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)     

Reproductive cycles of horses

Horses are long-day breeders. During the breeding season, cycle length is about 22 days with 5-7 days of oestrus. Gonadotroph cells are localized in the pars distalis as well the pars tuberalis of the pituitary and heterogeneity in the pattern of LH and FSH storage within the gonadotroph population is considered the basis for the differential regulation of gonadotrophin secretion throughout the reproductive cycle. No short and distinct periovulatory LH peak exists in the mare. The equine ovary has an extreme large size and weight. One to two major follicular waves develop per cycle. The preovulatory follicle reaches an average size of 40 mm. Only granulosa cells develop into luteal cells. Progesterone increases at the time of ovulation and reaches maximal concentrations on day 8. Functional luteolysis occurs around day 15 and is initiated by endometrial secretion of PGF(2α). In contrast to other species, no significant luteal oxytocin synthesis exists in the mare. During the oestrous cycle, uterus, vagina and endometrium undergo pronounced changes related to variations in the endocrine milieu. Seasonal reproductive activity is stimulated by photoperiod together with exogenous factors. The anovulatory season can be differentiated into an autumn transitional phase, a mid-anovulatory period and a spring transitional phase bringing the mare back into cyclic activity. During the mid-anovulatory period, follicular development is minimal. The beginning of the spring transitional period is characterized by the development of 1-3 anovulatory follicular waves before ovulation occurs and the most important factor for the re-initiation of ovulatory activity is the occurrence of repeated pronounced increases in circulating LH.     

The function of the growing follicle.

The follicle plays a major role in the dual function of the ovary--oocyte maturation and release and steroidogenesis required for regulating its own growth and providing the proper environment in reproductive organs for the transport of gametes and nidation. Some aspects of how follicles attain their functional competence following a series of developmental changes are discussed. The presentation is based on data obtained mainly in rodents in which follicular development occurs postnatally. The peak activity of follicular growth occurs during the 1st week of life, but not until the 5th day is follicular development clearly dependent upon gonadotrophin stimulation. The formation of the theca layer and zona pellucida, differentiation of the vascular system and competence to respond to gonadotrophins are acquired during the 2nd week. FSH alone is primarily responsible for granulosa cell proliferation and the integrity of the granulosa cell membrane, but has little differential effect on steroidogenic enzymes. Synergism of FSH and LH promotes an enrichment of the theca layer, enhancement of vascular development and antrum formation, and induces a marked differential stimulation of 20alpha-hydroxysteroid dehydrogenase, aromatizing and cholesterol side-chain cleavage systems. The number of gonadotrophin receptors on granulosa and theca cells increases with follicular development. Steroids secreted by the ovary seem to modulate follicular growth, not only by effects upon FSH and LH release but also by a local influence within the ovary. A number of physiological events related to follicular function are explained according to these observations.     

Inhibition of LHRH-induced LH and FSH release by gonadotrophin surge-attenuating factor (GnSAF) from human follicular fluid

It has been suggested that in superovulated women the endogenous LH surge is attenuated by a non-steroidal factor, called gonadotrophin surge-attenuating factor (GnSAF), which reduces gonadotrophin secretion in response to LHRH. To determine whether human follicular fluid (hFF) from superovulated women contains GnSAF activity, the secretion of LH and FSH by cultured sheep pituitaries was studied. After charcoal extraction of steroids, hFF was treated by heparin/Sepharose chromatography, which reversibly binds inhibin. The effects of whole hFF and the bound and unbound fractions on basal and LHRH-induced gonadotrophin secretion were then assessed. Steroid-free hFF significantly reduced basal FSH, but not basal LH, secretion, and significantly attenuated the LH and FSH responses to LHRH. The bound (inhibin) fraction significantly decreased both basal and LHRH-induced FSH secretion but did not affect LH release. The unbound fraction had no effect on basal LH or FSH secretion, but significantly attenuated LHRH-induced secretion of both LH and FSH. We conclude that the unbound fraction of hFF from superovulated women contains GnSAF. It has been demonstrated that GnSAF is a non-steroidal factor and its activity is distinct from that of inhibin.     

