fewer germ cells on BM/tubule (p < 0.05) in D9 hpg mice, whereas there was no significant difference for germ cells off BM/tubule and Sertoli cells/tubule between littermates. However, testicular weight was significantly reduced in D3-D9 hpg mice comparing to WT littermates. Gonadotrophin deficiency reduced D9 germ cells on BM indicating impaired gonocyte transformation into SSC. This suggests that gonadotrophins may mediate gonocyte transformation during minipuberty.

Gonadotropin-releasing hormone is prerequisite for the constitutive expression of pituitary annexin A5.

Annexin A5 (ANXA5), a member of the structurally related family of annexin proteins, is expressed in pituitary gonadotropes. We previously reported that ANXA5 expression is stimulated by gonadotropin-releasing hormone (GnRH). In the present study, we investigated ANXA5 expression in the anterior pituitary gland of GnRH-deficient mutant hypogonadal (hpg) mice. RT-PCR demonstrated that luteinizing hormone β subunit (LHβ) and ANXA5 mRNA levels were both lower in the pituitary gland of hpg mice than in wild-type mice. Immunohistochemistry showed that ANXA5 expression throughout the pituitary gland was very low in hpg mice, suggesting that ANXA5 is diminished in gonadotropes and also in other cell types. Subcutaneous administration of a GnRH analogue, des-gly10 (Pro9)-GnRH ethylamide (1 μg/day for 7 days), augmented the expression of LHβ and ANXA5 in the pituitary gland in hpg mice. However, LHβ- and ANXA5-positive cells did not show exactly matched spatial distributions. These findings suggest that GnRH is necessary for constitutive ANXA5 expression in the pituitary gland, not only in gonadotropes but also in other pituitary gland cell types. A close relationship between ANXA5 and LHβ expression was confirmed. It is suggested that a significant role of ANXA5 in the physiologic secretion of LH.

Restoration of testis function in hypogonadotropic hypogonadal mice harboring a misfolded GnRHR mutant by pharmacoperone drug therapy

Mutations in receptors, ion channels, and enzymes are frequently recognized by the cellular quality control system as misfolded and retained in the endoplasmic reticulum (ER) or otherwise misrouted. Retention results in loss of function at the normal site of biological activity and disease. Pharmacoperones are target-specific small molecules that diffuse into cells and serve as folding templates that enable mutant proteins to pass the criteria of the quality control system and route to their physiologic site of action. Pharmacoperones of the gonadotropin releasing hormone receptor (GnRHR) have efficacy in cell culture systems, and their cellular and biochemical mechanisms of action are known. Here, we show the efficacy of a pharmacoperone drug in a small animal model, a knock-in mouse, expressing a mutant GnRHR. This recessive mutation (GnRHR E(90)K) causes hypogonadotropic hypogonadism (failed puberty associated with low or apulsatile luteinizing hormone) in both humans and in the mouse model described. We find that pulsatile pharmacoperone therapy restores E(90)K from ER retention to the plasma membrane, concurrently with responsiveness to the endogenous natural ligand, gonadotropin releasing hormone, and an agonist that is specific for the mutant. Spermatogenesis, proteins associated with steroid transport and steroidogenesis, and androgen levels were restored in mutant male mice following pharmacoperone therapy. These results show the efficacy of pharmacoperone therapy in vivo by using physiological, molecular, genetic, endocrine and biochemical markers and optimization of pulsatile administration. We expect that this newly appreciated approach of protein rescue will benefit other disorders sharing pathologies based on misrouting of misfolded protein mutants.

Androgen Initiates Sertoli Cell Tight Junction Formation in the Hypogonadal (hpg) Mouse1

Sertoli cell tight junctions (TJs) form at puberty as a major component of the blood-testis barrier (BTB), which is essential for spermatogenesis. This study characterized the hormonal induction of functional Sertoli cell TJ formation in vivo using the gonadotropin-deficient hypogonadal (hpg) mouse that displays prepubertal spermatogenic arrest. Androgen actions were determined in hpg mice treated for 2 or 10 days with dihydrotestosterone (DHT). Follicle-stimulating hormone (FSH) actions were studied in hpg mice expressing transgenic human FSH (hpg+tgFSH) with or without DHT treatment. TJ formation was examined by mRNA expression and immunolocalization of TJ proteins claudin-3 and claudin-11, and barrier functionality was examined by biotin tracer permeability. Immunolocalization of claudin-3 and claudin-11 was extensive at wild-type (wt) Sertoli cell TJs, which functionally excluded permeability tracer. In contrast, seminiferous tubules of hpg testes lacked claudin-3, but claudin-11 protein was present in adluminal regions of Sertoli cells. Biotin tracer permeated throughout these tubules, demonstrating dysfunctional TJs. In hpg+tgFSH testes, claudin-3 was generally absent, but claudin-11 had redistributed basally toward the TJs, where function was variable. In hpg testes, DHT treatment stimulated the redistribution of claudin-11 protein toward the basal region of Sertoli cells by Day 2, increased Cldn3 and Cldn11 mRNA expression, then induced the formation of functional TJs containing both proteins by Day 10. In hpg+tgFSH testes, TJ protein redistribution was accelerated and functional TJs formed by Day 2 of DHT treatment. We conclude that androgen stimulates initial Sertoli cell TJ formation and function in mice, whereas FSH activity is insufficient alone, but augments androgen-induced TJ function.

