Rat Corpus Luteum Research Articles

Isozymes of cAMP-dependent protein kinase present in the rat corpus luteum.

Regulatory (R) subunits and their association with catalytic subunits to form cAMP-dependent protein kinase holoenzymes were investigated in corpora lutea of pregnant rats. Following separation by DEAE-cellulose chromatography, R subunits were identified by labeling with 8-N3[32P]cAMP and autophosphorylation on one and two-dimensional gel electrophoresis and by reactivity with antisera. DEAE-cellulose elution of R subunits with catalytic subunits as holoenzymes or without catalytic subunits was determined by sedimentation characteristics on sucrose density gradient centrifugation and by cAMP-stimulated kinase activation characteristics on Eadie-Scatchard analysis. We identified the presence of a type I holoenzyme containing RI alpha (Mr 47,000) subunits, a prominent type II holoenzyme containing RII beta (Mr 52,000) subunits, and a second more acidic type II holoenzyme peak containing both RII beta and RII alpha (Mr 54,000) subunits. However, the majority of total R subunit activity was associated with a catalytic subunit-free peak of RI alpha protein which on elution from DEAE-cellulose was associated with cAMP. This report establishes the more basic elution position from DEAE-cellulose of the prominent rat luteal RII beta holoenzyme in very close proximity to free RI alpha and presents one of the few reports of a normal tissue containing a large percentage of catalytic subunit-free RI alpha.

Quantitative changes in steroidogenic organelles in the corpus luteum of the pregnant rat in relation to progestin secretion on day 16 and in the morning and afternoon of day 22

The present study was carried out to determine whether the major steroidogenic organelles of luteal cells quantitatively reflect variations in ovarian steroid secretion rates during pregnancy in the rat. Assessments were made on day 16 and in the morning (AM) and afternoon (PM) of day 22 (term is day 23). Ovarian steroidogenesis differs both quantitatively and qualitatively between these stages of pregnancy, so together they provide an ideal physiological model to study structural-functional relationships in the ovary. Corpora lutea of five rats were examined at each stage after progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OHP) secretion rates had been determined by a venous outflow technique. Total progestin secretion (progesterone plus 20 alpha-OHP) fell from 32.5 +/- 5.2 to 9.8 +/- 1.2 micrograms/hr per ovary (mean +/- SEM) between day 16 and day 22 AM but then increased to 22.6 +/- 1.4 micrograms/hr per ovary by day 22 PM. The total volume of luteal cell cytoplasm was slightly greater at day 22 AM and PM than at day 16. Similarly, the volumes of smooth endoplasmic reticulum (SER), lipid droplets, and membrane-bound granules all increased, but the volume of mitochondria decreased slightly. In contrast, the surface areas of SER and the inner and outer mitochondrial membranes did not change between day 16 and day 22 AM but then increased substantially by day 22 PM. Therefore, steroid secretion rates per unit area of steroidogenic membrane showed no consistent pattern over the stages examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in Rat Luteal Ultrastructure and P45Oscc mRNA and Protein Content after in Vivo Treatment with a Gonadotropin-Releasing Hormone Agonist1

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.

Is the antiprogestin RU486 luteolytic in rats?

Subcutaneous injections of RU486 on Day 1, Day 4 or Days 3 and 4 of pregnancy in rats induced abortion of the embryo and transient vaginal cornification. Nevertheless, most corpora lutea appeared to be functional at autopsy on Day 12. The same treatments in pseudopregnant rats also produced transient vaginal cornification, but did not terminate pseudopregnancy. This suggests that transient antagonism of progesterone by RU486 does not terminate function of the corpus luteum in rats and that positive feedback by progesterone is not essential for continuing luteal function in rats.

Calcium-Calmodulin and Calcium-Phospholipid Dependent Phosphorylation of Membranous Fraction Proteins Related to the Tropic Regulation by Estradiol in the Corpus Luteum*

