Progesterone and estradiol secretion by porcine luteal cells is influenced by individual and combined treatment with prostaglandins E2 and F2alpha throughout the estrus cycle.

The present experiments were conducted to test whether the ratio of PGE2:PGF2alpha affects steroid secretion by porcine luteal cells. We examined the effect of separate and combined treatment with PGE2 and PGF2alpha on progesterone and estradiol secretion. Luteal cells were collected at three different stages of the luteal phase (1-3 days after ovulation; 10-12 days after ovulation and 14-16 days after ovulation). PGE2 alone in a dose dependent manner increased progesterone production by cells collected from mature corpora lutea. On the other hand, PGF2alpha in a dose dependent manner decreased progesterone secretion by cells of the same origin. Progesterone secretion by cells isolated from mature and regressing corpora lutea and treated with both prostaglandins increased in comparison to PGF2alpha-treated cultures. However, in cells collected from regressing corpora lutea PGE2 and PGF2alpha in a ratio of 2:1 and 4:1 increased estradiol production when compared to control and both ratios increased estradiol secretion in comparison to PGF2alpha-treated cells. These data 1) confirm the luteotropic effect of PGE2 and the luteolytic effect of PGF2alpha; 2) demonstrate that when the ratio of PGE2 to PGF2alpha changed from 1:1 to 2:1 or 4:1 cells were protected against the inhibitory effects of PGF2alpha on progesterone secretion by cells collected during the mid- and late luteal phase; and 3) suggest that elevated estradiol production by luteal cells, isolated during late luteal phase, under the influence of increased doses of PGE2 may serve as an additional source of estradiol to blastocysts, during early pregnancy in the pig.     

Progesterone and androgen secretion by isolated cultured bovine corpus luteum cells: effect of LH, hCG, PRL, estradiol 17beta and testosterone

Primary cell cultures of bovine corpora lutea were used in order to examine their morphology and secretion of progesterone and androgen in vitro. The cells were grown as monolayers up to 6 days at 37 degrees C medium 199 supplemented with 10% calf serum. The concentration of progesterone and androgen was measured using appropriate radioimmunoassays [1,3] respectively. Luteal cells were cultured with addition of the following amounts of hormones: 100 ng LH, 10 i.u. hCG, 100 ng PRL, 150 ng Estradiol 17 beta and 150 ng Testosterone/ml of culture medium. The luteal cells also created considerable amounts of androgens. It was found that only estradiol added to the culture medium caused an increase in the level of testosterone. Progesterone secretion following the addition of hormones increased under the influence of LH, T, and E2 in statistically significant manner while hCG and PRL had no statistically significant effects.     

Developmental programming: exogenous gonadotropin treatment rescues ovulatory function but does not completely normalize ovarian function in sheep treated prenatally with testosterone

Prenatal testosterone treatment leads to LH excess as well as ovarian follicular and ovulatory defects in the adult. These disruptions may stem from LH excess, abnormal FSH input, compromised ovarian sensitivity to gonadotropins, or intrinsic ovarian defects. To determine if exogenous gonadotropins rescue ovarian and ovulatory function of testosterone-treated sheep, the release of endogenous LH and biopotent FSH in control and prenatal testosterone-treated sheep was blocked with a GnRH antagonist during the first two breeding seasons and with LH/FSH coadministered in a manner approximating natural follicular phase. An acidic mix of FSH was administered the first 36 h at 2-h intervals and a less acidic mix for the next 12 h at 1-h intervals (different FSH preparations were used each year), and ovulation was induced with hCG. Circulating FSH and estradiol responses to gonadotropins measured in 2-h samples differed between treatment groups in Year 1 but not in Year 2. Ovarian follicular distribution and number of corpora lutea (in ewes that ovulated) tracked by ultrasonography and luteal progesterone responses were similar between control and prenatal testosterone-treated females but differed between years. Furthermore, hCG administration induced large cystic and luteinized follicles in both groups of females in Year 2, although the growth rate differed between control and prenatal testosterone-treated females. Our findings provide evidence that 1) ovulatory response in prenatal testosterone-treated females can be rescued with exogenous gonadotropins, 2) resultant follicular response is dependent on the nature of gonadotropic input, and 3) an abnormal follicular milieu may underlie differences in developmental trajectory of cystic follicles in prenatal testosterone-treated females.     

