Regulation of the proliferative activity of ovarian surface epithelial cells by follicular fluid

Despite critical roles of the ovarian surface epithelium (OSE) in ovulation and post-ovulatory wound repair, little is known about the physiological mechanism regulating OSE proliferation. A role of follicles and corpora lutea in locally regulating the proliferative activity of OSE has been suggested. In this study, the effects of follicular and luteal products on proliferation of cultured OSE cells were tested using cells obtained from seasonally anoestrous ewes. Follicular fluid but not luteal extracts induced OSE cell proliferation (2.5-fold relative to untreated controls; P < 0.0001). The response of OSE cells was not affected by follicle size or previous charcoal-extraction of follicular fluid (P > 0.1). Treatment with IGF-1 (2.2-fold; P < 0.01), EGF (1.9-fold; P < 0.01) and, to a lesser extent, FSH (P < 0.05) also induced OSE cell proliferation. In contrast, oestradiol or progesterone did not induce cell proliferation or enhance the effects of FSH on proliferation (P > 0.1). It was concluded that follicular fluid can directly stimulate ovine OSE cell proliferation and that this effect is attributable to non-steroidal mitogens.     

Seasonal variation of the ovarian follicular dynamics and luteal functions of sheep in the subtropics

This study was undertaken to describe the development of individual follicles and corpora lutea (CL) in Ossimi ewe lambs at different seasons of the year in the subtropics. Seven ewe lambs underwent daily ultrasonographic examination for 20 interovulatory periods (IOP) during spring, winter and autumn. Ovarian follicles >or=2 mm and corpora lutea were counted and measured. Blood samples were taken for progesterone (P(4)) analysis. All ewe lambs included, but one, were cyclic in all seasons studied. Three (65%) and two (35%) follicular waves were detected per estrous cycle. None of the characteristics of the large follicles was affected by season. Follicles >or=2 mm in diameter were significantly higher in winter. The CL developed slowly in autumn. Serum P(4) level was higher in autumn. Double ovulation was observed only in autumn. The data demonstrated that Ossimi sheep in the subtropics were cyclic in most seasons of the year. Season affected mainly the luteal functions with little influences on the follicular characteristics.     

The role of luteal steroid hormones in the regulation of the estrous cycle of high fecundity Olkuska sheep

The study was undertaken to investigate the steroid hormone production by sheep luteal cells. Corpora lutea were collected from 30 Olkuska sheep on Days 3, 6, 9, 12 and 15 of the estrous cycle during the reproductive season. In Experiment 1, steroid hormone concentration was estimated in extracts of CL. In Experiment 2, luteal cells were cultured in vitro for 24 h. Luteal cells isolated on Days 9 and 12 secreted high amounts of progesterone and androgens but smaller amounts of estradiol. Concentration of these steroids in CL extracts collected on the same days showed the same trend. In CL harvested on Day 15, a decrease in androgens and progesterone as well as a significant increase in estradiol were observed in culture media and in extracts. Judging from the high amounts of estradiol and low amounts of androgen observed at the end of the luteal phase, we speculate that the steroid hormones secreted by the regressing CL may play an active role in the regulation of the estrous cycle in the Olkuska sheep with autocrine influence on the luteal activity or a possible paracrine action on follicular growth.In the third Experiment, the possibility of heterogeneity in the multiple corpora lutea population of prolific Olkuska sheep was investigated. Differences were found in the level of progesterone and estradiol secretion by individual corpora lutea recovered from the same animal, which also varied in terms of weight. This is the first study which shows the existence of intra-ovarian and individual heterogeneity between corpora lutea recovered from ewes during the normal estrous cycle.     

