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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.

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.

The administration of prolactin to hypophysectomized rats results in regression of the corpora lutea, accompanied by immune-inflammatory events such as infiltration of monocytes and macrophages. Recent reports indicate an autocrine role for progesterone during the lifespan of the corpus luteum. In the present study, an inhibitor of 3beta-hydroxysteroid dehydrogenase, Trilostane, was used to investigate the hypothesis that a decrease in luteal tissue steroids precipitates the cascade of immune-inflammatory events leading to luteal regression in prolactin-treated hypophysectomized rats. Immature rats were induced to ovulate by administering eCG-hCG, and hypophysectomized on the day after ovulation (at 32 days of age). Rats were injected s.c. 9-11 days after hypophysectomy with (a) Trilostane (80 mg kg(-1) day(-1)), (b) ovine prolactin (500 mg day(-1)), (c) Trilostane plus prolactin, or (d) vehicle. Plasma and luteal tissue progesterone and 20alpha-dihydroprogesterone ('progestin') were quantified; luteal tissue monocytes-macrophages and apoptotic nuclei were counted, and luteal wet mass was determined. Rats treated with prolactin alone showed the expected markers of luteal regression: decreased plasma progestin, increased numbers of monocytes-macrophages and apoptotic nuclei in luteal tissue, and decreased luteal wet mass; however, progestin concentration in luteal tissue was unchanged. Treatment with Trilostane reduced plasma and luteal tissue progestin, but did not result in an infiltration of monocytes-macrophages or increased numbers of apoptotic nuclei in the corpora lutea, or any change in luteal wet mass. Trilostane in combination with prolactin reduced plasma and luteal tissue progestin and produced the expected markers of regression, with the exception of luteal tissue mass, which remained unchanged. In conclusion, inhibition of steroidogenesis does not initiate luteal regression or augment prolactin-induced luteal regression in hypophysectomized rats. Prolactin-induced infiltration of monocytes-macrophages is not accompanied by a decrease in luteal tissue progestin, at least in the early stages of luteal regression.

The role of LH in luteal function in pregnant dogs was investigated at two different periods during pregnancy: (i) the transitional period from apparent total independence of the corpus luteum to relative hormonal dependence (days 20-35); and (ii) the period of full hormonal dependence (days 35-40). At both periods, LH neutralization, LH inhibition and LH administration studies were conducted. At both periods LH immunoneutralization had no significant effect on the secretion pattern of progesterone or prolactin. GnRH antagonist treatment (Nal-Glu) decreased plasma LH below the detection limit in all treatment periods. Nal-Glu had no effect on prolactin. When GnRH antagonist osmotic pumps were implanted, a transient decrease in plasma progesterone concentrations occurred on days 21-22 but not during the remaining implantation period. When GnRH antagonist was injected, plasma progesterone temporarily decreased (24 h) after the beginning of treatment starting on day 20, but decreased for 5 days when the treatment started on day 35. When purified pig LH was injected i.v. twice a day for 2 consecutive days either from day 30 or from day 40, plasma progesterone concentrations remained constant during treatment. However, on days 40 and 41, an increase in prolactin was observed. These results indicate that LH immunoneutralization may not impair corpus luteum function. In addition, GnRH antagonist induces dose- and time-dependent effects. Only high doses resulted in a decrease in progesterone, the duration of which increased as pregnancy progressed. Continuous GnRH antagonist administration, even when associated with complete LH inhibition, was not associated with detectable effects on progesterone. Finally, LH administration does not stimulate progesterone but may modify prolactin in the last third of pregnancy. Other studies indicated a corpus luteum prolactin dependency. The present study indicates that, in pregnant bitches, LH may not be necessary to sustain progesterone synthesis but that its role may vary in a time-dependent manner.

The role of interferon in early pregnancy in red deer was investigated by (a) measuring production of interferon by the conceptus, (b) testing the anti-luteolytic effect of recombinant interferon-tau in non-pregnant hinds, and (c) treatment of hinds with interferon after asynchronous embryo transfer. Blastocysts were collected from 34 hinds by uterine flushing 14 (n = 2), 16 (n = 2), 18 (n = 8), 20 (n = 13) or 22 (n = 9) days after synchronization of oestrus with progesterone withdrawal. Interferon anti-viral activity was detectable in uterine flushings from day 16 to day 22, and increased with duration of gestation (P < 0.01) and developmental stage (P < 0.01). When interferon-tau was administered daily between day 14 and day 20 to non-pregnant hinds to mimic natural blastocyst production, luteolysis was delayed by a dose of 0.2 mg day(-1) (27.3 +/- 1.3 days after synchronization, n = 4 versus 21 +/- 0 days in control hinds, n = 3; P < 0.05). Interferon-tau was administered to hinds after asynchronous embryo transfer to determine whether it protects the conceptus against early pregnancy loss. Embryos (n = 24) collected on day 6 from naturally mated, superovulated donors (n = 15) were transferred into synchronized recipients on day 10 or day 11. Interferon-tau treatment (0.2 mg daily from day 14 to 20) increased calving rate from 0 to 64% in all recipients (0/11 versus 7/11, P < 0.005), and from 0 to 67% in day 10 recipients (0/8 versus 6/9, P < 0.01). The increased success rate of asynchronous embryo transfer after interferon-tau treatment in cervids may be of benefit where mismatched embryo-maternal signalling leads to failure in the establishment of pregnancy.

Recent evidence indicates that the cells of the immune system and their large network of secretory products, or cytokines, play an active role in the ovary throughout the oestrous cycle. In the present study, immune cell populations (T and B lymphocytes, macrophages, granulocytes and eosinophils) and expression of major histocompatibility complex (MHC) class II were investigated in corpora lutea from mares in early (days 2-4), mid- (days 7-10) and late (days 12-14) dioestrus, the post-luteolytic phase (days 16-17) and early pregnancy. The number of T lymphocytes within the corpus luteum increased in the late luteal phase. CD4+ cells did not increase until day 16, whereas the number of CD8+ cells increased before functional luteolysis; an apparently selective luteal infiltration of CD8+ cells was observed. MHC class II expression by non-steroidogenic cells was increased in samples from days 16-17, as was the number of infiltrating macrophages. Flow cytometry revealed very low expression of MHC class II by large luteal cells at all stages of the oestrous cycle. In early pregnancy, the number of CD4+ and CD8+ cells and macrophages decreased, as did MHC class II expression, compared with mid-dioestrous samples. B cells were present in very small numbers in all samples examined. Eosinophils were similarly sparsely distributed and numbers decreased further in pregnancy. After exogenous PGF2 alpha administration, populations of CD4+ cells and non-specific esterase staining cells were significantly smaller than after natural luteolysis, whereas eosinophil numbers were increased compared with samples from days 16-17. However, the number of CD8+ and CD5+ cells and MHC class II expression were not significantly different from those observed after natural luteolysis. These findings indicate that populations of immune cells in the equine corpus luteum vary during its lifespan. The selective increase in CD8+ cells before functional luteolysis indicates that they have a physiological role in the regression of the corpus luteum.