Changes in plasma gonadotrophin and prolactin concentrations following castration of the pony stallion

Concentrations of gonadotrophins and prolactin were recorded in pony stallions castrated during the early breeding season, to examine the regulatory role of the gonad at a time when testosterone has been postulated to exert positive feedback on LH secretion. Further, gonadotrophin concentrations in geldings are reported to return to values within the normal range of the entire stallion. In an attempt to characterize this species-specific reversal, the gonadotrophin concentrations of 6 male ponies castrated on 25 March were monitored for 4 months, and 4 stallions were used to generate control data. Blood samples were collected daily, from 3 d before to 10 d after castration (Day 0), and weekly thereafter until Day 122. The pituitary response to castration was immediate. Castration resulted in a previously unreported, dramatic (13-fold) but transient (3 d) surge in circulating concentrations of LH. Concentrations of LH and FSH increased in a logarithmically scaled (LH, R2 = 0.77; FSH, R2 = 0.93) manner over the subsequent 5 wk, during which temporal changes in concentrations of both hormones were strongly correlated (R2 = 0.97). The ratio of plasma gonadotrophin concentrations was consistent throughout (LH:FSH, 1.43 +/- 0.04). Maximal concentrations of LH (20.58 +/- 1.97 ng/mL, Day 34.8 +/- 3.2) were attained approximately 2 wk before the peak in FSH (16.99 +/- 1.97 ng/mL, Day 49.7 +/- 3.0). Plasma gonadotrophin concentrations exceeded those of entire stallions throughout the study. The equine testes inhibited LH secretion during the early breeding season, and no chronic decrease in plasma gonadotrophin concentrations was recorded. However, the LH surge evident for 3 d immediately afer castration, may be related to the dynamic seasonal interaction between gonadal steroids and the regulation of pituitary gonadotrophin release.     

Differential effects of amount of feeding on cell proliferation and progesterone production in response to gonadotrophins and insulin-like growth factor I by ovarian granulosa cells of broiler breeder chickens selected for fatness or leanness.

Strain differences in reproductive performance were demonstrated between broiler breeder female chickens selected for growth (GL line) or for food conversion efficiency (FC line) and the improvement in reproductive performance due to feed restriction also differed significantly. Feed allowance effects on the maturation of ovarian follicles, the incidence of atresia and egg production differed between the two lines exposed to similar feeding protocols. Feed restriction reduced body weights significantly and to a similar extent in both GL and FC lines. The number of normal and atretic yellow follicles was significantly higher under ad libitum feeding and in GL line than it was in the FC line. In both lines, feed restriction decreased multiple ovulation and increased egg production. In culture, granulosa cells from the three largest follicles (F1, F2 and F3) increased progesterone production in response to LH, FSH and insulin-like growth factor I but responses were different between the GL and FC lines fed either ad libitum or restricted diets. Granulosa cells from the two or three largest follicles in GL and FC (ad libitum) lines produced similar amounts of progesterone in response to LH, FSH and insulin-like growth factor I whereas, in restricted birds, the progesterone production was of the rank order F1 > F2 > F3 in both lines. The responsiveness of the GL line fed ad libitum was higher for LH than for either FSH or insulin-like growth factor I but in the GL line fed a restricted diet, it was high for all the hormones. In the FC line, responses to LH, FSH or insulin-like growth factor I were high in ad libitum-fed birds, but low in birds fed a restricted diet for all hormones. Insulin-like growth factor I combined with LH or FSH significantly increased the progesterone production of granulosa cells from birds fed restricted diets of both lines and this effect increased with increasing follicular size. There was a lack of interaction between insulin-like growth factor I and LH or FSH in the regulation of progesterone production by birds of both lines fed ad libitum. Insulin-like growth factor alone or in combination with LH or FSH increased granulosa cell proliferation in birds fed ad libitum more than it did in birds fed restricted diets. The greater proliferation rate of granulosa cells of chickens fed ad libitum, in response to insulin-like growth factor I alone or in combination with gonadotrophins, leading to the simultaneous differentiation of two or three large follicles with high progesterone production in response to LH or insulin-like growth factor I, accelerates the rate of maturation of follicles. This may also be the major cause of erratic and multiple ovulations in broiler breeder female chickens fed ad libitum. In conclusion, insulin-like growth factor I, alone or in combination with LH or FSH, is an important component in the control mechanisms for follicular development in broiler breeder hens. It is this component that is targeted by feed allowance and inadvertently altered by selection for growth.     

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.     

Are differences in FSH concentrations involved in the control of ovulation rate in Romanov and Ile-de-France ewes?