Evaluation of the physiological value of porcine luteal cells isolated in various stages of the luteal phase: Tissue culture approach

Porcine luteal cells were collected from corpora lutea in four different stages of the luteal phase and cultured as monolayers. Progesterone (P₄) secretion was assayed using radioimmunoassays (Gregoraszczuk, 1991). Luteal cells cultured from porcine corpora lutea collected in the early luteal phase maintained steroidogenic capacity for 6 days in culture until the time comparable with midluteal corpora lutea. Luteal cells collected from mature and regressing corpora lutea did not dedifferentiate during 2 days of culture. After this time secretion of progesterone decreased to undetectable amounts characteristic of old corpora lutea. The regression in the culture progressed. The results demonstrate that the degree of the decline of progesterone depends on the type of corpus luteum, which is connected to particular time intervals of the luteal phase. Before starting experiments it is necessary to take into consideration the stage of the luteal phase from which the material is collected for culture. This study provides evidence that long term culture is useful for investigating a variety of aspects of luteal function only if cells are collected in the early luteal phase. Short term culture is suitable for investigation of cells collected from mid and late luteal phase. Regulation of luteal function is dependent on stage of the luteal phase.     

Effect of growth hormone and insulin-like growth factor-I on spontaneous apoptosis in cultured luteal cells collected from early, mature, and regressing porcine corpora lutea

The aim of the present study was to test the hypothesis that growth hormone (GH) and insulin-like growth factor-I (IGF-I) act at a local level to inhibit luteal cell apoptosis. Luteal cells collected from the corpora lutea at different stages of the luteal phase were cultured for 24 h in M 199 medium supplemented with 5% of calf serum to cause attachment cells to the plastic. After 24 h, the media were changed and various concentrations of GH (10, 100 or 200 ng/ml) or IGF-I (30, 50 or 100 ng/ml) were added to the culture medium. Twenty-four hours later, cells were fixed for morphological assessment of apoptotic cells utilising a Hoechst staining technique. To support morphological observations, measurements of caspase-3 activity in cultured porcine luteal cells were performed. Increased incidence of apoptotic bodies and caspase-3 activity accompanied luteal regression and was associated with a decreased progesterone (P4) secretion by luteal cells. GH stimulated P4 secretion by luteal cells collected from developing (ELP) and mature (MLP) corpora lutea but had no effect on its secretion by cells collected from regressing corpora lutea (LLP). Moreover, it had no effect on the incidence of apoptotic bodies in all types of corpora lutea. However, suppression of caspase-3 activity was observed with 100 and 200 ng of GH/ml in all types of corpora lutea. IGF-I had a stimulatory effect on P4 secretion by ELP and MLP, decreased the incidence of apoptotic bodies and suppressed caspase-3 activity in cultures treated with all doses used. In conclusion, our results indicate that both GH and IGF-1 trigger anti-apoptotic effects either indirectly, by increasing progesterone secretion, or directly, through the inhibition of caspase-3 activity and subsequent prevention of apoptotic body formation.     

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.     

Low serum levels of FSH, LH and prolactin in luteal phase inadequacy

In order to elucidate the relationship between prolactin (PRL) levels and corpus luteum function in humans, assessment of temporal relationship between levels of PRL, LH, FSH, estradiol and progesterone was made in eleven normal cycling women and six short luteal women. All hormones were determined by specific radioimmunoassay. The mean PRL level in the luteal phase was higher than that in the follicular phase in normal women. On the other hand, no difference mean was seen between the PRL levels of follicular and luteal phases in short luteal women. In addition, follicular and luteal phase secretion of PRL in the short luteal phase (SLP) was lower than that in the normal control. LH and FSH in the follicular and luteal phases, estradiol secretion in the late follicular and early to mid-luteal phases in SLP were also lower than those in the control. These observations were consistent with the hypothesis that SLP is a sequel to aberrant folliculogenesis. In addition, it is inferred that low PRL levels in the SLP might be due to inadequate augmentation by estrogen, rather than giving PRL any positive controlling role in the maintenance of corpus luteum function.     