The polypeptide ubiquitin covalently binds to cytoplasmic proteins and marks them for proteolytic degradation. Ubiquitin is upregulated during apoptosis in some systems. Apoptosis increases during luteolysis but it is not known whether ubiquitin is expressed in regressing corpora lutea. Marmoset ovaries were removed on day 10 of the luteal phase from animals that had received either no treatment, treatment with the PGF2 alpha analogue cloprostenol 24 h earlier, or treatment with the GnRH antagonist antarelix for either 24 or 48 h before ovary collection. Ubiquitin was localized on ovarian sections by immunocytochemistry, and oligonucleosome formation characteristic of apoptosis was examined in isolated corpora lutea by electrophoresis of extracted [32P]DNA. Oligonucleosome formation was low in midluteal corpora lutea on day 10 but increased after induced luteal regression with PGF2 alpha and GnRH antagonist. Nuclear ubiquitin immunoreactivity was found in 1.66 +/- 0.66 steroidogenic cells and cytoplasmic staining was found in 0.4 +/- 0.3 steroidogenic cells (per x 40 field of view) in midluteal phase corpora lutea on day 10. Luteolytic induction with PGF2 alpha significantly increased the number of cells exhibiting cytoplasmic immunoreactivity to 12.24 +/- 1.6 (P < 0.05). Ubiquitin immunoreactivity was not observed after GnRH-induced luteal regression. Apoptotic oligonucleosome formation was found after induced luteal regression with both PGF2 alpha and GnRH antagonist, but ubiquitin upregulation only occurred after PGF2 alpha-induced regression. These results indicate that ubiquitin expression is not specific for luteolysis and is not an indicator of luteal apoptosis, but that the polypeptide does play a role in luteal cellular responses to PGF2 alpha.

Oxidative stress-induced apoptotic cell death has been implicated in the mechanisms of corpus luteum regression and follicular atresia. The objective of this investigation was to determine whether the antioxidant, oestradiol influenced apoptosis in pig luteal and follicular tissues exposed in vitro to hydrogen peroxide. Immunolocalization of fragmented DNA (a biochemical marker of apoptosis) in large steroidogenic luteal and granulosa cells was inhibited by oestradiol. Protection against apoptosis required relatively high concentrations of oestradiol (> or = 40 pg ml-1 incubation media) and was largely unmitigated by actinomycin D (suggesting a predominantly receptor-independent nongenomic mode of action). Ovarian cells were not protected by other (nonaromatizable) steroid hormones. It is suggested that oestradiol functions at the ovary as a reactive oxygen scavenger during pregnancy-mediated luteal rescue and folliculogenesis.

Prostaglandin F2 alpha (PGF2 alpha) decreases secretion of progesterone from the corpus luteum in domestic ruminants. However, it is less effective during the early part of the oestrous cycle (Louis et al., 1973) and at the time of maternal recognition of pregnancy (Silvia and Niswender, 1984; Lacroix and Kann, 1986). Decreased luteal responsiveness may be due to failure of PGF2 alpha to activate fully its normal second messenger system, protein kinase C (PKC). Alternatively, increased resistance of the corpus luteum to PGF2 alpha might be attributable to greater concentrations of recently identified biological inhibitors of PKC. These possibilities were addressed by measuring steady-state concentrations of mRNA encoding PGF2 alpha receptor and two inhibitors of PKC, protein kinase C inhibitor-1 (PKCI-1) and kinase C inhibitor protein-1 (KCIP-1, brain 14-3-3 protein), in corpora lutea collected from ewes on days 4, 10 and 15 of the oestrous cycle (n = 5 per day) and day 15 of pregnancy (n = 7). There were no differences in mean concentrations of mRNA encoding PGF2 alpha receptor among the groups. However, concentrations of mRNA encoding both inhibitors of PKC were higher (P < 0.01) on day 4 of the oestrous cycle compared with the other groups. Treatment of ewes with a luteolytic dose of PGF2 alpha, which activates PKC, did not change concentrations of mRNA encoding either PKCI-1 or KCIP-I up to 24 h later. Luteal expression of mRNA encoding the PKC inhibitors and PGF2 alpha receptor was also examined in ewes treated with oestradiol in vivo for 16 h in the midluteal phase. High concentrations of oestradiol in serum (20 and 70 pg ml-1) did not influence quantities of any of the mRNAs examined. Therefore, an increase in PKC inhibitors may be involved in resistance of the corpus luteum to PGF2 alpha during the early part of the oestrous cycle but does not appear to mediate the increased resistance of the corpus luteum to PGF2 alpha during maternal recognition of pregnancy. Neither PGF2 alpha nor oestradiol affected steady-state concentrations of mRNAs encoding PKCI-1 or KCIP-I.

The present study examined the effects of LH, prostaglandin E2 (PGE2), 8-bromo-cyclic AMP (cAMP) and forskolin on progesterone secretion by small and large pig luteal cells. Corpora lutea were isolated from gilts (n > or = 3 per day) on days 9, 12 and 14 of the oestrous cycle and days 9, 12, 14 and 30 of pregnancy. After enzymatic dissociation of the corpora lutea, small and large luteal cells were obtained by elutriation. Culture plates (24-well) were then seeded with 150,000 small luteal cells or 30,000 large luteal cells per well in 1 ml M199 medium in the absence or presence of LH, PGE2, LH plus PGE2, 8-bromo-cAMP or forskolin. After 12 h of incubation, culture plates were centrifuged, and the supernatant collected and frozen for subsequent assay of progesterone. Differences within day were not detected between cyclic and pregnant gilts, and thus, results were combined for days 9, 12 and 14. Basal progesterone secretion by small luteal cells was less (P < 0.05) on days 14 and 30 than days 9 and 12. Treatment with LH, PGE2, 8-bromo-cAMP or forskolin increased (P < 0.05) progesterone secretion by small luteal cells on days 9 and 12; however, treatments had no effect on days 14 and 30. Basal progesterone production by large luteal cells was less (P < 0.05) on day 30 compared with other days. PGE2 stimulated (P < 0.001) progesterone production by large luteal cells at all days. In contrast, 8-bromo-cAMP and forskolin inhibited progesterone production by large luteal cells on day 12 (P < 0.05), and day 14 (P < 0.001). These data show that pregnancy status does not alter luteal cell response to the aforementioned secretagogues. However, regulation of progesterone secretion differs between small and large luteal cells, and the age of the corpora lutea. Also, it is unlikely that the stimulatory actions of PGE2 involve increased cAMP production in pig large luteal cells.

