Previous studies indicate that as Siberian hamsters (Phodopus sungorus) age, they may lose their ... more Previous studies indicate that as Siberian hamsters (Phodopus sungorus) age, they may lose their ability to show gonadal regression in response to short days. In one study, hamsters that regressed on short days early in life failed to regress when exposed to short days a second time later in life. Thus, Siberian hamsters may experience age-related deficits in photoresponsiveness or may be incapable of regressing twice. In the present study, we attempted to discriminate between these possibilities by examining patterns of gonadal regression in hamsters transferred back and forth from long (1 6L:8D) to short days (6L:1 8D) every 6.5, 13, or 26 wk for a 2-yr period. A control group was maintained on long days and had enlarged gonads throughout the entire study. Hamsters alternating between 26 wk of long and short days exhibited complete gonadal regression during their initial but not during their second exposure to short days. Hamsters alternated between long and short days every 13 or 6.5 wk showed regression two to three times, respectively. After about 52 wk of age, the majority of animals in both groups did not regress when exposed to short days. Taken together, the results of this experiment indicate that male Siberian hamsters 1) can exhibit at least two rounds of short-day-induced gonadal regression and 2) fail to regress on short days after about 1 yr of age.
Follicle-stimulating hormone beta subunit (Fshb) expression is regulated by transforming growth f... more Follicle-stimulating hormone beta subunit (Fshb) expression is regulated by transforming growth factor beta superfamily ligands. Recently, we demonstrated that bone morphogenetic proteins (BMPs) stimulate Fshb transcription alone and in synergy with activins. Also, transfection of the BMP type II receptor (BMPR2) and constitutively active forms of the type I receptors (activin A receptor type I [ACVR1] or BMP receptor type IA [BMPR1A]) in immortalized gonadotroph cells (LbetaT2) stimulated murine Fshb promoter-reporter activity. A third type I receptor (BMP receptor type IB [BMPR1B]) is also expressed in LbetaT2 cells, but we did not previously assess its functional role. A point mutation in BMPR1B (Q249R) is associated with increased ovulation rates and elevated FSH levels in Booroola (FecB) sheep. Herein, we assessed whether BMPR1B can regulate Fshb transcription in LbetaT2 cells and whether its ability to do so is altered by the Q249R mutation. As with ACVR1 and BMPR1A, coexpression of BMPR1B with BMPR2 increased Fshb promoter-reporter activity in BMP2-dependent and BMP2-independent fashions. Unexpectedly, the BMPR1B-Q249R mutant was equivalent to the wild type in its ability to stimulate SMAD1/5 phosphorylation and Fshb transcription. Pharmacological inhibition of ACVR1, BMPR1A, and BMPR1B confirmed that one or more of these receptors are required for BMP2-stimulated SMAD1/5 phosphorylation and Fshb reporter activity. Knockdown of endogenous BMPR1A, but not ACVR1 or BMPR1B, significantly impaired the synergism of BMP2 with activin A. Collectively, these data suggest that BMPR1A is the preferred BMP2 type I receptor in LbetaT2 cells and that neither ACVR1 nor BMPR1B compensates for its loss. The specific mechanism(s) through which the Booroola FecB mutation alters BMPR1B function remains to be determined.
Precursor proteolysis is a crucial mechanism for regulating protein structure and function. Signa... more Precursor proteolysis is a crucial mechanism for regulating protein structure and function. Signal peptidase (SP) is an enzyme with a well defined role in cleaving N-terminal signal sequences but no demonstrated function in the proteolysis of cellular precursor proteins. We provide evidence that SP mediates intraprotein cleavage of IgSF1, a large cellular Ig domain protein that is processed into two separate Ig domain proteins. In addition, our results suggest the involvement of signal peptide peptidase (SPP), an intramembrane protease, which acts on substrates that have been previously cleaved by SP. We show that IgSF1 is processed through sequential proteolysis by SP and SPP. Cleavage is directed by an internal signal sequence and generates two separate Ig domain proteins from a polytopic precursor. Our findings suggest that SP and SPP function are not restricted to N-terminal signal sequence cleavage but also contribute to the processing of cellular transmembrane proteins.
Journal of Assisted Reproduction and Genetics, Jan 5, 2019
Infertility due to Gonadotropin-Resistant Ovary Syndrome (GROS) is a rare type of hypergonadotrop... more Infertility due to Gonadotropin-Resistant Ovary Syndrome (GROS) is a rare type of hypergonadotropic hypogonadism. Here, we report an original case of GROS, associated with compound heterozygous follicle-stimulating hormone receptor (FSHR) variants, in a woman who achieved a live birth by in vitro maturation (IVM) of her oocytes. This 31-year-old woman consulted our assisted reproduction center for a second opinion after having been advised, because of pervasive high serum follicle-stimulating hormone (FSH) levels, to pursue in vitro fertilization (IVF) with donor oocytes. She presented with primary infertility and progressively prolonged menstrual cycles. Her serum FSH levels were indeed found to be high, but in discordance with a normal anti-Müllerian hormone (AMH) level and antral follicle count. Genetic investigation found the patient to be compound heterozygous for two FSHR variants: I160T, a known pathologic variant, and N558H, which has never been previously reported. As there was no ovarian response to high daily doses of exogenous gonadotropins, IVM was proposed to the patient with success and she finally delivered at term a healthy boy. Effects of the receptor variants were analyzed in heterologous cells. Whereas the I160T mutation blocked FSHR membrane trafficking and FSH-stimulated cAMP-dependent signaling in transfected CHO cells, the novel variant, N558H, functioned equivalently to wild-type FSHR in the assays employed. In conclusion, IVM should always be offered as a first-line therapy to infertile women presenting with GROS. The N558H variant discovered in FSHR is novel, but its functional significance, if any, is unresolved and merits further investigation as it may be associated with a recessive FSHRrelated disorder.
