Rat Corpus Luteum Research Articles

Hormonal and immunological characterization of the 32 kilodalton ovarian-specific protein.

Recent studies from this laboratory have shown that the large luteal cell of the pregnant rat contains an abundant 32 kilodalton (32K) phosphoprotein which is up-regulated by estradiol. In order to assess the potential importance of this protein and to more fully understand its function, a specific polyclonal antibody was produced against the 32K and was used to examine its intraovarian localization, its tissue specificity, and its developmental regulation. Immunocytochemical localization of the 32K in the ovary of the pregnant rat found this protein to be selectively and abundantly expressed in the corpus luteum. Immunofluorescence study of small and large luteal cell populations clearly revealed an extensive localization of the 32K in the large luteal cells. Western blot analysis revealed that the 32K was absent from all steroidogenic and nonsteroidogenic tissues. Whereas this protein was absent from all other tissues examined in the rat, it was clearly expressed in corpora lutea of different animal species, including the mouse, hamster, cow, human, and pig. Although undetectable by immunohistochemistry, Western blot analysis showed this protein to be present in the follicle but at levels markedly lower than in the corpus luteum. Analysis of theca and granulosa cells revealed the presence of the 32K in both cell types. To further examine the developmental expression of this protein throughout gestation, Western blot analysis of microsomal fractions isolated from rat corpora lutea on days 3-21 of pregnancy was performed. The 32K was detected at low levels in early pregnancy, increased markedly on day 11, reached a peak on days 14-15, and remained elevated through day 21. Treatment with estradiol and PRL increased the content of the 32K in the corpus luteum. Human CG, known to cause follicular development to the preovulatory stage and to enhance luteal estradiol synthesis, also increased levels of the 32K in the corpus luteum, while it concomitantly decreased this protein in the follicle. In summary, the presence of a unique ovarian-specific 32 kilodalton protein has been established. This protein, which is present in low abundance in theca and granulosa cells, is localized to the large luteal cell and becomes abundantly expressed during midpregnancy, a time when there is a remarkable increase in luteal cell size and activity. Results of this study also demonstrate a multihormonal regulation of the 32K by tropic hormones. Thus, because of its apparent uniqueness and its timely and highly regionalized expression, the 32K may play a central role in the regulation of corpus luteum growth and function.

Prolactin stimulates the expression of luteinizing hormone/chorionic gonadotropin receptor messenger ribonucleic acid in the rat corpus luteum and rescues early pregnancy from bromocriptine-induced abortion.

Timed pseudopregnancy (psp) and pregnancy were induced in adult female rats by mating with infertile and fertile males, respectively. Corpora lutea (CL) and the residual parts of the ovaries were isolated and analyzed for luteinizing hormone/chorionic gonadotropin (LH/CG) receptor mRNA by Northern blot and solution hybridization analyses. Several LH/CG receptor mRNA transcripts were detected that could code for an intact functional receptor (6.8, 4.4, and 2.6 kb) as well as several smaller truncated transcripts. LH/CG receptor mRNA abundance in CL varied dramatically during both psp and pregnancy, with peak levels seen during the period of maximal progestational activity (Days 5-10 of psp and Days 7-14 of pregnancy). During the period of functional luteolysis, LH/CG receptor mRNA abundance decreased to low levels. The changes in LH/CG receptor expression could be explained by hormonal regulation. Bromocriptine treatment inhibited pituitary prolactin secretion. This treatment had a potent luteolytic effect by decreasing the levels of LH/CG receptor mRNA and plasma progesterone during early pregnancy, resulting in embryonal resorption in pregnant rats. Exogenous prolactin acted as a anti-luteolysin to reverse these effects by restoring LH/CG receptor mRNA abundance either by increasing gene expression or by stabilizing mRNA transcripts from degradation in young CL.

