In vitro secretion of progestins by rat luteal cells and their 20 alpha-hydroxysteroid dehydrogenase activity

Functionally active or regressing corpora lutea were harvested from pseudopregnant (psp) rats between days 5-8 of psp or day 15 of psp, respectively. They were enzymatically dispersed and cultured for 24 h to assess progestins in the medium and 20 alpha-hydroxysteroid dehydrogenase [20 alpha-HSD, catalyzing the conversion of progesterone to 20 alpha-dihydroprogesterone (20 alpha-OH-P)] activity in the cell. Though the active luteal cells retained low 20 alpha-HSD activity, they secreted 6-7 times more 20 alpha-OH-P than progesterone as the regressing luteal cells did. There was no significant difference between the total amounts of progestins in the 2 groups. When increasing doses of pregnenolone were added to the media, progesterone secretion from the active luteal cells was promoted and the progesterone to 20 alpha-OH-P ratio became comparable to the circulating progestins ratio during the mid-luteal phase. In contrast, from the regressing luteal cells only 20 alpha-OH-P secretion was promoted. These results indicate that an insufficient precursor supply results in the catabolism of a large part of synthesized progesterone before its release from luteal cells and suggest the presence of a high affinity but low capacity 20 alpha-HSD in active corpora lutea.     

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.     

Luteal expression of cytochrome P450 side-chain cleavage, steroidogenic acute regulatory protein, 3beta-hydroxysteroid dehydrogenase, and 20alpha-hydroxysteroid dehydrogenase genes in late pregnant rats: effect of luteinizing hormone and RU486

A decrease in serum progesterone at the end of pregnancy is essential for the induction of parturition in rats. We have previously demonstrated that LH participates in this process through: 1) inhibiting 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity and 2) stimulating progesterone catabolism by inducing 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) activity. The objective of this investigation was to determine the effect of LH and progesterone on the luteal expression of the steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (P450(scc)), 3beta-HSD, and 20alpha-HSD genes. Gene expression was analyzed by Northern blot analysis 24 and 48 h after administration of LH or vehicle on Day 19 of pregnancy. StAR and 3beta-HSD mRNA levels were lower in LH-treated rats than in rats administered with vehicle at both time points studied. P450(scc) mRNA levels were unaffected by LH. The 20alpha-HSD mRNA levels were not different between LH and control rats 24 h after treatment; however, greater expression of 20alpha-HSD, with respect to controls, was observed in LH-treated rats 48 h after treatment. Luteal progesterone content dropped in LH-treated rats at both time points studied, whereas serum progesterone decreased after 48 h only. In a second set of experiments, the anti-progesterone RU486 was injected intrabursally on Day 20 of pregnancy. RU486 had no effect on 3beta-HSD or P450(scc) expression but increased 20alpha-HSD mRNA levels after 8 h treatment. In conclusion, the luteolytic effect of LH is mediated by a drop in StAR and 3beta-HSD expression without effect on P450(scc) expression. We also provide the first in vivo evidence indicating that a decrease in luteal progesterone content may be an essential step toward the induction of 20alpha-HSD expression at the end of pregnancy in rats.     

Changes in rat luteal ultrastructure and P450scc mRNA and protein content after in vivo treatment with a gonadotropin-releasing hormone agonist

Previous studies have demonstrated that plasma progesterone levels decrease in pregnant rats treated in vivo with a gonadotropin-releasing hormone agonist (GnRH-Ag), without changes in testosterone or estradiol levels in ovarian vein plasma. The objective of this study was to determine the loci of GnRH-Ag disruption of progesterone synthesis by examining luteal mitochondria, lipid droplets, cellular composition, and P450 side-chain cleavage (P450scc) enzyme and mRNA content in the pregnant rat. On Day 7 or 11 of pregnancy, osmotic minipumps containing GnRH-Ag were implanted into 5-7 rats. Sham operations were performed on 5-6 controls at each time period. Five micrograms per day of GnRH-Ag were released for about 24 h, after which corpora lutea and jugular vein plasma were collected. The corpora lutea were prepared for microscopy or analyzed for P450scc enzyme and mRNA content. Plasma progesterone levels were measured by RIA. In those rats treated with GnRH-Ag, progesterone levels had decreased, and within the luteal cells, there was an increase in the number of lipid droplets and a decrease in the number of tubular cristae within the mitochondria. Concomitantly, P450scc enzyme and mRNA content decreased on both Day 8 and Day 12 of pregnancy. Also, GnRH-Ag treatment decreased the ratio of large to small steroidogenic luteal cells on Day 8 of pregnancy, but did not alter cellular ratios on Day 12 of pregnancy. These observations suggest that treatment with GnRH-Ag inhibits progesterone synthesis by decreasing the amount of P450scc mRNA and enzyme content, which may alter the mitochondrial cristae structure on Day 8 and Day 12 of pregnancy. The reduction in tubular cristae and P450scc enzyme in the mitochondria may account for the increase in lipid droplets, as less cholesterol is converted to pregnenolone. An additional mechanism of inhibition may be the reduction in the number of large steroidogenic luteal cells, which appear to be the major source of progesterone in the rat corpus luteum on Day 8 of pregnancy.     