Activins regulate FSH synthesis by stimulating the phosphorylation and nuclear accumulation of SM... more Activins regulate FSH synthesis by stimulating the phosphorylation and nuclear accumulation of SMAD2 and SMAD3, which bind to a consensus SMAD-binding element in the proximal murine FSHb (Fshb) subunit gene to drive transcription. Previous over-expression and in vitro DNA binding analyses suggested that SMAD4 participates in complexes with SMAD2 and SMAD3 to regulate Fshb expression. Here, we have characterized the role of endogenous SMAD4 in activin A induction of Fshb transcription in immortalized murine gonadotropes (LbT2). We identified five murine Smad4 mRNA isoforms, of which, four are newly described; however, the canonical full-length form predominated at both the mRNA and protein levels. Depletion of endogenous SMAD4 by RNA interference (RNAi) abolished activin A-induced Fshb promoter-reporter activity and greatly attenuated constitutively active activin type IB receptor-stimulated Fshb mRNA levels. The activin A response was rescued with an RNAi-resistant form of wild-type SMAD4, but not with a DNA-binding-deficient (Lys88Arg) SMAD4, suggesting that DNA binding by SMAD4 is necessary for activin induction of the Fshb gene. Though SMAD2 and SMAD3 are generally thought to partner with SMAD4 prior to accumulation in the nucleus, treatment with leptomycin B, an inhibitor of SMAD4 nuclear export, reduced but did not prevent activin A induction of Fshb mRNA levels or promoter activity. In addition, a constitutively nuclear form of SMAD4 rescued the effect of endogenous SMAD4 depletion. Collectively, these data demonstrate a necessary role for SMAD4 in activin A induction of the murine Fshb gene in immortalized gonadotropes.
Inhibins are gonadal hormones that act on pituitary gonadotrope cells to suppress FSH synthesis a... more Inhibins are gonadal hormones that act on pituitary gonadotrope cells to suppress FSH synthesis and secretion. Inhibin A and B are heterodimers of the inhibin ⍺-subunit disulfide-linked to one of two inhibin b-subunits. Homodimers or heterodimers of the inhibin b-subunits form the activins, which stimulate FSH production. Activins signal through complexes of type I and II receptor serine/ threonine kinases to increase transcription of the FSHb subunit gene. According to in vitro observations, inhibins impair FSH synthesis by competitively binding to activin type II receptors, particularly in the presence of the TGFb type III receptor (TGFBR3, or betaglycan). The role of TGFBR3 in inhibin action in vivo has not been determined. Here, we ablated Tgfbr3 specifically in murine gonadotropes. Conditional knockout females were supra-fertile, exhibiting enhanced folliculogenesis, numbers of ovulated eggs per cycle, and litter sizes relative to control mice. Despite these phenotypes, FSH levels appeared to be unaltered in knockout mice, and the mechanisms underlying their enhanced fertility remain unexplained. Inhibin B is the predominant form of the hormone in males and in females during most stages of the estrous cycle. Remarkably, inhibin A, but not inhibin B, suppression of FSH synthesis was impaired in cultured pituitaries of knockout mice, which may explain the absence of discernible changes in FSH levels in vivo. Collectively, these data challenge current dogma by demonstrating that TGFBR3 (betaglycan) functions as an inhibin A, but not an inhibin B, coreceptor in gonadotrope cells in vivo. Mechanisms of inhibin B action merit further investigation. (Endocrinology 159: 4077-4091, 2018) T he term inhibin was coined in the early 1930s to describe a hypothetical hormone from the testicular seminiferous epithelium that regulates the morphology of cells in the anterior pituitary gland (1). Four decades later, inhibin-like activity was discovered in ovarian follicular fluid and shown to selectively inhibit FSH secretion from pituitary gonadotrope cells while having no effects on the related LH (2). It took another decade before two forms of inhibin (A and B) were finally purified (3-7). Inhibins A and B are dynamically and differentially secreted from granulosa (and luteal) cells of the ovary across female reproductive cycles. In contrast, adult males of most species predominately secrete inhibin B from testicular Sertoli cells (8-11). Biochemical and molecular characterization of the inhibins revealed that they were heterodimeric TGFb superfamily ligands composed of the inhibin a-subunit disulfide-linked to one of two inhibin b-subunits (bA in inhibin A or bB in inhibin B). Shortly thereafter, it was discovered that inhibin b-subunits homodimerize or
Myostatin is a member of the transforming growth factor-b (TGF-b) family and a strong negative re... more Myostatin is a member of the transforming growth factor-b (TGF-b) family and a strong negative regulator of muscle growth. Here, we present the crystal structure of myostatin in complex with the antagonist follistatin 288 (Fst288). We find that the prehelix region of myostatin very closely resembles that of TGF-b class members and that this region alone can be swapped into activin A to confer signalling through the non-canonical type I receptor Alk5. Furthermore, the N-terminal domain of Fst288 undergoes conformational rearrangements to bind myostatin and likely acts as a site of specificity for the antagonist. In addition, a unique continuous electropositive surface is created when myostatin binds Fst288, which significantly increases the affinity for heparin. This translates into stronger interactions with the cell surface and enhanced myostatin degradation in the presence of either Fst288 or Fst315. Overall, we have identified several characteristics unique to myostatin that will be paramount to the rational design of myostatin inhibitors that could be used in the treatment of muscle-wasting disorders.
The glycoprotein FSH, a product of pituitary gonadotrope cells, regulates ovarian follicle develo... more The glycoprotein FSH, a product of pituitary gonadotrope cells, regulates ovarian follicle development in females and spermatogenesis in males. FSH is a heterodimer of the common a gonadotropin subunit and the hormone-specific FSHb subunit (a product of the Fshb gene). Using a conditional knockout approach (Cre-lox), we previously demonstrated that Fshb expression in mice depends on the transcription factors forkhead box L2 (FOXL2) and SMAD4. Deletion of Foxl2 or Smad4 alone led to FSH deficiency, female subfertility, and oligozoospermia in males. Simultaneous deletion of the two genes yielded a greater suppression of FSH and female sterility. The Cre-driver used previously was first active during embryonic development. Therefore, it is unclear whether FOXL2 and SMAD4 play important roles in the development or adult function of gonadotropes, or both. To address this question, we developed a tamoxifen-inducible Cre-driver line, which enabled Foxl2 and Smad4 gene deletions in gonadotropes of adult mice. After tamoxifen treatment, females with previously demonstrated fertility exhibited profound reductions in FSH levels, arrested ovarian follicle development, and sterility. FSH levels were comparably reduced in males 1 or 2 months after treatment; however, spermatogenesis was unaffected. These data indicate that (1) FOXL2 and SMAD4 are necessary to maintain FSH synthesis in gonadotrope cells of adult mice, (2) FSH is essential for female reproduction but appears to be unnecessary for the maintenance of spermatogenesis in adult male mice, and (3) the inducible Cre-driver line developed here provides a powerful tool to interrogate gene function in gonadotrope cells of adult mice.