Estradiol assumes a major role in the regulation of growth, vascularization, and progesterone synthesis in the midpregnant rat corpus luteum. To explore whether molecular events triggered by estradiol could be mediated, at least in part, by protein phosphorylation, we investigated whether estradiol treatment in vivo affects endogenous luteal protein phosphorylation systems detectable in vitro. Luteal nuclear, mitochondrial, and microsomal fractions were obtained by differential centrifugation from rats hypophysectomized and hysterectomized on day 12 of pregnancy and treated with or without estradiol for 72 h. Using [gamma-32P]ATP as phosphate donor, proteins were phosphorylated in the presence or absence of either calcium (Ca), Ca plus calmodulin, or Ca plus phospholipid. Phosphoproteins were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by autoradiography. The Coomassie blue stained proteins and phosphoprotein profiles were markedly different in the various fractions. Estradiol treatment in vivo caused an increase in the basal endogenous phosphorylation of several proteins in vitro. It also substantially enhanced the protein kinase C (PKC) and Ca-calmodulin kinase-dependent phosphorylation of selected proteins in subcellular fractions. The Ca-calmodulin kinase catalyzed phosphorylation of microsomal 56 and 60 kilodalton (kDa) proteins was remarkably increased by estradiol. Proteins (56 and 60 kDa) were also phosphorylated when Ca-calmodulin was added to the nuclear fraction, however, this phosphorylation did not appear to be affected by estradiol treatment. A major PKC substrate in the nuclear fraction was an 80 kDa protein whose phosphorylation was increased remarkably by estradiol treatment. In the mitochondrial fraction the most striking effect of estradiol was a marked increase in PKC-mediated phosphate transfer into a 76 kDa substrate. To determine whether estradiol action on protein phosphorylation was related to its tropic effect in the corpus luteum, the hormone was administered to day 10 hypophysectomized and hysterectomized pregnant rats. In this rat model, where estradiol has no stimulatory effect on either luteal steroidogenesis or growth, neither endogenous nor kinase-mediated phosphorylation was affected by this steroid. In summary, the present investigation has revealed that in vivo treatment with estradiol affects the PKC and the Ca-calmodulin dependent in vitro phosphorylation of selected proteins localized in different subcellular compartments and further suggests that phosphorylation systems are potential control points for estradiol regulation of rat corpus luteum function.

S-10-1 - Physiological activity and the receptor of basic fibroblast growth factor in the rat corpus luteum.

S-10-1 - Physiological activity and the receptor of basic fibroblast growth factor in the rat corpus luteum.

Effect of Tokishakuyakusan on Corpus Luteum Endothelin

The presence of endothelin-1 (ET) and effect of Tokishakuyakusan (TS) on ET in rat corpora lutea (CL) was investigated in superovulated ovaries, induced with pregnant mare's serum gonadotropin and human chorionic gonadotropin. A high concentration of ET was found in the CL. The level of ET was significantly lower in the CL from TS-treated rats than that in TS-untreated rats (402.68 versus 575.60 pg/g wet weight, p less than 0.05). In contrast, the ET levels in plasma were by far lower than those in CL. These data indicate an inhibitory effect of TS on ET, an intraovarian peptide, production or accumulation in the CL.

Blended Effects of Herbal Components of Tokishakuyakusan on Somatomedin C/Insulin-like Growth Factor 1 Level in Rat Corpus Luteum

The effect of herbal components of Tokishakuyakusan on somatomedin C/insulin-like growth factor I (IGF-1) level in medium from rat corpora lutea incubated in vitro was examined. Hoelen + poeny root + Japanese angelica root, hoelen + peony root, hoelen + Japanese angelica root or peony root + Japanese angelica root decreased the IGF-1 level. The data suggest that constituent herbal components of Tokishakuyakusan regulate the IGF-1 level by rat corpora lutea.

Vascular Endothelial Growth Factor is Expressed in Rat Corpus Luteum

In the course of the development of the ovarian follicle and differentiation of granulosa cells into corpus luteum (CL), extensive changes in the microvasculature of these structures take place. This suggests the local release of angiogenic factors. In the present work we examined whether a newly described secreted vascular endothelial growth factor (VEGF) is expressed in normal rat ovary by in situ hybridization. Our results demonstrate the expression of VEGF in the CL but not in mural granulosa cells, suggesting a temporal relation between VEGF expression and growth of capillary vessels. The hybridization pattern in the CL was consistent with localization of VEGF message to luteal cells. Expression of VEGF was detected also in cumulus oophorus cells. These findings suggest that VEGF is involved in the process of CL angiogenesis.

