Acute Follicle Stimulating Hormone Research Articles

The hypothalamic-pituitary-gonadal axis and thyroid hormone regulation interact to influence seasonal breeding in green anole lizards (Anolis carolinensis)

Reproductive physiology and behavior is mainly regulated by the hypothalamus-pituitary-gonad (HPG) axis, although abnormal thyroid hormone (TH) levels alter HPG axis activity. Seasonally breeding animals, such as green anole lizards (Anolis carolinensis), undergo drastic hormonal and behavioral changes between breeding and non-breeding seasons, with increased sex steroid hormones, larger gonads and increased reproductive behaviors during the breeding compared to non-breeding seasons. Relatively less is known regarding the regulation of gonadal TH in seasonal reproduction. We examined whether the gonadal expression of enzymes involved in TH activation are altered in concert with seasonal reproduction. Type 2 deiodinase (Dio2) mRNA, the TH activating enzyme, was upregulated in breeding compared to non-breeding testes, while type 3 deiodinase (Dio3) mRNA, the TH deactivating enzyme, was upregulated in breeding ovaries. To study the association between the HPG axis and local activation of TH, we manipulated the HPG axis during the non-breeding season by subcutaneously injecting luteinizing hormone (LH) and follicle stimulating hormone (FSH) in male lizards. We found that acute LH and FSH injections induced many aspects of breeding, with increased testes size and testosterone levels. Surprisingly, Dio3 was upregulated in the testes after LH and FSH injections, while Dio2 mRNA levels were unchanged. These results suggest that there might be different roles for local TH activation in developing and maintaining fully mature and functional gonads. Our findings continue to support the role for TH in regulating reproduction.

Suppression of Sertoli cell tumour development during the first wave of spermatogenesis in inhibin α-deficient mice.

A dynamic partnership between follicle-stimulating hormone (FSH) and activin is required for normal Sertoli cell development and fertility. Disruptions to this partnership trigger Sertoli cells to deviate from their normal developmental pathway, as observed in inhibin α-knockout (Inha-KO) mice, which feature Sertoli cell tumours in adulthood. Here, we identified the developmental windows by which adult Sertoli cell tumourigenesis is most FSH sensitive. FSH was suppressed for 7 days in Inha-KO mice and wild-type littermates during the 1st, 2nd or 4th week after birth and culled in the 5th week to assess the effect on adult Sertoli cell development. Tumour growth was profoundly reduced in adult Inha-KO mice in response to FSH suppression during Weeks 1 and 2, but not Week 4. Proliferative Sertoli cells were markedly reduced in adult Inha-KO mice following FSH suppression during Weeks 1, 2 or 4, resulting in levels similar to those in wild-type mice, with greatest effect observed at the 2 week time point. Apoptotic Sertoli cells increased in adult Inha-KO mice after FSH suppression during Week 4. In conclusion, acute FSH suppression during the 1st or 2nd week after birth in Inha-KO mice profoundly suppresses Sertoli cell tumour progression, probably by inhibiting proliferation in the adult, with early postnatal Sertoli cells being most sensitive to FSH action.

GRK-6 mediates FSH action synergistically enhanced by estrogen and the oocyte in rat granulosa cells

Estrogen is known to play a pivotal role in granulosa cell responses to follicle-stimulating hormone (FSH) that is critical for the establishment of dominant follicles and subsequent ovulation in mammals. Thus, elucidating the cellular and molecular mechanisms that regulate FSH activity is important to understand female fertility. We previously discovered that the oocyte is required for estrogen to exert its positive effects on FSH activity in rat granulosa cells. This finding supports the new concept that estrogen action in granulosa cells is mediated by the oocyte. In the current study, we explored the underlying mechanism. In the presence of oocytes, estrogens enhanced FSH-induced increases in aromatase, steroidogenic acute regulatory protein and FSH receptor mRNA expression as well as cAMP production. However, as forskolin did not mimic FSH activity this indicated that coexistence of estrogen/oocytes increases FSH activity at a site upstream of adenylate cyclase in granulosa cells. We therefore sought a possible involvement of the autoregulatory molecules for FSH receptor, G protein-coupled receptor kinases (GRKs) and ß-arrestins in enhancing FSH activity in response to the estrogen/oocyte co-treatment in granulosa cells. Among the seven known GRK and two ß-arrestin molecules, we found that estrogens with oocytes suppressed FSH-induced GRK-6 mRNA expression. Consistent with this finding, transfecting granulosa cells with small interfering RNA of GRK-6 significantly increased FSH induction of aromatase mRNA, suggesting that endogenous GRK-6 plays an inhibitory role in FSH-induced aromatase mRNA expression. Consequently, these findings strongly suggest that GRK-6 is involved in the mechanism by which estrogen and oocytes synergistically augment FSH activity in granulosa cells.