Reproductive Biology and Endocrinology, Dec 1, 2005
Background: Activins stimulate the synthesis of follicle stimulating hormone (FSH) in pituitary g... more Background: Activins stimulate the synthesis of follicle stimulating hormone (FSH) in pituitary gonadotropes, at least in part, by inducing transcription of its beta subunit (Fshb). Evidence from several laboratories studying transformed murine LbetaT2 gonadotropes indicates that activins signal through Smad-dependent and/or Smad-independent pathways, similar to those used by transforming growth factor beta-1 (TGFB1) in other cell types. Therefore, given common intracellular signaling mechanisms of these two ligands, we examined whether TGFBs can also induce transcription of Fshb in LbetaT2 cells as well as in purified primary murine gonadotropes. Methods: Murine Fshb promoter-reporter (-1990/+1 mFshb-luc) activity was measured in LbetaT2 cells treated with activin A or TGFB1, and in cells transfected with either activin or TGFB receptors. The ability of the ligands to stimulate phosphorylation of Smads 2 and 3 in LbetaT2 cells was measured by western blot analysis, and expression of TGFB type I and II receptors was assessed by reverse transcriptase polymerase chain reaction in both LbetaT2 cells and primary gonadotropes purified from male mice of different ages. Finally, regulation of endogenous murine Fshb mRNA levels by activin A and TGFB1 in purified gonadotropes and whole pituitary cultures was measured using quantitative RT-PCR. Results: Activin A dose-dependently stimulated -1990/+1 mFshb-luc activity in LbetaT2 cells, but TGFB1 had no effect at doses up to 5 nM. Similarly, activin A, but not TGFB1, stimulated Smad 2 and 3 phosphorylation in these cells. Constitutively active forms of the activin (Acvr1b-T206D) and TGFB (TGFBR1-T204D) type I receptors strongly stimulated -1990/+1 mFshb-luc activity, showing that mechanisms down stream of Tgfbr1 seem to be intact in LbetaT2 cells. RT-PCR analysis of LbetaT2 cells and whole adult murine pituitaries indicated that both expressed Tgfbr1 mRNA, but that Tgfbr2 was not detected in LbetaT2 cells. When cells were transfected with a human TGFBR2 expression construct, TGFB1 acquired the ability to significantly stimulate -1990/+1 mFshb-luc activity. In contrast to LbetaT2 cells, primary murine gonadotropes from young mice (8-10 weeks) contained low, but detectable levels of Tgfbr2 mRNA and these levels increased in older mice (1 yr). A second surprise was the finding that treatment of purified primary gonadotropes with TGFB1 decreased murine Fshb mRNA expression by 95% whereas activin A stimulated expression by 31-fold. These data indicate that TGFB1-insensitivity in LbetaT2 cells results from a deficiency in Tgfbr2 expression. In primary gonadotropes, however, expression of Tgfbr2 does occur, and its presence permits TGFB1 to inhibit Fshb transcription, whereas activin A stimulates it. These divergent actions of activin A and TGFB1 were unexpected and show that the two ligands may act through distinct pathways to cause opposing biological effects in primary murine gonadotropes.
Immunoglobulin superfamily, member 1 (IGSF1) is a transmembrane glycoprotein with high expression... more Immunoglobulin superfamily, member 1 (IGSF1) is a transmembrane glycoprotein with high expression in the mammalian pituitary gland. Mutations in the IGSF1 gene cause congenital central hypothyroidism in humans. The IGSF1 protein is co-translationally cleaved into N- and C-terminal domains (NTD and CTD), the latter of which is trafficked to the plasma membrane and appears to be the functional portion of the molecule. Though the IGSF1-NTD is retained in the endoplasmic reticulum and has no apparent function, it has a high degree of sequence identity with the IGSF1-CTD and is conserved across mammalian species. Based upon phylogenetic analyses, we propose that the ancestral IGSF1 gene encoded the IGSF1-CTD, which was duplicated and integrated immediately upstream of itself, yielding a larger protein encompassing the IGSF1-NTD and IGSF1-CTD. The selective pressures favoring the initial gene duplication and subsequent retention of a conserved IGSF1-NTD are unresolved.
Background: Loss-of-function mutations in immunoglobulin superfamily member 1 (IGSF1) cause an X-... more Background: Loss-of-function mutations in immunoglobulin superfamily member 1 (IGSF1) cause an X-linked syndrome of central hypothyroidism, macroorchidism, delayed pubertal testosterone rise, variable prolactin deficiency and variable partial GH deficiency in childhood. The clinical features and gene expression pattern suggest a pivotal role for IGSF1 in the pituitary, but detailed knowledge on pituitary hormone secretion in this syndrome is lacking. We therefore aimed to study the 24-hour pituitary hormone secretion in male patients with IGSF1 deficiency. Methods: We collected blood samples every 10 min for 24 h in eight adult male IGSF1-deficient patients and measured circulating TSH, prolactin and gonadotropins. Deconvolution, modified cosinor and approximate entropy analyses were applied to quantify secretion rates, diurnal rhythmicity and regularity of hormone release. Results were compared to healthy controls matched for age and body mass index. Results: Compared to healthy controls, IGSF1-deficient patients showed decreased pulsatile secretion of TSH with decreased disorderliness and reduced diurnal variation. Basal and pulsatile secretion of FSH was increased by over 200%, while LH secretion did not differ from healthy controls. We observed a bimodal distribution of prolactin secretion, i.e. severe deficiency in three and increased basal and total secretion in the other five patients. Conclusion: The altered TSH secretion pattern is consistent with the previously hypothesized defect in thyrotropin-releasing hormone signaling in IGSF1 deficiency. However, the phenotype is more extensive and includes increased FSH secretion without altered LH secretion as well as either undetectable or increased prolactin secretion.
