Antral follicle count reliably predicts number of morphologically healthy oocytes and follicles in ovaries of young adult cattle

Methods to predict numbers of healthy oocytes in the ovaries of young adults could have important diagnostic relevance in family planning and animal agriculture. We have observed that peak antral follicle count (AFC) determined by serial ovarian ultrasonography during follicular waves is very highly reproducible within individual young adult cattle, despite 7-fold variation among animals. Herein, we tested the hypothesis that AFC is positively associated with the number of morphologically healthy oocytes and follicles in ovaries and with serum concentrations of anti-Müllerian hormone (AMH), an indirect marker for number of healthy follicles and oocytes in ovaries. In the present study, age-matched young adult cattle (12-18 mo old) were subjected to serial ultrasonography to identify animals with a consistently high (> or =25 follicles that were > or =3 mm in diameter) or low (< or =15 follicles) AFC during follicular waves. Differences in serum AMH concentrations, ovary weight, and number of morphologically healthy and atretic follicles and oocytes were determined. The phenotypic classifications of cattle based on AFC during follicular waves or AMH concentrations both predict reliably the relative number of morphologically healthy follicles and oocytes in ovaries of age-matched young adult cattle.     

Ovarian dynamics and their associations with peripheral concentrations of gonadotropins,ovarian steroids,and inhibin during the estrous cycle in goats

Ovarian changes determined by daily transrectal ultrasound and its relationship with FSH, LH, estradiol-17beta, progesterone, and inhibin were investigated in six goats for three consecutive interovulatory intervals. Estrous cycles were synchronized using two injections of prostaglandin F2alpha analogue 11 days apart. All follicles 3 mm or greater in diameter and corpora lutea were measured daily. A follicular wave was defined as one or more follicles growing to 5 mm or greater in diameter. The day that the follicles reached 3 mm in diameter was defined as the day of wave emergence, and the first wave after ovulation was defined as wave 1. During the interovulatory interval (mean +/- SEM, 21.3 +/- 0.4 days; n = 18), follicular waves emerged at 0.3 +/- 0.5, 6.5 +/- 0.2, and 12.1 +/- 0.4 days for wave 1, wave 2, and wave 3, respectively, in goats with three waves of follicular development and at -0.6 +/- 0.3, 4.7 +/- 0.2, 9.4 +/- 0.5, and 13.4 +/- 0.5 days for wave 1, wave 2, wave 3, and wave 4, respectively, in goats with four waves of follicular development (Day 0 = the day of ovulation). The mean diameter of the largest follicle of the ovulatory wave was significantly larger than those of the largest follicles of the other waves. Corpora lutea could be identified ultrasonically at Day 3 postovulation and attained 12.1 +/- 0.3 mm in diameter on Day 8. Transient increases in plasma concentrations of FSH were detected around the day of follicular wave emergence. The level of FSH was negatively correlated with that of inhibin. These results demonstrated that follicular waves occurred in goats and that the predominant follicular wave pattern was four waves with ovulation from wave 4. These results also suggested that the emergence of follicular waves was closely associated with increased secretion of FSH.     

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.     

Pattern of follicular development in high fecundity Olkuska ewes during the estrous cycle

Folliculogenesis was studied daily in the 18 oestrous cycles in six prolific Olkuska ewes from October to December using transrectal ultrasonography to record the number and size of all ovarian follicles > or =2 mm in diameter. Blood samples were taken once a day and were analyzed for concentrations of FSH, LH, estradiol and progesterone. Follicular and hormonal data were analyzed for associations between different stages of development of the follicular waves and concentrations of FSH and estradiol. The first wave during which at least one follicle reached maximum diameter of > or =4 mm after ovulation, was defined as a wave 1, and the following waves were numbered sequentially. Waves 1, 2, 3, 4 and the ovulatory one emerged on days: -2 to 4, 4 to 8, 6 to 11, 10 to 12 and 11 to 15, respectively. The mean number of follicles per wave that reached diameter of > or =4 mm was 4.15 +/- 1.1 and 16.62 +/- 8.6 follicles per estrous cycle of a total 299 follicles were observed. Significantly more follicles (p> or =0.05) emerged on days 2, 8 and 13 than in other days. Serum FSH concentrations fluctuated from 0.11 ngml(-1) on day 2 to preovulatory maximum 1.81 ngml(-1) on day 17 of the estrous cycle. The emergence of follicular waves was associated with elevations of FSH concentrations in blood serum. The mean increase in FSH concentration was followed by the recruitment of follicles of the next wave. The mean daily FSH concentration and the mean number of follicles emerging each day were negatively correlated. The length of the interwave interval (4.4 +/- 1.6 days) did not differ significantly from the interval between pulses of FSH (4.8 +/- 0.3 days). The mean serum estradiol concentrations showed fluctuations until day 14 and then gradually increased from 5.47 +/- 0.3 pgml(-1) to reach a peak 13.14 +/- 0.2 pgml(-1) on the day before ovulation. To summarize, the growth of ovarian follicles during the estrous cycle in high fecundity Olkuska sheep exhibited a distinct wave-like pattern. Ovarian follicles emerged from the pool of 2 mm follicles. The preovulatory follicles originated from the large follicle population were present in the ovary at the time of luteal regression. The initial stages of the growth of the largest follicles appears to be controlled primarily by increases in FSH secretion.     

