Expression and regulation of ang-2 in murine ovaries during sexual maturation and development of corpus luteum

The aim of this study was to examine the expression and regulation of angiopoietin-2 (Ang-2) in murine ovaries during sexual maturation, gonadotropin treatment and luteal development by in situ hybridization and RT-PCR. By in situ hybridization Ang-2 mRNA was mainly localized in granulosa cells, thecal cells and corpus luteum, otherwise in oocytes. Moreover, Ang-2 mRNA was highly expressed in corpus luteum and granulosa cells of atretic follicles. According to RT-PCR data, Ang-2 mRNA was lowly expressed on day 10 after birth, then expression levels gradually increased and reached their highest values on day 25 after birth. In the superovulated model of immature mice, Ang-2 expression was strongly induced by equine chorionic gonadotropin (eCG) 48 h post the eCG injection, and was high from 0.5 to 13 h after hCG treatment. in situ hybridization showed that Ang-2 mRNA was highly expressed in corpus luteum from day 2 to 9 post the hCG injection, then the expression levels gradually declined on days 11 and 13 after hCG treatment. According to RT-PCR data, the levels of Ang-2 mRNA expression showed a decline after the hCG injection, with a nadir on day 3, followed by an increase, reaching the highest level on day 9 post-hCG injection. Then again Ang-2 expression gradually declined from day 11 to 15 after hCG injection. These results suggest that Ang-2 may be involved in follicular development, atresia, ovulation, and corpus luteum formation and regression.     

A combined radioimmunoassay and immunocytochemical study of ovarian oxytocin production during the periovulatory period in the ewe

Corpora lutea and follicles were taken from the ovaries of 12 ewes at intervals from the start of luteolysis until 3 days after ovulation. RIA analysis of the tissue oxytocin content showed that luteal oxytocin concentrations declined during luteolysis to reach basal values at about the time of the next ovulation. Oxytocin was first measurable in the walls of 3 out of 6 preovulatory follicles during the LH surge, with a small increase in concentration to 26.1 +/- 6.6 pg/mg before ovulation, and a further increase in the young corpus luteum to concentrations exceeding 1 ng/mg 2-3 days later. After the LH surge, oxytocin was also found in the follicular fluid at a concentration of 3.4 +/- 0.3 ng/ml. Using immunocytochemical techniques, oxytocin and neurophysin were first detected in the follicle wall immediately before ovulation, and were localized in the granulosa cells. After ovulation the stained cells initially formed strands which appeared to break down to clusters and then to individual cells as the corpus luteum matured. The immunocytochemical picture also suggested that neurophysin immunoreactivity increased within a few hours of ovulation but that processing to oxytocin may be delayed. Measurements of circulating oxytocin concentrations revealed a pulsatile release pattern throughout the follicular phase with the height of the pulses decreasing from 25 +/- 5 pg/ml during luteolysis to a minimum of 11 +/- 2 pg/ml during the LH surge.     

Sequential changes associated with the degeneration of preovulatory rat follicles.

In 26-day-old rats, follicles capable of ovulation were present 48 h after PMSG injection and they degenerated if not exposed to an ovulating dose of HCG. In such follicles 125I-labelled LH bound to the thecal and granulosa cells. By 60 h after PMSG, LH binding to the granulosa cells was reduced by 46% although these follicles retained their ability to ovulate. LH binding to the granulosa cells was lost in most follicles by 72 h and ovulation could not be induced. The thecal cells still possessed LH binding sites at 72 h after PMSG. HCG stimulation of these follicles resulted in disruption of the granulosa and the invasion of blood cells into the antrum.     

Steroidogenic and morphological characteristics of granulosa and thecal compartments of the differentiating rabbit corpus luteum in culture