The luteolytic effects of prostaglandin F2 alpha (PGF2 alpha) are thought to be mediated in part by the promotion of an increasingly oxidative cellular environment. Loss of antioxidants is one mechanism by which PGF2 alpha might induce or exacerbate oxidative damage within the corpus luteum. This study was performed to establish whether depletion of vitamin C is an acute effect of PGF2 alpha on the pig corpus luteum and to gain insight into the mechanism of luteal vitamin C loss at luteolysis. Gilts (n = 4) were anaesthetized and both utero-ovarian veins and an ear vein were catheterized. Each corpus luteum on the treated ovary received an intraluteal injection of PGF2 alpha (1 microgram) followed by a sustained release implant containing 100 micrograms of the prostaglandin. The other ovary served as the control and each corpus luteum received corresponding volumes of injection vehicle and blank implant. Blood was collected from the ear vein and both utero-ovarian veins every 15 min beginning 15 min before the onset of treatment. Collection of blood stopped when animals were ovariectomized and corpora lutea were collected at 2 h after treatment. Progesterone and vitamin C (ascorbate) concentrations were measured in tissue and plasma samples. PGF2 alpha-treated luteal tissue had similar progesterone, but significantly lower ascorbate, concentrations when compared with control corpora lutea. PGF2 alpha treatment resulted in a rapid and sustained increase in plasma ascorbate within the treatment-side utero-ovarian vein, while the control utero-ovarian vein and ear vein showed little change in plasma ascorbate during the experimental period. No effect of PGF2 alpha on plasma progesterone was evident. This finding suggests that PGF2 alpha depletes the pig corpus luteum of vitamin C by inducing secretion of the vitamin into the bloodstream. Further studies are necessary to determine whether the depletion of vitamin C that is induced by PGF2 alpha contributes to the demise of the pig corpus luteum.

The mechanism controlling luteal regression in primates is unknown but may involve cell death by apoptosis. Marmoset ovaries containing corpora lutea were studied at different stages of the normal ovarian cycle. Two additional groups of animals underwent induced luteolysis with either the prostaglandin F2 alpha analogue, cloprostenol, or the GnRH antagonist, antarelix, at the mid-luteal phase. Apoptosis in ovarian sections was estimated both by counting the number of cells exhibiting morphological features of apoptosis and by in situ labelling the 3' ends of the DNA fragments with digoxigenin-11-dUTP. Apoptosis was found to be significantly increased in corpora lutea in the early follicular phase (equivalent to the later stage of luteal lifespan) compared with the mid-luteal phase corpora lutea, as judged by either computerized morphometry or 3' end labelling. Apoptosis was also increased by the administration of either cloprostenol or antarelix when using the 3' end labelling end point, but only after cloprostenol when using computerized morphometry. A further form of cell death, characterized by the formation of cytoplasmic vacuoles, was also observed in corpora lutea undergoing both induced and spontaneous regression. These results demonstrate that apoptosis within the primate corpus luteum is increased in both physiological and induced luteal regression. In addition, they show that an alternative form of cell death is involved in both spontaneous and induced luteal regression, although the relative importance of the two mechanisms remains to be determined.

The regulation of mRNAs encoding insulin-like growth factor I (IGF-I) and the receptor for growth hormone (GH-R) in ovine luteal tissue by luteotrophic and luteolytic hormones was examined. In Expt 1, ewes were hypophysectomized (HPX) on day 5 of the oestrous cycle and administered saline (S), LH, GH, or LH + GH until day 12 of the oestrous cycle (n = 4 ewes per group). Concentrations of luteal mRNA encoding IGF-I in HPX + S ewes and pituitary-intact ewes at day 5 (n = 4) were approximately 60% (P < 0.05) of those in pituitary-intact ewes at day 12 (n = 4). Treatment of HPX ewes with GH or GH + LH, but not LH alone, increased concentrations of mRNA encoding IGF-I to values similar to those in pituitary-intact ewes at day 12. Hypophysectomy also reduced the mean concentration of mRNA encoding GH-R to approximately 60% (P < 0.05) of the values in pituitary-intact ewes (days 5 or 12). Treatment with LH, but not GH, increased (P < 0.05) concentrations of mRNA encoding GH-R to values observed in pituitary-intact ewes. In Expt 2, prostaglandin F2 alpha (PGF2 alpha; 1 mumole) injected into the ovarian artery on day 11 or day 12 of the oestrous cycle had no effect on luteal concentrations of mRNA for either IGF-I or GH-R. In Expt 3, concentrations of mRNA encoding IGF-I increased (P < 0.05) between days 3 and 6 and remained high for the duration (days 9, 12 and 15) of the oestrous cycle while luteal concentrations of mRNA encoding GH-R did not change. In conclusion, responsiveness of the corpus luteum to GH and luteal synthesis of IGF-I are likely regulators of luteal development and function. However, PGF2 alpha-induced luteolysis was not associated with a decrease in concentrations of mRNAs encoding either IGF-I or GH-R.

Extensive extracellular matrix remodelling occurs within the lifespan of the corpus luteum, particularly during corpus luteum formation and regression. A major mechanism for the regulation of extracellular matrix remodelling is via local production of specific proteinase inhibitors, such as the serine proteinase inhibitor plasminogen activator inhibitor type-1 (PAI-1). The objective of the present study was to characterize the localization, ontogeny and regulation of PAI-1 expression within ovine corpora lutea. Urokinase binding activity was detected within medium conditioned by ovine luteal cells. Production of PAI-1 by ovine luteal cells was confirmed by immunoprecipitating it from labelled proteins in culture medium. mRNA encoding PAI-1 was present within developing (day 3), mature (day 10) and regressing (30 h after prostaglandin F2 alpha injection on day 10 after the onset of oestrus) corpora lutea as demonstrated by in situ hybridization. The ontogeny of PAI-1 mRNA expression was characterized within corpora lutea collected on days 3, 7, 10, 13 and 16 after the onset of oestrus (n = 4, 4, 4, 3 and 4, respectively). Expression of PAI-1 mRNA did not differ during the luteal phase (P = 0.06), although a trend for an increase in the amount of PAI-1 mRNA was observed on day 16. Expression of PAI-1 mRNA was also examined during luteal regression in corpora lutea collected 0, 6, 12, 24 and 36 h after injection of prostaglandin F2 alpha on day 10 after the onset of oestrus (n = 4 at each time). Relative PAI-1 mRNA concentrations changed significantly during luteolysis induced by prostaglandin F2 alpha (P = 0.0002). Administration of prostaglandin F2 alpha resulted in a transient sevenfold increase in PAI-1 mRNA 6 h after injection (P = 0.0001) but by 12 h the amounts had returned to values similar to those detected on day 10. We conclude that PAI-1 is a major secretory product of ovine luteal cells and that a transient increase in PAI-1 mRNA occurs during luteolysis induced by prostaglandin F2 alpha. PAI-1 probably plays a key local role in the control of extracellular proteolysis during the luteal phase.

