MECHANISMS IN ENDOCRINOLOGY: Hormonal regulation of spermatogenesis: mutant mice challenging old paradigms.

The two pituitary gonadotrophins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and in particular LH-stimulated high intratesticular testosterone (ITT) concentration, are considered crucial for spermatogenesis. We have revisited these concepts in genetically modified mice, one being the LH receptor (R)-knockout mouse (LuRKO), the other a transgenic mouse expressing in Sertoli cells a highly constitutively active mutated Fshr (Fshr-CAM). It was found that full spermatogenesis was induced by exogenous testosterone treatment in LuRKO mice at doses that restored ITT concentration to a level corresponding to the normal circulating testosterone level in WT mice, ≈5 nmol/L, which is 1.4% of the normal high ITT concentration. When hypogonadal LuRKO and Fshr-CAM mice were crossed, the double-mutant mice with strong FSH signaling, but minimal testosterone production, showed near-normal spermatogenesis, even when their residual androgen action was blocked with the strong antiandrogen flutamide. In conclusion, our findings challenge two dogmas of the hormonal regulation of male fertility: (1) high ITT concentration is not necessary for spermatogenesis and (2) strong FSH stimulation can maintain spermatogenesis without testosterone. These findings have clinical relevance for the development of hormonal male contraception and for the treatment of idiopathic oligozoospermia.

Luteinizing Hormone and GATA4 Action in the Adrenocortical Tumorigenesis of Gonadectomized Female Mice.

BACKGROUND/AIMS: Physiological role of luteinizing hormone (LH) and its receptor (LHCGR) in adrenal remains unknown. In inhibin-α/Simian Virus 40 T antigen (SV40Tag) (inhα/Tag) mice, gonadectomy-induced (OVX) elevated LH triggers the growth of transcription factor GATA4 (GATA4)-positive adrenocortical tumors in a hyperplasia-adenoma-adenocarcinoma sequence. METHODS: We investigated the role of LHCGR in tumor induction, by crossbreeding inhα/Tag with Lhcgr knockout (LuRKO) mice. By knocking out Lhcgr and Gata4 in Cα1 adrenocortical cells (Lhcgr-ko, Gata4-ko) we tested their role in tumor progression. RESULTS: Adrenal tumors of OVX inhα/Tag mice develop from the hyperplastic cells localized in the topmost layer of zona fasciculata. OVX inhα/Tag/LuRKO only developed SV40Tag positive hyperplastic cells that were GATA4 negative, cleaved caspase-3 positive and did not progress into adenoma. In contrast to Lhcgr-ko, Gata4-ko Cα1 cells presented decreased proliferation, increased apoptosis, decreased expression of Inha, SV40Tag and Lhcgr tumor markers, as well as up-regulated adrenal- and down-regulated sex steroid gene expression. Both Gata4-ko and Lhcgr-ko Cα1 cells had decreased expression of steroidogenic genes resulting in decreased basal progesterone production. CONCLUSION: Our data indicate that LH/LHCGR signaling is critical for the adrenal cell reprogramming by GATA4 induction prompting adenoma formation and gonadal-like phenotype of the adrenocortical tumors in inhα/Tag mice.

The Presence of Human Chorionic Gonadotropin/Luteinizing Hormone Receptors in Pancreatic ß-Cells.

We investigated the possible presence of functional human chorionic gonadotropin (hCG)/luteinizing hormone (LH) receptors in ß-cells of pancreas, using a combination of techniques on hCG/LH receptor knockout mice, immortalized rat insulinoma cells, and human pancreatic islets. The results showed the presence of receptors and their activation resulted in a dose-dependent increase in glucose-induced release of insulin. These findings place hCG and LH among the regulators of insulin release with potential implications for insulin-level changes during the periods of altered hCG and LH secretion.

Consequences of genetic manipulations of gonadotrophins and gonadotrophin receptors in mice.