The cAMP-dependent signalling cascade in the two luteal cell types of the pregnant rat corpus luteum

Corpora lutea of rats, like those of many other species, contain two sub-populations of luteal cells. In this report we sought to determine whether the luteinizing hormone (LH)- and β-adrenergic cAMP signal transduction pathways known to be present in rat corpora lutea were segregated into separate luteal cell types. Results showed that large rat luteal cells, obtained on day 3 of pregnancy, exhibited elevated LH- and most notably epinephrine-stimulated adenylyl cyclase activities but equivalent cAMP-dependent catalytic protein kinase and total regulatory subunit cAMP binding activities compared to small luteal cells. Progesterone production by the large cell was greater than that by the small cell but both cells were equally sensitive to stimulation of progesterone by LH. However, neither the large nor the small rat luteal cell produced significant progesterone in response to epinephrine despite a marked epinephrine-stimulated adenylyl cyclase in both cell populations. The LH-stimulated progesterone synthetic response of the two sub-populations of rat luteal cells is more similar to that of the developing monkey corpus luteum and contrasts sharply with that of ruminants.

Structure and tissue-specific expression of 3ß-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase genes in human and rat classical and peripheral steroidogenic tissues

The enzyme 3ß-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3ß-HSD) catalyzes the oxidation and isomerization of 5-ene-3ß-hydroxypregnene and 5-ene-hydroxyandrostene steroid precursors into the corresponding 4-ene-ketosteroids necessary for the formation of all classes of steroid hormones. We have recently characterized two types of human 3ß-HSD cDNA clones and the corresponding genes which encode deduced proteins of 371 and 372 amino acids, respectively, and share 93.5% homology. The human 3ß-HSD genes containing 4 exons were assigned by in situ hybridization to the p11–p13 region of the short arm of chromosome 1. We have also recently elucidated the structure of three types of rat 3ß-HSD cDNAs as well as that of one type of 3ß-HSD from bovine and macaque ovary λgt11 cDNA libraries which all encode 372 amino acid proteins. The human type I 3ß-HSD is the almost exclusive mRNA species detected in the placenta and skin, while the human type II is the predominant mRNA species in the adrenals, ovaries and testes. The predicted rat type I and type II 3ß-HSD proteins expressed in adrenals, gonads and adipose tissue share 94% homology while they share 80% similarity with the liver-specific type III 3ß-HSD. Transient expression of human type I and type II as well as rat type I and type II 3ß-HSD cDNAs in Hela human cervical carcinoma cells reveals that 3ß-ol dehydrogenase and 5-ene-4-ene isomerase activities reside within a single protein and these cDNAs encode functional 3ß-HSD proteins that are capable of converting 3ß-hydroxy-5-ene-steroids into 3-keto-4-ene derivatives as well as the interconversion of 3ß-hydroxy and 3-keto-5α-androstane steroids. We have found that the rat type III mRNA species was below the detection limit in intact female liver while, following hypophysectomy, its accumulation increased to 55% of the levels measured in intact or HYPOX male rats, an increase which can be blocked by administration of ovine prolactin (oPRL). In addition, in female rats, treatment with oPRL for 10 days starting 15 days after HYPOX, markedly decreased ovarian 3ß-HSD mRNA accumulation accompanied by a similar decrease in 3ß-HSD activity and protein levels. Treatment with the gonadotropin hCG reversed the potent inhibitory effect of oPRL on these parameters and stimulated 3ß-HSD mRNA levels in ovarian interstitial cells. In intact females, hCG exerted marked trophic effects on rat corpora lutea with an increase in total ovarian 3ß-HSD expression and activity. We have also shown that treatment with hCG for 15 days in intact male rats caused a marked increase in testicular 3ß-HSD expression and activity while glucocorticoids exerted inhibitory effects on these parameters. We have also observed that the ontogeny of 3ß-HSD expression in human and rat adrenal gland, testis and ovary is closely correlated with steroid hormone biosynthesis, thus suggesting that regulation of the expression of 3ß-HSD is a limiting step in the biosynthesis of steroids in these tissues.

Regulation of ovarian antioxidant vitamins, reduced glutathione, and lipid peroxidation by luteinizing hormone and prostaglandin F2 alpha.

