Corpora Lutea Of Day Research Articles

Extended uterine receptivity for blastocyst implantation and full-term fetal development in mice with vitrified-warmed ovarian tissue autotransplantation.

Our previous study demonstrated that vitrified-warmed ovarian tissue autotransplantation (VOAT) into estrus cycle-ceased ovariectomized mice restored fertility to achieve full-term fetal development for transferred embryos, while less steroidogenesis in the corpus luteum was observed in VOAT mice. It has been reported that the window of uterine receptivity for blastocyst implantation is extended at lower estrogen levels. Therefore, we hypothesized that duration of the window in VOAT mice could be extended. Blastocysts were transferred into VOAT mice on day 5 of pseudopregnancy. Immunohistochemical analysis was performed to examine the potential in VOAT ovarian tissues. The rate of live birth pups from embryos transferred on day 5 of pseudopregnant VOAT mice was not different from that of embryos transferred on day 4 of pseudopregnancy in VOAT mice, while embryo transfer on day 5 into intact mice showed no pregnancy. Immunohistochemical analysis of the corpus luteum of day 8 pseudopregnant VOAT mice with uteri having decidualization induced on day 5 showed less steroidogenesis and blood vessel formation as compared to intact mice. Uterine receptivity was extended in VOAT mice. Less steroidogenesis and blood vessel formation in the transferred ovarian tissues may be associated with the extended uterine receptivity.

Maximal expression of suppressors of cytokine signaling in the rat ovary occurs in late pregnancy

Maintenance of the rodent corpus luteum (CL) during pregnancy requires prolactin receptor (PRLR) signal transduction via STAT5. At the end of pregnancy, prostaglandin F2alpha (PGF(2alpha)) induces luteal regression through many mechanisms, including downregulation of PRLR signaling. We have previously shown that a PGF(2alpha) analog upregulates suppressors of cytokine signaling (SOCS) proteins in the CL of day 19 pregnant rats leading to reduced STAT5 signaling. Here, we examined endogenous SOCS expression and STAT5 signaling in the rat ovary during normal pregnancy and luteolysis. The mRNA expression of Socs1, Socs2, and Socs3 and related cytokine-inducible SH2-containing protein (Cish) was low in early pregnancy (day 7), but significantly increased at mid-pregnancy (days 10 and 13) associated with increased endogenous tyrosine phosphorylation (TyrP) of STAT5. In support of the notion that these changes are due to increasing placental lactogen levels at this time, we found that treatment with exogenous PRL on day 7 increased TyrP of STAT5 and induced SOCS mRNA expression, except Socs3. After mid-pregnancy, further significant increases in Socs3 and Cish mRNA expression were observed. Such changes in mRNA expression correlated with protein levels, with protein levels of both SOCS3 and CISH being maximal in late pregnancy (days 19-21). In addition, a significant reduction in TyrP of STAT5 was first observed on day 20, with a further substantial decrease on day 21. Therefore, these results are consistent with the hypothesis that increased SOCS expression in the rat ovary during late pregnancy reduces STAT5 signaling, which may be important in PGF(2alpha)-induced luteolysis.

Exogenous estradiol enhances apoptosis in regressing post-partum rat corpora lutea possibly mediated by prolactin