On the day after ovulation, the thecal tissue and associated mural granulosa lutein cells of the rabbit corpus luteum were separated from the granulosa lutein 'core' by dissection and these tissues were cultured separately or together (whole corpus luteum) in defined medium for 10 days on stainless-steel grids. The medium was changed completely every 24 h. Replicate tissues were cultured with testosterone (10 ng/ml), but no other hormones were added to the medium. Progesterone production increased during the first 2 days of culture for whole corpus luteum, granulosa lutein cells and the thecal compartment which also included granulosa lutein cells. After 3 days, the production of progesterone declined gradually, but was still detectable on Day 10. The production of the metabolite, 20 alpha-dihydroprogesterone, by whole corpus luteum was equal to or greater than that of progesterone. Without the addition of testosterone, the granulosa lutein cells produced little (10 pg/culture) oestradiol during 1 day of culture, but the thecal compartment and whole corpus luteum each produced about 100 pg/culture on Day 1 and declining quantities over the next 2 days. In the presence of testosterone added to the medium, the formation of oestradiol was greatly increased for all tissues for 5-6 days of culture, after which time oestradiol was no longer detectable with or without testosterone in medium. Transmission electron microscopy of cells after 10-12 days of culture revealed fine structure that is characteristic of luteal cells, including abundant smooth endoplasmic reticulum, lipid droplets, and junctions between the luteal cells. The corpus luteum in culture resembles the corpus luteum in situ in that steroidogenesis and differentiation can proceed for a period after ovulation without extrinsic hormonal stimulation.     

Comparison of subpopulations of luteal cells obtained from cyclic and superovulated ewes

Peripheral blood samples were collected daily (Days 1-10 after ovulation) and analysed for progesterone content. Luteal tissue was collected on Day 10 after the LH surge, or Day 10 after hCG injection from cyclic and superovulated ewes, respectively. The tissue was enzymically dispersed and an aliquant was utilized for measurement of cell diameters, and staining for 3 beta-hydroxy-delta 5-steroid dehydrogenase-delta 5, delta 4-isomerase activity (3 beta-HSD). The remaining cell preparation was separated into small (10-22 micron) and large (greater than 22 micron) cell fractions by elutriation. Small and large cell suspensions were incubated (37 degrees C, 2 h) in the presence or absence or ovine LH (100 ng/ml) or dbcAMP (2 mM) and progesterone content of the medium was measured. Superovulation did not affect circulating progesterone concentrations, when expressed per mg luteal tissue recorded; basal progesterone production by small or large luteal cells; the unresponsiveness of large luteal cells to ovine LH or dbcAMP; the ratio of small:large cells recovered by dissociation the mean diameter of total cells; or the mean diameter of large cells. However, the mean cell diameter and LH stimulation of progesterone production by small cells were greater (P less than 0.05) in luteal tissue collected from superovulated than in that from cyclic ewes. These differences appear to be an amplification of basic function. Therefore, we conclude that corpora lutea obtained from superovulated ewes can be used to study functional aspects of small and large cells.     

Fate of the theca interna following ovulation in the ewe

Summary The fate of the theca interna after ovulation was studied in ewes, using light and electron microscopic histology and histochemistry. At the time of ovulation the theca interna was incorporated, apparently completely, into the margin of the developing corpus luteum and into the centres of many infoldings of the follicular wall. There was no evidence of degeneration of the more highly differentiated theca interna cells at or following the time of ovulation. Within 24 h of ovulation, cells derived from the theca interna began migrating from their original sites into the deeper, granulosa-derived areas of the luteal tissue. At later stages cells derived from the theca interna remained concentrated in septa derived from the follicular infoldings, but were also widely distributed throughout the luteal tissue. Structural evidence supported the view that the small luteal cells and fibroblasts of the corpus luteum were derived from the theca interna, and the large luteal cells from the membrana granulosa.The authors wish to thank Mrs. Linda Musk and Miss Anneke Veenstra for skilled technical assistanceDeceased on May 4, 1979     

Effect of short term passive immunization of the cyclic ewe against estradiol-17β

Three groups of 4 ewes were passively immunised against estradiol 17β during days 2 through 7, 10 through 17 and 15 through 20 of the estrous cycle respectively. The effects of the neutralisation of estradiol 17β at these specific times, on corpus luteum function, plasma concentrations of luteinising hormone (LH), estrus, and ovulation were observed.Passive immunisation at any phase of the oestrous cycle did not appear to effect the appearance of large ovarian follicles (5–15 mm diameter) at the next time of expected estrus, or the lifespan of the corpus luteum of that cycle. Treatment from day 15 to 20 of the cycle appeared to inhibit estral activity, ovulation and the preovulatory LH surge. Treatment from day 10 to 17 was less disruptive although some extension of the length of the estrous cycle was indicated, and treatment from day 2 to 7 produced little interference with cyclic reproductive activity.     