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.     

Function of abnormal corpora lutea in vitro after GnRH-induced ovulation in the anoestrous ewe

Normal and abnormal corpora lutea were recovered from anoestrous Romney Marsh ewes on Days 3, 4, 5 and 6 after treatment with small-dose (250 ng) multiple injections of GnRH followed by a bolus injection (125 micrograms) with (+P) and without (-P) progesterone pretreatment and a study made of their characteristics in vitro. Plasma progesterone concentrations initially rose concurrently in all animals but abnormal luteal function occurred in 70% of the -P ewes and was defined on Day 5 when plasma progesterone concentrations declined relative to those in the +P ewes. All corpora lutea recovered on Days 3 and 4 appeared macroscopically similar and there were no significant differences between the +P and -P groups in terms of luteal weight, progesterone content and binding of 125I-labelled hCG on these days. However, corpora lutea from the -P animals only exhibited a decline in progesterone production in vitro on Day 4 (P less than 0.01), and morphological differences became apparent on Days 5 and 6 when the abnormal corpora lutea from the -P animals also decreased in weight (P less than 0.01) and progesterone content (P less than 0.001). Binding of 125I-labelled hCG increased on Day 5 in the normal corpora lutea only. These results show that, although abnormal luteal function induced by GnRH treatment of anoestrous ewes could not be distinguished from normal corpora lutea before Day 5 by measurement of progesterone in peripheral plasma, a significant decline in progesterone production in vitro occurred on Day 4 in the abnormal corpora lutea. This was followed by significant decreases in weight and progesterone content and a failure to increase 125I-labelled hCG binding. Abnormal corpora lutea are therefore capable of some initial growth and progesterone production, before undergoing a rapid and premature regression from Day 4, which has similar characteristics to natural luteolysis.     

Participation of ovarian 20 alpha-hydroxysteroid dehydrogenase in luteotrophic and luteolytic processes during rat pseudopregnancy

In normal rats, before Day 12 of pseudopregnancy, minimal levels of 20 alpha-HSD activity were detected in functional CL whereas those in the residue were 3-5 times higher. When ovulation was blocked for more than 2 weeks by placing rats in a continuously lit environment before the induction of pseudopregnancy, only minimal levels of 20 alpha-HSD activity were detectable in the functional CL and residue before Day 12. In normal pseudopregnant rats, there was a linear increase in 20 alpha-HSD activity from Day 12 to 15 in the functional CL and residue, but the rate of elevation was much higher in functional CL. This tendency was much more clear-cut in rats in the continuous lighting. In immature rats in which pseudopregnancy was induced by PMSG and hCG treatment, 20 alpha-HSD activity peaked twice. The first small peak was attributed to the early regression of some of the large number of corpora lutea, and the changes in 20 alpha-HSD activity in most of the corpora lutea paralleled those in rats in continuous lighting. Bromocriptine abolished the prolactin surges, and in normal pseudopregnant rats an increase in 20 alpha-HSD activity in functional CL started from 12 h and the rate of the increase was accelerated from 36 h afterwards, while a relatively small increase was observed in the residue at 18 h and later.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of luteinizing hormone and prostaglandin F(2alpha) on progesterone secretion in superfused minced bovine luteal tissue in the early stage of the estrous cycle

