Expression of mRNA encoding insulin-like growth factors I and II and the type 1 IGF receptor in the bovine corpus luteum at defined stages of the oestrous cycle

Previous studies have implicated insulin-like growth factors I and II (IGF-I and -II), in the regulation of ovarian function. The present study investigated the localization of mRNA encoding IGF-I and -II and the type 1 IGF receptor using in situ hybridization to determine further the roles of the IGFs within the bovine corpus luteum at precise stages of the oestrous cycle. Luteal expression of mRNA encoding IGF-I and -II and the type 1 IGF receptor was detected throughout the oestrous cycle. The expression of IGF-I mRNAvaried significantly during the oestrous cycle. IGF-I mRNA concentrations were significantly higher on day 15 than on day 10, and IGF-I mRNA in the regressing corpus luteum at 48 h after administration of exogenous prostaglandin was significantly greater than in the early or mid-luteal phase (days 5 and 10). In contrast, there was no significant effect of day of the oestrous cycle on expression of mRNA for IGF-II and the type 1 IGF receptor in the corpus luteum. Expression of IGF-II mRNA was localized to a subset of steroidogenic luteal cells and was also associated with cells of the luteal vasculature. mRNA encoding the type 1 IGF receptor was widely expressed in a pattern indicative of expression in large and small luteal cells. These data demonstrate that the bovine corpus luteum is a site of IGF production and reception throughout the luteal phase. Furthermore, this study highlights the potential of IGF-II in addition to IGF-I in the autocrine and paracrine regulation of luteal function.     

Microvascular endothelial cells of the corpus luteum

The cyclic nature of the capillary bed in the corpus luteum offers a unique experimental model to examine the life cycle of endothelial cells, involving discrete physiologically regulated steps of angiogenesis, blood vessel maturation and blood vessel regression. The granulosa cells and theca cells of the developing antral follicle and the steroidogenic cells of the corpus luteum produce and respond to angiogenic factors and vasoactive peptides. Following ovulation the neovascularization during the early stages of corpus luteum development has been compared to the rapid angiogenesis observed during tumor formation. On the other end of the spectrum, the microvascular endothelial cells are the first cells to undergo apoptosis at the onset of corpus luteum regression. Important insights on the morphology and function of luteal endothelial cells have been gained from a combination of in vitro and in vivo studies on endothelial cells. Endothelial cells communicate with cells comprising the functional unit of the corpus luteum, i.e., other vascular cells, steroidogenic cells, and immune cells. This review is designed to provide an overview of the types of endothelial cells present in the corpus luteum and their involvement in corpus luteum development and regression. Available evidence indicates that microvascular endothelial cells of the corpus luteum are not alike, and may differ during the process of angiogenesis and angioregression. The contributions of vasoactive peptides generated by the luteal endothelin-1 and the renin-angiotensin systems are discussed in context with the function of endothelial cells during corpus luteum formation and regression. The ability of two cytokines, tumor necrosis factor alpha and interferon gamma, are evaluated as paracrine mediators of endothelial cell function during angioregression. Finally, chemokines are discussed as a vital endothelial cell secretory products that contribute to the recruitment of eosinophils and macrophages. The review highlights areas for future investigation of ovarian microvascular endothelial cells. The potential clinical applications of research directed on corpus luteum endothelial cells are intriguing considering reproductive processes in which vascular dysfunctions may play a role such as ovarian failure, polycystic ovary syndrome (PCOS), and ovarian hyperstimulation syndrome (OHSS).     

