Effect of the peripubertal pattern of LH and FSH secretion on in-vitro oestradiol-17 beta secretion and follicular growth within juvenile rat ovaries

Juvenile rat ovaries were placed in perfusion culture and exposed to (1) no gonadotrophin, (2) tonic NIH-FSH (200 ng RP-1 equiv./ml) or (3) NIH-FSH + NIH-LH pulses (2/h, amplitude = 80 ng RP-1 equiv./ml). After 3 h of perifusion, the ovaries were prepared for histological analysis and the perifusate assayed for oestradiol-17 beta. Since the NIH-FSH preparation is contaminated with LH, a second experiment was conducted using recombinant bovine LH and FSH. Ovaries were perifused for 3 h with (1) no hormones, (2) recombinant FSH (200 ng/ml) or (3) recombinant FSH plus 25 ng recombinant LH/ml. NIH-FSH alone increased the number of mid-size antral follicles (P less than 0.05) and decreased the number of small antral follicles (P less than 0.05). Pulsatile LH in the presence of FSH increased the number of mid-size antral follicles without reducing the number of small antral follicles. Studies with recombinant FSH and LH demonstrated that both FSH and LH are necessary to stimulate follicles to grow, indicating that the growth-promoting property of the NIH-FSH is due to the presence of both FSH and LH. Regardless of whether NIH or recombinant gonadotrophins were used, follicular growth was induced without increasing oestradiol secretion. These results demonstrate that enhanced oestradiol secretion is not essential for the induction of follicular growth, while both LH and FSH are necessary to stimulate small antral follicles to grow into mid-size antral follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of a GnRH agonist on follicular dynamics and response to FSH stimulation in prepubertal calves

Endocrine control of follicular growth was determined by observing the left ovary of prepubertal calves previously treated with a potent GnRH agonist for 13 days. The ovarian response to hormonal stimulation was determined using the right ovaries of the same animals. Three-month-old crossbred calves were assigned to one of the two following treatment groups: 1) saline control for 13 days, with purified porcine FSH for the last 3 days (n = 5); and 2) GnRHa for 13 days, with purified porcine FSH for the final 3 days (n = 5). The left ovaries were removed from all calves after 10 days, and the right ovaries were removed at the end of treatment. Plasma concentrations of FSH, LH and oestradiol-17 beta were followed up during the GnRHa and pFSH treatments. The maximum macroscopic diameter of the F1 follicle, as determined by daily ultrasonography, did not differ between GnRHa-treated calves (from 6.6 to 10.4 mm) and the saline control calves (from 6.7 to 10.3 mm). Histological analysis of the ovaries showed that the number of follicles > 0.40 mm in diameter varied greatly for calves of the two groups (from 11 to 220 at 10 days). GnRHa significantly increased the mean number of follicles (total and nonatretic) of size class > 5.4 mm as compared to saline control calves (P < 0.05). The FSH treatment significantly increased the mean number of follicles 3.00-5.4 and > 5.4 mm in diameter (P < 0.05), with no change in the number of follicles smaller than 3.00 mm. The rate of atresia of large follicles (3.01-5.40 mm) was significantly reduced by purified porcine FSH treatment in both groups (P < 0.05). In no case did the GnRHa induce ovulation or luteinization of follicles. The LH and FSH concentrations increased transiently after GnRHa treatment on the first day, but afterwards, both hormones increased to only one sixth of what was observed after the initial GnRHa injection treatment. This increase in LH and FSH was observed 1 h after GnRHa treatment on each consecutive day of the experiment and were significantly different in the control group (0 h versus 1 h versus 2 h x saline control versus GnRH agonists groups; P < 0.01). During the superovulatory treatment, FSH concentrations peaked at around 0.70 ng.mL-1 in both saline- and GnRHa-treated groups on the first day but on the last day of surovulatory treatment, FSH concentrations were higher in GnRHa agonist-treated calves than in the control calves (day 11 versus day 12 versus day 13 x saline control versus GnRH agonist treatment groups; P < 0.01). LH profiles were unchanged by surovulatory treatment. Concentrations of oestradiol-17 beta increased significantly over the three days (P < 0.001) of the superovulatory treatments in both groups (P < 0.01). These results indicate that GnRH agonist treatment allows recruited antral follicles to pursue their growth during the early selection process via sustained FSH and LH secretion allowing more than a single large follicle to maintain their growth without going to atresia.     