Altered sex hormone concentrations and gonadal mRNA expression levels of activin signaling factors in hatchling alligators from a contaminated Florida lake

Activins and estrogens participate in regulating the breakdown of ovarian germ cell nests and follicle assembly in mammals. In 1994, our group reported elevated frequencies of abnormal, multioocytic ovarian follicles in 6 month old, environmental contaminant-exposed female alligators after gonadotropin challenge. Here, we investigated if maternal contribution of endocrine disrupting contaminants to the egg subsequently alters estrogen/inhibin/activin signaling in hatchling female offspring, putatively predisposing an increased frequency of multioocytic follicle formation. We quantified basal and exogenous gonadotropin-stimulated concentrations of circulating plasma steroid hormones and ovarian activin signaling factor mRNA abundance in hatchling alligators from the same contaminated (Lake Apopka) and reference (Lake Woodruff) Florida lakes, as examined in 1994. Basal circulating plasma estradiol and testosterone concentrations were greater in alligators from the contaminated environment, whereas activin/inhibin betaA subunit and follistatin mRNA abundances were lower than values measured in ovaries from reference lake animals. Challenged, contaminant-exposed animals showed a more robust increase in plasma estradiol concentration following an acute follicle stimulating hormone (FSH) challenge compared with reference site alligators. Aromatase and follistatin mRNA levels increased in response to an extended FSH challenge in the reference site animals, but not in the contaminant-exposed animals. In hatchling alligators, ovarian follicles have not yet formed; therefore, these endocrine differences are likely to affect subsequent ovarian development, including ovarian follicle assembly.

Gene expression profiling of mouse Sertoli cell lines

The proliferation and differentiation of Sertoli cells is regulated by follicle-stimulating hormone (FSH). The molecular events following FSH stimulation are only partially known. To investigate FSH action in Sertoli cells, we established two novel FSH-responsive mouse Sertoli-cell-derived lines expressing human wild-type (WT) FSH receptor (FSHR) or overexpressing mutated (Asp567Gly) constitutively active FSHR (MUT). Gene expression profiling with commercially available cDNA arrays, including 588 mouse genes, revealed 146 genes expressed in both cell lines. Compared with the expression pattern of WT cells, 20 genes were identified as being either up- or down-regulated (>two-fold) in the MUT cells. We observed a strong differential expression of factors involved in cellular proliferation, e.g. cyclin D2 (repressed to nearly undetectable levels), proliferating cell nuclear antigen (2.5-fold repression) and Eps-8 (six-fold repression), and in genes involved in cellular differentiation, e.g. cytokeratin-18 (13-fold induction). The cDNA array results for six representative genes were confirmed by Northern blotting, which also included the parental SK-11 cell line devoid of FSHR expression. We found no further acute FSH- or forskolin-induced change in expression levels after 3-h stimulations, suggesting that the observed differences between the two cell lines is a consequence of mild, chronically increased, cAMP production in MUT cells. These results provide a platform for the further investigation of selected candidate genes in primary cultures and/or in vivo.