Minced luteal tissue of bovine corpora lutea from Day 4, 5, and 6 of the estrous cycle (n = 4 corpora lutea each) was superfused for 9 h, and the progesterone secretion under the influence of 100 ng luteinizing hormone (LH)/ml and/or 1,000 ng prostaglandin F(2alpha) (PGF(2alpha))/ml was determined. In vivo, this period of the estrous cycle is characterized by a transition from PGF(2alpha) refractoriness to PGF(2alpha) sensitivity. The investigations were carried out in order to examine whether this transition is reflected by a change in the hormone secretion pattern in vitro. The basal secretion was higher on Day 6 than on Day 4 and 5 (P < 0.01). PGF(2alpha) slightly increased the progesterone secretion, but there was no statistically significant difference (P > 0.05). LH, however, stimulated the progesterone secretion by about 30% in luteal tissue collected from Day 4 and 5 (P < 0.01). In luteal tissue collected from Day 6, the LH-induced increase in hormone secretion was not statistically significant due to two corpora lutea that showed no response at all to LH. The progesterone secretion of the two other corpora lutea, however, was increased by 30% (P < 0.01). When PGF(2alpha) and LH were simultaneously added, the LH-induced progesterone secretion was not inhibited; PGF(2alpha) even seemed to intensify the action of LH. The difference between the hormone secretion under the influence of LH alone and that under the influence of a combination of LH and PGF(2alpha), however, was not statistically significant. It is concluded that in cattle the end of the refractoriness to PGF(2alpha) in vivo is not reflected by a corresponding change of the hormone secretion pattern 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.     

Further studies on in vitro steroidogenesis by luteal cells from long-term hypophysectomized rats

Immature pregnant mare's serum gonadotropin-treated rats were hypophysectomized on the day of ovulation (Day 1 of luteal function), and luteal steroidogenesis and human chorionic gonadotropin (hCG) and prolactin (Prl) binding sites were determined on Days 5, 10, 20 and 30 (H5- H30 ) compared with intact rats on Days 5 or 10 (C5 or C10). On H5, dispersed luteal cells secreted large amounts of progesterone (P), 20 alpha-dihydroprogesterone (20 alpha-DHP), 17 alpha-hydroxyprogesterone (17 alpha-OHP), and small amounts of testosterone (T) and estradiol-17 beta (E2), but between H10 and H30 , reduced levels of all steroids were produced except for 20 alpha-DHP. Addition of large amount of pregnenolone (P5) or P (100-1000 ng) to dispersed luteal cells increased production of P and 20 alpha-DHP in C5 and H5 rats. The higher serum levels and basal in vitro production of 20 alpha-DHP from H5 to H30 indicates that 20 alpha-oxidoreductase persists in the corpora lutea (CL) at high levels and that 3 beta-ol-dehydrogenase is also present but with P rapidly shunted into its principal metabolite. From H5 to H30 , addition of 10 ng P to luteal cells increased the production of 17 alpha-OHP and addition of 10 ng androstenedione (A) or T enhanced production of T and E2, indicating that 17 alpha-oxidoreductase, 17 beta-hydroxysteroid dehydrogenase and aromatase also persist in the CL. In vitro addition of 10 ng LH significantly stimulated production of P from luteal cells on C5 and H5, whereas on C10 and H10, 100 ng LH was required and on H20 and H30 , 1 microgram LH produced a minimal increase in P.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Beta-adrenergic receptor concentration and subtype in the corpus luteum of the adult pseudopregnant rat

Luteal beta-adrenergic receptor concentration and subtype were determined in adult pseudopregnant rats during and after the period of the functional luteal phase. The specific beta-adrenergic receptor ligand (-)-3-[125I]iodocyanopindolol ([125I]ICYP) was used to determine the receptor concentration in corpora lutea of adult pseudopregnant rats. A 3-fold increase in beta-adrenergic receptor concentration was seen during the first 2-3 days of pseudopregnancy, whereafter the receptor concentration declined. During the functional luteal regression period (Day 12-15) the receptor levels were still low. In regressed (Day 16-22) corpora lutea a temporary increase in beta-receptor concentration was seen which may represent some role for beta-adrenergic mechanisms in the regulation of morphological regression in the corpus luteum. To determine the beta-adrenergic subtype, competition of [125I]ICYP-binding with selective beta 1- and beta 2-adrenergic antagonists was assessed in corpora lutea of different ages and in rat heart and uterus. The beta-adrenergic receptors in corpora lutea of adult pseudopregnant rats were shown to be solely of the subtype beta 2, regardless of the luteal age.     