Immune regulation of ovarian function in buffaloes (Bubalus bubalus)

We studied the infiltration of different subsets of immune system cells in the ovarian parenchyma of Egyptian buffaloes during follicular and luteal phases of the estrous cycle. All subsets of leukocytes infiltrated significantly more into corpora lutea (CL) than into Graafian follicles (GF) (P < 0.01) except for plasma cells that were abundant in the GF but not observed in the CL. The number of macrophages, lymphocytes, neutrophils and eosinophils were significantly greater in mature CL than in corpora hemorrhagica (CH) or regressing CL. Moreover, the regressing CL showed significantly more macrophages, lymphocytes and neutrophils than the CH. Large antral follicles were infiltrated with larger number of leukocytes than growing preantral atretic follicles. Macrophages and neutrophils observed in large antral follicles were significantly more abundant in the theca externa than the theca interna (P < 0.01). Only plasma cells were significantly greater in number in the theca intema (P < 0.01). Leukocytes infiltrated significantly more into large mature follicles than large, growing, preantral atretic follicles (P < 0.01). Results of this study reveal the calling of leukocytes in a significant numbers inside the ovarian tissue of buffaloes around the time of ovulation and at luteolysis. It is possible that leukocytes with their powerful bioactive cytokines (IL-1, TNFalpha, GM-CSF, and INF-gamma) may assist in ovarian functions such as ovulation and luteolysis.     

Relationship between progesterone and estradiol contents of follicular fluid and the morphological appearance of granulosa cells of follicles coexisting with corpora lutea in bovine ovaries

Bovine ovaries (n=149) bearing follicles (>5 mm) coexisting with mature corpora lutea (CL;>10 mm) were obtained at a local abattoir without regard for the reproductive status of the donor cows. Most corpora lutea were 21 to 25 mm in diameter, and nearly half of the largest follicles were 11 to 15 mm in diameter. When oocytes were aspirated from follicles 16 to 30 mm in diameter, approximately 60% of them proved to be degenerated. Concentrations of progesterone (P4) and estradiol-17beta (E2) in the follicular fluid of 23 follicles (>10 mm) were determined. Progesterone and estradiol-17beta were found to be the major hormone in 16 (69.6%) and 7 (30.4%) of the follicles, respectively. Light-microscope observations of the granulosa cells of the same 23 follicles showed that 7 were deficient in mural granulosa cells, and that 15 of the remaining 16 follicles were atretic or luteinizing. Ultrastructural observations of granulosa cells revealed many lipid droplets in the cytoplasm of follicles coexisting with mature CL, suggesting the initiation of luteinization. These results show that approximately 70% of the follicles were P4-dominant and that more than 95% of them were morphologically degenerated. Thus it is suggested that morphological signs of atresia precede changes in the concentrations of hormones in the follicular fluid of follicles coexisting with corpora lutea (>10 mm) during the middle of the estrous cycle.     

Steroid production and hCG binding by ram-induced ovarian follicles in seasonally anoestrous ewes

The introduction of rams to a group of previously isolated anoestrous ewes has been shown to stimulate ovarian follicular development and ovulation. The present experiment was carried out to determine the ability of follicles arising from this ram stimulus to produce steroids and bind hCG. Seasonally anoestrous Southdown ewes were exposed to rams for 24 h, 40 h, 3 days, 10 days or 20 days before ovariectomy. Steroid production and the concentration of hCG binding sites in follicles dissected from the ovaries were measured in vitro. The presence of a ram caused ovulation and enhanced oestradiol production by follicles, but had little effect on total androgen production or the number of hCG binding sites present in the follicles when compared to follicles from anoestrous ewes. The oestradiol concentrations in large follicles were not as high as in preovulatory follicles from cyclic ewes reported in other studies. Follicles continued to develop through the ram contact period and when incubated after 40 h and 10 days of ram contact produced high levels of progesterone, indicating partial luteinization, although the corpora lutea (CL) resulting from the induced ovulations regressed prematurely. We suggest that the lack of hCG binding sites in ram-induced follicles may be the cause of poor luteinization and suboptimal development of luteal tissue after induced ovulation in ewes during seasonal anoestrus.     

