Endocrine regulation of ovarian antral follicle development in cattle

Antral follicle growth in cattle occurs in two distinct phases; the first 'slow' growth phase spans the time from antrum acquisition to a size of approximately 3 mm detectable by transrectal ultrasound, and the second 'fast' phase is gondadotrophin-dependent and includes cohort growth, dominant follicle (DF) selection, and DF growth. This review summarises current concepts of the relative roles FSH and LH, ovarian and metabolic hormones play mainly in the second phase of antral follicle growth in animals of different reproductive and nutritional states. It is proposed that differential FSH response may enable one cohort follicle to become selected, and that follicular secretions, particularly inhibin, suppress FSH and thus are responsible for DF selection and dominance. Acute dependence of the DF on LH pulses will determine DF lifespan, and the LH pulse profile can be influenced by metabolic hormones such as leptin, providing one possible link for nutritional state and reproduction. Direct ovarian effects of acute and chronic changes in growth hormone, insulin and insulin-like growth factor (IGF)-I have been described on cohort follicles, DF oestrogen activity and on DF growth. Influences of metabolic hormones on early antral follicles undergoing their first 'slow' growth phase are less well described, yet metabolic hormones appear to enhance growth into the cohort available for FSH-induced emergence, and may influence subsequent developmental competence of oocytes.     

Kinetics of follicle growth in the prepubertal gilt.

Follicular growth rates were determined by histological examination of ovaries of five prepubertal gilts following treatment with the stathmokinetic agent colchicine. One ovary from each of five gilts was removed surgically and then colchicine (n = 3) or saline (n = 2) was infused i.v. Precisely 2 h after treatment with colchicine, the remaining ovary was removed. Ovaries were processed for histological analyses and sectioned at 10 microns; every twentieth section was stained with hematoxylin and periodic acid-Schiffe's. Sections were viewed with a projection microscope and individual follicles were measured. Eight classes of follicles were established such that the number of granulosa cells per cross section doubled in each class. Diameters of follicles for each class were as follows: 1) less than 106 microns, 2) 106-148 microns, 3) 148-206 microns, 4) 206-287 microns, 5) 287-400 microns, 6) 400-657 microns, 7) 657-1480 microns, and 8) 1480-3130 microns. A layer of thecal cells was first seen in class 2 follicles, and 76% of class 3 follicles had a thecal layer. Oocyte diameter increased through the first four classes and reached a maximum diameter of approximately 110 microns. Almost all follicles greater than 400 microns had an antrum. Preantral follicles had a lower mitotic index and a higher mitotic time and class time than antral follicles. Growth rate increased with increasing size of follicles. Preantral follicles grew at a rate of 5.2 microns/day whereas antral follicles grew at 313 microns/day.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of indomethacin on ovarian follicle rupture--a morphological observation

The purpose of this investigation was to study the role played by indomethacin in blocking ovulation. Immature Wistar rats induced to maturation by PMSG and HCG and normal mature rats were used. Changes in follicle wall of preovulatory follicles occurred after indomethacin treatment were studied both by light and electron microscopy, and were compared with those in controls. 94% of PMSG and HCG stimulated rats, then followed indomethacin injection (3 mg/rat), were inhibited to ovulate; while rats only given hormonal stimulation ovulated in 100%. Adult females in proestrus were treated with indomethacin in doses either of 5 mg or 7.5 mg, none of them ovulated. Whereas, ova were found in the ampullae of normal controls. Ovarian histological examinations of indomethacin treated rats showed that ovum frequently went through the stratum granulosa, however, the theca or the albuginea failed to rupture. The electron microscopy examinations showed that a large amount of collagen fibers scattered under the albuginea layer and interwove with cells of albuginea and theca externa. These two layers, due to containing abundant collagen fibers, thus became barriers for an ovum escaping from a follicle. Follicle walls near the gap of ovulated follicles in controls only had a small quantity of collagen fibers which were more or less with obscure appearance. Cytolysis in albuginea and theca externa layers was also noted. Theca interna cells and granulosa cells, with well developed Golgi bodies and more smooth endoplasmic reticulum in experimental rats revealed that these two tissue components still had a normal endocrine function in spite of receiving indomethacin treatment. The possible effects of prostaglandins on degradation of collagen fibers and contraction of preovulatory follicles were also discussed.     

