Comparison of in vitro follicle-stimulating hormone (FSH) activity of equine gonadotropins (luteinizing hormone, FSH, and chorionic gonadotropin) in male and female rats

Not only equine FSH (eFSH) but also equine LH (eLH) and equine CG (eCG/PMSG) exhibit FSH activity in the rat. The concomitant loss of LH and FSH activities upon dissociation at acidic pH of eLH demonstrates that its FSH activity is intrinsic to the molecule and not due to contamination by FSH. Indeed, this latter hormone dissociates at a much higher pH. The binding activity as well as the in vitro biological activity of the equine gonadotropins were determined on rat gonadal cells from both male and female rats using the homologous hormone rat FSH as a reference. In the female, the biological activity of all of the gonadotropins is strictly related to their binding activity. In the male, this is true for all hormones except eFSH. Indeed, the biological activity of eFSH on rat Sertoli cells is higher than expected from its binding activity. Moreover, the FSH from other species (rat, ovine, and porcine) as well as eLH and eCG inhibit the response elicited by a submaximal dose of eFSH. These data indicate that eFSH acts as a superagonist of rat FSH in rat Sertoli cells, since it triggers cell activation at a much lower concentration than expected from its relative binding activity.     

Investigation of binding sites for follicle-stimulating hormone and chorionic gonadotropin in human ovarian cancers

The binding of FSH and hCG to ovarian tumor biopsies from 31 women was analyzed to determine whether ovarian cancers contain receptors for gonadotropic hormones. For comparative purposes, gonadotropin binding to receptor-positive control tissues was also assessed. Specific binding was defined as [125I]hFSH and [125I]hCG binding prevented by coincubation of tissue samples with an excess of unlabeled gonadotropin. Various types of epithelium-derived tumors (serous, mucinous, endometrioid, and undifferentiated; n = 29) had low levels of specific FSH and hCG binding (less than 15% of receptor-positive control tissues) which were not dependent on tissue concentration and not saturable. A germ cell tumor had similar binding characteristics. In contrast, specific FSH binding to a granulosa cell-theca cell (GC-TC) tumor was directly proportional to tissue concentration, and binding was maximal in the presence of 100 ng/ml [125I]FSH. Scatchard analyses of FSH binding yielded a linear plot. Although the FSH-binding capacity of the GC-TC tumor (6.3 fmol/mg) exceeded that of control tissues, their binding affinities (Kd = 1.4 X 10(-9)M) were similar. Specific hCG binding to the GC-TC tumor was low and not dependent on tissue concentration. We conclude that the specific binding of FSH to the GC-TC tumor was characteristic of the interaction of gonadotropin with receptors in target tissues. However, the low level of FSH and hCG binding to tumors of epithelial origin did not represent gonadotropin-receptor binding. Thus, the common ovarian tumors of epithelial origin may not respond directly to gonadotropin, but malignancies of sex cord-stromal origin, such as GC-TC tumors, could be modulated by FSH.     

Subunit interaction of human chorionic gonadotropin (hCG) with rat ovarian luteinizing hormone (LH)/CG receptor.

The subunit interaction of hCG with its rat ovarian LH/CG receptor was studied by cross-linking the solubilized receptor-hormone complex with glutaraldehyde (GA), disuccinimidyl suberate (DSS) or dithiobis(succinimidyl propionate) (DSP) and analyzing the complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. The hormone was labeled either in its alpha-subunit (125I-hCG) or in its beta-subunit (3H-hCG) or the label (3H) was introduced into the receptor molecule instead of the hormone. All of the labeling procedures led to the detection of only the receptor-(alpha,beta)hCG and receptor-(alpha)hCG complexes on the autoradiograms. The sizes of these complexes were 137,000 and 106,000, respectively, under reducing conditions. These results suggest that the receptor binds one hormone molecule, and that hCG interacts with the receptor mainly through its alpha-subunit. In addition, polyclonal antibodies directed against the LH/CG receptor and the alpha- and beta-subunits of hCG were used to detect the non-reduced receptor-(alpha,beta)hCG complex in immunoblotting. As antibodies directed against both the alpha-subunit and the beta-subunit were able to detect the Mr 130,000 complex, it is conceivable that both of the subunits are at least partially exposed on the receptor-hormone complex. 125I-hCG was also cross-linked to the membrane-bound receptor. The membrane-bound complex had an Mr of 144,000 under reducing conditions, i.e. approximately 7000 higher than that of the solubilized complex (Mr 137,000). This may indicate that the membrane-bound receptor is covalently modified or differs in conformation from the solubilized receptor.     