Reactive oxygen species are generated by the rat ovary, and they evoke marked antigonadotropic responses in ovarian cells. Protection against reactive oxygen species is provided by antioxidants such as vitamins C, E, and A, and reduced glutathione (GSH). Our objectives were to establish the ovarian levels of these antioxidants during development and regression of the corpus luteum of the pseudopregnant rat and to determine whether these levels were changed by an acute treatment with either a luteotropic (LH) or luteolytic (prostaglandin [PG] F2 alpha) agent. In addition, we evaluated the extent of oxidative activity in the ovary by determining the level of lipid peroxidation. Follicular development was associated with a significant increase in ovarian levels of vitamin A and GSH, whereas levels of vitamins E and C were unchanged. During the luteal phase, vitamin E levels tended to increase, whereas vitamin A and GSH levels decreased. Luteal regression was associated with a marked increase in ovarian levels of vitamins E and A, whereas GSH levels increased only transiently. Acute treatment with LH in the midluteal phase produced a transient decrease in vitamin C levels that was maximal at 4 h. Luteal vitamin E levels were markedly increased 24 h after LH treatment, whereas vitamin A levels were unchanged, and no evidence of lipid peroxidation was seen. Acute treatment with PGF2 alpha produced a transient decrease in luteal vitamin C levels coincident with transient lipid peroxidation and a sustained fall in serum progesterone levels. Ovarian vitamin A levels were elevated 24 h after PGF2 alpha treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation, characterization, and culture of cell subpopulations forming the pregnant rat corpus luteum.

The aim of this investigation was to isolate, characterize, and culture the small and large luteal cell subpopulations forming the corpus luteum of the pregnant rat. Since the large luteal cells are extremely fragile and do not survive standard cell dispersion, a method which allows the survival and the long-term culture in serum-free media of small and large cells was developed. The two luteal cell populations differed not only by their size but also by their morphology in culture. The small luteal cells (12-20 mu in diameter) are characterized by a large oval nucleus, contain few lipid droplets and have a stellate shape. In contrast, the large luteal cells have a smaller spherical nucleus, high lipid content, and do not flatten out completely in culture, most probably due to the abundance of lipid droplets. Both luteal cell types express 3 beta HSD and the cytochrome P450 enzymes involved in steroidogenesis. However, it is the lipid filled large luteal cells that secrete the most progesterone, androgen, and estradiol; express greater amounts of P450scc and P450AROM; and possess more PRL and LH receptors. Despite the greater expression of LH receptor in the large luteal cells, small and large luteal cells responded to LH with equal increase in steroidogenic output. In serum free culture, luteal cells produced progesterone for up to 20 days; however, an exogenous source of cholesterol was a prerequisite for maximal progesterone secretion. The pattern of progesterone secretion by cultures of small and large luteal cells differed remarkably from that of mixed cell population. When nonsteroidogenic corpus luteum cells were cocultured with the large luteal cells, a severalfold increase in progesterone secretion was observed. This stimulation occurred even when cells were cocultured in the absence of exogenous source of cholesterol. In summary, a successful method was developed to disperse, isolate, and independently culture the two luteal cell populations forming the rat corpus lutem. The results indicate that the marked difference in the steroidogenic capacity of these two cell populations is due, in large part, to the difference in their size rather than to their origin in the follicle. In addition, the results have revealed an important effect of the nonsteroidogenic cells forming the corpus luteum on luteal cell steroidogenesis.

Isolation, characterization, and culture of cell subpopulations forming the pregnant rat corpus luteum

Isolation, characterization, and culture of cell subpopulations forming the pregnant rat corpus luteum

Different effects of the antiprogesterone RU486 on progesterone secretion by the corpus luteum of rats with 4- and 5-day oestrous cycles

Administration of the antiprogesterone RU486 (2 mg/day) for 14 days to rats with a 5-day reproductive cycle resulted in an increase in both ovarian and pituitary weight in contrast with rats with a 4-day oestrous cycle. Luteal progesterone production decreased earlier in 4-day than in 5-day cyclic rats. Treatment of 5-day cyclic rats with antiprogesterone from the day of metoestrus onwards resulted in the advancement of the preovulatory prolactin surge by 24 h. Progesterone production by the corpus luteum was, however, not affected, indicating that in 5-day cyclic rats the corpora lutea are still functionally active at the time of the preovulatory surge of prolactin. They become, therefore, stimulated both in size and progesterone production. In contrast, the corpora lutea in 4-day cyclic rats are functionally inactive at the time of the preovulatory surge of prolactin, and prolactin acts luteolytically. In conclusion, the advancement of the preovulatory surge of prolactin by 24 h accounts, at least in part, for the increase in ovarian weight in 5-day cyclic rats after treatment with antiprogesterone. The results of these experiments do not agree with a direct effect of the antiprogesterone RU486 on progesterone secretion by the corpus luteum.