Effect of exogenous gonadotrophin (PMSG) on the antral follicle population in the sheep.

Follicles were obtained from the ovaries of four groups of 15 ewes. Ewes in the control group were ovariectomized on the 12th day of the oestrous cycle. The other ewes were all given PMSG on the 12th day of the cycle; some were ovariectomized 24 or 40 h later, the others were given prostaglandin followed by hCG and were ovariectomized 6 or 12 h after the hCG injection. All follicles greater than 2 mm in diameter were measured and examined macroscopically for signs of atresia. Some were subjected to detailed morphological examination, the pattern of steroid secretion was determined in others. All the evidence from these three approaches suggested that, in vivo, reversal of the atretic process ('rescue') plays no part in the increase in the number of follicles observed following administration of PMSG.     

Ovarian follicular dynamics after cauterization of the dominant follicle in anestrous ewes

An experiment was conducted to ascertain if follicles could reach ovulatory size after the largest follicle (dominant) has been removed at different times during a progestin treatment in anestrous ewes, and secondly to determine if these new follicles could respond to an hCG-induced ovulation and have similar function as corpora lutea. Mature crossbred sheep (n=44) in anestrous were treated with an intravaginal sponge containing 40 mg of FGA (day 0=sponge insertion) for 9 days. Treatments consisted of cauterization of the largest follicle on the experimental day 3 (T1), day 6 (T2) and day 9 (T3); day 12 to ascertain the size of the largest follicle in control ewes. During laparotomies, the diameters of the largest follicle (DF), and those of the second and third largest follicles (SF1 and SF2, respectively) were determined. On day 12, a second laparotomy was performed for those ewes which had their DF cauterized on days 3, 6 and 9, a fourth group was left intact and only laparotomized on day 12. At this time, the size of the new DF, SF1 and SF2 were determined. Immediately after the laparotomy on day 12, all the ewes were treated with 1000 i.u. of hCG to induce ovulation. Blood samples were collected daily from day 0 to 50 and samples were analyzed for progesterone concentrations. The size of the DF at the time of sponge removal was smaller that those observed on day 3 or 6 of sponge suggesting that follicles in ewes treated with this progestin regress and a new wave of follicular development ensues between day 6 and the time of sponge removal. The size of the DF on day 12 was also smaller in ewes that have the largest follicle removed at the time of sponge removal reflecting that these follicles had a shorter period of growth; however, the rate of growth was greater for these follicles than for follicles arising after cauterization on day 3 or 6 after sponge insertion. There were no differences among treatments, in the number of ewes that formed a corpus luteum (CL) in response to hCG. Life span of the corpora lutea did not differ among ewes having their DF removed on day 6 or 9 or those that served as controls, however, ewes that had their DF removed on day 3 developed longer lived CL in a larger proportion of animals. Average progesterone concentration during the life span of the induced corpora lutea was greater in control ewes than in any other experimental group. These observations allow us to conclude that, (a) the follicular dynamics observed in anestrous ewes treated with a progestin intravaginal sponge resembles that observed during the normal estrous cycle in the ewe; (b) the effects of progesterone on life span of the corpus luteum could not be only related to direct effects at the follicle but also involve changes in other components of the uterine-ovarian-hypothalamic axis; (c) the mechanisms controlling luteal life span seem to be different to those mechanisms controlling the function of the induced corpus luteum.     