Effects of human chorionic gonadotropin (hCG) and prolactin (PRL) on 3β-hydroxy-5-ene-steroid dehydrogenase/ Δ5- Δ4 isomerase (3β-HSD) expression and activity in the rat ovary

Using a recently cloned rat ovary 3β-HSD cDNA and antibodies raised against purified human placental 3β-HSD, we have studied the effects of treatment with human chorionic gonadotropin (hCG) and hyperprolactinemia achieved by pituitary implants, alone or in combination, on the expression and activity of ovarian 3 β-hydroxysteroid dehydrogenase/ Δ 5- Δ 4 isomerase (3β-HSD) in intact adult rats. 32P-and 35S-labeled cDNA probes were used to evaluate the effects of treatments on 3β-HSD mRNA levels by dot blot and in situ hybridization, respectively, while enzymatic activity was measured by the conversion of [ 14C]dehydroepiandrosterone into [ 14C]androstenedione. The present data show that hCG exerts a marked trophic effect on rat corpora lutea with an increase in total ovarian 3β-HSD mRNA levels, 3β-HSD protein content as well as enzymatic activity, resulting in an increase in serum progesterone levels. Prolactin-secreting pituitary implants alone, on the other hand, while exerting small effects on 3β-HSD expression and activity, led to a marked potentiation of the stimulatory effect of hCG on all parameters. The present data show that hCG and PRL act synergistically to stimulate ovarian progesterone secretion via an increase in 3β-HSD mRNA levels, protein content and enzymatic activity.

Luteal steroidogenesis and regression in the rat: Effects of human chorionic gonadotrophin and phospholipase A2 on cells and plasma membranes

Progesterone production in vitro was measured in response to human chorionic gonadotrophin and phospholipase A2 using dispersed luteal cells from control and prostaglandin F2 alpha (PGF2 alpha)-treated rats. Highly purified luteal plasma membrane suspensions were prepared by partition distribution in dextranpolyethylene glycol, and membrane fluidity was measured by fluorescence polarization using the probe, trans-parinaric acid. Fluorescence polarization was highly dependent upon calcium, and was markedly affected by PGF2 alpha treatment. No support was found for the theory that bulk membrane fluidity has a role in receptor or enzyme hindrance in the induction of luteal cell regression. However, membrane phase transitions induced by calcium interaction with anionic phospholipids and free radical damage may be involved in regression of the rat corpus luteum.

Mechanism for Control of HydroxymethylglutaryL Coenzyme A Reductase and Cytochrome P-450 Side Chain Cleavage Message and Enzyme in the Corpus Luteum*

The present study was undertaken to investigate the mechanism for control of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase and cytochrome P-450 side chain cleavage (P-450scc), enzymes involved in the synthesis and processing of cholesterol in the corpus luteum of pregnant rats. The role of sterol and estradiol were investigated by administering 4-aminopyrazolo-[3,4d]pyrimidine (4-APP), aminoglutethimide and/or estradiol to day 12 hypophysectomized-hysterectomized pregnant rats. Estradiol treatment markedly increased mRNA levels of HMG-CoA reductase in the corpus luteum. This stimulatory effect of estradiol was specific for the reductase since the mRNA for luteal P-450scc did not increase following estradiol treatment. To determine whether estradiol's action on HMG-CoA reductase mRNA was mediated by changes in cellular free cholesterol, the separate and combined action of estradiol and either 4-APP or aminoglutethimide on both sterol content and HMG-CoA reductase expression was examined. The estradiol induced rise in HMG-CoA reductase was not accompanied by a decrease in luteal cholesterol. 4-APP treatment depleted cholesterol in the corpus luteum and induced a greater increase in HMG-CoA reductase mRNA than estradiol. Combined treatment with estradiol and 4-APP resulted in a synergistic increase in HMG-CoA reductase mRNA despite the fact that estradiol did not further reduce the 4-APP induced decrease in luteal sterol content. This indicates that luteal cells possess an estradiol-mediated mechanism for up-regulation of HMG-CoA reductase gene expression which is distinct from the cholesterol negative feedback regulation. However, estradiol stimulation of HMG-CoA reductase was totally inhibited when sterol content was elevated by aminoglutethimide, suggesting that estradiol stimulation of HMG-CoA reductase expression is not sufficient to overcome the negative regulatory effect of cholesterol. In contrast to HMG-CoA reductase mRNA which appears to be controlled by sterol and estradiol, P-450scc mRNA levels remained constant in corpora lutea despite wide changes in sterol and steroid content induced by either 4-APP, aminoglutethimide or estradiol treatment. However, interestingly, aminoglutethimide increased substantially the content of P-450scc protein. This increase in P-450scc enzyme was not due to a greater amount of translatable message since similar levels of immunoprecipitable 35S-P-450scc were translated in vitro from luteal mRNA of rats treated with or without aminoglutethimide. In summary, results of this investigation have revealed that the expression of HMG-CoA reductase and cytochrome P-450scc are controlled by totally different mechanisms in the rat corpus luteum.(ABSTRACT TRUNCATED AT 400 WORDS)

