Effects of neonatal progestin exposure on female reproductive tract structure and function in the adult ewe

Endometrial glands are present in all mammalian uteri and produce secretions that are hypothesized to support conceptus (i.e., embryo/fetus and placental membranes) survival and development. In sheep, endometrial gland morphogenesis occurs postnatally and can be epigenetically ablated by chronic neonatal exposure to a progestin from birth, thereby producing an adult uterine gland knock-out (UGKO) phenotype. This study determined the long-term effects of neonatal progestin exposure on adult ovine reproductive tract structure and function. Neonatal ewes were exposed to norgestomet (Nor) from birth to 32 wk of age. Unexposed ewes served as controls. After puberty, adult Nor-treated (n = 6) and control (n = 6) ewes were repeatedly bred at estrus (Day 0) to intact rams of proven fertility. In contrast to a pregnancy rate of 80% for control ewes, pregnancy was never detected on Day 25 after mating (or thereafter) in bred UGKO ewes. Control and Nor-treated ewes were then bred and necropsied on Day 9. Similar numbers of hatched blastocysts were present in uterine flushings from control and Nor-treated ewes. Weights of the ovaries and cervices were not affected by treatment. No histoarchitectural differences between control and Nor-treated ewes were detected for ovaries, oviducts, cervices, or vaginae. However, uterocervical and uterine weight as well as uterine horn length were less for Nor-treated ewes. The uteri of Nor-treated ewes were devoid of endometrial glands and lacked the stromal delineation characteristic of intercaruncular endometrium in control ewes. Endometrial width, area, and lumenal epithelial length were decreased in uteri from Nor-treated ewes, but myometrial width and morphology were not affected. Expression of a number of mRNAs that are expressed predominantly in the endometrial epithelia was not different between uteri from control and from Nor-treated ewes. Collectively, these results indicate that neonatal exposure of ewes to a progestin from birth appears to only affect development of the uterus and not any extrauterine reproductive tract tissues. The infertility of the UGKO ewes appears to result from a lack of endometrial glands and, by extension, of their secretions that are required to support growth and development of peri-implantation conceptuses.     

Atresia of large ovarian follicles of the rat

In the rat, at the beginning of pregnancy a cohort of antral follicles develops until the preovulatory stage. However, these follicles, differentiating in the hyperprolactinemic milieu, produce only small amount of estradiol, do not ovulate and undergo rapid degeneration. They constitute an interesting physiological model of atresia. In the present study, we analysed the development and subsequent degeneration of such follicles. The study was performed on Wistar female rats killed in succession between days 1-9 of pregnancy. Excised ovaries were submitted to a routine histological procedure. Paraffin sections were subjected to hematoxylin and eosin staining or in situ DNA labelling. Histological and TUNEL staining revealed that the investigated group of follicles grew slower than that on the corresponding days of the estrous cycle and reached a preovulatory size and morphological appearance on day 5 of pregnancy. They did not ovulate and between days 6 and 9 of pregnancy an increasing number of apoptotic cells appeared within these follicles. They were localized predominantly in the antral granulosa layer, especially near the cumulus oophorus complex (COC) and in the region linking the COC with the follicular wall. The COC and the theca layer were much less affected. In late stages of atresia, also cumulus cells became apoptotic but degenerating oocytes did not exhibit positive TUNEL staining. Only limited number of the theca cells have undergone apoptosis and generally they were not hypertrophied. Our findings indicate that much smaller than normal amount of intrafollicular estradiol was sufficient to support a normal, according to the morphological criteria, although slower development of antral follicles to the late preovulatory stage.     

The expression of angiogenic growth factors and their receptors in ovarian follicles throughout the estrous cycle in the ewe