A study was conducted to determine the effects of pregnancy-specific protein B (PSPB) and prostaglandin F2 alpha (PGF2 alpha) on bovine luteal cell progesterone, prostaglandin E2 (PGE2) and oxytocin production in vitro. Corpora lutea were enucleated from multiparous cows with normal oestrous cycles during the mid-luteal (days 10-12; n = 5) or late-luteal (days 17-18; n = 5) stage. Mixed large and small cells (1.5 x 10(5) cells per well) were incubated in 500 microliters modified Ham's F-12 medium. Cells were incubated for 18 h before treatments were added. Cells were treated with PSPB (0, 2.5, 5.0 micrograms) and PGF2 alpha (0, 100, 200 ng) in a 3 x 3 factorial arrangement. After treatments were added, media samples were collected at 6 and 12 h. During the 18 h pretreatment incubation, progesterone, PGE2 and oxytocin production was similar between the prospective treatment groups. Progesterone production was greater (P < 0.001) by mid-stage than by late-stage cells. In addition, progesterone decreased (P < 0.001) as incubation time increased. Progesterone production was not affected by PGF2 alpha, but PSPB increased (P < 0.02) progesterone at the 5.0 micrograms dose. Late-stage luteal cells produced more (P < 0.001) PGE2 than did mid-stage cells; PGE2 production decreased (P < 0.001) with increased incubation time. Luteal PGE2 production increased in response to PSPB treatment (P < 0.01) and PGF2 alpha treatment (P < 0.001). Luteal oxytocin production was greater (P < 0.01) by mid-stage compared with late-stage cells. Oxytocin production decreased (P < 0.001) with incubation time in mid-stage cells, but in late-stage cells oxytocin production was similar over time. Neither PSPB nor PGF2 alpha had an effect on oxytocin. These results indicate that PSPB does not affect luteal oxytocin, but does increase progesterone and PGE2 production. In addition, PGF2 alpha increases luteal PGE2, but does not affect progesterone or oxytocin production. These data do not show an interaction between PSPB and PGF2 alpha in regulating bovine luteal cell endocrine function.

The effect of exogenous oestrogen on blood flow and quantitative histology of the corpus luteum were studied in pseudopregnant rabbits. Pseudopregnancy was induced with human chorionic gonadotrophin and an oestradiol capsule was implanted on day 0 of pseudopregnancy. Regression of the corpus luteum was initiated in the mid-luteal phase by removal of the capsule. Thirty-two New Zealand white rabbits were randomly allocated to four groups (eight rabbits per group) for studies of blood flow--control: oestradiol capsule not removed: -24 h: oestradiol capsule removed 24 h before blood flow measurements; -48 h: oestradiol capsule removed for 48 h; and -48 h/+48 h: oestradiol capsule removed for 48 h and then reimplanted for an additional 48 h. All blood flow studies were performed on day 11 of pseudopregnancy with radioactive microspheres. Quantitative histology was performed on a separate group of 16 rabbits allocated to the same groups as above (four rabbits per group). Blood flow to the corpus luteum was unchanged in the -24 h group, but declined by 44% in the -48 h group. Blood flow returned to control values in the -48 h/+48 h group. Quantitative histology revealed no changes in the -24 h group, but a significant decrease in volume of the corpus luteum and volume of the luteal cell cytoplasm in the -48 h group. Corpus luteum volume was fully restored and luteal cell cytoplasm volume was partially restored in the -48 h/+48 h group. In contrast, the number of luteal cells, vessel space volume and capillary surface area did not change with oestrogen withdrawal.(ABSTRACT TRUNCATED AT 250 WORDS)

The ability of luteal tissue from rhesus monkeys, collected from spontaneous and prostaglandin F2 alpha (PGF2 alpha) induced luteolytic cycles, to secrete progesterone in response to hCG or dibutyryl cAMP in vitro was assessed. It was expected that the corpus luteum exposed to PGF2 alpha would behave in a similar manner to the corpus luteum of normal cycles undergoing luteal regression. PGF2 alpha (10 ng microliters-1 h-1) or vehicle (1 microliter h-1) was infused into the corpus luteum from 7 days after the preovulatory oestradiol surge. Lutectomy was performed at 2, 3 and 4 days after the start of the intraluteal infusion and at menses. Luteal progesterone content was determined and the secretion of progesterone in response to hCG (10 miu ml-1) or dibutyryl cAMP (5 mmol l-) was evaluated in vitro. A third group of monkeys underwent lutectomy sequentially during the luteal phase and were grouped by luteal age from days after the preovulatory oestradiol surge: early luteal (days 4-6), mid-luteal (days 7-9), late luteal (days 10-14) and menses. Immediately before luteal excision, an ovarian venous blood sample was taken for progesterone determination. Approximately 2-5 mg of minced luteal tissue was incubated at 37 degrees C in 2 ml of Krebs-Ringer bicarbonate buffer for 2 h in the presence or absence of hCG or dibutyryl cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

This study examined the effects of LH and PGE2 on progesterone secretion by small and large porcine luteal cells with or without low-density lipoproteins. Corpora lutea were isolated from gilts 13-14 days after administering gonadotrophins; enzymatically dissociated and small and large cells were isolated by elutriation. Culture plates, 24-well, were then seeded with 150,000 small or 30,000 large luteal cells suspended in 1 ml M199 medium supplemented with 5 micrograms insulin ml-1, 40 ng hydrocortisone ml-1 and with or without low-density lipoproteins (50 micrograms cholesterol ml-1) or PGE2. Cells were cultured for up to 24 h in a humidified incubator at 37 degrees C under 5% CO2 in air. The low-density lipoproteins stimulated (P < 0.05) progesterone secretion by large, but not small, luteal cells. Prostaglandin E2 stimulated (P < 0.05) progesterone production by large luteal cells in a dose-dependent manner, and the stimulatory effects of PGE2 were greater (P < 0.05) in the presence than in the absence of low-density lipoproteins. Progesterone secretion by small luteal cells was not significantly affected by PGE2. Progesterone production by small luteal cells was enhanced (P < 0.05) by LH, and the stimulatory effects of LH were greater (P < 0.05) in the presence than in the absence of low-density lipoproteins. In the absence of these lipoproteins, LH had no effect on progesterone secretion by large luteal cells; however, in the presence of low-density lipoproteins, LH increased (P < 0.05) progesterone secretion by large cells, though to a lesser (P < 0.05) extent than the effect of LH on small cells. These data demonstrate that progesterone secretion by porcine luteal cells is stimulated differentially by LH and PGE2 and that small luteal cells are more responsive to LH and PGE2 acts primarily on large luteal cells.

The aim of this study was to determine whether daily increasing doses of hCG could overcome luteal regression induced by PGF2 alpha in rhesus monkeys. Prostaglandin F2 alpha (10 ng microliters-1 h-1) or vehicle (tham buffer; 1 microliter h-1) was infused directly into the corpus luteum for 7 days, beginning 7 days after the preovulatory oestradiol surge. hCG was injected i.m. in increasing doses (15, 30, 60, 90, 180, 360 and 720 iu) for 7 days, starting on the eighth day after the preovulatory oestradiol surge, or 1 day after the initiation of luteal infusion of PGF2 alpha or vehicle. Monkeys receiving vehicle plus hCG on the same days served as controls. Daily progesterone, oestradiol and hCG concentrations were determined from blood drawn from the saphenous or femoral vein, and the duration of the luteal phases were recorded. Where intraluteally infused PGF2 alpha resulted in premature, functional luteolysis, hCG always inhibited the luteolytic effect of PGF2 alpha; the secretory patterns of progesterone and oestradiol were augmented, and peak values were reached in concert with the highest concentration of hCG in the blood, and the luteal phase was significantly increased compared with those of untreated monkeys or with monkeys treated with PGF2 alpha alone or vehicle. Treatment with hCG alone or with PGF2 alpha vehicle also resulted in maintained luteal function and a significantly longer luteal phase, but both progesterone and oestradiol concentrations began to decline before hCG reached peak values in the circulation.(ABSTRACT TRUNCATED AT 250 WORDS)