Effects of repeated removal of large ovarian follicles and treatment with progestin on ovarian function in the ewe

Preovulatory follicles were removed from ewes during estrus to determine hormonal, ovarian and behavioral responses. In Experiment 1, new follicles were recruited and ovulated within 4 days, and a second estrous period was observed in most ewes. In Experiment 2, follicles were removed at Day 0 (estrus), Day 3.5 and Day 7.0 to determine responses to repeated follicular removal in the absence of a corpus luteum (CL). Ewes in two groups were given exogenous progestin at the time of first or second surgery. Each follicular removal was followed by a surge of follicle-stimulating hormone (FSH) and follicular growth, and in many cases, behavioral estrus and/or a surge of luteinizing hormone (LH) was detected around the time of the next follicular removal. Although not necessary for display of estrus, treatment with progestin during follicular maturation increased the number of ewes showing estrus. When the newly developing follicles were allowed to ovulate, resulting corpora lutea produced low levels of progesterone or had a short life span.     

Hormonal regulation and cell-specific expression of endothelin-converting enzyme 1 isoforms in bovine ovarian endothelial and steroidogenic cells

Endothelin-converting enzyme 1 (ECE-1) is a key enzyme in the biosynthesis of endothelin 1 (ET-1), a potent regulator of ovarian function. Different ECE-1 isoforms are localized in distinct intracellular compartments. Thus, the spatial and temporal pattern of ECE-1 expression determines the site of big ET-1 activation and the bioavailability of ET-1. This study was undertaken to investigate the hormonal regulation and cell-specific expression of ECE-1 isoforms in endothelial and steroidogenic cells of bovine follicles and corpora lutea (CL). Using enriched follicular and luteal cell subpopulations and in situ hybridization techniques, we showed that the ECE-1 gene is expressed by both endothelial and steroidogenic cells; however, the intracellular ECE-1a isoform was present only in ET-1-expressing endothelial cells. Steroidogenic cells in follicles or in CL, deficient in ET-1, expressed only the plasma membrane ECE-1b isoform. The intensity of antisense ECE-1 labeling in the granulosa cell layer increased with follicular size; insulin-like growth factor I and insulin upregulated ECE-1 expression when cultured with granulosa cells, suggesting that these growth factors may increase ECE-1 in growing follicles. In contrast, ET-1 and LH downregulated ECE-1 in steroidogenic cells. This effect could account for low ECE (and ET-1) levels, which characterize the early luteal phase. These findings suggest that ECE-1 is regulated during different stages of the cycle in a physiologically relevant manner. The hormonal regulation and intracellular localization of bovine ECE-1 isoforms revealed in this study may provide new insights into ET-1 biosynthesis and mode of action in different cellular microenvironments within the ovary.     

Changes in the levels of pituitary and steroid hormones in ovine and human ovarian follicular fluid

Changes in the protein and steroid hormones of follicular fluid, aspirated from different follicles of sheep and human ovaries, have been measured and correlated with the size of the follicles. As the fluid contains a number of proteins, steroids have been measured directly and after ether extraction. The follicular fluid concentrations of progesterone and 17 beta-oestradiol measured directly in the fluid increased with the size of the follicles. The levels of free testosterone remained constant in all sizes of follicles, while those of bound hormone showed a 10- to 15-fold increase over the free testosterone concentrations in both the sheep and human follicular fluid. A decrease in the levels of bound testosterone in the fluid of large follicles (LFFL) coincided with the increase in bound 17 beta-oestradiol, suggesting the possible conversion of bound testosterone to oestrogen as the follicle attained maturity. The ratio of follicle-stimulating hormone (FSH) to luteinizing hormone (LH) varied in the fluid obtained from different size follicles, being 1:7 in small (SFFL), 1.3.5 in medium (MFFL) and 1:2.3 in large (LFFL) follicles of sheep ovaries. The LH content of follicular fluid of different size follicles appeared to be the same, with LFFL showing a minor increase over SFFL. In the human, the fluid from medium follicles contained very little LH compared to LFFL. These differences in the pattern of LH levels present in the fluid from different size follicles between human and sheep ovaries presumably reflect species variations in the entry of LH into the follicles.     