Ontogeny and cellular localization of 125I-labeled insulin-like growth factor-I, 125I-labeled follicle-stimulating hormone, and 125I-labeled human chorionic gonadotropin binding sites in ovaries from bovine fetuses and neonatal calves.

In a previous study we reported that ovaries from bovine fetuses, which consist mainly of preantral follicles with few antral follicles, are weakly responsive to gonadotropins (FSH and LH). Insulin-like growth factor-I (IGF-I) is known to enhance gonadotropin responsiveness in vitro, but there is a lack of consistent data on the involvement of IGF-I, FSH, and LH during early stages of folliculogenesis in cattle. In the study reported here, we assessed autoradiographically the ontogeny of 125I-gonadotropin and 125I-IGF-I binding activities during preantral and early antral stages in cattle. Follicular growth was initiated around Day 180 of gestation in fetuses. The density of 125I-FSH binding was high in granulosa cells from primary (mean +/- SEM 10.5 +/- 0.7 grains/cell, 0.05-mm diam.) and secondary follicles (10.8 +/- 0.8 to 13.6 +/- 1.2 grains/cell, 0.06-0.15 mm) but increased significantly (p < 0.05) in early antral follicles (18.2 +/- 1.1 grains/cell, 0.16-3.0 mm). Specific 125I-IGF-I binding levels were low in granulosa cells from preantral follicles, averaging 2.5 +/- 0.6-3.1 +/- 0.9 grains/cell. However, after antrum formation, the density of 125I-IGF-I binding increased significantly (p < 0.05) with follicular diameter in granulosa cells and was 5.7 +/- 0.7 and 9.1 +/- 0.6 grains/cell for antral I (0.16-0.5 mm) and antral II (0.6-3.0 mm) follicles, respectively. 125I-FSH and 125I-IGF-I binding densities were low in theca cells from preantral and early antral follicles as well as in the interstitial tissue and granulosa cells from atretic follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Follicular and FSH dynamics in ewes with a history of high and low ovulation rates

Daily transrectal ultrasound scanning and twice-daily blood sampling were used to monitor the temporal relationships between FSH concentrations and follicle development during complete interovulatory intervals for ewes in which the ovulation rate in each of the 2 previous years was high or low (> or = 3 and < or = 2 ovulations, respectively). Follicles that reached > or = 5 mm were used to define a follicular wave and were tracked retrospectively to 3 mm (emergence). The hypothesis that FSH surges (identified with a computer program) and follicular waves (retrospectively determined based on ultrasound scanning) are temporally associated was supported in both groups by the emergence of an anovulatory or ovulatory follicular wave near the peak of an FSH surge. Further support for the hypothesis was a significant increase in FSH concentrations before and a significant decrease after follicular-wave emergence in both groups independent of the identification of FSH surges. Ewes with a history of high ovulation rates had smaller follicles (anovulatory and ovulatory) and more ovulations, but the 2 groups were similar in the number of ovulatory follicular waves and associated FSH surges, number and characteristics of the FSH surges, and mean FSH concentrations per interovulatory interval. Surges of FSH were periodic (every 3 or 4 d) regardless of the ovulation-rate group or follicle response. In ewes with a low ovulation rate, the nonovulatory FSH surges were most frequently associated with emergence of detected anovulatory follicular waves. In ewes with a high ovulation rate, more FSH surges were not associated with a detected follicular wave, as defined, presumably because the largest follicle did not reach 5 mm. The results indicated that the factors resulting in a high ovulation rate were not exerted through circulatory patterns or concentrations of FSH but involved a shorter growth phase and smaller maximal diameter of follicles.     