Decline in circulating estradiol during the periovulatory period is correlated with decreases in estradiol and androgen, and in messenger RNA for p450 aromatase and p450 17alpha-hydroxylase, in bovine preovulatory follicles

The preovulatory surge of gonadotropins induces meiotic maturation of the oocyte, the follicular/luteal phase shift in hormone production, and ovulation. This complex and rapid series of developmental changes is difficult to study in large mammals, such as primates and ruminants, because variability in the length of individual reproductive cycles makes it virtually impossible to predict the time of the LH surge. We have validated an experimental model for inducing the LH surge and ovulation in cattle and used it to study the sequence of changes in hormone secretion and some of the mechanisms of these changes. Luteolysis and a follicular phase were induced by injection of prostaglandin F(2alpha); injection of a GnRH analogue 36 h later induced an LH surge and ovulation. The LH surge peaked 2 h after GnRH and ovulation followed 22-31 h after the surge, consistent with the periovulatory interval in natural cycles. The ensuing luteal phase was normal, both in length and in concentrations of circulating progesterone. In experiment I, the uteroovarian effluent was collected, via cannulation of the vena cava, at frequent intervals relative to GnRH injection. Circulating estradiol declined progressively after GnRH, reaching a nadir by 8-10 h before ovulation, whereas concentrations of androstenedione and testosterone remained constant. In experiment II, preovulatory follicles were obtained at 0, 3.5, 6, 12, 18, or 24 h after GNRH: Concentrations of androgens and estradiol were measured in follicular fluid and medium from cultures of follicle wall (theca + granulosa cells); steady-state levels of mRNA for 17alpha-hydroxylase (17alphaOH) and P450 aromatase were measured in follicular tissue. Shortly after the LH surge (3.5 h post-GnRH) there was an acute increase in the capacity of follicular tissue to secrete androstenedione, but not estradiol, in vitro. Thereafter, both androgens and estradiol declined, both in follicular fluid and in medium collected from cultures of follicle wall. Levels of mRNA for 17alphaOH and aromatase in follicle wall decreased significantly by 6 h after GnRH, suggesting that declining levels of these enzymes underlie the decreases in steroid production by follicular cells. These results show that in cattle the preovulatory decrease in follicular estradiol production is mediated by redundant mechanisms, because androgen production and the capacity of granulosa cells to convert androgens to estradiol decline coordinately, in concert with decreases in mRNA for 17alphaOH and P450 aromatase.     

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.     

Synergistic effects of insulin-like growth factor I and gonadotrophins on relaxin and progesterone secretion by ageing corpora lutea of pigs.

Insulin-like growth factor I (IGF-I) is involved in paracrine/autocrine regulation of gonadal steroidogenesis and peptide hormone biosynthesis. This study was designed to determine whether IGF-I alone, or an interaction of IGF-I, is involved in augmenting the actions of luteinizing hormone (LH) and prolactin in controlling relaxin and progesterone secretion from ageing corpora lutea of hysterectomized gilts at days 110, 113 and 116 after oestrus. Luteal tissue slices were incubated for 8 h with IGF-I (0, 50, 300 ng ml-1), LH (0, 100, 1000 ng ml-1), and prolactin (0, 100, 1000 ng ml-1) alone or in combination. Progesterone and relaxin concentrations were determined by radioimmunoassay of spent medium and of homogenates from luteal tissue slices before and after incubation. Porcine luteal tissue from day 110 had a net output of 25 ng progesterone and 26 ng relaxin in the control and of 65 ng progesterone and 2125 ng relaxin in the combined IGF-I, LH and prolactin treatment mg-1 of luteal tissue, respectively. IGF-I, LH and prolactin alone or in combination significantly increased (P < 0.01) progesterone production by luteal tissue from day 110, but they were partially effective at day 113 and ineffective at day 116. By contrast, the same hormone treatments increased relaxin production by luteal tissue from days 110 and 113. Even at day 116, prolactin alone or with LH or IGF-I continued to stimulate relaxin production. In conclusion, IGF-I augments the ability of prolactin and LH to increase relaxin production by ageing corpora lutea; however, a decrease in progesterone secretion and an increase in relaxin secretion at day 113 indicate that different mechanisms control progesterone and relaxin secretion in pigs.     