Translation of rat ovarian mRNA to two 20 alpha-hydroxysteroid dehydrogenase isozymes in Xenopus oocytes.

20 alpha-Hydroxysteroid dehydrogenase (20 alpha-HSD) in rat luteal tissue catalyzes the conversion of progesterone into a biologically inactive steroid, 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OHP) and depletes the output of progesterone into the circulation. An increase in 20 alpha-HSD activity in luteal tissue is therefore a prerequisite for the regression of functional corpora lutea in rats. We have reported that ovarian 20 alpha-HSD is composed of two isoforms (HSD1 and HSD2). In this study, among batches of ovaries collected randomly during the estrous cycle, we selected two batches (batches A and B): the cytosol preparation from batch A contained both HSD1 and HSD2 activities, whereas that from batch B contained only HSD1 activity. From these 2 batches, we extracted mRNA, and each mRNA preparation was subjected to translation in Xenopus oocytes. The translation products of batch A exhibited both HSD1 and HSD2 activities, and those of batch B only HSD1 activity in accordance with the enzymatic activities observed in the respective cytosolic preparations. The results are compatible with the presence of two distinct mRNAs coding HSD1 and HSD2, and if so their transcription will be regulated separately according to the functional state of the ovary.

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.

Expression, action, and steroidal regulation of insulin-like growth factor-I (IGF-I) and IGF-I receptor in the rat corpus luteum: their differential role in the two cell populations forming the corpus luteum.

The overall aim of this investigation was to examine the expression and steroidal regulation of insulin-like growth factor-I (IGF-I) and the IGF-I receptor in the rat corpus luteum and to examine the specificity of IGF-I action in the two luteal cell populations. We first examined whether the corpus luteum expresses the IGF-I and IGF-I receptor genes. Using a solution hybridization/RNase protection assay, IGF-I and IGF-I receptor mRNAs were represented by protected bands 224 and 265 bases in length, respectively. In addition, Northern blot analysis showed that, as in liver, rat IGF-I and IGF-I receptor cDNAs hybridized with 7.5-, 1.8-, and 0.8- to 1.2-kilobase transcripts and an 11-kilobase transcript, respectively. Both IGF-I and IGF-I receptor mRNAs were detected on all days of pregnancy tested (days 5-21). Since the rat corpus luteum increases remarkably in size and steroidogenic capacity at midpregnancy due to estradiol stimulation, we determined whether these developmental changes are accompanied by an increased expression of the IGF-I and/or IGF-I receptor genes. Total RNA was isolated from corpora lutea of day 12 hypophysectomized-hysterectomized rats treated with or without estradiol for 3 days. Estradiol caused a clear and marked reduction in IGF-I and IGF-I receptor mRNA. [125I]IGF-I bound with high specificity and affinity to luteal cell membranes. Large and small cell populations forming corpora lutea of day 3 and 14 pregnant rats were separated by elutriation and used for the determination of binding activity and for cell culture, respectively. IGF-I receptors were found to be localized principally in the large luteal cell population. The small luteal cells had approximately 6.5-fold less IGF-I-binding activity. The difference in binding activity in both cell populations was reflected in the ability of both cell types to respond to IGF-I. IGF-I (25 ng/ml) had a profound effect on the production of progesterone by the large luteal cells. No stimulatory effect of IGF-I on the small luteal cells was observed. Addition of estradiol (10 ng/ml) to the cell culture remarkably enhanced IGF-I stimulation of progesterone biosynthesis by the large luteal cells. In summary, the results of this investigation have revealed that the corpus luteum of the pregnant rat is a major site of expression of both the IGF-I and IGF-I receptor genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of rate of change of luteinizing hormone concentration on in-vitro progesterone secretion within rat corpora lutea during differentiation