First postpartum ovulations and corpora lutea in ewes which lamb in the breeding season

Two experiments involving crossbred ewes which lambed during the breeding season were performed to determine whether: (a) the interval to first postpartum ovulation could be reduced by weaning or mastectomy; (b) there are differences in luteal structure and luteinizing hormone (LH) receptor concentration between first postpartum corpora lutea induced with GnRH and normal cycling corpora lutea and (c) pretreatment of postpartum ewes with progesterone would affect luteal LH receptor concentration and luteal phase serum progesterone concentration.In experiment I, the mean interval (±SEM) to the first postpartum ovulation was 22.3 ± 1.1 days and was not significantly altered by weaning or mastectomy. More than half of the ewes had small, short-lived peaks of serum progesterone associated with short-lived corpora lutea prior to the normal luteal phase rise of serum progesterone. In experiment II, 2 h after GnRH injection on day 18 postpartum, serum LH concentrations were higher in ewes which received progesterone treatment on days 13 and 14 than in control ewes. Progesterone treatment did not affect mean corpus luteum weight (157 mg) or concentration of LH receptors (0.95 fmol/mg) in first postpartum corpora lutea, but progesterone-treated ewes had significantly higher endogenous serum progesterone concentrations on days 21–24. GnRH-induced corpora lutea from postpartum ewes were lighter in weight, paler in color, had lower LH receptor concentrations and had a more regressed histological appearance than corpora lutea of a similar age from normal, cycling ewes.     

Morphometric estimation of the numbers of granulosa cells in preovulatory follicles of the ewe

Several lines of evidence suggest that follicular granulosa cells give rise to the large luteal cells of the corpus luteum in the sheep. To further investigate this suggestion, numbers of granulosa cells in preovulatory follicles were estimated by morphometric methods for comparison with a previous estimate of numbers of large luteal cells (9.6 +/- 0.9 x 10(6)). Preovulatory follicles from five Corriedale ewes were obtained after synchronization of the oestrous cycle with the prostaglandin analogue cloprostenol. Morphometry was undertaken using light microscopy of plastic-embedded tissue sectioned at 1 micron. Mitotic index in the membrana granulosa was 0.05 +/- s.e.m. 0.05%. Mean follicular diameter was 6.25 +/- 0.25 mm and there were 7.68 +/- 0.53 x 10(6) granulosa cells per follicle. These results demonstrate a similarity between the number of granulosa cells per follicle and the number of large luteal cells per corpus luteum and thus support the hypothesis that large luteal cells are derived from granulosa cells.     

Effect of oxytocin infusion on secretion of progesterone and luteinizing hormone and the concentration of uterine oxytocin receptors during the periovulatory period in cloprostenol-treated ewes.

Oxytocin infusions were initiated on day 10 of the oestrous cycle in ewes, and luteal regression was induced by injection of 100 micrograms cloprostenol on day 12. Blood samples were collected at frequent intervals via an indwelling jugular vein cannula to measure concentrations of progesterone and luteinizing hormone (LH) during the luteal and follicular phases in saline (n = 6) and oxytocin (n = 5) infused animals. The oxytocin infusion maintained peripheral plasma concentrations of 53 +/- 3.2 pg oxytocin ml-1 (mean +/- SEM) compared with values of about 1 pg ml-1 during oestrus in control ewes. Oxytocin infusion had no effect on luteal phase progesterone concentrations, the timing of luteolysis, basal luteinizing hormone (LH) secretion, LH pulse frequency, or the timing or height of the LH surge. Treated ewes came into oestrus significantly earlier than controls (P < 0.05) but ovulated normally. Uterine samples collected 96 h after cloprostenol injection (approximately day 2 of the cycle) showed that oxytocin receptor concentrations were significantly higher in the endometrium in ewes that had been given a 5 day oxytocin infusion than in control animals (556 and 262 fmol mg-1 protein, respectively: geometric means from ANOVA, P < 0.001), whereas myometrial receptor concentrations were not affected (113 and 162 fmol mg-1 protein, respectively). We conclude that the previously reported delay in luteal development caused by oxytocin infusion (Wathes et al., 1991) is not due to the inhibition or delay of ovulation, but must instead occur via a direct influence on the developing corpus luteum.(ABSTRACT TRUNCATED AT 250 WORDS)     