The effect of the absence or presence of a corpus luteum on the ovarian follicular population and serum oestradiol concentrations during the estrous cycle in Sanjabi ewes

The aim of this study was to determine if the presence or absence of a corpus luteum (CL) during estrous synchronization in ewes can affect the ovarian follicular population and the serum oestradiol concentrations. The estrous cycles of 197 Sanjabi ewes were synchronized using a 12-day treatment with intravaginal progestagen sponges (Chronogest^®). Estrus was detected in 144 ewes, 27–39 h after sponge removal. Blood samples were taken daily from day 2 and continued for 19 days and analyzed for serum oestradiol concentration. Nine ewes were slaughtered on each experimental day (days 1–16 after estrus) for ovary collection. The ovaries per ewe were classified as those without, or with one or two CL's, for each slaughter day. Visible follicles on the surface of the ovaries were classified, based on their diameter, into (i) very small (<2 mm), (ii) small (2–3.4 mm), (iii) medium (3.5–5 mm) and (iv) large (>5 mm) categories, and the respective numbers recorded. Results indicated, the number of ovarian follicles to decrease (P < 0.01) from days 1 to 5 of the cycle and showed a significant increase on day 7. Numbers were high again on day 11 and decreased (P < 0.01) on day 16 of the estrous cycle. The serum oestradiol concentrations were significantly higher (P < 0.001) in the double than in the single ovulating animals (one or two CL's, respectively) on days 2–0. However serum levels were also significantly higher (P < 0.001) in single, than twin ovulating animals on days 4–5 and 12–16 of the estrous cycle. There were no significant differences in the total number of very small follicles between animals without and those with two CL's. The number of small, medium and large follicles in ewes, with or without a CL on the ovary was significantly higher (P < 0.01) than ewes with two ovulations at certain stages of the estrous cycle. The present study provides evidence of differences in the follicular ovarian population in ovaries without CL's and double ovulations. The existence of an intraovarian effect of the CL numbers on follicular population is demonstrated.     

Localization of androgen receptor and cytochrome P450 aromatase in the follicle and corpus luteum of the porcine ovary

The following study was undertaken to localize androgen receptors (AR) and aromatase cytochrome P450 (P450arom) in porcine ovarian tissue because ovarian androgens may act locally to modulate follicular and luteal function in various species. Androgen receptor was detected immunohistochemically in granulosa and theca cells of preantral as well as in growing antral follicles. The most intensive staining was observed in the antral granulosa layer. Luteinizing granulosa cells of preovulatory follicles, and luteal cells from the early and midluteal phases stained weakly for the androgen receptor. Fully regressed corpora lutea in the early follicular phase of the next cycle did not stain for androgen receptor. In contrast, granulosa cells were very weakly stained for aromatase in early stages of follicular development. The P450arom was maximally expressed with the same intensity in mural and antral layers in large ovulatory follicles. Corpora lutea from the early luteal phase showed positive staining, whereas those from midluteal phase did not stain for aromatase, some cells of regressed corpora lutea unexpectedly exhibited aromatase staining.     

Pituitary control of ovarian activity in the lizard, Agama agama

The principal ovarian structures in Agama agama are non-yolky and yolky follicles, corpora lutea and atretic follicles. Hypophysectomy affects all stages of vitellogenesis and follicular atresia results. Atresia involves resorption and digestion of yolk by the granulosa cells, which eventually undergo a fatty degeneration. Immature follicles and corpora lutea are not affected by hypophysectomy.
Injections of 0.2 mg FSH (NIH-FSH-S3 ovine) on alternate days for 14 days not only maintain vitellogenesis in hypophysectomized animals, but also tend to overstimulate the small follicles to synthesize yolk. A similar dose of LH(NIH-LH-SII ovine) fails to maintain vitellogenesis in hypophysectomized animals, while a higher dose of 0.4 mg LH tends to overstimulate the small follicles. An unusual rupture of yolky follicles in FSH injected lizards is reported and a possible development of such follicles into corpora lutea is discussed.     

