Prolactin-induced expression of intercellular adhesion molecule-1 and the accumulation of monocytes/macrophages during regression of the rat corpus luteum.
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Intercellular adhesion molecule-1 (ICAM-1) is thought to facilitate the recruitment and migration of monocytes/macrophages to sites of inflammation. Here we investigated whether the luteolytic effect of prolactin in the hypophysectomized rat is associated with the expression of ICAM-1. In addition, we examined the effect of exogenous testosterone (or its potential conversion to estradiol endogenously) on the corpus luteum to address recent speculation that ovarian steroids might augment luteal regression. Immature, 30-day-old rats were ovulated with eCG and hCG and then hypophysectomized; this resulted in a single cohort of persistent corpora lutea. The rats were assigned randomly into four treatment groups: vehicle treatment without or with testosterone (VEH-T4, VEH+T4) and prolactin treatment without or with testosterone (PRL-T4, PRL+T4). Corpora lutea of control rats exhibited minimal ICAM-1 staining and contained relatively few monocytes/macrophages. In contrast, corpora lutea of prolactin-treated rats exhibited prominent ICAM-1 staining and contained numerous monocytes/macrophages. Testosterone did not overtly affect ICAM-1 staining, numbers of monocytes/macrophages, or concentrations of plasma progestins (progesterone and 20alpha-dihydroprogesterone) in either VEH or prolactin treatment groups; notwithstanding, luteal weights increased significantly in response to testosterone in VEH+T4 rats compared to VEH-T4 rats and prolactin-treated rats. We conclude that ICAM-1 expression and monocyte/macrophage accumulation are associated with prolactin-induced luteal regression in the rat and that these aspects are not influenced by testosterone. (+info)
Progesterone on an oestrogen background enhances prolactin-induced apoptosis in regressing corpora lutea in the cyclic rat: possible involvement of luteal endothelial cell progesterone receptors.
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Preovulatory surges of both prolactin (PRL) and progesterone have been suggested to be necessary for the induction of apoptosis in the regressing corpus luteum of the cyclic rat. The aim of these experiments was to study whether the administration of PRL and/or progesterone on the morning of pro-oestrus reproduces the regressive changes that happen in the cyclic corpus luteum (CL) during the transition from pro-oestrus to oestrus, and to analyse the temporal relationships between two characteristic features of structural luteolysis (luteal cell apoptosis and accumulation of macrophages). Cyclic rats (treated at 0900 h with an LHRH antagonist to block LH secretion) were injected at 1000 h with PRL and progesterone and killed at 0, 30, 60, 90 and 180 min after treatment. The number of apoptotic cells increased progressively from 60 min after treatment onward in hormone-treated rats, whereas the number of macrophages did not change throughout the period of time considered. Rats injected with PRL plus progesterone showed significantly greater numbers of apoptotic cells than those injected with PRL alone. The luteolytic effects of progesterone were in keeping with the presence of luteal endothelial cells showing progesterone receptor (PR) immunoreactivity in pro-oestrus. Treatment of rats during dioestrus and pro-oestrus with the specific antioestrogens LY117018 and RU58668 decreased the luteolytic effects of PRL and progesterone and the number of luteal endothelial cells immunostained for PR. These results strongly suggest that the preovulatory PRL surge and the preovulatory increase in progesterone together trigger structural regression of the corpus luteum. This seems to be dependent on oestrogen-driven cyclic changes in PRs in luteal endothelial cells. (+info)
Downregulation of long-form prolactin receptor mRNA during prolactin-induced luteal regression.
