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Pulsatility of systemic FSH and LH concentrations during follicular-wave development in cattle.

Changes in systemic FSH and LH pulsatility in temporal association with follicular-wave emergence and follicle deviation were studied in cattle. Wave emergence was defined as occurring when the future dominant follicle first reached 4 mm, as established retrospectively. Follicle deviation was defined as the beginning of a change in growth rates between the 2 largest follicles and occurred 60.4 +/- 4.2 h after wave emergence. Follicles were tracked by transrectal ultrasound scanning every 8 h, and blood samples for pulse characterization were collected every 20 min from before emergence until after deviation. Pulses were characterized by the Pulsar program applied to each 8-h increment, centered on the hour of follicle scanning in each heifer (n = 6). Pulsatility of FSH was not detected for any of the 8-h increments. The mean FSH concentrations for the 24 samples per 8 h increased (P < 0.05) between 8 h before and 8 h after wave emergence, followed by a decrease 40 to 16 h before deviation. The low mean values continued for 24 h after deviation. Pulses of LH were detected for all 8-h increments. The LH mean of all concentrations per 8 h and pulse frequency increased (P < 0.05) between the hour of wave emergence and 32 h after emergence and then pulse frequency plateaued at a mean interpeak interval of 1.3 h. Increased LH means for all concentrations per 8 h and basal concentration were reached 32 h before deviation. The results indicated that elevated concentrations of LH and reduced concentrations of FSH were present 32 to 16 h before to at least 24 h after the beginning of follicle deviation. However, an abrupt, short-term change in FSH concentrations or in LH pulsatility in close temporal association with follicle deviation that could act as an acute stimulus to initiate deviation was not found.

Animals↗

Development and application of competitive ELISA assays for rat LH and FSH.

Rat LH (rLH) and FSH (rFSH) were measured by sensitive and specific competition ELISAs. The rat LH ELISA used rLH-I-9 coated plates, an antiserum against rLH and an antibody against rabbit IgG labeled with peroxidase. Using rLH-RP-3 as a standard, rat LH was determined by binding of the anti-LH antibody to rLH-I-9 coated plates. The sensitivity of the assay was 0.8 ng/mL. Similarly, the rat FSH-ELISA used rFSH-I-8 coated plates, an antiserum against rFSH and an antibody against rabbit IgG labeled with peroxidase. Using rFSH-RP-3 as a standard, the FSH-ELISA was also determined by binding of the anti-FSH antibody to rFSH-I-8 coated plates. The sensitivity of this assay was 1.25 ng/mL. Both rat LH and FSH ELISA assays are highly specific and provide accurate determination of gonadotrophins in buffers, sera, cell culture media, and anterior pituitary extracts. These assays were used for monitoring the gonadotrophin surge-attenuating factor (GnSAF) and inhibin activities present in human follicular fluid (hFF). The 2 new ELISA procedures have practical advantages (safety, convenience, economy) over the RIA methods, and they perform as well as the RIA techniques at the same range of concentrations.

Animals↗

Human FSH isoforms: carbohydrate complexity as determinant of in-vitro bioactivity.

Differences in sialic acid content of the hormone have been considered the main determinant of FSH polymorphism. The aim of the present study was to investigate the effect of variations in the oligosaccharide structure of the intrapituitary human FSH (hFSH) glycosylation variants on their intrinsic biological activity. FSH charge isoforms obtained after chromatofocusing were further separated by lectin affinity chromatography [Concanavalin A (ConA), Wheat germ agglutinin (WGA), Lentil lectin (LcH)]. Isolated isoforms were separately tested for in-vitro bioactivity in a rat Sertoli cell aromatization bioassay. Our results show that: (1) FSH microheterogeneity is due not only to variations in the sialic acid content of the hormone but also to differences in the internal structure of the carbohydrate chains, and (2) variations in the sialic acid content as well as differences in the complexity of the glycans determine the full biological expression of FSH glycosylation variants.

Animals↗

Estradiol enhances the stimulatory effect of FSH on testicular maturation and contributes to precocious initiation of spermatogenesis.

