The effects of peritoneal macrophages on monolayered luteal cell progestin secretion in the rat

Functionally active or regressing luteal cells were obtained from pseudo-pregnant (psp) rats between days 5-8 of psp or on day 15 of psp, respectively. They were monolayer-cultured (10(6)/dish) in the presence of 0.2 micrograms/ml LH 2.0 micrograms/ml PRL and 10 micrograms/ml pregnenolone for 4 days with or without macrophages, although functionally active luteal cells secreted progesterone dominantly during day 1 of culture (Day 1), the amounts of progesterone and 20 alpha-OH-P secreted were inverted on Day 2, and the dominance of 20 alpha-OH-P continued from Day 2 to Day 4. In the functionally regressing luteal cell culture, more 20 alpha-OH-P than progesterone was secreted throughout the culture period. The addition of peritoneal macrophages (2.5 X 10(6] to the active luteal cell monolayer lengthened the dominance of progesterone secretion for an additional day and the inversion occurred on Day 3. The progestin ratio (progesterone/20 alpha-OH-P) on Day 2 was maintained significantly higher. The daily addition of macrophages maintained the progesterone dominance throughout the culture period. On the other hand, macrophages had no effect on luteal cells already functionally regressing. These results indicate that macrophages are effective in maintaining the progesterone secreting activity of luteal cells in vitro.     

Inhibition of induction of 20 alpha-hydroxysteroid dehydrogenase activity in rat corpora lutea in vitro by the progesterone antagonist RU486.

To determine if the antiprogestagen RU486 has a direct effect on luteal progesterone secretion, whole corpora lutea or dispersed luteal cells were incubated in the presence of RU486. Whole corpora lutea, isolated from rats at day 5 of pseudopregnancy, were incubated individually in hormone-free medium. The concentrations of progesterone and 20 alpha-dihydroprogesterone in the medium plus corpus luteum was measured before and after 24 h of incubation. In the absence of RU486 the concentration of 20 alpha-dihydro-progesterone increased, while that of progesterone remained unchanged. In the presence of RU486 (230 microM) the concentration of both progesterone and 20 alpha-dihydro-progesterone was increased. Dispersed luteal cells were incubated for 24 h in the presence of various amounts of RU486. In the absence and in the presence of 0.2 and 2.3 microM RU486 a high ratio between 20 alpha-dihydro-progesterone and progesterone was found, while in the presence of 23 microM RU486 the concentrations of progesterone and 20 alpha-dihydro-progesterone were equal. 20 alpha-Hydroxysteroid dehydrogenase (20 alpha-HSD) activity measured in luteal homogenates started to increase between 6 and 12 h of incubation. This increase could be prevented after incubation of the corpora lutea in the presence of 23 or 230 microM RU486 for 24 hrs. It is concluded that the progesterone antagonist RU486 can have a direct effect on luteal progesterone production. RU486 prevents the increase in 20 alpha-HSD activity that normally occurs during in vitro incubation. However, since these effects in vitro can only be obtained with high concentrations of RU486, it is unlikely that this antiluteolytic effect plays a role after injection of RU486 in vivo.     

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.     

Progesterone receptor is not required for progesterone action in the rat corpus luteum of pregnancy

In this study, we investigated whether progesterone exerts a local action regulating the function of the corpus luteum of pregnancy in rats. The luteal activities of the enzymes 3beta-hydroxysteroid dehydrogenase (3beta-HSD), involved in progesterone biosynthesis, and 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), that catabolizes progesterone and reduces progesterone secretion by the corpus luteum, were evaluated after intrabursal ovarian administration of progesterone in pregnant rats that had received a luteolytic dose of prostaglandin F2alpha (PGF2alpha). Luteal 3beta-HSD activity decreased and 20alpha-HSD activity increased after PGF2alpha treatment (100 microg x 2 intraperitoneally on Day 19 of pregnancy at 12:00 p.m. and 4:00 p.m.) when compared with controls sacrificed at 8:00 p.m. on Day 20 of pregnancy. This effect of PGF2alpha on the luteal 3beta-HSD and 20alpha-HSD activities was abolished in animals that also received an intraovarian dose of progesterone (3 microg/ovary on Day 19 of pregnancy at 8:00-9:00 a.m.). In a second functional study, luteal cells obtained from 19-day pregnant rats responded to the synthetic progestin promegestone (R5020) in a dose-dependent manner, with an increase in the progesterone output. In addition, the glucocorticoid agent hydrocortisone did not affect progesterone accumulation in the same luteal cell culture. We also examined by immunocytochemistry the expression of progesterone receptors (PR) in the corpora lutea during pregnancy and demonstrated the absence of PR in this endocrine gland in all the days of pregnancy studied. In the same pregnant rats, positive staining for PR was observed in cells within the uteroplacental unit, such as cells of the decidua basalis and trophoblast giant cells of the junctional zone. In addition, positive PR staining was observed in the ovarian granulosa and theca cells of growing follicles, but not in corpora lutea of ovaries obtained from cycling rats at proestrus. In summary, this report provides further evidence of a local action of progesterone regulating luteal function in the rat despite the absence of a classic PR.     