We have produced over the years several genetically modified mouse models (transgenic [TG], knockout [KO] and knockin [KI]) for the study of normal and aberrant functions of gonadotrophins and their receptors. We summarise in the present review some of our recent findings on these animal models. One is the cascade of extragonadal phenotypes triggered by ovarian hyperstimulation in TG mice overexpressing the human choriongonadotrophin (hCG) beta-subunit and presenting with elevated levels of serum luteinising hormone (LH)/hCG bioactivity. Massively elevated levels of serum progesterone, rather than oestrogens, are responsible for the induction of pituitary prolactinomas and the subsequently elevated prolactin (PRL) levels. Along with normal oestradiol and elevated progesterone levels, the increased concentration of PRL induces lobuloalveolar development of the mammary gland, with ultimate formation of oestrogen and progesterone receptor-negative malignant tumours. Another TG mouse model expressing a constitutively activating mutant form of the follicle-stimulating hormone receptor (FSHR) presents with a strong ovarian phenotype inducing advanced follicular development and depletion, haemorrhagic follicles, teratomas and infertility. A third TG mouse model, coexpressing binding- and signalling-deficient mutants of LHCGR in the KO background for the same receptor (R) gene provided convincing evidence that functional complementation through homo-di/oligomerisation is a physiologically relevant mode of activation of class A G protein-coupled receptors (GPCR). Taken together, genetically modified mouse models provide powerful tools for the elucidation of normal and pathological functions of gonadotrophins and their R.

Luteinizing hormone receptor deficiency increases the susceptibility to alkylating agent-induced lymphomagenesis in mice.

Previous studies have revealed a close link between luteinizing hormone (LH)/human chorionic gonadotropin (hCG) signaling and oncogenesis in gonadal and nongonadal tissues. To investigate whether genetic ablation of LH receptor (Lhr) affects the animal's oncogenic susceptibility, adult female wild-type (wt), heterozygous, and homozygous Lhr knockout (LhrKO) mice were intraperitoneally injected with an alkylating agent, N-methyl-N-nitrosourea (MNU, 50 mg/kg of body weight). The mice were sacrificed when they were short of breath or 10 months after the injection. The results showed that MNU induced non-Hodgkin's thymic and lymphonodus lymphomas in 70.6% and 100% of heterozygous and homozygous animals, respectively, compared with 35.7% in wt siblings. The tumor development was rapid; they were more aggressive and metastasized to the spleen, liver, and kidney in Lhr-deficient mice compared to wt siblings. All tumors were immunostained-positive for a T-cell specific marker, CD3, but not for a B-cell marker, CD22, suggesting that all the lymphomas arose from T-cells, which are known to be LH/hCG receptor-positive. There was no rearrangement of the Lhr gene locus or differences in thymic cell proliferation among the genotypes. However, apoptosis was lower in the Lhr-deficient thymuses. The thymic Bcl-2 levels were elevated and caspase-3 activation was reduced in Lhr heterozygous and homozygous animals. In conclusion, MNU induced a higher incidence and an earlier onset of aggressive lymphomas in LhrKO animals, which may be associated with a reduction in apoptosis of thymocytes.

Mutations along the pituitary-gonadal axis affecting sexual maturation: novel information from transgenic and knockout mice.

During the last 10 years, numerous activating and inactivating mutations have been detected in the genes encoding the two gonadotrophins, luteinising hormone (LH) and follicle-stimulating hormone (FSH), as well as their cognate receptors (R), LHR and FSHR. Because activation of the hypothalamic-pituitary-gonadal axis is a crucial event in the onset and progression of puberty, mutations affecting gonadotrophin action have major influence on this developmental process. Many of the phenotypic effects observed have been expected on the basis of the existing information about gonadotrophin action (e.g. delayed puberty), but also many unexpected findings have been made, including the lack of phenotype in women with activating LHR mutations, and the discrepancy in phenotypes of men with inactivating mutations of FSHbeta (azoospermia and infertility) and FSHR (oligozoospermia and subfertility). Some of the possible mutations, such as inactivating LHbeta and activating FSHR mutations in women, have not yet been detected. Genetically modified mice provide relevant phenocopies for the human mutations and serve as good models for studies on molecular pathogenesis of these conditions. They may also predict phenotypes of the mutations that have not yet been detected in humans. We review here briefly the effects of gonadotrophin subunit and receptor mutations on puberty in humans and contrast the information with findings on genetically modified mice with similar mutations.

Knockout of luteinizing hormone receptor abolishes the effects of follicle-stimulating hormone on preovulatory maturation and ovulation of mouse graafian follicles.

