The aim of this study was to search for direct biochemical effects of highly purified FSH on isolated ovarian follicular theca in vitro. Granulosa cells (GC; approximately 1 × 105 cells per follicle) were flushed from isolated follicles of pro-oestrous rats. The remaining theca layer and the isolated GC were incubated with highly purified ovine FSH. Prostaglandin E (PGE) accumulation was measured by radioimmunoassay. Follicle-stimulating hormone induced a 15-fold increase in PGE accumulation over the basal level in the follicular theca, the stimulated rate exceeding threefold that observed in the GC fraction derived from the same follicle. Follicle-stimulating hormone caused no significant increase in cyclic AMP level or steroidogenesis in the theca layer, but was active on these parameters in the GC. In contrast, LH increased the accumulation of cyclic AMP, progesterone and testosterone, as well as of PGE, in follicular theca. Exogenous 8-bromo cyclic AMP or cyclic GMP also stimulated PGE production in follicular theca or GC, but FSH was without any effect on the level of endogenous cyclic GMP in GC or follicular theca. Antibodies to FSH prevented the effect of FSH (but not that of LH) on PGE formation by follicular theca and GC, while antibodies to the β-subunit of LH blocked the effect of LH but not of FSH. We conclude that highly purified FSH has a stimulatory effect on PGE formation by the follicular theca.
U. Zor, B. Strulovici, R. Braw, H. R. Lindner and A. Tsafriri
T. R. Koiter, G. C. J. van der Schaaf-Verdonk, H. Kuiper, N. Pols-Valkhof and G. A. Schuiling
The effects of steroid-free bovine follicular fluid (bFF) and sodium phenobarbitone on spontaneous LH releasing hormone (LHRH)-induced secretion of FSH and LH were studied in ovariectomized rats. Luteinizing hormone releasing hormone was administered by infusion to rats anaesthetized with phenobarbitone. Bovine follicular fluid reduced FSH release and synthesis. Luteinizing hormone release remained unaffected after bFF treatment. Phenobarbitone reduced both FSH and LH release. The observed suppressive effects of bFF and phenobarbitone on FSH secretion were additive, suggesting that the basal release of FSH has an LHRH-dependent and an LHRH-independent component. Furthermore, bFF did not affect pituitary responsiveness of LH secretion to LHRH and reduced the responsiveness of FSH secretion only when administered some time before the LHRH challenge. The present observations support the view that in the ovariectomized rat the pituitary gland is the only site of action of inhibin-like activity as present in bFF.
P. NEAL and T. G. BAKER
The response of mouse ovaries maintained in organ culture to follicle-stimulating hormone (FSH), luteinizing hormone (LH) and human chorionic gonadotrophin (HCG) was assessed using quantitative histological and radioimmunoassay techniques.
In terms of the induction of preovulatory maturation in follicular oocytes, 1 μg FSH/ml medium was as effective as 10 μg LH/ml. The lowest doses of HCG and LH used (0·2 i.u./ml and 1 μg/ml respectively) had no effect on oocyte maturation, whereas the response to FSH was virtually unchanged irrespective of dose (1–10 μg/ml). When the level of progesterone in the medium at the end of organ culture was used as an index of ovarian response, LH was more effective than FSH and HCG, although all the hormones induced a significant increase, irrespective of dose.
These results are discussed in terms of the mode of action of gonadotrophins in the processes culminating in ovulation.
B. T. DONOVAN and A. N. LOCKHART
The induction of ovulation and follicular luteinization in the guinea-pig by follicle-stimulating (FSH) and luteinizing (LH) hormones was studied in the intact and in the hypophysectomized animal. Follicular luteinization could be produced as early as day 8 of the oestrous cycle by 250 mu. FSH, but not before day 10 by 400 mu. LH, while either FSH or LH caused ovulation when given on day 12 or later. When injected on day 13, luteinization could be induced with 62·5 mu. FSH or 200 mu. LH. Luteinization of follicles also occurred when 400 mu. LH or 250 mu. FSH was given immediately after hypophysectomy on day 13, but when hormone treatment was delayed for 4·5 h after surgery the incidence of luteinization was greatly reduced. Combined treatment with 200 mu. LH and 125 mu. FSH, by contrast, readily caused luteinization. It is concluded that ovarian follicles are resistant to luteinization during the first half of the oestrous cycle and that the response to LH late in the cycle is dependent upon the maintenance of follicular responsiveness by endogenous gonadotrophin.