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OBJECTIVE: Prolactin is capable of both trophic and lytic actions in rat corpora lutea. In corpora lutea responding to a trophic prolactin signal, the long form of the prolactin receptor is the dominant form and is upregulated by prolactin. We investigated whether mRNA for the short form of the prolactin receptor was dominant in corpora lutea responding to a lytic prolactin signal, and whether the relative concentrations of the mRNAs for both forms of the prolactin receptor were changed during this response. DESIGN AND METHODS: Immature rats were ovulated by injection of 5 IU equine chorionic gonadotrophin and 5 IU human chorionic gonadotrophin, and were hypophysectomized shortly after ovulation. Nine days after hypophysectomy, rats were injected with prolactin (500 microg/day) or vehicle for 24 (n=6, n=6) or 72 h (n=13, n=5). Total RNA was isolated from corpora lutea and mRNA for both types of prolactin receptor were analyzed by semiquantitative RT-PCR using the ribosomal protein S16 as the internal control. RESULTS: The intensities of the long- and short-form prolactin receptor signals were normalized to the S16 internal control and expressed as relative densitometric units. The normalized values at 24h for prolactin-treated vs vehicle-treated rats were 0.23 +/- 0.05 vs 0.49 +/- 0.15 (P>0.05) for the short form and 4.04 +/- 0.8 vs 4.23 +/- 0. 6 (P>0.05) for the long form. The values for 72 h were 0.30 +/- 0.05 vs 0.24 +/- 0.05 (P>0.05) for the short form and 2.76 +/- 0.4 vs 5. 53 +/- 0.3 (P<0.01) for the long form respectively. CONCLUSION: The long form of the prolactin receptor is the dominant form at both time-points; however, the concentration of mRNA for this receptor isoform was specifically downregulated by prolactin treatment. Our results suggest that the short form of the prolactin receptor alone is unlikely to mediate the luteolytic action of prolactin, but that luteolytic events may be influenced via a change in the ratio of the two receptor isoforms. (+info)
Participation of reactive oxygen species in PGF2alpha-induced apoptosis in rat luteal cells.
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Prostaglandin F(2alpha) (PGF(2alpha)) is implicated in the process of luteal regression in many species. Treatment of rat luteal tissue with PGF(2alpha) increases the generation of reactive oxygen species. Since reactive oxygen species have been implicated in apoptosis, the present study was undertaken to determine whether reactive oxygen species play a role in the PGF(2alpha)-induced apoptosis of rat luteal cells. Rat luteal cells were loaded with 6-carboxy-2, 7'-dichlorodihydro-fluorescein (CDCFH) diacetate, di (acetomethyl ester), which can be oxidized by reactive oxygen species to yield CDCF, a fluorescent molecule, and the cells were treated with different doses of PGF(2alpha). Incubation with 100 micromol PGF(2alpha) l(-1) induced an increase in CDCF fluorescence (P < 0. 05). Treatment of cells with PGF(2alpha) for 48 h in serum-free medium induced a dose-dependent increase in cell death, and these cells exhibited the morphological characteristics typical of apoptosis, including condensed or fragmented nuclei and fragmentation of internucleosomal DNA. Pretreatment of these cells with ascorbic acid, N,N'-dimethylthiourea, or superoxide dismutase, which acts as an antioxidant or a radical scavenger, prevented the PGF(2alpha)-induced apoptosis. These results demonstrate that PGF(2alpha) produces reactive oxygen species and induces apoptosis in rat luteal cells, indicating that the reactive oxygen species may induce apoptotic cell death during luteolysis. (+info)
Relationship between the secretion of the corpus luteum and the length of the follicular phase of the ovarian cycle.
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The variation in the length of the follicular phase in many mammals may be related to the cellular origin of oestradiol secreted during the luteal phase. In all species the time taken for a small developing follicle (4-5 layers of granulosa cells) to mature to a preovulatory follicle may be the same as that which has been found experimentally in the mouse (10-17 days). In animals such as the sheep, in which there is no source of oestradiol other than the Graafian follicle, follicular development proceeds unimpaired throughout the luteal phase, and the 'follicular phse' which involves only the final stages of maturation of the Graafian follicle is relatively short. In primates, however, in which there is an extrafollicular source of oestrogen from the CL, the secretion of gonadotrophins is suppressed during each luteal phase to a level too low to initiate and maintain follicular development. At the end of each luteal phase and the beginning of the subsequent follicular phase, therefore, it is necessary to initiate the growth of a new crop of small follicles. The time taken for these follicles to develop inot preovulatory follicles determines the length of the follicular phase. (+info)
Role of tumor necrosis factor alpha and its type I receptor in luteal regression: induction of programmed cell death in bovine corpus luteum-derived endothelial cells.