LH, FSH, PRL and ACTH cells in pituitary glands of cows with ovarian cysts

We investigated morphometrically and densitometrically LH, FSH, PRL, and ACTH cells in pituitary glands in cows with follicular cysts and normal animals. Three groups were considered: cows in diestrus, cows in periestrus and cows with follicular cysts. The periestrous group was obtained by merging pro- and metaestrous groups, since the two did not differ when evaluated separately. The attribution to groups was confirmed by plasma progesterone measurements and by postmortem examination of the ovaries. After immunocytochemical labeling the pituitary cells were counted, and their area and total immunoreactivity were measured. The results suggest hypofunction of the LH cells (P < 0.05) and hyperfunction of the ACTH cells (P < 0.05) from the cows of the follicular cyst group. The FSH and PRL cells were similar in the three groups. These changes are consistent with the reduced LH release reported by most authors in cows with follicular cysts and should be relevant to the pathogenesis of the bovine follicular cysts. The subnormal activity of LH cells might be secondary to the activation of the ACTH cells.     

Intrafollicular steroids and anti-mullerian hormone during normal and cystic ovarian follicular development in the cow

Development of follicular cysts is a frequent ovarian dysfunction in cattle. Functional changes that precede cyst formation are unknown, but a role for anti-Müllerian hormone (AMH) in the development of follicular cysts has been suggested in humans. This study aimed to characterize intrafollicular steroids and AMH during follicular growth in a strain of beef cows exhibiting a high incidence of occurrence of follicular cysts. Normal follicular growth and cyst development were assessed by ovarian ultrasonography scanning during the 8 days before slaughtering. Experimental regression of cysts was followed by rapid growth of follicles that reached the size of cysts within 3-5 days. These young cysts exhibited higher intrafollicular concentrations of testosterone, estradiol-17beta, and progesterone than large early dominant follicles did in normal ovaries, but they exhibited similar concentrations of AMH. Later-stage cysts were characterized by hypertrophy of theca interna cells, high intrafollicular progesterone concentration, and high steroidogenic acute regulatory protein mRNA expression in granulosa cells. Progesterone and AMH concentrations in the largest follicles (> or =10 mm) and cysts were negatively correlated (r = -0.45, P < 0.01). Smaller follicles (<10 mm) exhibited higher intrafollicular testosterone and estradiol-17beta concentrations in ovaries with cysts compared to normal ovaries. During follicular growth, AMH concentration dropped in follicles larger than 5 mm in diameter and in a similar way in ovaries with and without cysts. In conclusion, enhanced growth and steroidogenesis in antral follicles <10 mm preceded cyst formation in cow ovaries. Intrafollicular AMH was not a marker of cystic development in the cow, but low AMH concentrations in cysts were associated with luteinization.     

Superstimulation of ovarian follicular growth with FSH oocyte recovery, and embryo production from Zebu (Bos indicus) calves: effects of treatment with a GnRH agonist or antagonist

The capacity of heifer calves of a late sexually maturing Zebu (Bos indicus) genotype to respond to superstimulation with FSH at a young age and in vitro oocyte development were examined. Some calves were treated with a GnRH agonist (deslorelin) or antagonist (cetrorelix) to determine whether altering plasma concentrations of LH would influence follicular responses to FSH and oocyte developmental competency. Brahman calves (3-mo-old; 140 +/- 3 kg) were randomly assigned to 3 groups: control (n = 10); deslorelin treatment from Day -8 to 3 (n = 10); and cetrorelix treatment from Day -3 to 2 (n = 10). All calves were stimulated with FSH from Day 0 to 2, and were ovariectomized on Day 3 to determine follicular responses to FSH and to recover oocytes for in vitro procedures. Before treatment with FSH, heifers receiving deslorelin had greater (P < 0.001) plasma LH (0.30 +/- 0.01 ng/ml) than control heifers (0.17 +/- 0.02 ng/ml), while plasma LH was reduced (P < 0.05) in heifers treated with cetrorelix (0.13 +/- 0.01 ng/ml). Control heifers had a surge release of LH during treatment with FSH, but this did not occur in heifers treated with deslorelin or cetrorelix. All heifers had large numbers of follicles > or = 2 mm (approximately 60 follicles) after superstimulation with FSH, and there were no differences (P > 0.10) between groups. Total numbers of oocytes recovered and cultured also did not differ (P > 0.05) for control heifers and heifers treated with deslorelin or cetrorelix. Fertilization and cleavage rates were similar for the 3 groups, and developmental rates to blastocysts were also similar. Zebu heifers respond well to superstimulation with FSH at a young age, and their oocytes are developmentally competent.     