Despite differences in FSH concentrations ranging from 1.5 ng/ml (Romanov ewes) to 4 ng/ml (Ile-de-France ewes) between the follicular and luteal phases, follicular growth (numbers of follicles growing, growth rates, maximum size reached) was morphologically similar between the two stages of the cycle. Injection of 750 i.u. hCG at Day 6 or 16 of the cycle triggered ovulation of 4.1 +/- 0.7 and 4.0 +/- 1.3 follicles in Romanov and 2.2 +/- 0.5 and 1.7 +/- 0.5 follicles in Ile-de-France ewes, respectively, demonstrating that functional differentiation was similar between the two stages of the cycle. As gonadotrophin environment differs between these two stages of the cycle, this suggests that there is a wide flexibility in the amount of gonadotrophins required to trigger terminal follicular growth and that ovarian requirements for gonadotrophins might work through thresholds. When Romanov and Ile-de-France ewes were given similar amounts of exogenous gonadotrophins (1250 i.u. PMSG, 750 i.u. hCG) after hypophysectomy, ovulation rates were close to the usual values (Romanov, 5.5 +/- 3.9; Ile-de-France, 1.4 +/- 0.5), demonstrating that differences in gonadotrophin concentrations during the follicular phase do not play a major role in the high ovulation of the Romanov compared to the Ile-de-France ewes.     

In vitro regulation of sheep ovarian surface epithelium (OSE) proliferation by local ovarian factors

The ovarian surface epithelium (OSE) forms a lining around the entire ovary and actively participates in the ovulatory cycle. To investigate how specific growth factors and hormones affect OSE proliferation, the present study used sheep as a model to examine the effects of follicular and luteal products on the proliferation of sheep OSE cells in culture, and to analyse the influences of large antral follicles and corpora lutea (CL) on the expression of gonadotrophin receptors (FSHR and LHR) in the OSE. In the present study, follicular fluids from medium and large follicles, and extracts of corpora lutea stimulated the growth of OSE cells. The results of the present study showed that factors in the follicular fluid can induce OSE proliferative activity, and this stimulation effect could not be attributed to steroids in the follicular fluid since oestrogen and progesterone treatments failed to stimulate OSE cells. The expression of LH and FSH receptors over large follicles (5 mm or larger) was two and four times higher than those over stroma and CL, respectively. In conclusion, OSE proliferation in cycling sheep is associated with underlying developing follicles and CL, mediated by, at least in part, the up-regulation of gonadotrophin receptors, and facilitated by the action of mitogenic glycopeptides and growth factors, but not steroids.     

Relationships between luteinizing hormone, follicle-stimulating hormone and prolactin secretion and ovarian follicular development in the weaned sow

Folliculogenesis was studied by assessing development of the largest 10 follicles obtained from 10 sows 48 h after weaning and by analyzing changes in plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL) for 24 h before weaning until 48 h after weaning. Follicular diameter, follicular fluid volume, and concentrations of estradiol and testosterone and granulosa cell numbers were determined in all follicles, and 125I-hCG binding to theca and granulosa and maximal aromatase activity in vitro was determined in five follicles/sow. Overall, a significant rise in LH, but not in FSH, occurred at weaning, although in individual sows an increase in LH was not necessarily related to subsequent estrogenic activity of follicles. In 9/10 sows, PRL fell precipitously after weaning. In lactation, LH was negatively, and after weaning, positively, correlated with FSH and PRL. Marked variability in follicular development existed within and between sows. Overall, most follicular characteristics were positively correlated to follicular diameter; however, in larger follicles the number of granulosa cells was variable and unrelated to estrogenic activity, which--together with theca and granulosa binding of hCG--increased abruptly at particular stages of follicular development. Differences in maturation of similarly sized follicles from different sows were related to estrogenic activity of the dominant follicles but not to consistent differences in LH, FSH or PRL secretion. Both the dynamics and the control of folliculogenesis in the sow, therefore, appear to be complex.     

Secretion of gonadotrophins change during the luteal phase of the bovine oestrous cycle in the absence of corresponding changes in progesterone or 17β-oestradiol

