Tumor necrosis factor-alpha and its receptor in bovine corpus luteum throughout the estrous cycle.
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The objective of this study was to investigate tumor necrosis factor alpha (TNF-alpha) expression, the presence of functional TNF-alpha receptors, and expression of TNF receptor type I (TNF-RI) mRNA in the bovine corpus luteum (CL) during different stages of the estrous cycle. Reverse transcription (RT)-polymerase chain reaction (PCR) showed no difference in TNF-alpha mRNA expression during the estrous cycle. Concentrations of TNF-alpha in the CL tissue increased significantly from the mid to the late luteal stage and decreased thereafter (P < 0.05). An RT-PCR analysis showed higher levels of TNF-RI mRNA in CL of Days 3-7 than of other stages (P < 0.05). (125)I-TNF-alpha binding to the membranes of bovine CL was maximal after incubation at 38 degrees C for 48 h. The binding was much greater for TNF-alpha than for related peptides. A Scatchard analysis revealed the presence of a high-affinity binding site in the CL membranes collected at each phase of the estrous cycle (dissociation constant: 3.60 +/- 0.58-5.79 +/- 0.19 nM). In contrast to TNF-RI mRNA expression, the levels of receptor protein were similar at each stage of the estrous cycle. When cultured cells of all luteal stages were exposed to TNF-alpha (1-100 ng/ml), TNF-alpha stimulated prostaglandin F(2alpha) and prostaglandin E(2) secretion by the cells in a dose-dependent fashion (P < 0.01), especially during the early luteal phase, although it did not affect progesterone secretion. These results indicate the local production of TNF-alpha and the presence of functional TNF-RI in bovine CL throughout the estrous cycle, and suggest that TNF-alpha plays some roles in regulating bovine CL function throughout the estrous cycle. (+info)
Follicular and luteal phase characteristics following early cessation of gonadotrophin-releasing hormone agonist during ovarian stimulation for in-vitro fertilization.
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Gonadotrophin-releasing hormone agonists (GnRHa) are widely used in in-vitro fertilization (IVF) for the prevention of a premature rise in luteinizing hormone (LH) concentrations. However, the administration of GnRHa during the follicular phase may also impair subsequent luteal function due to retarded recovery of pituitary gonadotrophin secretion. Therefore, luteal supplementation is generally applied. The present study was designed to determine whether a premature LH surge would still be prevented after early cessation of GnRHa during ovarian stimulation and whether subsequent luteal phase LH production would be sufficient to support progesterone synthesis by the corpus luteum. Sixty patients were randomized for three groups: (i) A long GnRHa/human menopausal gonadotrophin (HMG) protocol with luteal support by repeated human chorionic gonadotrophin (HCG) (n = 20), (ii) early follicular phase cessation of GnRHa without luteal support (n = 20), and (iii) a long GnRHa protocol without luteal support (n = 20). Frequent ultrasound and blood sampling was performed during the entire IVF cycle. Forty normo-ovulatory women served as controls. No premature LH surges were found after early cessation of GnRHa. In this group, some pituitary recovery occurred during the late luteal phase, but this did not affect corpus luteum function. Progesterone concentrations were shown to be dependent on disappearance of the pre-ovulatory bolus of HCG. Pregnancies occurred in all three groups. In conclusion, early follicular phase cessation of GnRHa is still effective in the prevention of a premature rise in LH. Although some pituitary recovery was observed thereafter, corpus luteum function is still abnormal due to early luteolysis. (+info)
Superoxide dismutase expression in the human corpus luteum during the menstrual cycle and in early pregnancy.
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To investigate the possible role of the superoxide radical and its scavenging system in the human corpus luteum, superoxide dismutase (SOD) values and lipid peroxide concentrations were analysed in the corpora lutea during the menstrual cycle and in early pregnancy. Copper-zinc SOD (Cu,Zn-SOD) activities increased from the early to mid-luteal phase, and gradually decreased thereafter and were the lowest in the regression phase. In pregnant corpus luteum, Cu,Zn-SOD activities were significantly higher than those in the mid-luteal phase. In contrast, manganese SOD (Mn-SOD) activities were low in the mid-luteal phase and increased toward the regression phase. Changes in mRNA expression of both types of SOD were similar to changes in their activities. Lipid peroxide concentrations were the highest in the regression phase whereas they were remarkably low in pregnant corpus luteum. The effects of human chorionic gonadotrophin (HCG) on luteal SOD were studied in vitro. HCG significantly increased Cu,Zn-SOD expression in mid-luteal phase corpora lutea, but not in late luteal phase corpora lutea. In conclusion, the present study suggests that the superoxide radical and its scavenging system, especially Cu,Zn-SOD, play important roles in the regulation of human luteal function. The stimulation of luteal Cu, Zn-SOD expression by HCG may be important in maintaining luteal cell integrity when pregnancy occurs. (+info)
Mechanisms controlling the function and life span of the corpus luteum.