Healthy follicles are highly vascularized whereas those undergoing atresia have poor vascularity, suggesting a relationship between follicular vascularization and follicular function. Vascularization is regulated by angiogenic factors, and among them vascular endothelial growth factor (VEGF) and angiopoietin-Tie (Ang-Tie) systems are of central importance. The objectives of this study were to determine if VEGF, VEGF receptor-2 (VEGFR-2), and components of the Ang-Tie system are expressed in ovarian follicles at both the protein and mRNA levels and to explore if their expression is related to the stage of the estrous cycle in the ewe. Ovaries from cyclic ewes were collected during the luteal phase (n = 5) or before (n = 5), during (n = 4), and after (n = 4) the preovulatory luteinizing hormone (LH) surge. After fixation, ovaries were wax-embedded, serially sectioned, and analyzed for both protein and mRNA expression of VEGF, VEGFR-2, angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), Tie-1 (mRNA only), and Tie-2. mRNA was studied by in situ hybridization using digoxigenin-11-UTP-labeled ovine riboprobes. A similar pattern of expression was observed for mRNA and protein for all of the factors. Both mRNA and protein expression of VEGF, VEGFR-2, Ang-1, Ang-2, Tie-1 (mRNA only), and Tie-2 in the granulosa and theca cells of follicles ≥2 mm in diameter was significantly different among the stages of the estrous cycle, with the highest expression detected at the post-LH surge stage. Theca cells expressed significantly greater levels of the six angiogenic factors compared with granulosa cells at all stages of the estrous cycle. Expression levels in granulosa and theca cells were comparable between small (2.0 to 2.5 mm) and medium (2.5 to 4.0 mm) follicles, but large follicles (>4.0 mm) expressed higher mRNA and protein levels (all P < 0.05) for all factors at all stages of the estrous cycle. These data show (i) that VEGF, VEGFR-2, and the Ang-Tie system are present in both granulosa and theca cells of the ovarian follicle, (ii) that thecal cells consistently express greater levels of all of these factors compared with granulosa cells, and (iii) that their levels of expression are related to the stage of the estrous cycle and to follicle size.     

A rapid technique for the histological examination of large ovarian follicles

A rapid technique for counting and classifying large ovarian follicles of domestic animals is described. Using a cryostat, 250-micrograms thick sections were cut from the frozen ovary; an image of the surface of each ovarian section was recorded on videotape. By replaying the videotape, the largest profile of each follicle larger than 1 mm in diameter was readily identified and measured. The presence or absence of atresia was determined by applying standard histological methods to fragments of individual follicles taken from the frozen sections. The results obtained are similar to those found using previous methods and demand only one-quarter of the time.     

Ultrasonographic study of ovarian function during early pregnancy and after parturition in the ewe

Transrectal ultrasonography of ovaries was performed in 11 ewes from Days 10 to 26 and on Day 30 of pregnancy to record the number, size, and position of ovarian follicles > or = 3 mm in diameter and corpora lutea (CL). Transrectal and/or transabdominal ultrasonography of the uterus was performed on Days 10 to 26, 30, 35, 40, 45 and 50 of gestation to ascertain the number and position of the conceptuses. In a second experiment, ultrasonography was conducted in 15 ewes on Days 10, 25, 30, 45 and 50 of pregnancy and from Days 13 to 29 after parturition. Ovarian data were classified into ovaries without CL (Group 1), ovaries with the CL in 1 ovary (Group 2), and ovaries with the CL in both ovaries during pregnancy (Group 3). In early pregnant ewes, the total number of follicles and the diameter of all follicles > or = 3 mm were smaller (P < 0.05) in the CL-bearing ovaries (both Group 2, n = 7 and Group 3, n = 8) than in the non-CL-bearing ovaries (Group 1, n = 7), while the largest follicle diameter was significantly smaller in Group 3 than in Group 1 or 2 ovaries. The number of 3-mm follicles in Group 2 ovaries was lower (P < 0.05) than in Group 1 or 3 ovaries, but the mean number of follicles > or = 5 mm in diameter was significantly lower in Group 3 than in Group 1. The total luteal volume per ovary was higher (P < 0.001) in Group 2 than in Group 3 ovaries of early pregnant ewes. The total follicle diameter and the number of follicles growing from 3 to > or = 5 mm in diameter was lower (P < 0.05) for Group 2 ovaries of ewes that carried twins (n = 3) compared with Group 2 ovaries from ewes with singletons (n = 4). There were no differences in follicular dynamics between Group 3 ovaries and the ovaries of Group 2 in ewes that carried twins. No follicles > 3 mm were seen in the ovaries of postpartum ewes that contained CL during gestation, until Days 21 and 25 postpartum for Groups 2 (n = 10) and 3 (n = 8), respectively, and follicles reaching > or = 5 mm in diameter were detected in only 2 ovaries (Group 2), on Days 27 and 28 postpartum, respectively. We conclude that during early pregnancy in ewes there is a suppression of antral follicle growth which appears to be exerted primarily by the developing conceptus but remains confined to CL-bearing ovaries. Residual local inhibition of follicular development extends into the postpartum period.     