Immature rats were injected with pregnant mares' serum gonadotrophin followed by human chorionic gonadotrophin (hCG). Ovaries were removed 0, 2, 5 or 8 days after hCG and either prepared for morphometric analysis or perifused with 0, 5 or 30 ng luteinizing hormone (LH)/min. In a second study, ovaries were removed on Day 2 or 8 and perifused with 0.1 mg 8-br-cyclic adenosine 5'-phosphate/ml (8-br-cAMP). On Day 0, the granulosa cells of the preovulatory follicles were small (53 +/- 0.5 microns2) with a cytoplasmic to nuclear (Cy:Nu) ratio less than or equal to 1.5. By Day 2, corpora lutea (CL) were present and composed of 95% small luteal cells (diameter less than 125 microns2, Cy:Nu greater than or equal to 3.0) and 5% large luteal cells (diameter greater than 125 microns2, Cy:Nu ratio greater than or equal to 3.0). The percentage of large luteal cells increased to 36 +/- 7% by Day 5, suggesting that they are derived from a select population of small luteal cells. Basal progesterone secretion increased from 38 +/- 5 on Day 0 to 1010 +/- 48 pg/mg/ml on Day 8. The rate of 5 ng LH/min stimulated progesterone secretion on Days 0, 2 and 8; 30 ng LH/min stimulated progesterone secretion on Days 0, 2 and 8, but not on Day 5; 8-br-cAMP stimulated progesterone secretion on both Days 2 and 8. These data demonstrate that once granulosa cells are induced to luteinize they lose their capacity to secrete progesterone in response to 5 ng LH/min and do not regain their responsiveness to LH rate until they completely differentiate. The loss of this LH responsiveness appears to be due to an inability to stimulate sufficient intracellular cAMP concentrations, since cAMP stimulates progesterone secretion on both Days 2 and 8.

Effect of pulse amplitude of luteinizing hormone, duration and rate of change on progesterone secretion from rat corpora lutea

Corpora lutea (CL) were obtained from immature rats primed with pregnant mares' serum gonadotrophin followed by human chorionic gonadotrophin (hCG). Two days after hCG, CL were isolated, placed in perifusion culture and exposed to control medium or specific pulses of luteinizing hormone (LH). In Expt 1, a frequency of 1 pulse LH/h (amplitude 500 pg/ml, duration 40 min, 30 ng/min) increased progesterone secretion compared with control values (P less than 0.05). In Expt 2, LH rate was held constant and the amplitude and duration of a single LH pulse varied; 250 and 500 ng LH/ml initially stimulated progesterone secretion equivalently, but increasing the duration of the LH pulse prolonged high progesterone secretion. These observations suggest that at less than or equal to 500 ng LH/ml, once a stimulatory amplitude is obtained, higher amplitudes do not further increase progesterone secretion, while increasing pulse duration further enhances progesterone secretion. In Expt 3, the LH pulse amplitude was 250 ng/ml and the rate set at 0, 5 or 30 ng LH/min; only 30 ng LH/min resulted in sustained stimulation of progesterone (P less than 0.05). Taken together, these data demonstrate that the characteristics which determine whether an LH pulse will be stimulatory include not only amplitude and duration but also the rate at which an amplitude is obtained.

Acute action of luteinizing hormone on mouse Leydig cells: Accumulation of mitochondrial phosphoproteins and stimulation of testosterone synthesis

Upon stimulation of Leydig cells with luteinizing hormone (LH) or dibutyryl-3',5'-cyclic AMP (Bt 2cAMP) at 37°C, two mitochondrial phosphoproteins accumulate with the same stimulant dose response as the increased rate of testosterone synthesis. The proteins pp32 and pp30 have apparent isoelectric points of 6.6 and 6.5 and molecular weights of approximately 32 and 30 kDa respectively, as determined by two-dimensional polyacrylamide gel electrophoresis. These two phosphoproteins are not detected in mouse adipose or liver cells nor in the total testicular cell population, of which Leydig cells constitute a small percentage. However, both proteins are also observed in mouse adrenal cells stimulated by ACTH or Bt 2cAMP. The appearance of pp32 and pp30 is prevented by inhibitors of cytosolic protein translation, indicating that only newly synthesized protein is available as a substrate for phosphorylation. Proteolytic peptide mapping indicates that both of these mouse Leydig and adrenal proteins have structural similarity to pp30 (formerly denoted as i b), the 30 kDa mitochondrial phosphoprotein that we have observed previously in peptide hormone or Bt 2cAMP-stimulated rat adrenal cortex (Pon, L.A, Hartigan, J.A. and Orme-Johnson, N.R. (1986) J. Biol. Chem. 261, 13309–13316; Alberta, J.A., Epstein, L.F., Pon, L.A. and Orme-Johnson, N.R. (1989) J. Biol. Chem. 264, 2368–2372) and rat corpus luteum cells (Pon, L.A. and Orme-Johnson, N.R. (1986) J. Biol. Chem. 261, 6694–6599). Since pp32 is a larger mitochondrial protein of similar primary structure to pp30, it is a potential precursor of this protein. Finally, the detection of the mitochondrial phosphoprotein pp30 in a third steroidogenic tissue type and a third species provides further correlative evidence that the production of pp30 may be an integral part of the subcellular mechanism by which peptide hormones stimulate steroid hormone biosynthesis.