The hypothesis in the present study was that changes in circulating luteinizing hormone (LH) and follicle stimulating hormone (FSH) would occur during the luteal phase of the oestrous cycle (Days 4–19; Day 0, day of behaviourial oestrus) that were not related to corresponding changes in concentrations of progesterone and 17β-oestradiol. The stage of the oestrous cycle of cows (n = 18) was synchronised to obtain cows that were on alternate days of the cycle. Blood samples were collected every other day at 15 min intervals for 12 h from all cows: Days 4, 6, 8, 10, 12, 14, 16, 18 (n = 9) and Days 5, 7, 9, 11, 13, 15, 17, 19 (n = 9). Concentrations of LH, FSH, 17β-oestradiol and progesterone were determined in these samples. Data were compared across days to determine when significant changes occurred in concentrations or patterns of secretion of the gonadotrophins and ovarian steroid hormones during the oestrous cycle. There were significant changes in mean concentrations of FSH in circulation between Days 6 and 12. The most striking changes in secretion of gonadotrophins that could not be explained by changes in gonadal steroids were the fluctuations in amplitude of LH pulses between Days 7 and 12. Amplitude of LH pulses increased between Days 7 and 11 and subsequently decreased between Days 11 and 12 of the oestrous cycle. Some changes in gonadotrophin secretion that occurred in the present study can be explained by fluctuations in concentrations of progesterone and 17β-oestradiol in circulation. Other changes cannot be explained by fluctuations in circulating concentrations of these steroids. We accept our hypothesis because the concomitant changes in mean concentration of FSH between Days 6 and 11 and amplitude of LH pulses between Days 7 and 12 of the bovine oestrous cycle cannot be explained by changes in circulating concentrations of progesterone and 17β-oestradiol.     

Ovarian morphology and the concentration of steroids, and of gonadotrophins during the breeding season in ewes actively immunized against oestradiol-17 beta or oestrone

Ewes were actively immunized against oestrone-6-(O-carboxymethyl)-oxime-bovine serum albumin, 17 beta-oestradiol-6-(O-carboxymethyl)oxime-bovine serum albumin or bovine serum albumin (controls). All 4 control ewes, 1 of 5 oestradiol-immunized ewes and 1 of 5 oestrone-immunized ewes had regular oestrous cycles. The other animals displayed oestrus irregularly or remained anoestrous. The plasma concentrations of LH and, to a lesser degree, FSH were increased relative to those in control ewes on Days 11-12 after oestrus or a similar total period after progestagen treatment in ewes not showing oestrus. The ovaries were examined and jugular venous blood, ovarian venous blood and follicular fluid were collected at laparotomy on Days 9-10 of the oestrous cycle. The ovaries of immunized ewes were heavier than those of control ewes. There were no CL in 5 of the immunized ewes but in the other 5 there were more CL than in the control ewes. Ovaries from 4 of 5 oestrone-immunized ewes contained luteinized follicles, while ovaries from 4 of 5 oestradiol-immunized ewes contained very large follicles with a degenerated granulosa and a hyperplastic theca interna. Both types of follicles produced progesterone, detectable in ovarian venous plasma and production of other steroids, particularly androstenedione, was also increased. The steroid-binding capacity of plasma was increased in the immunized ewes. The binding capacity of follicular fluid for oestradiol-17 beta and oestrone was similar to that of jugular venous plasma from the same ewes. These results suggest that immunization against oestrogens disrupts reproductive function by interfering with the feedback mechanisms controlling gonadotrophin secretion.     

Modulating peripheral gonadotrophin levels affects follicular expression of mRNAs encoding insulin-like growth factors and receptors in sheep

Evidence suggests that the insulin-like growth factor (IGF) system is involved in follicular growth and development in sheep. However, little information exists as to the role that key peripheral factors play in regulating the expression of IGF components within the follicle. The present study investigated the regulatory effects of FSH and LH on gene expression for IGF ligands and receptors in ovine follicles, using bovine follicular fluid (bFF) and gonadotrophin-releasing hormone antagonist (GnRHa) model systems to perturb endogenous gonadotrophin secretion. Gene expression studies were carried out using in-situ hybridisation with sheep-specific ribonucleotide probes. Treatment of ewes with bFF had no effect on IGF-I mRNA levels. However, IGF-II mRNA levels, particularly in small follicles, and follicular type II IGF-R gene expression significantly increased following bFF administration (P<0.001). Conversely, there was a significant (P<0.001) decrease in type I IGF-R mRNA levels after only 12h of bFF treatment, especially in healthy follicles, although this was transient and was followed by a significant (P<0.01) increase in gene expression levels by 60 h of bFF treatment. Treatment of ewes with GnRHa resulted in a significant increase in mRNA levels encoding IGF-I (P<0.001), IGF-II in early atretic and large follicles (P<0.05), and type II IGF-R in healthy and early atretic follicles (P<0.001). In contrast, GnRHa administration decreased type I IGF-R gene expression levels after 60 h of treatment (P<0.001). These data highlight the roles that endogenous FSH and LH play in regulating IGF ligand and receptor gene expression in the sheep follicle.     