In Vivo Levels of Prostaglandin F2α, E2 and Prostacyclin in the Corpus Luteum of Pregnant and Pseudopregnant Rats1

Prostaglandin F2 alpha (PGF2 alpha) is a well-known luteolytic factor in the rat corpus luteum. To investigate a possible luteal origin of PGF2 alpha, measurements of this prostaglandin were performed in different luteal tissues in vivo. Prostaglandin E2 (PGE2) and the stable metabolite of prostacyclin, 6-keto-PGF1 alpha, were assayed simultaneously. Corpora lutea of different ages from 57 pregnant and pseudopregnant rats (mated with sterile males) were rapidly excised, dissected in 0 degree C indomethacin solution, homogenized, and extracted for prostaglandins with solid-phase extraction cartridges. Prostaglandins were determined by radioimmunoassay. Plasma levels of progesterone and 20 alpha-dihydroprogesterone were also monitored. In the adult pseudopregnant rat model, luteolysis occurs at Day 13 +/- 1, and maximal levels of all three prostaglandins were detected on Day 13 of pseudopregnancy: 0.40 +/- 0.02, 2.6 +/- 0.29, and 1.76 +/- 0.24 pmol/mg protein (mean +/- SEM, n=7) for PGF2 alpha, PGE2, and 6-keto-PGF1 alpha respectively. In pregnant rats, on the corresponding day, levels were considerably lower: 0.15 +/- 0.02, 0.90 +/- 0.13, and 0.50 +/- 0.06 pmol/mg protein (mean +/- SEM, n=9, p less than 0.0001), respectively. Luteal levels in pregnant rats showed a continuous decline on Days 13 and 19 for all prostaglandins measured, whereas in pseudopregnant rats an increment of PGF2 alpha was noted between Days 7 and 13 and remained high on Day 19. PGE2 closely followed levels of PGF2 alpha, but at a 5- to 10-fold higher level. The coefficient of correlation between PGF2 alpha and PGE2 in the luteal compartment of both models was 0.87 (p less than 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and Characterization of an Abundant Phosphoprotein Specific to the Large Luteal Cell*

An abundant protein with a relative mol wt of 32K present specifically in the large cells of the pregnant rat corpus luteum has been identified. Separation of large and small luteal cells by elutriation, followed by protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), have revealed that the 32K protein was present as a major protein in the large luteal cells but was practically absent in the small cell population. This protein appears to be highly tissue and cell specific and resolves into three protein species by two-dimensional SDS-PAGE with the major protein having an isoelectric point (pI) greater than or equal to 8.5. It was not detected in preantral follicles or placentas of the same pregnant rats, or in any other tissue examined. After subcellular fractionation, the 32K protein(s) was found in the particulate fraction and was localized principally in the microsomal compartment. Autoradiographic analysis of 35S-amino acid-labeled tissue demonstrated that the 32K protein(s) is synthesized in the corpus luteum. When particulate fractions from small and large cells were incubated in the presence of [gamma-32P]ATP followed by SDS-PAGE, phosphorylation of the 32K protein was apparent. Phosphorylation of this protein was not enhanced by the addition of cofactors for cAMP, Ca2(+)-calmodulin- or Ca2(+)-phospholipid-dependent kinases. Experimental inhibition of steroidogenesis with amino-glutethimide caused a remarkable reduction in the luteal content of this 32K protein whereas estradiol and human CG treatment increased its content. In summary, we have discovered and partially characterized a unique 32K protein(s) which is expressed and phosphorylated only in large luteal cells of the corpus luteum. This protein(s), which is regulated by estradiol formed locally, may serve as a powerful marker for both the large luteal cell and estrogen action in the corpus luteum.

Morphologically and Functionally Distinct Subpopulations of Steroidogenic Cells in Corpora Lutea During Pregnancy in Rats.