To assess the effects of prostaglandins and prolactin on corpus luteum function and regression, sterile-mated adult pseudopregnant rats hysterectomized on day 5 after mating were injected with indomethacin or prolactin. Daily samples of blood were collected via the tail, from day 12 to day 21, and assayed for serum concentrations of progesterone, 20 alpha-dihydroprogesterone and LH, whereafter corpora lutea and the remainder of ovaries were separated and the tissue content of PGF2 alpha, PGE2 and LH receptor mRNA were measured. Injections of prolactin (8 iu) s.c. or a low dose of indomethacin (200 micrograms kg-1) s.c. were administered twice a day, beginning on day 13 after mating. Both indomethacin and prolactin significantly increased serum progesterone concentrations (P < 0.05; n = 8), and extended the period of functional corpora lutea when compared with controls. Indomethacin, but not prolactin, lowered the concentration of serum 20 alpha-dihydroprogesterone. In the corpora lutea of indomethacin-treated animals, collected on day 21, both prostaglandins measured were reduced in concentration by 50% or more, compared with controls (P < 0.05; n = 8), whereas prolactin had no effect. Both prolactin and indomethacin treatment caused a substantial (tenfold) increase in the concentration of LH receptor mRNA, confined solely to the luteal compartment. These findings in vivo provide further evidence for a luteolytic role of locally synthesized prostaglandins in the rat ovary. Furthermore, prolactin can sustain corpus luteum function by exerting a luteotrophic effect during the late luteal phase, as judged by the stimulation of progesterone synthesis and the expression of LH receptors.

The relationship between the corpus luteum and the uterus in terms of the secretion of oxytocin and PGF2 alpha was investigated in free-living and captive roe deer Capreolus capreolus. During the breeding season the corpus luteum contained oxytocin and oxytocin-neurophysin mRNA, and secreted oxytocin in response to administration of the PGF2 alpha analogue cloprostenol. The oxytocin receptor was present in the uterus during the bleeding season and during delayed implantation; however, in contrast to the situation observed in other ruminants in which it has been studied, administered oxytocin did not stimulate uterine secretion of PGF2 alpha. Trophoblast interferon was undetectable at any stage of conceptus development. The absence of the mechanism underlying episodic uterine secretion of PGF2 alpha during luteolysis, which may account for the monoestry of roe deer, is consistent with the previously observed luteolytic effect of the PGF2 alpha analogue.

This study was undertaken to investigate the relationship between the inhibitory effect of restraint stress and the protective effect of the feto-placental luteotrophic factors on luteal function during mid-pregnancy in rats. The number of conceptuses was adjusted to one (1C group) or more than ten (FC group) on day 7 of pregnancy, and each rat received restraint stress from day 12 to day 17 of pregnancy. Restraint stress consisted of placing a rat individually in a small plastic holder three times a day for 1 h each time. Restraint stress significantly decreased serum progesterone concentration on day 17 of pregnancy in the 1C group, but not in the FC group. Restraint stress also decreased serum progesterone concentration on day 17 of pregnancy in the 1C group which received bilateral adrenalectomy on day 12 of pregnancy. The number of animals with fetal resorption in this group of rats (10 out of 14 animals) was significantly greater than in any other group of rats. The number of animals with fetal resorption in the adrenalectomized 1C group was significantly lower after daily injections of 4 mg progesterone from day 12 to day 17 of pregnancy. In the FC group of rats, even in adrenalectomized rats, restraint stress did not cause any changes in serum progesterone concentration or fetal loss. These data indicate that restraint stress is luteolytic and causes fetal loss during mid-pregnancy; this effect can be blocked by some factors from conceptuses, as occurred in the FC group.

In Schreibers' long-fingered bat (Miniopterus schreibersii) from South Africa, implantation is delayed from May to August during which time the corpus luteum is inactive and plasma progesterone and LH concentrations low. Treatment of pregnant long-fingered bats with exogenous prolactin for 10 days in early delayed implantation (May) resulted in changes in luteal ultrastructure that were consistent with increased steroidogenesis, high plasma progesterone concentrations and the initiation of implantation. Treatment of bats with hCG for 10 days in early delayed implantation induced similar changes in luteal ultrastructure and plasma progesterone concentrations, but did not initiate implantation. Treatment with exogenous progesterone during early delayed implantation had no influence on the corpus luteum or the conceptus. We conclude that prolactin is the most important pituitary hormone in the control of delayed implantation, although the route via which it induces implantation is unknown.

Although the decrease of progesterone in serum and in luteal tissue during luteal regression is well characterized, relatively little is known about changes in proteins produced by the corpus luteum during this time. The first objective was to examine changes in patterns of protein secretion that might be associated with functional and structural luteal regression. The second objective was to characterize the expression of two major secretory products of regressing corpora lutea. Thirty normally cyclic heifers were randomly assigned at day 15-16 of the oestrous cycle (oestrus = day 0) to be ovariectomized at 0 h (no PGF2 alpha; n = 5) or at 4, 8, 12, 24 or 48 h after PGF2 alpha-induced luteal regression (n = 5 per time point). Total cellular RNA was isolated from tissue frozen at the time of ovariectomy. Thin slices (< 1 mm) of tissue were placed in methionine-deficient minimum essential media with [35S]methionine and placed in a humidified CO2 incubator at 38 degrees C. Media and tissues were collected 6 h later. Changes in profiles of secreted proteins were analysed by one-dimensional SDS-PAGE. A number of proteins (relative molecular mass ranging from 14,300 to 200,000) were produced by luteal tissue at each time point (0-48 h). The major secretory proteins had relative molecular masses of approximately 21,500, 28,200, 43,700 and 46,000. Secretion of the relative molecular mass 46,000 protein(s) increased (P < 0.05) between 4 and 24 h after PGF2 alpha injection compared with the 0 h group.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin F2 alpha (PGF2 alpha)-induced release of ovarian oxytocin was investigated to determine whether the effect in vivo was local. [3H]PGF2 alpha infused downstream into a single ovarian lymphatic was transferred into the adjacent ovarian vasculature (estimated transfer 1.1 and 1.7%, two experiments). When unlabelled PGF2 alpha was infused in a similar manner (76 pmol min-1), there was a prompt eightfold increase in ovarian oxytocin release from the adjacent ovary containing a corpus luteum, but no effect on the opposite corpus luteum, showing that the effect was local. Instillation of 2% lignocaine into the ovarian vascular pedicle did not affect PGF2 alpha-induced oxytocin release, supporting the idea that neural mechanisms are not involved. Repeated doses of PGF2 alpha given close-arterially produced a successive reduction in oxytocin release. This effect was prevented by a prior infusion of insulin-like growth factor-I (IGF-I), which itself gave a small, but significant, increase in oxytocin release. The results show that PGF2 alpha in ovarian lymphatics acts locally and directly to stimulate ovarian oxytocin secretion, that repeated exposure of the corpus luteum to pulses of PGF2 alpha can result in tachyphylaxis, and that this latter effect can be ameliorated by IGF-I infused in vivo.

