Surges of FSH during the follicular and early luteal phases of the estrous cycle in heifers

Surges of FSH were characterized in each of 12 Holstein heifers using a computerized cycle detector program, and as mean changes averaged over all heifers. Blood samples were collected 6 times a day at 4-h intervals beginning at late diestrus. Concentrations of FSH were adjusted relative to the preovulatory LH peak (Hour 0) and profiled beginning 48 h before and ending 120 h after the LH peak. Peak concentrations of FSH and LH occurred synchronously in 11 of 12 (92%) heifers, and only a 4-h interval separated peak concentrations in the remaining heifer. The FSH surge that was synchronous with the LH surge was designated FSH Surge 1 and was used as a reference to designate other FSH surges. Surge -1 of FSH was detected in 58% of the heifers at mean Hour -21.2, and Surges 2, 3 and 4 were detected in 92%, 92% and 75% of the heifers, respectively, at mean Hours 25.1, 57.8 and 78.7. Mean peak levels and duration of FSH Surges-1, 2, 3 and 4 were significantly lower than for FSH Surge 1. Mean concentrations of FSH significantly increased and decreased before and after the LH peak, resulting from the synchrony between FSH Surge 1 and the LH surge in individual heifers. Additionally, there was a tendency (P < 0.08) for a second and third increase in mean FSH concentrations at Hours 24 and 60, which was attributed to FSH Surges 2 and 3 that occurred in individuals. Peak FSH concentrations of Surge 2 occurred (mean, Hour 25.1) within 8 h of maximal mean concentrations at Hour 24 in 91% of the heifers. Correspondingly, peak FSH concentrations of Surge 3 occurred (mean, Hour 57.8) within 8 h of maximal mean concentrations at Hour 60 in 64% of the heifers. Surges -1 and 4 of FSH occurred less frequently and at various times within and among heifers compared with Surges 1 to 3; therefore, they were not detected as mean increases in FSH concentrations but were masked as a result of concentrations being averaged over all heifers. In summary, FSH surges were detected in individual heifers before and after the combined FSH/LH surge. The interpeak intervals for FSH Surges 1 to 2 (25 h), 2 to 3 (33 h) and 3 to 4 (21 h) suggests a rhythmic nature to the surges.     

Synchronous fluctuations of LH and FSH in plasma samples collected daily during the estrous cycle in mares

Circulating concentrations of LH and FSH in each of 12 mares were measured in daily blood samples from 3 d before until 3 d after an interovulatory interval (ovulation=Day 0). The interval was normalized to its mean length (22 d) and partitioned into periods relative to high and low (first significant increase and decrease: Days 3 and 14, respectively) mean FSH concentrations. The resulting experimental periods were as follows: 1) Days −3 to 2 corresponding to the periovulatory period, 2) Days 3 to 14 corresponding to the luteal period, and 3) Days −7 to 3 corresponding to the follicular-periovulatory period. An adaptive threshold method was used to detect peak concentrations of LH and FSH fluctuations. There was no significant difference in the number of detected LH fluctuations per mare among the 3 periods (means, 1.2, 1.8, 1.6 fluctuations, respectively). However, more (P<0.05) FSH fluctuations per mare were detected during the luteal period (mean, 2.4 fluctuations) than during the periovulatory period (mean, 0.5 fluctuations) and follicular-periovulatory period (mean, 1.2 fluctuations). Synchronous LH and FSH fluctuations, defined as the simultaneous detection of peak concentrations of fluctuations, occurred more (P<0.05) often per mare during the luteal period (mean, 1.3 fluctuations) than during the periovulatory period (mean, 0.1 fluctuations) and follicular-periovulatory period (mean, 0.2 fluctuations). During the luteal period, concentrations of LH peaked (P<0.05) during FSH fluctuations and, conversely, concentrations of FSH peaked (P<0.05) during LH fluctuations, indicating a high degree of coupling between the 2 gonadotropins. In summary, fluctuations of LH and FSH occurred in synchrony with a high degree of coupling between them during the luteal period, but not during the periovulatory and follicular-periovulatory periods.     