Number of oocytes obtained from cows by OPU in early, but not late lactation increased with plasma insulin and estradiol concentrations and expression of mRNA of the FSH receptor in granulosa cells

The effects of lactation stage and hormonal profile on the quality and quantity of oocytes and the gonadotrophic sensitivity of granulosa cells (GC) from small antral follicles obtained by sequential aspirations from ovaries of high producing dairy cows were examined. Cows in late lactation (263(+/- 60) days postpartum) and 98(+/- 16) days pregnant in positive energy balance (EB) showed no significant changes in plasma concentrations of estradiol, progesterone, insulin, IGF1 or expression of mRNA of the FSH receptor in GC from small antral follicles during the 49 days experimental period. There were no changes in the number and quality of oocytes obtained from each aspiration. In cows in early lactation (72.8 +/- 6 days postpartum), plasma insulin concentrations increased and were positively correlated with plasma estradiol concentration. Due to the sequential aspirations progesterone blood concentrations were low in early lactation cows. Expression of mRNA of the FSH receptor increased in GC from small antral follicles of early lactation cows together with the number of oocytes obtained with aspiration sessions. No differences were found in morphological quality or function between oocytes obtained from small antral follicles from cows in early or late lactation. In early, but not late lactation, the number of oocytes was correlated with both insulin and E2 plasma concentrations. Improved EB and sensitivity of GC to FSH may be involved in oocyte recruitment in early lactation.     

Immunocytochemical study of anti-Müllerian hormone in sheep ovarian follicles during fetal and post-natal development

Anti-Müllerian hormone (AMH) was detected in perinatal and postnatal sheep ovaries, using avidin-biotin immunohistochemistry with a monoclonal antibody specific for ruminant AMH. Immunoreactivity was limited to granulosa cells, and was influenced both by the degree of follicular development, and by the age of the animal. In the fetus, only the most advanced follicles exhibited a faint immunoreactivity at 120 days gestation, and no reaction was observed in younger animals. Immediately before and after birth, primordial follicles were still negative, but a faint reaction was elicited in young growing follicles, increasing with follicle size. Strong immunoreactivity was visible in antral follicles, especially in the innermost granulosa cell layers, close to the oocyte and lining the antral cavity.     

Suppression of plasma FSH concentrations with bovine follicular fluid blocks ovulation in GnRH-treated seasonally anoestrous ewes

The specific requirement for FSH in the final stages of preovulatory follicle development was assessed in seasonally anoestrous ewes given 2-h injections of GnRH (250 ng/injection), with (N = 10) or without (N = 10) concurrent treatment with bovine follicular fluid (bFF: 2 ml given i.v. at 8-h intervals). Treatment with bFF significantly (P less than 0.01) suppressed plasma FSH concentrations, but, at least for the first 30 h of treatment, did not influence the magnitude of GnRH-induced LH episodes (mean max. conc. 3.00 +/- 0.39 and 3.63 +/- 0.51 ng/ml for bFF-treated and control ewes, respectively). Of 10 animals treated with GnRH for 72 h, 5/5 control ewes showed oestrus and ovulated whereas 0/5 bFF-treated ewes showed oestrus or ovulated in response to GnRH treatment. There was, however, a transient (13.2 +/- 1.0 h) increase in plasma LH concentrations in the ewes given bFF (mean max. conc. 4.64 +/- 1.57 ng/ml), which was coincident with the preovulatory LH surge recorded in animals given GnRH alone. In 10 GnRH-treated ewes slaughtered after 32 h of treatment, the mean diameter of the largest antral follicle was significantly (P less than 0.001) greater in control ewes (5.92 +/- 0.17 mm) than in animals that were also given bFF (3.94 +/- 0.14 mm). In addition, the incidence of atresia in the 3 largest antral follicles present at this time was greater in bFF-treated ewes. These results show that, when plasma FSH concentrations are suppressed by administration of bFF, although the magnitude of GnRH-induced LH episodes is unchanged, preovulatory follicular development is impaired and ovulation does not occur.(ABSTRACT TRUNCATED AT 250 WORDS)     

