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.     

Quantitative cell composition of human and bovine corpora lutea from various reproductive states

The cell composition of human and bovine corpora lutea (CL) from various reproductive states was investigated by computerized video-based interactive Bioquant image analysis system IV and by light microscope immunocytochemistry. Human and bovine CL contained more nonluteal cells than luteal cells. Human CL contained a lower number of luteal and a greater number of nonluteal cells than bovine CL. Regardless of the reproductive state, human CL contained more small luteal cells than large luteal cells. In all reproductive states except in the late luteal phase, the bovine CL also contained more small luteal cells than large luteal cells. The average sizes of all the cells in human CL were smaller than in bovine CL. Human CL contained more vascular space than bovine CL during mid and late luteal phases. The number of luteal cells increased and nonluteal cells decreased from early to mid luteal phase, and then luteal cells decreased and nonluteal cells increased in late luteal phase and in degenerating human and bovine CL. While the change of number of small and large luteal cells first occurred from early to mid luteal phase in human CL, it did not take place until the late luteal phase in bovine CL. The average size of large luteal cells in humans and of small luteal cells in cattle did not change, whereas size of the other cells changed in different reproductive states in both human and bovine CL. The cell composition of term pregnancy human CL was similar to mid or late luteal phase, whereas the cell composition of early pregnancy bovine CL was similar to mid luteal phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of the adenylyl cyclase system in the regulation of corpus luteum function in the human and in nonhuman primates

We have reviewed the properties of luteinizing hormone/human chorionic gonadotropic (LH/hCG)-sensitive adenylyl cyclase (AC) of human corpus luteum (CL) and its regulation by several hormones and nonhormonal activators. We have also described the changes in enzyme activity in membrane preparations of human and cynomolgus monkey CL obtained at various stages of the menstrual cycle and pregnancy. The data have been analyzed with respect to the functional status of the luteal tissue and to the species differences among primate CL. In the menstrual cycle, luteal AC responsiveness to LH/hCG was detectable during the midluteal phase, but not during the late luteal phase or in the follicular phase of the following cycle. In addition, nonhormonal stimulation was high in CL obtained during the midluteal and late luteal phases, but declined drastically by the follicular phase of the next cycle. In early pregnancy, the enzyme was unresponsive to LH/hCG stimulation, yet its sensitivity to nonhormonal stimulation was similar, if not identical, to that of midluteal phase CL. Functional activity was also evident at the end of pregnancy. These results demonstrate that expression of AC activity in primate luteal membrane changes significantly with varying hormonal status under physiologic conditions. It is concluded that the AC system in luteal membranes is an effective model to study the mechanisms that regulate function and life span of the human and nonhuman primate CL.     

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.     

Immunocytochemical localization of relaxin in human corpora lutea: cellular and subcellular distribution and dependence on reproductive state

Relaxin is one of the hormones present during pregnancy and it is synthesized primarily by corpora lutea (CL). Other reproductive tissues including CL of the menstrual cycle may also synthesize this hormone. Very little is known, however, about the cellular and subcellular distribution of relaxin in human CL and dependence of luteal relaxin on the reproductive state. The light and electron microscope immunocytochemical studies described here were undertaken to obtain this information using antisera to porcine and human relaxin. Immunostaining was found in large luteal cells (17-30 microns) but not in small luteal cells (7-16 microns) or in nonluteal cells in any of the reproductive states or in human hepatocytes. Luteal immunostaining was low in early luteal phase; it increased progressively, reaching the highest level in late luteal phase, and then decreased greatly in corpora albicantia. Term pregnancy CL contained similar immunostaining as early luteal phase CL. Mid luteal phase CL contained more immunostained cells than late luteal phase CL, but the late luteal phase CL contained a greater amount of immunostaining per cell than mid luteal phase CL. The immunogold particles due to relaxin were primarily present in secretory granules and to a small extent in rough endoplasmic reticulum. Quantitation revealed that secretory granules contained a much higher number of gold particles than did rough endoplasmic reticulum. These two organelles from late luteal phase CL contained greater numbers of gold particles than those from mid luteal phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

The presence of pregnancy-associated plasma protein-A in human corpora lutea: cellular and subcellular distribution and dependence on reproductive state.