Prolactin Action on Luteal Protein Expression in the Corpus Luteum*

Although it is well established that PRL is essential for luteal cell steroidogenesis and growth in the rat, its exact mechanism of action remains unknown. Whereas PRL stimulates growth and induces the content of specific proteins in several target tissues, its effect on proteins in the corpus luteum has not been examined. To determine whether PRL affects the synthesis of specific luteal protein(s), corpora lutea were obtained from rats hypophysectomized on day 3 of pregnancy and then treated with or without PRL (125 micrograms x 2/day) for 4 days. In this rat model, corpora lutea are exquisitely responsive to PRL and produce high levels of progesterone in response to PRL stimulation. Analysis of cytosolic proteins on one- and two-dimensional gel electrophoresis revealed a dramatic inhibitory effect of PRL on a 37,000 mol wt (MW) protein. This PRL-regulated protein (PRP) resolved into five separate isoelectric variants (pIs 5.5, 5.6, 6.15, 6.6, and 6.85). Since differences in phosphorylation state can alter the isoelectric point of proteins, we examined the possibility that the 37,000 MW PRP is a substrate for phosphorylation. Luteal homogenates were incubated with [32P]ATP in the presence or absence of Ca2+, Ca2+/calmodulin, Ca2+/phospholipid, or cAMP. Phosphorylation of PRP was not affected by PRL treatment or the addition of specific cofactors. The least abundant isoelectric species (pI 6.85) was identified by Western blot analysis as annexin I, a known regulator of phospholipase A2 and prostaglandin synthesis. To determine whether the other four isoelectric species represent variants of the same protein, we developed a polyclonal antibody to the 6.15 isoelectric species which is remarkably regulated by PRL. In the absence of PRL, the 6.15 isoelectric species (37K) is the major protein of the PRP band. Conversely, PRL treatment resulted in the virtual disappearance of this luteal protein. The antibody recognized the 37K alone, indicating that the other 37,000 MW isoelectric species were distinct proteins. To examine the time course of PRL action on the expression of the 37K, luteal cells from day 3 pregnant rats were cultured with different doses of PRL from 6 h to 5 days. Western blot analysis of luteal cellular proteins indicated that PRL caused a decrease in the expression of the 37K within 6 h of treatment. Although it is well known that estradiol together with PRL is required for optimal growth of the rat corpus luteum, estradiol alone had no inhibitory action on the 37K nor did it affect the inhibitory action of PRL.(ABSTRACT TRUNCATED AT 400 WORDS)

Endothelin-1 in luteal tissue

The aim of this study was to immunologically and biologically detect endothelin-1 (ET-1) in rat corpora lutea (CL). Recently, we established a highly sensitive and specific sandwich-enzyme immunoassay (EIA) for ET-1. Using this assay, the presence of ET-1 was investigated in superovulated ovaries, induced with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), and ovaries from pseudopregnant rats, induced by cervical stimulation. A high concentration of immunoreactive endothelin-1 (ir-ET-1) was found in the CL. On reverse phase-high performance liquid chromatography (RP-HPLC) coupled with EIA, ir-ET-1 was exclusively eluted at the same position as synthetic ET-1, indicating that ir-ET-1 is identical to ET-1. The level of ir-ET-1 was significantly ( P < 0.001) higher in the CL 7 days after hCG injection than it was 4 days after hCG injection. On day 7 of pseudopregnancy (PSP), the ir-ET-1 level was also significantly ( P < 0.001) higher than on day 4 of PSP. These results demonstrated that ET-1 is present in a high concentration in the CL, suggesting a new intraovarian peptide which may have a physiological function in the ovary and which may vary in quantity according to the age of the CL.