K. J. Teerds, J. Closset, F. F. G. Rommerts, D. G. de Rooij, D. M. Stocco, B. Colenbrander, C. J. G. Wensing and G. Hennen
The effects of pure FSH and/or LH preparations on the number of Leydig cells and their function in immature hypophysectomized rats have been investigated. As a result of hypophysectomy at the age of 17–18 days, the number of recognizable Leydig cells per testis decreased, as did the steroidogenic capacity in vivo and in vitro. Treatment with 64 μg FSH on both 22 and 23 days of age, did not affect the number of recognizable Leydig cells. In contrast, two injections of LH (10 μg) caused a sixfold increase in the number of Leydig cells, but had a negative effect on spermatogenesis. These stimulatory and inhibitory effects of LH diminished when FSH was added. Treatment with FSH for 7 days caused a twofold increase in the number of Leydig cells when compared with hypophysectomized controls. 3β-Hydroxysteroid dehydrogenase (3β-HSD) and esterase activity in Leydig cells also increased under the influence of FSH. The pregnenolone production per Leydig cell in the presence of 5-cholesten-3β,22(R)-diol (22R-hydroxycholesterol) as substrate showed a sevenfold increase. Plasma testosterone levels 2 h after injection of human chorionic gonadotrophin in intact rats and hypophysectomized FSH-treated rats were the same. Following LH treatment for 7 days, the number of Leydig cells proved to be 11 times higher, and 3β-HSD and esterase activity were not different from intact controls. The testicular pregnenolone production was four- to fivefold higher when compared with untreated hypophysectomized rats. However, pregnenolone production per Leydig cell in LH-treated rats was only slightly different from the hypophysectomized controls.
In conclusion, FSH treatment caused an increase in the number and steroidogenic activity of Leydig cells, and LH had a major effect on the number of Leydig cells, but did not stimulate the steroidogenic capacity.
Journal of Endocrinology (1989) 120, 97–106
S. G. Hillier
The gonadotrophins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were first isolated in more or less pure forms over half a century ago. Purified pituitary gonadotrophins were used by Fevold (1941) and Greep, Van Dyke & Chow (1942) in their classic experiments which demonstrated unequivocally the need for both FSH and LH to stimulate normal ovarian follicular development and oestrogen secretion in hypophysectomized rats. Human pituitary gonadotrophins of equivalent purity have never been widely available for clinical use, but within the next few years pharmaceutical grades of human recombinant FSH and LH are both likely to become so (e.g. Keene, Matzuk, Otani et al. 1989). Taking account of recent advances in our understanding of gonadotrophin action at the cellular level, it should be possible to use these pure human gonadotrophins to devise improved strategies for stimulating ovarian function in infertile women.
Development-dependent follicular responsiveness to gonadotrophins
FSH receptors are located
V Sriraman, MR Sairam and AJ Rao
The relative role of LH and FSH in regulation of differentiation of Leydig cells was assessed using an ethane 1,2-dimethylsulfonate (EDS)-treated rat model in which endogenous LH or FSH was neutralized from day 3 to day 22 following EDS treatment. Serum testosterone and the in vitro response of the purified Leydig cells to human chorionic gonadotropin (hCG) was monitored. In addition RNA was isolated from the Leydig cells to monitor the steady-state mRNA levels by RT-PCR for 17alpha-hydroxylase, side chain cleavage enzyme, steroidogenic acute regulatory protein (StAR), LH receptor, estrogen receptor (ER-alpha) and cyclophilin (internal control). Serum testosterone was undetected and the isolated Leydig cells secreted negligible amount of testosterone on stimulation with hCG in the group of rats that were treated with LH antiserum following EDS treatment. RT-PCR analysis revealed the absence of message for cholesterol side chain cleavage enzyme and 17alpha-hydroxylase although ER-alpha and LH receptor mRNA could be detected, indicating the presence of undifferentiated precursor Leydig cells. In contrast, the effects following deprival of endogenous FSH were not as drastic as seen following LH neutralization. Deprival of endogenous FSH in EDS-treated rats led to a significant decrease in serum testosterone and in vitro response to hCG by the Leydig cells. Also, there was a significant decrease in the steady-state mRNA levels of 17alpha-hydroxylase, cholesterol side chain cleavage enzyme, LH receptor and StAR as assessed by a semiquantitative RT-PCR. These results establish that while LH is obligatory for the functional differentiation of Leydig cells, repopulation of precursor Leydig cells is independent of LH, and also unequivocally establish an important role for FSH in regulation of Leydig cell function.