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The primary function of the corpus luteum is secretion of the hormone progesterone, which is required for maintenance of normal pregnancy in mammals. The corpus luteum develops from residual follicular granulosal and thecal cells after ovulation. Luteinizing hormone (LH) from the anterior pituitary is important for normal development and function of the corpus luteum in most mammals, although growth hormone, prolactin, and estradiol also play a role in several species. The mature corpus luteum is composed of at least two steroidogenic cell types based on morphological and biochemical criteria and on the follicular source of origin. Small luteal cells appear to be of thecal cell origin and respond to LH with increased secretion of progesterone. LH directly stimulates the secretion of progesterone from small luteal cells via activation of the protein kinase A second messenger pathway. Large luteal cells are of granulosal cell origin and contain receptors for PGF(2alpha) and appear to mediate the luteolytic actions of this hormone. If pregnancy does not occur, the corpus luteum must regress to allow follicular growth and ovulation and the reproductive cycle begins again. Luteal regression is initiated by PGF(2alpha) of uterine origin in most subprimate species. The role played by PGF(2alpha) in primates remains controversial. In primates, if PGF(2alpha) plays a role in luteolysis, it appears to be of ovarian origin. The antisteroidogenic effects of PGF(2alpha) appear to be mediated by the protein kinase C second messenger pathway, whereas loss of luteal cells appears to follow an influx of calcium, activation of endonucleases, and an apoptotic form of cell death. If the female becomes pregnant, continued secretion of progesterone from the corpus luteum is required to provide an appropriate uterine environment for maintenance of pregnancy. The mechanisms whereby the pregnant uterus signals the corpus luteum that a conceptus is present varies from secretion of a chorionic gonadotropin (primates and equids), to secretion of an antiluteolytic factor (domestic ruminants), and to a neuroendocrine reflex arc that modifies the secretory patterns of hormones from the anterior pituitary (most rodents). (+info)
Decreased progesterone levels and progesterone receptor antagonists promote apoptotic cell death in bovine luteal cells.
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We tested the hypothesis that progesterone (P(4)) acts at a local level to inhibit luteal apoptosis. Initial experiments employed aminoglutethimide, a P450 cholesterol side-chain cleavage inhibitor, to inhibit steroid synthesis. Cultured bovine luteal cells were treated with aminoglutethimide (0.15 mM) +/- P(4) (500 ng/ml) for 48 h. Luteal cells were recovered and snap frozen for isolation and analysis of oligonucleosomal DNA fragmentation or fixed for morphological analysis. Medium was collected for analysis of P(4) levels by RIA. Aminoglutethimide inhibited P(4) synthesis by > 95% and increased the level of apoptosis as evidenced by (32)P-labeled oligonucleosomal DNA fragmentation (> 40%). P(4) supplementation inhibited the onset of apoptosis that was induced by aminoglutethimide. These data were further supported by morphological assessment of apoptotic cells utilizing a Hoechst staining technique and together strongly suggest that P(4) has anti-apoptotic capacity. Using reverse transcription-polymerase chain reaction, we were able to isolate a 380-base pair cDNA from the bovine corpus luteum (CL) that was 100% homologous to the progesterone receptor (PR) previously found in bovine oviductal tissue. Furthermore, PR transcripts were present in large and small luteal cells. Immunohistochemistry also revealed that PR protein was present in both large and small luteal cells. To determine whether the anti-apoptotic effect of P(4) was regulated at the receptor level, luteal cells were cultured in the presence of PR antagonists, RU-486 and onapristone, for 48 h. Both antagonists caused approximately a 40% increase in (32)P-labeled oligonucleosomal DNA fragmentation. Interestingly, there was no difference (P >/= 0.05) in P(4) levels after treatment with PR antagonists. These observations support the concept that P(4) represses the onset of apoptosis in the CL by a PR-dependent mechanism. (+info)
Rescue of the corpus luteum and an increase in luteal superoxide dismutase expression induced by placental luteotropins in the rat: action of testosterone without conversion to estrogen.