The Journal of Clinical Endocrinology and Metabolism, Apr 1, 2016
on behalf of the IGSF1 Clinical Care Group † Context: Mutations in the immunoglobulin superfamily... more on behalf of the IGSF1 Clinical Care Group † Context: Mutations in the immunoglobulin superfamily, member 1 (IGSF1) gene cause the X-linked IGSF1 deficiency syndrome consisting of central hypothyroidism, delayed pubertal testosterone rise, adult macroorchidism, variable prolactin deficiency, and occasionally transient partial GH deficiency. Since our first reports, we discovered 20 new families with 18 new pathogenic IGSF1 mutations. Objective: We aimed to share data on the largest cohort of patients with IGSF1 deficiency to date and formulate recommendations for clinical management. Methods: We collected clinical and biochemical characteristics of 69 male patients (35 children, 34 adults) and 56 female IGSF1 mutation carriers (three children, 53 adults) from 30 unrelated families according to a standardized clinical protocol. At evaluation, boys were treated with levothyroxine in 89%, adult males in 44%, and females in 5% of cases. Results: Additional symptoms in male patients included small thyroid gland volume (74%), high birth weight (25%), and large head circumference (20%). In general, the timing of pubertal testicular growth was normal or even premature, in contrast to a late rise in T levels. Late adrenarche was observed in patients with prolactin deficiency, and adult dehydroepiandrosterone concentrations were decreased in 40%. Hypocortisolism was observed in 6 of 28 evaluated newborns, although cortisol concentrations were normal later. Waist circumference of male patients was increased in 60%, but blood lipids were normal. Female carriers showed low free T 4 (FT 4) and low-normal FT 4 in 18% and 60%, respectively, delayed age at menarche in 31%, mild prolactin deficiency in 22%, increased waist circumference in 57%, and a negative correlation between FT 4 concentrations and metabolic parameters. Conclusion: IGSF1 deficiency represents the most common genetic cause of central hypothyroidism and is associated with multiple other characteristics. Based on these results, we provide recommendations for mutational analysis, endocrine work-up, and long-term care.
Immunoglobulin superfamily, member 1 (IGSF1) is a transmembrane glycoprotein highly expressed in ... more Immunoglobulin superfamily, member 1 (IGSF1) is a transmembrane glycoprotein highly expressed in the mammalian pituitary gland. Shortly after its discovery in 1998, the protein was proposed to function as a coreceptor for inhibins (and was even temporarily renamed inhibin binding protein). However, subsequent investigations, both in vitro and in vivo, failed to support a role for IGSF1 in inhibin action. Research on IGSF1 nearly ground to a halt until 2011, when next-generation sequencing identified mutations in the X-linked IGSF1 gene in boys and men with congenital central hypothyroidism. IGSF1 was localized to thyrotrope cells, implicating the protein in pituitary control of the thyroid. Investigations in two Igsf1 knockout mouse models converged to show that IGSF1 deficiency leads to reduced expression of the receptor for thyrotropin-releasing hormone (TRH) and impaired TRH stimulation of thyrotropin secretion, providing a candidate mechanism for the central hypothyroidism observed in patients. Nevertheless, the normal functions of IGSF1 in thyrotropes and other cells remain unresolved. Moreover, IGSF1 mutations are also commonly associated with other clinical phenotypes, including prolactin and growth hormone dysregulation, and macroorchidism. How the loss of IGSF1 produces these characteristics is unknown. Although early studies of IGSF1 ran into roadblocks and blind alleys, armed with the results of detailed clinical investigations, powerful mouse models, and new reagents, the field is now poised to discover IGSF1's function in endocrine tissues, including the pituitary and testes.
Follicle-stimulating hormone (FSH), a dimeric glycoprotein produced by pituitary gonadotrope cell... more Follicle-stimulating hormone (FSH), a dimeric glycoprotein produced by pituitary gonadotrope cells, regulates spermatogenesis in males and ovarian follicle growth in females. Hypothalamic gonadotropin-releasing hormone (GnRH) stimulates FSHβ subunit gene (Fshb) transcription, though the underlying mechanisms are poorly understood. To address this gap in knowledge, we examined changes in pituitary gene expression in GnRH-deficient mice (hpg) treated with a regimen of exogenous GnRH that increases pituitary Fshb but not luteinizing hormone β (Lhb) messenger RNA levels. Activating transcription factor 3 (Atf3) was among the most upregulated genes. Activating transcription factor 3 (ATF3) can heterodimerize with members of the activator protein 1 family to regulate gene transcription. Co-expression of ATF3 with JunB stimulated murine Fshb, but not Lhb, promoter-reporter activity in homologous LβT2b cells. ATF3 also synergized with a constitutively active activin type I receptor to incre...
Inhibins are transforming growth factor-β family heterodimers that suppress follicle-stimulating ... more Inhibins are transforming growth factor-β family heterodimers that suppress follicle-stimulating hormone (FSH) secretion by antagonizing activin class ligands. Inhibins share a common β chain with activin ligands. Follistatin is another activin antagonist, known to bind the common β chain of both activins and inhibins. In this study, we characterized the antagonist-antagonist complex of inhibin A and follistatin to determine if their interaction impacted activin A antagonism. We isolated the inhibin A:follistatin 288 complex, showing that it forms in a 1:1 stoichiometric ratio, different from previously reported homodimeric ligand:follistatin complexes, which bind in a 1:2 ratio. Small angle X-ray scattering coupled with modeling provided a low-resolution structure of inhibin A in complex with follistatin 288. Inhibin binds follistatin via the shared activin β chain, leaving the α chain free and flexible. The inhibin A:follistatin 288 complex was also shown to bind heparin with lowe...
Activin ligands are formed from two disulfide-linked inhibin β subunit chains. They exist as homo... more Activin ligands are formed from two disulfide-linked inhibin β subunit chains. They exist as homodimeric proteins, as in the case of activin A (ActA; InhβA/InhβA) or activin C (ActC; InhβC/InhβC), or as heterodimers, as with activin AC (ActAC; InhβA:InhβC). While the biological functions of ActA and activin B (ActB) have been well-characterized, little is known about the biological function of ActC or ActAC. One thought is that the InhβC chain functions to interfere with ActA production by forming less active ActAC heterodimers. Here, we assessed and characterized the signaling capacity of ligands containing the InhβC chain. ActC and ActAC activated SMAD2/3-dependent signaling via the type I receptor, activin receptor-like kinase 7 (ALK7). Relative to ActA and ActB, ActC exhibited lower affinity for the cognate activin type II receptors and was resistant to neutralization by the extracellular antagonist, follistatin. In mature adipocytes, which exhibit high ALK7 expression, ActC eli...
Follicle-stimulating hormone (FSH) regulates gonadal function and fertility. Measurement of FSH i... more Follicle-stimulating hormone (FSH) regulates gonadal function and fertility. Measurement of FSH in bodily fluids and tissues is possible with radioimmunoassays and enzyme-linked immunosorbent assays (ELISAs). Recently, several novel assays were developed to measure pituitary hormones including growth hormone, prolactin, and luteinizing hormone in mice from small sample volumes. Here, we describe a novel and sensitive ELISA that enables the accurate measurement of FSH in serum, plasma, and whole blood from female and male mice. The assay can also be used to measure FSH in murine pituitary lysates and cell culture media. In summary, the new methodology described here will enable investigators to measure FSH from a variety of biological samples in mice accurately, at low cost, and in their own laboratories.