BackgroundIn pregnant rats, structural luteal regression takes place after parturition and is associated with cell death by apoptosis. We have recently shown that the hormonal environment is responsible for the fate of the corpora lutea (CL). Changing the levels of circulating hormones in post-partum rats, either by injecting androgen, progesterone, or by allowing dams to suckle, was coupled with a delay in the onset of apoptosis in the CL. The objectives of the present investigation were: i) to examine the effect of exogenous estradiol on apoptosis of the rat CL during post-partum luteal regression; and ii) to evaluate the post-partum luteal expression of the estrogen receptor (ER) genes.MethodsIn a first experiment, rats after parturition were separated from their pups and injected daily with vehicle or estradiol benzoate for 4 days. On day 4 post-partum, animals were sacrificed, blood samples were taken to determine serum concentrations of hormones, and the ovaries were isolated to study apoptosis in situ. In a second experiment, non-lactating rats after parturition received vehicle, estradiol benzoate or estradiol benzoate plus bromoergocryptine for 4 days, and their CL were isolated and used to study apoptosis ex vivo. In a third experiment, we obtained CL from rats on day 15 of pregnancy and from non-lactating rats on day 4 post-partum, and studied the expression of the messenger RNAs (mRNAs) encoding the ERalpha and ERbeta genes.ResultsExogenous administration of estradiol benzoate induced an increase in the number of apoptotic cells within the CL on day 4 post-partum when compared with animals receiving vehicle alone. Animals treated with the estrogen had higher serum prolactin and progesterone concentrations, with no changes in serum androstenedione. Administration of bromoergocryptine blocked the increase in serum prolactin and progesterone concentrations, and DNA fragmentation induced by the estrogen treatment. ERalpha and ERbeta mRNAs were expressed in CL of day 4 post-partum animals at levels similar to those found in CL of day 15 pregnant animals.ConclusionWe have established that estradiol accelerates apoptosis in the CL during post-partum luteal regression through a mechanism that possibly involves the secretion of pituitary prolactin. We have also shown that the post-partum rat CL express ERalpha and ERbeta mRNAs suggesting that they can be targeted by estrogen.

Clinostat Rotation Induces Apoptosis in Luteal Cells of the Pregnant Rat1

Recent studies have shown that microgravity induces changes at the cellular level, including apoptosis. However, it is unknown whether microgravity affects luteal cell function. This study was performed to assess whether microgravity conditions generated by clinostat rotation induce apoptosis and affect steroidogenesis by luteal cells. Luteal cells isolated from the corpora lutea of Day 8 pregnant rats were placed in equal numbers in slide flasks (chamber slides). One slide flask was placed in the clinostat and the other served as a stationary control. At 48 h in the clinostat, whereas the levels of progesterone and total cellular protein decreased, the number of shrunken cells increased. To determine whether apoptosis occurred in shrunken cells, Comet and TUNEL assays were performed. At 48 h, the percentage of apoptotic cells in the clinostat increased compared with that in the control. To investigate how the microgravity conditions induce apoptosis, the active mitochondria in luteal cells were detected with JC-1 dye. Cells in the control consisted of many active mitochondria, which were evenly distributed throughout the cell. In contrast, cells in the clinostat displayed fewer active mitochondria, which were distributed either to the outer edge of the cell or around the nucleus. These results suggest that mitochondrial dysfunction induced by clinostat rotation could lead to apoptosis in luteal cells and suppression of progesterone production.

Control of ovarian follicular growth and maturation by the corpus luteum and the placenta during pregnancy in sheep

Ovarian follicular growth and maturation and its control throughout pregnancy have not been described fully in sheep. Experiment 1 characterized the size and maturation (steroid production in vitro and aromatase activity) of ovarian follicles obtained at days 20, 50, 80 and 110 of pregnancy compared with those obtained at day 12 of the oestrous cycle. There was no difference in the number of small follicles (< 3 mm in diameter) between cyclic and pregnant ewes, regardless of the stage of pregnancy. There was a marked reduction (P < 0.01) in the number of medium follicles (3-5 mm) starting at day 80 of pregnancy. Large follicles (> 5 mm) were not detected at day 110 of pregnancy. In vitro testosterone output by follicles was constant throughout pregnancy. Oestradiol output remained steady until day 80, but decreased markedly at day 110 of pregnancy. This decrease was associated with a reduction in aromatase activity in follicles obtained at this stage. Experiment 2 examined the effect of administration of high concentrations of progesterone between day 100 and day 120 after mating on resumption of follicular growth in ewes that underwent Caesarean section at day 99 of pregnancy. In ewes that underwent Caesarean section, progesterone supplementation was successful in mimicking the profile found in pregnant ewes, but did not prevent re-initiation of follicular growth, as demonstrated by the presence of large follicles (> 5 mm) at day 120 after mating. Experiment 3 examined the effects of PGF(2alpha)-induced regression of the corpus luteum of day 100 of pregnancy on resumption of follicular growth. High concentrations of PGF(2alpha) (0.28 mg kg(-1) body weight) administrated at day 100 of pregnancy were required to initiate regression of the corpus luteum. At day 120 after mating, the mean (+/- SEM) diameter of the largest follicle in PGF(2alpha)-treated ewes (3.40 +/- 0.47 mm) was significantly greater (P < 0.05) than that in control pregnant ewes (2.52 +/- 0.34 mm). Experiment 4 examined the effect of removal of the fetus and of the corpus luteum at day 100 of pregnancy on resumption of ovulation. Removal of the corpus luteum by PGF(2alpha) treatment at the time of removal of the fetus resulted in earlier occurrence of short luteal phases (27.8 versus 40.6 days, PGF(2alpha)-treated versus non-treated) but did not alter the timing of the first normal luteal phases (41 days). In conclusion, the results from these experiments indicate that placental compounds play a major role in inhibiting follicular growth and maturation during late pregnancy in sheep.