Immunization of ewes against the amino-terminal peptide (alpha N) of the pro-alpha-subunit of inhibin has been shown to reduce fertility, thought to be due to disruption of ovulation. The aims of this study were to examine the effects of active immunization of ewes against alpha N on circulating concentrations of FSH, LH and on ovarian inhibin and progesterone, and to relate these observations to number of corpora lutea and oocyte recovery rates. Ewes were immunized against one or both of two recombinant full length bovine-alpha N immunogens (FP1 and FP2). Three experiments were performed in which jugular venous plasma was sampled from control and immunized ewes: (1) hourly across the oestrous surge of gonadotrophins (Expt 1); (2) daily for one entire oestrous cycle, and in the subsequent cycle, oviducts were flushed to recover ovulated eggs (Expt 2); and (3) samples were taken at 10 min intervals during the follicular and luteal phases (Expt 3). Binding of 125I-labelled alpha N1-26 to serum was greater (P < 0.05) in immunized groups than in controls for all experiments. The number of eggs per corpus luteum recovered from the oviducts was lower (P < 0.05) in the alpha N-immunized groups (39%) than in controls (88%). There were more (P < 0.05) corpora lutea per ewe in FP2 immunized groups 4 (1.8 +/- 0.45) and 5 (1.75 +/- 0.5) than in the control group (1.13 +/- 0.13), but no increase in group 3 (FP1; 1.4 +/- 0.24).(ABSTRACT TRUNCATED AT 250 WORDS)

The objective of this study was to clarify the role of oestradiol in luteal function by examining its effect on the oxytocin stimulation of 15-keto-13,14-dihydro-prostaglandin F2 alpha (PGFM) concentrations in cyclic mares. In the first experiment, three groups of mares (4 per group) were given a bolus injection of 17 alpha-oestradiol (1 mg), oestradiol (1 mg) or vehicle on days 7, 9, 11, 13 and 15 of the cycle. Six hours later the mares were challenged with 10 iu oxytocin intravenously and frequent blood samples were taken from 15 min before to 15 min after for measurement of PGFM. Results showed a significant stimulatory effect of oestradiol (five times greater than controls at day 11; P < 0.05), but not of 17 alpha-oestradiol, on the oxytocin stimulation of PGFM. As a relatively large dose was given systemically in this experiment, a second experiment was performed to introduce a dose that was more physiological into the uterus. Small Silastic spheres (1 cm diameter) were impregnated with or without oestradiol at a concentration that gave a release rate similar to that of embryos at day 12 (10 ng h-1). These were inserted (one per mare) into the uterus of two groups of mares (five per group) on day 7. The mares were challenged with oxytocin on days 9, 11, 13 and 15 of the cycle and blood samples were taken as before for determination of PGFM. The results showed that oestradiol enhanced (four times greater than controls at day 13; P < 0.05) the oxytocin stimulation of PGFM concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

The luteolytic response to a prostaglandin F2 alpha analogue, cloprostenol, was investigated in vivo and in vitro at defined stages of the luteal phase. In vivo administration of cloprostenol to female marmoset monkeys on day 3 after ovulation had no effect on plasma progesterone concentrations, whereas administration on day 14 after ovulation reduced plasma progesterone to preovulatory concentrations within 4 h. To identify the cellular basis for this luteolytic action, marmoset luteal tissue obtained on days 3, 6 and 14 after ovulation was incubated in vitro and progesterone production, cAMP accumulation and phosphoinositide (PI) turnover measured in response to cloprostenol, human chorionic gonadotrophin (hCG) with or without cloprostenol, or dibutyryl-cAMP with or without cloprostenol. Progesterone production was stimulated by both hCG and dbcAMP at all stages of the luteal phase. Although neither hCG nor dbcAMP had any significant effects on PI turnover, hCG also increased cAMP accumulation. In marmoset luteal tissue obtained on day 3 after ovulation, cloprostenol had no significant effect on basal or hCG/dbcAMP-stimulated progesterone production but significantly stimulated PI turnover. In contrast, on days 6 and 14 after ovulation, cloprostenol significantly inhibited hCG- and dbcAMP-stimulated progesterone production and the cAMP response to hCG, but had no significant effect on PI turnover. Since progesterone production by the marmoset corpus luteum depends on the luteotrophic support of luteinizing hormone (LH), these observations suggest that the luteolytic action of cloprostenol in vivo involves the inhibition of LH/hCG action at sites both prior and subsequent to cAMP accumulation. However, such luteolytic effects do not appear to require the generation of inositol phosphates by increased PI turnover.

Mature heifers (n = 31) were infused with either saline or noradrenaline (0.3 microgram kg-1 min-1) via a cannula inserted into the aorta abdominalis through the coccygeal artery (cranial to the origin of the ovarian artery). Noradrenaline was infused for three periods of 30 min on days 11 and 12 and on days 15 and 16 of the oestrous cycle. After the noradrenaline treatment, saline was given for 1 h in the same way. During each infusion, peripheral blood samples were collected for progesterone and oxytocin determination every 5-10 min and then once a day until oestrus. As a control, four heifers were infused with saline in latin square design on days 11, 12 and on days 15, 16; they were bled once a day until oestrus. Other heifers were infused on the same days, but 1 h after the last infusion of noradrenaline, 500 micrograms of prostaglandin F2 alpha (PGF2 alpha) analogue was injected, to measure any remaining luteal oxytocin. For comparison four heifers were injected with PGF2 alpha analogue alone on day 12 and four others on day 16. Blood samples were taken as described previously. Each infusion of noradrenaline stimulated (P < 0.01) progesterone secretion. There was a significant (P < 0.05) response of oxytocin to each noradrenaline stimulation on days 11 and 12, although on each day the response to the second infusion was reduced and further reduced after the third infusion. On days 15 and 16, only the first noradrenaline infusion caused a clear surge of oxytocin with much smaller increases in oxytocin secretion after subsequent infusions.(ABSTRACT TRUNCATED AT 250 WORDS)

Activities of superoxide dismutase (SOD) and lipid peroxide (LPO) were studied in corpora lutea of pregnant rats. SOD activities, both Mn-SOD and Cu,Zn-SOD, gradually increased in the corpora lutea until day 15 of pregnancy and decreased thereafter until day 21 of pregnancy, in a similar manner to serum progesterone concentration. LPO activities remained low until day 15 of pregnancy, but increased rapidly after day 15 to day 21 of pregnancy. Incubation of the dispersed luteal cells from day 15 of pregnancy in vitro showed that FeSO4 and ascorbic acid, which induce lipid peroxidation, significantly inhibited progesterone secretion. The inhibitory effects of FeSO4 and ascorbic acid were blocked by the simultaneous addition of alpha-tocopherol. These results suggest important roles for SOD and LPO in regulating luteal function during pregnancy.