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The superoxide radical and its scavenger, superoxide dismutase (SOD), play important roles in the regulation of corpus luteum function. The present study was undertaken to investigate whether SOD is related to pregnancy-induced maintenance of corpus luteum function. Placentae obtained from rats on Day 12 of pregnancy were incubated for 24 h, and the supernatant was used as placental luteotropins. Pseudopregnant rats were given the placental incubation medium from Day 9 to Day 12 of pseudopregnancy. The treatment significantly increased serum progesterone concentrations on Day 12 of pseudopregnancy. Both activities and mRNA levels of copper-zinc SOD (Cu,Zn-SOD) and manganese SOD (Mn-SOD) in the corpus luteum were also increased on Day 12 of pseudopregnancy. Treating the placental incubation medium with charcoal significantly eliminated the stimulatory effects of placental incubation medium on serum progesterone concentrations and luteal Mn-SOD expression, but not on Cu,Zn-SOD expression. The inhibitory effect of the charcoal treatment on luteal Mn-SOD expression was reversed by supplementation with testosterone or dihydrotestosterone (DHT), but serum progesterone concentrations were recovered only by DHT. Testosterone or DHT alone had no effect on serum progesterone concentrations and luteal SOD expression. In conclusion, placental luteotropins increased SOD expression in the corpus luteum and stimulated progesterone production, suggesting that SOD is involved in the maintenance of the corpus luteum function by placental luteotropins. In addition, androgen, with other placental luteotropins, acted to stimulate progesterone production and Mn-SOD expression in pseudopregnant rats. (+info)
Evidence for the presence of luteinizing hormone-chorionic gonadotrophin receptors in the pig umbilical cord.
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Pig umbilical cord, like that of humans, contains two arteries and a vein surrounded by Wharton's jelly with amnion covering the exterior surface. The aim of the present study was to investigate whether LH-hCG receptors are present in the pig umbilical cord, using light microscope immunohistochemistry, semiquantitative autoradiography, western blotting and reverse transcription-polymerase chain reaction. Umbilical cords were collected on days 48, 71 and 103 of fetal life (n = 6). Monoclonal and polyclonal anti-LH receptor antibodies were used to study receptor distribution. Immunoreactivity was observed in the umbilical blood vessels, the epithelium of umbilical amnion and cells in the Wharton's jelly. No differences in LH-hCG receptor distribution related to the sex of the fetus, period of fetal life or section of the umbilical cord were observed. Strong immunostaining was observed in umbilical vein and in umbilical arteries. However, in the arteries, the tunica media expressed weaker receptor immunostaining than did the tunica intima and tunica adventitia. No immunoactivity was detected in non-target tissue (skeletal muscle) but LH receptors were immunostained in the pig ovary. Topical autoradiography showed that vein and arteries in the umbilical cord bind 125I-labelled hCG, which was highly diminished after co-incubation with an excess of unlabelled hCG. The binding of 125I-labelled hCG to the Wharton's jelly and epithelial amnion was less intense than it was to vessels. Gonadotrophin binding sites were not present in the skeletal muscle. The pig umbilical arteries, vein and Wharton's jelly contained a 75 kDa immunoreactive LH-hCG receptor protein similar to that found in corpora lutea. Southern blot analysis of reverse transcription-polymerase chain reaction products, performed to enhance the sensitivity and specificity of LH receptor transcripts determination in umbilical cord tissues, revealed that the expected fragments of 740 and 470 bp were present in the arteries, vein, Wharton's jelly and corpora lutea (positive control). An additional product of 670 bp was found in the corpora lutea and arteries of umbilical cord, but not in the vein and Wharton's jelly. This is probably the first reported evidence of the presence of LH-hCG receptors in the umbilical cord of a non-human female mammal. (+info)
Nitric oxide synthase activity and progesterone release by isolated corpora lutea of rabbits in the early and mid-luteal phases of pseudopregnancy are modulated differently by prostaglandin E-2 and prostaglandin F-2alpha via adenylate cyclase and phospholipase C.
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By examining in vitro the effects of prostaglandin E-2 (PGE-2) and prostaglandin F-2alpha (PGF-2alpha) induced in the corpora lutea (CL) of pseudopregnant rabbits, we have demonstrated that these prostaglandins modulate luteal nitric oxide synthase (NOS) activity and progesterone production differently, depending on the age of the CL. On CL obtained on day 4 of pseudopregnancy (day-4), PGE-2 was found to depress NOS total activity to 13% of control and to significantly increase basal progesterone secretion by 61%, while PGF-2alpha had no effect. On day-9 CL, PGE-2 was ineffective, but PGF-2alpha caused a 2.5-fold increase of NOS activity and a marked decrease in progesterone production. Using specific inhibitors, we found that the regulatory actions of PGE-2 in vitro are mediated via the adenyl cyclase/protein kinase A (PKA) second messenger system, while the PGF-2alpha-induced luteolytic effects on day-9 CL depend upon activation of the phospholipase C/protein kinase C (PKC) system. The different responsiveness of day-4 and day-9 CL to PGE-2 and PGF-2alpha could depend on receptor availability for these two prostaglandins, even if other cellular mechanisms cannot be excluded. The present study supports a functional role for NOS in regulating the steroidogenic capacity of rabbit CL, and reveals a novel interaction between a stimulatory G-protein-coupled receptor and PKC/PKA-mediated signal transduction modulating NOS activity. (+info)