Control of ovarian follicular growth and maturation by the corpus luteum and the placenta during pregnancy in sheep

Ovarian follicular growth and maturation and its control throughout pregnancy have not been described fully in sheep. Experiment 1 characterized the size and maturation (steroid production in vitro and aromatase activity) of ovarian follicles obtained at days 20, 50, 80 and 110 of pregnancy compared with those obtained at day 12 of the oestrous cycle. There was no difference in the number of small follicles (< 3 mm in diameter) between cyclic and pregnant ewes, regardless of the stage of pregnancy. There was a marked reduction (P < 0.01) in the number of medium follicles (3-5 mm) starting at day 80 of pregnancy. Large follicles (> 5 mm) were not detected at day 110 of pregnancy. In vitro testosterone output by follicles was constant throughout pregnancy. Oestradiol output remained steady until day 80, but decreased markedly at day 110 of pregnancy. This decrease was associated with a reduction in aromatase activity in follicles obtained at this stage. Experiment 2 examined the effect of administration of high concentrations of progesterone between day 100 and day 120 after mating on resumption of follicular growth in ewes that underwent Caesarean section at day 99 of pregnancy. In ewes that underwent Caesarean section, progesterone supplementation was successful in mimicking the profile found in pregnant ewes, but did not prevent re-initiation of follicular growth, as demonstrated by the presence of large follicles (> 5 mm) at day 120 after mating. Experiment 3 examined the effects of PGF(2alpha)-induced regression of the corpus luteum of day 100 of pregnancy on resumption of follicular growth. High concentrations of PGF(2alpha) (0.28 mg kg(-1) body weight) administrated at day 100 of pregnancy were required to initiate regression of the corpus luteum. At day 120 after mating, the mean (+/- SEM) diameter of the largest follicle in PGF(2alpha)-treated ewes (3.40 +/- 0.47 mm) was significantly greater (P < 0.05) than that in control pregnant ewes (2.52 +/- 0.34 mm). Experiment 4 examined the effect of removal of the fetus and of the corpus luteum at day 100 of pregnancy on resumption of ovulation. Removal of the corpus luteum by PGF(2alpha) treatment at the time of removal of the fetus resulted in earlier occurrence of short luteal phases (27.8 versus 40.6 days, PGF(2alpha)-treated versus non-treated) but did not alter the timing of the first normal luteal phases (41 days). In conclusion, the results from these experiments indicate that placental compounds play a major role in inhibiting follicular growth and maturation during late pregnancy in sheep.

Expression and regulation of mRNAs for insulin-like growth factor-I receptor and LH receptor in corpora lutea

Relationship between insulin-like growth factor-I receptor (IGF-IR) and luteinizing hormone receptor (LHR) mRNA expression as well as their regulation was determined in rat corpora lutea (CL). In the CL of estrous cycle rat, LHR mRNA positive CL expressed high level of mRNA of IGF-IR. While the expression of LHR mRNA decreased on estrus, the CL still expressed relatively high level of IGF-IR mRNA. In pseudopregnant rat CL, the expression level of LHR mRNA was low on day 1, the most intense signals were detected on day 8, the signals of LHR mRNA became undetectable on day 14. In contrast to LHR expression, the high level of IGF-IR mRNA was observed in pseudopregnant CL of day 1, and thereafter its signals were detected from day 2 to day 14. Pregnant rat CL expressed both LHR and IGF-IR mRNAs. IGF-I stimulated LHR expression in CL. PGF(2alpha) inhibited expression of IGF-IR and LHR. PGE(2) negated the inhibiting effects of PGF(2alpha). These data suggest that IGF-I may be involved in regulating CL function, and maintaining CL structure through changes in expression of its receptors. Inhibited expression of IGF-IR by PGF(2alpha) may be part of mechanisms for regression of CL.