Altered Expression of Alzheimer's Disease-Related Proteins in Male Hypogonadal Mice

Age-related depletion of estrogens and androgens is associated with an increase in Alzheimer's disease (AD) brain pathology and diminished cognitive function. Here we investigated AD-associated molecular and cellular changes in brains of aged hypogonadal (hpg) male and female mice. hpg Mice have a spontaneous, inactivating genetic mutation in the GnRH gene resulting in life-long deficiency of gonadotropins and gonadal sex hormones. Western blot analysis revealed low levels of amyloid precursor protein and high levels of presenilin 1, amyloid precursor protein C-terminal fragment, and β-amyloid 42 in brains of aged male, but not female, hpg mice. Changes were confined to the hippocampus and were not evident in the cerebellum or other brain tissues. Male hpg mice tended to have lower levels of IL-1β protein than male littermate controls. Immunohistochemical staining of the basal forebrain revealed that male hpg mice had lower choline acetyltransferase levels per neuron compared with controls. These AD-like changes specific to male hpg mice supports a link between androgen depletion and the development of AD pathology.

Follicle-stimulating hormone increases bone mass in female mice

Elevated follicle-stimulating hormone (FSH) activity is proposed to directly cause bone loss independent of estradiol deficiency in aging women. Using transgenic female mice expressing human FSH (TgFSH), we now reveal that TgFSH dose-dependently increased bone mass, markedly elevating tibial and vertebral trabecular bone volume. Furthermore, TgFSH stimulated a striking accrual of bone mass in hypogonadal mice lacking endogenous FSH and luteinizing hormone (LH) function, showing that FSH-induced bone mass occurred independently of background LH or estradiol levels. Higher TgFSH levels increased osteoblast surfaces in trabecular bone and stimulated de novo bone formation, filling marrow spaces with woven rather than lamellar bone, reflective of a strong anabolic stimulus. Trabecular bone volume correlated positively with ovarian-derived serum inhibin A or testosterone levels in TgFSH mice, and ovariectomy abolished TgFSH-induced bone formation, proving that FSH effects on bone require an ovary-dependent pathway. No detectable FSH receptor mRNA in mouse bone or cultured osteoblasts or osteoclasts indicated that FSH did not directly stimulate bone. Therefore, contrary to proposed FSH-induced bone loss, our findings demonstrate that FSH has dose-dependent anabolic effects on bone via an ovary-dependent mechanism, which is independent of LH activity, and does not involve direct FSH actions on bone cells.

Hypogonadalマウス下垂体におけるアネキシンA5の発現と分布にゴナドトロピン放出ホルモン（GnRH）が及ぼす影響

Hypogonadalマウス下垂体におけるアネキシンA5の発現と分布にゴナドトロピン放出ホルモン（GnRH）が及ぼす影響

Reproductive Hormone-Dependent and -Independent Contributions to Developmental Changes in Kisspeptin in GnRH-Deficient Hypogonadal Mice