Glycomic analyses of ovarian follicles during development and atresia

To examine the detailed composition of glycosaminoglycans during bovine ovarian follicular development and atresia, the specialized stromal theca layers were separated from the stratified epithelial granulosa cells of healthy (n = 6) and atretic (n = 6) follicles in each of three size ranges: small (3–5 mm), medium (6-9 mm) and large (10 mm or more) (n = 29 animals). Fluorophore-assisted carbohydrate electrophoresis analyses (on a per cell basis) and immunohistochemistry (n = 14) were undertaken. We identified the major disaccharides in thecal layers and the membrana granulosa as chondroitin sulfate-derived ?uronic acid with 4-sulfated N-acetylgalactosamine and ?uronic acid with 6-sulfated N-acetylgalactosamine and the heparan sulfate-derived Δuronic acid with N-acetlyglucosamine, with elevated levels in the thecal layers. Increasing follicle size and atresia was associated with increased levels of some disaccharides. We concluded that versican contains 4-sulfated N-acetylgalactosamine and it is the predominant 4-sulfated N-acetylgalactosamine proteoglycan in antral follicles. At least one other non- or 6-sulfated N-acetylgalactosamine proteoglycan(s), which is not decorin or an inter-α-trypsin inhibitor family member, is present in bovine antral follicles and associated with hitherto unknown groups of cells around some larger blood vessels. These areas stained positively for chondroitin/dermatan sulfate epitopes [antibodies 7D4, 3C5, and 4C3], similar to stem cell niches observed in other tissues. The sulfation pattern of heparan sulfate glycosaminoglycans appears uniform across follicles of different sizes and in healthy and atretic follicles. The heparan sulfate products detected in the follicles are likely to be associated with perlecan, collagen XVIII or betaglycan.     

Changes in the expression of cytochrome P450c17 associated with ovarian cystic follicles. An immunocytochemical and enzymatic analysis of porcine ovaries.

The steroidogenic activity of normal preovulatory and cystic follicles, and corpora lutea of porcine ovaries was investigated by immunocytochemical and radioenzymatic techniques. Using a specific antibody to porcine cytochrome P450c17, immunocytochemical staining was specifically localized in the theca interna layer of normal follicles and undetectable in the granulosa layer. The theca interna layers of non-luteinized cystic follicles were immunoreactive while those of luteinized follicles were not. Corpora lutea cells were essentially negative. The 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase activity was similar in luteinized cystic follicular and corpora lutea tissues, which had 8 times higher activity than found in normal preovulatory follicles. The formation of either corpora lutea or luteinized cysts led to a profound decline (12- to 15-fold) in 17 alpha-hydroxylase and 17,20 lyase activities compared to normal preovulatory follicles. In agreement with these enzyme findings, radioimmunoassays revealed very high levels of progesterone with nearly undetectable levels of androgens in the luteinized cysts. These studies demonstrate the functional similarities between cells of luteinized cysts and those of normal corpora lutea and suggest a pathology associated suppression of P450c17 expression in porcine cystic follicles.     

Morphology and histochemistry of ovarian changes in the field rat, Millardia meltada