Periodic fluctuations in FSH concentrations during the ovine estrous cycle

Heterologous radioimmunoassays for ovine LH and ovine FSH were validated and used to examine the concentrations of gonadotropins during the estrous cycle. Concentrations of LH were maximal on the day of estrus as previously reported. Concentrations of FSH were minimal 1 or 2 days before estrus, increased markedly during estrus, and fluctuated widely during diestrus. Most ewes ($\text{11}\text{13}$) had periodic waves of FSH occurring at short intervals (3.5–6 days, $\text{3}\text{13}\text{ewes}$), long intervals (10–18 days, $\text{3}\text{13}\text{ewes}$), or at both long and short intervals ($\text{5}\text{13}\text{ewes}$).     

Secretion of LH, FSH and oestradiol-17 beta during the follicular phase of the oestrous cycle in the ewe

Plasma concentrations of LH, FSH and oestradiol-17 beta were measured in blood samples taken at 15 min intervals for 48 h during the follicular phase of four Merino ewes. The amplitude of pulses of LH and the mean concentration of LH were higher at the beginning of the follicular phase, 36-24 h before the preovulatory surge of LH (amplitude 2.4 ng ml-1, mean concentration 3.9 ng ml-1), than at the end, 24-0 h before the preovulatory surge (amplitude 1.2 +/- 0.1 ng ml-1; mean concentration 1.4 +/- 0.1 ng ml-1). There was no change in the inter-pulse interval during this time (mean 74 +/- 5 min). Over the same period, oestradiol levels increased from 7-8 pg ml-1 to a peak of 10-15 pg ml-1. Mean FSH concentrations declined (36-24 h: 3.6 ng ml-1 vs 24-0 h: 1.8 +/- 0.3 ng ml-1) before rising at the time of the preovulatory surge of LH and again 24 h later. It was concluded that the biphasic response of LH to oestrogen that is seen in ovariectomized ewes may also operate during the follicular phase of the oestrous cycle in entire ewes.     

Plasma FSH and LH in prepubertal Booroola ewe lambs

Basal plasma concentrations (four 30-min samples) and GnRH-induced release of gonadotrophins were measured every 15 days between 30 and 90 days and at 110 days of age in Merino ewe lambs from the prolific Booroola ('B') flock (n = 18-23), the medium prolificacy ('T') flock (n = 14-20), and the 'O' flock (n = 4-8) of low prolificacy. At ages of 30 and 45 days B ewe lambs had mean basal plasma FSH concentrations of 145 and 122 ng/ml which were significantly higher (P less than 0.01) than those seen in T (45 and 53 ng/ml), and O (39 and 38 ng/ml) flock ewes. Between 60 and 110 days of age there were no significant differences between genotypes. The increment in FSH concentrations above basal levels induced by the subcutaneous injection of 100 micrograms synthetic GnRH was only significantly (P less than 0.05) greater in B than T and O genotype ewe lambs at 110 days of age but not at other ages. The basal plasma FSH differences between the B, T and O genotypes at 30 and 45 days of age were not consistently related to the size of litter in which lambs were born. At 30 days of age the mean plasma LH concentration of B, T, and O flock lambs were 2.6 +/- 0.5, 1.2 +/- 0.6 and 0.7 +/- 0.8 ng/ml respectively. These differences were not significant. At later ages there were also no significant differences between the genotypes with respect to basal LH, and the increase in LH induced by exogenous GnRH was always similar for the three genotypes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pituitary luteinizing hormone (LH) and follicle-stimulating hormone (FSH) responses to gonadotropin-releasing hormone during the rat estrous cycle: an increased ratio of FSH to LH is secreted during the secondary FSH surge

The hormonal interactions required for the generation of a secondary surge of FSH on the evening of proestrus have not been clearly defined. The role of GnRH in driving a surge of FSH has been questioned by findings in previous studies. In the current study, gonadotropin secretion was measured from pituitary fragments obtained from rats at 0900 and 2400 h on each day of the estrous cycle. Pituitary fragments were perifused in basal (unstimulated) conditions or in the presence of GnRH pulses to determine whether a selective increase in basal release of FSH and/or an increase in the responsiveness to GnRH occurs during the secondary FSH surge. Each anterior pituitary was cut into eighths and placed into a microchamber for perifusion. Seven pulses of GnRH (peak amplitude = 50 ng/ml; duration = approximately 2 min) were administered at a rate of one per hour starting at 30 min. Fractions of perfusate were collected every 5 min and frozen until RIA for LH and FSH. The mean total amount of LH or FSH secreted during the hour interval following each of the last six pulses of GnRH (or the corresponding basal hour) was calculated. Analysis of variance with repeated measures indicated that the evening secretion of LH on proestrus (2400 h) dropped significantly (p less than 0.05) from a maximum on the morning of proestrus (0900 h), whereas the FSH secretion remained elevated at this time. Therefore, the ratio of FSH to LH secreted in response to GnRH pulses was highest during the secondary FSH surge and lowest on the morning of proestrus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluctuations in the plasma levels of the follicle-stimulating hormone during estrous cycle, anestrus, gestation and lactation in the ewe: evidence for an endogenous rhythm of FSH release