Proliferation of Luteal Steroidogenic Cells in Cattle

The rapid growth of the corpus luteum (CL) after ovulation is believed to be mainly due to an increase in the size of luteal cells (hypertrophy) rather than an increase in their number. However, the relationship between luteal growth and the proliferation of luteal steroidogenic cells (LSCs) is not fully understood. One goal of the present study was to determine whether LSCs proliferate during CL growth. A second goal was to determine whether luteinizing hormone (LH), which is known have roles in the proliferation and differentiation of follicular cells, also affects the proliferation of LSCs. Ki-67 (a cell proliferation marker) was expressed during the early, developing and mid luteal stages and some Ki-67-positive cells co-expressed HSD3B (a steroidogenic marker). DNA content in LSCs isolated from the developing CL increased much more rapidly (indicating rapid growth) than did DNA content in LSCs isolated from the mid CL. The cell cycle-progressive genes CCND2 (cyclin D2) and CCNE1 (cyclin E1) mRNA were expressed more strongly in the small luteal cells than in the large luteal cells. LH decreased the rate of increase of DNA in LSCs isolated from the mid luteal stage but not in LSCs from the developing stage. LH suppressed CCND2 expression in LSCs from the mid luteal stage but not from the developing luteal stage. Furthermore, LH receptor (LHCGR) mRNA expression was higher at the mid luteal stage than at the developing luteal stage. The overall results suggest that the growth of the bovine CL is due to not only hypertrophy of LSCs but also an increase in their number, and that the proliferative ability of luteal steroidogenic cells decreases between the developing and mid luteal stages.     

Changes in the size distribution of goat steroidogenic luteal cells during pregnancy

Experiments were conducted to investigate the size distribution of goat steroidogenic luteal cells throughout pregnancy. Corpora lutea were collected from very early (<6 weeks), early (6–8 weeks), middle (9–14 weeks) or late (15–18 weeks) stages of pregnancy. Luteal tissue was dissociated into single-cell suspension by enzyme treatments. Cells were stained for 3β-hydroxysteroid dehydrogenase (3β-HSD) activity, a marker for steroidogenic cells. The steroidogenic cells covered a wide spectrum of size ranging from 5 to 45 μm in diameter. There was a significant increase in mean cell diameter (P>0.01) as pregnancy progressed. Mean diameter of 3β-HSD positive cells increased from 14.73±0.35 μm in the corpus luteum of very early pregnancy to 24.20±0.45 μm in the corpus luteum of late pregnancy. The ratio of large (>20 μm in diameter) to small (5–20 μm in diameter) luteal cells was 0.28:1.0 in very early pregnancy, with the 7.5–15 μm cell size class being dominant. However, the ratio of large-to-small luteal cells was increased to 1.77:1.0 μm as pregnancy advanced and 25–35 μm cell sizes became predominant. It is likely that small luteal cells could develop into large cells as pregnancy progresses. Development of pregnancy is also associated with an increase in size of steroidogenic luteal cells.     

Cholesterol transport and steroidogenesis by the corpus luteum

The synthesis of progesterone by the corpus luteum is essential for the establishment and maintenance of early pregnancy. Regulation of luteal steroidogenesis can be broken down into three major events; luteinization (i.e., conversion of an ovulatory follicle), luteal regression, and pregnancy induced luteal maintenance/rescue. While the factors that control these events and dictate the final steroid end products are widely varied among different species, the composition of the corpus luteum (luteinized thecal and granulosa cells) and the enzymes and proteins involved in the steroidogenic pathway are relatively similar among all species. The key factors involved in luteal steroidogenesis and several new exciting observations regarding regulation of luteal steroidogenic function are discussed in this review.     

Evidence for the existence of a luteal cell type that is steroidogenic and releases relaxin

Secretion of relaxin from cultured luteal cells derived from pregnant sows was detected by a reverse hemolytic plaque assay. In this method, luteal cells are cultured in monolayers together with protein-A-conjugated ovine red blood cells. In the presence of porcine relaxin anti-serum and complement, relaxin-releasing cells become surrounded by an area of hemolysis--a plaque--which can be microscopically visualized. After fixation, these same luteal cells in monolayers were stained for the presence of 3 beta-hydroxysteroid dehydrogenase, an enzyme marker for steroidogenic cells. Cells could then be classified by their ability to form plaques (relaxin-releasing cells) and/or steroidogenic capability (positive staining). Dual-secretors (large luteal cells that were steroidogenic and released relaxin) could be identified in dispersed luteal cells derived from pigs at all stages of pregnancy examined (Day 22-112 of gestation, n = 9; term is Day 114 +/- 2 days). In addition, luteal cells were detected that were either steroidogenic only or released relaxin, and finally, cells that appeared to possess neither endocrine capability. Frequency analysis of functional subtypes indicated approximately equal representation of each in the first half of pregnancy, but an apparent fall in relaxin-releasing cells in the preparturient period. It is suggested that dual-secretors may represent one mechanism that allows the corpus luteum to express multiple endocrine function during pregnancy without the requirement for increased cell numbers.     

