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Although a number of studies have shown that the occurrence and progression of osteoarthritis (OA) is related to endocrine system dysfunction, there is limited evidence about what roles sex hormones play. The aim of the present study was to examine the capacity of 17β-estradiol (ED) and follicle stimulating hormone (FSH) to alter the differentiation of bone marrow (BM) cells in arthritic mice. The experiments were conducted in collagenase-induced osteoarthritis in mice. Cartilage degradation was observed by safranin and toluidine blue staining. Flow cytometry was used to define different BM and synovial cell populations. The influence of FSH and ED on osteoclastogenesis was studied in BM cultures and on the osteoblastogenesis in primary calvarial cultures. The levels of IL-8, TNF-α, FSH, and osteocalcin were estimated by ELISA. FSH increased cartilage degradation and serum osteocalcin levels, while ED abolished it and lowered serum osteocalcin. FSH elevated the percentage of monocytoid CD14+/RANK+ and B cell CD19+/RANK+ cells in contrast to ED which inhibited the accumulation of these osteogenic populations. Also, ED changed the percentage of CD105+/F4/80+ and CD11c+ cells in the synovium. FSH augmented and ED suppressed macrophage colony-stimulating factor (M-CSF) + receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast (OC) formation, and this correlated with a respective increase and decrease of IL-8 secretion. FSH did not influence osteoblast (OB) formation while ED enhanced this process in association with changes of TNF-α, IL-8, and osteocalcin production. ED reduced osteoclast generation in bone. The key outcome of the current study is that both hormones influenced BM cell differentiation, with FSH favoring osteoclast formation and ED favoring osteoblast accumulation.

Blocking the action of FSH genetically or pharmacologically in mice reduces body fat, lowers serum cholesterol, and increases bone mass, making an anti-FSH agent a potential therapeutic for three global epidemics: obesity, osteoporosis, and hypercholesterolemia. Here, we report the generation, structure, and function of a first-in-class, fully humanized, epitope-specific FSH blocking antibody with a K D of 7 nM. Protein thermal shift, molecular dynamics, and fine mapping of the FSH-FSH receptor interface confirm stable binding of the Fab domain to two of five receptor-interacting residues of the FSHβ subunit, which is sufficient to block its interaction with the FSH receptor. In doing so, the humanized antibody profoundly inhibited FSH action in cell-based assays, a prelude to further preclinical and clinical testing., Competing Interests: The authors declare no competing interest.

Follicle-stimulating hormone (FSH) has been newly demonstrated to play a great role in promoting fat accumulation, providing a potential to target FSH for controlling fat accumulation and treating obesity. A short, 13-amino acid of FSHβ (FSHβ13AA) was indentified to be the FSH receptor-binding epitope in both humans and mice. By conservation analysis, we found such FSHβ13AA is highly conserved across species. Accordingly, we designed a new FSH antigen by synthesizing a tandem of FSHβ13AA (LVYKDPARPNIQK) and then conjugating it to ovalbumin (FSHβ13AA-T-OVA). Then, we tested its efficacy in suppressing fat accumulation in both ovariectomized and intact mouse models. Vaccination with this novel antigen emulsified in mild adjuvant, Specol, was highly effective in preventing ovariectomy-induced body weight gain and fat accumulation in mice (P < 0.01). Mechanistically, FSH vaccination treatment inhibited lipid biosynthesis by inactivating PPARγ adipogenic signaling pathway and simultaneously enhanced adipocyte themogenesis via upregulating UCP1 expression in both visceral and subcutaneous adipose tissues. Moreover, injection of this novel FSH vaccine also substantially reduced (P < 0.05) fat accumulation in both intact male and female mice. These actions result from the specific binding of the generated antibody to the β-subunit to block its action, rather than lowering the circulating levels of FSH, as evidenced by nearly no alterations in serum FSH levels in mice following FSH vaccination. Overall, we developed a novel FSH antigen and vaccine, and demonstrated it is highly efficacious in suppressing fat accumulation., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Follicle stimulating hormone (FSH) plays a critical role in female reproductive development and homeostasis. The blood/serum concentration of FSH is an important marker for reporting multiple endocrinal functions. The standardized method for mouse FSH (mFSH) quantification based on radioimmunoassay (RIA) suffers from long assay time (∼2 days), relatively low sensitivity, larger sample volume (60 μL), and small dynamic range (2-60 ng/mL); thus, it is insufficient for monitoring fast developing events with relatively small mFSH fluctuations (e.g., estrous cycles of mammals). Here, we developed an automated microfluidic chemiluminescent ELISA device along with the disposal sensor array and the corresponding detection protocol for rapid and quantitative analysis of mFSH from mouse tail serum samples. With this technology, highly sensitive quantification of mFSH can be accomplished within 30 min using only 8 μL of the serum sample. It is further shown that our technique is able to generate results comparable to RIA but has a significantly improved dynamic range that covers 0.5-250 ng/mL. The performance of this technology was evaluated with blood samples collected from ovariectomized animals and animals with reimplanted ovarian tissues, which restored ovarian endocrine function and correlated with estrus cycle analysis study.

Problem: Gonadotrophin hormones are used for the controlled ovarian stimulation (COS) as part of the in vitro fertilization techniques. Therapeutic proteins have the potential to induce an unwanted immune response., Method of Study: The presence of anti-FSH, anti-LH and anti-hCG antibodies were determined in patients from two different clinical trials after the repeated administration of hMG or FSH., Results: In the first study, 27 subjects were screening for the presence of anti-FSH antibodies. From the 27 patients, only one patient showed the presence of low levels of antibodies. In a second study, 25 patients were screened for the presence of anti-FSH, anti-LH and anti-hCG antibodies. At the end of the study, no patients showed the presence of antibodies., Conclusion: The results of this study suggest that repeated treatment cycles with FSH or hMG in patients undergoing COS for in vitro fertilization can be safely and effectively applied without concerns for immunogenicity., (© 2017 IBSA Institut Biochimque SA. American Journal of Reproductive Immunology published by John Wiley & Sons Ltd.)

We prepared monoclonal antibodies (mAbs) against a recombinant tethered follicle-stimulating hormone (rec-FSH) from Japanese eel Anguilla japonica that was produced in Escherichia coli. Positive hybridomas (clones eFA-C5, eFA-C10, eFA-C11, eFA-C12, eFA-C13, and eFB-C14) were selected by using the eel FSH antigen in ELISA, and anti-eel FSH mAbs were purified from culture supernatants by performing affinity chromatography. Three of the 6mAbs were characterized and their isotypes were identified as IgG2b (eFA-C5 and eFA-C11) and IgG1 (eFB-C14). In western blotting assays, the mAbs recognized the antigen as a 24.3-kDa band, and further detected bands of 34 and 32kDa in the supernatants of CHO cells transfected with cDNA encoding tethered eel FSHβ/α and LHβ/α, respectively. PNase F-mediated deglycosylation of the recombinant proteins resulted in a drastic reduction in their molecular weight, to 7-9kDa. The mAbs eFA-C5 and eFA-C11 recognized the eel FSHα-subunit that is commonly encoded among glycoprotein hormones, whereas eFB-C14 recognized the eel FSHβ-subunit, and immunohistochemical analysis revealed that the staining by these mAbs was specifically localized in the eel pituitary. We also established an ELISA system for detecting rec-tethered FSHβ/α and LHβ/α produced from CHO cell lines. Measurement of biological activities in vitro revealed that only weak activity of rec-FSHβ/α was detected. The activity of rec-LHβ/α was found to be increased in a dose-dependent manner for eel oocyte maturation., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