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.     

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.     

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.     

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.     

Dependence on prolactin of the luteolytic effect of prostaglandin F2alpha in rat luteal cell cultures

Luteal regression is a multistep, prolonged process, and long-term luteal cultures are required for studying it in vitro. Cell suspensions from ovaries of superovulated rats were enriched with steroidogenic cells, seeded on laminin or fibronectin, and maintained in defined medium for up to 10 days. Progesterone secretion was much lower than that of 20alpha-dihydroprogesterone, a product of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD). Prolactin added throughout the incubation period gradually increased the percent progesterone out of total progestins to fourfold, while reducing 20alpha-HSD mRNA by 73%. Luteinizing hormone accelerated the establishment of higher percent progesterone by prolactin but by itself had no effect. Prolactin did not increase total progestin production or cytochrome P450 side-chain cleavage (P450(scc)) mRNA. Cell viability was unaffected by prolactin and/or LH. Prostaglandin F2alpha (PGF2alpha) was added 7-8 days after seeding. In prolactin-treated cells, PGF2alpha reduced steroidogenesis after 4-45 h, and at 45 h total progestins and P450(scc) mRNA were reduced by 45%. At 8-45 h PGF2alpha reduced the percent progesterone out of total progestins, and at 45 h 20alpha-HSD mRNA was doubled. In contrast, in prolactin-deprived cultures, PGF2alpha had little effect on total progestins or 20alpha-HSD mRNA but doubled P450(scc) mRNA. Phospholipase C activity was stimulated by PGF2alpha regardless of prolactin. Thus, when prolactin-treated, our cultures are a good model for mature corpora lutea challenged with PGF2alpha; the finding that without prolactin PGF2alpha has an alternative set of actions could help in identifying the signaling pathways of PGF2alpha responsible for its luteolytic effects.     

Effect of RU 486 on luteal function in the early pregnant rat

A dose of 30 mg RU 486/kg, an antiprogesterone, was administered to pregnant rats on Day 2 (Group 1) or Day 4 (Group 2) of pregnancy. RU 486 significantly changed serum progesterone and oestradiol concentrations and luteal 3 beta-HSD and 20 alpha-HSD activities in Group 1, and implantation was significantly inhibited. The luteal 3 beta-HSD activity in Group 2 rats on Day 6 was significantly (P less than 0.01) lower than the control value (7.5 +/- 0.6 and 10.1 +/- 0.6 mU/mg protein respectively). This decline in the 3 beta-HSD activity was followed by a marked decrease in the serum progesterone concentration, resulting in a significant decrease of the progesterone/oestradiol ratio and implantation was completely inhibited. The 20 alpha-HSD activity, which could not be detected on Day 6 in the control rats, was twice as great in Group 2 than in Group 1 rats (17.5 +/- 1.2 and 7.4 +/- 3.1 mU/mg protein respectively). Ultrastructural examination of corpora lutea of Group 2 rats confirmed luteolysis. These results suggest that RU 486 has a luteolytic effect and its anti-implantation effect is concomitant with luteolysis of the corpora lutea of pregnancy.     

Progestin regulation of progesterone biosynthetic enzymes in cultured rat granulosa cells

Progestins have recently been shown to augment gonadotropin-stimulated progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) biosynthesis in cultured rat granulosa cells. The mechanism by which progestins autoregulate ovarian progestin biosynthesis was investigated by studying the modulation of pregnenolone biosynthesis as well as the activities of the enzymes 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD). Granulosa cells obtained from immature hypophysectomized, estrogen-treated rats were cultured with FSH and/or progestins. Pregnenolone production was measured in the presence of cyanoketone (10(-6) M) to inhibit 3 beta-HSD activity. Enzymatic activities of 3 beta-HSD and 20 alpha-HSD were determined in cell homogenates by direct enzyme assays. FSH stimulated pregnenolone production, while treatment with progesterone or R5020 alone was ineffective. Concomitant treatment with the progestins further enhanced FSH-stimulated pregnenolone production in a dose-dependent manner with minimal effective doses of 10(-8) and 10(-7) M for R5020 and progesterone, respectively. In FSH-primed cells, LH increased pregnenolone accumulation, and concomitant treatment with R5020 also enhanced the LH action. Furthermore, the gonadotropins stimulated the activity of 3 beta-HSD, and this effect was further enhanced by concomitant treatment with either R5020 or progesterone in a dose-dependent manner. In addition, the 20 alpha-HSD activities were enhanced by progestins in cells treated with FSH but not with LH. Thus, both natural and synthetic progestins stimulate the gonadotropin-induced progesterone production in cultured granulosa cells via enhancing the 3 beta-HSD enzyme as well as pregnenolone biosynthesis.     