Patterns of plasma progesterone, androgen and oestrogen concentrations and in-vitro ovarian steroidogenesis during embryonic diapause and implantation in the mink (Mustela vison)

Peripheral plasma progesterone concentrations exhibited an increase 10 days before implantation, coinciding with the resumption of blastocyst growth and with a decrease in plasma androgen values (DHA, androstenedione, testosterone). No definite pattern of oestrone was observed and oestradiol concentrations remained undetectable. The production of steroids by dispersed luteal cells showed that the growth of the corpora lutea paralleled that of blastocysts and resulted in hypertrophy followed by hyperplasia of the luteal cell. The production of progesterone in the medium increased with blastocyst size up to implantation; it was enhanced by mink charcoal-treated serum, but prolactin, LH, FSH or a combination of these hormones did not affect the progesterone production, whatever the stage of diapause. DHA and androstenedione secretion increased in the two last stages of blastocyst growth and was enhanced by LH. The conversion of androstenedione and testosterone into oestrone and oestradiol was observed at all stages of embryonic diapause, indicating that corpora lutea contain aromatase activity even at an early stage. The secretion of oestrone was higher than that of oestradiol. The non-luteal tissue contributed up to 50% of the steroid production; while progesterone and androgen production remained constant, that of oestradiol decreased at the end of the delay period. These results indicated a change in the size and the secretory capacity of the luteal cell related to blastocyst development and implantation. Although progesterone was the main product of the corpora lutea, androgens and oestrogens were also secreted.     

The effect of the presence and pattern of luteinizing hormone stimulation on ovulatory follicle development in sheep

The aim of this study was to examine the role of LH on the growth of the large preovulatory follicle and its secretion of hormones in sheep. Ewes with ovarian autotransplants were treated with GnRH-antagonist at the time of luteal regression and different LH regimes applied for 60-66 h before administration of an ovulatory stimulus (hCG). In Experiment 1 (N = 24; n = 8), ewes received either no LH or constant or pulsatile infusion of LH at the same dose (1.25 microg/h). In Experiment 2 (N = 12, n = 6), LH was constantly infused at a rate of 1.25 microg or 2.5 microg oLH/h. In Experiment 1, animals receiving either pulsatile or constant LH exhibited increases in estradiol and inhibin A secretion (P < 0.001) and a depression in FSH (P < 0.001) that resembled the normal follicular phase. Similarly in Experiment 2, doubling the dose of LH resulted in a two-fold increase in ovarian estradiol secretion (P < 0.05) but no other changes. All animals receiving LH, regardless of the pattern of stimulation, ovulated and established a normal luteal phase. In contrast, no LH treatment resulted in constant immuno-active LH without pulses, unchanged FSH and inhibin A concentrations (P < 0.05), and basal estradiol secretion (P < 0.001). Morphologically normal large antral follicles were observed in this group and although corpora lutea formed in response to hCG, progesterone profiles were abnormal. In conclusion, these results suggest that LH is an essential requirement for normal ovulatory follicle development and subsequent luteal function and show that a pulsatile mode of LH stimulation is not required by ovulatory follicles.     

Mating stimulates estradiol production by ovaries of the musk shrew (Suncus murinus).

Asian musk shrews (Suncus murinus) are induced ovulators, but exhibit no cyclic changes in reproductive structures or in sexual behavior. Mating behavior is induced by contact with a male. To determine if mating induces changes in ovarian steroidogenesis, ovaries removed from unmated animals and at 3, 10, 15, and 36 h after mating were cultured for 4 h in the presence or absence of gonadotropins (LH + FSH, 1 microgram/ml). Histological analysis revealed no obvious changes in follicular size or appearance at the end of culture in ovaries cultured at 3 and 10 h post-mating, as compared with ovaries from unmated shrews, and mating did not stimulate any discernable changes in steroid secretion in these two groups. However, at the end of the culture period, ovulation had occurred or was occurring in ovaries from 35% of the animals ovariectomized at 15 h after mating, and corpora lutea (CLs) were present in 39% of ovarian pairs obtained 36 h after mating. At 15 h post-mating, ovaries with ovulations secreted three times more estradiol than did ovaries that showed no evidence of stimulation by mating, but there were no differences in testosterone or progesterone production. In contrast, ovaries isolated 36 h post-mating with CLs secreted dramatically more of all three steroids than ovaries without CLs (23, 13, and 52 times more estradiol, testosterone, and progesterone, respectively). These data are consistent with plasma concentrations of estradiol at the time of ovariectomy, which were twice as high at both 15 and 36 h after mating, in animals whose ovaries showed evidence of ovarian stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the synthesis of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the bovine ovary in vivo and in vitro