The Role of Phosphoinositide Metabolism and Intracellular Calcium in Oocyte Meiotic Resumption Induced by Follicle Stimulating Hormone (FSH) and Follicular Fluid Meiosis-Activating Sterol (FF-MAS)

Objective: To make in vitro maturation a successful treatment, it is essential to achieve a more profound insight into the function of the oocyte. It has been shown that active induction of meiotic resumption by FSH in mouse cumulus-enclosed oocytes (CEO) depends on phosphoinositide metabolism and intracellular calcium. However, it is not known whether these pathways play a role in cumulus cells or the oocyte. This study was conducted to assess whether in denuded oocytes phosphoinositide metabolism and intracellular calcium are also involved in the reinitiation of meiosis promoted by FF-MAS, a naturally occurring sterol synthesized by follicle cells. Design: CEO and naked oocytes (NkO), cultured under conditions which prevent spontaneous meiotic resumption, were exposed to FSH or FF-MAS, respectively, in the presence or absence of phosphoinositide metabolism inhibitors (neomycin and LiCl) or an intracellular calcium chelator (BAPTA/AM). Materials and Methods: Oocytes were collected from large antral follicles of three-week-old female mice stimulated with 5 IU of pregnant mare’s serum gonadotropin, and were cultured in α-MEM medium supplemented with 4 mM hypoxanthine, to prevent spontaneous germinal vesicle breakdown (GVBD). CEO were stimulated with FSH (0.1 IU.ml−1) whereas NkO were stimulated with FF-MAS (10 μM). A proportion of oocytes was also exposed to the continued presence of either neomycin (1 mM) or LiCl (10 mM), or loaded with BAPTA/AM (15 μM). After 20 h, the rate of GVBD was assessed by visual examination and compared between the different groups. Results: Oocytes cultured in the presence of hypoxanthine alone were unable to resume meiosis. As expected, FSH and FF-MAS were able to induce meiotic resumption in CEO and NkO, respectively (81.1% and 84.2% GVBD). Neomycin and LiCl significantly inhibited FSH-mediated GVBD in CEO (12.9% and 15.2% GVBD, respectively). Similarly, loading oocytes with BAPTA/AM repressed meiotic resumption promoted by FSH (29.7% GVBD). However, in NkO stimulated with FF-MAS, the two phosphoinositide inhibitors as well as the calcium chelator failed to prevent meiotic resumption, GVBD occurring at a high rate, comparable to the control group (82.8%, 91.2% and 81.9%, respectively). Conclusions: In CEO arrested at the GV stage, FSH-induced meiotic resumption requires activation of phosphoinositide metabolism and release of intracellular calcium. However FF-MAS, which is thought to be synthesized by cumulus cells following acute FSH stimulation, can promote GVBD in NkO irrespective of the presence of neomycin, LiCl and BAPTA/AM. This suggests that, during meiotic resumption, these signaling pathways are involved in the response of cumulus cells to FSH. On the contrary, they do not appear to play a role in the mechanism which regulates the reinitiation of the meiotic cycle within the oocyte.

Follicle-stimulating hormone is required for the initial phase of spermatogenic restoration in adult rats following gonadotropin suppression.

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.

Role of follicle stimulating hormone in the induction of hyperplasia during compensatory ovarian hypertrophy

In adult rats, removal of one ovary leads to an acute albeit transient rise in serum follicle stimulating hormone and an increase in the weight of the remaining ovary. In an attempt to correlate the high titre of endogenous follicle stimulating hormone with the changes taking place at the macromolecular level, the phenomenon of compensatory ovarian hypertrophy was studied for one cycle after hemiovariectomy at metoestrus in the adult, cycling female rats derived from the Holtzman strain. The significant finding with respect to hormonal changes was an acute follicle stimulating hormone surge commencing 6h post-unilateral ovariectomy, reaching a maximum at 12 h and declining thereafter, hitherto not reported in the Holtzman strain. Serum luteinizing hormone, prolactin, oestradiol-17β and testosterone remained unaltered while progesterone showed a decline at 6 h after surgery. There was an increase in the number of healthy class III (> 350 µm) follicles with a concomitant drop in atretic class III follicles 24 h post-unilateral ovariectomy. Analysis for DNA, RNA and protein content showed that all three constituents registered a continuous rise in the hypertrophying ovary up to 120h after surgery. When expressed as ¼g/mg ovarian weight, the increase in DNA reached a maximum at 24 h and declined thereafter. The kinetics of DNA synthesis was followed by pulse labelling with [3H] thymidine at 18, 24, 36 and 48 h after unilateral ovariectomy. Maximum incorporation occurred at 36 h. Autoradiographic studies showed that the granulosa cells of healthy follicles preferentially incorporated the label. In an extension of this study, it was found that labelling index registered a significant increase following ovariectomy, the maximum being reached at 24 h especially in classIII follicles. The results clearly point out the crucial role of hyperplasia in the response of the contralateral ovary to the surgery and implicate the rise in follicle stimulating hormone as the primary signal for initiation of such a response. This raises the question whether in compensatory ovarian hypertrophy follicle stimulating hormone has a mitogenic role