Superovulation of dairy cows with purified FSH supplemented with defined amounts of LH

This study investigated the effects of a purified follicle stimulating hormone (FSH) preparation supplemented with three different amounts of bovine luteinizing hormone (bLH) and a commercially available FSH with a high LH contamination on superovulatory response, plasma LH and milk progesterone levels in dairy cows. A total of 112 lactating Holstein-Friesian crossbred dairy cows were used for these experiments; the cows were randomly assigned to treatment groups consisting of purified porcine FSH (pFSH) supplemented with bLH. Group 1 was given 0.052 IU LH 40 mg armour units (AU) FSH (n = 6); Group 2 was given 0.069 IU LH (n = 32); Group 3 received 0.423 IU LH (n = 34); while Group 4 cows (n = 36) were superovulated with a commercially available FSH-P((R)). This compound appeared to contain 8.5 IU LH 40 mg AU FSH according to bioassay measurement. All animals received a total of 40 mg AU FSH at a constant dose twice daily over a 4-d period. Levels of milk progesterone and plasma LH were determined during the course of superovulatory treatment. The Group 1 treatment did not reveal multiple follicular growth, and no embryos were obtained. Superovulation of Group 3 cows resulted in significantly (P<0.05) more corpora lutea (CL; 12.6+/-1.1) and fertilized ova (5.1+/-1.3) compared with Groups 2 and 4 (10.1+/-0.9 and 2.6+/-0.6, 9.0+/-0.9 and 2.7+/-0.5, respectively). Due to a high percentage of degenerated embryos (33%) Group 3 yielded only one more transferable embryo than Groups 2 and 4. Among groups, LH levels differed in the period prior to induction of luteolysis and were similar thereafter. The progesterone pattern following FSH LH administration reflected the amount of LH supplementation. Milk progesterone levels on the day prior to embryo collection were correlated to the number of CLs and recovered embryos. It is concluded that under the conditions of our experiment superovulation with 0.423 IU LH 40 mg AU FSH may yield a significantly improved superovulatory response in dairy cows. It is further suggested that LH supplementation exerts its effects mainly on follicular and oocyte maturation during the period prior to luteolysis.     

Circulating gonadotropins levels and contribution of different large antral follicles to isofolliculia in sheep

The aim of this study was first to search for isofolliculia in right and left ovaries during postnatal development of Ouled Djellel ewe lambs, a non-seasonal breed of sheep. In addition, the contribution of different sizes of large antral follicles to this phenomenon was studied, and finally the variations in both plasma FSH and LH levels during this period of life were determined. Plasma was collected from groups of four ewe lambs at 0 (<24 h), 1 week, and every 2 weeks from 4 to 14 weeks of age. Thereafter, each group was slaughtered, right and left ovaries recovered, weighed and their length and width measured. One ovary was fixed in Bouin–Holland's solution and prepared for histological study. The other one was immediately frozen and cut in a cryostat and prepared for histochemical study. This latter method was used to detect the activity of alkaline phosphatase, lactate dehydrogenase, NADH₂-tetrasolium reductase, and glucose-6-phosphate dehydrogenase enzymes. Number and size of antral follicles and their contribution to isofolliculia were determined from ovarian sections of both studies. Isofolliculia was seen in right and left ovaries of Ouled Djellel ewe lambs at 4, 6, 8, and 10 weeks of age. This phenomenon was characterized by the presence of large antral follicles almost equal in size and total enzymatic inactivity in the interstitium. Weight and dimensions of right and left ovaries increased rapidly from birth to 4 weeks of age, and then rose gradually to week 8 and then rising again to week 10, followed by a decline at 12 and 14 weeks of age. All large antral follicles contributed to isofolliculia in right and left ovaries but, the percentage of antral follicles <2 mm at 4, 6, 8, and 10 weeks of age were significantly greater than the percentage of follicles ≥2 and <3 mm and the contribution of follicles ≥3 mm was the lowest. FSH levels increased slowly from birth to 6 weeks of age then, increased rapidly to week 10, followed by a decline at weeks 12 and 14. LH was low at birth and the level increased slowly to 8 weeks of age, followed by a further rapid increase at 10 weeks of age. All parameters studied did not show any significant differences between the right and left ovary. It was concluded that isofolliculia occurred between 4 and 10 weeks of age in left and right ovaries of Ouled Djellel ewe lambs. This phenomenon was characterized by the increase of both ovarian weights and dimensions, and of plasma FSH and LH levels. All large antral follicles ≥1 mm in diameter contributed to isofolliculia but the contribution of antral follicles <2 mm was greater than the contribution of antral follicles ≥2.     