Heterogeneity among ovarian blood vessels: Endogenous hepatic lipase is concentrated in blood vessels of rat corpora lutea

We used indirect immunofluorescence and immunogold light microscopy to examine the distribution of hepatic lipase, an enzyme involved in lipoprotein metabolism, in ovaries of gonadotropin-treated immature rats. Antibodies utilized were rabbit anti-rat hepatic lipase IgG, anti-rat von Willebrand factor (VWF, an endothelial cell marker), and goat anti-rabbit IgG conjugated to gold particles or rhodamine. Immunoreagents were applied to fresh frozen sections of unfixed ovary or liver (positive control) or were delivered to ovaries by vascular perfusion before fixation in situ and silver-enhancement of sections. Appropriate controls verified that the immunolocalizations were specific. Immunofluorescence implied that luteal but not stromal blood vessels of ovaries were positive for hepatic lipase, whereas luteal and stromal blood vessels bore VWF. The improved morphology gained by perfusing ovaries with antibodies allowed precise localization of the enzyme. Hepatic lipase was concentrated within thin-walled vessels of corpora lutea but not those of stroma in ovaries at the time of peak steroidogenic activity. Quantification of hepatic lipase-labeled vessels in stromal and luteal compartments confirmed our visual impression. Many images suggested that stromal vessels lacking hepatic lipase gained this enzyme upon contact with luteal tissue. Perfusion of ovaries with cationized ferritin labeled all ovarian vessels equally well, ruling out the possibility that the observed distribution of hepatic lipase was artifactual. These findings demonstrate that ovarian blood vessels are heterogeneous for hepatic lipase. Moreover, they imply that luteal tissue, perhaps luteal cells, may influence expression of hepatic lipase binding sites by endothelial cells.

Inhibition and stimulation of rat luteal protein phosphorylation by protein kinase effectors

Estradiol-17ß (E 2) predetermined protein phosphorylation systems have been identified recently in midpregnant rat corpus luteum. Major type protein kinase activities in these systems were explored here using as probes protein kinase inhibitors. Luteal nuclear, mitochondrial, microsomal and cytosolic fractions were obtained from rats hysterectomized and hypophysectomized on day 12 of pregnancy and then treated for 72 h with E 2. In vitro phosphate transfer from [ γ- 32P]ATP was monitored by SDS-PAGE followed by autoradiography. Polymyxin B (PMB), 1–200 μM, a PKC inhibitor, completely blocked, in a dose dependent manner, the Ca 2+ phospholipid (PL) stimulated radiolabeling of nuclear fraction M r 79 000 substrate(s) as expected. Similarly, the calmodulin (CaM) antagonist compound 48 80 , 1–20 μg/ml, inhibited the Ca 2+/CaM-dependent phosphorylation of the microsomal fraction M r 60 000 and M r 56 000 proteins. The Ca 2+ PL-enhanced labeling of mitochondrial fraction M r 76 000 substrate(s) was only partially susceptible to inhibition by PMB or compound 48/80. Studies of microsomal fraction phosphoprotein bands not stimulated by added factors indicated that the radiolabeling of M r 75000 protein(s) was partially blocked by compound 48 80 but not by PMB. Phosphate transfer to M r 41 000 protein(s) was refractory to all inhibitors kinase protein inhibitor (PKI), while the phosphorylation of M r 31 000 protein(s) was refractory to all inhibitors employed here. Surprisingly, regardless of hormonal pretreatment, PMB and compound 48/80 activated in every subcellular fraction the cofactor independent appearance of at least one phosphoprotein band, between M r 87000–99000. This novel observation should be instrumental in understanding the actions of these compounds towards living cells.