PGE receptor characteristics on porcine luteal cells during the estrous cycle and early pregnancy

This study examined the affinities and concentrations of prostaglandin E (PGE) receptors on porcine luteal cells during the estrous cycle and early pregnancy. Corpora lutea (CL) were obtained from nonpregnant gilts at days 9 ( n = 4), 12 ( n = 3), and 14 ( n = 6); three gilts possessed red, vascular CL and three gilts had white nonvascular CL) of the estrous cycle, and days 9 ( n = 4), 12 ( n = 3), 14 ( n = 5), and 30 ( n = 5) of pregnancy. The CL were dissociated enzymatically to disperse single cells and the red blood cells were removed by elutriation. The luteal cells were assayed for specific PGE binding by displacement analysis with use of [ 3H] PGE 2 and varying concentrations of unlabeled PGE 2. The specific binding of [ 3H] PGE 2 to luteal cells decreased ( p < 0.05) from days 9 to 14 of the estrous cycle, but only decreased ( p < 0.05) from days 9 to 12 of pregnancy. Specific binding was higher ( p < 0.05) on day 14 of pregnancy than the comparable stage of the estrous cycle. The affinities of PGE receptors decreased ( p < 0.05) only on the luteal cells dissociated from red, vascular CL of day 14 nonpregnant gilts compared with those of other days of the estrous cycle and pregnancy. The number of PGE receptors on porcine luteal cells was similar ( p > 0.05) in pregnant and nonpregnant gilts, but decreased ( p < 0.05) on days 12–14 postestrus. During early pregnancy, it was evident that high affinity PGE receptors are sustained on porcine luteal cells; however, the role of the PGE receptors in maternal recognition of pregnancy remains speculative.

Establishment and Characterization of a Simian Virus 40-Transformed Temperature-Sensitive Rat Luteal Cell Line

The primary culture of rat luteal cells and their long-term maintenance have been difficult. Low cellular yields have limited the possibility for the study of gene regulation in luteal cells. The goal of this study was to develop a cell line to serve as a model by which to study the expression and regulation of various genes specific to luteal cells. We attempted to develop a luteal cell line by transformation of large luteal cells through infection with a temperature-sensitive simian virus (SV-40 tsA209) mutant that has a temperature-sensitive mutation required for the maintenance of cell transformation. We report here the successful establishment of such a cell line, designated GG-CL cells. Large luteal cells were purified to homogeneity by flow cytometry from corpora lutea of day 14 pregnant rats, cultured for 24 h, and then infected with the SV-40 tsA209 mutant virus. Transformed cells were maintained at the permissive temperature (33 C) until colonies were identified. Several colonies of transformed cells were isolated and passaged. They multiplied at 33 C and formed multilayers. At the nonpermissive temperature (40 C), cells reverted to the normal differentiated phenotype similar to the primary luteal cells in culture. To determine whether GG-CL cells express the genes found in normal luteal cells, messenger RNA (mRNA) expression was examined by either Northern analysis or RT-PCR with primers specific to each mRNA. GG-CL cells were found to express receptors for interleukin-6 and glucocorticoid, as well as the newly discovered estrogen receptor-β (ER-β) and the orphan nuclear receptor nur 77. No receptors for ER-α, progesterone, LH, or PRL could be detected. This cell line also expressed 20α-hydroxysteroid dehydrogenase (20α-HSD), but not cholesterol side-chain cleavage cytochrome P450 (P450scc), 3β-hydroxysteroid dehydrogenase, or aromatase cytochrome P450 (P450arom). Although the cells did not express the PRL receptor, they did express Janus kinase (JAK2) and signal transducers and activators of transcription (Stat5b), and, when transfected with the PRL receptor, they responded to PRL with a marked inhibition in 20α-HSD mRNA expression. In addition, estradiol enhanced ER-β expression in a dose-dependent manner whereas cAMP stimulation caused a marked and rapid increase in the expression of the orphan receptor nur 77. In summary, a temperature-sensitive cell line was successfully established from the large luteal cells of rat corpora lutea. These cells express key genes encoding enzymes and receptors inherent to this defined luteal cell population and respond to stimulation by PRL, estradiol, and cAMP.