Pregnancy-associated plasma protein-A (PAPP-A) is a high-molecular-weight glycoprotein primarily secreted by syncytiotrophoblasts of human placenta. It is not known, however, whether human CL of menstrual cycle or pregnancy also contain this protein. Therefore, light and electron microscope immunocytochemical studies were undertaken to investigate the presence, cellular and subcellular distribution, and dependence of luteal PAPP-A content on reproductive state. Human CL from early, mid, and late luteal phases and from term pregnancies immunostained specifically for PAPP-A. Immunostaining was found in large luteal cells (17-30 microns) but not in small luteal cells (7-16 microns) or in nonluteal cells in any of the reproductive states. Immunostaining was not found in negative control tissues, i.e. human liver or bovine CL of pregnancy. As expected however, term-pregnancy human placenta used for a positive control tissue immunostained intensely for PAPP-A. The luteal immunostaining was highest in early luteal phase, decreased progressively from early to mid and from mid to late luteal phases, and then disappeared in corpora albicantia. The relative intensity of immunostaining in early luteal phase human CL was similar to that in term-pregnancy human placenta and higher than in term-pregnancy human CL. The immunogold particles due to PAPP-A were primarily associated with secretory granules of large luteal cells. A small number of gold particles were also found in rough endoplasmic reticulum and cytoplasm. In conclusion, human CL contain immunoreactive PAPP-A. The luteal content varies with reproductive state, with the highest amount found in early luteal phase CL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of mRNA and proteins for GnRH I and II and their receptors in primate corpus luteum during menstrual cycle

The differential expression of mRNA and protein of GnRH I, II and their receptors (RI and RII) in the monkey corpus luteum (CL) were measured during different stages of the luteal phase of the menstrual cycle as an initial step towards considering the role and regulation of GnRH (I and II) system during luteinization and luteolysis in primates. RT-PCR confirmed the sequence identity of PCR products and real time PCR quantified specific mRNA expressions. Proteins were localized by immunohistochemistry (IHC). Changes in mRNA expression patterns of GnRH I and II (increased) and GnRH RII (decreased) were maximal at mid-late to late stages, that is, at CL regression, where as GnRH RI was low during the entire luteal phase. However, RT-PCR and IHC studies confirmed the presence of GnRH RI at both mRNA and protein levels, respectively. IHC results showed the presence of GnRH I, II and their receptors in steroidogenic cells (granulose-luteal cells and thecal-luteal cells) across the luteal phase. Hence, GnRH I and II systems may have a role on both luteinization (from early to mid stages of CL) and luteolysis (from mid-late to very-late stages of CL). These novel findings suggest that monkey luteal GnRH system may have a role in fertility regulation in paracrine and/or autocrine manner.     

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)     

Induction of relaxin secretion in rhesus monkeys by human chorionic gonadotropin: dependence on the age of the corpus luteum of the menstrual cycle

Peripheral concentrations of immunoreactive relaxin are undetectable in primates during the nonfertile menstrual cycle, but become measurable during the interval when chorionic gonadotropin (CG) rises in early pregnancy. The objectives of the current study were to determine if exogenous CG, administered in a dosage regimen which invoked patterns and concentrations resembling those of early pregnancy, would induce relaxin secretion in nonpregnant rhesus monkeys, and whether the induction was dependent on the age of the corpus luteum (CL) at the onset of treatment. Female rhesus monkeys received twice-daily i.m. injections of increasing doses of human CG (hCG) for 10 days beginning in the early (n = 4), mid (n = 6) or late (n = 4) luteal phase of the menstrual cycle [5.3 +/- 0.3, 8.3 +/- 0.5, and 12.0 +/- 0.4 days after the midcycle luteinizing hormone (LH) surge, respectively; means +/- SEM]. Whereas immunoreactive relaxin was nondetectable in the luteal phase of posttreatment cycles, detectable levels of relaxin were observed in 2 of 4, 5 of 6, and 3 of 4 monkeys during hCG treatment in the early, mid and late luteal phase, respectively. Although CG treatment rapidly enhance progesterone levels, the appearance of relaxin was deferred; relaxin was first detectable 9.0 +/- 1.0 and 4.7 +/- 1.9 days after the onset of CG treatment at early and late luteal phases. Patterns of relaxin concentrations differed among groups (P less than 0.05, ANOVA; split plot design) and relaxin levels were lowest (P less than 0.01) in monkeys treated during the early luteal phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Equine luteal function regulation may depend on the interaction between cytokines and vascular endothelial growth factor: an in vitro study