Hormonal regulation of estradiol biosynthesis, aromatase activity, and aromatase mRNA in rat ovarian follicles and corpora lutea

To determine the molecular basis for changes in aromatase (P450arom) activity in rat ovarian follicles and corpora lutea, seven clones for rat P450arom cDNA have been identified and isolated from a rat granulosa cell λgtll cDNA expression library using a 62 mer deoxyoligonucleotide probe (derived from an amino acid sequence of purified human placental aromatase) and a human placental P450arom cDNA probe. One of the rat P450arom cDNA clones contained an insert 1.2 kb in size. Both the human 1.8 kb cDNA and the rat 1.2 kb cDNA probes hybridized to a single species of P450arom mRNA that was 2.6 kb in size. Northern blot analysis revealed that corpora lutea isolated on day 15 of pregnancy contained high amounts of P450arom mRNA, whereas granulosa cells of antral follicles of hormonally primed, hypophysectomized rats (i.e., those from which mRNA was isolated to construct the cDNA library) contained only low amounts of P450arom mRNA. The lower amounts of P450arom in granulosa cells of preovulatory follicles in the estradiol-follicle-stimulating hormone primed hypophysectomized rats were unexpected because follicles incubated in medium containing testosterone substrate produce more estradiol than do corpora lutea isolated on day 15 of pregnancy and incubated under similar conditions. Additional studies will determine the hormonal events responsible for the elevated amounts and constitutive maintenance of P450arom mRNA and aromatase activity in luteal cells in vivo and in vitro.     

Prostaglandin f(2alpha) induces distinct physiological responses in porcine corpora lutea after acquisition of luteolytic capacity

This study examines differences in intracellular responses to cloprostenol, a prostaglandin (PG)F(2alpha) analog, in porcine corpora lutea (CL) before (Day 9 of estrous cycle) and after (Day 17 of pseudopregnancy) acquisition of luteolytic capacity. Pigs on Day 9 or Day 17 were treated with saline or 500 microgram cloprostenol, and CL were collected 10 h (experiment I) or 0.5 h (experiment III) after treatment. Some CL were cut into small pieces and cultured to measure progesterone and PGF(2alpha) secretion. In experiment I, progesterone remained high and PGF(2alpha) low in luteal incubations from either Day 9 or Day 17 saline-treated pigs. Cloprostenol increased PGF(2alpha) production 465% and decreased progesterone production 87% only from Day 17 luteal tissue. Cloprostenol induced prostaglandin G/H synthase (PGHS)-2 mRNA (0.5 h) and protein (10 h) in both groups. In cell culture, cloprostenol or phorbol 12, 13-didecanoate (PDD) (protein kinase C activator), induced PGHS-2 mRNA in luteal cells from both groups. However, acute cloprostenol treatment (10 min) decreased progesterone production and increased PGF(2alpha) production only from Day 17 luteal cells. Thus, PGF(2alpha) production is induced by cloprostenol in porcine CL with luteolytic capacity (Day 17) but not in CL without luteolytic capacity (Day 9). However, this change in PGF(2alpha) production is not explained by a difference in induction of PGHS-2 mRNA or protein.     

Characterization and physiological variation of estrogen receptors in rabbit corpora lutea throughout pregnancy and pseudopregnancy: the effect of hysterectomy and sustained estradiol treatment

Previous studies suggest that regression of the rabbit corpus luteum is associated with a uterine-induced loss of responsiveness to estradiol. To determine if this is due to loss of estrogen receptor, cytoplasmic and nuclear estrogen receptors were measured in pseudopregnant, hysterectomized-pseudopregnant and pregnant rabbits throughout luteal life. Estrogen receptor levels were higher in corpora lutea than in nonluteal tissue and were generally higher in nuclei compared to cytosol. Estrogen receptor levels were low on Day 3, increased 2- to 3-fold by Day 6-8, reached peak levels by Days 8-10, and then gradually decreased in a pattern similar to the pattern of serum progesterone typical of each group. Hysterectomy was not associated with elevated cytoplasmic or nuclear estradiol receptor levels. When hysterectomized rabbits were treated with estradiol-filled Silastic implant on Day 1, nuclear estradiol receptor levels fell by Day 20 to levels seen in untreated hysterectomized rabbits. Despite substantial losses in nuclear estrogen receptor, serum progesterone remained elevated on Days 16 and 20. Thus, the ability of estradiol to maintain serum progesterone in hysterectomized rabbits did not correlate directly with the level of estrogen receptor.     

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.     

Lipoprotein lipase activity in the bovine corpus luteum during the estrous cycle and early pregnancy.