Effects of estradiol and progesterone on circulating LH and FSH secretion,and ovarian antral follicle growth in anestrous ewes

In the ewe, ovarian antral follicles emerge or grow in a wave-like pattern and each wave is preceded by a peak in the serum FSH level. The purpose of the current study was to investigate whether in anestrous Western White Face ewes, a combination of progesterone and estradiol affects the circulating FSH peak secretion and the number of small ovarian follicles. Five ewes were treated with subcutaneous silastic rubber implants (10 cm × 0.47 cm), containing 10% estradiol-17β w/w (controls) and 5 ewes were treated with the same estradiol implant, along with subcutaneous implants (11 cm × 0.48 cm) containing 10% progesterone w/w for 12 days. Daily transrectal ovarian ultrasonography and blood sampling was performed from 5 days before, to 9 days after the period of implantation. Blood samples were also taken every 12 min for a 6 h period on day −2, 6 and 13 prior to or after implant insertion (day 0, day of implant insertion). Pulsatility in the serum LH levels was eliminated by the implants (P < 0.05). During the implantation period, the serum FSH peak amplitude was lower in ewes treated with implants releasing estradiol and progesterone, compared to ewes treated with implants releasing only estradiol (P < 0.05). No follicular waves emerged during implant treatment in both groups (P < 0.05) and the number of serum FSH peaks did not differ during implantation, compared to before implantation. During the implantation period, the number of small follicles did not differ in ewes with implants releasing estradiol and progesterone, compared to ewes treated with implants releasing only estradiol. To conclude, supra-physiological concentrations of estradiol completely eliminated the serum LH pulsatality and suppressed the follicular wave emergence, while the FSH secretory peaks that preceded the follicular waves were not affected. Supra-physiological concentrations of estradiol-17β with physiological concentrations of progesterone decreased the serum FSH peak amplitude, eliminated the serum LH pulses, but did not decrease the size of the small follicle pool in anestrous ewes.     

Peptide regulators in the ovarian follicle

Data generated within the last several years have shown that follicular fluid contains substances, presumably peptide in nature, which exert potentially important effects on granulosa cells and the oocyte. This review briefly summarizes the current evidence concerning the nature and importance of these putative regulators including the luteinization inhibitor, oocyte maturation inhibitor, inhibitors of the binding of luteinizing hormone and follicle stimulating hormone, stimulators of ornithine decarboxylase and intrafollicular peptides with gonadotrophin-releasing activity. Although the existence of such activities has been clearly demonstrated, the evidence for a regulatory role of these agents in the control of ovarian physiology is not compelling. However, their occurrence must now be taken into account in our attempts to understand the mechanism of follicular growth, differentiation and atresia.     

Localization of nucleoside phosphatases in the antral follicle of the guinea pig ovary

Ultrastructural localization of nucleozidphosphatases (5'-nucleotidase, adenosin triphosphatase (ATPase) and beta-glicerophosphatase) in antral follicles of the guinea-pig ovary has been studied. Certain heterogeneity has been found in distribution of the enzymes: the cells in the follicular tunic possess the greatest 5'-nucleotidase and ATPase activity. When 5'-adenosin monophosphate (5'-AMP) is used as a substrate, the lead phosphate residue is mainly revealed in the external surface of plasmolemma and as "caps" in the margical zone of nucleoplasm. ATPase activity is chiefly observed in nucleoli of granular cells and in those of the external follicular tunic cells. Histochemical reaction with 5'-AMP proceeds most intensively in the lucid tunic and in processes of the granular cells contacting with the oocyte. A possibility is discussed on participation of the metabolic enzymes, that localize in these structures, in the mechanisms controlling the oocyte maturation.     