Kisspeptin is a potent activator of GnRH-induced gonadotropin secretion and is a proposed central regulator of pubertal onset. In mice, there is a neuroanatomical separation of two discrete kisspeptin neuronal populations, which are sexually dimorphic and are believed to make distinct contributions to reproductive physiology. Within these kisspeptin neuron populations, Kiss1 expression is directly regulated by sex hormones, thereby confounding the roles of sex differences and early activational events that drive the establishment of kisspeptin neurons. In order to better understand sex steroid hormone-dependent and -independent effects on the maturation of kisspeptin neurons, hypogonadal (hpg) mice deficient in GnRH and its downstream effectors were used to determine changes in the developmental kisspeptin expression. In hpg mice, sex differences in Kiss1 mRNA levels and kisspeptin immunoreactivity, typically present at 30 days of age, were absent in the anteroventral periventricular nucleus (AVPV). Although immunoreactive kisspeptin increased from 10 to 30 days of age to levels intermediate between wild type (WT) females and males, corresponding increases in Kiss1 mRNA were not detected. In contrast, the hpg arcuate nucleus (ARC) demonstrated a 10-fold increase in Kiss1 mRNA between 10 and 30 days in both females and males, suggesting that the ARC is a significant center for sex steroid-independent pubertal kisspeptin expression. Interestingly, the normal positive feedback response of AVPV kisspeptin neurons to estrogen observed in WT mice was lost in hpg females, suggesting that exposure to reproductive hormones during development may contribute to the establishment of the ovulatory gonadotropin surge mechanism. Overall, these studies suggest that the onset of pubertal kisspeptin expression is not dependent on reproductive hormones, but that gonadal sex steroids critically shape the hypothalamic kisspeptin neuronal subpopulations to make distinct contributions to the activation and control of the reproductive hormone cascade at the time of puberty.

An in vitro comparative study of growth media, sera and FSH effects on the growth and maturation of Syrian mice preantral follicles and enclosed-oocytes

Summary Developing a culture system for preantral follicles has important biotechnological implications due to the potential to produce a large number of oocytes for embryo production and transfer. To accomplish this goal, the present study was aimed to culture preantral follicles in the presence of different media, sera and FSH concentrations. Six-week-old preantral follicles (95 ± 5 µm) were cultured in North Carolina State University medium 23 (NCSU23), tissue culture medium 199 (TCM199) and leibovitz-15 medium (L-15) for 6 days. Tissue culture medium 199 showed a significant increase in the follicle diameter (115 µm), survival (39%), oocyte maturation (32%) and germinal vesicle breakdown (GVBD) (29%) rates as compared to L-15 and NCSU23 (P<0.05). A 6-day culture showed increased follicular growth as compared to 2, 4 and 8-days (P<0.05). When the experiment was run with 1, 2, 5 and 10% fetal calf serum (FCS), prepubertal gilt serum (PGS), embryonic stem cell fetal calf serum (ESFCS) and hypogonadal mouse serum (hpgMS), the 5% FCS showed increased follicle diameter (134 µm), survival (52%), oocyte maturation (49%) and GVBD (45%) as compared to control and other types of sera used (P<0.05). While 100 mIU/ml FSH + 5% FCS in TCM199 showed a significant increase in follicle diameter (197 µm), survival (96%), oocyte maturation (91%) and GVBD (67%: P<0.0001). So, it is concluded that the TCM199 medium, with the addition of 100 mIU/ml FSH and 5% FCS, is appropriate for the optimal in vitro growth of Syrian mice preantral follicles and enclosed oocytes.

Mechanisms of FSH synthesis: what we know, what we don't, and why you should care

The pituitary gonadotropin hormones, FSH and LH, are key regulators of reproductive physiology. Though the two hormones are produced by the same cell type, often in response to the same endocrine and paracrine regulators, they sub-serve different biological functions and their synthesis and secretion are differentially regulated. This stems largely from differences in transcriptional, post-transcriptional, and post-translational regulation of their unique beta subunits. That is, both hormones are dimeric glycoproteins and share a common alpha subunit. Their unique beta subunits, however, derive from different genes encoding distinct proteins. Past and recent research indicates synthesis and release of the two hormones are subject to extensive and independent regulation. LH appears to be secreted predominantly via the regulated secretory pathway, whereas FSH release is largely constitutive. As such, investigations of FSH-beta subunit synthesis may lend direct insight into mechanisms underlying patterns of secreted FSH, more so than investigations of the LHbeta subunit. Here, we review recent investigations of transcriptional regulation of the FSH-beta subunit gene from different mammalian species, including humans. The results reveal both conserved and species-specific regulatory mechanisms that might contribute to inter-species variation in FSH release.

Direct Action through the Sertoli Cells Is Essential for Androgen Stimulation of Spermatogenesis

ABSTRACT Androgens act to stimulate spermatogenesis through androgen receptors (ARs) on the Sertoli cells and peritubular myoid cells. Specific ablation of the AR in either cell type will cause a severe disruption of spermatogenesis. To determine whether androgens can stimulate spermatogenesis through direct action on the peritubular myoid cells alone or whether action on the Sertoli cells is essential, we crossed hypogonadal (hpg) mice that lack gonadotrophins and intratesticular androgen with mice lacking ARs either ubiquitously (ARKO) or specifically on the Sertoli cells (SCARKO). These hpg.ARKO and hpg.SCARKO mice were treated with testosterone (T) or dihydrotestosterone (DHT) for 7 d and testicular morphology and cell numbers assessed. Androgen treatment did not affect Sertoli cell numbers in any animal group. Both T and DHT increased numbers of spermatogonia and spermatocytes in hpg mice, but DHT has no effect on germ cell numbers in hpg.SCARKO and hpg.ARKO mice. T increased germ cell numbers in hpg.SCARKO and hpg.ARKO mice, but this was associated with stimulation of FSH release. Results show that androgen stimulation of spermatogenesis requires direct androgen action on the Sertoli cells.