Oxytocin infusions were initiated on day 10 of the oestrous cycle in ewes, and luteal regression was induced by injection of 100 micrograms cloprostenol on day 12. Blood samples were collected at frequent intervals via an indwelling jugular vein cannula to measure concentrations of progesterone and luteinizing hormone (LH) during the luteal and follicular phases in saline (n = 6) and oxytocin (n = 5) infused animals. The oxytocin infusion maintained peripheral plasma concentrations of 53 +/- 3.2 pg oxytocin ml-1 (mean +/- SEM) compared with values of about 1 pg ml-1 during oestrus in control ewes. Oxytocin infusion had no effect on luteal phase progesterone concentrations, the timing of luteolysis, basal luteinizing hormone (LH) secretion, LH pulse frequency, or the timing or height of the LH surge. Treated ewes came into oestrus significantly earlier than controls (P < 0.05) but ovulated normally. Uterine samples collected 96 h after cloprostenol injection (approximately day 2 of the cycle) showed that oxytocin receptor concentrations were significantly higher in the endometrium in ewes that had been given a 5 day oxytocin infusion than in control animals (556 and 262 fmol mg-1 protein, respectively: geometric means from ANOVA, P < 0.001), whereas myometrial receptor concentrations were not affected (113 and 162 fmol mg-1 protein, respectively). We conclude that the previously reported delay in luteal development caused by oxytocin infusion (Wathes et al., 1991) is not due to the inhibition or delay of ovulation, but must instead occur via a direct influence on the developing corpus luteum.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor I (IGF-I) is involved in paracrine/autocrine regulation of gonadal steroidogenesis and peptide hormone biosynthesis. This study was designed to determine whether IGF-I alone, or an interaction of IGF-I, is involved in augmenting the actions of luteinizing hormone (LH) and prolactin in controlling relaxin and progesterone secretion from ageing corpora lutea of hysterectomized gilts at days 110, 113 and 116 after oestrus. Luteal tissue slices were incubated for 8 h with IGF-I (0, 50, 300 ng ml-1), LH (0, 100, 1000 ng ml-1), and prolactin (0, 100, 1000 ng ml-1) alone or in combination. Progesterone and relaxin concentrations were determined by radioimmunoassay of spent medium and of homogenates from luteal tissue slices before and after incubation. Porcine luteal tissue from day 110 had a net output of 25 ng progesterone and 26 ng relaxin in the control and of 65 ng progesterone and 2125 ng relaxin in the combined IGF-I, LH and prolactin treatment mg-1 of luteal tissue, respectively. IGF-I, LH and prolactin alone or in combination significantly increased (P < 0.01) progesterone production by luteal tissue from day 110, but they were partially effective at day 113 and ineffective at day 116. By contrast, the same hormone treatments increased relaxin production by luteal tissue from days 110 and 113. Even at day 116, prolactin alone or with LH or IGF-I continued to stimulate relaxin production. In conclusion, IGF-I augments the ability of prolactin and LH to increase relaxin production by ageing corpora lutea; however, a decrease in progesterone secretion and an increase in relaxin secretion at day 113 indicate that different mechanisms control progesterone and relaxin secretion in pigs.

The effect of insulin-like growth factors (IGFs) and insulin on the release of progesterone and oxytocin from bovine corpus luteum was investigated at early (days 5-7), mid- (days 8-12) and late (days 15-18) luteal phases of the oestrous cycle in an in vitro microdialysis system. The expression of specific receptors was evaluated in bovine corpora lutea of the respective luteal stages. A 30 min infusion of IGF-1, IGF-2 (1.3, 13 and 130 nmol l-1) or insulin (13, 130 and 1300 nmol l-1) caused a stimulation of the release of progesterone (P < 0.05). IGF-1 was most effective in releasing progesterone. Oxytocin release from corpora lutea was stimulated by insulin at all doses tested (13-1300 nmol l-1), whereas the IGFs were only effective at the highest dose (130 nmol l-1) applied. The high doses of IGFs (130 nmol l-1) and insulin (1300 nmol l-1) stimulated the release of progesterone and oxytocin throughout the luteal phase (P < 0.05). For all three peptides, greatest stimulation was seen during the late luteal phase (days 15-18 of the oestrous cycle) with the peak of progesterone release directly related to peptide infusion (P < 0.05). In addition, IGF-1 stimulated total release of progesterone (units in 4 h) after the beginning of the stimulation during this phase (P < 0.05). IGF-1 caused a gradual increase of progesterone even beyond the time of peptide perfusion, whereas IGF-2 and insulin stimulated progesterone release only during the peptide perfusion. Distinct receptors for IGF-1 and IGF-2 were present in corpora lutea membrane preparations at all stages investigated. Specific binding for insulin was also seen in all stages of the cycle without any cycle-dependent changes in the amount of binding. The displacement of labelled insulin by unlabelled IGF-1 and IGF-2 did not show the rank of order that has been described as typical for insulin receptors (i.e. insulin > IGF-1 > IGF-2), but comparable binding affinities were observed for the three unlabelled ligands. Specific binding of IGF-2 was markedly higher than that of IGF-1 or insulin throughout the cycle (1.9- and 4.9-fold higher compared with IGF-1 and insulin, respectively). Receptor specificity did not change during luteal development. Binding affinity and capacity of IGF-1 receptor was constant throughout the oestrous cycle. Specific IGF-2 binding increased and showed a positive co-operativity towards the end of the cycle. Specific binding of insulin was not significantly different in the three luteal stages examined.

Concentrations of progesterone and luteinizing hormone in plasma were analysed for two consecutive years in samples from nonpregnant female roe deer. Three animals were treated with monthly prostaglandin injections (325 micrograms cloprostenol) from October 1989 to April 1990 and from October 1990 to March 1991, and three were kept as controls. In control animals, a small increase in progesterone concentrations in July 1990 occurred at the same time as the commencement of the rut in other husbanded roe deer. In prostaglandin-treated animals, progesterone concentration was high at the time of the rut and remained so until late February 1990. After the next rut (August 1990), progesterone concentration remained high until March 1991. Between October and February-March, injections of prostaglandins induced dramatic, but temporary (lasting 72 h), decreases in plasma progesterone concentrations, indicating luteal regression and subsequent ovulation. We infer that roe deer can ovulate repeatedly and should therefore not be regarded as an obligate monoestrous species.

Luteal cells were obtained by digestion of luteal tissue of cyclic (day 12) and early pregnant (days 12, 20 and 30) pigs. Suspensions of the dispersed luteal cells (5 x 10(4) cells ml-1) were incubated for 2 h in minimum essential medium (MEM) alone (control) and MEM with different concentrations of prostaglandin F2 alpha (PGF2 alpha) and PGE2 (0.01, 0.1, 1, 10, 100 and 1000 ng ml-1) and luteinizing hormone (LH) 100 and 1000 ng ml-1, or with combinations of LH + PGF2 alpha and LH + PGE2. Net progesterone production was measured in the incubation media by direct radioimmunoassay. The overall response pattern of the luteal cells to exogenous hormones on day 12 of the oestrous cycle and pregnancy differed (P < 0.05) from treatment on day 20 and 30 of pregnancy. In general progesterone production was higher (P < 0.05) and the response to PGF2 alpha and PGE2 treatment was most obvious on day 12 of the oestrous cycle and pregnancy. Overall, PGF2 alpha stimulated progesterone production in a dose-dependent manner (P < 0.05). The response to PGE2 was of a quadratic nature (P < 0.05) in which the lowest and the highest doses of PGE2 were associated with a greater production of progesterone than were the intermediate doses. Treatment of luteal cells with PGF2 alpha + LH or PGE2 + LH caused overall inhibition (P < 0.05) of progesterone production compared with treatment with each hormone alone. This interaction was not affected by the dose of LH used. These findings indicate that PGF2 alpha and PGE2 are involved in the autocrine control of corpus luteum function.