Evaluation of antral follicle growth in the macaque ovary during the menstrual cycle and controlled ovarian stimulation by high‐resolution ultrasonography

To date, ultrasonography of monkey ovaries is rare and typically of low resolution. The objectives of this study were to use state‐of‐the‐art, high‐resolution, transabdominal ultrasonography with real‐time Doppler capabilities to: (1) determine whether one can reliably detect in real time the large dominant follicle, the corpus luteum (CL), and small (<2 mm) antral follicles on the ovaries of rhesus monkeys during the natural menstrual cycle; and (2) predict the follicular response of rhesus ovaries to controlled ovarian stimulation (COS) protocols. Rhesus monkeys were selected for transabdominal ultrasonography using a GE Voluson 730 Expert Doppler System at discrete stages of the menstrual cycle. Subsequently, serial ultrasound scanning was employed to observe growth of antral follicles and the CL. Finally, females were scanned to assess follicular growth during COS. The dominant structure and small antral follicles (<2 mm) were reliably visualized in real time. The follicle destined to ovulate could be identified by size differential by day 3 of the follicular phase. The number of small antral follicles present before onset of COS protocol correlated positively with the number of metaphase II‐stage oocytes collected after treatment. The results of this study demonstrate that the population dynamics of antral follicle pools can be noninvasively evaluated in monkeys during natural and pharmacologic ovarian cycles. Am. J. Primatol. 71:384–392, 2009. © 2009 Wiley‐Liss, Inc.     

Suppression of ovarian activity in the gilt and reversal by exogenous gonadotrophin administration

The aim of the current experiment was to study the regulation of follicle development in the pig using a potent GnRH agonist (GnRH-A) to initially suppress follicle development. Large-White hybrid gilts (n = 8) were treated during the luteal phase with GnRH-A. Four of these GnRH-A treated gilts and four control gilts were given a GnRH bolus on days 14 and 28 after GnRH-A administration or during the luteal phase in control gilts. Blood samples were collected for 10 h for FSH and LH, after which 1500 IU PMSG were administered and the ovaries and uteri recovered 72 h later. A further four GnRH-A treated gilts and four control gilts were slaughtered either 28 days after GnRH-A administration or during the luteal phase respectively, and all follicles > or = 1 mm diameter were dissected. The mean basal plasma FSH level was lower (P < 0.01) in GnRH-A treated than control gilts and showed no response to the GnRH challenge although levels increased (P < 0.01) in control gilts. The mean basal plasma LH levels were similar (P > 0.1) in GnRH-A treated and control gilts. Whilst in GnRH-A treated gilts plasma LH levels showed no response to the GnRH challenge, plasma LH levels were increased (P < 0.01) in control gilts. Pulsatile LH secretion was abolished in GnRH-A treated but not in control gilts. Plasma oestradiol levels were lower (P < 0.001) in GnRH-A treated gilts than in control gilts, but nevertheless both GnRH-A treated and control gilts responded to PMSG with increased plasma oestradiol levels. Treatment with GnRH-A reduced both the mean (2.1 vs. 2.7 mm; P < 0.01) and the maximal follicle diameter (4 vs. 6 mm) and reduced (P < 0.01) the total number of follicles > or = 2 mm diameter compared with control gilts. Administration of PMSG increased both mean follicle diameter (5.1 vs. 4.4 mm; P < 0.01) and maximal follicle diameter (7 vs. 9 mm) and caused a reduction (P < 0.001) in the total number of follicles > or = 2 mm diameter in both GnRH-A treated and control gilts. In summary, this study has demonstrated, for the first time in the pig, that the inhibition of follicle development as a result of pituitary down regulation/desensitisation can be reversed by exogenous gonadotrophin treatment. This model will be a powerful tool with which to investigate the precise regulation of follicle development in the pig.     