Lipolytic activity measured at pH 8.6 in bovine corpora lutea exhibited classical properties of lipoprotein lipase (LPL) in terms of serum and heparin stimulation and NaCl inhibition. LPL activity was measured in 23 corpora lutea collected at different stages of the estrous cycle and early pregnancy. The LPL activity in cyclic corpora lutea (mumole FA released/hr/100 mg acetone powder) was low at Days 4-8 of the estrous cycle (3.1 +/- 1.5: mean +/- SE) and at Days 19-20 (1.6 +/- 0.6). However, high activity of the enzyme was found at Days 12-15 of the cycle (11.8 +/- 1.8); these concentrations were significantly (P less than 0.01) elevated over those found at Days 4-8 and 19-20. The enzyme activity began to decline at Days 16-18 of the estrous cycle (5.1 +/- 1.7). Low enzyme activity was found in the corpora lutea removed from two cows at Day 22 of pregnancy. Progesterone concentrations were measured in 16 of the 23 corpora lutea and a good correlation (r = 0.75, P less than 0.01) was found between lipoprotein lipase and progesterone concentrations of the tissue. The data suggest that LPL may be involved in controlling the transfer of fatty acids, including arachidonic, from plasma lipoproteins to luteal tissue.     

Comparison of PGF2α-induced luteolysis in early pregnant and estrogen-treated ‘pseudopregnant’ gilts

Conceptus estrogen clearly plays a major role in luteal maintenance in the pig; however, other conceptus-derived substances or conceptus-induced uterine secretory products appear to have a local luteotrophic/anti-luteolytic effect on the corpora lutea (CL) and likely may play a key role in maternal recognition of pregnancy in the pig. The objective of these studies was to compare PGF₂α-induced luteolysis in estrogen-treated ‘pseudopregnant’ gilts versus pregnant gilts during the period of maternal recognition of pregnancy. In Experiment 1, doses of PGF₂α ranging from 1 to 100 μg were administered via intraluteal silastic implants to pseudopregnant gilts to determine the dose necessary to cause functional (progesterone) and structural (weight) luteal regression similar to that observed during the natural estrous cycle. Luteal sensitivity to this minimally effective luteolytic dose of PGF₂α was then determined for both pseudopregnant and pregnant gilts in Experiment 2. Experiment 3 investigated whether Day 13 porcine conceptus tissue could directly prevent PGF₂α-induced luteolysis at the level of the CL. The minimally effective luteolytic dose of PGF₂α (100 μg) determined in the pseudopregnant pig caused a similar decline in progesterone concentration and weight of CL in pregnant gilts, suggesting that the susceptibility of CL of pregnant and pseudopregnant pigs to PGF₂α is similar. However, luteal weight was greater (P<0.05) for the pregnant gilts than for pseudopregnant gilts, suggesting that estrogen treatment alone cannot mimic the conceptus effects on CL growth and development. Experiment 3 demonstrated that lyophilized Day 13 conceptus tissue implanted directly into individual CL could partially inhibit PGF₂α-induced luteolysis, providing for the first time direct evidence that porcine conceptuses as early as Day 13 contain factors which can directly (i. e. at the level of the CL) prevent luteal regression.     

Effect of pregnancy, injection of oestradiol benzoate or hCG on steroid concentration and release by pig luteal cells

Corpora lutea were obtained from pig ovaries on Day 18 of pregnancy or pseudopregnancy. Pseudopregnancy was induced by the administration of oestradiol benzoate on Days 11-15 of the oestrous cycle or by the administration of hCG on Day 12. The luteal cells were prepared for morphometric analysis and investigation of steroid production in vitro by dispersion with 0.25% trypsin. A blood sample from each sow was collected at slaughter for measurement of progesterone, oestradiol-17 beta and testosterone. The concentrations of these steroids were also estimated in luteal tissue and in the medium after incubation. Progesterone concentration was significantly higher (P less than 0.01) in luteal tissue and in plasma of pregnant than of pseudopregnant sows. Testosterone content of luteal tissue from all sows was 20-fold higher than oestradiol, although plasma concentrations of these hormones were not different. The luteal cells from hCG-treated sows produced more progesterone (P less than 0.01) in vitro than did those from the other groups. The luteal cells from oestradiol-treated sows generally released smaller amounts of steroids during incubation. Treatment with hCG increased the proportion of large luteal cells and decreased the proportion of small luteal cells. These results demonstrate that hCG or oestradiol benzoate injections altered the steroidogenic activity of luteal cells and that treatment with hCG was also associated with changes in the diameter of the luteal cells and thus in the ratio of small to large luteal cells.