Modulatory action of FSH on LH-induced follicular growth in rats

Three to four ovaries from rats on the day of di-oestrus I were placed in perifusion culture at 10:30 h and exposed to (1) no gonadotrophin (in-vitro controls); (2) tonic FSH (200 ng RP-1/ml); (3) tonic LH (30 ng RP-1/ml); (4) tonic FSH + tonic LH; or (5) tonic FSH plus hourly pulses of LH (amplitude = 50 ng/ml). The total amount of LH administered was 3060 ng RP-1 regardless of mode of delivery. After culture for 3 h, the ovaries were prepared for histological analysis. Compared to in-vitro controls, tonic LH stimulation increased the number of follicles with greater than 1.6 X 10(5) granulosa cells (P less than 0.05); it was estimated that each follicle in the larger size class increased by 5.5 +/- 2.7 X 10(4) cells. Tonic FSH or tonic FSH + tonic LH treatment did not promote growth into the 1.6 X 10(5) cell class. In the presence of tonic FSH, hourly LH pulses induced follicular growth similar to that observed after tonic LH treatment. The data demonstrate that LH promotes the growth of follicles in vitro. FSH modulates this stimulatory action of LH, allowing it to be expressed when LH is administered in hourly pulses.     

Distribution of the intermediate filament proteins vimentin,keratin, and desmin in the bovine ovary

The distribution of the intermediate filament (IF) proteins desmin, keratin, and vimentin was studied immunohistochemically in bovine ovaries. Special attention was paid to granulosa cells to examine possible marked changes of IF distribution in relation to folliculogenesis during ovarian development. Therefore, ovaries were used from fetuses from 3 months of gestation onward, calves, heifers, and cows. In all ovaries, desmin immunoreactivity was restricted to smooth muscle cells in blood vessel walls. Keratin appeared a characteristic of the ovarian surface epithelium. Co-localization of keratin and vimentin was observed in the epithelium of rete ovarii tubules in fetuses and calves, and in cortical cord epithelium and pregranulosa cells of primordial follicles in fetuses at 3–7 months of gestation. Vimentin was demonstrated in endothelium and in fibroblasts. In addition, vimentin immunoreactivity was present in granulosa cells of primary, secondary, and antral follicles. In antral follicles, these granulosa cells mainly had an elongated appearance and either contained an oblong or a round nucleus. Those with an oblong nucleus were characteristic for atretic antral follicles. In nonatretic follicles, numerous vimentin immunore-active, elongated granulosa cells with a round nucleus were observed, especially in the peripheral granulosa layer and in small (<3 mm in diameter) antral follicles. Additionally, in antral follicles, protrusions of vimentin-positive corona radiata cells were observed, that penetrated the zona pellucida to contact the oocyte. The data show that the distribution of vimentin containing IFs is associated with various aspects of granulosa cell activity, as mitosis, atresia, and intercellular transport. © 1995 Wiley-Liss, Inc.     