Changes in peripheral levels of bioactive and immunoreactive inhibin, estradiol-17 beta, progesterone, luteinizing hormone, and follicle-stimulating hormone associated with follicular development in cows induced to superovulate with equine chorionic gonadotropin.

Changes in concentrations of bioactive and immunoreactive (ir-) inhibin, estradiol-17 beta, progesterone, LH, and FSH in peripheral blood were determined in cows induced to superovulate with eCG. The pattern of follicular growth was also characterized by daily ultrasonographic examination. Hormonal profiles and follicular development during the intact estrous cycle of the same animals before eCG treatment served as controls. Equine CG increased the number of follicles of various sizes (small, greater than or equal to 4 less than 7, medium, greater than or equal to 7 less than 10; large, greater than or equal to 10 mm in diameter) by 4 days after administration. The second growth of large follicles occurred within 1 day after superovulation. Inhibin bioactivity in jugular vein blood was detectable 48 h after eCG injection (44 h before LH peak), whereas it was not detected before administration of eCG or during control cycles. Circulating levels of bioactive inhibin further increased during the two waves of growth of large follicles. The highest activity of inhibin was noted at the time of the preovulatory LH peak (0 h). Thereafter, bioactivity of inhibin in peripheral plasma dropped from 0 to 24 h after the LH peak, and the activity increased again at 72 h compared to the value at -44 h. Plasma levels of ir-inhibin showed a pattern similar to changes in bioactive inhibin in the eCG-treated cows. Plasma concentrations of estradiol-17 beta also increased concomitantly with two waves of growth of large follicles. There was no correlation between plasma levels of progesterone and inhibin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of recombinant bovine somatotropin on ovarian function in heifers: follicular populations and peripheral hormones.

The objective of this study was to investigate the possible effect of recombinant bovine somatotropin (BST) on ovarian folliculogenesis and ovulation rate. Twelve Hereford x Friesian heifers received daily injections of either 25 mg BST (6 heifers) or vehicle (6 heifers) for a period of two estrous cycles until slaughter. Blood samples were collected three times a week for measurements of peripheral growth hormone (GH), insulin-like growth factor I (IGF-I), FSH, LH, estradiol, and progesterone. Serial blood samples were also taken every 10 min for 8 h on Days 12 and 19 of the second estrous cycle to monitor GH, IGF-I, FSH, and LH profiles. At the end of treatment (Day 7 of the third estrous cycle), the heifers were killed and their ovaries were collected. Ovulation rate was determined by counting the number of fresh corpora lutea (CL). All antral follicles greater than or equal to 2 mm in diameter were dissected to assess antral follicle populations. Granulosa and thecal cells from the three largest follicles and CL from each heifer were collected for FSH and LH binding measurements. All heifers had a single ovulation. The treated heifers had significantly more antral follicles (60.2 +/- 6.7) than did the animals in the control group (33.2 +/- 3.2) (p less than 0.001). When follicles were grouped according to diameter, the mean numbers of follicles greater than 10 mm, 5-10 mm, and 2-5 mm in diameter were 0.8 +/- 0.2, 6.8 +/- 1.4, and 52.5 +/- 6.5 for the treated group, and 0.8 +/- 0.2, 6.5 +/- 1.0, and 25.8 +/- 2.7 for controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in follicle-stimulating hormone and follicle populations during the ovarian cycle of the common marmoset