Effect of estradiol valerate on ovarian follicle dynamics and superovulatory response in progestin-treated cattle

Three experiments evaluated the effects of estradiol valerate (EV) on ovarian follicular and CL dynamics, intervals to estrus and ovulation, and superovulatory response in cattle. Experiment 1 compared the efficacy of two norgestomet ear implants (Crestar and Syncro-Mate B; SMB) for 9 d (with PGF at implant removal), combined with either 5 mg estradiol-17beta and 100 mg progesterone (EP) or 5 mg EV and 3mg norgestomet (EN) im at the time of implant insertion on CL diameter and follicular wave dynamics. Ovaries were monitored by ultrasonography. There was no effect of norgestomet implant. Diameter of the CL decreased following EN treatment (P < 0.01). Mean (+/- S.D.) day of follicular wave emergence (FWE) was earlier (P < 0.0001) and less variable (P < 0.0001) in EP- (3.6 +/- 0.5 d) than in EN- (5.7 +/- 1.5 d) treated heifers. Intervals from implant removal to estrus (P < 0.001) and ovulation (P < 0.01) were shorter in EN- (45.7 +/- 11.7 and 74.3 +/- 12.6 h, respectively) than in EP- (56.4 +/- 14.1 and 83.3 +/- 17.0 h, respectively) treated heifers. Experiment 2 compared the efficacy of EP versus EN in synchronizing FWE for superovulation in SMB-implanted cows. At random stages of the estrous cycle, Holstein cows (n = 78) received two SMB implants (Day 0) and were randomly assigned to receive EN on Day 0 or EP on Day 1. Folltropin-V treatments were initiated on the evening of Day 5, with PGF in the morning and evening of Day 8, when SMB were removed. Cows were inseminated after the onset of estrus and embryos were recovered 7 d later. Non-lactating cows had more CL (16.7 +/- 11.3 versus 8.3 +/- 4.9) and total ova/embryos (14.7 +/- 9.5 versus 7.9 +/- 4.6) than lactating cows (P < 0.05). EP-treated cows tended (P = 0.09) to yield more transferable embryos (5.6 +/- 5.2) than EN-treated cows (4.0 +/- 3.7). Experiment 3 compared the effect of dose of EV on ovarian follicle and CL growth profiles and synchrony of estrus and ovulation in CIDR-treated beef cows (n = 43). At random stages of the estrous cycle (Day 0), cows received a CIDR and no further treatment (Control), or an injection of 1, 2, or 5 mg im of EV. On Day 7, CIDR were removed and cows received PGF. Follicular wave emergence occurred within 7 d in 7/10 Control cows and 31/32 EV-treated cows (P < 0.05). In responding cows, interval from treatment to FWE was longer (P < 0.05) in those treated with 5 mg EV (4.8 +/- 1.2 d) than in those treated with 1 mg (3.2 +/- 0.9 d) or 2 mg (3.4 +/- 0.8 d) EV, while Control cows were intermediate (3.8 +/- 2.0 d). Diameter of the dominant follicle was smaller (P < 0.05) at CIDR removal and tended (P = 0.08) to be smaller just prior to ovulation in the 5 mg EV group (8.5 +/- 2.2 and 13.2 +/- 0.6 mm, respectively) than in the Control (11.8 +/- 4.6 and 15.5 +/- 2.9 mm, respectively) or 1mg EV (11.7 +/- 2.5 and 15.1 +/- 2.2 mm, respectively) groups, with the 2mg EV group (10.7 +/- 1.5 and 14.3 +/- 1.7 mm, respectively) intermediate. Diameter of the dominant follicle at CIDR removal was less variable (P < 0.01) in the 2 and 5mg EV groups than in the Control group, and intermediate in the 1mg EV group. In summary, treatment with 5mg EV resulted in a longer and more variable interval to follicular wave emergence than treatment with 5mg estradiol-17beta, which affected preovulatory dominant follicle size following progestin removal, and may have also affected superstimulatory response in Holstein cows. Additionally, 5 mg EV appeared to induce luteolysis in heifers, reducing the interval to ovulation following norgestomet removal. Conversely, intervals to, and synchrony of, follicular wave emergence, estrus and ovulation following treatment with 1 or 2 mg EV suggested that reduced doses of EV may be more useful for the synchronization of follicular wave emergence in progestogen-treated cattle.     