The temporal relationship between patterns of LH and FSH secretion, and development of ovulatory-sized follicles during the mid- to late-luteal phase of sheep

The aim of the present study was to investigate the temporal relationship between the secretory pattern of serum LH and FSH concentrations and waves of ovarian antral follicles during the luteal phase of the estrous cycle in sheep. The growth pattern of ovarian antral follicles and CL were monitored by transrectal ultrasonography and gonadotropin concentrations were measured in blood samples collected every 12 min for 6 h/d from 7 to 14 d after ovulation. There were two follicular waves (penultimate and final waves of the cycle) emerging and growing during the period of intensive blood sampling. Mean and basal LH concentrations and LH pulse frequency increased (P < 0.001) with decreasing progesterone concentration at the end of the cycle. Mean and basal FSH concentrations reached a peak (P < 0.01) on the day of follicular wave emergence before declining to a nadir by 2 d after emergence. None of the parameters of pulsatile LH secretion varied significantly with either the emergence of the final follicular wave or with the end of the growth phase of the largest follicle of the penultimate wave of the cycle. However, mean and basal LH concentrations did increase (P < 0.05) after the end of the growth phase of the largest follicle of the final follicular wave of the cycle. Furthermore, the end of the growth phase of the largest follicle of the final wave coincided with functional luteolysis. In summary, there was no abrupt or short-term change in pulsatile LH secretion in association with the emergence or growth of the largest follicle of a wave. We concluded that the emergence and growth of ovarian antral follicles in follicular waves do not require changes in LH secretion, but may involve changes in sensitivity of ovarian follicles to serum LH concentrations.     

Quantitative autoradiographic study of FSH binding sites in prepubertal ovaries of three strains of rats

Iodinated FSH was injected to 18- and 36-day-old rats of 3 strains (03, 04 and 12) with different sensitivity to FSH (12 less than 03 less than 04) and autoradiography was performed on histological sections of the labelled ovaries. Specific labelling was quantified by microphotometry on histological slides, on granulosa cells of individual follicles with different sizes (greater than 80 micron diameter) and qualities. In small preantral follicles (less than 160 micron diameter) the labelling was low and homogeneous within the granulosa; it increased between 18 and 36 days of age in the 3 strains. At 36 days, ovaries were characterized by the presence of large preantral and antral follicles with a higher labelling in the outer layers of granulosa (near the theca), compared to the inner layers. In definitely atretic follicles, a loss of binding sites was detected in the outer layers. In rats of Strains 03 and 04, the number of binding sites for FSH in the outer layers of granulosa of follicles with a diameter of greater than 160 micron increased with follicular size; no change was detected in follicles of Strain 12 rats. The low number of binding sites for FSH and the lack of terminal maturation which characterize the follicles of strain 12 rats can be related to the poor and delayed follicular development, the low sensitivity to exogenous FSH and the low fertility of the animals of this strain.     

Anti-mullerian hormone is an endocrine marker of ovarian gonadotropin-responsive follicles and can help to predict superovulatory responses in the cow

The major limitation to the development of embryo production in cattle is the strong between-animal variability in ovulatory response to FSH-induced superovulation, mainly due to differences in ovarian activity at the time of treatment. This study aimed to establish whether anti-Müllerian hormone (AMH) was an endocrine marker of follicular populations in the cow, as in human, and a possible predictor of the ovarian response to superovulation. Anti-Müllerian hormone concentrations in plasma varied 10-fold between cows before treatment and were found to be highly correlated with the numbers of 3- to 7-mm antral follicles detected by ovarian ultrasonography before treatment (r=0.79, P<0.001) and the numbers of ovulations after treatment (r=0.64, P<0.01). Between-animal differences in AMH concentrations were found to be unchanged after a 3-mo delay (r=0.87, P<0.01), indicating that AMH endocrine levels were characteristic of each animal on a long-term period. The population of healthy 3- to 7-mm follicles was the main target of superovulatory treatments, contained the highest AMH concentrations and AMH mRNA levels compared with larger follicles, and contributed importantly to AMH endocrine levels. In conclusion, AMH was found to be a reliable endocrine marker of the population of small antral gonadotropin-responsive follicles in the cow. Moreover, AMH concentrations in the plasma of individuals were indicative of their ability to respond to superovulatory treatments.     