On days 5, 10, 15 and 20 of pregnancy, rat corpora lutea (CL) were dissected and dissociated into single cell suspensions by enzyme treatments. The suspended luteal cells were allowed to sediment in a BSA gradient at 4 degrees C for 3.5 hours. Five fractions were collected from the top (Fraction (Fr.) 1) to the bottom (Fr. 5) of the gradient. Cells were incubated in serum-free DME-F12 for 20 hours with or without hCG (100 ng/ml) to test them functionally, and the accumulation of progesterone and testosterone was determined by radioimmunoassay. To assess 3,3-hydroxysteroid dehydrogenase (HSD) activity, a histochemical suspension-staining procedure was used. Cells were examined by light microscopy, and the percentage of cells containing dark blue formazan deposits and their diameters were determined in at least 40 microscopic fields. The number of cells staining for 3 beta-HSD did not vary by day 15 but decreased from 141.6 +/- 16.5 X 10(3) cells/CL on day 15 to 113.8 +/- 13.2 X 10(3) cells/CL on day 20 of pregnancy. However, 3 beta-HSD-positive cells maintained the same levels of progesterone secretion until the advent of luteolysis, then they increased in size progressively throughout pregnancy. In BSA gradients, the relatively larger 3 beta-HSD-positive cells migrated faster than the smaller 3 beta-HSD-positive cells on each day of pregnancy. The diameters of 3 beta-HSD-positive cells differed significantly in Frs. 2, 3, 4 and 5 on days 15 and 20 of pregnancy. On day 15 of pregnancy, less progesterone accumulated in wells containing 3 beta-HSD-positive cells from Fr. 2 (mean diameter; 24.96 microns) than from Fr. 3, Fr. 4 and Fr. 5 (mean diameters; 27.20, 30.79 and 31.28 microns, respectively) but the Fr. 2 cells responded more to hCG stimulation. Fr. 2 also showed a higher ratio of testosterone accumulation to progesterone accumulation than the other fractions. The response to hCG stimulation of cells in Fr. 2 tended to be higher than that in Fr. 3 on day 20 of pregnancy. These data suggest that the steroidogenic rat luteal cells are comprised of morphologically and functionally different cell types after day 15 of pregnancy. No stimulating nor inhibiting effects were observed in co-incubation of cells from Fr. 2 with cells from Fr. 3 or Fr. 4 on days 15 and 20 of pregnancy.

Estradiol Regulation of Sterol Carrier Protein-2 Independent of Cytochrome P450 Side-Chain Cleavage Expression in the Rat Corpus Luteum*

A major action of estradiol in the corpus luteum of the pregnant rat is to increase the supply of cholesterol substrate for progesterone production by stimulating both cholesterol synthesis and uptake. To determine whether this steroid also affects cholesterol metabolism and transport, estradiol's action on the expression of cytochrome P450 side-chain cleavage enzyme (P450scc) and the cholesterol transport protein, sterol carrier protein-2 (SCP2), was examined. Mitochondria isolated from corpora lutea of estradiol-treated rats secreted significantly more progestagen than mitochondria of control corpora lutea. Several findings indicate that estradiol enhances cholesterol transport and availability to the P450scc rather than affects the expression of this enzyme: 1) the difference in mitochondrial progestagen synthesis induced by estradiol was obliterated by the presence of 25-hydroxycholesterol; 2) immunoblotting of P450scc indicated no stimulatory effect of estradiol on the amount of enzyme; and 3) levels of P450scc mRNA were not increased by estradiol. Whereas estradiol had no stimulatory effect on P450scc it caused a mark (3-fold) increase in the mitochondrial content of SCP2. Thus, the increase in luteal progestagen synthesis stimulated by estradiol appears to be associated with an increase in mitochondrial SCP2 and is independent of luteal P450 content or message.

Aromatase Cytochrome P450 and Cholesterol Side-Chain Cleavage Cytochrome P450 in Corpora Lutea of Pregnant Rats: Diverse Regulation by Peptide and Steroid Hormones*