Effect of treatment of estrous ewes with indomethacin on the distribution of ovarian blood to the periovulatory follicle

The level of prostaglandin (PG) F2 alpha increases within the wall of the ovine follicle pending ovulation. Coincidently, the quantity of ovarian blood distributed to the follicular wall progressively declines. A potential cause(PGF2 alpha)-and-effect (impaired follicular blood supply) relationship was considered. At an early stage of estrus, ewes were injected systemically either with vehicle or indomethacin (an inhibitor of biosynthesis of prostaglandins). Abdominal laparotomies were carried out and the ovaries examined near the expected time of ovulation. The ovary containing the largest follicle or an ovulation point was perfused with radioactive microspheres via the ovarian artery. The periovulatory follicle was isolated from the ovary and the content of radioactivity monitored with respect to that of the whole ovary. Follicular tissue was analyzed for PGF2 alpha. Treatment with the drug was associated with: 1) failure of follicular rupture; 2) follicular hyperemia and edema; and 3) suppressed synthesis of PGF2 alpha. A reduction of the supply of ovarian blood reaching the preovulatory follicle, and a mediatory task of follicular prostaglandins in this process, could be a critical determinant of ovulation.     

Effect of intrauterine infusion of sperm antigens on gilt fertility

A total of 149 maiden gilts (Landrace x Yorkshire) were assigned at random to one of three treatment groups at the onset of puberty. During the first and second estrus, two groups of gilts were inseminated with either 50 ml attenuated semen (n = 50) or physiological saline (n = 50). The control gilts (n = 49) remained untreated. At the third estrus, all gilts were inseminated with fresh extended semen. Intrauterine infusion with attenuated semen prior to breeding significantly increased conception rate of gilts compared with controls (82.0% vs 63.3%; P<0.05). The average interval from puberty to breeding and the age at breeding were lower in gilts treated with attenuated semen (42.75 +/- 0.89 d and 210.39 +/- 2.98 d) than control gilts (45.62 +/- 1.75 d and 218.29 +/- 3.08 d), but these differences were not significant. Litter size and weights at birth and weaning were not influenced by treatment. Results from this study indicate that presensitization to sperm antigens prior to breeding may be a useful and practical method of improving the fertility of maiden gilts.     

Lipoprotein receptor expression during luteinization of the ovarian follicle

Ovarian follicles luteinize after ovulation, requiring structural and molecular remodeling along with exponential increases in steroidogenesis. Cholesterol substrates for luteal steroidogenesis are imported via scavenger receptor-BI (SR-BI) and the low-density lipoprotein (LDL) receptor from circulating high-density lipoproteins and LDL. SR-BI mRNA is expressed in pig ovaries at all stages of folliculogenesis and in the corpus luteum (CL). An 82-kDa form of SR-BI predominates throughout, is weakly present in granulosa cells, and is robustly expressed in the CL, along with the less abundant 57-kDa form. Digestion of N-linked carbohydrates substantially reduced the SR-BI mass in luteal cells, indicating that differences between forms is attributable to glycosylation. Immunohistochemistry revealed SR-BI to be concentrated in the cytoplasm of follicular granulosa cells, although found mostly at the periphery of luteal cells. To examine receptor dynamics during gonadotropin-induced luteinization, pigs were treated with an ovulatory stimulus, and ovaries were collected at intervals to ovulation. SR-BI in granulosa cell cytoplasm increased through the periovulatory period, with migration to the cell periphery as the CL matured. In vitro culture of follicles with human chorionic gonadotropin induced time-dependent upregulation of 82-kDa SR-BI in granulosa cells. SR-BI and LDL receptor were reciprocally expressed, with the latter highest in follicular granulosa cells, declining precipitously with CL formation. We conclude that luteinization causes upregulation of SR-BI expression, its posttranslational maturation by glycosylation, and insertion into luteal cell membranes. Expression of the LDL receptor is extinguished during luteinization, indicating dynamic regulation of cholesterol importation to maintain elevated steroid output by the CL.