T. Rajendra Kumar

ANTI-MULLERIAN HORMONE ATTENUATES THE EFFECTS OF FSH ON FOLLICLE DEVELOPMENT IN THE MOUSE OVARY

Although ovarian follicle growth is under the influence of many growth factors and hormones of which FSH remains one of the most prominent regulators. Therefore, factors affecting the sensitivity of ovarian follicles to FSH are also important for follicle growth. The aim of the present study was to investigate whether anti-Mullerian hormone (AMH) has an inhibitory effect on follicle growth by decreasing the sensitivity of ovarian follicles to FSH. Furthermore, the combined action of AMH and FSH on ovarian follicle development was examined. Three different experiments were performed. Using an in vitro follicle culture system it was shown that FSH-stimulated preantral follicle growth is attenuated in the presence of AMH. This observation was confirmed by an in vivo experiment showing that in immature AMH-deficient females, more follicles start to grow under the influence of exogenous FSH than in their wild-type littermates. In a third experiment, examination of the follicle population of 4-month-old wild-ty...

Follicle-stimulating hormone stimulates TNF production from immune cells to enhance osteoblast and osteoclast formation

Declining estrogen production after menopause causes osteoporosis in which the resorption of bone exceeds the increase in bone formation. We recently found that mice deficient in the β-subunit of follicle-stimulating hormone (FSHβ) are protected from bone loss despite severe estrogen deficiency. Here we show that FSHβ-deficient mice have lowered TNFα levels. However, TNFα-deficient mice are resistant to hypogonadal bone loss despite having elevated FSH, suggesting that TNFα is critical to the effect of FSH on bone mass. We find that FSH directly stimulates TNFα production from bone marrow granulocytes and macrophages. We also explore how TNFα up-regulation induces bone loss. By modeling the known actions of TNFα, we attribute the high-turnover bone loss to an expanded osteoclast precursor pool, together with enhanced osteoblast formation. TNFα inhibits osteoblastogenesis in the presence of ascorbic acid in culture medium, but in its absence this effect becomes stimulatory; thus, ascorbic acid reverses the true action of TNFα. Likewise, ascorbic acid blunts the effects of TNFα in stimulating osteoclast formation. We propose that hypogonadal bone loss is caused, at least in part, by enhanced FSH secretion, which in turn increases TNFα production to expand the number of bone marrow osteoclast precursors. Ascorbic acid may prevent FSH-induced hypogonadal bone loss by modulating the catabolic actions of TNFα.

Analysis of Ovarian Gene Expression in Follicle-Stimulating Hormone β Knockout Mice1

FSH is a heterodimeric glycoprotein hormone that is produced in the gonadotroph cells of the anterior pituitary. It acts on Sertoli cells of the testis and granulosa cells of the ovary. We previously demonstrated that FSHβ knockout female mice are infertile due to a block in folliculogenesis preceding antral stage development. To investigate aberrations of ovarian gene regulation in the absence of FSH, we analyzed the expression of several important marker genes using Northern blot and in situ hybridization techniques. Key findings are as follows: 1) Follicles of FSHβ knockout mice develop a well organized thecal layer, which is positive for P450 17α-hydroxylase and LH receptor messenger RNAs (mRNAs). This indicates that theca recruitment is completed autonomously with respect to FSH. 2) Granulosa cells in FSH-deficient mice demonstrate an increase in FSH receptor mRNA, and decreases in P450 aromatase, serum/glucocorticoid-induced kinase, and inhibin/activin subunit mRNAs. These data support studies that ...

Genetic Rescue of Follicle-Stimulating Hormone β-Deficient Mice1

FSH is an α:β heterodimeric pituitary glycoprotein that shares a common α-subunit with LH and TSH. To study the role of FSH in mammalian reproduction, we have previously generated an FSH-deficient mouse model using embryonic stem (ES) cell technology by introducing a null mutation in the unique FSHβ gene. Male mice deficient in FSH are fertile despite their small testes and reduced sperm number and motility. In contrast, FSH-deficient female mice are infertile due to a block in folliculogenesis at the preantral stage. In this set of experiments, we have rescued the mutant phenotypes of FSHβ-deficient mice by two genetic strategies. In the type I rescue mice, we introduced into the FSHβ-deficient background a 10-kb human FSHβ transgene that is selectively expressed in pituitary gonadotropes. The presence of this transgene [and thus the interspecies hybrid (i.e. mouse α:human FSHβ hormone)] in the background of mouse FSHβ deficiency completely restored the testis size, sperm number, and motility defects to ...