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.     

An ultrasound-aided study of temporal relationships between the patterns of LH/FSH secretion, development of ovulatory-sized antral follicles and formation of corpora lutea in ewes

To characterize the pulsatile secretion of LH and FSH and their relationships with various stages of follicular wave development (follicles growing from 3 to > or =5 mm) and formation of corpora lutea (CL), 6 Western white-faced ewes underwent ovarian ultrasonography and intensive blood sampling (every 12 min for 6 h) each day, for 10 and 8 consecutive days, commencing 1 and 2 d after estrus, respectively. Basal serum concentrations of LH and LH pulse frequency declined, whereas LH pulse duration and FSH pulse frequency increased by Day 7 after ovulation (P<0.05). LH pulse amplitude increased (P<0.05) at the end of the growth phase of the largest ovarian follicles in the first follicular wave of the cycle. The amplitude and duration of LH pulses rose (P<0.05) 1 d after CL detection. Mean and basal serum FSH concentrations increased (P<0.05) on the day of emergence of the second follicular wave, and also at the beginning of the static phase of the largest ovarian follicles in the first follicular wave of the cycle. FSH pulse frequency increased (P<0.05) during the growth phase of emergent follicles in the second follicle wave. The detection of CL was associated with a transient decrease in mean and basal serum concentrations of FSH (P<0.05), and it was followed by a transient decline in FSH pulse frequency (P<0.05). These results indicate that LH secretion during the luteal phase of the sheep estrous cycle reflects primarily the stage of development of the CL, and only a rise in LH pulse amplitude may be linked to the end of the growth phase of the largest follicles of waves. Increases in mean and basal serum concentrations of FSH are tightly coupled with the days of follicular wave emergence, and they also coincide with the end of the growth phase of the largest follicles in a previous wave, but FSH pulse frequency increases during the follicle growth phase, especially at mid-cycle.     

Influence of ovaries and photoperiod on reproductive function in the mare.

A 16 h daily photoperiod hastened the onset of the ovulatory season (first ovulation); gonadotrophin and follicular changes prior to the onset were similar in intact light-treated and control mares. A preovulatory decline in FSH concentrations before the onset of the ovulatory season preceded the decrease in number of follicles (15--25 mm) and the rise in LH concentrations which was temporally associated with the growth of an ovulatory follicle. Seasonal changes of FSH and LH concentrations were found in ovariectomized mares and were influenced by photoperiod. During the anovulatory season, there was no ovarian influence on gonadotrophin concentrations. However, during the ovulatory season the ovaries exerted a positive influence on seasonally elevated LH concentrations during oestrus and a negative influence during dioestrus. The ovaries exerted a negative influence on seasonally elevated FSH concentrations throughout the oestrous cycle. The onset of the ovulatory season occurred at the time of the first sustained increase in LH concentrations resulting from positive seasonal (increasing photoperiod) and ovarian influences.     

Hormonal requirements for the growth and differentiation of hamster preantral follicles in long-term culture

Preantral follicles from pro-oestrous and oestrous hamsters were isolated enzymically (Stages 1-5) and by microdissection (Stage 6) and cultured for up to 168 h in the absence or presence of 100 ng ovine FSH or LH separately or combined or 1 or 10 micrograms progesterone or estradiol-17 beta in serum-free defined medium and exposed to 1 muCi [3H]thymidine for 24 h before termination. In the presence of insulin and hydrocortisone but not gonadotrophins, the morphology of follicles from pro-oestrous animals at Stages 1-4 (1-4 layers granulosa cells; no theca) were unaffected for up to 48 h whereas for Stages 5 (5-6 layers granulosa cells and developing theca) and 6 (7-8 layers granulosa cells and theca), atresia was prominent by 24 h. FSH significantly reduced the percentage of atretic follicles in Stages 1-5 throughout the culture period; but was effective only up to 96 h for Stage-6 follicles. LH was also effective, albeit to a lesser extent. FSH increased follicular labelling indexes during every 24-h labelling period and, during a pulse-chase period, follicular DNA content and granulosa cell numbers. FSH, but not LH, induced differentiation by 96 h of preantral follicles at Stage 6 into small antral stages (Stages 7-8). FSH and LH together induced almost the same effect as FSH alone. However, neither progesterone nor oestradiol had any significant long-term effects on DNA synthesis and oestradiol induced atresia beyond 24 h. Both FSH and LH induced follicular maturation in vitro as evident from increases in progesterone, androstenedione and oestradiol production. Follicles (Stages 1-4) collected from oestrous hamsters responded to FSH to a lesser extent than did those from pro-oestrous animals, possibly because of in-vivo exposure to periovulatory changes in gonadotrophins; however, an antrum formed in Stage-6 follicles by 72 h.     