The common marmoset (Callithrix jacchus) belongs to the family Callitrichidae, the only anthropoid primates with a high and variable number of ovulations (one to four). An understanding of folliculogenesis in this species may provide some insight into factors regulating multiple follicular growth in primates. The aims of this study were to characterize in detail changes in the antral follicle population at different stages of the ovarian cycle, to characterize the marmoset FSH profile, and to relate cyclic changes in FSH to changes in follicle sizes and circulating estradiol concentrations. Fifty-five pairs of ovaries were collected (32 of which were at five distinct stages of the cycle) from adult marmosets, and antral follicles were manually excised and separated into four size groups. Daily urinary FSH and plasma estradiol and progesterone concentrations from Day 0 of the follicular phase to 2 days postovulation were measured in 22 marmosets using enzyme immunoassays. The FSH profile revealed two distinct peaks, on Days 2 and 6, during the 10-day follicular phase, with a marginal periovulatory increase on Days 9 and 10. Estradiol levels rose significantly (P: < 0.05) above baseline (Days 1-4) on Day 5 and continuously increased to a peak on the day preceding ovulation (Days 8 and 9). Follicle dissection revealed a high (mean = 68) and variable (range, 14-158) total number of antral follicles >0.6 mm. The number of antral follicles significantly declined (P: < 0.001) with age. The number of preovulatory follicles (>2 mm) was positively correlated with the number of antral follicles (P: < 0. 001) and tended to be negatively related to age (P: = 0.06). The number of antral follicles did not vary significantly with stage of the ovarian cycle, although the follicle size distribution was cycle-stage dependent (P: < 0.05). Follicles >1.0 mm appeared only in the follicular phase, and preovulatory follicles (>2.0 mm) appeared only at the end of the follicular phase (Days 7-9). The Day 2 FSH peak corresponded to emergence of a population of medium-size antral follicles, and the Day 6 peak was consistent with rising estradiol levels and appearance of the preovulatory follicles. These results suggest that some aspects of marmoset folliculogenesis are comparable to those in Old World primates, including the absence of multiple follicular waves and the appearance of an identifiable dominant follicle in the midfollicular phase. However, the midphase FSH peak, multiple dominant follicles, and abundance of nonovulatory antral follicles differ strongly from the pattern in Old World primates and humans. The findings are discussed in relation to the regulation of growth of multiple ovulatory follicles and provide the basis for further studies on factors influencing the dynamics of follicular growth and development in this species.     

Alterations in peripheral concentrations of inhibin A in cattle studied using a time-resolved immunofluorometric assay: relationship with estradiol and follicle-stimulating hormone in various reproductive conditions

The aims of this study were to develop a sensitive and specific assay for bovine inhibin A using europium and to investigate the endocrine role of inhibin A in various reproductive conditions by characterizing the relationship between profiles of inhibin A, FSH, and estradiol and follicle growth during the postpartum period, during the intact estrous cycle, and in cows with follicular cysts. The time-resolved immunofluorometric assay (Tr-IFMA) for bovine inhibin A, using purified polyclonal antibodies to alpha and beta(A) subunits, was specific for bovine inhibin A and did not cross-react with bovine activin A, activin AB, activin B, pro-alphaC or human recombinant inhibin B. The detection limit of the IFMA was 3.3 pg/ml expressed in terms of bovine 32-kDa inhibin A. Dose-response curves of plasma samples obtained from intact and FSH-stimulated cows and cystic cows were parallel to the standard without any preassay processing of samples. Plasma inhibin A levels increased (P < 0.01) concomitant with emergence of nonovulatory or ovulatory follicular waves during the postpartum period. In cystic cows, plasma inhibin A was sustained at high levels for a longer period, associated with growth of persistent dominant follicles. The highest levels of inhibin A were noted during the growth phase of normal and persistent dominant follicles; however, inhibin A levels declined (P < 0.01) as these dominant follicles ceased to grow or ovulated. An inverse relationship between patterns of plasma inhibin A and FSH existed during each follicular wave in the three physiologic conditions. Increases in plasma inhibin A levels were associated with increases in plasma estradiol levels during most follicular waves; however, there was no increase in plasma estradiol level and no relationship between patterns of estradiol and FSH during follicular waves observed during the early postpartum period or midluteal phase of the estrous cycle. In conclusion, the Tr-IFMA does not require pretreatment of samples and can be used for precise measurement of bovine inhibin A without interference with free inhibin alpha subunits. Inhibin A, produced primarily during growth of the dominant follicle, functions as a negative feedback regulator for FSH secretion throughout the postpartum period and the estrous cycle, whereas estradiol appears to have a minor role in regulation of FSH compared with inhibin A, especially during the early postpartum period and midluteal phase of the estrous cycle. The results also indicate that a persistent dominant follicle sustains inhibin A production for a longer period than the dominant follicle emerging in the estrous cycle and establishes long-term dominance by suppressing emergence of a new follicular wave.     