In fish, both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play important roles in reproduction. Here we explored the structure and differential specificity of tilapia (t) gonadotropins (GTHs) to delineate their physiological relevance and the nature of their regulation. We generated structural models of tGTHs and GTH receptors (R) that enabled us to better understand the hormone-receptor interacting region. In tilapia, FSH release is under the control of the hypothalamic decapeptide GnRH, an effect that was abolished by specific bioneutralizing antisera [anti-recombinant (r) tFSHβ]. These antisera also reduced the basal secretion and delayed GnRH-stimulated production of 11-ketotestosterone (11KT), and dramatically reduced LH levels. Immunoneutralization of tLH using anti-rtLHβ significantly reduced its GnRH-stimulated levels. Basal 11KT and FSH levels were also reduced. Taken together, these results suggest a feedback mechanism between FSH and LH release in tilapia., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Recent evidences have highlighted that the pituitary hormones have profound effects on bone, so that the pituitary-bone axis is now becoming an important issue in the skeletal biology. Here, we discuss the topical evidence about the dysfunction of the pituitary-bone axis that leads to osteoporotic bone loss. We will explore the context of FSH and TSH hormones arguing their direct or indirect role in bone loss. In addition, we will focus on the knowledge that both FSH and TSH have influence on proinflammatory and proosteoclastogenic cytokine expression, such as TNF α and IL-1, underlining the correlation of pituitary-bone axis to the immune system.

Aims: Anti-follicle-stimulating hormone (FSH) autoantibody was found to highly correlate with oligospermia and asthenospermia, but the actual effect of FSH autoantibody on spermatogenesis is still unknown., Main Methods: In this study, 21-day rats were immunized seven times with FSH peptides linked with Keyhole Limpet Hemocyanin (KLH) (experimental group) or KLH (control group) every 2 weeks. Luteinizing hormone (LH) and inhibin B level in the immunized rat sera were measured by enzyme-linked immunosorbent assay (ELISA). Apoptosis of spermatogenic cells in the testis was detected by in situ end labeling method (TUNEL), and the mRNAs of Bax, Bcl-2 and Caspase-3 in testis were detected by fluorescent Quantitative PCR., Key Findings: Compared with the control, serum inhibin B level was significantly decreased at all time points (34.49%, 23.20%, and 37.00%) (p<0.05). There was no difference in the serum LH level between experimental and control groups. FSH peptide immunization increased the apoptosis of spermatogenic cells in the testis that was associated with an imbalance of Bax and Bcl-2 expression and upregulation of Caspase-3., Significance: These results suggest that FSH autoantibody could cause the reduction of inhibin B, thereby inducing hypospermatogenesis via augment of spermatogenic cell apoptosis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Luteinizing hormone (Lh) and follicle-stimulating hormone (Fsh) control many aspects of gonadal development and function in teleosts. In the present paper, the specific antisera against ricefield eel Lhb (Lh beta subunit), Fshb (Fsh beta subunit), and Cga (the common pituitary glycoprotein hormone alpha subunit) were generated, and the cellular localization, initial appearance, and subsequent development of gonadotrophs in relation to early ovarian differentiation and development in the ricefield eel, a protogynous sex-changing teleost, were examined with immunochemistry. Lhb- and Fshb-immunoreactive signals were identified in distinct pituitary cells that occupied primarily the peripheral regions of the adenohypophysis. During ontogeny, Lhb-immunoreactive signals were first detected in the pituitary around 40 days after hatching (dah) when the oogonia transitioned into early primary growth oocytes, and the intensity of immunoreactivity increased concomitantly with the growth of primary oocytes from 60 to 140 dah. During overwintering from 170 to 230 dah, Lhb-immunoreactive signals were significantly decreased when a large proportion of perinucleolus oocytes contained intense Balbiani bodies. In contrast, Fshb-immunoreactive signals were not detectable in the pituitary until around 230 dah (in the spring after hatching) and slightly increased from 285 dah when the late perinucleolus oocytes began to enter the secondary growth phase. Both Lhb- and Fshb-immunoreactive cells were increased when the early cortical alveoli oocytes emerged at 300 dah. The mRNA expression of lhb and fshb coincided with their immunoreactive signals. Taken together, these results suggest that only Lh is involved in primary oocyte growth in ricefield eels, but both Fsh and Lh are important for the secondary ooctye growth.

Female fertility can be affected by diseases or dysfunctions of reproductive tract, neuroendocrine system, and immune system. Reproductive autoimmune failure can be associated with overall activation of immune system or with immune system reactions specifically directed against ovarian antigens. Majority of the antiovarian autoantibodies are directed against β-subunit of follicle stimulating hormone (anti-FSH). This paper summarizes a current clinical classification of female infertility in the context of general activation of autoimmunity and antiovarian autoimmunity by describing serum anti-FSH. The presence of naturally occurring anti-FSH in healthy women will be discussed. In addition, the putative impairment of ovarian folliculogenesis in case of increased production of those antibodies in infertile women will be characterized.

PROBLEM  In this baseline study, our aim is to show the relationship of parameters and gonad hormones in menopausal and postmenopausal women. METHOD  Blood samples were taken from menopausal and postmenopausal women (12-14 months and ≥10 years, respectfully, since their last menstruation). Adolescents aged 13.7 ± 0.7 were used as controls. Hormones were measured by ELISA and percentages of CD45, CD4, CD8, CD3, CD19, IL-2, CD25 and HLA-DR were measured by flow cytometry. RESULTS Both groups showed an increase in the percentage of CD3, CD4 and CD8. Levels of CD19 were significantly lower in the postmenopausal group. However, changes in immunologic parameters during menopause were less marked than the hormonal changes observed in these groups. Most of the correlations LH × CD3 (-ve), LH × IL2R (-ve) and E2 × CD19 (+ve) suggesting how menopausal women with particularly high LH or low E2 levels may be affected. Only CD3 and HLA-DR correlated with the hormonal changes in the postmenopausal group. IL-2 levels were high in the menopausal group and low in the postmenopausal group; however, no correlation was observed. DISCUSSION  Menopause is characterized by increased levels of IL-2, which has critical immune-modulatory effects. These changes may be related to the overall hormonal change process observed during menopause., (© 2011 John Wiley & Sons A/S.)

Interference in immunoassay caused by endogenous immunoglobulin is a cause of incorrect laboratory results that can drastically affect patient management. Two cases of immunoglobulin interference in serum follicle-stimulating hormone (FSH) assays are presented. These cases illustrate two common mechanisms for false-positive interference in two-site (sandwich) immunoassays. The first case describes a circulating autoimmune FSH immunoglobulin complex ('macro'-FSH), which has not been previously described for FSH, and the second a cross-linking antibody directed against the assay reagents. Immunoglobulin interference was detected and characterized using a combination of method comparison, immunosubtraction and size exclusion chromatography.