Overexpression of Human Chorionic Gonadotropin Causes Multiple Reproductive Defects in Transgenic Mice

Abstract Human CG is a pregnancy marker secreted by the placenta, and it utilizes the same receptors as does LH. Human CG is a heterodimer, and its subunits are expressed in tissues other than placenta. Similarly, LH/hCG receptors are also expressed in multiple tissues; however, the physiological significance of this expression is unknown. Free hCGβ is efficiently secreted in vitro in transfected cells and is highly expressed in many human cancers; however, the biological effects of free hCGβ in vivo are unknown. To study in vivo consequences of elevated levels of free hCGβ and hCG dimer in both male and female reproductive physiology, we used mouse metallothionein 1 promoter to generate multiple lines of transgenic mice that overexpressed either one or both subunits of hCG. Although mice expressing the glycoprotein hormone α subunit are normal and fertile, both male and female transgenic mice overexpressing only the hormone-specific hCGβ subunit are infertile. The hCGβ subunit-expressing transgenic female mice progressively develop cystic ovaries, whereas the male transgenic mice are infertile but otherwise are not phenotypically discernible. In contrast, both the male and female transgenic mice coexpressing high levels of the hCG subunits (i.e., the hCG dimer) demonstrate multiple reproductive defects. The male transgenic mice have Leydig cell hyperplasia, very high levels of serum testosterone, reduced testis size, and dramatically enlarged seminal vesicles and are infertile and display overly aggressive behavior when caged with females. The female transgenic mice are also infertile, have elevated levels of serum estradiol, and progressively develop hemorrhagic and cystic ovaries with thecal layer enlargement and stromal cell proliferation and degenerating kidneys. These results suggest that the in vivo biological effects of ectopically expressed free hCGβ subunit are distinct from those of the hCG dimer and are gender specific. These transgenic mice are useful models for studying the biology of free hCGβ subunit, for further analyzing the gain of function effects of hCG during early Leydig cell development, and for studying the roles of hCG in ovarian and kidney pathophysiology and function.

Analysis of the Testicular Phenotype of the Follicle-Stimulating Hormone β-Subunit Knockout and the Activin Type II Receptor Knockout Mice by Stereological Analysis1

This study evaluated the role of FSH and activin A on testicular function using quantitative stereological analysis of testicular cell types in mice with targeted disruption of genes encoding the FSHβ -subunit and the activin type IIA receptor (ActRIIA). Using the optical dissector technique, the numbers of Sertoli cells and germ cells per testis were determined. Testis weights in homozygous males lacking the FSHβ gene or the ActRIIA gene were decreased approximately 60% compared with wild-type or respective heterozygotes. Sertoli cell numbers decreased in both homozygous mice by 30–39%, and there was a comparable decline in germ cell numbers in both models. The degree of germ cell attrition increased in the later stages of spermatogenesis from a 46% reduction of spermatogonia to a 60% decrease in round spermatids. As the FSH levels are decreased in both models, the cellular lesion in both is most likely due to the FSH deficiency. Although the decrease in the Sertoli cell complement represents one cause o...

Male Reproductive Phenotypes in Double Mutant Mice Lacking Both FSHβ and Activin Receptor IIA

Activins are known to signal through two serine/threonine kinase type II receptors. Activin receptor IIA is widely expressed in the male reproductive axis, including the pituitary and testis. Our previous studies using gene knockout mice have confirmed the essential in vivo role of activin receptor IIA in FSH homeostasis. Activin receptor IIA-null male mice are fertile, have suppressed pituitary and serum FSH levels, and demonstrate a decrease in testis size as a result of reduced Sertoli cells and germ cells. Similarly, FSHβ null male mice are fertile despite reduced testis size and Sertoli cell number. To define the direct roles of activin receptor IIA signaling locally in the testis, independent of its effects on FSH homeostasis, we generated double mutant mice lacking both activin receptor IIA and FSH by a genetic intercross and analyzed the male reproductive phenotypes. The double mutant male mice lacking both FSH and activin receptor IIA are fertile, demonstrate no significant reduction in testis si...