We hypothesized that cytokines influence luteal angiogenesis in mares, while angiogenic factors themselves can also regulate luteal secretory capacity. Therefore, the purpose of this study was to evaluate the role of cytokines--tumor necrosis factor alpha (TNF), interferon gamma (IFNG) and Fas ligand (FASL)--on in vitro modulation of angiogenic activity and mRNA level of vascular endothelial growth factor A (VEGF), its receptor VEGFR2, thrombospondin 1 (TSP1), and its receptor CD36 in equine corpus luteum (CL) throughout the luteal phase. After treatment, VEGF protein expression was determined in midluteal phase (mid) CL cells. The role of VEGF on regulation of luteal secretory capacity was assessed by progesterone (P(4)) and prostaglandin E(2) (PGE(2)) production and by mRNA levels for steroidogenic enzymes 3-beta-hydroxysteroid dehydrogenase (3betaHSD) and PGE synthase (PGES). In early CL cells, TNF increased angiogenic activity (bovine aortic endothelial cell viability) and VEGF and VEGFR2 mRNA levels and decreased CD36 (real-time PCR relative quantification). In mid-CL cells, TNF increased VEGF mRNA and protein expression (Western blot analysis) and reduced CD36 mRNA levels, while FASL and TNF+IFNG+FASL decreased VEGF protein expression. In late CL cells, TNF and TNF+IFNG+FASL reduced VEGFR2 mRNA, but TNF+IFNG+FASL increased TSP1 and CD36 mRNA. VEGF treatment increased mRNA levels of 3betaHSD and PGES and secretion of P(4) and PGE(2). In conclusion, these findings suggest a novel auto/paracrine action of cytokines, specifically TNF, on the up-regulation of VEGF for angiogenesis stimulation in equine early CL, while at luteolysis, cytokines down-regulated angiogenesis. Additionally, VEGF stimulated P(4) and PGE(2) production, which may be crucial for CL establishment.     

Effect of prostaglandin E2 alpha on the hCG-stimulated progesterone production by human corpora lutea

The effect of prostaglandin PGF2 alpha on the hCG stimulated and basal progesterone production by human corpora lutea was examined in vitro. hCG (40 i.u./ml) stimulated progesterone formation in corpora lutea of early (days 16-19 of a normal 28 day cycle), mid (days 20-22) and late (days 23-27) luteal phases. This stimulation was inhibited by PGF2 alpha (10 micrograms/ml) in corpora lutea of mid and late luteal phases. PGF2 alpha alone did not show a consistent effect on basal progesterone production. The inhibition of hCG stimulated progesterone production by PGF2 alpha at times corresponding to luteolysis indicates a role for that prostaglandin in the process of luteolysis in the human corpus luteum.     

Nuclear volume and chromatin conformation of small and large bovine luteal cells: effect of gonadotropins and prostaglandins and dependence on luteal phase

Summary Change in nuclear volume and chromatin conformation are generally considered to reflect altered gene expression in eukaryotic cells. The present studies were undertaken to investigate whether these nuclear parameters of luteal cells can be altered by hormone treatment in vitro or change during the estrous cycle. The nuclear volume of small luteal cells was significantly lower than that of large luteal cells during the cycle and pregnancy. The nuclear volumes of small and large luteal cells from pregnancy did not change during incubation without any hormone or with 10 nM prostaglandin (PG)F₂. However, incubation with 1 nM human chorionic gonadotropin (hCG) or 10 nM PGE₁ resulted in a significant increase of nuclear volume of small luteal cells by 4 h and that of large luteal cells by 6 h. Small cells were more responsive to hCG than large luteal cells. The nuclear volumes of small and large luteal cells also significantly increased from early to mid luteal phase with no further change in late luteal phase. hCG and PGE₁, as well as PGF₂, treatment resulted in a change of chromatin conformation of small and large luteal cells. Dibutyryl cyclic AMP (10 mM) mimicked the hormones by increasing nuclear volumes and changing the chromatin conformation of small and large luteal cells. Chromatin conformation of small and large luteal cells also changed from early to mid luteal phase and mid to late luteal phase. In conclusion, in vitro, hCG and PGs can regulate nuclear volume and/or chromatin conformation of small as well as large bovine luteal cells. In vivo, these nuclear changes occur during the periods of luteal growth, development and regression in the estrous cycle.     

Administration of prostaglandin f(2 alpha) during the early bovine luteal phase does not alter the expression of ET-1 and of its type A receptor: a possible cause for corpus luteum refractoriness