Interrelationships of estradiol, inhibin, and gonadotropins during follicle deviation in pony mares

The changes in circulating concentrations of FSH, LH, estradiol, and total inhibin associated with the beginning of follicle diameter deviation were compared among the last anovulatory follicular wave of the year and the first and second ovulatory waves in pony mares ( n=7 ). Follicle diameters and circulating hormone concentrations for each wave were normalized to the observed beginning of deviation (Day 0). Follicle deviation was demonstrated during the anovulatory wave as well as during the ovulatory waves, and the diameter of the future dominant follicle at the beginning of deviation was similar for the three waves (overall mean: 23.7+/-0.6 mm). Circulating estradiol concentrations did not increase during the last anovulatory wave but increased similarly for the two ovulatory waves, beginning near the onset of deviation. There were no differences among waves in concentrations of inhibin encompassing deviation. The FSH concentrations for the wave-stimulating FSH surge did not differ significantly among the three waves; combined for the three waves, concentrations decreased between Days -3 and 7. Circulating LH did not increase during the last anovulatory wave but increased during the first and second ovulatory waves beginning on Days 6 and -2, respectively. Results indicated that the increase in circulating estradiol at the beginning of deviation was not required for suppression of the wave-stimulating FSH surge and the initiation of deviation, based on an estradiol increase in association with deviation during the ovulatory waves but not during the anovulatory wave. Concentrations of inhibin were similar among waves and, therefore on a temporal basis, the similar suppression of FSH was attributable to inhibin. The later increase in LH before the first ovulation was not attributable to estradiol, based on the similarity between the two ovulatory waves in the increasing estradiol concentrations.     

Concentrations of immunoreactive inhibin in ovarian and peripheral venous plasma and follicular fluid of Booroola ewes that are homozygous carriers or non-carriers of the FecB gene.

No gene-specific differences were found during either the luteal or follicular phases of the oestrous cycle in the venous secretion rates of ovaries or in concentrations of immunoreactive inhibin in peripheral plasma between Booroola ewes that were homozygous carriers (BB) or non-carriers (++) of the FecB gene. In three experiments in which concentrations of plasma inhibin and follicle-stimulating hormone (FSH) were compared, gene-specific differences were noted for FSH (P less than 0.05), but no significant correlations were noted between FSH and inhibin for either genotype. Granulosa cells and follicular fluid, but not theca interna, stroma or corpora lutea, were the major intra-ovarian sites of inhibin; no gene-specific differences were noted for inhibin concentrations in follicular fluid or in any of the intra-ovarian tissues. The mean concentrations of inhibin in follicular fluid remained constant irrespective of follicular diameter whereas the mean total contents of inhibin increased significantly with increasing diameter (P less than 0.05). Inhibin secretion rates were four times higher in ovaries with oestrogen-enriched follicles (i.e. greater than or equal to 50 ng oestradiol ml-1) than in ovaries with no such follicles (P less than 0.01). Moreover, inhibin concentrations were higher in follicular fluid of oestrogen-enriched follicles than in those with low oestrogen (i.e. less than 50 ng ml-1; P less than 0.05). Ovariectomy resulted in a significant reduction in concentrations of immunoreactive inhibin from plasma (P less than 0.01). The residual plasma inhibin in some Booroola ewes was not associated with genotype. It is concluded that, although antral follicles are a major source of inhibin in Booroola ewes, immunoreactive inhibin is not associated with the FecB gene and is not responsible for the gene-specific differences in concentrations of FSH in plasma.     

Ovarian antral follicular dynamics and their associations with peripheral concentrations of gonadotropins and ovarian steroids in anoestrous Finnish Landrace ewes