Reproductive Defects in γ-Glutamyl Transpeptidase-Deficient Mice1

Mice deficient in g-glutamyl transpeptidase (GGT) are growth retarded as a result of cysteine deficiency secondary to excessive glutathione excretion in urine and display coat color defects and cataracts. Although GGT is widely expressed throughout the mouse reproductive axis, little is known about its role in reproduction. Here, we present an analysis of the reproductive phenotypes of GGT-deficient mice. Mutant male mice have reduced testis and seminal vesicle size and suppressed serum insulin-like growth factor I and FSH levels and are infertile. Although these mice are severely oligospermic, histological analysis of testes reveals grossly normal stages of spermatogenesis, including late stage spermatids, but the tubule diameter is reduced. GGT-deficient female mice are also hypogonadal and infertile. At 6 weeks of age, the ovaries of mutant mice are histologically indistinguishable from those of its wild-type counterpart. However, the absence of antral follicles and corpora lutea and follicular degeneration are apparent by 11‐13 weeks. In addition, immature female mutant mice (at 21‐23 days) are insensitive to exogenous gonadotropin administration and fail to superovulate, suggesting an intraovarian defect. Consistent with these mutant phenotypes, HPLC analysis of adult mutant testes and ovaries showed a reduction in intracellular cysteine levels. Administration of N-acetylcysteine in the drinking water beginning on day 21 to mutant mice for 2 weeks restored testis, seminal vesicle, and ovary sizes to values comparable to those in wild-type mice. Furthermore, N-acetylcysteine-fed (continuously) mutant male and female mice were fertile and produced normal numbers of offspring when mated to wild-type control mice. These results demonstrate that GGT itself is not necessary for reproductive function. However, GGT plays an important role in cysteine homeostasis within the mouse reproductive axis. (Endocrinology 141: 4270 ‐ 4277, 2000)

Regulation of FSHβ and GnRH receptor gene expression in activin receptor II knockout male mice

To examine in vivo, the local effects of inhibins and activins within the anterior pituitary, independent of their endocrine effects exerted from the gonad, in mediating FSH homeostasis, we used castrated knockout mice lacking either inhibin alpha or activin receptor II (ACVR2) alone or in combination. Compared to castrated wild-type (WT) mice, FSHbeta mRNA levels in the pituitaries of Acvr2 null mice were significantly downregulated in the absence of gonadal feedback. FSHbeta mRNA levels were not significantly higher in the pituitaries of castrated inhibin alpha null mice compared to those in Acvr2 null mice and remained the same in the pituitaries of castrated double mutant mice lacking both inhibin and ACVR2. In contrast to FSHbeta mRNA expression changes, pituitary FSH content was significantly reduced in Acvr2 null mice whereas it was only slightly upregulated in inhibin alpha null mice. Combined absence of both ACVR2 signaling and inhibins caused a decrease in FSH content compared to that in the absence of inhibins alone. These changes in pituitary content were in parallel to those in serum FSH levels in these three groups of castrated mice, suggesting that the unopposed actions of locally produced inhibins are dominant over those effects mediated by ACVR2 signaling to regulate FSH biosynthesis and secretion. Thus, our in vivo results demonstrate that within the pituitary, locally produced activins and inhibins exert their actions at distinct phases of FSH homeostasis. In an independent set of experiments, we tested whether in vivo signaling via ACVR2 is necessary for hypothalamic GnRH biosynthesis and for GnRH receptor expression. Our results demonstrate that in contrast to previous in vitro studies, signaling through ACVR2 is neither required for hypothalamic synthesis of GnRH peptide nor for expression of GnRH receptors in the anterior pituitary. We conclude that within the hypothalamic-pituitary short loop, ACVR2 signaling is critical only for FSH homeostasis and not for GnRH biosynthesis or induction of pituitary GnRH receptor expression. Our studies confirm the importance of using in vivo genetic models for studying regulation of the hypothalamic-pituitary-gonadal axis.

Blocking FSH induces thermogenic adipose tissue and reduces body fat

Menopause is associated with bone loss and enhanced visceral adiposity. A polyclonal antibody that targets the β-subunit of the pituitary hormone follicle-stimulating hormone (Fsh) increases bone mass in mice. Here, we report that this antibody sharply reduces adipose tissue in wild-type mice, phenocopying genetic haploinsufficiency for the Fsh receptor gene Fshr. The antibody also causes profound beiging, increases cellular mitochondrial density, activates brown adipose tissue and enhances thermogenesis. These actions result from the specific binding of the antibody to the β-subunit of Fsh to block its action. Our studies uncover opportunities for simultaneously treating obesity and osteoporosis.