Steroidogenic and morphological characteristics of granulosa and thecal compartments of the differentiating rabbit corpus luteum in culture

On the day after ovulation, the thecal tissue and associated mural granulosa lutein cells of the rabbit corpus luteum were separated from the granulosa lutein 'core' by dissection and these tissues were cultured separately or together (whole corpus luteum) in defined medium for 10 days on stainless-steel grids. The medium was changed completely every 24 h. Replicate tissues were cultured with testosterone (10 ng/ml), but no other hormones were added to the medium. Progesterone production increased during the first 2 days of culture for whole corpus luteum, granulosa lutein cells and the thecal compartment which also included granulosa lutein cells. After 3 days, the production of progesterone declined gradually, but was still detectable on Day 10. The production of the metabolite, 20 alpha-dihydroprogesterone, by whole corpus luteum was equal to or greater than that of progesterone. Without the addition of testosterone, the granulosa lutein cells produced little (10 pg/culture) oestradiol during 1 day of culture, but the thecal compartment and whole corpus luteum each produced about 100 pg/culture on Day 1 and declining quantities over the next 2 days. In the presence of testosterone added to the medium, the formation of oestradiol was greatly increased for all tissues for 5-6 days of culture, after which time oestradiol was no longer detectable with or without testosterone in medium. Transmission electron microscopy of cells after 10-12 days of culture revealed fine structure that is characteristic of luteal cells, including abundant smooth endoplasmic reticulum, lipid droplets, and junctions between the luteal cells. The corpus luteum in culture resembles the corpus luteum in situ in that steroidogenesis and differentiation can proceed for a period after ovulation without extrinsic hormonal stimulation.     

Progesterone is a cell death suppressor that downregulates Fas expression in rat corpus luteum

In female rats, apoptotic cell death in the corpus luteum is induced by the prolactin (PRL) surge occurring in the proestrous afternoon during the estrous cycle. We have previously shown that this luteolytic action of PRL is mediated by the Fas/Fas ligand (FasL) system. During pregnancy or pseudopregnancy, apoptosis does not occur in the corpus luteum. Progesterone (P4), a steroid hormone secreted from luteal steroidogenic cells, attenuated PRL-induced apoptosis in cultured luteal cells in a dose-dependent manner. P4 significantly decreased the expression of mRNA of Fas, but not FasL, in cultured luteal cells prepared from both proestrous and mid-pseudopregnant rats. These data indicate that P4 suppresses PRL-induced luteal cell apoptosis via reduction of the expression level of Fas mRNA in the corpus luteum, suggesting that P4 acts as an important factor that can change the sensitivity of corpus luteum to PRL.     

Corpus luteum size and plasma progesterone concentration in cows

It is often assumed that a larger corpus luteum will produce more progesterone and generate higher circulating plasma concentrations. The aim of the study was to determine whether the size of the corpus luteum does actually determine circulating plasma progesterone concentrations. Data were collated from a number of studies on various aspects of luteal function in non-lactating dairy cows to allow comparisons to be made between corpus luteum weight and plasma progesterone concentration across the luteal phase. In these studies oestrous cycles had been synchronised and animals slaughtered on day 5, day 8 or day 16 following oestrus. Both corpus luteum weight and plasma progesterone concentration increased between day 5 and day 8. Plasma progesterone concentration but not luteal weight also increased between day 8 and day 16. On day 5 there was a strong relationship between corpus luteum weight and plasma progesterone (R² = 0.64; P < 0.001). However, no such relationship was present on day 8 or day 16. These results indicate that while during the early stage of corpus luteum development a relationship between size and progesterone is present, by day 8 of the cycle, the size of the corpus luteum is no longer of importance in determining circulating progesterone concentrations.     