The effect of hCG treatment on Day 12 post-mating on ovarian function and reproductive performance of ewes and ewe lambs

The objectives of this study were to determine the effect of a single intramuscular injection of 200 IU hCG (Chorulon) on Day 12 post-mating on ovarian function and subsequent lambing performance in ewes and ewe lambs bred at synchronised oestrus during the breeding season and on the lambing performance of ewes induced to breed during late anoestrus. All animals were mated to rams at synchronised oestrus and on Day 12 post-mating given normal saline or 200 IU hCG.In Experiment 1, laparoscopic results showed that hCG treatment induced accessory corpora lutea in ewes (control = 0/7; hCG = 5/7) but not in ewe lambs (control = 0/7; hCG = 0/7).In Experiment 2, hCG treatment did not improve the lambing rate (control = 50; hCG = 57) or the litter size (control = 1.80; hCG = 1.96) in ewes (control = 100; hCG = 91). However, hCG treatment significantly (P > 0.05) improved the lambing rate (control = 29; hCG = 58; P < 0.05) in ewes conceiving at the first oestrus after treatment. hCG treatment (control = 42; hCG = 42) also failed to improve the lambing rate in ewe lambs (control = 48; hCG = 41).In Experiment 3, hCG treatment had no significant (P > 0.05) effect on the lambing rate (control = 72; hCG = 62) or the litter size (control = 1.59; hCG = 1.58) in ewes (control = 111; hCG = 115) induced to breed during anoestrus or on ewes returning to oestrus and conceiving after treatment (lambing rate: control = 86; hCG = 72; litter size: control = 1.44; hCG = 1.35). In conclusion, the data obtained in this study suggest that during the breeding season hCG may, by stimulating ovarian function, improve embryo survival in ewes conceiving at the first post-treatment oestrus. This effect, however was not observed in ewe lambs.     

Estrogen metabolites in equine ovarian follicles: gas chromatographic-mass spectrometric determinations in relation to follicular ultrastructure and progestin content

Equine follicular fluid was aspirated at various developmental stages (viable, preovulatory and atretic) determined by ultrastructural study. Estrogens and progestins were analyzed by gas chromatography-mass spectrometry associated with stable isotope dilution. Progesterone and 17-hydroxyprogesterone were the principal progestins of the preovulatory and viable follicles. Among the catechol estrogens, 2-hydroxy-estradiol was particularly abundant in the preovulatory follicle and its definitive identification was made by the scan of a full mass spectrum.     

Chromatographic comparison of chondroitin-containing proteoglycan from small and large bovine ovarian follicles

Acetone precipitates were obtained from bovine follicular fluid and chromatographed using Sephacryl S-300 and Sepharose CL-2B. Glycosaminoglycan side chains were precipitated with alcohol after hydrolysis of proteoglycan by protease. Concentrations of glycosaminoglycans were quantitated by high performance liquid chromatography. Results showed that heparan sulfate was not covalently attached to protein and concentrations did not vary between small and large follicles. In contrast, virtually all of the chondroitin sulfate was bound to protein and concentrations were lower in follicular fluid from large follicles. Sulfate content of chondroitin did not change.     

Effect of dihydrotestosterone on the growth and function of ovarian follicles in intact immature female rats primed with PMSG

Intact, immature female rats were primed with PMSG and treated with 4 injections of DHT. DHT given at 0, 12, 24 and 36 h caused a significant decrease in the ovulation rate 72 h after the PMSG treatment. Concurrent treatment with oestrogen reversed the inhibitory effects of the androgen. The androgen effect was apparently exerted directly on the ovary since DHT did not alter the surge of LH and FSH which occurred at 58 h after PMSG treatment. The DHT inhibition of ovulation was observed in the treatment cycle as well as in subsequent cycles which followed a second PMSG injection. This finding suggests that intermediate size follicles were also adversely affected by the androgen. To confirm that androgen affects follicles of all size ranges, follicles less than 200 microns, 200-400 microns and greater than 400 microns in diameter were isolated from the ovaries of rats treated with PMSG and DHT or the vehicle. The follicles were isolated by density gradient separation of follicles followed by filtration with pre-calibrated Teflon sieves. In some experiments, granulosa cells were also harvested from isolated follicles. DHT treatment did not affect the numbers of follicles of any size but did reduce the oestrogen content of follicles of all sizes. Follicles from DHT-treated animals contained fewer granulosa cells and the cells from treated animals had lower aromatase activity than did cells from control rats. Taken together, these findings suggest that DHT reduces the ovulation rate by decreasing the number of granulosa cells/follicle and by altering the oestrogen synthetic abilities of the cells. All follicles, regardless of size, were sensitive to androgen treatment.     