Prolonged stimulation by follicle-stimulating hormone is required for the induction of ovarian follicular cysts by human chorionic gonadotropin in hypophysectomized rats

Combined stimulation by follicule-stimulating hormone (FSH) and subovulatory doses of human chorionic gonadotropin (hCG, luteinizing hormone [LH]-like activity) produces large ovarian follicular cysts in hypophysectomized (HYPOXD) immature rats. To obtain a better understanding of the extent to which stimulation by FSH is required in order for hCG to induce these ovarian cysts, immature HYPOXD rats were given subcutaneous (sc) injections of 1 IU hCG twice daily for 9 d, either alone or with daily injections of 2 μg of highly purified ovine FSH on 1. Day one of hCG treatment; 2. Days one and two of hCG treatment; 3. Days one through five of hCG treatment; or 4. All 9 d of hCG treatment. Ovaries and serum samples were collected on the morning of d 10 of treatment. Animals that were treated for 9 d with hCG, but that received either no FSH or only 1 or 2 d of FSH treatment, did not display antral follicles on day 10 of treatment. The largest cross-sectional areas for the ovaries from animals that received 1 or 2 d of FSH treatments ranged between 6.84±0.51 mm(2) and 8.94±0.89 mm(2). The diameters of the largest preantral follicles in the ovaries of these two groups ranged between 0.278±0.011 and 0.320±0.028 mm, respectively. In contrast, ovaries from hCG-treated HYPOXD rats that received FSH treatments for either 5 or 9 d displayed follicular cysts by the morning of day 10. The largest cross-sectional areas for the ovaries from these two treatment groups were similar (15.68±1.61 and 18.7±5.13 mm(2), respectively), as were the mean diameters of the cystic follicles in these two groups (0.929±0.096 and 0.830±0.063 mm, respectively). Although serum androstenedione and testosterone concentrations were greater for HYPOXD rats that received combined FSH+hCG treatments than for animals that received hCG treatments alone, these concentrations did not increase with increasing numbers of days of FSH treatment. As with serum androstenedione and testosterone concentrations, serum estradiol and estrone concentrations for HYPOXD rats treated with hCG alone were limited (0.002±0.001 and 0.004±0.002 ng/mL, respectively), but had increased by day 10 after a single injection of FSH on day one of treatment. In contrast to serum androgen concentrations, serum estradiol and estrone concentrations continued to increase as the number of days of combined FSH+hCG treatment increased. These observations indicate that, in the rat, a significant period of exposure to tonic stimulation by both FSH and LH-like activity is required for the development of large ovarian cysts. Further, this period of exposure to FSH appears to be linked to increased peripheral serum estrogen concentrations, rather than to increased androgen concentrations. Therefore, the data provide indirect support for the concept that estrogens play a direct role, at the level of the ovary, in the induction of large ovarian cysts in the rat.     

Ovarian responses of pregnant mare serum gonadotropin- and human chorionic gondotropin-primed rats: desensitizing, luteolytic, and ovulatory effects of a single dose of human chorionic gonadotropin.

We conducted a study to determine the morphological appearance and functional responsiveness of ovarian tissues after administration of hCG to 28-day-old rats primed 65 h earlier with PMS gonadotropin (PMSG) and after administration of a second dose of hCG 5 days later, i.e. to 33-day-old rats containing heavily luteinized ovaries. Sixty-five hours after the administration of 50 IU PMSG sc to 25-day-old rats, ovaries already contained an abundance of luteinized follicles and an adenylyl cyclase (AC) system that was responsive to LH, epinephrine, and NaF. The administration of 50 IU hCG sc at this time initially resulted in a loss of LH-responsive ovarian AC. Within 4 days of the hCG injection, the ovaries of the now 32-day-old rats were heavily luteinized, and ovarian AC was highly responsive to LH, epinephrine, and NaF. The administration of a single sc dose of 200 IU hCG to 33-day-old PMSC- and hCG-primed rats with luteinized ovaries resulted in a rapid desensitization of the ovarian AC to LH and a drop in serum progesterone levels, During the subsequent 7 days, serum progesterone levels continued to decline, while total ovarian AC reacquired responsiveness to LH by days 4--5 after the densensitizing dose of hCG. Dissection of ovarian components revealed, however, that the AC system of the corpora lutea originally present at the time of the second hCG injection remained permanently refractory to LH and that the AC in corpora lutea newly formed from freshly ovulated follicles exhibited a significant responsiveness to LH, epinephrine, and NaF. However, these new corpora lutea were not fully active, since serum progesterone never rose. Subcutaneous administration of 50 IU hCG to 33-day-old PMSG- and hCG-primed rats also promoted a rapid loss of AC responsiveness to LH. This lower concentration of hCG was not sufficient to promote follicular development or ovulation, and the ovarian AC remained refractory to LH for at least 7 days. Intravenous administration of 75 IU hCG to 33-day-old PMSG- and hCG-primed rats similarly promoted a rapid and permanent loss of luteal AC responsiveness to LH; again, follicles did not mature to a preovulatory state and, in fact, appeared to undergo atresia rather than ovulation. These results indicate that in heavily luteinized ovaries 1) hCG promotes desensitization of rat luteal AC to LH, 2) Desensitization of AC to LH stimulation in corpora lutea is permanent and irreversible, and 3) only under conditions where follicles mature and ovulate and new corpora lutea are formed does total ovarian AC reacqure responsiveness during the subsequent week.     