This study investigated the in vitro immune-modulating activities of recombinant versus highly purified urinary follicle-stimulating hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotropin (hCG) at the cellular level. CD4(+) T cells were isolated from peripheral blood mononuclear cells obtained from ten healthy women (aged 19-30 years) with regular menstrual cycles during the follicular phase of their cycle. CD4(+) T cells were stimulated with anti-CD3/CD28 monoclonal antibodies as a T cell-specific mitogen. Proliferative and cytokine responses were analyzed at standard time points (72h). Recombinant FSH (r-FSH) and LH (r-LH) alone showed a modest capacity to influence proliferation and cytokine release by CD4(+) T cells. Conversely, their addition to T cells in combination with recombinant hCG (r-hCG) induced a powerful down-modulation of T cell proliferation, decreased interferon-gamma (IFN-gamma) secretion and increased interleukin-10 (IL-10) production. These immune-modulating activities were not present when CD4(+) T cells were stimulated either in the presence of urinary-purified FSH (u-FSH) or human menopausal gonadotropin (HMG), alone or in combination with recombinant hCG. We are the first to suggest that urinary-purified gonadotropins do not display profound immune-modulating activities as compared with the recombinant preparations, despite their endocrine effects. Therefore, the use of the recombinant preparations in assisted reproductive techniques might be relevant not only for their well-documented endocrine actions but also for their impact on the transient immune tolerance known to favour embryo implantation and progression of pregnancy., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Single- and multi-analyte detection of two gonadotropic hormones (follicle stimulating hormone (hFSH) and luteinizing hormone (hLH)) was achieved by a Surface Plasmon Resonance (SPR) immunoassay on untreated human urine samples. Multi-analyte detection was accomplished using two alternative formats which are based in the individual or simultaneous immobilization of the hormones on the sensor surface. The lowest detection limit for both hormones in urine was found to be 1 ng mL(-1), which in international units (IU) in terms of the World Health Organization (WHO) standards represents 8 mIU mL(-1) of hLH and 14 mIU mL(-1) of hFSH, respectively. The reliability of the assay was demonstrated by intra- and inter-assay variabilities < 6%, chip-to-chip variabilities < 5%, recoveries in the range of 80-120% and stability of the sensor response through more than 100 measurements. The sensitivity of this biosensing methodology renders it in a useful technique for the diagnosis of reproductive disorders, as well as for fertility monitoring.

Sex hormones regulate cholangiocyte hyperplasia in bile duct-ligated (BDL) rats. We studied whether follicle-stimulating hormone (FSH) regulates cholangiocyte proliferation. FSH receptor (FSHR) and FSH expression was evaluated in liver sections, purified cholangiocytes, and cholangiocyte cultures (NRICC). In vivo, normal female and male rats were treated with FSH or immediately after BDL with antide (a gonadotropin-releasing hormone antagonist blocking FSH secretion) or a neutralizing FSH antibody for 1 wk. We evaluated 1) cholangiocyte proliferation in sections and cholangiocytes and 2) changes in secretin-stimulated cAMP (functional index of cholangiocyte growth) levels, and ERK1/2 and Elk-1 phosphorylation. NRICC were stimulated with FSH before evaluation of proliferation, cAMP/IP(3) levels, and ERK1/2 and Elk-1 phosphorylation. To determine whether FSH regulates cholangiocyte proliferation by an autocrine mechanism, we evaluated the effects of 1) cholangiocyte supernatant (containing FSH) on NRICC proliferation and 2) FSH silencing in NRICC before measuring proliferation and ERK1/2 and Elk-1 phosphorylation. Cholangiocytes and NRICC express FSHR and FSH and secrete FSH. In vivo administration of FSH to normal rats increased, whereas administration of antide and anti-FSH antibody to BDL rats decreased 1) ductal mass and 2) secretin-stimulated cAMP levels, proliferation, and ERK1/2 and Elk-1 phosphorylation in cholangiocytes compared with controls. In NRICC, FSH increased cholangiocyte proliferation, cAMP levels, and ERK1/2 and Elk-1 phosphorylation. The supernatant of cholangiocytes increased NRICC proliferation, inhibited by preincubation with anti-FSH antibody. Silencing of FSH gene decreases cholangiocyte proliferation and ERK1/2 and Elk-1 phosphorylation. Modulation of cholangiocyte FSH expression may be important for the management of cholangiopathies.

A detailed characterization regarding the chromatographic and biochemical properties of an immunoaffinity column for sample pretreatment using a follicle stimulating hormone (FSH) beta-unit specific mAb has been performed. A maximum binding capacity of approximately 300 microg/mL was determined. A K(D) value of 4.5x10(-8) M could be obtained in batch isotherm measurements. The dynamic binding capacity at 10% did not change when residence time was at least 6 min. High selectivity could be demonstrated using crude culture supernatant from FSH producing Chinese hamster ovary (CHO) cells. A great stability of the matrix was confirmed by performing 100 consecutive chromatographic runs without loss of capacity.

Aim: To detect the anti-follicle-stimulating hormone (FSH) antibody in idiopathic infertile patients and fertile subjects in order to determine the role of this antibody in patients with spermatogenic dysfunction., Methods: The anti-FSH antibody in serum was detected by an enzyme-linked immunosorbent assay (ELISA). The functional and structural integrity of the sperm membrane was evaluated with hypo-osmotic swelling (HOS) test and the ultrastructure of the spermatozoa was investigated by transmission electron microscopy (TEM)., Results: The extent of positive FSH antibody in the patients with oligozoospermia and/or asthenozoospermia was significantly higher than that in the fertile subjects and infertile patients with normal sperm concentration and motility, but it was significantly lower than that in the patients with azoospermia. The extent of anti-FSH antibody in the patients with azoospermia was significantly greater than that in patients with oligospermia and/or asthenospermia, infertile people with normal sperm density and motility and fertile people. The hypo-osmotic swelling test showed that the percentage of HOS-positive spermatozoa (swollen) was 45.1 mu 3.5% in the FSH antibody-positive group and 59.1% micro 6.2% in the FSH antibody-negative control group. The percentage of functional membrane damage to spermatozoa was significantly higher in the anti-FSH antibody-positive group than in the control group. TEM showed that the outer acrosomal membrane was located far from the nucleus, and detachment of the acrosome was found in the FSH autoantibody-positive group., Conclusion: These data suggest that the presence of anti-FSH antibody is strongly correlated with the sperm quantity and quality in idiopathic male infertility. Anti-FSH antibody may be an important factor causing spermatogenic dysfunction and infertility., ((c) 2008, Asian Journal of Andrology, SIMM and SJTU. All rights reserved.)

The growth and function of the epididymis are regulated by testosterone produced by Leydig cells in the testes. In the present study it was observed that neutralization of endogenous follicle stimulating hormone (FSH) in immature rats using a highly specific antiserum to ovine FSH resulted in changes in the histology of the epididymis along with a decrease (50-60%) in its weight compared with the normal serum-treated controls. These changes were observed in both rat and monkey epididymis without any decrease in serum testosterone, on which epididymis is known to be dependent. A detailed study was therefore carried out on the effects of deprivation of FSH or testosterone on the histology of epididymis. The changes in epididymal histology following FSH deprivation included a decrease in the size of the tubule lumen in the rat as well as in the adult male bonnet monkey in which the antiserum against ovine FSH was raised. Intensive vacuolization and uneven surface of the luminal epithelium was also observed. In contrast, the effect of deprivation of testosterone support by way of administration of LH antiserum or fiutamide resulted in a decrease in the size of the lumen and degenerative changes. These results suggest that cauda epididymidis is a target for FSH action.

Follicle-stimulating hormone plays a key role in spermatogonial development in adult rats via poorly understood mechanisms. We aimed to identify the role of this hormone in the regulation of germ cell apoptosis and proliferation in adult rats by suppression of FSH action following passive immunoneutralization with a rat FSH antibody for 4 and 7 days. Apoptosis and proliferation were identified by TUNEL and proliferating cell nuclear antigen labeling methods, respectively. Intrinsic and extrinsic apoptotic pathways were identified by immunohistochemistry, stereological techniques, and RT-PCR by assessing pathway-specific proteins and genes. Following FSH suppression for 4 and 7 days, we have previously reported a 30% decrease in spermatogonial number, with increased apoptosis in a stage-specific manner. The present study also shows stage-specific increases in apoptosis with no changes in proliferation. This increase in apoptosis was attributable to an increase in spermatogonial apoptosis via the intrinsic rather than extrinsic pathway, as shown by increased activated caspase 9-positive spermatogonia. The concomitant suppression of FSH and LH/testosterone showed that testosterone alone or together with FSH was more important in spermatocyte and spermatid survival by regulating both apoptotic pathways. A reduction in the level of the intrinsic pathway transcript Bcl2l2 (apoptosis suppressor gene) following FSH suppression for 4 days shows that FSH regulates some components of the intrinsic pathway. This study reveals that FSH predominantly acts as a survival factor for spermatogonia by regulating the intrinsic pathway while having no affect on germ cell proliferation in rats in vivo.