Previous studies indicate that as Siberian hamsters (Phodopus sungorus) age, they may lose their ... more Previous studies indicate that as Siberian hamsters (Phodopus sungorus) age, they may lose their ability to show gonadal regression in response to short days. In one study, hamsters that regressed on short days early in life failed to regress when exposed to short days a second time later in life. Thus, Siberian hamsters may experience age-related deficits in photoresponsiveness or may be incapable of regressing twice. In the present study, we attempted to discriminate between these possibilities by examining patterns of gonadal regression in hamsters transferred back and forth from long (1 6L:8D) to short days (6L:1 8D) every 6.5, 13, or 26 wk for a 2-yr period. A control group was maintained on long days and had enlarged gonads throughout the entire study. Hamsters alternating between 26 wk of long and short days exhibited complete gonadal regression during their initial but not during their second exposure to short days. Hamsters alternated between long and short days every 13 or 6.5 wk showed regression two to three times, respectively. After about 52 wk of age, the majority of animals in both groups did not regress when exposed to short days. Taken together, the results of this experiment indicate that male Siberian hamsters 1) can exhibit at least two rounds of short-day-induced gonadal regression and 2) fail to regress on short days after about 1 yr of age.
Follicle-stimulating hormone beta subunit (Fshb) expression is regulated by transforming growth f... more Follicle-stimulating hormone beta subunit (Fshb) expression is regulated by transforming growth factor beta superfamily ligands. Recently, we demonstrated that bone morphogenetic proteins (BMPs) stimulate Fshb transcription alone and in synergy with activins. Also, transfection of the BMP type II receptor (BMPR2) and constitutively active forms of the type I receptors (activin A receptor type I [ACVR1] or BMP receptor type IA [BMPR1A]) in immortalized gonadotroph cells (LbetaT2) stimulated murine Fshb promoter-reporter activity. A third type I receptor (BMP receptor type IB [BMPR1B]) is also expressed in LbetaT2 cells, but we did not previously assess its functional role. A point mutation in BMPR1B (Q249R) is associated with increased ovulation rates and elevated FSH levels in Booroola (FecB) sheep. Herein, we assessed whether BMPR1B can regulate Fshb transcription in LbetaT2 cells and whether its ability to do so is altered by the Q249R mutation. As with ACVR1 and BMPR1A, coexpression of BMPR1B with BMPR2 increased Fshb promoter-reporter activity in BMP2-dependent and BMP2-independent fashions. Unexpectedly, the BMPR1B-Q249R mutant was equivalent to the wild type in its ability to stimulate SMAD1/5 phosphorylation and Fshb transcription. Pharmacological inhibition of ACVR1, BMPR1A, and BMPR1B confirmed that one or more of these receptors are required for BMP2-stimulated SMAD1/5 phosphorylation and Fshb reporter activity. Knockdown of endogenous BMPR1A, but not ACVR1 or BMPR1B, significantly impaired the synergism of BMP2 with activin A. Collectively, these data suggest that BMPR1A is the preferred BMP2 type I receptor in LbetaT2 cells and that neither ACVR1 nor BMPR1B compensates for its loss. The specific mechanism(s) through which the Booroola FecB mutation alters BMPR1B function remains to be determined.
Precursor proteolysis is a crucial mechanism for regulating protein structure and function. Signa... more Precursor proteolysis is a crucial mechanism for regulating protein structure and function. Signal peptidase (SP) is an enzyme with a well defined role in cleaving N-terminal signal sequences but no demonstrated function in the proteolysis of cellular precursor proteins. We provide evidence that SP mediates intraprotein cleavage of IgSF1, a large cellular Ig domain protein that is processed into two separate Ig domain proteins. In addition, our results suggest the involvement of signal peptide peptidase (SPP), an intramembrane protease, which acts on substrates that have been previously cleaved by SP. We show that IgSF1 is processed through sequential proteolysis by SP and SPP. Cleavage is directed by an internal signal sequence and generates two separate Ig domain proteins from a polytopic precursor. Our findings suggest that SP and SPP function are not restricted to N-terminal signal sequence cleavage but also contribute to the processing of cellular transmembrane proteins.
Journal of Assisted Reproduction and Genetics, Jan 5, 2019
Infertility due to Gonadotropin-Resistant Ovary Syndrome (GROS) is a rare type of hypergonadotrop... more Infertility due to Gonadotropin-Resistant Ovary Syndrome (GROS) is a rare type of hypergonadotropic hypogonadism. Here, we report an original case of GROS, associated with compound heterozygous follicle-stimulating hormone receptor (FSHR) variants, in a woman who achieved a live birth by in vitro maturation (IVM) of her oocytes. This 31-year-old woman consulted our assisted reproduction center for a second opinion after having been advised, because of pervasive high serum follicle-stimulating hormone (FSH) levels, to pursue in vitro fertilization (IVF) with donor oocytes. She presented with primary infertility and progressively prolonged menstrual cycles. Her serum FSH levels were indeed found to be high, but in discordance with a normal anti-Müllerian hormone (AMH) level and antral follicle count. Genetic investigation found the patient to be compound heterozygous for two FSHR variants: I160T, a known pathologic variant, and N558H, which has never been previously reported. As there was no ovarian response to high daily doses of exogenous gonadotropins, IVM was proposed to the patient with success and she finally delivered at term a healthy boy. Effects of the receptor variants were analyzed in heterologous cells. Whereas the I160T mutation blocked FSHR membrane trafficking and FSH-stimulated cAMP-dependent signaling in transfected CHO cells, the novel variant, N558H, functioned equivalently to wild-type FSHR in the assays employed. In conclusion, IVM should always be offered as a first-line therapy to infertile women presenting with GROS. The N558H variant discovered in FSHR is novel, but its functional significance, if any, is unresolved and merits further investigation as it may be associated with a recessive FSHRrelated disorder.