Male rats were daily injected with human FSH (hFSH) or estradiol benzoate (EB) or hFSH+EB between day 5 and 15 of life and autopsied on day 16. hFSH accelerated testicular growth, increased number of spermatogonia and serum level of testosterone. hFSH stimulated also spermatogonia differentiation, which resulted in 5-fold increase of the number of spermatocytes. EB given alone induced adverse, inhibitory effects on spermatogenesis and serum testosterone, did not influence serum FSH and LH but increased 14-fold the level of prolactin. Except from testosterone, EB given with hFSH not only overcame inhibitions, but multiplied hFSH stimulatory effects on spermatogenesis up to 30-times of control values. In addition, after FSH+EB premeiotic germ cell ratio reached adult type value precociously. Estradiol may play regulatory roles in testicular maturation (1) inhibitory, direct one or resulting from decrease in testosterone secretion; (2) stimulatory, by enhancement of FSH action, with a possible involvement of prolactin that may act in concert with FSH.

Animals↗

A 361 base pair region of the rat FSH-beta promoter contains multiple progesterone receptor-binding sequences and confers progesterone responsiveness.

The rat is frequently used as a model to study the role of progesterone (P) in regulating FSH secretion and synthesis. The ability of P to modulate rat FSH-beta mRNA levels suggests the presence of a functional hormone response element. We have found three PRE-like sequences upstream of the transcription start site in the rat FSH-beta gene. These sequences are herein referred to as PRE-like sequence #1, #2 and #3 with #1 being most distal from the start site. The current studies determined whether these PRE-like sequences bound P receptor (PR) and were functional in regulating the induction of expression by P. Electrophoretic mobility shift assays (EMSA) demonstrated that a single 289 base pair (bp) DNA fragment encompassing all three PRE-like sequences specifically bound PR. Further, PR bound with high affinity to double-stranded oligonucleotides representing individual PRE-like sequences #1, #2 and, with lower affinity to a double-stranded oligonucleotide representing PRE-like sequence, #3. We have cloned a 361 bp sequence from the promoter region of the rat FSH-beta gene encompassing all three PRE-like sequences into a luciferase reporter vector (pGL3-promoter) yielding pFSHbeta361-luc+ which when transiently transfected into primary rat pituitary cell cultures, conferred P-responsiveness to a heterologous promoter. P-responsiveness was dependent upon the presence of PR and was blocked by the PR antagonist RU-486. These data strongly suggest the presence of functional PRE's in the rat FSH-beta gene promoter.

Animals↗

Cloning and sequence analysis of FSH and LH in the giant panda (Ailuropoda melanoleuca).

The giant panda (Ailuropoda melanoleuca) is an endangered species and indigenous to China. It has been proposed that it has a highly specialized reproductive pattern with low fecundity, but little is known about its basic reproductive biology at the molecular level. In this report the genes encoding gonadotropin subunits alpha, follicle-stimulating hormone (FSH) beta and luteinizing hormone (LH) beta of the giant panda were amplified for the first time by RT-PCR from pituitary total RNA, and were cloned, sequenced and analyzed. The results revealed that the open reading region (ORF) of gonadotropin subunits alpha, FSH beta and LH beta are 363, 390 and 426 bp long, respectively. They displayed a reasonably high degree (74-94, 85-93, 75-91%, for alpha, FSH beta and LH beta subunits, respectively) of identity when deduced amino acids were compared with homologous sequences from partial available mammals including human, cattle, sheep, pig, rat, mouse. Three distinct differences were found at the site of 59 aa of the alpha subunit and 55 aa, 68 aa of FSH beta subunit. Our results provide an insight into understanding the mechanism of reproduction regulation and genetic characteristics of giant panda which will make an actual contribution to its conservation. In addition they lay a foundation for a further study towards producing recombinant panda FSH and LH which can be used in artificial breeding aimed to increase its captive reproductive efficiency.