Two experiments were carried out to analyse FSH secretion in the ewe. The first was a long-term study during which four ewes under controlled photoperiods were checked for plasma concentrations of FSH twice daily for a period of 16 months. They were successively anestrous, cycling, gestating and lactating. The results suggested that an endogenous secretion rhythm of FSH persisted throughout each of the physiological states of the ewes. The periodic cycles of FSH production lasted about 5 days during anestrus and gestation but extended to about 6 days during estrus. One of the three waves of secretion we noted during one cycle was represented by the two periovulatory surges, the first coincident with the LH peak, the second occuring 30-40 h later. Plasma levels of FSH were similar during estrous cycles and anestrus, whereas the FSH secretion decreased gradually throughoug gestation. During lactation, large differences were observed among animals before the recovery of cyclic ovarian activity. The second experiment consisted of frequent blood sampling (every ten minutes) of eight ewes for 6 hours during anestrus. FSH was secreted differently compared to LH. No pulsatile production of FSH was demonstrated and no increase in FSH levels was seen at the time of the episodic LH surge.     

LH pulse frequency and the emergence and growth of ovarian antral follicular waves in the ewe during the luteal phase of the estrous cycle

Background

Oviduct epithelial cells (OEC) co-culture promotes in vitro fertilization (IVF) in human, bovine and porcine species, but no data are available from equine species. Yet, despite numerous attempts, equine IVF rates remain low. Our first aim was to verify a beneficial effect of the OEC on equine IVF. In mammals, oviductal proteins have been shown to interact with gametes and play a role in fertilization. Thus, our second aim was to identify the proteins involved in fertilization in the horse.

Methods & results

In the first experiment, we co-incubated fresh equine spermatozoa treated with calcium ionophore and in vitro matured equine oocytes with or without porcine OEC. We showed that the presence of OEC increases the IVF rates. In the subsequent experiments, we co-incubated equine gametes with OEC and we showed that the IVF rates were not significantly different between 1) gametes co-incubated with equine vs porcine OEC, 2) intact cumulus-oocyte complexes vs denuded oocytes, 3) OEC previously stimulated with human Chorionic Gonadotropin, Luteinizing Hormone and/or oestradiol vs non stimulated OEC, 4) in vivo vs in vitro matured oocytes. In order to identify the proteins responsible for the positive effect of OEC, we first searched for the presence of the genes encoding oviductin, osteopontin and atrial natriuretic peptide A (ANP A) in the equine genome. We showed that the genes coding for osteopontin and ANP A are present. But the one for oviductin either has become a pseudogene during evolution of horse genome or has been not well annotated in horse genome sequence. We then showed that osteopontin and ANP A proteins are present in the equine oviduct using a surface plasmon resonance biosensor, and we analyzed their expression during oestrus cycle by Western blot. Finally, we co-incubated equine gametes with or without purified osteopontin or synthesized ANP A. No significant effect of osteopontin or ANP A was observed, though osteopontin slightly increased the IVF rates.

Conclusion

Our study shows a beneficial effect of homologous and heterologous oviduct cells on equine IVF rates, though the rates remain low. Furthers studies are necessary to identify the proteins involved. We showed that the surface plasmon resonance technique is efficient and powerful to analyze molecular interactions during fertilization.     

LH pulse frequency and the emergence and growth of ovarian antral follicular waves in the ewe during the luteal phase of the estrous cycle

Background  

In the ewe, ovarian antral follicles emerge or grow from a pool of 2–3 mm follicles in a wave like pattern, reaching greater than or equal to 5 mm in diameter before regression or ovulation. There are 3 or 4 such follicular waves during each estrous cycle. Each wave is preceded by a peak in serum FSH concentrations. The role of pulsatile LH in ovarian antral follicular emergence and growth is unclear; therefore, the purpose of the present study was to further define this role.     