In order to investigate the pattern of ovarian cholesterol biosynthesis during the bovine estrous cycle, tissue concentrations of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme in the synthesis of cholesterol, were determined by immunoblot techniques. Medium-sized (9-11 mm) and large (14-18 mm) follicles, after removal of follicular fluid by centrifugation, and corpora lutea from the early, early-mid, late-mid, and late stages of the luteal phase were used (n = 5 per group). The specific content (per microgram of tissue homogenate protein) and total content of HMG-CoA reductase in medium-sized and large follicles were substantially lower than those of corpora lutea of the early-mid and late-mid luteal phase. The specific content was elevated in a number of the corpora lutea from the early luteal phase and was low in regressing corpora lutea. Thus during the midluteal phase, when steroid hormone production is elevated, the total and specific contents of HMG-CoA reductase are also elevated. To investigate the mechanisms whereby the levels of HMG-CoA reductase are regulated, primary monolayer cultures of bovine luteal cells (early-mid and late-mid luteal phase) were used. Cells were cultured for 24 h in Dulbecco's modified Eagle's medium containing lipoprotein-poor fetal calf serum (2% vol/vol). At this concentration there was no stimulation of the production of progesterone above that seen with no addition of serum. Under these conditions the total and specific contents, and the synthesis, of HMG-CoA reductase were stimulated by treatment with (Bu)2cAMP (1 mM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of immunization against progesterone on oestrus, cycle length, ovulation rate, luteal regression and LH secretion in the ewe

The effects of active immunization against progesterone on reproductive activity were studied in Merino ewes. Immunization against progesterone caused a shortening (P less than 0.01) of the interval between ovulations from 17-18 days (controls) to between 6 and 10 days (immunized group); this was associated with a corresponding reduction in the interval between LH surges. The immunized ewes also had higher (P less than 0.05) ovulation rates (1.72) than controls (1.25) and exhibited a reduced (P less than 0.01) incidence of oestrus (26% v. 95%). Many immunized ewes continued to ovulate despite the persistence of corpora lutea from earlier ovulations which led to an accumulation on the ovaries of many corpora lutea of different ages. The frequency of LH pulses in ewes immunized against progesterone (1.8 +/- 0.2 pulses/4 h) was significantly (P less than 0.001) higher than that of control ewes (0.3 +/- 0.1 pulses/4 h). This study highlights the importance of progesterone in the control of oestrus, ovulation, ovulation rate, luteal regression and the secretion of LH in the ewe.     

Estradiol-17 beta and androgen secretion by isolated porcine ovarian follicular cells in vitro

The cellular sources and gonadotropic regulation of porcine ovarian estrogen and androgen were assessed by culturing isolated granulosa cells and thecal cells from medium size follicles (4-6 mm diameter) separately for 24 h in a chemically defined medium containing gonadotropins and (or) testosterone. At the end of the culture period, estradiol-17 beta (estradiol) and androgens in the media were determined by radioimmunoassays. Production of estradiol by granulosa cells without an exogenous aromatizable androgen was low in the absence or presence of a highly purified preparation of either follicle-stimulating hormone (FSH. 0.25 microgram/mL) or luteinizing hormone (LH. 1 microgram/mL). Addition of testosterone or androstenedione (0.5 microM), but not dihydrotestosterone or pregnenolone, significantly increased estradiol secretion. Additional increases were observed when FSH, LH, prostaglandin E2, or dibutyryl cyclic 3'.5'-adenosine monophosphate was present. Production of estradiol by thecal cells was low in the presence or absence of exogenous testosterone, and was essentially unaffected by the presence of gonadotropins. Thecal cells, however, released large amounts of androstenedione and smaller amounts of testosterone and other androgens during 24-h culture and the production of these androgens was stimulated by LH but not by FSH. Androgen secretion by granulosa cells was negligible when compared with the theca and was unaffected by gonadotropins. It is concluded that the theca is the prime site for follicular androgen biosynthesis by the porcine ovarian follicle, and, upon LH stimulation, may provide androgen precursors for estradiol production by granulosa cells.     