FSH is required for spontaneous folliculogenesis and is widely used in ovarian stimulation in IVF. Previously, increased concentrations of antibodies against FSH (anti-FSH) have been demonstrated in infertile women. This study aimed to: (i) assess the possible association of anti-FSH with an adverse outcome of IVF with regard to clinical parameters characterizing the ovarian reserve; and (ii) compare serum and follicular fluid (FF) anti-FSH concentrations in relation to follicle size and endocrine markers. IVF patients (n = 182) subjected to gonadotrophin-releasing hormone-antagonist protocol were assessed for anti-FSH using enzyme-linked immunosorbent assay. Increased concentrations of serum anti-FSH immunoglobulin (Ig)G and IgA were associated with impaired ovarian stimulation outcome, with cut-off values <1.0 arbitrary units predicting poor ovarian response (

In fish, FSH is generally important for early gonadal development and vitellogenesis. As in mammals, FSH is a heterodimer composed of an alpha subunit that is noncovalently associated with the hormone-specific beta subunit. The objective of the present study was to express glycosylated, properly folded, and biologically active tilapia FSH (tFSH) using the Pichia pastoris expression system. Using this material, we aimed to develop a specific ELISA and to enable the study of FSH response to GnRH. The methylotrophic yeast P. pastoris was used to coexpress recombinant genes formed by fusion of mating factor alpha leader and tilapia fshb and cga coding sequences. Western blot analysis of tilapia pituitary FSH, resolved by SDS-PAGE, yielded a band of 15 kDa, while recombinant tFSH beta (rtFSH beta) and rtFSH beta alpha had molecular masses of 17-18 kDa and 26-30 kDa, respectively. Recombinant tFSH beta alpha was found to bear only N-linked carbohydrates. Recombinant tFSH beta alpha significantly enhanced 11-ketotestosterone (11-KT) and estradiol secretion from tilapia testes and ovaries, respectively, in a dose-dependent manner (similar to tilapia pituitary extract, affinity-purified pituitary FSH, and porcine FSH). Using antibodies raised against rtFSH beta, FSH-containing cells were localized adjacent to hypothalamic nerve fibers ramifying in the proximal pars distalis (PPD), while LH cells were localized in a more peripheral region of the PPD. Moreover, FSH is under the control of hypothalamic decapeptide GnRH, an effect that was abolished through the use of specific bioneutralizing antisera, anti-rtFSH beta. It also reduced basal secretion of 11-KT.

Objective: To establish rat models of FSH autoantibody and to investigate the effect of FSH autoantibody on the spermatogenic capability of rat testis., Methods: Thirsty 21-day old SD rats were randomly divided into an experimental and a control group of equal number. A specific polypeptide corresponding to the rat FSHbeta subunit was synthesized and coupled to (keyhole limpet hemocyanin) KLH. The rats in the experimental group were immunized with polypeptide-KLH and these in the control group with KLH. Further immunization was performed every 2 weeks for 7 times. On the 77th, 91st and 105th day of the immunization, 5 rats from the experimental group and another 5 from the control group were killed. Then the structures of the seminiferous tubule and epididymal sperm were observed by light and electron microscope, respectively. Meanwhile, the counts of sperms and the percentage of swelled sperm were calculated. And the level of serum testosterone was detected by enzyme-linked immunospecific assay (ELISA)., Results: The titer of the anti-polypeptide antibody was 1:200 on the 49th day of the immunization, and reached 1:400 on the 63rd. Compared with the control group, the percentage of swelled sperm significantly decreased on the 91st day (60.4 +/- 6.23 vs 50.60 +/- 3.05, P < 0.05), and the number of spermatogenic cells and sperms in seminiferous tubules reduced on the 105th day in the experimental group, the counts of sperms (46.08 +/- 6.56 vs 32.53 +/- 3.41) and the percentage of swelled sperm (60.60 +/- 5.86 vs 48.60 +/- 3.85) significantly lower (P < 0.05), while the level of serum T significantly higher than that in the control group (P < 0.05)., Conclusion: FSH autoantibody might cause testis dyszoospermia.

Problem: We have previously demonstrated the presence of naturally occurring antibodies against follicle-stimulating hormone (FSH) in patients with endometriosis and polycystic ovary syndrome (PCOS). Here, we investigated the parameters associated with anti-FSH antibodies in in vitro fertilization (IVF) patients., Methods of Study: The following parameters were studied in 135 patients: peripheral FSH levels, FSH beta-subunit gene (FSHB) haplotypes, history of previous IVF, and susceptibility to autoimmune reactions in general [seven common autoantibodies (against nuclear antigens on human and rodent substrates, smooth muscle, gastric parietal cells, beta2-glycoprotein I, cardiolipin, and thyroid peroxidase) and HLA-DQB1 alleles]., Results: Although the anti-FSH levels were higher in patients when compared with controls, those higher levels were not associated with FSHB haplotypes. The anti-FSH IgM associated with (i) the levels of FSH in women with male and tubal factor infertility; (ii) the history of IVF in patients with PCOS, endometriosis, and unexplained infertility; and (iii) the production of common autoantibodies among all IVF patients. The anti-FSH IgA associated with HLA-DQB1*03. The anti-FSH IgG correlated with the values of anti-FSH IgA and IgM., Conclusion: Anti-FSH may be naturally occurring antibodies associated with peripheral FSH concentrations, but increased in infertile women with dysregulation of immune reactions and repeatedly performed IVF.

The critical influence of follicle stimulating hormone (FSH) on male fertility relates both to its impact on Sertoli cell proliferation in perinatal life and to its influence on the synthesis of Sertoli cell-derived products essential for germ cell survival and function in the developing adult testis. The nature and timing of this shift of germ cells to their reliance on specific Sertoli cell-derived products are not defined. Based on existing data, it is apparent that the dominant function of FSH shifts between 9 and 18 day postpartum (dpp) during the first wave of spermatogenesis from driving Sertoli cell proliferation to support germ cells. To enable comprehensive analysis of the impact of acute in vivo FSH suppression on Sertoli and germ cell development, FSH was selectively suppressed in Sprague-Dawley rats by passive immunisation for 2 days and/or 4 days prior to testis collection at 3, 9 and 18 dpp. The 3 dpp samples displayed no measurable changes, while 4 days of FSH suppression decreased Sertoli cell proliferation and numbers in 9 dpp, but not 18 dpp, animals. In contrast, germ cell numbers were unaffected at 9 dpp but decreased at 18 dpp following FSH suppression, with a corresponding increase in germ cell apoptosis measured at 18 dpp. Sixty transcripts were measured as changed at 18 dpp in response to 4 days of FSH suppression, as assessed using Affymetrix microarrays. Some of these are known as Sertoli cell-derived FSH-responsive genes (e.g. StAR, cathepsin L, insulin-like growth factor binding protein-3), while others encode proteins involved in cell cycle and survival regulation (e.g. cyclin D1, scavenger receptor class B 1). These data demonstrate that FSH differentially affects Sertoli and germ cells in an age-dependent manner in vivo, promoting Sertoli cell mitosis at day 9, and supporting germ cell viability at day 18. This model has enabled identification of candidate genes that contribute to the FSH-mediated pathway by which Sertoli cells support germ cells.