Amino Acid Sequence↗

Inhibin is involved in the suppression of FSH secretion in the growth phase of the dominant follicle during the early luteal phase in cows.

This study was carried out to examine the involvement of inhibin in the regulation of FSH secretion during the growth phase of the dominant follicle in the early luteal phase of cows. Six cows were given a single i.v. bolus injection of 100 ml inhibin antiserum raised against bovine 32-kDa inhibin in a castrated male goat, and five animals received the same amount of castrated male goat serum (control serum) on Day 5 of the estrous cycle (Day 0 = estrus). All animals in each group experienced a wave of follicular development after ovulation, and the dominant follicle was over 8.5 mm in diameter on Day 5. The corpus luteum was identified for each group on Day 5. Plasma concentrations of estradiol and progesterone gradually increased after ovulation and reached around 3.7 pg/ml and 3.0 ng/ml on Day 5, respectively, suggesting that the dominant follicle and corpus luteum were functional. Administration of inhibin antiserum produced a clear increase (P < 0.001) in plasma FSH within 8 hr compared with that in control animals. Plasma levels of luteizing hormone showed a moderate increase during 40 hr after the injection of antiserum (P = 0.08). A large number of antral follicles (4 mm in diameter) developed after the hypersecretion of FSH, coupled with the rise in plasma estradiol levels. These results clearly demonstrated that inhibin neutralization during the early luteal phase produces hypersecretion of FSH with a coincident stimulation of follicular development, indicating that inhibin is an important factor for the negative regulation of FSH secretion during the early luteal phase when secretion of estradiol and progesterone are normally high.

Analysis of Variance↗

Effects of human pituitary FSH isoforms on mouse follicles in vitro.

The effects of human FSH glycoforms on mouse follicle development and function in vitro were analysed, and an attempt was made to relate markers of follicular maturation to the expression of immunolocalized connexin (Cx) 43 and Cx26-based gap junctions. Three FSH fractions comprising discrete pI ranges [7.10-5.99 (pool I), pI 5.62-4.95 (pool II) and <3.75 (pool III)] were studied. Pool I produced the strongest effect on preantral granulosa cell proliferation and oestradiol production, and was highly effective for stimulating antral formation; this isoform also evoked a peripheral distribution of Cx43-containing gap junctions. Pool II was effective in promoting preantral granulosa cell proliferation but required higher FSH doses. This particular isoform provoked a more central distribution of Cx43-containing gap junctions, which was associated with a lower oestradiol production and less effective antral formation. Pool III was the least active for all markers of follicle development, and this was associated with minimal induction of Cx43-based gap junctions. The effects of the three FSH isoform pools on Cx26 expression were similar. The pattern of differences strongly suggests that FSH isoforms have complementary and specific actions on developing follicles, and that a shifting stage specific balance of isoforms is required for optimal follicle development.

Animals↗

Comparison of the effectiveness of recombinant and urinary FSH preparations in the achievement of follicular selection in chronic anovulation.

In order to compare the effectiveness of urinary and recombinant FSH (rFSH) preparations in achieving the threshold of follicular growth, stimulated cycles from patients with chronic anovulation, treated with a constant dose of FSH until the emergence of a selected follicle, were retrospectively analysed. Sixty-four cycles were performed using a similar starting dose of either urinary FSH (group 1) or rFSH (group 2), which was kept constant up to the time of follicular selection, assessed on ultrasound (follicular diameter >10 mm). The results of this study showed that, while the number of selected follicles was similar, the mean daily FSH dose required to achieve the threshold of follicular selection was significantly lower in group 2 (70.4 +/- 3.4 IU/day) than in group 1 (86.5 +/- 4 IU/day; P < 0.005). Furthermore, at the time of human chorionic gonadotrophin (HCG) administration, the total FSH dose was significantly lower in group 2 than in group 1, but plasma oestradiol values were equivalent. These data suggest that the higher effectiveness of rFSH preparations over urinary ones may be explained by a lower threshold dose required to achieve follicular selection.

Journal Article↗

Sheep embryos derived from FSH/eCG treatment have a lower in vitro viability after vitrification than those derived from FSH treatment.