The interaction of testosterone and gonadotropins in stimulating estradiol and progesterone secretion by cultures of corpus luteum cells isolated from pigs in early and midluteal phase.

The first objective of this research was to define the capacity of corpora lutea of pig to secrete estradiol in the presence of an androgen substrate which was testosterone. The second objective was to define the synergism between gonadotropic hormones such as LH, FSH, and PRL and testosterone as measured by estradiol and progesterone secretion by two types of porcine luteal cells. Luteal cells were collected from newly forming corpora lutea (0-3 days after ovulation) and from mature corpora lutea (8-10 days after ovulation). After dispersion, luteal cells were suspended in medium M199 supplemented with 10% of calf serum and grown as monolayers at 37 degrees C. Control cultures were grown in medium alone while other cultures were supplemented with either testosterone alone at a concentration of 1 x 10(-7) M or with 10, 100, 500 ng LH plus testosterone, 10, 100, 500 ng FSH plus testosterone or 10, 100, 500 ng PRL plus testosterone. After 2 days of cultivation all cultures were terminated and media were frozen at 20 degrees C for further steroid analysis. Testosterone added to the culture medium in the absence of gonadotropins was without effect on estradiol and progesterone secretion by luteal cells collected in the corpora lutea of the early luteal phase. On the other hand testosterone added to the medium significantly increased progesterone and estradiol secretion by cultured luteal cells collected in the midluteal phase of the cycle. No additive stimulatory action of gonadotropins and testosterone on progesterone secretion was observed in cultures of luteal cells from the early luteal phase but this was not the case in cultures of luteal cells from the midluteal phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunolocalization of cholesterol side-chain-cleavage cytochrome P-450 and ultrastructural studies of bovine corpora lutea

Corpora lutea were collected from cows at four stages of the luteal phase and prepared for immunostaining at the light microscope level. Other corpora lutea, which were fully developed, were dispersed by collagenase treatment and freshly isolated and cultured cells were processed for immunostaining. Electron microscopy was carried out on mature corpora lutea and freshly isolated cells. Positive staining for cholesterol side-chain-cleavage cytochrome P-450 (P-450scc), an inner-mitochondrial membrane enzyme considered to catalyse the rate-limiting step in the conversion of cholesterol to progesterone, was observed in all corpora lutea. The intensity of staining was much greater in mature corpora lutea than in young or regressing corpora lutea. Only small and large luteal cells stained positively and cells of the vasculature and other connective tissue elements did not. When cells were cultured and had become flatter, the intensity of immunostaining was observed to be greater in large luteal cells than in small luteal cells which was interpreted to be due, in part, to the greater volume density of mitochondria in these cells. In some cultured small luteal cells the pattern of immunostaining appeared as whorls of strands encircling the nucleus. This pattern was interpreted as a three-dimensional network of mitochondria organized into 'strands', more than one mitochondrion in cross-section, perhaps formed during the process of attachment and elongation of the cells. Further observations made at the electron microscope level, included the presence of close (5-8 nm) contacts with interconnecting septa between small luteal cells in tissue.     

Sex-related expression of 20alpha-hydroxysteroid dehydrogenase mRNA in the adult mouse.

The enzyme 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) catalyzes the conversion of progesterone into its inactive form, 20alpha-hydroxyprogesterone. To gain information about the exact sites of 20alpha-HSD mRNA expression, we performed in situ hybridization using a (35)S-labeled cRNA probe in tissues of adult mice of both sexes. 20alpha-HSD mRNA was expressed in both male and female gonads. In the ovary, high expression was found in luteal cells of corpora lutea, while much lower expression could be detected in granulosa cells of growing follicles. In the testis, a specific hybridization signal was detected only in Leydig cells. In the female reproductive tract, 20alpha-HSD mRNA was found in the epithelial cells of the uterine cervix. In the adrenal cortex, only the zona reticularis exhibited specific radiolabeling, the expression being very high in the female and very low in the male. In the skin, specific labeling was restricted to sebaceous glands, the hybridization signal being much higher in the female than in the male. In the liver, 20alpha-HSD mRNA was found in hepatocytes, with a higher degree of expression in the female. In the kidney, specific labeling was observed in the epithelial cells of distal convoluted tubules, the signal being also much more striking in the female than in the male. In non-reproductive tissues, it clearly appears that the expression of 20alpha-HSD mRNA is higher in the female than in the male, suggesting that 20alpha-HSD may play an important role in reducing the intracellular concentration of progesterone originating from the circulation at a much higher level in the female.     