Relationships between FSH surges and follicular waves during the estrous cycle in mares

Individual follicles >/=15 mm were monitored daily by ultrasonography in 12 mares during the estrous cycle. Follicular waves were designated as major waves (primary and secondary) and minor waves based on maximum diameter of the largest follicle of a wave (major waves, 34 to 47 mm; minor waves, 18 to 25 mm). Dominance of the largest follicle of major waves was indicated by a wide difference (mean, 18 mm) in maximum diameter relative to the second largest follicle. Dominant follicles of primary waves (n=12) emerged (attained 15 mm) at a mean of Day 12 and resulted in the ovulations associated with estrus (ovulation=Day 0). The dominant follicle of a secondary wave (n=1) emerged on Day 2 and subsequently ovulated in synchrony with the dominant follicle of the primary wave, which emerged on Day 9. The largest follicles of minor waves (n=4) emerged at a mean of Day 5, reached a mean maximum diameter 3 days later, and subsequently regressed. There was a significant increase in mean daily FSH concentrations either 6 days (primary wave) or 4 days (minor waves) before the emergence of a wave. Mean concentrations of FSH decreased significantly 2 days after emergence of the primary wave. Divergence between diameter of the dominant and largest subordinate follicle of the primary wave was indicated by a significantly greater mean diameter of the dominant follicle than of the largest subordinate follicle 3 days after wave emergence and by the cessation of growth of the largest subordinate follicle beginning 4 days after the emergence of a wave. Surges of FSH were identified in individual mares by a cycle-detection program; surges occurred every 3 to 7 days. Elevated mean FSH concentrations over the 6 days prior to emergence of the primary wave was attributable to a significantly greater frequency of individual FSH surges before wave emergence than after emergence and to an increase in magnitude of peak concentrations of FSH associated with individual surges.     

Myometrial LH/hCG receptors during the estrous cycle and pregnancy in pigs

Receptors for luteinizing hormone/human chorionic gonadotropin (LH/hCG) have been identified in porcine, rabbit, rat, and human myometrium. To determine the estrous cycle and pregnancy related changes in the receptor capacity and affinity, radioreceptor assays were performed with membrane homogenates of porcine uterine tissues. Cycling gilts were divided into four experimental groups: I (n=6), day 1–2; II (n=5), day 6–7; III (n=5), day 11–12; and IV (n=6), day 18–20 of the estrous cycle. Pregnant pigs were divided into three experimental groups: I (n=5), day 35–40; II (n=5), day 65–70; and III (n=4), day 95–105 of pregnancy. The concentrations [femtomoles/mg protein (fmol/mg protein)] and affinities of unoccupied LH/hCG binding sites were characterized in all samples of myometrium. Receptor concentrations were highest (P<0.01) in groups II and III (19.3±2.5 and 35.8±2.1 fmol/mg protein, respectively), and was lowest in groups I and IV (5.3±1.4 and 7.5±0.7 fmol/mg protein, respectively). Receptor affinity constants (K_a) were consistent (P>0.05) throughout the estrous cycle [I, (5.1±1.5)×10⁹; II, (3.0±0.8)×10⁹; III, (3.2±0.9)×10⁹; IV, 5.5±0.7×10⁹ lm⁻¹]. Plasma hormone concentrations of progesterone, estrogen and LH were typical of values noted at these times. During pregnancy, receptor concentrations were greatest (P<0.05) in group II (85.4±18.5 fmol/mg protein). In groups I and III receptor numbers were 10.8±2.3 and 26.7±6.6 fmol/mg protein, respectively. The K_a in group I was 10 times greater (P<0.05) than K_a in groups II and III, (I, 3.1±0.9×10¹⁰ lm⁻¹; II, 3.4±0.3×10⁹ lm⁻¹; III, 3.3±1.1×10⁹ lm⁻¹). Plasma hormone concentrations typically found during pregnancy were noted. The function of these LH/hCG binding sites remains unknown; however, changes in receptor capacity during the estrous cycle and pregnancy support a role for modulation of the receptor by hormonal factors.