Number Of Preantral Follicles Research Articles

The recovery of preantral follicles from ovaries of domestic cats and their characterisation before and after culture

The mechanical dissection of ovaries from domestic cats provided high numbers of preantral follicles (300–24 500 per ovary) with diameters of 40–90 μm. The high variability between the individual queens was not correlated to age, cycle, breed or season. The number of primordial and primary follicles was significantly dependent upon the total recovery rate (r = 0.997; P < 0.0001). The growing population (secondary follicles) was relatively constant until the isolation rate dropped below 1000 per ovary. Then the number of growing follicles was correlated to the resting pool. Follicle viability, as estimated by trypan blue staining, dropped immediately after isolation to 32.5%. After 1 week of culture, follicle viability varied depending on the method of culture (in monolayer, 33.3%; co-culture with granulosa cells, 37.5%; in collagen gels, 30.8%; in conditioned medium, 9.3%; on agarose pad, 17.9%). A 10μm increase in diameter was observed in approximately 65% of growing follicles (61.3–68.6% depending on growth conditions). The number of surrounding granulosa cells decreased and 32% of the oocytes were without granulosa cells. The addition of epidermal growth factor (EGF) and insulin-transferrin-selenite (ITS) to the culture medium prevented these alterations in structure. In the control group only 22% of intrafollicular oocytes had an intact germinal vesicle as opposed to ITS-medium (57%) and EGF-medium (33%). Cryopreservation of preantral follicles resulted in a 60% reduction in follicle viability.

Synergistic effects of steroids with FSH on folliculogenesis, steroidogenesis and FSH- and hCG-receptors in hypophysectomized mice

Injection of ovine FSH (4 micrograms day-1) for 4 days into hypophysectomized mice does not restore folliculogenesis to normal cyclic values. This may be due to insufficient production of oestradiol. The present study was designed to determine whether FSH- and LH-induced oestradiol was critical for growth and differentiation of follicles at all stages. Twelve days after hypophysectomy, mice were injected s.c. with 10, 50 or 250 micrograms oestradiol cyclopentylpropionate daily with or without ovine FSH (4 micrograms day-1) for 1-4 days. One ovary from each animal was used for histology. From the second ovary, follicles were isolated at different stages and incubated with [3H]thymidine for 3 h to determine the rate of DNA synthesis. Incubation medium and serum were used for steroid determinations. After oestradiol treatment alone, there was a dose-dependent response in serum oestradiol, but ovarian and uterine weights did not increase further with the increasing doses of oestradiol administered. This finding was consistent with an increase in the number of preantral follicles and small antral follicles but excluding the development of preovulatory follicles. Treatment with 10 and 50 micrograms of oestradiol cyclopentylpropionate did not prevent antral follicles from undergoing atresia. The higher dose (250 micrograms day-1) of oestradiol cyclopentylpropionate delayed atresia of antral follicles and maintained more large healthy antral follicles. After concurrent injection of oestradiol and FSH, ovarian weight was 2-3 times greater than with either FSH or oestradiol alone; the number of follicles and follicular DNA synthesis at all stages of development increased without any signs of atresia; the in vitro accumulation of oestradiol also increased. Oestradiol alone induced FSH receptors in granulosa cells, but did not induce hCG receptors in any ovarian compartment; FSH alone induced FSH and hCG receptors in granulosa cells but not in thecal-interstitial tissues, whereas, oestradiol plus FSH enhanced FSH receptors in granulosa cells and LH/hCG receptors in granulosa and thecal-interstitial tissues. The synergistic effect of oestradiol with FSH was mimicked by the same dose of diethylstilboestrol, testosterone or dihydrotestosterone, but the latter steroids increased only the number of antral follicles, presumably because of their shorter half-lives. These results indicate that in mice oestradiol stimulates the growth of preantral and antral follicles and delays follicular atresia; oestrogens and androgens act synergistically with FSH to enhance follicular proliferation and differentiation, and prevent follicles from undergoing atresia.