An improved and cost effective method to isolate FSH from buffalo pituitary glands is described here. The buFSH activity was monitored throughout by a highly sensitive heterologous radioimmunoassay (sensitivity 0.2 ng oFSH/mL) and the in vivo biological activity of the final preparation was also established. A biologically active buFSH-enriched preparation with a moderate recovery (42%) was obtained. The yield of the final buFSH-enriched preparation was 26.5 mg/kg of buffalo pituitary gland. In SDS-PAGE, the purified buFSH resolved as a heterodimer of 30 kDa molecular size, with a 21 kDa presumptive alpha-subunit. This preparation was also characterized in terms of biological and some of its physicochemical properties. A high-titer antiserum to buFSH was also raised in rabbit using this preparation. The reagents generated, buFSH and buFSH-specific polyclonal antisera, have possible diagnostic and therapeutic usage for improvement of reproductive health of water buffaloes.

Sensitive and specific measurement of FSH is critical to research in reproductive biology, and the increasing availability of transgenic mouse models has created a need for a robust, sensitive, and specific mouse (m) FSH assay. The present study evaluated a time-resolved immunofluorometric assay (IFMA) for mFSH using monoclonal antibody to human (h) FSHbeta as a capture antibody and a biotinylated polyclonal antibody to rat alpha subunit as a detection probe, with signaling amplified by europium-labeled streptavidin. The mFSH IFMA lowered the detection limit 34-fold (5 vs. 170 pg/sample) compared with standard mFSH RIA. The mFSH IFMA demonstrated parallelism of response to dilutions of castrated mouse serum and rat FSH but no cross-reactivity with hFSH and mLH or hLH, whereas the RIA demonstrated nonparallel cross-reactivity with hFSH. The IFMA has a wide analytical range, with a good precision profile for within- and between-assay reproducibility. Because the IFMA is a sandwich-type assay with strict dimer-specificity by design, the lower readings and recovery obtained were compared with the RIA when both assays used a pituitary-purified mFSH assay standard that contained isolated or fragmented subunits as well as intact dimeric FSH. When used with mouse serum sample, the mFSH IFMA demonstrated the expected increases following orchidectomy as well as markedly enhanced sensitivity to very low levels of endogenous mFSH in gonadotropin-deficient mice. Furthermore, the IFMA measured mFSH with fidelity in both intact and orchidectomized male mice without any interference from transgenic hFSH. The greatly enhanced sensitivity, specificity, and technical convenience of this mFSH IFMA will allow wider application of FSH measurements to very small blood samples in immature and mature mice as well as transgenic models.

The role of follicle stimulating hormone (FSH) in Leydig cell function was evaluated by a passive neutralization approach at different stages of Leydig cell development. Neutralization of endogenous FSH in neonatal rats (10-day-old) resulted in reduction of testes weight, however the testicular testosterone levels and in vitro testosterone production by purified Leydig cells were elevated. Administration of FSH antiserum to immature (25-28-day-old) and adult (90-day-old) rats did not have any effect on testes weight, serum testosterone and testicular testosterone. Interestingly, there was a significant reduction in testosterone production by isolated Leydig cells under hCG stimulated and 22-R-hydroxycholesterol (22-R-OH CHOL) saturated conditions. In support of this observation administration of recombinant FSH to immature and adult rats resulted in significant increase in testosterone production by Leydig cells following incubations in presence of hCG and saturating concentrations of 22-R-OH CHOL, although there was no change in serum and testicular testosterone levels. The role of FSH in immature rats was also confirmed employing FSH receptor antiserum which was raised against the unique domains of FSH receptor. RT-PCR analysis revealed a significant reduction in the mRNA levels of StAR and IGF-1 following blockade of FSH action by FSH receptor antiserum. The results of our studies suggest a stage specific function for FSH in regulation of Leydig cell development by modulating the LH responsiveness and steroidogenesis.

Objective: To detect the anti-FSH antibody using ELISA, and further probe into the role of anti-FSH in infertile patients., Methods: The anti-FSH antibody was detected using ELISA in the serum of patients with spermatogenesis dysfunction, of infertile patients with normal sperm density and motility, and of normal fertile males., Results: The positive rate of anti-FSH antibody in the patients with oligospermia and/or asthenospermia [22.4% (22/98)] was significantly higher than that in the normal fertile [4% (2/50)] (P < 0.05) and that in the infertile patients with normal sperm density and motility [6.7% (2/30)] (P < 0.05). The positive rate of anti-FSH antibody in the patients with oligospermia and/or asthenospermia was lower than that in the patients with azoospermia [54.5% (12/22)] (P < 0.05). There was no significant difference in the positive rate between the normal control and the sterile males with normal sperm density and motility., Conclusion: The anti-FSH antibody may be an important factor to cause spermatogenesis dysfunction by combining FSH to form immune compound and depress the activation of FSH.

Antibodies of appropriate specificity are able to block the action of hormones which are obligatory for successful reproduction. Thus, if immunisation using such hormones can provoke adequate titres of bioneutralizing antibodies in sexually mature individuals, the vaccinee becomes infertile ('immunocontraception') for as long as sufficient titres of the antibodies are maintained. In the case of hormones that are required for the development of sexual maturity in the male, immunisation of young animals can prevent sexual maturation ('immunocastration'). The hormones which have been targeted are gonadotropin-releasing hormone (GnRH) for both immunocastration and immunocontraception, and follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG) for immunocontraception.

Accumulated data suggest that endothelial cells express specific receptors for several peptide and (glyco)protein hormones that may transport hormones across the cell to be delivered to the interstitial fluid and tissue target cells. Surprisingly, very little information is available on the actual endothelial organelles involved in this cellular process. In the present study the transfer of follicle-stimulating hormone (FSH) through the endothelial barrier of rat testes was examined by analysing the binding and transport of gold-tagged recombinant human (rh)FSH under various conditions using electron microscopy. At 4 degrees C the probe bound specifically to the luminal surface of the endothelial cells without internalization. The use of 125I-rhFSH, which allows precise quantitation of the binding, confirmed the specificity of hormone interaction with the testicular microvasculature. At 37 degrees C the hormone was internalized via coated pits and vesicles into an extensive subluminal tubulo-vesicular compartment and was transported across the endothelium via a system of tubules and vesicles. Moreover, monoclonal antibodies against the FSH receptor ectodomain coupled to colloidal gold followed the same route. In contrast, a non specific, fluid-phase uptake via caveolae was observed for a major plasma protein - rat serum albumin and a fluid-phase tracer - peroxidase. These results suggest that FSH transcytosis across the testicular endothelial barrier is receptor-mediated and involves luminal uptake via coated pits/vesicles, sorting at the level of luminal early endosomes, and transcellular transport through transcytotic tubulo-vesicular organelles. Similar receptor-mediated pathways are likely to be involved in the physiological functioning of a number of other protein and peptide hormones that must translocate specifically from blood to the target cells.