Daily transrectal ultrasonography of ovaries was done in seven Finn ewes during three 17-day periods from May to July. Blood samples were collected each day for estimation of the serum follicle-stimulating hormone (FSH), oestradiol and progesterone concentrations, and also every 15 min for 6 h, halfway through each period of ultrasonographic examination, to determine the patterns of gonadotropic hormone secretion. Four ewes ceased cycling from March to mid-April (ewes entering anoestrus early) and three in May (ewes entering anoestrus late). In all ewes cyclicity resumed during the period from mid-August to mid-September. The growth of ovarian antral follicles to periovulatory sizes of >/=5 mm in diameter was seen at all stages of anoestrus. An average of four waves of follicular development (follicles growing from 3 to >/=5 mm in diameter before regression) with a periodicity of 4 days were recorded during each of the three scanning periods. There was a close temporal relationship between days of follicular wave emergence and peaks of successive FSH fluctuations. Ewes entering anoestrus late exceeded ewes that became anoestrus early in numbers of large (>/=5 mm in diameter) ovarian antral follicles and maximum follicle diameter. Peak concentrations of transient FSH increases were higher (P<0.05) in ewes entering anoestrus late than in ewes entering anoestrus early. The secretion of luteinising hormone, (LH; mean and basal level, and LH pulse frequency, but not amplitude) was lowest during the month of June in all ewes. Oestradiol production was markedly suppressed throughout anoestrus. Peaks of progesterone secretion appeared to occur at regular intervals and were associated with the end of the growth phase of the largest follicles of sequential waves. In conclusion, the growth of ovarian follicles to ostensibly ovulatory diameters is maintained throughout anoestrus in Finn ewes and periodic emergence of follicular waves is correlated with an endogenous rhythm of FSH secretion. The present study also provides evidence for the inverse relationship between the time of the onset of seasonal anoestrus and the number and size of antral follicles developing throughout anoestrus in Finn ewes, and indicates that differences exist in both the secretion of and ovarian responsiveness to gonadotropic hormones among early and late anoestrous ewes.     

Use of high-resolution transrectal ultrasonography to assess changes in numbers of small ovarian antral follicles and their relationships to the emergence of follicular waves in cyclic ewes

Transrectal ovarian ultrasonographic studies have shown that, in cattle, follicular wave emergence is associated with a large increase in the number of small antral follicles (4-6mm in diameter); an analogous association has not been found for small follicles (2-3mm in diameter) in the ewe. In previous studies in ewes, accurate assessment of the number of follicles has been limited to follicles > or =2 or 3mm in size. Newer, high-resolution equipment allowed us to identify follicles > or =0.4mm and to quantify all antral follicles > or =1mm in diameter in seven cyclic Western White Face ewes. This allowed us to expand the small follicle pool examined, from 1 to 4 follicles/day (2-3.5mm in diameter) in earlier studies, to 8-18 follicles/day (1-3mm in diameter). Total number of small follicles (> or =1 and < or =3mm in diameter) increased between Days -1 and 0 (Day 0=day of ovulation), and declined between Days 1 and 3 (P<0.05). There were no significant changes in the number of small or medium (4mm in diameter) follicles around days of follicle wave emergence (+/-2 days). The 1-3 follicles in the 2-3mm size range, which constituted a follicle wave (i.e. grew to > or =5mm in size before regression or ovulation), were the only small follicles to emerge in an orderly succession during the estrous cycle, approximately every 3-5 days. Thus, unlike in cattle, there is no apparent increase in numbers of small follicles at follicle wave emergence in cyclic sheep, and little evidence for selection of recruited follicles and follicular dominance.     

Decreased oocyte DAZL expression in mice results in increased litter size by modulating follicle-stimulating hormone-induced follicular growth

While the germ cell-specific RNA binding protein, DAZL, is essential for oocytes to survive meiotic arrest, DAZL heterozygous (het) mice have an increased ovulation rate that is associated with elevated inhibin B and decreased plasma follicle-stimulating hormone (FSH). The relationship between decreased oocyte DAZL expression and enhanced follicular development in het mice was investigated using in vitro follicle cultures and in vivo modulation of endogenous FSH, by treating mice with inhibin and exogenous FSH. In vitro, follicles from het mice are more sensitive to FSH than those of wild-type (wt) mice and can grow in FSH concentrations that are deleterious to wild-type follicles. In vivo, despite no differences between genotypes in follicle population profiles, analysis of granulosa cell areas in antral follicles identified a significantly greater number of antral follicles with increased granulosa cell area in het ovaries. Modulation of FSH in vivo, using decreasing doses of FSH or ovine follicular fluid as a source of inhibin, confirmed the increased responsiveness of het antral follicles to FSH. Significantly more follicles expressing aromatase protein confirmed the earlier maturation of granulosa cells in het mice. In conclusion, it is suggested that DAZL expression represses specific unknown genes that regulate the response of granulosa cells to FSH. If this repression is reduced, as in DAZL het mice, then follicles can grow to the late follicular stage despite declining levels of circulating FSH, thus leading to more follicles ovulating and increased litter size.     