Hypophysectomy of the cyclic mouse. II. Effects of follicle-stimulating hormone (FSH) and luteinizing hormone on folliculogenesis, FSH and human chorionic gonadotropin receptors, and steroidogenesis.

This study was designed to determine the effects of FSH and LH on ovarian follicular development in adult hypophysectomized (HX) mice. Twelve days after HX, the animals received s.c. injections of ovine FSH (oFSH; 4 micrograms/day) or oFSH (4 micrograms/day) plus ovine LH (oLH; 2 micrograms/day) twice a day for 1 to 4 days. After 4 days of treatment with FSH alone, the number of preantral follicles (stages 1-3) increased significantly compared to that in HX controls and reached cyclic numbers; however, incorporation of [3H]thymidine into these preantral follicles as compared to HX controls did not increase. The number of healthy antral follicles (stages 4-5) and incorporation of [3H]thymidine into stage 5 follicles started to increase after only 1 day of treatment with FSH, and the number of atretic follicles concomitantly decreased. Treatment with both FSH and LH for 1 to 4 days increased the number of healthy follicles and restored DNA synthesis at all stages (1-5) to normal levels. Two days of replacement with FSH or FSH plus LH was required for follicles to attain preovulatory size (stage 6). FSH alone induced FSH and hCG receptors in granulosa cells, but without the induction of thecal LH/hCG receptors; FSH induced production of progesterone and androstenedione by stage 6 follicles, but not estradiol (E2) accumulation in the incubation medium or in the serum. Combined FSH and LH induced hCG receptors in the theca and interstitium, and also restored follicular E2 production to proestrous values. LH alone increased only the number of stage 2-3 follicles. Unexpectedly, LH alone also induced thecal hCG receptors as well as FSH receptors in granulosa cells of preantral and antral follicles. The present results demonstrate that FSH is essential for follicular growth at all stages and for prevention of atretic antral follicles. Both FSH and LH are necessary for regulation of follicular development and differentiation from the earliest preantral to preovulatory stages.

Early Folliculogenesis in Primate Ovaries: Testing the Role of Estrogen1

The purpose of this study was to examine the effect of an exogenous estrogen, diethylstilbestrol (DES), on follicle development in the ovary of a juvenile primate. The immature cynomolgus monkey (12-22 mo) was used as a model since ovaries at this age lack endogenous gonadotropin support but are capable of responding to exogenous hormonal stimulation. In addition, the pituitary gland receives virtually no GnRH stimulation and under these conditions lacks responsiveness to estrogen feedback. Two groups of three monkeys each received DES for 14 days. Members of the second group also were given GnRH antagonist to assure no GnRH action upon the gonadotropes. The left ovary of each monkey was removed just prior to Day 1 of DES treatment and served as the control. The right ovary was removed on Day 14 of treatment. Both ovaries from each monkey were prepared for evaluation by light microscopy. Results indicated that both the number of preantral follicles and the mean number of medium-sized (0.5-1 mm in diameter) developing antral follicles decreased significantly (p less than 0.05) in the DES-treated ovaries with no increase in early-atretic antral follicles. These data suggest that DES, at the amount administered, inhibits the growth of both preantral and medium-sized antral follicles in the primate. Whether these effects are manifest directly at the follicle level or are mediated by other mechanisms remains to be determined.

Comparison of the total ovarian follicular populations at day 140 of pregnancy and at day 5 postpartum in ewes

The total ovarian follicular populations were determined in ewes at Day 140 of pregnancy and at Day 5 postpartum. The right and left ovaries of five pregnant and five non-suckling ewes of the Préalpes-du-Sud breed were used in this study. All the ovaries were serially sectioned at a thickness of 7 μm, and every section was examined microscopically. The mean numbers of preantral follicles per ovary increased (P<0.005) at Day 5 postapartum as compared to Day 140 of pregnancy. The distribution of preantral non-atretic follicles into different size classes clearly showed a sharp increase in the mean number of follicles per size class at Day 5 postpartum, especially those leaving the reserve of primordial follicles. No difference was detected between both groups of ewes in the mean number of antral follicles. The diameter of the largest antral follicle at Day 140 of pregnancy does not exceed 1.5 mm. However, at Day 5 postpartum, a population of large follicles ≥ 1.5 mm was observed, reaching 2–4 mm in diameter. We conclude that although the pattern of normal follicular development is inhibited during late pregnancy, the ovary at this time is not quiescent, and ovarian follicular development starts well before parturition. The increasing number of preantral follicles, as well as the enlargement of antral follicle diameter observed at Day 5 postpartum, may be correlated with increasing secretion of FSH after lambing.