The active immunization of the cyclic ewe against an estrone protein conjugate

Four mature, cyclic ewes were given injections (I.M.) of a conjugate of 1,3,5 (10)-estratrien-3-ol-6,17-dione, 6 carboxyoxime bovine serum albumin (immunized ewes) on day 3 after estrus, and at days 10, 20, 40, 58, 91 and 134 after this initial treatment. Six control ewes treated with carrier emulsion alone continued to cycle normally. Three of the immunized ewes failed to exhibit estrus, an associated preovulatory surge of LH and ovulation. One ewe showed 1 abnormally short estrous period and then became anestrus. Injection of an estrone-protein-conjugate at days 3 and 13 after estrus did not appear to interfere with the rate of structural luteolysis of the corpus luteum present, but plasma concentrations of progesterone reached abnormally high luteal phase levels and in 2 ewes failed, subsequently to decline to normal follicular phase levels. Estrone binding capacity rose as early as day 9 after first treatment, and concentrations of LH rose as early as day 14. Subsequently, plasma levels of LH, estrone and progesterone and antisera titer rose; the only significant cross reaction of the antisera was with estradiol 17beta (11.32 +/- 2.80%).     

Comparative expression of luteinizing hormone and follicle-stimulating hormone receptors in ovarian follicles from high and low prolific sheep breeds

Expression of gonadotropin receptors and granulosa cell sensitivity to gonadotropin hormones by small (1-3 mm) and large (3.5-7 mm) follicles were compared in Romanov (ROM, ovulation rate = 3) and Ile-de-France (IF, ovulation rate = 1) ewes in the early and late follicular phase. In healthy follicles, LH receptor levels in granulosa cells increased with increasing follicular size (p < 0. 001) while FSH receptor levels decreased (p < 0.05). In granulosa cells of large follicles, LH receptor (LHR) mRNA levels were greater in the late than in the early follicular phase (p < 0.001, p < 0.05, for ROM and IF, respectively). In the early follicular phase, LHR levels in granulosa (p < 0.001) and theca cells (p < 0.05) of small follicles were greater in ROM than in IF ewes. FSH receptor mRNA levels in granulosa cells of small and large ROM follicles were greater than in the corresponding IF follicles (p < 0.05). Finally, a greater responsiveness (increase in cAMP secretion) to both FSH and hCG was observed by granulosa cells collected during the early follicular phase from ROM vs. IF ewes. Data provide evidence that the greater ovulation rate in the ROM as compared to the IF breed is associated with a greater gonadotropin responsiveness during the early follicular phase.     

Response to gonadotropic stimulation of cultured rat luteal cells isolated from corpora lutea of early pregnancy. Cytochemical and biochemical studies

The hormonal activity of corpora lutea isolated from pregnant rat was examined on 1, 2, 3, 4, 5, 6, 15, and 20th day of pregnancy. The cells were grown as a monolayers up to 6 days at 37 degrees C in Medium 199 supplemented with 10% calf serum. The concentrations of progesterone and estrogens were measured using appropriate radioimmunoassays [1, 7] respectively. Luteal cells were cultured with the addition of the following amounts of hormones: 100 ng LH, 10 i.u. HCG, 100 ng PRL and 150 ng estradiol 17 beta. Cytochemical and histochemical observation of the activity of delta 5, 3 beta-hydroxysteroid dehydrogenase (delta 5, 3 beta-HSD) were also carried out. The addition of LH and HCG to culture medium of cells collected on day 1 and 2 of pregnancy caused increased histochemical reaction for delta 5, 3 beta-HSD and progesterone secretion. It was only on day 3 of pregnancy that the influence of PRL was observed. On day 4 corpus luteum cells began to respond to exogenous estradiol. On day 5 the sensitivity of corpus luteum to exogenous hormones disappeared but the intensive hormonal activity of the corpus luteum marked by the high level of progesterone, was maintained.     

Functional and morphological characteristics of the first corpus luteum formed after parturition in ewes

The ability of sheep luteal cells from the first corpus luteum formed after parturition (Group F) to secrete progesterone in the presence or absence of LH was compared with that of luteal cells obtained from normal cyclic ewes (Group C). Luteal concentrations of receptors for LH and prostaglandins (PG) F-2 alpha (PGF-2 alpha) and the cellular composition of corpora lutea from Groups F and C were also compared. Luteal cells from Group F secreted less progesterone in either the presence or absence of LH (P less than 0.01). There was no difference in the number of receptors for LH or PGF-2 alpha per luteal cell between Groups F and C (P greater than 0.1), nor was there a difference in the number of large or small steroidogenic luteal cells (P greater than 0.1). It was concluded that, if short-lived corpora lutea are insensitive to gonadotrophins, this response is not mediated by decreased numbers of receptors for LH. In addition, if the first corpus luteum formed post partum in ewes is more sensitive to the luteolytic effects of PGF-2 alpha, this effect is not mediated by an increased number of receptors for PGF-2 alpha or an increased proportion of PGF-2 alpha-sensitive large luteal cells.     