In the non breeding period, the effect of two superovulatory treatments (eCG/FSH in single dose or FSH alone in four decreasing doses) on the production of embryo quality following in vitro viability after vitrification procedures was investigated using forty-four adult Sarda breed ewes. In sheep treated with eCG/FSH, the mean number of corpora lutea was significantly (P < 0.05) higher (11.8+/-4.0 vs. 8.05+/-3.8), although the recovery rate was significantly (P < 0.01) lower (74.6 vs. 59.9) than with FSH alone. After vitrification (ethylene glycol and glycerol) was repeated three times, the rates of re-expansion at first and second warming were significantly (P < 0.01) higher in embryos derived from FSH alone than in those with both gonadotrophins (94.9 and 41.9 vs. 72.8 and 18.6) and after the last vitrification the hatched blastocyst rates were 22.5 and 7.6. After differential stain, blastocysts derived from FSH alone showed a mean number of cells significantly higher than blastocysts from eCG/FSH (184.2 vs. 157.7). It was concluded that superovulatory treatment with eCG/FSH may increase the ovarian responses compared with FSH alone, but these embryos showed a reduction in viability rates after repeated vitrification.

Animals↗

Pituitary adenylate cyclase-activating polypeptide directly stimulates LH and FSH secretion by human pituitary gonadotrophinomas.

The effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on LH and FSH secretion by human pituitary gonadotrophinomas in cell culture was studied. PACAP (1-38 peptide, 0.2-20 nmol/L) dose-dependently stimulated both LH and FSH secretion after 24 hours incubation. Of 11 tumours studied, PACAP (20 nmol/L) stimulated LH and/or FSH secretion by 1.7-4 fold in 9 cases. Two tumours did not respond to PACAP, although LHRH was stimulatory in these. None of the 11 tumours contained gsp mutations, excluding the possibility that these were the cause of the occassionally observed non-responsiveness to PACAP. A combination of PACAP (20 nmol/L) together with TRH (25 nmol/L) resulted in greater stimulatory effects on LH and FSH secretion than exerted by either peptide alone, but this was not observed with LHRH. In 3 tumours tested, PACAP stimulated cAMP production 2-3 fold by cultured human pituitary gonadotrophinomas but had no effect on rate of phosphatidylinositol (PI) turnover. These results indicate that PACAP can directly stimulate LH and FSH secretion by human pituitary gonadotrophs and that PACAP-receptors in gonadotrophin-secreting tumours are coupled with adenylate cyclase but not the PI second messenger system. We conclude that PACAP may play a role in controlling gonadotroph function in the human pituitary gland.

Adult↗

Electrophysiological changes of Sertoli cells produced by the acute administration of amino acid and FSH.

Electrophysiological studies were carried out using seminiferous tubules of "Sertoli cell enriched testes" of rats irradiated in utero. Sertoli cells were inserted with glass microelectrodes in a superfusion chamber continuously perfused with KRb buffer. The topical application of FSH (4.0 mU/ml) produced a biphasic effect characterized by a rapid hyperpolarization (less than 3 s) followed by depolarization. The depolarizing effect of FSH was prolonged and potentiated in the presence of 5 mmol/l alpha-methylamino-isobutyric acid in the bath medium of the superfusion chamber. Verapamil, at a dose (250 mumol/l) that nullified the stimulatory action of FSH in the amino acid transport, suppressed the depolarizing effect of FSH. It was concluded that in immature rat testes FSH produces electrophysiological changes that mediate the stimulatory action of the amino acid transport.

Amino Acids↗

FSH-TSH functional overlap in cockerel testicle. Durable amplification of the hormone receptors by treatment at hatching.