Immediate and delayed effects of heat stress on follicular development and its association with plasma FSH and inhibin concentration in cows

The aim of this study was to characterize the immediate effects of heat stress on plasma FSH and inhibin concentrations, and its involvement in follicular dynamics during a complete oestrous cycle, and to examine a possible delayed effect of heat stress on follicular development. Holstein dairy cows were oestrous synchronized and randomly assigned to either cooled (n = 7) or heat-stressed (n = 6) treatment groups. During a complete oestrous cycle, control cows, which were cooled, maintained normothermia, whereas heat-stressed cows, which were exposed to direct solar radiation, developed hyperthermia. At the end of this oestrous cycle (treated cycle), both groups were cooled and maintained normothermia for the first 10 days of the subsequent oestrous cycle. Throughout this period, follicular development was examined by ultrasonography, and plasma samples were collected. During the second follicular wave of the treated oestrous cycle, a significantly larger cohort of medium sized follicles (6-9 mm) was found in heat-stressed cows than in cooled cows (P < 0.05). The enhanced growth of follicles in this wave in heat-stressed cows was associated with a higher plasma FSH increase which lasted 4 more days (days 8-13 of the oestrous cycle; P < 0.05), and coincided with a decrease in the plasma concentration of immunoreactive inhibin (days 5-18 of the oestrous cycle; P < 0.05). During the follicular phase (days 17-20 of the treated cycle), heat-stressed cows showed an increase in the number of large follicles (>/= 10 mm), and the preovulatory plasma FSH surge was significantly higher in heat-stressed cows than in cooled cows (P < 0.01). The effect of heat stress was also observed during the first follicular wave of the subsequent cycle: the postovulatory plasma FSH concentration was higher (P < 0.01), but fewer medium follicles developed, and the first follicular wave decreased at a slower rate in previously heat-stressed cows than in cooled cows (0.40 and 0.71 follicles per day, respectively). This study shows both immediate and delayed effects of heat stress on follicular dynamics, which were associated with high FSH and low inhibin concentrations in plasma. These alterations may have physiological significance that could be associated with low fertility of cattle during the summer and autumn.     

Effects of FSH and LH on incorporation of [3H]thymidine into follicular DNA

To assess the roles of FSH and LH on follicular growth, after various experimental manipulations, hamster follicles were sorted into 10 stages and incubated for 4 h with [3H]thymidine. Stages 1-4 correspond to follicles with 1-4 layers of granulosa cells, respectively; Stage 5 = 5 or 6 layers of granulosa cells plus theca; Stage 6 = 7-8 layers of granulosa cells plus theca; Stage 7 = early formation of the antrum; Stages 8-10 = small, intermediate and large antral follicles, respectively. Phenobarbitone sodium injected at 13:00 h on pro-oestrus blocked the normal rise of blood FSH and LH concentrations at 15:00 h and prevented the increase of [3H]thymidine incorporation into follicles of Stages 1-9. The optimal treatment to reverse the effects of phenobarbitone was 1 microgram FSH and 2 micrograms LH injected i.p. at 13:00 h which restored DNA replication to follicles of Stages 2-10: FSH acted primarily on Stages 2-5 and LH on Stages 5-10. Injection of phenobarbitone at 13:00 h on prooestrus followed by 2.5 micrograms FSH at 22:00 h restored DNA synthesis by the next morning to follicles at Stages 1-8. In hamsters hypophysectomized at 09:00 h on the day of oestrus (Day 1), injection on Day 4 of 2.5 micrograms FSH restored DNA synthesis 6 h later to Stage 2-6 follicles. Unilateral ovariectomy on Day 3 resulted 6 h later in an acute rise in FSH and LH and change of follicles from Stage 4 to Stage 5 but, paradoxically, there was decreased synthesis of DNA in follicles of Stages 5-10.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of the peripubertal pattern of FSH, LH and prolactin secretion in regulating in-vitro steroidogenesis and follicular growth within juvenile rat ovaries