Changes in activities of superoxide dismutase, nitric oxide synthase, glutathione-dependent enzymes and the incidence of apoptosis in sheep corpus luteum during the estrous cycle

Anti-oxidative enzymes play a role in protecting cells from oxidative stress-induced cell death. The present study was conducted to evaluate whether the anti-oxidant and pro-oxidant enzymatic capacities of the sheep corpus luteum (CL) are correlated with steroidogenic and structural status of the gland during the estrous cycle. Steroidogenic activity, apoptosis and superoxide dismutase (SOD1 and SOD2), nitric oxide synthase (NOS), glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferase (GST) activities were determined in the CL at specific developmental stages of the luteal phase. The intensity of apoptotic DNA fragmentation, characteristic of physiological cell death, was much greater in CL at late luteal phase than at early and mid-luteal phase, concomitantly with the diminution in the plasma progesterone concentrations from mid-to late luteal phase. SOD1 and GPX activities increased from early to mid-luteal phase, and increased further at late luteal phase. SOD2 and GST activities were not different between early and mid-luteal phase, but increased at late luteal phase. GSR activity was not different between any luteal phase examined. NOS activity decreased from early to mid- and late luteal phase. These results show that the activities of SOD1, SOD2, NOS, GPX, GSR and GST in the sheep CL are subject to major changes during the estrous cycle, and that the anti-oxidant and pro-oxidant enzymatic capacities of luteal cells are not correlated with cell steroidogenic status and integrity during the late luteal phase.     

Evaluation of the physiological value of porcine luteal cells isolated in various stages of the luteal phase: Tissue culture approach

Porcine luteal cells were collected from corpora lutea in four different stages of the luteal phase and cultured as monolayers. Progesterone (P₄) secretion was assayed using radioimmunoassays (Gregoraszczuk, 1991). Luteal cells cultured from porcine corpora lutea collected in the early luteal phase maintained steroidogenic capacity for 6 days in culture until the time comparable with midluteal corpora lutea. Luteal cells collected from mature and regressing corpora lutea did not dedifferentiate during 2 days of culture. After this time secretion of progesterone decreased to undetectable amounts characteristic of old corpora lutea. The regression in the culture progressed. The results demonstrate that the degree of the decline of progesterone depends on the type of corpus luteum, which is connected to particular time intervals of the luteal phase. Before starting experiments it is necessary to take into consideration the stage of the luteal phase from which the material is collected for culture. This study provides evidence that long term culture is useful for investigating a variety of aspects of luteal function only if cells are collected in the early luteal phase. Short term culture is suitable for investigation of cells collected from mid and late luteal phase. Regulation of luteal function is dependent on stage of the luteal phase.     

Progesterone production by monkey luteal cell subpopulations at different stages of the menstrual cycle: changes in agonist responsiveness.

Small (less than or equal to 15 microns diameter) and large (greater than 20 microns diam.) luteal cells of the rhesus monkey have been separated by flow cytometry based on light scatter properties. To determine whether the steroidogenic ability and agonist responsiveness of luteal cell subpopulations vary during the life span of the corpus luteum, small and large cells were obtained at early (Days 3-5), mid (Days 7-8), mid-late (Days 11-12), and late (Days 14-15) luteal phase of the cycle. Cells (n = 4 exp./group) were incubated in Ham's F-10 medium + 0.1% BSA for 3 h at 37 degrees C with or without hCG (100 ng/ml), prostaglandin E2 (PGE2; 14 microM), dibutyryl-cAMP (db-cAMP; 5 mM), or pregnenolone (1 microM). Basal progesterone (P) production by large cells was up to 30-fold that by small cells depending on the stage of the cycle. HCG stimulated (p less than 0.05) P secretion by both small (1.8 +/- 0.2-fold) and large (3.7 +/- 0.7-fold) cells in the early luteal phase. HCG responsiveness declined during the luteal lifespan; P production by small cells was not significantly enhanced by hCG by mid luteal phase, whereas that by large cells was stimulated 1.7 +/- 0.2-fold (p less than 0.05) even at late luteal phase. Cell responses to db-cAMP were similar to those for hCG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Luteolysis in the rhesus monkey: Ovarian venous estrogen, progesterone, and prostaglandin F2α-metabolite