Functional overlap of FSH and TSH in cockerel testicle is expressed by alteration of testicular weight and canalicular diameter length. While on neonatal exposure TSH was more active than FSH, at five weeks of age both hormones developed an equal action. A single neonatal exposure to FSH had a durable effect on testicle weight and canalicular diameter, while the effect of post-hatching TSH persisted only in respect of the second parameter. A single neonatal exposure to TSH failed to amplify TSH effect on a second exposure at five weeks of age, while neonatal TSH and FSH treat equally enhanced the effect of FSH given at five weeks. This suggests that under given conditions, perinatal hormone excess may account for lasting amplification of the receptor(s) not only in respect of the adequate hormone, but also in respect of a related hormone, hormone-like material or analogon.

Age Factors↗

Lighting regimens and plasma LH and FSH in broiler breeders.

Egg production by meat-type fowl is markedly inferior to that from commercial laying hens, and so, to assess the degree to which photorefractoriness might be a contributing factor, male- and female-line broiler breeders were maintained on 8-, 11- or 16-h photoperiods. In addition, to determine the age-related rate of change in response to an increment in photoperiod, other birds were transferred from 8- to 16-h photoperiods at 67 or 124 d. Blood samples were taken from all groups, except those on constant 11-h photoperiods, in both genotypes at 67, 69, 124 and 126 d, and from all lighting groups in the female line at 58 weeks (end of trial), and the plasma was assayed for plasma luteinising hormone (LH) and follicle stimulating hormone (FSH) concentration to investigate possible correlations with rate of sexual maturity, total egg numbers and terminal rates of lay. Prepubertal LH was consistently higher for the female line than for the male line, and higher for 16-h birds than for 8-h birds. At 69 and 126 d, LH values were not significantly different from those 2 d earlier for 8-h birds, but significantly reduced for 16-h birds. There was an increase in LH following photostimulation at 67 d, but no significant change after the 124-d light increase. There were no significant differences in FSH between the two genetic lines, nor any effect of photostimulation at 67 or 124 d. There was a tendency for FSH in 8-h birds to be higher than for 16-h birds, and this difference became significant for male-line birds at 67 d. At 58 weeks, LH was higher for constant 11- and 16-h birds and for birds photostimulated at 67 d than for constant 8-h controls or birds transferred from 8 to 16h at 124 d. Neither baseline nor photoinduced prepubertal changes in plasma LH nor FSH were found to be of value for predicting age at sexual maturity or subsequent rates of egg production. At 58 weeks, LH was not generally correlated with sexual maturity, total eggs or terminal rates of lay, however, there was a negative correlation with age at first egg in birds photostimulated at 124 d. It must be concluded that plasma LH and FSH concentrations are of minimal value to the broiler breeder industry for predicting the degree of photorefractoriness, the age at sexual maturity, or subsequent egg production.

Aging↗

Induction of aromatase activity in porcine granulosa cells by FSH and cyclic AMP.

This paper examines the involvement of mRNA and protein synthesis in the induction of aromatase activity by FSH and also the site of action of androgens on FSH induction of aromatase activity in porcine granulosa cells. Treatment of cells with FSH or dbcAMP for an initial 48 h (induction period) resulted in an induction of aromatase activity, as measured by the production of estradiol during a subsequent 6h test period. Addition of testosterone to cells cultured with dbcAMP during the induction period enhanced the aromatase activity during both the induction and test periods. However, while DHT has no effect during the induction period, it significantly inhibited the aromatase activity during the test period. Culturing the granulosa cells with actinomycin D or cycloheximide during the induction period resulted in a dose-dependent inhibition of FSH induction of aromatase activity, suggestive of the synthesis of new protein(s). These results indicate clearly that FSH induction of aromatase activity involves the synthesis of mRNA and proteins.

1-Methyl-3-isobutylxanthine↗

Cytochrome P-450 levels of immature rat ovaries during early follicular development: quantification, cellular and subcellular distribution, enzymatic activity and response to FSH in vivo and in vitro.