K Soumano, JG Lussier and CA Price
This study tested the hypothesis that luteal LH receptor (LHr) and follicular LHr and FSH receptor (FSHr) steady-state mRNA levels are greater during superovulation with equine chorionic gonadotrophin (eCG) compared with that with FSH. Heifers were stimulated with eCG (n = 10) or FSH (n = 10), and ovaries were recovered the day before and at 12 and 24 h after luteolysis was induced with prostaglandin F2 alpha (PGF2 alpha). Total RNA was purified from individual follicles and corpora lutea. Steady-state levels of LHr and FSHr mRNA were assessed by slot blot analysis employing homologous cDNA probes. There were no differences in luteal LHr between FSH- and eCG-stimulated animals before luteolysis, and hybridization signals were detected in only one of six animals by 12 h after injection of PGF2 alpha. After PGF2 alpha injection, steady-state levels of follicular LHr were 4-fold lower (P < 0.05) and follicular FSHr mRNA levels were 2.4-fold lower (P < 0.05) in eCG- compared with FSH-treated cattle. In eCG-treated animals, induction of luteolysis led to a significant increase in follicular LHr mRNA levels (P < 0.01) and a significant decrease in follicular FSHr mRNA levels (P < 0.01). There was no such effect of luteolysis in FSH-treated animals. We conclude that superovulation with eCG, compared with FSH, results in lower follicular levels of LHr and FSHr mRNA but does not affect luteal LHr mRNA levels.
H. M. Picton and A. S. McNeilly
Ewes chronically treated with gonadotrophin-releasing hormone (GnRH) agonist were used to investigate the importance of the peripheral concentration of LH in FSH-stimulated follicular development. Twenty-four Welsh Mountain ewes were treated with two agonist implants containing 3·3 mg buserelin. During week 6 of treatment all the ewes were given a 72-h continuous infusion of ovine FSH alone (3 μg/h) or FSH with large (7·5 μg)- or small (2·5 μg) amplitude pulses of ovine LH delivered at 4-hourly intervals. The importance of baseline LH throughout the FSH infusion was evaluated in six animals which were treated with a specific antiserum against bovine LH (LH-AS) 15–20 h before the start of FSH treatment.
In the absence of LH-AS, infusion of FSH alone or with large or small pulses of LH stimulated the development of a normal number of small follicles (≤ 2·5 mm in diameter) and large follicles (> 2·5 mm in diameter). These follicles had normal diameter and steroid secretion compared with control ewes on day 8 of the luteal phase. In contrast, the animals pretreated with LH-AS developed no follicles > 2·0 mm in diameter but the number of small follicles per ewe was significantly (P < 0·05) increased.
These results support the hypothesis that FSH in the absence of pulsatile LH release stimulates preovulatory follicular development in ewes treated with GnRH agonist. The follicular response to LH pulses of different amplitude is dependent on both the stage of development of the follicle and the peripheral concentration of FSH. The endogenous basal level of LH present throughout the FSH infusion is essential for FSH to induce follicle growth beyond > 2·5 mm in diameter.
Journal of Endocrinology (1991) 128, 449–456
M Chopineau, N Martinat, C Troispoux, H Marichatou, Y Combarnous, F Stewart and F Guillou
Horse (Equus caballus) luteinizing hormone (eLH) and chorionic gonadotrophin (eCG), which have the same amino acid sequence, are unusual in that, although they express only LH activity in equids, they express dual LH and FSH activities in all other species tested. Donkey (Equus asinus) LH (dkLH) and CG (dkCG), which also share an identical peptide backbone, have been less well characterized and conflicting results concerning their FSH activity in heterologous species have appeared in the literature. In order to assess and compare the intrinsic LH and FSH activities of the horse and donkey LHs in heterologous species, recombinant eLH (r.eLH/CG) and recombinant dkLH (r.dkLH/CG) were expressed, for the first time, in COS-7 cells. Their LH activities were assessed in a rat Leydig cell bioassay, and their FSH activities were estimated in a bioassay using Y1 cells stably expressing the human FSH receptor. Human CG (hCG) was expressed (r.hCG) and analysed in the same system. The results showed that, whereas r.dkLH/CG was about twice as active as r.eLH/CG in the LH bioassay, it was five times less active than r.eLH/CG in the FSH bioassay; r.hCG was about three times less active than r.eLH/CG in the LH bioassay but was completely inactive in the FSH bioassay. These results confirm that dkLH/CG possesses significant FSH activity in heterologous species that is not attributable to contamination with FSH.
Journal of Endocrinology (1997) 152, 371–377