Direct Action through the Sertoli Cells Is Essential for Androgen Stimulation of Spermatogenesis

Androgens act to stimulate spermatogenesis through androgen receptors (ARs) on the Sertoli cells and peritubular myoid cells. Specific ablation of the AR in either cell type will cause a severe disruption of spermatogenesis. To determine whether androgens can stimulate spermatogenesis through direct action on the peritubular myoid cells alone or whether action on the Sertoli cells is essential, we crossed hypogonadal (hpg) mice that lack gonadotrophins and intratesticular androgen with mice lacking ARs either ubiquitously (ARKO) or specifically on the Sertoli cells (SCARKO). These hpg.ARKO and hpg.SCARKO mice were treated with testosterone (T) or dihydrotestosterone (DHT) for 7 d and testicular morphology and cell numbers assessed. Androgen treatment did not affect Sertoli cell numbers in any animal group. Both T and DHT increased numbers of spermatogonia and spermatocytes in hpg mice, but DHT has no effect on germ cell numbers in hpg.SCARKO and hpg.ARKO mice. T increased germ cell numbers in hpg.SCARKO and hpg.ARKO mice, but this was associated with stimulation of FSH release. Results show that androgen stimulation of spermatogenesis requires direct androgen action on the Sertoli cells.

Effect of FSH on testicular morphology and spermatogenesis in gonadotrophin-deficient hypogonadal mice lacking androgen receptors

FSH and androgen act to stimulate and maintain spermatogenesis. FSH acts directly on the Sertoli cells to stimulate germ cell number and acts indirectly to increase androgen production by the Leydig cells. In order to differentiate between the direct effects of FSH on spermatogenesis and those mediated indirectly through androgen action, we have crossed hypogonadal (hpg) mice, which lack gonadotrophins, with mice lacking androgen receptors (AR) either ubiquitously (ARKO) or specifically on the Sertoli cells (SCARKO). These hpg.ARKO and hpg.SCARKO mice were treated with recombinant FSH for 7 days and testicular morphology and cell numbers were assessed. In untreated hpg and hpg.SCARKO mice, germ cell development was limited and did not progress beyond the pachytene stage. In hpg.ARKO mice, testes were smaller with fewer Sertoli cells and germ cells compared to hpg mice. Treatment with FSH had no effect on Sertoli cell number but significantly increased germ cell numbers in all groups. In hpg mice, FSH increased the numbers of spermatogonia and spermatocytes, and induced round spermatid formation. In hpg.SCARKO and hpg.ARKO mice, in contrast, only spermatogonial and spermatocyte numbers were increased with no formation of spermatids. Leydig cell numbers were increased by FSH in hpg and hpg.SCARKO mice but not in hpg.ARKO mice. Results show that in rodents 1) FSH acts to stimulate spermatogenesis through an increase in spermatogonial number and subsequent entry of these cells into meiosis, 2) FSH has no direct effect on the completion of meiosis and 3) FSH effects on Leydig cell number are mediated through interstitial ARs.

Occurrence of testicular microlithiasis in androgen insensitive hypogonadal mice

BackgroundTesticular microliths are calcifications found within the seminiferous tubules. In humans, testicular microlithiasis (TM) has an unknown etiology but may be significantly associated with testicular germ cell tumors. Factors inducing microlith development may also, therefore, act as susceptibility factors for malignant testicular conditions. Studies to identify the mechanisms of microlith development have been hampered by the lack of suitable animal models for TM.MethodsThis was an observational study of the testicular phenotype of different mouse models. The mouse models were: cryptorchid mice, mice lacking androgen receptors (ARs) on the Sertoli cells (SCARKO), mice with a ubiquitous loss of androgen ARs (ARKO), hypogonadal (hpg) mice which lack circulating gonadotrophins, and hpg mice crossed with SCARKO (hpg.SCARKO) and ARKO (hpg.ARKO) mice.ResultsMicroscopic TM was seen in 94% of hpg.ARKO mice (n = 16) and the mean number of microliths per testis was 81 +/- 54. Occasional small microliths were seen in 36% (n = 11) of hpg testes (mean 2 +/- 0.5 per testis) and 30% (n = 10) of hpg.SCARKO testes (mean 8 +/- 6 per testis). No microliths were seen in cryptorchid, ARKO or SCARKO mice. There was no significant effect of FSH or androgen on TM in hpg.ARKO mice.ConclusionWe have identified a mouse model of TM and show that lack of endocrine stimulation is a cause of TM. Importantly, this model will provide a means with which to identify the mechanisms of TM development and the underlying changes in protein and gene expression.