Effect of changes in FSH induced by bovine follicular fluid and FSH infusion in the preovulatory phase on subsequent ovulation rate and corpus luteum function in the ewe

Treatment of Damline ewes with i.v. injections of various doses (2, 5 or 10 ml) of bovine follicular fluid for 72 h after prostaglandin-induced luteal regression resulted in a significant decrease in plasma concentrations of FSH after a 1.5-2 h delay but did not affect LH. The half life of this decrease in plasma FSH levels (156 min) after injection of follicular fluid was similar to that for clearance (159 min) of ovine FSH after infusion. A significant rebound increase in plasma FSH levels occurred by 13 h after all follicular fluid injections, and the magnitude of this rebound was inversely related to the dose of follicular fluid injected. A significant delay in the onset of oestrus occurred only with 5 and 10 ml bovine follicular fluid. There was no significant effect on ovulation rate or subsequent corpus luteum function as measured by plasma concentrations of progesterone. Infusion of ovine FSH (50 micrograms/h for 48 h) during the period of follicular fluid treatment prevented the delay in onset of oestrus and resulted in a substantial (2-10-fold) increase in ovulation rate. Corpus luteum function in terms of progesterone secretion was also enhanced. These results show that (1) intermittent suppression of FSH during the preovulatory period in the ewe does not affect subsequent ovulation rate or corpus luteum function and (2) the delay in the onset of oestrus induced by bovine follicular fluid can be prevented by exogenous FSH.     

Post ovulatory follicles of the domestic duck.

Histogenesis of the post-ovulatory follicles in relation to the regression process has been studied in the Indian domestic duck. The activities of the alkaline phosphatase, acid phosphatase, sudan black-B and periodic acid Schiff reaction have been demonstrated histochemically. No lutein cells comparable to the mammalian corpus luteum are found. The hypertrophy or proliferation of granulosa cells are not observed in this bird. The functional significance of different histochemically demonstrable material and steroidogenesis in the avian post-ovulatory follicles have been briefly discussed.     

Effect of estrogen on postovulatory LH surge in baboons.

In normally cycling female baboons, an LH surge appeared prior to ovulation, in addition, another LH surge (postovulatory LH surge) was observed within two days after ovulation. An attempt was then made to determine the effect of postovulatory LH on the luteinization of corpus luteum in baboons. Injections of 300 micrograms estradiol benzoate were given at 09.00 and 16.00 hr daily for 5 days following ovulation; the plasma level of LH was increased, but plasma progestin was suppressed. These results infer that the injected estrogen (estradiol benzoate) may inhibit the luteotrophic effect of postovulatory LH on the corpus luteum, therefore, plasma progestin remains lower even though postovulatory LH is elevated.     

In-vivo and in-vitro steroidogenic activity of post-ovulatory ovarian follicles of the rabbit

Pseudopregnancy was induced in rabbits by injection of 50 i.u. hCG into the lateral ear vein. Blood was collected on Days 0, 1, 2 and 6 after hCG from the lateral ear vein and, in some studies, from the ovarian vein as well. Ovaries were collected on Days 0, 1, 2 or 6 after hCG injection, and follicles (greater than 0.8 mm diam.) were obtained by microdissection. Concentrations of oestradiol-17 beta, testosterone and progesterone in blood and follicular homogenates were measured by RIA. Follicular steroidogenesis was increased significantly on Day 6 after ovulation, at the time when the corpus luteum transforms into an oestrogen-dependent tissue. The ability of developing post-ovulatory follicles to secrete steroids in vitro in response to a gonadotrophic stimulus also increased over this same time interval. Follicular oestradiol production on Days 1 and 2 of pseudopregnancy may synergize with the post-ovulatory secretion of FSH to promote further follicular growth in the pseudopregnant rabbit.