Spermatogenesis is a complex process, and previous studies have clearly established the role for testosterone in its maintenance. However, the role of FSH remains controversial, although several lines of evidence suggest its importance in initiation of spermatogenesis. In the present study, the relative roles of FSH and LH have been evaluated using specific antisera capable of neutralizing endogenous hormones in adult male rats following ethanedimethylsulphonate (EDS) treatment. Restoration of spermatogenesis in EDS-treated rats was monitored following FSH or LH deprivation by histological analysis and flow cytometry. Deprivation of FSH resulted in a reduction of seminiferous tubule diameter and spermatogonial number, which was much more drastic than that observed following LH deprivation. More importantly, FSH deprivation was associated with a significant reduction in the number of pachytene spermatocytes. These results provide evidence for a definite role for FSH in regulation of spermatogenesis, in addition to confirming the role of LH in spermatogenesis via testosterone.

This experiment was conducted to investigate the use of monoclonal mouse anti-human FSH as a primary antibody for the immunohistochemical staining on chicken adenohypophysis paraffin sections. The use of monoclonal mouse anti-human primary antibody on chicken tissues has been developed. The protocol has been modified by comparing the results of the different groups. Briefly, this present protocol suggests modification in two areas; first, the antigen retrieval step, by placing the slides into the buffered citrate in microwave oven at 700 watt for 5 min, 3 times, and antigen-antibody reactions. Secondly, the incubation with the primary antibody (dilution 1/100 for anti-FSH) at 37 degrees C for 90 min.

Testosterone is metabolised to the more potent androgen, dihydrotestosterone, by the 5alpha-reductase (5alphaR) enzyme. We previously showed that 5alpha-reduced androgens are important for maintaining androgen action on rat spermatogenesis when testicular testosterone concentrations are reduced. This study investigated expression and activity of the 5alphaR isoforms, type 1 (5alphaR-1) and type 2 (5alphaR-2), in the rat during hormone manipulation in order to understand the factors that regulate the testicular concentration of 5alphaR and testicular 5alpha-reduced androgen biosynthesis. Testicular 5alphaR-1 and 5alphaR-2 mRNA and enzyme activity were measured by real-time PCR and specific enzyme assays respectively. Hormone levels were first suppressed using two models of gonadotrophin suppression: testosterone and oestradiol treatment (LH/testosterone deficiency) or GnRH immunisation (LH/testosterone and FSH deficiency). Hormones were then either restored or suppressed for 6 days by a variety of hormonal treatments. 5alphaR-1 mRNA and enzyme activity increased when testosterone was suppressed, yet restoration of testosterone decreased 5alphaR-1 mRNA and enzyme activity, suggesting that testosterone negatively regulates 5alphaR-1. suppression of FSH decreased 5alphaR-1 mRNA yet FSH administration increased 5alphaR-1 mRNA, but no changes in 5alphaR-1 activity were observed within the 6 day period. In contrast to 5alphaR-1, testosterone did not affect the testicular concentration of 5alphaR-2 mRNA or activity, but there was evidence for modulation of 5alphaR-2 activity by FSH. Measurement of testicular androgens revealed that 5alphaR-1 was primarily responsible for the production of 5alpha-reduced metabolites. It is concluded that the 5alphaR isoforms in rat testis are differentially regulated by testosterone and FSH: testosterone negatively regulated 5alphaR-1 mRNA and enzyme activity but had no affect on 5alphaR-2, whereas FSH positively regulated 5alphaR-1 mRNA and appeared to regulate 5alphaR-2.

Objective: The purpose of the present study was to evaluate the hormonal profile of patients of postmenopausal age during estrogen replacement therapy (ERT) with special reference to the serum levels of biologically active FSH (B-FSH) in a self-adjusted ERT model., Design: The hormonal values found have been correlated to climateric symptoms reported by the patients (scored by the Kupperman menopausal index (KI))., Methods: B-FSH was measured using an assay based on a cell system transfected permanently with FSH receptor cDNA. All women (n=32) applied estradiol percutaneously using 1 mg estradiol-17beta (E(2)) as an initial dose and were encouraged to increase the daily dose until they felt comfortable according to a specific scheme. Twelve of the 32 women were hysterectomized and treated, accordingly, with ERT only; 20 women received megestrol acetate monthly for 10 days., Results: The initial average KI was 30 (range 10-54). A high degree of correlation (r=0.83; P<0.001) was observed between B-FSH and immunologically active FSH (I-FSH). Serum I-FSH and E(2) correlated negatively (r=-0.21; P<0.001); similarly, a negative correlation (r=-0.15; P<0.01) was observed between serum B-FSH and E(2) levels. Serum I-FSH and KI showed modest but significant positive correlation (r=0.13; P<0.01); a somewhat higher degree of correlation (r=0.19; P<0.005) was observed when B-FSH and KI were compared. E(2) showed positive correlation to serum sex-hormone binding globulin levels (r=0.22; P<0.001)., Conclusions: This study shows that the transdermal self-adjusted hormone replacement therapy (HRT) model introduced is suitable for studies on endocrine changes during postmenopausal ERT. The finding of poor correlation between serum E(2) levels and KI emphasizes the importance of hormonal measurements during postmenopausal HRT.

Neuropeptide Y (NPY) is a putative neuroregulator of the reproductive axis in the central nervous system. In this study we evaluated the effects of central infusion of exogenous NPY on the secretory activity of pituitary gonadotrophic cells in prepubertal lambs. Immature female Merino sheep (n=12) were infused of Ringer solution (control) or 50 microg of NPY to the third ventricle for 5 min and then slaughtered 3 h later. Immunoreactive luteinizing hormone (LH) and follicle stimulating hormone (FSH) cells were localised by immunohistochemistry using antibody raised against LHbeta and FSHbeta. Messenger RNA analyses were performed by in situ hybridisation using sense and antisense riboprobes produced from beta subunits of LH and FSH cDNA clones. The results were generated by computer image analysis to determine the area fraction occupied by immunoreactive and/or hybridising cells and optical density for immunostaining and hybridisation signal. LH in the blood plasma was determined by radioimmunoassay. It was found, that in the lambs infused with NPY the area fraction and optical density for immunoreactive LH cells and mRNA LHbeta-expressing cells increased significantly (P<0.001), compared to the vehicle-infused animals. The concentration of LH in the blood plasma did not differ between control and treated groups. The NPY infusions had no effect on the immunoreactivity of FSH cells or on expression of mRNA for FSHbeta. In conclusion we suggest that NPY may be an important component of mechanisms stimulating the synthesis and storage but not the release of LH in the pituitary gonadotrophs from prepubertal female sheep. In addition, this effect is specific for LH, no such effect was apparent on FSH.

The purpose of this study was to identify autoantigens contained in human ovary extracts. Serum samples from 36 infertile women with anti-ovary antibodies as detected with an ELISA technique were tested in Western blot against human ovary extracts. A reactive protein with a molecular mass matching that of the FSH was detected in 34 cases. These serum samples also reacted strongly in Western blot and ELISA with purified FSH and, in immunofluorescence, with pituitary cells. Using the Pepscan approach, with overlapping peptides matching the amino acid sequence of the human FSH beta-chain, several immunoreactive regions were evidenced. The 78-93 amino acid sequence of the human FSH beta-chain appeared as one of the major epitopes. Synthetic peptides of this region were prepared and demonstrated to react with human serum samples from women with anti-ovary antibodies. These data demonstrate that FSH can be an autoantigen, recognized by autoantibodies associated with infertility., ((c)2001 Elsevier Science.)