It is considered a dogma that a secretory peak of LH is indispensable as the trigger of ovulation. However, earlier studies on hypophysectomized rodents have shown that stimulation with recombinant FSH, devoid of any LH activity, is able to boost the final stages of follicular maturation and trigger ovulation. As the expression of ovarian LH receptors (LHRs) still persists after hypophysectomy, such studies cannot totally exclude the possibility that LHR activation is involved in the apparently pure FSH effects. To revisit this question, we analyzed in LHR knockout (LuRKO) mice the progression of folliculogenesis and induction of ovulation by human chorionic gonadotropin and human recombinant FSH treatments. The results provide clear evidence that follicular development and ovulation could not be induced by high doses of FSH in the absence of LHR expression. Ovarian histology and oocyte analyses indicated that follicular maturation did not advance in LuRKO mice beyond the antral follicle stage. Neither were ovulations detected in LuRKO ovaries after any of the gonadotropin treatments. The ovarian resistance to FSH treatment in the absence of LHR was confirmed by real-time RT-PCR and immunohistochemical analyses of a number of gonadotropin-dependent genes, which only responded to the treatments in wild-type control mice. Negative findings were not altered by estradiol priming preceding the gonadotropin stimulations. Hence, the present study shows that, in addition to ovulation, the expression of LHR is essential for follicular maturation in the progression from antral to preovulatory stage.

Luteinizing hormone receptor knockout (LuRKO) mice and transgenic human chorionic gonadotropin (hCG)-overexpressing mice (hCG alphabeta+) have bone phenotypes.

Considerable attention has been paid to the role of sex steroids during periods of major skeletal turnover, but the interaction of the gonadotropic hormones, which include LH, FSH, and human chorionic gonadotropin (hCG), within bone tissue have been overlooked. The question is pertinent due to the recent detection of extragonadal expression of gonadotropin receptors. Western blotting, immunolocalization, and RT-PCR supported the presence of osteoblast LH receptors. However, osteoblast cells failed to bind [(125)I]hCG and treatment with hCG failed to generate either cAMP or phosphorylated ERK 1/2. Bone mineral density (BMD) and bone histomorphometry were examined in the following models: 1) LH receptor null mutant (LuRKO) mice; 2) transgenic mice overexpressing hCG (hCG alphabeta+); and 3) ovariectomized (OVX) hCG alphabeta+ model. Male LuRKO mice showed a decrease in BMD after 5 months, apparently secondary to suppressed gonadal steroid production. Similarly, 9- to 10-wk-old female LuRKO mice exhibited decreases in histomorphometric parameters tested. The data indicate that loss of LH signaling results in a reduction in bone formation or an increase in bone resorption. By contrast, there were significant increases in BMD and histomorphometric indices for female, but not male, hCG alphabeta+ mice, indicating that chronic exposure to hCG results in bone formation or a decrease in bone resorption. However, OVX of the hCG alphabeta+ mice resulted in a significant reduction in BMD comparable to OVX WT controls. Although gonadotropin levels are tightly linked to sex steroid titers, it appears that their effects on the skeleton are indirect.

Effects of deletion of the prolactin receptor on ovarian gene expression.

Prolactin (PRL) exerts pleiotropic physiological effects in various cells and tissues, and is mainly considered as a regulator of reproduction and cell growth. Null mutation of the PRL receptor (R) gene leads to female sterility due to a complete failure of embryo implantation. Pre-implantatory egg development, implantation and decidualization in the mouse appear to be dependent on ovarian rather than uterine PRLR expression, since progesterone replacement permits the rescue of normal implantation and early pregnancy. To better understand PRL receptor deficiency, we analyzed in detail ovarian and corpora lutea development of PRLR-/- females. The present study demonstrates that the ovulation rate is not different between PRLR+/+ and PRLR-/- mice. The corpus luteum is formed but an elevated level of apoptosis and extensive inhibition of angiogenesis occur during the luteal transition in the absence of prolactin signaling. These modifications lead to the decrease of LH receptor expression and consequently to a loss of the enzymatic cascades necessary to produce adequate levels of progesterone which are required for the maintenance of pregnancy.

The role of follicle-stimulating hormone in spermatogenesis: lessons from knockout animal models.