The role of prostaglandin F₂α (PGF₂α) in luteolysis in the non-human primate is poorly understood. We have recently reported that chronic PGF₂α infusion to the corpus luteum via Alzet pump, induced premature, functional luteolysis in the rhesus monkey. In the present study we sought to determine the ovarian events leading to spontaneous luteolysis in the monkey. Rhesus monkeys underwent laparotomy during the early luteal (4–5 days after the preovulatory estradiol surge, PES), mid-luteal (7–9 days PES), and late luteal (10–14 days PES) phases or at the first day of menses (M). Concentrations of progesterone, estradiol, estrone, and 13, 14-dihydro-15-keto-PGF₂α (PGFM) were measured in the ovarian venous effluents ipsilateral and contralateral to the ovary bearing the corpus luteum. Steroid levels in the ovarian vein on the corpus luteum side were significantly higher than the non-corpus luteum side throughout the cycle. PGFM levels were similar on both sides until the late luteal phase, when the effluent of the ovary bearing the corpus luteum contained significantly more PGFM (206±3) vs. 123±9 pg/ml, mean±sem); this disparity increased further at the time of menses (241±38 vs. 111±22 pg/ml). These data are the first to show an asymmetric secretion of PGFM in the ovarian venous effluent in the primate and suggest that PGF₂α of ovarian and possibly of corpus luteum origin may be directly involved in luteal demise.     

Prostaglandin F and progesterone secretion by porcine endometrium and corpus luteum in vitro

Slices of porcine endometrium and corpus luteum tissue obtained from mature sows throughout the luteal phase of the oestrous cycle were incubated in culture medium which was analysed at regular intervals over a period of 8 hours for prostaglandin F and progesterone. Prostaglandin F secretion was greatest by endometrium obtained during the mid III to late I luteal stage of the cycle and the increased levels secreted by this tissue were paralleled by high levels of secretion from corpus luteum tissue. The addition of indomethacin (10 μg/ml) to the culture medium completely abolished prostaglandin F secretion by both endometrium and luteal tissue indicating that the high levels of the prostaglandin were due to synthesis. Progesterone secretion by the corpus luteum was maximal from early luteal tissue and had declined to considerably lower levels by late stage tissue when prostaglandin secretion was greatest. The possible physiological significance of luteal prostaglandin F secretion is discussed.     

Effects of prostaglandin F2 alpha-induced luteolysis on the populations of cells in the ovine corpus luteum

Receptors for prostaglandin (PG) F2 alpha in the ovine corpus luteum are localized on large steroidogenic luteal cells. Therefore, it was hypothesized that during luteolysis, the first demonstrable effects of PGF2 alpha would occur in the population of large luteal cells. To test this hypothesis, the numbers and sizes of large and small luteal cells, fibroblasts, capillary endothelial cells, and pericytes were determined in corpora lutea collected 12, 24, or 36 h (6 animals/group) following administration of PGF2 alpha on Day 10 postestrus and from untreated ewes on Days 10 and 12 postestrus. The numbers and sizes of luteal cells were determined after enzymatic dissociation of the luteal tissue into single cell suspensions and by morphometric analysis of luteal slices. Serum levels of progesterone decreased (p less than 0.05) within 12 h of treatment, indicating that luteolysis was induced. Recovery of the two types of steroidogenic luteal cells following enzymatic dissociation was different (p less than 0.05). Recovery of both steroidogenic cell types decreased with time after PGF2 alpha treatment, suggesting that they had become more fragile. As determined by morphometry, the number of large luteal cells was not different at any time point examined; however, by 36 h after treatment, the average diameter of large luteal cells had decreased (p less than 0.05). In contrast, by 24 h after treatment, there was a decrease in the number of small luteal cells (p less than 0.05) but no change in their diameter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Corpus luteum function during the early postpartum interval in lactating rhesus monkeys: in vivo and in vitro response to exogenous gonadotropin.