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.     

Effect of prostaglandin F2 alpha on release of progesterone and leukotriene B-4 by cells from corpora lutea of mares.

Corpora lutea were recovered from mares either 4 to 5 days or 12 to 13 days after ovulation. Mixed populations of luteal cells were prepared by collagenase digestion and were incubated for 24 h in the presence or absence of prostaglandin (PG) F-2 alpha (250 ng/ml). PGF-2 alpha significantly (P = 0.03) reduced progesterone secretion by cells from late diestrous corpora lutea and tended (P = 0.06) to reduce secretion by early diestrous cells. PGF-2 alpha had no significant effect on leukotriene B-4 (LTB-4) production by cells from early diestrous corpora lutea, but significantly (P = 0.03) increased LTB-4 production by late diestrous luteal cells. It seems possible that LTB-4 could play a role as an intermediary in the action of PGF-2 alpha in luteolysis in the mare.     

Gonadotropin-releasing hormone 1 directly affects corpora lutea lifespan in Mediterranean buffalo (Bubalus bubalis) during diestrus: presence and in vitro effects on enzymatic and hormonal activities

The expression of gonadotropin-releasing hormone (GNRH) receptor (GNRHR) and the direct role of GNRH1 on corpora lutea function were studied in Mediterranean buffalo during diestrus. Immunohistochemistry evidenced at early, mid, and late luteal stages the presence of GNRHR only in large luteal cells and GNRH1 in both small and large luteal cells. Real-time PCR revealed GNRHR and GNRH1 mRNA at the three luteal stages, with lowest values in late corpora lutea. In vitro corpora lutea progesterone production was greater in mid stages and lesser in late luteal phases, whereas prostaglandin F2 alpha (PGF2alpha) increased from early to late stages, and PGE2 was greater in the earlier-luteal phase. Cyclooxygenase 1 (prostaglandin-endoperoxide synthase 1; PTGS1) activity did not change during diestrus, whereas PTGS2 increased from early to late stages, and PGE2-9-ketoreductase (PGE2-9-K) was greater in late corpora lutea. PTGS1 activity was greater than PTGS2 in early corpora lutea and lesser in late luteal phase. In corpora lutea cultured in vitro, the GNRH1 analog (buserelin) reduced progesterone secretion and increased PGF2alpha secretion as well as PTGS2 and PGE2-9-K activities at mid and late stages. PGE2 release and PTGS1 activity were increased by buserelin only in late corpora lutea. These results suggest that GNRH is expressed in all luteal cells of buffalo, whereas GNRHR is only expressed in large luteal phase. Additionally, GNRH directly down-regulates corpora lutea progesterone release, with the concomitant increases of PGF2alpha production and PTGS2 and PGE2-9-K enzymatic activities.     

Relationship between adenohypophyseal and steroid hormones and variations in serum and urinary melatonin levels during the ovarian cycle, perimenopause and menopause in healthy women

Morning levels of serum melatonin, FSH, LH, prolactin (PRL), progesterone and estradiol were studied by RIA during the ovarian cycle, perimenopause and menopause in 79 healthy women. FSH and LH levels showed a slight nonsignificant increase from the fertile period to perimenopause, exhibiting a significantly greater increase during menopause. PRL, progesterone and estradiol showed parallel changes, reaching lower levels during menopause. Serum melatonin levels decreased with age, attaining minimum levels in menopause. FSH and estradiol were significantly correlated with melatonin in the follicular phase, while in the luteal phase a negative correlation was found between melatonin, progesterone and estradiol. No significant correlations were noted between serum hormone levels during the perimenopausal period. In menopause, as during the follicular phase, melatonin and FSH were negatively correlated. As expected, a significant positive correlation was found between morning serum levels of melatonin and nocturnal urinary excretion of this indoleamine in all groups studied.