The development of knockout mouse models for the FSH-beta subunit, the FSH receptor, and LH-receptor performed in different laboratories has confirmed and extended our knowledge concerning the critical role of these hormone-signaling systems in spermatogenesis. In this article, we summarize the phenotypic changes observed in male FSH receptor knockout (FORKO) mice. Young FORKO males have underdeveloped testis with 50% reduction in Sertoli cells, suggesting that FSH-R signaling is required very early for gonadal development, maturity, and function. These mice experience delayed puberty with postponement in the formation of round spermatids. Adult males show reduction in serum testosterone levels despite normal circulating LH concentration, indicating disturbances in Sertoli-Leydig cell communication. As a consequence of reduced sperm production and sperm quality, adult FORKO males have reduced fertility. Aberrant sperm from FORKO males have retention of cytoplasmic droplets and inadequate DNA compaction, hallmarks of infertility in many species including man. Interestingly, these changes are also experimentally inducible in FSH- and/or FSH-R-immunized male bonnet monkeys, creating a state of infertility. Reports of human mutations in FSH-beta and the FSH receptor also indicate that spermatogenesis is dependent on this system. Further investigations in FORKO males should be helpful in uncovering the downstream genes involved in sustaining Sertoli cell function and maintenance of the quantitative and qualitative aspects of spermatogenesis. This might pave the way for treatment of male infertility and contraception.

O papel dos receptores das gonadotrofinas na reproduçäo feminina / Role of gonadotropins receptors in female reproduction

As açöes fundamentais das gonadotrofinas hipofisárias na vida sexual reprodutiva de ambos os sexos dependem da integridade estrutural e funcional dos seus respectivos receptores, Os receptores das gonadotrofinas localizados na membrana citoplasmàtica säo membros da grande família dos receptores acoplados à proteína G e apresentam uma estrutura comum caracterizada por uma extensa porçäo extracelular e setes hélices transmembranas. A recente identificaçäo de mutaçöes inativadoras e ativadoras de ocorrência natural nos genes dos receptores do LH e do FSH contribuíram para a maior compreensäo de estados patológicos gonadais. Neste trabalho, revisamos os aspectos moleculares dos defeitos dos genes dos receptores das gonadotrofinas e suas implicaçöes fenotípicas no sexo feminino. Nas mulheres com mutaçöes inativadoras em homozigose nestes genes, sintomas freqüentes como alteraçöes menstruais (amenorréia secundária e oligoamenorréia) e infertilidade podem alertar o endocrinologista para o estabelecimento do diagnõsti-co definitivo da resistência ovariana ao LH ou ao FSH.

Targeted disruption of luteinizing hormone/human chorionic gonadotropin receptor gene.

LH/hCG receptors were disrupted by gene targeting in embryonic stem cells. The disruption resulted in infertility in both sexes. The gonads contained no receptor mRNA or receptor protein. Serum LH levels were greatly elevated, and FSH levels were moderately elevated in both sexes; estradiol and progesterone levels decreased but were not totally suppressed in females; testosterone levels were dramatically decreased and estradiol levels moderately elevated in males. The external and internal genitalia were grossly underdeveloped in both sexes. Abnormalities included ambiguous vaginal opening, abdominal testes, micropenis, dramatically decreased weights of the gonads and reproductive tract, arrested follicular growth beyond antral stage, disarray of seminiferous tubules, diminished number and hypotrophy of Leydig cells, and spermatogenic arrest beyond the round spermatid stage. LH/hCG receptor gene disruption had no effect on FSH receptor mRNA levels in ovaries and testes, progesterone receptor (PR) levels in ovaries and androgen receptor (AR) levels in testes. However, it caused a dramatic decrease in StAR and estrogen receptor-alpha (ERalpha) mRNA levels and an increase in ERbeta mRNA levels in both ovaries and testes. Estradiol and progesterone replacement therapy in females and testosterone replacement in males, to determine whether phenotype and biochemical changes were a consequence of decreased gonadal steroid levels or due to a loss of LH signaling, revealed complete restoration of some and partial restoration of others. Nevertheless, the animals remained infertile. It is anticipated that the LH receptor knockout animals will increase our current understanding of gonadal and nongonadal actions of LH and hCG.