A study has been made of the morphological and histochemical changes of the ovary of the field rat, Millardia meltada during its oestrous cycle and pregnancy. The follicular growth and atresia, ovulation and formation of corpora lutea occur throughout the year except severe winter months (December and January). Fluctuations in the follicular development occur on different days of the oestrous cycle and pregnancy. The granulosa cells show a progressive increase in their size in successive stages of follicle growth. The granulosae of normal follicles show some sparsely scattered lipid bodies which consist of phospholipids. Theca interna cells during follicular growth develop diffuse lipoproteins and lipid droplets consisting of triglycerides, phospholipids and cholesterol and/or its esters. The luteal cells of corpora lutea are formed by the granulosa cells as the theca interna cells degenerate and disappear. The fibroblast-like cells of thecal origin, alongwith the blood vessels, invade the luteal cell mass. The luteal cells during metoestrus, dioestrus and first half of pregnancy show abundant diffuse lipoproteins and a few lipid droplets composed mainly of phospholipids and some triglycerides, which are indicative of active steroidogenesis. The details of degenerative histological and histochemical alterations of corpora lutea during oestrous cycle and pregnancy are also described and discussed. Morphological and histochemical changes of follicular atresia are described. The granulosa cells of atretic follicle degenerate and disappear leaving behind theca interna cells which form patches of interstitial gland cells during the reproductive activity of the present rat. Interstitial gland cells show diffusely distributed sudanophilic lipoproteins and lipid droplets consisting of triglycerides, cholesterol and/or its esters and some phospholipids, which are indicative of steroidogenesis. The functional significance of histological and histochemical changes, which occur in various components of the ovary during oestrous cycle and pregnancy, has been discussed.     

Effects of testosterone and 2-hydroxyflutamide on progesterone receptor expression in porcine ovarian follicles in vitro

The purpose of the study was to test the possible role of the androgen receptor (AR) agonist (testosterone; T), an AR antagonist (2-hydroxyflutamide; 2-Hf) or combination of both (T + 2-Hf) on progesterone receptor (PGR) expression in cultured porcine granulosa cells (GCs) or whole follicles. GCs isolated from mature pig follicles (6–8 mm in diameter) were cultured for 48 h. Experimental cultures were carried out with the addition of T (10^?7 M), 2-Hf (1.7 × 10^?4 M) or both T and 2-Hf for the last 24 h of culture. To better imitate in vivo conditions, isolated whole porcine follicles (6–8 mm in diameter) were cultured for 24 h in an organ culture system, with the addition of the same factors. The cells or sections obtained from cultured follicles were processed for PGR immunocytochemical or immuno-histochemical staining. In addition, expression of PGR protein was determined by Western blot and progesterone (P₄) concentrations in the culture media were measured by a radioimmunoassay. We found that isoform A of PGR is expressed in both granulosal and follicular cultures. The 2-Hf in the presence of T increased PGR protein expression in porcine GCs and whole follicles. In both granulosal and follicular cultures, 2-Hf or T alone inhibited P₄ secretion, but simultaneous addition of 2-Hf and T increased P₄ secretion. Our results indicate that androgens may be involved in the control of PGR expression in porcine GCs in vitro. Moreover, we suggest a potential auto/paracrine regulation of the follicular function by androgen-dependent signaling pathway.     

Localization of an activin/activin receptor system in the porcine ovary.

The aim of this study was to locate a possible activin/activin receptor system within porcine ovaries containing functional corpora lutea. In situ hybridization was used to assess the gene expression of beta(A)- and beta(B)-activin subunits, and immunohistochemical studies were done to detect activin-A protein and activin receptor type II. mRNA expression of the beta(A)- and beta(B)-activin subunits was found in the granulosa from the unilaminar follicle stage onward, in the developing thecal layer of multilaminar and small antral follicles, in the theca interna of mid-sized antral follicles, in corpora lutea, and in the ovarian surface epithelium. Immunoreactive activin A protein could be detected at the same ovarian sites, but in thecal tissue of small antral follicles only. This protein was also demonstrated at the peripheral zone of oocytes from multilaminar and antral follicles. A positive immunoreaction for activin receptor was found in granulosa cells from multilaminar and older follicles and in oocytes from the earliest stages of follicular development onward. In late multilaminar follicles and in antral follicles, the oolemma was stained. Except for small antral follicles, a positive activin receptor immunoreaction was absent in the follicular theca. Activin receptor immunoreaction was furthermore present in corpora lutea and in the ovarian surface epithelium. It is concluded that, within porcine ovaries containing functional corpora lutea, an activin/activin receptor system is present in all intact follicles, the corpora lutea and the surface epithelium. Within follicles, granulosa and theca cells are the main sites of activin synthesis, while oocytes and granulosa cells are the main activin binding sites.