Characterization of ovarian follicular wave dynamics in women

A wave phenomenon of ovarian follicular development in women has recently been documented in our laboratory. The objective of the present study was to characterize follicular waves to determine whether women exhibit major and minor wave patterns of follicle development during the interovulatory interval (IOI). The ovaries of 50 women with clinically normal menstrual cycles were examined daily using transvaginal ultrasonography for one IOI. Profiles of the diameters of all follicles >or=4 mm and the numbers of follicles >or=5 mm were graphed during the IOI. Major waves were defined as those in which one follicle grew to >or=10 mm and exceeded all other follicles by >or=2 mm. Minor waves were defined as those in which follicles developed to a diameter of <10 mm and follicle dominance was not manifest. Blood samples were drawn to measure serum concentrations of estradiol-17beta, LH, and FSH. Women exhibited major and minor patterns of follicular wave dynamics during the IOI. Of the 50 women evaluated, 29/34 women with two follicle waves (85.3%) exhibited a minor-major wave pattern of follicle development and 5 women (14.7%) exhibited a major-major wave pattern. Ten of the 16 women with three follicle waves (62.5%) exhibited a minor-minor-major wave pattern, 3 women (18.8%) exhibited a minor-major-major wave pattern, and 3 women (18.8%) exhibited a major-major-major wave pattern. Documentation of major and minor follicular waves during the menstrual cycle challenges the traditional theory that a single cohort of antral follicles grows only during the follicular phase of the menstrual cycle.     

Differential effects of various estradiol-17beta treatments on follicle-stimulating hormone peaks, luteinizing hormone pulses, basal gonadotropin concentrations, and antral follicle and luteal development in cyclic ewes

In a previous study, 10-day estradiol implant treatment truncated the FSH peaks that precede follicular waves in sheep, but subsequent ovine FSH (oFSH) injection reinitiated wave emergence. The present study's objectives were to examine the effects of a 20-day estradiol and progesterone treatment on FSH peaks, follicle waves, and responsiveness to oFSH injection. Also, different estradiol doses were given to see whether a model that differentially suppressed FSH peaks, LH pulses, or basal gonadotropin secretion could be produced in order to study effects of these changes on follicular dynamics. Mean estradiol concentrations were 11.8 +/- 0.4 pg/ml, FSH peaks were truncated, wave emergence was halted, and the number of small follicles (2-3 mm in diameter) was reduced (P < 0.05) in cyclic ewes given estradiol and progesterone implants (experiment 1). On Day 15 of treatment, oFSH injection failed to induce wave emergence. With three different estradiol implant sizes (experiment 2), estradiol concentrations were 5.2, 19.0, 27.5, and 34.8 (+/-4.6) pg/ml in control and treated ewes, respectively. All estradiol treatments truncated FSH peaks, except those that created the highest estradiol concentrations. Experiment 2-treated ewes had significantly reduced mean and basal FSH concentrations and LH pulse amplitude and frequency. We concluded that 20-day estradiol treatment truncated FSH peaks, blocking wave emergence, and reduced the small-follicle pool, rendering the ovary unresponsive to oFSH injection in terms of wave emergence. Varying the steroid treatment created differential FSH peak regulation compared with other gonadotropin secretory parameters. This provides a useful model for future studies of the endocrine regulation of ovine antral follicular dynamics.     

Inhibin a increases apoptosis in early ovarian antral follicles of diethylstilbestrol-treated rats

The purpose of this study was to evaluate the role of inhibin A in follicular development and apoptosis-related mechanisms in preantral and early antral follicles from prepubertal diethylstilbestrol (DES)-treated rats. Granulosa cells isolated from the ovaries of 23- to 25-day-old rats were cultured in serum-free medium containing FSH (20 ng/ml), transforming growth factor beta (5 ng/ml), and estradiol (50 ng/ml) in the presence or absence of different concentrations of recombinant human inhibin A. (3)H-Thymidine incorporation was decreased in the presence of Inh, but no significant changes were observed in progesterone and estradiol levels in culture medium. An increase in low molecular weight DNA fragmentation indicative of apoptosis and an increase in the levels of Bax protein with no changes in Bcl-2 protein levels were evident in early antral follicles incubated for 24 h with Inh. For each animal, Inh (0.5 micro g/ovary) was injected intrabursally in one ovary, and the contralateral ovary served as a control. Ovarian histology revealed an inhibitory effect of Inh treatment on the follicular development induced by DES. At 24 h after Inh injection, the number of preantral follicles was increased compared with controls, whereas the number of early antral follicles was decreased. In addition, in vivo Inh treatment caused an increase in the percentage of apoptotic cells in preantral and early antral follicles. These results suggest that inhibin produced by the dominant follicle may act as a paracrine factor inhibiting the growth of neighboring follicles, thus participating in the mechanism of follicular selection.     