Conversion of 5(10)-oestrene-3β,17β-diol to 19-nor-4-ene-3-ketosteroids by luteal cells in vitro: possible involvement of the 3β-hydroxysteroid dehydrogenase/isomerase

We have previously suggested that in porcine granulosa cells, a putative intermediate, 5(10)-oestrene-3,17-dione is involved in 4-oestrene-3,17-dione (19-norandrostenedione; 19-norA) and 4-oestren-17 beta-ol-3-one (19-nortestosterone: 19-norT) formation from C19 aromatizable androgens. In this study, luteal cells prepared from porcine, bovine and rat corpora lutea by centrifugal elutriation were used as a source of 3 beta-hydroxysteroid dehydrogenase/isomerase in order to investigate the role of this enzyme in the biosynthesis of 19-norsteroids. Small porcine luteal cells made mainly 19-norT and large porcine luteal cells 19-norA from 5(10)-oestrene-3 beta,17 beta-diol, the reduced product of the putative intermediate 5(10)-oestrene-3,17-dione. However, neither small nor large cells metabolized androstenedione to 19-norsteroids. Serum and serum plus LH significantly stimulated formation of both 19-norA and 19-norT from 5(10)-oestrene-3 beta,17 beta-diol, compared with controls. Inhibitors of the 3 beta-hydroxysteroid dehydrogenase/isomerase (trilostane and cyanoketone) significantly reduced formation of 19-norT in small porcine luteal cells and 19-norA in large porcine luteal cells, although they were effective at different concentrations in each cell type. In parallel incubations, formation of [4-14C]androstenedione from added [4-14C]dehydroepiandrosterone was also inhibited by cyanoketone in both small and large porcine luteal cells in a dose-dependent manner; however, trilostane (up to 100 mumol/l) did not inhibit androstenedione formation in large porcine luteal cells. In addition, the decrease in progesterone synthesis induced by trilostane and cyanoketone (100 mumol/l each) was accompanied by a parallel accumulation of pregnenolone in both cell types. These results suggest that 3 beta-hydroxysteroid dehydrogenase/isomerase, or a closely related enzyme, present in small and large porcine luteal cells can convert added 5(10)-3 beta-hydroxysteroids into 19-nor-4(5)-3-ketosteroids in vitro. In the porcine ovarian follicle, therefore, formation of 19-norA from androstenedione can be envisaged as a two-step enzymatic process: 19-demethylation of androstenedione to produce the putative intermediate 5(10)-oestrene-3,17-dione, and subsequent isomerization to 19-norA. In contrast to granulosa cells, porcine luteal cells synthesized 19-norA or 19-norT only when provided with the appropriate substrate. Unfractionated rat luteal cells also metabolized 5(10)-oestrene-3 beta,17 beta-diol to a mixture of 19-norA and 19-norT; conversion was inhibited by trilostane. In addition, small bovine luteal cells synthesized mainly 19-norT and formation was also inhibited by trilostane and cyanoketone.(ABSTRACT TRUNCATED AT 400 WORDS)

In Vivo Generation of Hydrogen Peroxide in the Rat Corpus Luteum during Luteolysis*

The hypothesis that hydrogen peroxide generation occurs in the corpora lutea of superovulated rats during luteolysis was tested using a peroxide-dependent inhibitor of catalase, 3-amino-1,2,4-triazole (AT). Luteal regression was induced during midpseudopregnancy by injection of 500 micrograms prostaglandin F2 alpha (PGF2 alpha) 1 h before administration of AT (0.1 g/kg, ip) and was confirmed by progesterone analysis of peripheral blood serum. Within groups of both PGF2 alpha-treated and untreated control rats, other rats also received ethanol (0.2 g/kg, ip), which prevents hydrogen peroxide-mediated inhibition of catalase by AT. Diluted homogenates of ovaries removed 1 h after AT administration were assayed for catalase activity by measuring the decrease in absorbance at 240 nm for 30 sec after the addition of hydrogen peroxide (10 mM). Ethanol-sensitive catalase inhibition by AT was significantly higher (47.9 +/- 3.38%) in samples from PGF2 alpha-treated groups than in controls (23.1 +/- 4.82%; P less than 0.01; n = 9). Similar increases in catalase inhibition by AT were found in luteal tissue of rats treated with PGF2 alpha 24 h earlier and in rats in which luteolysis was allowed to occur spontaneously in late pseudopregnancy. Hemoglobin an AT assays revealed that the changes in catalase activity were not the result of altered blood contamination or AT concentration in the luteal homogenates. Since catalase inhibition by AT is only seen in the presence of hydrogen peroxide, these results support the conclusion that an early and sustained component of corpus luteum regression is the generation of hydrogen peroxide in luteal tissue.