Estrogen receptor alpha signaling pathways differentially regulate gonadotropin subunit gene expression and serum follicle-stimulating hormone in the female mouse

Estrogen, acting via estrogen receptor (ER)alpha, regulates serum gonadotropin levels and pituitary gonadotropin subunit expression. However, the cellular pathways mediating this regulation are unknown. ERalpha signals through classical estrogen response element (ERE)-dependent genomic as well as nonclassical ERE-independent genomic and nongenomic pathways. Using targeted mutagenesis in mice to disrupt ERalpha DNA binding activity, we previously demonstrated that ERE-independent signaling is sufficient to suppress serum LH levels. In this study, we examined the relative roles of ERE-dependent and -independent estrogen signaling in estrogen regulation of LH, FSH, prolactin, and activin/inhibin subunit gene expression, pituitary LH and FSH protein content, and serum FSH levels. ERE-independent signaling was not sufficient for estrogen to induce pituitary prolactin mRNA or suppress pituitary LHbeta mRNA, LH content, or serum FSH in estrogen-treated ovariectomized mice. However, ERE-independent signaling was sufficient to reduce pituitary glycoprotein hormone alpha-subunit, FSHbeta, and activin-betaB mRNA expression. Together with previous serum LH results, these findings suggest ERE-independent ERalpha signaling suppresses serum LH via reduced secretion, not synthesis. Additionally, ERE-dependent and ERE-independent ERalpha pathways may distinctly regulate steps involved in the synthesis and secretion of FSH.     

Stimulatory effects of recombinant follicle-stimulating hormone on Leydig cell function and spermatogenesis in immature hypophysectomized rats

Although earlier reports suggest a stimulatory effect of FSH on Leydig cell function, controversy exists due to unavailability of FSH preparations free of contaminating LH. Recent availability of recombinant human FSH preparations made it possible to reinvestigate this question. Immature male rats were hypophysectomized (21-22 days old at surgery) and implanted with osmotic minipumps releasing 8 IU recombinant FSH or 18 IU purified human pituitary FSH (hpFSH)/day, whereas control animals received vehicle alone. After 7 days of treatment, testicular weight increased in the recombinant FSH and hpFSH-treated animals to values 2.3- and 2.5-fold those of controls, respectively. Analyses of the steroidogenic capacity of Leydig cells in testes of rats treated with recombinant FSH or hpFSH also revealed 2.9- and 3.8-fold androgen production in vitro compared to controls. In these rats recombinant FSH and hpFSH increased the LH receptor number in testicular homogenate by 50% and 70%, respectively. The increase in LH receptor number was associated with increases in the LH receptor mRNA levels. In hypophysectomized control rats, small seminiferous tubules contained spermatogonia and zygotene/early pachytene spermatocytes. In contrast, treatment with either FSH preparation enhanced the progression of meiosis, as evidenced by large number of pachytene spermatocytes and appearance of round spermatids. The present results show that LH-free recombinant FSH, like purified pituitary FSH, is capable of increasing the LH receptor content and steroidogenic responsiveness of Leydig cells through paracrine mechanisms together with a stimulatory effect on spermatogenesis. These observations suggest that prepubertal elevation of FSH secretion may be important for increasing Leydig cell steroidogenic capacity and spermatogenic progression.