The response of the postpartum corpus luteum to exogenous gonadotropin was studied in 12 lactating rhesus monkeys given daily injections of either human chorionic gonadotropin (HCG, n = 6) or saline (control, n = 6) for 4 days immediately following parturition. Peripheral blood samples were collected daily. On the 5th day postpartum, luteectomy was performed progesterone production by dispersed luteal cells was examined. Whereas progesterone in the peripheral circulation of control monkeys progressively declined between days 1 and 5 postpartum, progesterone levels increased significantly (p less than 0.025) with the onset of HCG treatment and remained significantly (p less than 0.025) elevated above the controls throughout the period of HCG treatment. However, despite the daily administration of HCG, circulating progesterone levels declined (p less than 0.05) between days 3 and 5 postpartum. The weight of the corpus luteum excised from HCG-treated macaques was significantly (p less than 0.005) greater than that of the controls. Dispersed cells from corpora lutea of saline-treated monkeys produced progesterone in vitro under control conditions (nutrient medium alone) and responded to the addition of high (100 ng/ml), but not low (1 ng/ml), levels of HCG with increased steroidogenesis. Although luteal cells from HCG-treated macaques tended to produce more progesterone in vitro than cells from control monkeys, they also exhibited a 50-fold reduction in sensitivity to HCG in vitro. These data suggest that the corpus luteum of lactating postpartum rhesus monkeys exhibited steroidogenic function which was stimulated by exogenous gonadotropin. However, prolonged exposure of the corpus luteum to high levels of exogenous gonadotropin appeared to produce a state of refractoriness to additional gonadotropic stimuli.     

Hypoxia regulates cell proliferation and steroidogenesis through protein kinase A signaling in bovine corpus luteum

Hypoxia is an important physiological process which ensures corpus luteum (CL) formation and development, thus playing an important role in steroidogenesis. Recent studies have shown that CL develops in an analogous to tumorigenesis by accumulation of hypoxia-inducible factor-1 alpha subunit (HIF1A) in response to hypoxia. To investigate the relationship among hypoxia, steroidogenesis, and cell proliferation during CL lifespan, histological and steroidogenic analyses of CL were performed at various CL stages in non-pregnant Holstein. Also, the hypoxia-mediated steroidogenesis and cell proliferation were studied in vitro with both primary luteal and luteinized granulosa cells. Our results showed that progesterone (P(4)) concentration increased with the upregulation of steroidogenic protein including steroidogenic acute regulatory protein (STAR) and CYP11A1 (P450scc) in the middle luteal stage. On the other hand, the cell proliferation- or hypoxia-associated proteins were upregulated in the early stage, including the proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor A (VEGFA), HIF1A, and aryl hydrocarbon receptor nuclear translocator (ARNT). In primary culture, phospho-protein kinase A (p-PKA) was downregulated, as were P(4) secretion and steroidogenic proteins both under oxygen-conditioned hypoxia in luteal cells and cobalt chloride-induced hypoxia in luteinized granulosa cells. However, under the treatment of hypoxia, PCNA, which was downregulated in luteal cells, was upregulated together with HIF1A and VEGFA in luteinized granulosa cells. Taken together, present study suggested that hypoxia downregulated steroidogenesis through PKA signaling and that the hypoxia-regulated cell proliferation could be activated during CL formation.     

Changes in the distribution of sizes of ovine luteal cells during the estrous cycle

The ovine corpus luteum is composed of two types of steroidogenic cells, which are referred to as small and large luteal cells. In this study, the size and number of steroidogenic cells were determined in corpora lutea collected on Days 4, 8, 12, and 16 of the estrous cycle. Corpora lutea were dissociated into single-cell suspensions that were stained for 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity, a marker for steroidogenic cells. The size of 3 beta-HSD-positive cells was measured with a Zeiss Videoplan Image Analyzer. On Day 4, most of the 3 beta-HSD-positive cells were less than 18 microns in diameter, the median being 11.2 microns. By Day 8, the number of 3 beta-HSD-positive cells increased 3-fold, and the median diameter increased to 12.8 microns. Although the number of 3 beta-HSD-positive cells was reduced by approximately 50% on Day 16, the median size on Days 12 and 16 was 14.6 and 16.8 microns, respectively. The ratio of large (greater than 18 microns) to small (less than 18 microns) luteal cells was 0.11 +/- 0.03 on Day 4; the ratio increased linearly to 0.67 +/- 0.09 by Day 16. This increase between Days 4 and 12 was attributable to an overall increase in the size of the cells; the increase between Days 12 and 16, however, was due to a loss of small luteal cells. When the experiment was conducted near the end of the breeding season, before animals became anestrous, the median size of the luteal cells did not change at different times of the estrous cycle but remained constant throughout. These data suggest that development of the corpus luteum is associated with an increase in the size and number of steroidogenic luteal cells, and that luteolysis is associated with a preferential loss of small luteal cells.