Mouse ovarian follicles secrete factors affecting the growth and development of like-sized ovarian follicles in vitro

A series of experiments have been carried out to determine whether follicles secrete factors able to affect the growth and development of other, like-sized follicles. Late preantral mouse ovarian follicles were either cocultured or cultured in media conditioned by previously cultured follicles. In particular, the experiments examined whether follicles do secrete such factors, whether the level of FSH in the culture media can affect that process, and what the nature of such secretory factor(s) might be. First, pairs of follicles were cocultured across a polycarbonate membrane containing pores. This showed that communication between the follicles resulted in the stimulation of growth and that the stimulation was due, at least in part, to the production of secretory factor(s). In subsequent experiments, follicles were cultured in media that had been preconditioned by previously cultured follicles. The concentration of FSH in the cultures determined the effect of the conditioned media: conditioned media was stimulatory to follicle growth when levels of FSH remained high throughout the culture, but inhibitory when FSH levels were dropped midway through the cultures. Heat inactivation removed this inhibitory effect, showing that the factor was likely to be a protein; addition of follistatin to the conditioned media did not alter its effect, indicating that the factor was unlikely to be activin. We have shown through a series of culture experiments that mouse follicles secrete factor(s) that can affect the development of other like-sized follicles when cultured from the late preantral to Graafian stages. Furthermore, we have shown that the effect (or production) of such factors is dependent on the FSH environment of the follicles.     

Luteal function in cows following destruction of ovarian follicles at midcycle

Luteal function was studied in the absence of non-ovulatory ovarian follicles to determine if these follicles are involved in luteal regression in cattle. After at least one estrous cycle, cows were assigned randomly to treatment (n=5) or control (n=5). All cows were laparotomized on day 10 postestrus (Estrus = day 0). During laparotomy of treated cows, all visible follicles on both ovaries were destroyed by electrocautery, and follicular growth was prevented by ovarian x-irradiation. In controls, laparotomy and ovarian manipulation were as in treated cows but follicles were not destroyed and ovaries were not irradiated. On day 22 postestrus, ovaries of 4 treated cows contained no visible follicles and concentrations of estradiol-17beta in jugular plasma (0.4 +/- 0.1 pg/ml) were less (P<0.05) than in controls (3.2 +/- 0.4 pg/ml). Daily mean concentrations of LH from surgery to day 22 postestrus in treated cows did not differ from controls. On day 22 postestrus, progesterone in jugular plasma and weights of corpora lutea in treated cows were greater (P<0.05) than in controls. Between days 12 and 18 postestrus, concentrations of estradiol-17beta and PGF(2)alpha in utero-ovarian venous plasma of controls increased prior to detectable declines in concentrations of progesterone. Therefore, non-ovulatory ovarian follicles present during mid to late diestrus are necessary for luteal regression in non-pregnant cattle.     

Ovarian follicles during infancy in Romanov and Ile-de-France ewe lambs

The ovaries of new born lambs (15 Ile-de-France and 19 Romanov, 34 ovaries) and of 4-week-old lambs (6 Ile-de-France and 12 Romanov, 18 ovaries) were examined histologically to compare ovarian follicular development in infant lambs of breeds differing in their prolificacy. Breed was the major factor affecting follicular population at birth. Ile-de-France lambs had a higher total number of growing follicles (P less than 0.001), and more preantral (P less than 0.001) and antral (P less than 0.005) follicles than did Romanov lambs. Furthermore, the size of the largest follicles was also reduced in Romanov compared to Ile-de-France lambs. At 4 weeks of age, most of the features of the ovarian follicular population except the mean size of the third largest follicle were similar between the two breeds. However, atresia of antral follicles had appeared only in Ile-de-France and not in Romanov lambs. When a challenge with exogenous gonadotrophins (1000 i.u. PMSG followed by 1500 i.u. hCG) was attempted, ovulation was triggered in 2/6 and 0/12 Ile-de-France and Romanov lambs respectively. Massive follicular development was noted in 3/6 Ile-de-France lambs but in none of 12 Romanov lambs. Retardation of follicular development together with retardation in the establishment of ovarian sensitivity to gonadotrophins are therefore features typical of the ovaries of Romanov lambs compared to Ile-de-France lambs during the post-natal period.