In previous studies we have shown that aromatase cytochrome P450 (P450arom) mRNA and protein increase markedly in luteal tissue between days 10-19 of gestation, whereas cholesterol side-chain cleavage cytochrome P450 (P450scc) appears to be constitutively maintained regardless of hormonal changes occurring during pregnancy. To identify pituitary and placental hormones that regulate these two P450 enzymes in the rat corpus luteum, serum LH activity and pituitary PRL release were selectively inhibited by administration of LH antiserum (LH-Ab) or CB-154, respectively. Placental hormones were removed by hysterectomy. Hormonal activities were replaced by the administration of hCG, PRL, testosterone (T), or estradiol (E), given individually or in combination. Induction of aromatase mRNA transcripts (3.3, 2.6, and 1.9 kilobases) and protein (54,000 mol wt) between days 10-15 of gestation was blocked by either surgical hysterectomy or LH-Ab treatment. Hysterectomy on day 10 combined with CB-154 abolished not only aromatase mRNA, but also markedly reduced P450scc mRNA (2.0 kilobases) by day 12. Induction of aromatase was partially restored in the day 10-15 hysterectomized rats by treatment with PRL plus E (most effective), PRL plus T, or PRL alone, but not by either T or E alone. Similar results were observed 2 days after hysterectomy (day 12), except that hysterectomy alone caused a transient 3.5-fold increase in P450arom mRNA and protein, most likely due to a transient release of pituitary LH. Aromatase mRNA and protein were also increased in intact pregnant rats treated with hCG between days 10-12. However, no effect of hCG was observed before (days 8-10) or after (days 13-19) midgestation. Likewise, LH-Ab had no effect if given after day 13. Despite hormone-specific regulation of the content of aromatase protein, E biosynthesis in vitro was not strictly related to aromatase enzyme content. We conclude that aromatase mRNA and protein are maintained by PRL at a low level of expression in the first half of pregnancy, can be modulated by LH at midgestation, and are subsequently induced to high levels in the second half of gestation by placental factors (rat placental lactogen-1 and T) and the conversion of T to E in the corpus luteum. P450scc appears to be constitutively maintained. Thus, two P450 genes known to be regulated by LH/cAMP in the rat follicle are controlled by diverse peptide and steroid signal transduction mechanisms in the corpus luteum.

-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.

The effects of prolactin and prostaglandin F2 alpha on plasma membrane changes during luteolysis in the rat.

The ability of PRL to modify prostaglandin F2 alpha (PGF2 alpha)-induced membrane changes during functional corpus luteum regression was examined in the pseudopregnant rat. Fluorescence polarization studies conducted 24 h after sc injection of PGF2 alpha revealed a marked and significant increase in the polarization parameter, which is suggestive of reduced plasma membrane fluidity. At the same time, there was a decrease in hCG binding and a significant increase in apparent phospholipase-A2 activity during incubation of dispersed rat luteal cells. Each of these changes was attenuated when the animals were pretreated with PRL 30 min before PGF2 alpha. The decrease in plasma progesterone caused by PGF2 alpha treatment was also inhibited by PRL. PGF2 alpha also stimulated a significant polarization increase in dispersed cells prepared from ovaries removed 1 h after injection of this luteolytic agent, although this effect could not be demonstrated in plasma membrane samples. These results indicate that PRL and PGF2 alpha affect the same membrane pathway in the rat corpus luteum and that this pathway appears to be closely coupled to luteal cell function.

Steroid concentrations in rat corpora lutea isolated during the oestrous cycle and pseudopregnancy: effect of induction of ovulation at dioestrus.

Corpora lutea could be identified under the dissection microscope up to 7 days after formation. They were isolated during the oestrous cycle and pseudopregnancy and the progesterone and 20 alpha-OH-progesterone contents were compared with serum values of these steroids. The pattern of progesterone in serum resembled that found in the corpora lutea. However, the pattern of 20 alpha-OH-progesterone concentrations in serum and corpora lutea were different. While 20 alpha-OH-progesterone concentrations in the corpora lutea showed large variations during the cycle, changes in serum concentrations of 20 alpha-OH-progesterone were relatively small. Measurement of hormone concentrations in isolated corpora lutea is therefore a sensitive method for studying corpus luteum activity. To study whether corpora lutea derived after ovulation of immature follicles showed deficient luteal activity, rats at dioestrus (2 days before pro-oestrus) were induced to ovulate by the injection of 10 IU human chorionic gonadotrophin (hCG) and subsequent luteal activity was studied by measuring hormone concentrations in the corpora lutea on day 5 of pseudopregnancy. Concentrations of progesterone, but not of 20 alpha-OH-progesterone, in corpora lutea derived from follicles induced to ovulate at dioestrus-day 1 were significantly lower than those in corpora lutea derived from follicles induced to ovulate at proestrus. This difference was observed not only when pseudopregnancy was induced by cervical stimulation but also when it was induced by implantation of a pituitary gland under the kidney capsule. However, in the latter case, corpora lutea already present on the day of hCG injection also became activated.(ABSTRACT TRUNCATED AT 250 WORDS)