Short- and long-term effects of hypophysectomy and unilateral ovariectomy on ovarian follicular populations in sheep.

The effects of hypophysectomy and unilateral ovariectomy on the total number of follicles with greater than 3 layers of granulosa cells were determined at 4 and 70 days following treatment. The population of preantral follicles (less than 0.23 min diam.) was found to be under the control of gonadotrophins but such control was only evident on a long-term basis. At 70 days after unilateral ovariectomy there was a large increase in the number of preantral follicles but at 70 days after hypophysectomy there was a large decrease. The population of antral follicles (greater than 0.23 mm diam.) was under the immediate control of gonadotrophins. By 4 days after hypophysectomy all large antral follicles had become atretic and the number of antral follicles was further decreased at 70 days after treatment. At 70 days after unilateral ovariectomy there was an increase in the number of antral follicles. The follicular growth rates at 70 days following treatment were decreased in hypophysectomized ewes but increased in ewes after unilateral ovariectomy.

SERUM GONADOTROPHINS AND FOLLICULAR DEVELOPMENT IN IMMATURE RATS AFTER EARLY ANDROGEN ADMINISTRATION

Follicular development and serum gonadotrophin levels were studied in female rats after neonatal androgen administration. After injection of 1250 mug testosterone propionate (TP) on day 5 after birth the composition of the follicular population was altered: at nearly all ages the number of pre-antral follicles (follicular volume 2-20 X 10(5) mum2) was lower than in oil-treated rats, in some cases the number of small antral follicles (21-249 X 10(5) mum3) was also lower. Furthermore levels of serum follicle-stimulating hormone and luteinizing hormone were decreased from day 7 to day 20 suggesting that the high gonadotrophin levels before day 20 are of importance for normal follicular development. In contrast, final follicular maturation in TP-treated rats was enhanced; at day 35 more large antral follicles (follicular volume greater than or equal to 500 X 10(5) mum3) were present in TP-treated rats than in oil-treated rats. The presence of more large antral follicles was accompanied by higher plasma oestradiol concentrations, higher uterine weights and advanced vaginal opening. These results demonstrate an inhibition of normal follicular growth and an acceleration of ovarian maturation after neonatal androgen administration.

Quantitative aspects of follicular development in the untreated and PMS-treated cyclic hamster.

AbstractHamsters injected at 0900 on day 1 of the cycle (metestrus) with either 0, 5 or 15 IU pregnant mare's serum (PMS) were killed at 1500 of days 1 to 4 of the cycle and the ovaries prepared for light microscopy and for a quantitative evaluation of follicular development. In the untreated cyclic hamster, the maximal number of preantral follicles with eight or more layers of granulosa cells occurred between the afternoon of day 4 (proestrus) and day 1, coinciding with the highest blood levels of FSH and LH. It is concluded that the elevated preovulatory levels of gonadotropins not only induce the ovulation of the mature antral follicles but at the same time recruit the next set of follicles for development during the new cycle.By the afternoon of day 1, treatment with either 5 or 15 IU PMS recruited more follicles into large preantral and incipient antral stages than in the untreated hamsters. However, by day 2 the pattern of follicular distribution was similar between the 5 IU PMS and untreated group whereas considerably more antral follicles had differentiated in the animals given 15 IU PMS. The ability of 15 IU PMS to elicit superovulation therefore depends on the levels being initially high enough to mature more follicles at critical stages of their development; the prolonged biological half life of PMS then sustains these follicles throughout the cycle.