Establishment and characterization of a simian virus 40-transformed temperature-sensitive rat luteal cell line.

The primary culture of rat luteal cells and their long-term maintenance have been difficult. Low cellular yields have limited the possibility for the study of gene regulation in luteal cells. The goal of this study was to develop a cell line to serve as a model by which to study the expression and regulation of various genes specific to luteal cells. We attempted to develop a luteal cell line by transformation of large luteal cells through infection with a temperature-sensitive simian virus (SV-40 tsA209) mutant that has a temperature-sensitive mutation required for the maintenance of cell transformation. We report here the successful establishment of such a cell line, designated GG-CL cells. Large luteal cells were purified to homogeneity by flow cytometry from corpora lutea of day 14 pregnant rats, cultured for 24 h, and then infected with the SV-40 tsA209 mutant virus. Transformed cells were maintained at the permissive temperature (33 C) until colonies were identified. Several colonies of transformed cells were isolated and passaged. They multiplied at 33 C and formed multilayers. At the nonpermissive temperature (40 C), cells reverted to the normal differentiated phenotype similar to the primary luteal cells in culture. To determine whether GG-CL cells express the genes found in normal luteal cells, messenger RNA (mRNA) expression was examined by either Northern analysis or RT-PCR with primers specific to each mRNA. GG-CL cells were found to express receptors for interleukin-6 and glucocorticoid, as well as the newly discovered estrogen receptor-beta (ER-beta) and the orphan nuclear receptor nur 77. No receptors for ER-alpha, progesterone, LH, or PRL could be detected. This cell line also expressed 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), but not cholesterol side-chain cleavage cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase, or aromatase cytochrome P450 (P450arom). Although the cells did not express the PRL receptor, they did express Janus kinase (JAK2) and signal transducers and activators of transcription (Stat5b), and, when transfected with the PRL receptor, they responded to PRL with a marked inhibition in 20alpha-HSD mRNA expression. In addition, estradiol enhanced ER-beta expression in a dose-dependent manner whereas cAMP stimulation caused a marked and rapid increase in the expression of the orphan receptor nur 77. In summary, a temperature-sensitive cell line was successfully established from the large luteal cells of rat corpora lutea. These cells express key genes encoding enzymes and receptors inherent to this defined luteal cell population and respond to stimulation by PRL, estradiol, and cAMP.

Serotonin as a possible physiological regulator of ovine luteal function

Serotonin (5-hydroxytryptamine, 5-HT) has been found to significantly ( P < 0.05) stimulate basal progesterone production by dispersed ovine luteal cells harvested on Days 3 and 10 of the oestrous cycle; these effects were inhibited by mianserin. There was no stimulation of cells from corpora lutea of Day 14 pregnant sheep. High performance liquid chromatography (HPLC) analysis demonstrated the presence of 5-HT in corpora lutea (CL), with the highest concentration in early (Day 3) dispersed luteal cells. However, 5-hydroxyindoleacetic acid, a major metabolite of 5-HT, was not detected in either midcycle (Day 10) or early pregnant (Day 14) CL. Furthermore, 5-HT could not be observed immunohistochemically in any of the CL. These results suggest that serotonin may only have a minor role, if any, in the regulation of ovine steroidogenesis.

Evidence that a non-steroidal factor from corpus luteum of pregnant sheep inhibits aromatase activity of ovarian follicles in vitro.