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The role of tumor necrosis factor alpha (TNF alpha) and its type I receptor (TNFRI) in structural luteolysis was investigated. A semiquatitative reverse-transcription polymerase chain reaction (RT-PCR) was used to characterize the pattern of TNFRI mRNA expression within the corpus luteum (CL) throughout the estrous cycle and its cellular distribution. Increase in TNFRI mRNA levels was recorded both in regressed luteal tissue and in CL of cows injected with prostaglandin F(2 alpha). All three major cell types composing the CL, steroidogenic (large and small) and endothelial cells expressed the TNFRI gene. A densitometric analysis of TNFRI mRNA expression revealed that resident endothelial cells had significantly higher levels of TNFRI mRNA than steroidogenic luteal cells. The physiological effects associated with TNFRI expression were investigated in the various luteal cell types. TNF alpha-induced programmed cell death (PCD) in dose- and time-dependent manners of cultured luteal endothelial cells (LECs) but not of in vitro luteinized steroidogenic cells. Several lines of evidence are provided to show that progesterone regulates luteal cell survival: 1) CL and LECs express progesterone receptor mRNA, 2) physiological levels of the steroid abolished TNF alpha-induced PCD of LECs, and 3) progesterone-producing cells are protected from PCD. In conclusion, this study suggests that TNF alpha-induced PCD during structural luteolysis is mediated by TNFRI, primarily affects endothelial cells, and that the decline in progesterone, preceding structural luteolysis, is a prerequisite for the initiation of apoptosis in endothelial cells. (+info)
Regulation of lipid peroxidation by nitric oxide and PGF2alpha during luteal regression in rats.
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Corpus luteum regression is related to an increased generation of reactive oxygen species. Although several studies indicate that PGF(2alpha) is involved in regression of the corpus luteum in mammalian species through an increase in reactive oxygen species, the exact mechanism remains unknown. In the present study, the relationship between nitric oxide and PGF(2alpha) in regulation of lipid peroxidation was studied. Ovarian tissue from pseudopregnant rats at mid- (day 5) or late phase or at the time of regression (day 9 of pseudopregnancy) of corpus luteum development was used. Thiobarbituric acid reactants, used as a lipid peroxidation index, were higher on day 9 of pseudopregnancy than on day 5. In contrast, glutathione content (an antioxidant metabolite) was lower on day 9 than on day 5 of pseudopregnancy. These results indicate that there was an enhanced oxidative status in ovarian tissue during luteolysis. Administration of N(omega)-nitro-L-arginine methyl ester (L-NAME: 600 micromol l(-1)), a competitive nitric oxide synthase (NOS) inhibitor, led to a decrease in basal thiobarbituric acid reactant content in ovarian tissue from rats on day 9 of pseudopregnancy only, indicating that during regression of the corpus luteum, NO could act as intermediary in ovarian lipid peroxidation. Administration of a luteolytic dose (3 microg kg(-1) body weight i.p.) of a synthetic PGF(2alpha) increased thiobarbituric acid reactant content in ovaries from rats on day 9 of pseudopregnancy. As this effect was reversed partially by L-NAME, it is proposed that during regression of corpora lutea, PGF(2alpha) and NO are involved in regulation of lipid peroxidation. As this effect was only reversed partially, it is possible that there is another mechanism involving PGF(2alpha) (but not the NO-NOS pathway) in regulation of ovarian lipid peroxidation. Furthermore, the administration of PGF(2alpha) enhanced ovarian NOS activity, whereas cyclooxygenase inhibition (by indomethacin treatment in vivo) reduced it. As western blotting of ovarian homogenates obtained from PGF(2alpha)-injected rats increased inducible NOS (iNOS) content, it is concluded that PGF(2alpha) enhances both activity and synthesis of NO in rat ovarian tissues during luteolysis. Taken together, these results indicate that in ovaries with regressing corpora lutea, both NO and PGF(2alpha) are involved in part in regulation of lipid peroxidation. (+info)
Endothelin-1 mediates prostaglandin F(2alpha)-induced luteal regression in the ewe.
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A diversified series of experiments was conducted to determine the potential role of endothelin-1 (ET-1) in ovine luteal function. Endothelin-1 inhibited basal and LH-stimulated progesterone production by dispersed ovine luteal cells during a 2-h incubation. This inhibition was removed when cells were preincubated with cyclo-D-Asp-Pro-D-Val-Leu-D-Trp (BQ123), a highly specific endothelin ET(A) receptor antagonist. Administration of a luteolytic dose of prostaglandin F(2alpha) (PGF(2alpha)) rapidly stimulated gene expression for ET-1 in ovine corpora lutea (CL) collected at midcycle. Intraluteal administration of a single dose of BQ123 to ewes on Day 8 or 9 of the estrous cycle mitigated the luteolytic effect of PGF(2alpha). Intramuscular administration of 100 microg ET-1 to ewes at midcycle reduced plasma progesterone concentrations for the remainder of the estrous cycle. Following pretreatment with a subluteolytic dose of PGF(2alpha), i.m. administration of 100 microg ET-1 caused a rapid decline in plasma progesterone and shortened the length of the estrous cycle. These data complement and extend previously published reports in the bovine CL and are the strongest evidence presented to date in support of a role for ET-1 in PGF(2alpha)-mediated luteal function in domestic ruminants. (+info)