Our hypothesis was that patients developing ovarian hyperstimulation syndrome (OHSS) might have a disturbed responsiveness or delayed activation of the immunosuppresive cytokine system. In a prospective cohort study, women (n=428) undergoing in vitro fertilisation (IVF) treatment were subjected to repeated blood sampling and collection of clinical data. Fifteen patients, who developed severe OHSS, were compared with matched (age, follicle numbers, pregnancy) control patients. Samples of serum and plasma were collected throughout the stimulation and up to 7 days after embryo transfer as well as during hospitalisation for OHSS. Levels of IL-4, IL-10, IL-13, oestradiol and progesterone were measured. Significantly lower levels of IL-10 were seen at the start of gonadotrophin stimulation in OHSS patients, with an increase seen after the development of OHSS. In these OHSS patients, a negative correlation between IL-10 levels and number of follicles at time of aspiration existed, but there were no correlations between steroid and IL-10 levels. Levels of IL-13 and IL-4 were low in both groups and did not change during stimulation. The lower levels of IL-10 at start of stimulation in OHSS patients, as compared with controls, may be of pathophysiological importance by allowing for an enhanced Th-1 type immune response during gonadotrophin stimulation and thereby increased and generalised inflammation. The increase in IL-10 after development of OHSS indicates that IL-10 at that time is induced in a systemic attempt to suppress the inflammation of OHSS.

Whereas FSH action is critical for the growth of preantral follicles, its role in the development of primordial follicles is controversial. The objective of the present study was to evaluate whether perinatal (fetal through early postnatal) FSH action is needed for the formation of primordial follicles, which first appear in the hamster ovary on the 7th to 8th day of postnatal life. A single dose of FSH-specific polyclonal antibody was injected into pregnant hamsters on the 12th, 13th, or 14th day of gestation and into newborn hamsters. Some of the antibody-exposed postnatal hamsters were injected with a single dose of equine CG (eCG) to check the reversibility of the antibody action. Ovaries were collected on D8pn or D12pn, and the percentage of primordial, primary, and secondary follicles was quantitated morphometrically. Ovaries of 8-day-old hamsters that were born to mothers treated with a single s.c. dose of the anti-FSH-antibody on day 12 of gestation had significantly reduced numbers of primordial follicles, compared with those treated with preimmune serum or saline (2.4% vs. 25%); however, the antibody inhibition was nearly completely reversed (approximately 18%) by a single injection of eCG on the first day of life. Delaying antibody treatment during late gestation caused a time-dependent block in granulosa cell differentiation, with a consequent proportional increase in the percentage of primordial follicles. This indicates that FSH-induction of primordial follicle development begins at a critical time of ovarian development. On the other hand, shortening the postnatal duration of eCG exposure reduced the degree of reversal, suggesting that prolonged perinatal FSH action is essential for developing the full gamut of primordial follicles. These results provide the first direct evidence that FSH action during fetal ovarian development is critical for the onset of primordial follicle formation.

The immunopotency and in-vitro biopotency of clinical batches of Gonal-F((R)) and Puregon((R)) (recombinant human follicle stimulating hormones) were compared and their carbohydrate chains investigated for charge heterogeneity and internal carbohydrate complexity. Immunopotency (IU/pmol) for both Gonal-F and Puregon was 0.35 +/- 0.01 and biopotency (ED(50), pmol/l) was similar, being 7.3 +/- 0.6 and 5.4 +/- 0.2 respectively. Charge distributions were essentially the same with no difference either in median isoelectric point (pI) (between 4.26 and 4.50), or in the bulk of material fractionated between pI 4 and 5 (66.0 +/- 1.8% Gonal-F and 72.0 +/- 1.8% Puregon). However, there were minor differences in charge at extremes of pI, Gonal-F being slightly more acidic: 18.2% Gonal-F versus 9.8% Puregon at pI 3.5-4.0 (P: = 0.03) and 6.7% Gonal-F versus 10.7% Puregon at pI 5.0-5.5 (P: = 0.03). Carbohydrate complexity was the same: 9.3 versus 10.9 (complex), 76.6 versus 78.6 (intermediate) and 14.1 versus 10.5% (simple). In summary, Gonal-F and Puregon have similar immunopotency, in-vitro biopotency and internal carbohydrate complexity, differing slightly in charge heterogeneity, Gonal-F having more acidic glycoforms. We conclude them to be intrinsically very similar, expecting no difference in clinical efficacy on the basis of respective structure.

The quality of sperm ejaculated by bonnet monkeys and normal, healthy proven fertile volunteer men, both actively immunized with ovine follicle-stimulating hormone (oFSH), was examined at different times of study for chromatin packaging and acrosomal glycoprotein concentration by flow cytometry. Susceptibility of sperm nuclear DNA to dithiothreitol (DTT)-induced decondensation, as measured by ethidium bromide binding, was markedly high compared with values at day 0 in men and monkeys during periods when FSH antibody titer was high. Sperm chromatin structure assay yields alphat values, which is another index of chromatin packaging. Higher alphat values, signifying poor packaging, occurred in both species following immunization with heterologous pituitary FSH. The binding of fluorosceinated pisum sativum agglutinin (PSA-FITC) to acrosome of sperm of monkeys and men was significantly low, compared with values at day 0 (control) during periods when cross-reactive FSH antibody titer was high and endogenous FSH was not detectable. Blockade of FSH function in monkeys by active immunization with a recombinant oFSH receptor protein corresponding to a naturally occurring messenger RNA (mRNA) also resulted in production of sperm with similar defects in chromatin packaging and reduced acrosomal glycoprotein concentration. Thus, it appears that in monkeys and men, lack of FSH signaling results in production of sperm that exhibit defective chromatin packaging and reduction in acrosomal glycoprotein content. These characteristics are similar to that exhibited by sperm of some class of infertile men. Interestingly, these alterations in sperm quality occur well ahead of decreased sperm counts in the ejaculate.

Follicle-stimulating hormone (FSH) plays an important part in testicular development. Its role in the regulation of spermatogenesis in the adult, however, remains controversial. This study aimed to explore the role of FSH in the maintenance of adult rat spermatogenesis by using immunoneutralization to selectively withdraw FSH action for periods of up to 8.5 days and then assessing the outcome by quantification of germ cell number. Adult rats received either an ovine polyclonal rat FSH antibody (FSHAb, 2 mg/kg subcutaneous daily-a dosage known to neutralize >90% of FSH in serum) for 2, 4, 7, or 8.5 days or a control sheep immunoglobulin (ConAb, 2 mg/kg) for 8.5 days. Testes were perfusion fixed, and germ cell numbers per testis were quantified using the optical disector (sic) stereological method. The percentage of seminiferous tubules displaying apoptotic cells was determined by the in situ end labeling method (TUNEL). FSHAb treatment for 4, 7, or 8.5 days significantly reduced the number of type A/intermediate spermatogonia (approximately 74% of control values) associated with stages I-IV. Similar reductions were seen in type B spermatogonial and preleptotene spermatocyte numbers after 8.5 days of FSHAb treatment (approximately 69% of control values; P < 0.05). Decreases (P < 0.05) in the numbers of pachytene spermatocytes in stages I-III and VIII, round spermatids in stages I-III, VII, and VIII (approximately 70% of control values), and step 19 elongated spermatids in stage VII (51% of control values) were achieved after 8.5 days of FSHAb treatment. Compared with control, FSHAb treatment increased the percentage of stage XIV-III tubules containing TUNEL-positive cells by about twofold after 7 days of FSHAb treatment (P < 0.05). This study supports a role for FSH in the maintenance of quantitatively normal adult rat spermatogenesis, specifically by regulating A3 and A4 spermatogonial subtypes. FSH may act on these spermatogonia by enhancing the stage-dependent survival of type A spermatogonia. Effects at other sites in spermatogenesis are suggested by the changes in spermatocyte and spermatid populations. However, to clarify these effects, selective FSH withdrawal would need to be prolonged until steady state had been achieved.