Porcine and ovine follicular tissues were used to investigate, in vitro, the effect of charcoal-treated aqueous extract from ovine corpora lutea of pregnancy on aromatase activity as determined by the conversion of [3H]testosterone to oestradiol by follicular walls and measurement of 3H2O release. Extract (500 micrograms protein) prepared from corpora lutea of day 112 of pregnancy but not extract (500 micrograms) prepared from ovine fetal cotyledonary tissue obtained at a similar time significantly decreased (P < 0.02) aromatase activity of pig follicles in the absence of FSH. These results demonstrate that a non-steroidal factor in the corpora lutea of late pregnancy directly inhibits aromatase activity. When the effects of different doses (300, 600 or 1200 micrograms) of luteal extract from corpora lutea of day 100 of pregnancy on aromatase activity of pig follicles were studied, the dose by treatment (presence or absence of FSH) interaction was not significant. Luteal extract dose at 300 micrograms did not affect aromatase activity but a significant decrease in activity occurred at 600 micrograms of luteal extract (600 versus 300 micrograms, P < 0.02). There was no further significant increase in the inhibitory effect with 1200 micrograms luteal extract. When the effects of 600 micrograms luteal extract from corpora lutea of days 15, 75 or 100 of pregnancy on aromatase activity of pig follicles were studied, a significant (P < 0.05) stage of pregnancy effect was detected, but the stage of pregnancy by treatment (presence or absence of FSH) interaction was not significant. No effect was noted with day 15 or day 75 luteal extract.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a gonadal nonsteroidal factor that specifically inhibits release of luteinizing hormone in ewes.

Bilaterally ovariectomized ewes were used to investigate the effect of systemic administration (i.v.) of charcoal-treated aqueous luteal extracts from ovine corpora lutea on plasma concentrations of pituitary gonadotrophins. Jugular blood samples were taken every 15 min at least 5 h before (control period) and 5 h after (treatment period) injection. In Expt 1, the administration of luteal extract from corpora lutea of days 70-76 of pregnancy, but not of the extract prepared from muscular tissue, resulted in a significant decrease of mean concentrations of luteinizing hormone (LH) (P < 0.02) and frequency of LH pulses (P < 0.01). Plasma follicle-stimulating hormone (FSH) concentrations were not affected by injections of either extract. These findings provide the first demonstration of the presence of a nonsteroidal factor in the corpus luteum of midpregnancy that selectively suppresses the secretion of LH. In Expt 2, mean concentrations of LH and FSH and frequency of LH pulses were unaffected by injections of luteal extracts from ovine corpora lutea of days 10-12 of the oestrous cycle or day 15 of pregnancy. These data suggest that some factor(s), probably from the fetoplacental endocrine unit, is required to ensure the production of a significant quantity of the luteal LH-inhibiting factor after day 15 of pregnancy. In Expt 3, treatment of luteal extract from corpora lutea of day 70 of pregnancy with proteolytic enzymes destroyed the LH-inhibiting activity, suggesting the proteic nature of the luteal LH-inhibiting factor. In Expt 4, plasma concentrations of LH were not affected by injection of charcoal-treated extract prepared from fetal cotyledonary tissue of days 110-120 of pregnancy suggesting that the LH-inhibiting factor exclusively originates from the corpus luteum during pregnancy. These experiments provide the first direct evidence for the existence of a potent nonsteroidal factor of luteal origin that specifically inhibits pulsatile secretion of LH, without influencing FSH release in female animals. We propose the term LH-release-inhibiting factor (LH-RIF) to describe this activity.

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)

Human corpus luteum: immunocytochemical localization of epidermal growth factor

Six corpora lutea (day 17 to day 25) of the menstrual cycle and four ovarian stromal tissues from seven cycling women were examined for the presence of epidermal growth factor (EGF) by immunocytochemistry with the use of the indirect peroxidase-antiperoxidase (PAP) procedure. After tissue sections of 4 micron were mounted, endogenous peroxidases were removed with hydrogen peroxide, and the sections were incubated for 1 hour at room temperature followed by 16 hours at 4 degrees C with a highly specific antisera for mouse EGF (mEGF), nonimmunized normal rabbit serum, or antiserum preadsorbed with excess antigen. After the reaction with the second antibody (goat antirabbit IgG) for 1 hour at room temperature, the peptide was localized by use of PAP and 3.3'-diaminobenzidine as the chromogen. EGF could be localized in the luteal cells of five corpora lutea (day 17 to day 25) but not in a corpus luteum of day 22 and all ovarian stromal tissue examined. The localization of immunoreactive EGF in the human corpus luteum demonstrates directly for the first time the presence of this growth-promoting peptide in this tissue, which suggests its local production.