Luteal regression is initiated by prostaglandin F(2 alpha) (PGF(2 alpha)). In domestic species and primates, demise of the corpus luteum (CL) enables development of a new preovulatory follicle. However, during early stages of the cycle, which are characterized by massive neovascularization, the CL is refractory to PGF(2 alpha). Our previous studies showed that endothelin-1 (ET-1), which is produced by the endothelial cells lining these blood vessels, plays a crucial role during PGF(2 alpha)-induced luteolysis. Therefore, in this study, we compared the effects of PGF(2 alpha) administered at the early and mid luteal phases on ET-1 and its type A receptors (ETA-R) along with plasma ET-1 and progesterone concentrations, and the mRNA levels of PGF(2 alpha) receptors (PGF(2 alpha)-R) and steroidogenic genes. As expected, ET-1 and ETA-R mRNA levels were markedly induced in midcycle CL exposed to luteolytic dose of PGF(2 alpha) analogue (Cloprostenol). In contrast, neither ET-1 mRNA nor its receptors were elevated when the same dose of PGF(2 alpha) analogue was administered on Day 4 of the cycle. In accordance with ET-1 expression within the CL, plasma ET-1 concentrations were significantly elevated 24 h after PGF(2 alpha) injection only on Day 10 of the cycle. The steroidogenic capacity of the CL (plasma progesterone as well as the mRNA levels of steroidogenic acute regulatory protein and cytochrome P450(scc)) was only affected when PGF(2 alpha) was administered during midcycle. Nevertheless, PGF(2 alpha) elicited certain responses in the early CL: progesterone and oxytocin secretion were elevated, and PGF(2 alpha)-R was transiently affected. Such effects probably result from PGF(2 alpha) acting on luteal steroidogenic cells. These findings may suggest, however, that the cell type mediating the luteolytic actions of PGF(2 alpha), possibly the endothelium, could yet be nonresponsive during the early luteal phase.     

Cellular distribution and cycle phase dependency of gonadotropin and eicosanoid binding sites in bovine corpora lutea.

Bovine luteal functions are regulated by gonadotropins and eicosanoids. The specific binding sites that presumably mediate the actions of these regulatory agents have previously been characterized in bovine luteal tissue. However, the cellular distribution and/or the cycle phase dependency of these binding sites have never been investigated. In the present study, we investigated these parameters by using quantitative light microscope autoradiography. The results showed that both small and large luteal cells contained binding sites for LH/hCG, prostaglandin (PG)E2, PGF2 alpha, PGI2, and leukotriene (LT)C4. In addition, luteal blood vessels contained LH/hCG and LTC4 binding sites and luteal fibroblasts contained PGE2 binding sites. On a per cell basis, there were more binding sites for all ligands in large luteal cells as compared to small or nonluteal cells. After correction for the cellular area differences, small luteal cells contained more LH/hCG, PGE2, PGI2, and LTC4 binding sites, while large luteal cells contained more PGF2 alpha binding sites. The small and large luteal cell binding of hCG, PGE2, PGI2, and LTC4 increased from early to mid luteal phase, followed by a decline in the late luteal phase. PGF2 alpha binding, on the other hand, increased from early to late luteal phase. In contrast to luteal cells, binding of hCG and LTC4 to luteal blood vessels and binding of PGE2 to luteal fibroblasts did not change during the cycle. These results suggest that LH/hCG and eicosanoid regulation of luteal function is more complex than previously envisioned and it involves both small and large luteal cells and, in some cases, also nonluteal cells.     

Effect of prostaglandin F2α on the hCG-stimulated progesterone production by human corpora lutea

The effect of prostaglandin PGF_2α on the hCG stimulated and basal progesterone production by human corpora lutea was examined . hCG (40 i.u./ml) stimulated progesterone formation in corpora lutea of early (days 16–19 of a normal 28 day cycle), mid (days 20–22) and late (days 23–27) luteal phases. This stimulation was inhibited by PGF_2α (10 μg/ml) in corpora lutea of mid and late luteal phases. PGF_2α alone did not show a consistent effect on basal progesterone production. The inhibition of hCG stimulated progesterone production by PGF_2α at times corresponding to luteolysis indicates a role for that prostaglandin in the process of luteolysis in the human corpus luteum.     

Influence of high-density lipoprotein on estradiol stimulation of luteal steroidogenesis

The aim of this investigation was to determine whether luteal cells utilize cholesterol derived from high-density lipoprotein (HDL) for steroidogenesis and whether estrogen enhances luteal utilization of exogenous sterol. Incubation of Day 15 corpora lutea (CL) with different doses of human HDL resulted in a dose-dependent increase in progesterone production. HDL in vitro enhanced the overall steroidogenic capacity. However, the percentage of increases in 17 alpha-hydroxyprogesterone, testosterone and estradiol were significantly less than that of progesterone. Day 12 hypophysectomized and hysterectomized pregnant rats were treated with either estradiol, testosterone or vehicle for 72 h. Serum pregnenolone and progesterone were markedly increased by the steroid treatment, yet in vitro production of progesterone by CL in all the groups was similar. However, in the presence of HDL in the media, only luteal tissues from steroid-treated rats increased their progesterone output. The reduced production of progesterone by luteal cells of vehicle-treated rats was not due to an accumulation of pregnenolone but to an overall reduction in exogenous sterol utilization. In summary, results of this investigation suggest 1) luteal cells of pregnant rats effectively utilize cholesterol from HDL for maximal steroidogenesis, and 2) estradiol may stimulate luteal steroidogenesis, at least in part, by affecting the incorporation or utilization of cholesterol from HDL into the cell.