The role of follicle-stimulating hormone (FSH) in adult rat spermatogenesis is unclear. Although exogenous testosterone (T) restores spermatogenesis following gonadotropin-releasing hormone (GnRH) immunization or T plus estradiol (TE) treatments, an assessment of the independent action of T and FSH was not possible, as exogenous T treatment maintains serum FSH levels. We have used passive immunization against FSH to determine whether T alone is capable of reinitiating spermatogenesis after chronic and acute FSH withdrawal. Adult rats received T-filled Silastic implants 6 cm (T6) or 8 cm (T24) in length for 7 days in combination with either a polyclonal sheep antisera raised against rat FSH (FSHAb, 2 mg/kg SC daily) or control sheep immunoglobulin (ConAb) after either GnRH immunization (12 weeks) or TE treatment (9 weeks). The neutralizing capacity of the FSHAb was determined using a FSH in vitro bioassay; this analysis demonstrated that administration of FSHAb in vivo reduced FSH levels by >90%. Testes were fixed and germ cell number per testis quantified using the optical dissector. GnRH immunization reduced spermatogonia, pachytene spermatocytes, and round spermatids to 50, 13, and <1% of normal, respectively. T6 and T24 Silastic implants with the inclusion of the FSHAb did not increase the number of spermatogonia, pachytene spermatocytes, and round spermatids (50, 15, and 1% of normal, respectively). T6+ConAb treatment increased spermatogonial, pachytene spermatocyte, and round spermatid numbers to 74, 30, and 3% of normal, respectively (P < 0.05). No further increases were seen with T24 implants. TE treatment suppressed pachytene spermatocytes and round spermatids to 33 and 1% of normal, respectively (P < 0.05). T6+FSHAb treatment did not increase the number of pachytene spermatocytes and round spermatids (36 and 8%, respectively), whereas T6+ConAb treatment increased pachytene spermatocyte and round spermatid number to 50 and 28% of normal, respectively (P < 0.05). T24+FSHAb treatment increased the number of pachytene spermatocyte and round spermatids (56 and 22% of normal, respectively; P < 0.05), whereas T24+ConAb treatment increased these cells forms to 79 and 31% of normal, respectively. In conclusion, T alone is unable to restore spermatogenic cell populations in the setting of chronic FSH withdrawal. Although acute FSH withdrawal markedly impairs the restoration process, higher doses of T can partially compensate for the lack of FSH. These data suggest that FSH is important for the initial phase of spermatogenic restoration.

The physiological role of follicle stimulating hormone (FSH) in the regulation of the release of the putative ovarian factor gonadotrophin surge-inhibiting or -attenuating factor (GnSIF/AF) was investigated. Blood FSH concentrations were immunoneutralized in female rats during different days of the ovarian cycle. FSH antiserum was injected in different protocols to neutralize the FSH surge(s) and/or basal FSH concentrations. All animals were killed on the morning of the day of pro-oestrus of the control rats. Hemi-pituitaries were incubated and gonadotrophin-releasing hormone (GnRH) stimulated and basal luteinizing hormone (LH) and FSH releases and pituitary contents were measured. The biological effectiveness of the FSH antiserum was established by monitoring the histology of vaginal smears and inhibin concentrations in trunk-blood. The predominance of small leukocytes in the smears (low oestradiol bioactivity) and the decreased (bioactive) inhibin secretion imply that anti-FSH neutralizes FSH bioactivity. Simultaneously, the results showed that all anti-FSH injections did not affect the biphasic LH response to GnRH in vitro, which shows an unaltered presence of GnSIF/AF bioactivity in the blood circulation. The results suggest that under physiological conditions GnSIF/AF bioactivity, which keeps the LH responsiveness of the pituitary gland suppressed to the action of GnRH, is not supported by inhibin and not controlled by FSH.

Problem: Hormone immunoneutralization is hampered by immunologic cross-reactivity caused by close-sequence homology between related molecules. One solution is to use smaller fragments to induce antibodies of greater specificity., Method of Study: A number of peptides selected from beta-follicle-stimulating hormone (FSH) were conjugated to tetanus toxoid and were used to immunize female rats. The antisera were examined for FSH cross-reactivity by immunoassays and in an in vitro bioassay., Results: In the immunoassays, the antisera did not react with FSH but did react with their respective peptides. In the bioassay, sera from VYKDPARPC- and CDSLYTYP-immunized animals inhibited FSH-receptor interaction by 73% and 68%, respectively. These animals also showed reduced estradiol levels. Sequences were synthesized around VYKDPARPC and were tested on a FSH-receptor-bearing Chinese hamster ovary cell line. LVYKDPARPC, VYKDPARPC, YKDPARPIC, CLVYKDPARP, and LVYKDPARP inhibited FSH-receptor interaction by greater than 50%. In female mice, TRDLVYKDPARPKI and LVYKDPARP disrupted estrous cycling in all animals; LVYKDPARPC and CLVYKDPARP disrupted cycling in three of five animals, whereas VYKDPARPC disrupted cycling in one of four animals., Conclusions: Peptides from two areas of beta-FSH (VYKDPARP and DSLYTYP) were shown to raise FSH-neutralizing antibodies, which were able to suppress estradiol levels. An additional leucine residue to VYKDPARP greatly enhanced the peptide's ability to inhibit FSH-receptor binding and caused fertility disruption in vivo.

The effect of FSH on the development of the testis in the ram lamb was examined in two experiments where lambs were passively immunized against ovine beta-FSH from birth until 100 or 160 d. In both experiments, immunization resulted in a slower testicular growth relative to that of controls. This effect became apparent at around the start of the period of rapid testicular growth (60-70 d of age) and resulted in testicular weights at the end of treatment ranging from 37 to 51% of those of control groups. Within the testis, this was reflected in shorter seminiferous tubules (48-64% of controls) and in lower numbers of Sertoli cells per testis (57-82%). In the rams immunized until 160 d of age, spermatogenesis had begun and immunization against FSH provoked a lower production of germinal cells which was not solely due to the lower number of Sertoli cells but also due to fewer germinal cells being supported by each Sertoli cell. However, the numbers of A0 spermatogonia per testis and the daily production of the A1 spermatogonia were unaffected by immunization, but the production of the B2 spermatogonia and, as a consequence, of leptotene and pachytene spermatocytes and of round spermatids were all markedly lower (43-47% of controls). These effects were not due to any decreases in the secretion of LH or testosterone as seen in the blood levels of these two hormones. These results show that, in the ram lamb, FSH is essential for normal testicular development and for the establishment of a normal population of Sertoli cells. They also confirm that, once spermatogenesis is established, FSH is necessary for a normal production of germinal cells, with one or more of the divisions between the A1 and B2 spermatogonia being sensitive to suppression of FSH.

Objective: To develop and validate an enzyme-linked immunosorbent assay (ELISA) for total urinary follicle-stimulating hormone (FSH) that can be used to evaluate frozen-thawed urine samples., Design: A method was developed for dissociating urinary FSH into subunits by boiling. An ELISA for the free beta subunit of FSH was used to measure total FSH in urine samples collected from male and female volunteers. The urinary FSH values were compared with serum FSH concentrations in paired samples., Setting: A clinical research laboratory., Patient(s): Serum and urine samples from normal male and female volunteers and from men with infertility were used to develop and validate an assay for total urinary FSH., Intervention(s): None., Result(s): Urinary FSH concentrations in frozen-thawed urine were highly correlated to matched serum FSH concentrations., Conclusion(s): A microtiter plate ELISA for FSH beta subunit provides an economical method for evaluating FSH secretion in men and women. This method has practical applications for population-based epidemiologic studies in which urine samples are collected in the field and stored frozen before analysis.