Endocrine, paracrine and autocrine control of follicular development

Development of a single follicle during the menstrual cycle is under control of hormones stimulating follicular maturation, ovulation and luteogenesis. Several factors intervene locally to prevent other follicles from developing at the same time as dominant follicle. These other follicles remain quiescent or evaluate to atresia. Atresia results from the action of several endocrine, paracrine and autocrine mechanisms which synergistically inhibit aromatase activity. The subsequent lack of estrogens reduces granulosa cell multiplication. The oocyte will not become fertilizable before the preovulatory peak of LH, after the resumption of meiosis and after reaching metaphase of the second meiotic division. Several factors are involved in the inhibition of spontaneous resumption of meiosis: cyclic nucleotides, sex steroids, somatostatin and oocyte maturation inhibitor(s) (OMI). Ovulation is related to breakdown of connective tissue synthesized by granulosa cells under the influence of FSH. Connective tissue lysis is dependent on proteolytic enzymes which are released and activated by FSH, LH and relaxin. A paracrine control could be involved in ovulation: LH induces the production of prostaglandin and relaxin by theca cells which, in turn, stimulate collagenase and proteoglycanase secretion by granulosa cells.     

Growth,antrum formation,and estradiol production of bovine preantral follicles cultured in a serum-free medium

The objective of this study was to identify factors that would allow the establishment of a serum-free culture system that could support follicular and oocyte growth, antrum formation, and estradiol-17beta (E(2)) production in preantral follicles of bovine ovaries. Large preantral follicles (145-170 micro m in diameter) were microsurgically dissected from ovaries, embedded in 0.15% type I collagen gels, and maintained in a serum-free medium for up to 13 days at 38.5 degrees C in 5% CO(2) in air. This culture environment allowed most preantral follicles to maintain a three-dimensional structure with the presence of a thecal layer and basement membrane surrounding the granulosa cells throughout the entire culture period. The effects of insulin, insulin-like growth factor (IGF)-I, IGF-II, FSH, and LH on preantral follicle growth were investigated in serum-free medium. Follicular diameters were significantly larger in the presence of insulin, IGF-I, IGF-II, or FSH after 13 days in culture. Oocyte diameters were also significantly larger in the presence of all hormones tested. The single addition of insulin, IGF-I, or FSH induced antrum formation between Days 7 and 13 of culture. Insulin progressively induced E(2) secretion by follicles after antrum formation, but IGF-I and FSH had no apparent effect. FSH and LH caused an increase in oocyte diameter in the presence of insulin. The addition of three hormones (insulin, FSH, and LH) initiated antrum formation and E(2) production earlier than insulin-containing medium alone. Furthermore, maximal E(2) secretion was maintained steadily between 7 and 13 days in this culture condition. From these results, we have demonstrated that insulin, FSH, and LH play substantial roles in the growth and development of bovine large preantral follicles in a serum-free medium.     

A model for follicular selection and ovulation: lessons from superovulation

A model for selection of the preovulatory follicle during the normal ovarian cycle is proposed. During menstruation the concentration of FSH rises to a level high enough to "activate" a single small antral follicle (2-4 mm dia.) so that it can produce large amounts of oestradiol. As the follicle develops, the concentration of FSH is suppressed below this threshold level by the secretion of oestradiol and inhibin. The dominant follicle becomes increasingly sensitive to FSH so that it continues to develop in an environment which inhibits development of other follicles. Multiple ovulation can be achieved by extending the period during which the level of FSH remains above this threshold level (e.g. during treatment with clomiphene or gonadotrophins). Although multiple ovulation occurs when the gate is widened in this way, the follicles are never completely synchronous as they continue to grow at approximately the same rate. Current evidence suggests that ovulation occurs at random between the two ovaries in successive cycles and that the corpus luteum exerts an inhibitory effect on folliculogenesis by suppressing the secretion of FSH and LH. These observations are compatible with the hypothesis that while small antral follicles are recruited continuously, at all stages of reproductive life, selection of the dominant follicle requires the unique gonadotrophic environment which is only present in the early follicular phase. The follicle of the month is, therefore, selected by chance because it is at the right place at the right time.