Activins regulate FSH synthesis by stimulating the phosphorylation and nuclear accumulation of SM... more Activins regulate FSH synthesis by stimulating the phosphorylation and nuclear accumulation of SMAD2 and SMAD3, which bind to a consensus SMAD-binding element in the proximal murine FSHb (Fshb) subunit gene to drive transcription. Previous over-expression and in vitro DNA binding analyses suggested that SMAD4 participates in complexes with SMAD2 and SMAD3 to regulate Fshb expression. Here, we have characterized the role of endogenous SMAD4 in activin A induction of Fshb transcription in immortalized murine gonadotropes (LbT2). We identified five murine Smad4 mRNA isoforms, of which, four are newly described; however, the canonical full-length form predominated at both the mRNA and protein levels. Depletion of endogenous SMAD4 by RNA interference (RNAi) abolished activin A-induced Fshb promoter-reporter activity and greatly attenuated constitutively active activin type IB receptor-stimulated Fshb mRNA levels. The activin A response was rescued with an RNAi-resistant form of wild-type SMAD4, but not with a DNA-binding-deficient (Lys88Arg) SMAD4, suggesting that DNA binding by SMAD4 is necessary for activin induction of the Fshb gene. Though SMAD2 and SMAD3 are generally thought to partner with SMAD4 prior to accumulation in the nucleus, treatment with leptomycin B, an inhibitor of SMAD4 nuclear export, reduced but did not prevent activin A induction of Fshb mRNA levels or promoter activity. In addition, a constitutively nuclear form of SMAD4 rescued the effect of endogenous SMAD4 depletion. Collectively, these data demonstrate a necessary role for SMAD4 in activin A induction of the murine Fshb gene in immortalized gonadotropes.
Inhibins are gonadal hormones that act on pituitary gonadotrope cells to suppress FSH synthesis a... more Inhibins are gonadal hormones that act on pituitary gonadotrope cells to suppress FSH synthesis and secretion. Inhibin A and B are heterodimers of the inhibin ⍺-subunit disulfide-linked to one of two inhibin b-subunits. Homodimers or heterodimers of the inhibin b-subunits form the activins, which stimulate FSH production. Activins signal through complexes of type I and II receptor serine/ threonine kinases to increase transcription of the FSHb subunit gene. According to in vitro observations, inhibins impair FSH synthesis by competitively binding to activin type II receptors, particularly in the presence of the TGFb type III receptor (TGFBR3, or betaglycan). The role of TGFBR3 in inhibin action in vivo has not been determined. Here, we ablated Tgfbr3 specifically in murine gonadotropes. Conditional knockout females were supra-fertile, exhibiting enhanced folliculogenesis, numbers of ovulated eggs per cycle, and litter sizes relative to control mice. Despite these phenotypes, FSH levels appeared to be unaltered in knockout mice, and the mechanisms underlying their enhanced fertility remain unexplained. Inhibin B is the predominant form of the hormone in males and in females during most stages of the estrous cycle. Remarkably, inhibin A, but not inhibin B, suppression of FSH synthesis was impaired in cultured pituitaries of knockout mice, which may explain the absence of discernible changes in FSH levels in vivo. Collectively, these data challenge current dogma by demonstrating that TGFBR3 (betaglycan) functions as an inhibin A, but not an inhibin B, coreceptor in gonadotrope cells in vivo. Mechanisms of inhibin B action merit further investigation. (Endocrinology 159: 4077-4091, 2018) T he term inhibin was coined in the early 1930s to describe a hypothetical hormone from the testicular seminiferous epithelium that regulates the morphology of cells in the anterior pituitary gland (1). Four decades later, inhibin-like activity was discovered in ovarian follicular fluid and shown to selectively inhibit FSH secretion from pituitary gonadotrope cells while having no effects on the related LH (2). It took another decade before two forms of inhibin (A and B) were finally purified (3-7). Inhibins A and B are dynamically and differentially secreted from granulosa (and luteal) cells of the ovary across female reproductive cycles. In contrast, adult males of most species predominately secrete inhibin B from testicular Sertoli cells (8-11). Biochemical and molecular characterization of the inhibins revealed that they were heterodimeric TGFb superfamily ligands composed of the inhibin a-subunit disulfide-linked to one of two inhibin b-subunits (bA in inhibin A or bB in inhibin B). Shortly thereafter, it was discovered that inhibin b-subunits homodimerize or
Myostatin is a member of the transforming growth factor-b (TGF-b) family and a strong negative re... more Myostatin is a member of the transforming growth factor-b (TGF-b) family and a strong negative regulator of muscle growth. Here, we present the crystal structure of myostatin in complex with the antagonist follistatin 288 (Fst288). We find that the prehelix region of myostatin very closely resembles that of TGF-b class members and that this region alone can be swapped into activin A to confer signalling through the non-canonical type I receptor Alk5. Furthermore, the N-terminal domain of Fst288 undergoes conformational rearrangements to bind myostatin and likely acts as a site of specificity for the antagonist. In addition, a unique continuous electropositive surface is created when myostatin binds Fst288, which significantly increases the affinity for heparin. This translates into stronger interactions with the cell surface and enhanced myostatin degradation in the presence of either Fst288 or Fst315. Overall, we have identified several characteristics unique to myostatin that will be paramount to the rational design of myostatin inhibitors that could be used in the treatment of muscle-wasting disorders.
The glycoprotein FSH, a product of pituitary gonadotrope cells, regulates ovarian follicle develo... more The glycoprotein FSH, a product of pituitary gonadotrope cells, regulates ovarian follicle development in females and spermatogenesis in males. FSH is a heterodimer of the common a gonadotropin subunit and the hormone-specific FSHb subunit (a product of the Fshb gene). Using a conditional knockout approach (Cre-lox), we previously demonstrated that Fshb expression in mice depends on the transcription factors forkhead box L2 (FOXL2) and SMAD4. Deletion of Foxl2 or Smad4 alone led to FSH deficiency, female subfertility, and oligozoospermia in males. Simultaneous deletion of the two genes yielded a greater suppression of FSH and female sterility. The Cre-driver used previously was first active during embryonic development. Therefore, it is unclear whether FOXL2 and SMAD4 play important roles in the development or adult function of gonadotropes, or both. To address this question, we developed a tamoxifen-inducible Cre-driver line, which enabled Foxl2 and Smad4 gene deletions in gonadotropes of adult mice. After tamoxifen treatment, females with previously demonstrated fertility exhibited profound reductions in FSH levels, arrested ovarian follicle development, and sterility. FSH levels were comparably reduced in males 1 or 2 months after treatment; however, spermatogenesis was unaffected. These data indicate that (1) FOXL2 and SMAD4 are necessary to maintain FSH synthesis in gonadotrope cells of adult mice, (2) FSH is essential for female reproduction but appears to be unnecessary for the maintenance of spermatogenesis in adult male mice, and (3) the inducible Cre-driver line developed here provides a powerful tool to interrogate gene function in gonadotrope cells of adult mice.