Hypersecretion of Follicle-Stimulating Hormone (FSH) after Ovariectomy of Hypophysectomized, Pituitary-Grafted Rats: Implications for Local Regulatory Control of FSH*

Previous observations have shown that a portion of the acute (less than 12 h) FSH hypersecretion after ovariectomy (OVX) is LHRH independent, thereby suggesting that mechanisms governing the acute FSH hypersecretory response to OVX may reside largely within the anterior pituitary gland. Accordingly, the present studies were conducted to determine whether acute OVX-induced FSH hypersecretion can be elicited in an animal model in which the anterior pituitary gland is isolated from diencephalic chemical signals, and if so, whether the hypersecretion could be abated by the FSH-suppressing protein, follistatin. Adult female rats hypophysectomized (H) 1 week earlier received anterior pituitary grafts (H/G) (one to three glands per rat) under the kidney capsule. In order to increase ovarian secretion of negative feedback effectors substances (i.e. estrogen, inhibin), some H/G rats were injected sc with 30 IU PMSG 4-6 days after receiving pituitary transplants, whereas other rats were given the saline vehicle. Two days later (0830 h), a blood sample was obtained via an indwelling atrial catheter inserted the previous day. H/G rats given saline or PMSG then were further subdivided and either castrated or sham castrated. Additional blood samples were obtained from the catheter, and trunk blood was collected from decapitated rats 24 h after OVX for measurement of serum estradiol and PRL levels. For comparison, H rats not receiving renal pituitary transplants were subdivided into similar experimental groups as the H/G rats. Blood samples were also obtained after sham OVX or OVX of pituitary-intact, 4-day cycling rats on diestrous day 1. Ovariectomy of PMSG-treated H rats receiving either one or three pituitary allografts resulted in a significant (P less than 0.01) increase in serum FSH levels by 12 h after OVX followed by a 2- to 3-fold increase in FSH levels by 24 h relative to either the pre-OVX FSH levels measured in this group or the FSH levels measured in PMSG-treated H/G rats 24 h after sham OVX. In contrast, OVX of saline-treated H/G rats failed to elicit FSH hypersecretion. Similarly, FSH hypersecretion was not observed after OVX of saline- or PMSG-treated H rats. Whereas serum LH levels were increased 24 h after OVX of diestrous rats, no such increases were detected 24 h after OVX of any H or H/G rats. In an additional experiment, H rats receiving two pituitary allografts were treated with PMSG and subsequently castrated. Twenty-four hours later, rats were injected iv with either 60 micrograms purified porcine follistatin or saline.(ABSTRACT TRUNCATED AT 400 WORDS)

FSH and androgen regulation of Sertoli cell function in the immature rat.

The effects of follicle stimulating hormone (FSH) in immature rats during the period when androgen binding protein (ABP) secretion is initiated (about 16 days of age) were studied. 14-day-old rats were treated with luteinizing hormone (LH) FSH or testosterone (T) following unilateral gonadectomy. At various intervals after injections rats were sacrificed and the other testis removed and weighed. Testes were analyzed for ABP. Treatment with FSH for 3 days in 15-day-old animals increased ABP secretion into the epididymis. In rats given 1 mg testosterone propionate daily for 2 days prior to treatment with FSH ABP secretion in response to FSH was blocked completely in the epididymis although it was enhanced in the testis. LH treatment gave similar results. Antibody inhibition of FSH completely blocked the acute FSH effect on ABP. These studies demonstrate that FSH stimulates Sertoli cell secretion of ABP in immature rats.