Follicle selection in cattle: dynamics of follicular fluid factors during development of follicle dominance.

Follicle diameter deviation during follicular waves in cattle begins with a reduction in growth rates of developing subordinate follicles, in contrast to the maintenance of a constant growth rate by a developing dominant follicle. In experiment 1, the temporal changes encompassing deviation in concentrations of follicular fluid factors relative to one another in the three largest follicles (F1, F2, and F3) were studied. Follicular fluid samples were collected when F1 reached diameter ranges of 7.0-7.9, 8.0-8.9, 9.0-9.9, and 10.0-10.9 mm (n = 12 per range). The first increase (P < 0.05) in the difference between F1 and F2 for estradiol occurred at the 8.0- to 8.9-mm range, which was one range earlier than for diameter (P < 0.05). Free insulin-like growth factor (IGF)-1 concentrations in F1 were similar among diameter ranges, but concentrations in F1 were higher (P < 0.05) than in F2 for each range except 7.0-7.9 mm. Concentrations of free IGF-1 in F2 decreased (P < 0.05). No significant differences were detected in concentrations of progesterone, androstenedione, total inhibin, and inhibin-A. Averaged over follicles, inhibin-B decreased (P < 0.05) between the 8.0- to 8.9- and 10.0- to 10.9-mm ranges, and activin-A increased (P < 0.05) between the 7.0- to 7.9- and 9.0- to 9.9-mm ranges. However, no differences were found among follicles. In experiment 2, changes associated with the development of dominance by F2 were studied using ablation of F1 at the beginning of expected deviation (F1, 8.5 mm; Hour 0) as the reference point. Follicular fluid factors were compared at Hour 12 between F2 of a control group (F1 intact; n = 10) and an ablated group (F1 ablated; n = 10). Diameter (P < 0.02), estradiol (P < 0.001), free IGF-1 (P < 0.002), and progesterone (P < 0.003) were greater and IGF-binding protein-2 was lower (P < 0.01) in F2 of the ablated group at Hour 12. No differences were detected in concentrations of androstenedione, total inhibin, and inhibin-A. The results of the two experiments indicated, on a temporal basis, that intrafollicular changes in estradiol and the IGF system, but not in the inhibin/activin system, could account for a reported greater FSH responsiveness by the future dominant follicle than by the future subordinate follicles by the beginning of diameter deviation in cattle.     

Concentrations of insulin-like growth factor-I, estradiol and progesterone in follicular fluid of ovarian follicles during early pregnancy in cattle

To determine if the presence of the developing conceptus is associated with changes in intrafollicular concentrations of insulin-like growth factor-I (IGF-I), estradiol (E2) and/or progesterone during early pregnancy in cattle, either pregnant (n=16) or nonpregnant (n=15) cows were slaughtered on Day 10, 15 or 18 postestrus. Ovaries and follicular fluid were collected. Follicles were grouped by diameter: 1.0 to 3.9 mm (small; n=63), 4.0 to 7.9 mm (medium; n=128), and >/= 8.0 mm (large; n=38). The average diameter of large follicles was greater (P<0.05) in pregnant than in nonpregnant cows on Day 10, but on Day 18 it was greater (P<0.05) in nonpregnant than in pregnant cows (11.3 vs 9.7 mm). Status (pregnant vs nonpregnant) did not affect (P>0.10) follicular fluid progesterone nor IGF-I concentrations. In contrast, the status and days postestrus affected (P<0.05) follicular fluid E2 concentrations. Follicular fluid E2 levels in the three follicle size-categories on Day 10 did not differ (P>0.10) between pregnant and nonpregnant cows. However, on Days 15 and 18 postestrus, follicular fluid E2 concentrations in pregnant cows was lower (P<0.05) in large follicles than in nonpregnant cows. We conclude that the presence of a developing conceptus early in pregnancy may alter follicular growth and inhibit follicular E2 production in cattle. These effects appear to be mediated by factors other than IGF-I.