Reproductive Biology and Endocrinology, Dec 1, 2005
Background: Activins stimulate the synthesis of follicle stimulating hormone (FSH) in pituitary g... more Background: Activins stimulate the synthesis of follicle stimulating hormone (FSH) in pituitary gonadotropes, at least in part, by inducing transcription of its beta subunit (Fshb). Evidence from several laboratories studying transformed murine LbetaT2 gonadotropes indicates that activins signal through Smad-dependent and/or Smad-independent pathways, similar to those used by transforming growth factor beta-1 (TGFB1) in other cell types. Therefore, given common intracellular signaling mechanisms of these two ligands, we examined whether TGFBs can also induce transcription of Fshb in LbetaT2 cells as well as in purified primary murine gonadotropes. Methods: Murine Fshb promoter-reporter (-1990/+1 mFshb-luc) activity was measured in LbetaT2 cells treated with activin A or TGFB1, and in cells transfected with either activin or TGFB receptors. The ability of the ligands to stimulate phosphorylation of Smads 2 and 3 in LbetaT2 cells was measured by western blot analysis, and expression of TGFB type I and II receptors was assessed by reverse transcriptase polymerase chain reaction in both LbetaT2 cells and primary gonadotropes purified from male mice of different ages. Finally, regulation of endogenous murine Fshb mRNA levels by activin A and TGFB1 in purified gonadotropes and whole pituitary cultures was measured using quantitative RT-PCR. Results: Activin A dose-dependently stimulated -1990/+1 mFshb-luc activity in LbetaT2 cells, but TGFB1 had no effect at doses up to 5 nM. Similarly, activin A, but not TGFB1, stimulated Smad 2 and 3 phosphorylation in these cells. Constitutively active forms of the activin (Acvr1b-T206D) and TGFB (TGFBR1-T204D) type I receptors strongly stimulated -1990/+1 mFshb-luc activity, showing that mechanisms down stream of Tgfbr1 seem to be intact in LbetaT2 cells. RT-PCR analysis of LbetaT2 cells and whole adult murine pituitaries indicated that both expressed Tgfbr1 mRNA, but that Tgfbr2 was not detected in LbetaT2 cells. When cells were transfected with a human TGFBR2 expression construct, TGFB1 acquired the ability to significantly stimulate -1990/+1 mFshb-luc activity. In contrast to LbetaT2 cells, primary murine gonadotropes from young mice (8-10 weeks) contained low, but detectable levels of Tgfbr2 mRNA and these levels increased in older mice (1 yr). A second surprise was the finding that treatment of purified primary gonadotropes with TGFB1 decreased murine Fshb mRNA expression by 95% whereas activin A stimulated expression by 31-fold. These data indicate that TGFB1-insensitivity in LbetaT2 cells results from a deficiency in Tgfbr2 expression. In primary gonadotropes, however, expression of Tgfbr2 does occur, and its presence permits TGFB1 to inhibit Fshb transcription, whereas activin A stimulates it. These divergent actions of activin A and TGFB1 were unexpected and show that the two ligands may act through distinct pathways to cause opposing biological effects in primary murine gonadotropes.
Immunoglobulin superfamily, member 1 (IGSF1) is a transmembrane glycoprotein with high expression... more Immunoglobulin superfamily, member 1 (IGSF1) is a transmembrane glycoprotein with high expression in the mammalian pituitary gland. Mutations in the IGSF1 gene cause congenital central hypothyroidism in humans. The IGSF1 protein is co-translationally cleaved into N- and C-terminal domains (NTD and CTD), the latter of which is trafficked to the plasma membrane and appears to be the functional portion of the molecule. Though the IGSF1-NTD is retained in the endoplasmic reticulum and has no apparent function, it has a high degree of sequence identity with the IGSF1-CTD and is conserved across mammalian species. Based upon phylogenetic analyses, we propose that the ancestral IGSF1 gene encoded the IGSF1-CTD, which was duplicated and integrated immediately upstream of itself, yielding a larger protein encompassing the IGSF1-NTD and IGSF1-CTD. The selective pressures favoring the initial gene duplication and subsequent retention of a conserved IGSF1-NTD are unresolved.
Background: Loss-of-function mutations in immunoglobulin superfamily member 1 (IGSF1) cause an X-... more Background: Loss-of-function mutations in immunoglobulin superfamily member 1 (IGSF1) cause an X-linked syndrome of central hypothyroidism, macroorchidism, delayed pubertal testosterone rise, variable prolactin deficiency and variable partial GH deficiency in childhood. The clinical features and gene expression pattern suggest a pivotal role for IGSF1 in the pituitary, but detailed knowledge on pituitary hormone secretion in this syndrome is lacking. We therefore aimed to study the 24-hour pituitary hormone secretion in male patients with IGSF1 deficiency. Methods: We collected blood samples every 10 min for 24 h in eight adult male IGSF1-deficient patients and measured circulating TSH, prolactin and gonadotropins. Deconvolution, modified cosinor and approximate entropy analyses were applied to quantify secretion rates, diurnal rhythmicity and regularity of hormone release. Results were compared to healthy controls matched for age and body mass index. Results: Compared to healthy controls, IGSF1-deficient patients showed decreased pulsatile secretion of TSH with decreased disorderliness and reduced diurnal variation. Basal and pulsatile secretion of FSH was increased by over 200%, while LH secretion did not differ from healthy controls. We observed a bimodal distribution of prolactin secretion, i.e. severe deficiency in three and increased basal and total secretion in the other five patients. Conclusion: The altered TSH secretion pattern is consistent with the previously hypothesized defect in thyrotropin-releasing hormone signaling in IGSF1 deficiency. However, the phenotype is more extensive and includes increased FSH secretion without altered LH secretion as well as either undetectable or increased prolactin secretion.