Juvenile rat ovaries were placed in perifusion culture and exposed to (1) tonic FSH (200 ng PR-1 equiv./ml), (2) LH pulses (2/h, amplitude = 80 ng RP-1 equiv./ml), (3) tonic FSH and LH pulses, (4) tonic FSH with LH mini-surges, or (5) tonic FSH with LH and prolactin mini-surges. The LH mini-surge consisted of a series of 80 ng/ml pulses (2/h) with LH increasing to 180 ng/ml for 2 h then returning to the 80 ng/ml pulses. The prolactin mini-surge consisted of a series of 15 ng/ml pulses (2/h) with prolactin increasing to 40 ng/ml for 2 h before returning to the 15 ng/ml pulses. The LH mini-surge occurred at 14:00 h daily while a prolactin mini-surge occurred at 14:00 h and 06:00 h daily. Ovaries were perifused for 0 (in-vivo control), 24 or 48 h, incubated for 1 h in hormone-free medium to assess steroid secretion and subsequently prepared for histological analysis. After a 24 h exposure to FSH, oestradiol secretion was increased, while exposure to LH pulses enhanced progesterone secretion. Treatment with FSH, LH pulses or FSH plus LH pulses decreased the number of small antral follicles by 24 h of perifusion compared to control (P less than 0.05). The LH mini-surge maintained the small and medium-sized antral follicles after 24 h and increased the number of preovulatory-sized follicles over controls by 48 h (P less than 0.05). Prolactin/LH mini-surges increased the number of preovulatory-sized follicles within 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antral follicle growth and endocrine changes in prepubertal cattle,sheep and goats

In the growing heifer calve, there is an early post-natal, gonadotrophin driven increase in ovarian antral follicle growth. The endocrine regulation of and reason for this initial stimulation of ovarian follicular development are not fully understood. This initial endocrine activity appears to be later held in check by negative feedback suppression mechanisms until the heifer is of a sufficient body size to initiate oestrous cycles and to reproduce. There is increasing evidence from recent ultrasonographic studies, performed in the same groups of prepubertal heifer calves, that the development of ovarian antral follicles and tubular genitalia occur in parallel. There appear to be two distinct periods of enhanced development of the reproductive organs, from 2 to 14 weeks of age and again from 34 to 60 weeks of age, or just prior to puberty. First ovulation in heifers is preceded by a gradual increase in pulsed LH secretion, which results in enhanced antral follicle development and oestrogen production. It was demonstrated that prepubertal heifers produced recurrent antral follicular waves; maximum sizes and life span of the dominant follicles of waves, as well as periodicity and FSH dependency of wave emergence were similar to those in adult cattle. In does, no Graafian follicles are seen at birth and total follicle numbers increase to 2 months of age, and then decline to 5 months of age. In ewe lambs, studies using transrectal ovarian ultrasonography showed that antral follicle recruitment and growth increased after the first 2 months of age and just before puberty. This bi-phasic pattern of changes in ovarian follicle recruitment and growth is strikingly similar to that in heifer calves, but it contrasts with earlier post-mortem examinations of ovaries in ewe lambs. Unlike in cattle and adult ewes, the rhythmic pattern of follicular wave emergence was not established in pre- and peripubertal ewe lambs. The early increase in antral follicle numbers and size in ewe lambs may be, at least in part, due to changes in FSH release and potency, and enhanced follicle production prior to first ovulation is probably caused by an increase in the frequency of LH pulses.     

Role of intrafollicular regulators and FSH in growth and development of large antral follicles in sheep

Manipulation of circulating concentrations of hormones and ovarian follicle status was carried out on Day 11-12 of the oestrous cycle in sheep. All follicles visible on the ovary were ablated by cautery and ewes were treated with oestradiol or ovine follicular fluid (oFF) to suppress FSH or with PMSG to increase circulating gonadotrophic activity. One group underwent unilateral ovariectomy which greatly increased endogenous FSH and was the only treatment which significantly affected LH pulse frequency. The size distribution of antral follicles, the extent of atresia and the mitotic index of granulosa cells of follicles on Day 15 showed that (a) treatment with oFF inhibited the growth of follicles beyond 2 mm diameter by suppressing the mitotic index of the granulosa cells and (b) the concentration of FSH in peripheral plasma was related to the ability of small antral follicles to grow during the late luteal-early follicular phase of the cycle. Subsequently, it was demonstrated that oFF inhibits, in a dose-dependent manner, folliculogenesis sustained by PMSG in ewes on Days 12-15. Inhibition of folliculogenesis was represented by a decrease in those follicles greater than 4 mm, an increase in the relative proportion of follicles less than 2 mm, and minimal change in the average number of follicles visible on the ovarian surface, and a decrease in the mitotic index of granulosa cells of follicles less than 2 mm. There was no change in the extent of atresia.(ABSTRACT TRUNCATED AT 250 WORDS)