Absolute values of cytochrome P-450 (cyt. P-450) content were determined by dual beam spectrophotometry in mitochondrial and microsomal fractions of granulosa cells and stroma (theca and interstitial cells) obtained from ovaries of immature rats treated with diethylstilbestrol (DES) or with DES and FSH. Cholesterol side-chain cleavage activity and aromatase activity were also measured in relevant cell fractions. The cyt. P-450 content in the granulosa cell mitochondria was significantly increased in FSH-treated animals (cyt.P-450 in nmols/mg protein, mean +/- SE: DES-treated 0.027 +/- 0.00213, N = 9; DES/FSH-treated 0.0668 +/- 0.0120, N = 13, P = 0.014). The increased cyt. P-450 content was associated with an increase in cholesterol side-chain cleavage activity confirming the functional relevance of the spectrophotometric measurements. The increase in cyt. P-450 content in stroma after FSH treatment was not statistically significant but there was a significant increase in cholesterol side-chain cleavage activity. The cholesterol side-chain cleavage activity of intact granulosa cells from secondary follicles of DES-treated rats was significantly stimulated by in vitro exposure to FSH during 2 hr assay incubations. A significant cyt. P-450 content was detected in the microsomal fraction of granulosa cells from DES-treated rats which was not associated with aromatase activity. Granulosa cell microsomal cyt. P-450 content was not significantly altered by in vivo FSH treatment despite a dramatic increase in aromatase activity of such microsomal preparations.

Animals↗

Crystalline dihydrotestosterone implants in the lateral septum of male rats. A positive effect on LH and FSH.

Previous investigations in our laboratory have shown that testosterone implanted into the lateral septum in male rats increases LH and FSH secretion. However, it was unclear whether the effect of testosterone was direct via androgen receptor, or indirect via the estrogen receptor after conversion by aromatization to estradiol. To answer this question, we implanted either testosterone or the non-aromatizable androgen 5 alpha-dihydrotestosterone (DHT), into the lateral septum of adult male rats and measured plasma levels of LH and FSH by radioimmunoassay 2 days after implantation. Both testosterone and DHT significantly increased the plasma LH and FSH concentrations. Mean concentration of LH in control animals was 0.21 +/- 0.06 ng/ml, a figure that increased to 0.7 +/- 0.12 and 0.55 +/- 0.1 ng/ml after DHT or testosterone implantation respectively. Mean concentration of FSH in control animals was 1.5 +/- 0.3 ng/ml; this figure increased to 3 +/- 0.3 and 2.9 +/- 0.3 ng/ml after DHT or testosterone implantation. Neither plasma DHT (64.0 +/- 5.6 vs. 52 +/- 5 ng/100ml) nor plasma testosterone levels (4.1 +/- 0.38 vs. 3.3 +/- 0.18 ng/ml) were significantly affected by the implants. We conclude that androgens independently of conversion to estrogen acting in the lateral septum facilitates the release of LH and FSH.

Animals↗

Increased serum FSH in female fragile X premutation carriers with either regular menstrual cycles or on oral contraceptives.

Fragile X premutations are known to be a risk factor for diminished ovarian function at a relatively young age. We studied endocrine profiles of female fragile X family members (n = 79) at risk of premature ovarian failure (POF). Of these 79 women aged <40 years, 45 had menstrual cycles, and 34 were using oral contraceptives. Of the women with menstrual cycles, the premutation carriers had higher serum FSH concentrations than women who were not carrying the premutation. Even premutation carriers with regular cycles showed increased serum FSH concentrations. Moreover, premutation carriers using oral contraceptives also demonstrated increased serum FSH concentrations. Irrespective of whether oral contraceptives were used, a serum FSH concentration of > or =15 IU/l was more common in the premutation carriers than in the other women. One premutation carrier using oral contraceptives had a serum FSH concentration of >40 IU/l, the threshold that defines POF. We confirmed that premutation carriers with menstrual cycles demonstrate premature ovarian dysfunction. However, we also found endocrine signs of unrecognized ovarian dysfunction in premutation carriers using oral contraceptives, despite endocrine alterations by oral contraceptives. Premutation carriers may have a poorer prognosis for future pregnancy, either achieved spontaneously or by assisted reproductive technology. We recommend that premutation carriers should be counselled not to wait too long if they wish to start a family.

Adult↗