The Journal of Clinical Endocrinology and Metabolism, Apr 1, 2016
on behalf of the IGSF1 Clinical Care Group † Context: Mutations in the immunoglobulin superfamily... more on behalf of the IGSF1 Clinical Care Group † Context: Mutations in the immunoglobulin superfamily, member 1 (IGSF1) gene cause the X-linked IGSF1 deficiency syndrome consisting of central hypothyroidism, delayed pubertal testosterone rise, adult macroorchidism, variable prolactin deficiency, and occasionally transient partial GH deficiency. Since our first reports, we discovered 20 new families with 18 new pathogenic IGSF1 mutations. Objective: We aimed to share data on the largest cohort of patients with IGSF1 deficiency to date and formulate recommendations for clinical management. Methods: We collected clinical and biochemical characteristics of 69 male patients (35 children, 34 adults) and 56 female IGSF1 mutation carriers (three children, 53 adults) from 30 unrelated families according to a standardized clinical protocol. At evaluation, boys were treated with levothyroxine in 89%, adult males in 44%, and females in 5% of cases. Results: Additional symptoms in male patients included small thyroid gland volume (74%), high birth weight (25%), and large head circumference (20%). In general, the timing of pubertal testicular growth was normal or even premature, in contrast to a late rise in T levels. Late adrenarche was observed in patients with prolactin deficiency, and adult dehydroepiandrosterone concentrations were decreased in 40%. Hypocortisolism was observed in 6 of 28 evaluated newborns, although cortisol concentrations were normal later. Waist circumference of male patients was increased in 60%, but blood lipids were normal. Female carriers showed low free T 4 (FT 4) and low-normal FT 4 in 18% and 60%, respectively, delayed age at menarche in 31%, mild prolactin deficiency in 22%, increased waist circumference in 57%, and a negative correlation between FT 4 concentrations and metabolic parameters. Conclusion: IGSF1 deficiency represents the most common genetic cause of central hypothyroidism and is associated with multiple other characteristics. Based on these results, we provide recommendations for mutational analysis, endocrine work-up, and long-term care.
Immunoglobulin superfamily, member 1 (IGSF1) is a transmembrane glycoprotein highly expressed in ... more Immunoglobulin superfamily, member 1 (IGSF1) is a transmembrane glycoprotein highly expressed in the mammalian pituitary gland. Shortly after its discovery in 1998, the protein was proposed to function as a coreceptor for inhibins (and was even temporarily renamed inhibin binding protein). However, subsequent investigations, both in vitro and in vivo, failed to support a role for IGSF1 in inhibin action. Research on IGSF1 nearly ground to a halt until 2011, when next-generation sequencing identified mutations in the X-linked IGSF1 gene in boys and men with congenital central hypothyroidism. IGSF1 was localized to thyrotrope cells, implicating the protein in pituitary control of the thyroid. Investigations in two Igsf1 knockout mouse models converged to show that IGSF1 deficiency leads to reduced expression of the receptor for thyrotropin-releasing hormone (TRH) and impaired TRH stimulation of thyrotropin secretion, providing a candidate mechanism for the central hypothyroidism observed in patients. Nevertheless, the normal functions of IGSF1 in thyrotropes and other cells remain unresolved. Moreover, IGSF1 mutations are also commonly associated with other clinical phenotypes, including prolactin and growth hormone dysregulation, and macroorchidism. How the loss of IGSF1 produces these characteristics is unknown. Although early studies of IGSF1 ran into roadblocks and blind alleys, armed with the results of detailed clinical investigations, powerful mouse models, and new reagents, the field is now poised to discover IGSF1's function in endocrine tissues, including the pituitary and testes.
Follicle-stimulating hormone (FSH), a dimeric glycoprotein produced by pituitary gonadotrope cell... more Follicle-stimulating hormone (FSH), a dimeric glycoprotein produced by pituitary gonadotrope cells, regulates spermatogenesis in males and ovarian follicle growth in females. Hypothalamic gonadotropin-releasing hormone (GnRH) stimulates FSHβ subunit gene (Fshb) transcription, though the underlying mechanisms are poorly understood. To address this gap in knowledge, we examined changes in pituitary gene expression in GnRH-deficient mice (hpg) treated with a regimen of exogenous GnRH that increases pituitary Fshb but not luteinizing hormone β (Lhb) messenger RNA levels. Activating transcription factor 3 (Atf3) was among the most upregulated genes. Activating transcription factor 3 (ATF3) can heterodimerize with members of the activator protein 1 family to regulate gene transcription. Co-expression of ATF3 with JunB stimulated murine Fshb, but not Lhb, promoter-reporter activity in homologous LβT2b cells. ATF3 also synergized with a constitutively active activin type I receptor to incre...
Inhibins are transforming growth factor-β family heterodimers that suppress follicle-stimulating ... more Inhibins are transforming growth factor-β family heterodimers that suppress follicle-stimulating hormone (FSH) secretion by antagonizing activin class ligands. Inhibins share a common β chain with activin ligands. Follistatin is another activin antagonist, known to bind the common β chain of both activins and inhibins. In this study, we characterized the antagonist-antagonist complex of inhibin A and follistatin to determine if their interaction impacted activin A antagonism. We isolated the inhibin A:follistatin 288 complex, showing that it forms in a 1:1 stoichiometric ratio, different from previously reported homodimeric ligand:follistatin complexes, which bind in a 1:2 ratio. Small angle X-ray scattering coupled with modeling provided a low-resolution structure of inhibin A in complex with follistatin 288. Inhibin binds follistatin via the shared activin β chain, leaving the α chain free and flexible. The inhibin A:follistatin 288 complex was also shown to bind heparin with lowe...
Activin ligands are formed from two disulfide-linked inhibin β subunit chains. They exist as homo... more Activin ligands are formed from two disulfide-linked inhibin β subunit chains. They exist as homodimeric proteins, as in the case of activin A (ActA; InhβA/InhβA) or activin C (ActC; InhβC/InhβC), or as heterodimers, as with activin AC (ActAC; InhβA:InhβC). While the biological functions of ActA and activin B (ActB) have been well-characterized, little is known about the biological function of ActC or ActAC. One thought is that the InhβC chain functions to interfere with ActA production by forming less active ActAC heterodimers. Here, we assessed and characterized the signaling capacity of ligands containing the InhβC chain. ActC and ActAC activated SMAD2/3-dependent signaling via the type I receptor, activin receptor-like kinase 7 (ALK7). Relative to ActA and ActB, ActC exhibited lower affinity for the cognate activin type II receptors and was resistant to neutralization by the extracellular antagonist, follistatin. In mature adipocytes, which exhibit high ALK7 expression, ActC eli...
Follicle-stimulating hormone (FSH) regulates gonadal function and fertility. Measurement of FSH i... more Follicle-stimulating hormone (FSH) regulates gonadal function and fertility. Measurement of FSH in bodily fluids and tissues is possible with radioimmunoassays and enzyme-linked immunosorbent assays (ELISAs). Recently, several novel assays were developed to measure pituitary hormones including growth hormone, prolactin, and luteinizing hormone in mice from small sample volumes. Here, we describe a novel and sensitive ELISA that enables the accurate measurement of FSH in serum, plasma, and whole blood from female and male mice. The assay can also be used to measure FSH in murine pituitary lysates and cell culture media. In summary, the new methodology described here will enable investigators to measure FSH from a variety of biological samples in mice accurately, at low cost, and in their own laboratories.
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Papers by Daniel Bernard