Alzheimer-like changes are found in male, but not female hypogonadal mice and appear exacerbated after injury

Alzheimer-like changes are found in male, but not female hypogonadal mice and appear exacerbated after injury

Transgenic mutant D567G but not wild-type human FSH receptor overexpression provides FSH-independent and promiscuous glycoprotein hormone Sertoli cell signaling

We have characterized the in vivo actions of human wild-type FSH receptor (FSHR) overexpressed in Sertoli cells of transgenic (Tg) mice (TgFSHRwt) compared with transgenic overexpression of the human activated mutant FSHR*D567G (TgFSHR*D567G). Testicular TgFSHRwt expression significantly elevated specific FSH binding (>2-fold, P < 0.01) relative to nontransgenic testes, similar to increased FSH binding in TgFSHR*D567G testes. Isolated TgFSHRwt Sertoli cells exhibited higher FSH-stimulated cAMP levels compared with non-Tg or TgFSHR*D567G cells but did not display the elevated FSH-independent basal cAMP levels found in TgFSHR*D567G Sertoli cells. Furthermore, Sertoli cell overexpression of TgFSHR*D567G but not TgFSHRwt allowed promiscuous cAMP responses to human chorionic gonadotropin (300 IU/ml) and TSH (30 mIU/ml), demonstrating increased constitutive signaling and altered glycoprotein hormone specificity via the intracellular D567G substitution rather than FSHR overexpression. Despite elevating Sertoli cell FSH sensitivity, overexpression of TgFSHRwt had no detectable effect upon normal testis function and did not stimulate Sertoli and germ cell development in testes of gonadotropin-deficient hypogonadal (hpg) mice, in contrast to the increased meiotic and postmeiotic germ cell development in TgFSHR*D567G hpg testes. Increased steroidogenic potential of TgFSHR*D567G hpg testes was demonstrated by elevated Cyp11a1 and Star expression, which was not detected in TgFSHRwt hpg testes. Androgen-regulated and Sertoli cell-specific Rhox5 gene expression was increased in TgFSHR*D567G but not TgFSHRwt hpg testes, providing evidence of elevated LH-independent androgen activity due to mutant FSHR*D567G. Hence, transgenic FSHR overexpression in Sertoli cells revealed that the D567G mutation confers autonomous signaling and steroidogenic activity in vivo as well as promiscuous glycoprotein hormone receptor activation, independently of FSHR overexpression alone.

Effects of FSH on testicular mRNA transcript levels in the hypogonadal mouse.

FSH acts through the Sertoli cell to ensure normal testicular development and function. To identify transcriptional mechanisms through which FSH acts in the testis, we have treated gonadotrophin-deficient hypogonadal (hpg) mice with recombinant FSH and measured changes in testicular transcript levels using microarrays and real-time PCR 12, 24 and 72 h after the start of treatment. Approximately 400 transcripts were significantly altered at each time point by FSH treatment. At 12 h, there was a clear increase in the levels of a number of known Sertoli cell transcripts (e.g. Fabp5, Lgals1, Tesc, Scara5, Aqp5). Additionally, levels of Leydig cell transcripts were also markedly increased (e.g. Ren1, Cyp17a1, Akr1b7, Star, Nr4a1). This was associated with a small but significant rise in testosterone at 24 and 72 h. At 24 h, androgen-dependent Sertoli cell transcripts were up-regulated (e.g. Rhox5, Drd4, Spinlw1, Tubb3 and Tsx) and this trend continued up to 72 h. By contrast with the somatic cells, only five germ cell transcripts (Dkkl1, Hdc, Pou5f1, Zfp541 and 1700021K02Rik) were altered by FSH within the time-course of the experiment. Analysis of canonical pathways showed that FSH induced a general decline in transcripts related to formation and regulation of tight junctions. Results show that FSH acts directly and indirectly to induce rapid changes in Sertoli cell and Leydig cell transcript levels in the hpg mouse but that effects on germ cell development must occur over a longer time-span.