Contrasting effects of oestradiol-17 beta and human chorionic gonadotrophin on steroidogenesis in the rabbit corpus luteum

On Day 10 of pseudopregnancy, rabbits were given an i.v. injection of hCG (10-20 i.u.) that was sufficient to cause new ovulations and the loss of follicular oestradiol secretion. There was an immediate 3-4-fold rise in serum progesterone which returned to near prestimulation values (approximately 27 ng/ml) within 12 h in the presence of an implant containing oestradiol-17 beta. In the absence of oestradiol, serum progesterone continued to decline to reach low values (approximately 4 ng/ml) within 24 h and the original corpora lutea subsequently regressed. The administration of oestradiol 24 h after injection of hCG, when progesterone secretion was low, arrested any further decline in progesterone and then restored serum progesterone to normal values. This steroidogenic effect of oestradiol in vivo was a function of enhanced luteal steroidogenesis; corpora lutea removed and incubated for 12 h produced progesterone at high, linear rates, whereas the corpora lutea from animals that did not receive oestradiol produced low or insignificant quantities of progesterone in vitro. We conclude that hCG at these doses is compatible with continued responsiveness of the corpora lutea to oestrogen and that hCG produces its luteolytic effect primarily by ovulating follicles, thus stopping the secretion of the luteotrophic hormone, oestradiol.     

Variations in oxytocin, vasopressin and neurophysin concentrations in the bovine ovary during the oestrous cycle and pregnancy

Bovine ovaries were obtained from the abattoir and corpora lutea were classified as: (1) early luteal phase (approximately Days 1-4); (2) mid-luteal phase (Days 5-10); (3) late luteal phase (Days 11-17); (4) regressing (Days 18-20) and (5) pregnant (Days 90-230). In addition, preovulatory follicles and whole ovaries without luteal tissue were collected. Concentrations of oxytocin, vasopressin, bovine neurophysin I and progesterone were measured in each corpus luteum by radioimmunoassay. Progesterone and neurophysin I levels increased from Stage 1 to Stage 2, plateaued during Stage 3 and declined by Stage 4. Oxytocin and vasopressin concentrations increased from Stage 1 to Stage 2 but declined during Stage 3 and were low (oxytocin) or undetectable (vasopressin) in follicles, whole ovaries and pregnancy corpora lutea. Therefore the concentrations of both peptide hormones were maximal during the first half of the cycle and declined before those of progesterone. The high concentration of oxytocin within the corpus luteum coupled with the presence of bovine neurophysin I suggests that oxytocin is synthesized locally.     

Expression of activities of two 20 alpha-hydroxysteroid dehydrogenase isozymes in rat corpora lutea.

The rat ovary contains two isozymes of 20 alpha-hydroxysteroid dehydrogenase (HSD-1 and HSD-2). In this study, the expression of activity of each isozyme was investigated in ovaries that contained a single generation of corpora lutea during pseudopregnancy. This condition was induced by cervical stimulation in rats that had been rendered anovulatory by housing them in a continuously lit environment. The total activity of cytosolic 20 alpha-HSD was lower in the ovaries of these pseudopregnant rats than in ovaries containing multiple generations of corpora lutea. In normal pseudopregnancy, HSD-1 activity was low on days 5 and 9 and increased markedly on day 15, whereas HSD-2 was lower than HSD-1 and did not vary throughout pseudopregnancy. However, on days 5 and 9 of continuous-light pseudopregnancy, low activity of HSD-1 only was detected; by day 15, HSD-1 activity had increased sixfold and HSD-2 activity could be detected. Immunohistochemical methods using a specific antibody recognizing both HSD-1 and HSD-2 revealed that the number of 20 alpha-HSD-positive luteal cells increased by day 15. Thus, the increase in total enzyme activity and appearance of HSD-2 activity observed at late pseudopregnancy was accompanied by an increase in the number of 20 alpha-HSD-positive luteal cells.     

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)