Prostaglandin F2 alpha (PGF2 alpha) inhibits lipoprotein-stimulated progesterone production by bovine luteal cells in vitro and the objective of this study was to localize the site of action of PGF2 alpha. Cultured bovine luteal cells were treated with PGF2 alpha for seven days, and then with either lipoproteins or 25-hydroxycholesterol in the presence of aminoglutethimide (which inhibits cholesterol side-chain cleavage) for the final 48 h. The effects of PGF2 alpha on progesterone production, cellular cholesterol content, mitochondrial cholesterol content and cholesterol side-chain cleavage activity were determined. As expected, PGF2 alpha inhibited (P less than 0.05) lipoprotein-stimulated progesterone production. However, PGF2 alpha did not inhibit low-density lipoprotein-stimulated, or high density lipoprotein-stimulated, increases in cellular cholesterol (P less than 0.05) or inhibit lipoprotein-induced increases in mitochondrial cholesterol content (P less than 0.05). Additionally, cholesterol content of mitochondria increased (P less than 0.05) in the presence of PGF2 alpha alone. To determine if the PGF2 alpha-induced inhibition of steroidogenesis occurred at, or after, the side-chain cleavage reaction, we treated cells with the readily diffusable sterol, 25-hydroxycholesterol. Prostaglandin F2 alpha did not inhibit 25-hydroxycholesterol-stimulated progesterone production (P less than 0.05). Prostaglandin F2 alpha may therefore exert its luteolytic effect at a site after cholesterol transport to the mitochondria but before cholesterol side-chain cleavage.

This study was conducted to determine whether intrauterine infusion of recombinant bovine interferon-alpha I1 (rboIFN-alpha I1), which has 70% sequence identity to bovine trophoblast protein-1, will prevent regression of corpora lutea anticipated to have a short lifespan. Twenty-six beef cows in good body condition were allotted to four treatment groups at parturition in a 2 x 2 factorial design. Treatments were: group 1, saline; group 2, rboIFN-alpha I1; group 3, norgestomet-saline; and group 4, norgestomet-rboIFN-alpha I1. Norgestomet implants were inserted on days 21-24 postpartum and removed 9 days later (before injection of human chorionic gonadotrophin (hCG)). Ovulation was induced 30 to 33 days postpartum with 5000 or 10,000 iu hCG. Groups 1 (n = 7) and 3 (n = 5) were given intrauterine infusions (rectocervical approach) twice daily with saline on days 1-12 or 13-24 after hCG injection, respectively. Cows allotted to groups 2 (n = 8) and 4 (n = 6) were given intrauterine infusions (rectocervical approach) of 2 mg rboIFN-alpha I1 twice daily on days 1-12 or 13-24 after hCG injection, respectively. Treatment with both norgestomet and rboIFN-alpha I1 delayed (P less than 0.01) luteolysis. Lengths of luteal phases (days; mean +/- SEM) were 8.4 +/- 0.7 (group 1, saline), 14.1 +/- 1.0 (group 2, rboIFN-alpha I1), 18.6 +/- 1.3 (group 3, norgestomet-saline) and 20.8 +/- 1.2 (group 4, norgestomet-rboIFN-alpha I1). Concentration of progesterone in serum was similar among all groups the first 6 days following hCG-induced ovulation, but differed (P less than 0.01) thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous studies have suggested that prostaglandin F-2 alpha (PGF-2 alpha) may have a role in luteolysis in rabbits. Rabbits (4-6/group) were given a single injection of saline, or 100, 500 or 2500 micrograms PGF-2 alpha (i.m.) on Day 7, 9, 12 or 15 of pregnancy or pseudopregnancy. Daily blood samples were taken via the marginal ear vein before and for 3 days after the PGF-2 alpha injection. Concentrations of serum progesterone were determined by radioimmunoassay in pseudopregnant rabbits. There were no significant differences between PGF-2 alpha-treated and control rabbits on Days 7 or 9. On Day 12 of pseudopregnancy, progesterone concentration was significantly (P less than 0.05) lower in treated than in control rabbits, the effect being dose dependent. On Day 15 of pseudopregnancy, it was not possible to distinguish between controls and treated groups because luteolysis occurred in all rabbits. In contrast, on Days 7 and 9 of pregnancy, the concentration of progesterone in treated groups was lower than in the control groups (P less than 0.05), the effect being dose dependent. This difference was maintained throughout the sampling period and resulted in termination of pregnancy. By Day 12 of pregnancy, the response to PGF-2 alpha was transient, with a significant decline in progesterone for only 2 days, followed by a return to control concentrations and normal delivery of litters. On Day 15 of pregnancy, no treatment with PGF-2 alpha significantly altered progesterone concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

After parturition, eight sows were zero weaned by removing all piglets 6 h after birth; a further 18 sows suckled at least ten piglets each. Blood samples were collected on Day 4 after zero weaning or on Days 4, 14 and 21 of lactation and the sampling frequency increased during suckling bouts. Ovaries were recovered from sows on these days and corpora lutea were either extracted for estimation of relaxin and progesterone concentration, fixed for immunohistochemical analysis or incubated in vitro in the presence or absence of luteinizing hormone (LH) or oxytocin. Luteal weight and progesterone were higher in the zero-weaned sows than in lactating sows (P less than 0.05 and less than 0.001, respectively); relaxin content was below detection by Day 14. This was supported by immunohistochemical staining for relaxin, which showed limited immunostaining in zero-weaned and Day 4 sows, but none in the tissue recovered on Days 14 and 21, which showed typical signs of regression. Secretion of progesterone and relaxin by luteal tissue in vitro was highest in zero-weaned sows (P less than 0.05), decreased as lactation progressed and neither LH nor oxytocin had any significant effect. Concentrations of plasma relaxin were all less than 0.2 ng/ml in three of the four zero-weaned and Day-4-suckled sows assayed; there was no detectable increase during suckling bouts. It was concluded that during lactation the old corpus luteum of pregnancy is not able to release relaxin in response to suckling in vivo or to oxytocin treatment in vitro.

Early corpus luteum development in nonpregnant and pregnant goats was characterized by a steady increase in peripheral plasma concentrations of progesterone and a high release of prostacyclin (PGI-2) but low release of prostaglandin F-2 alpha (PGF-2 alpha). Jugular administration of oxytocin antagonist (OXA) (0.2 microgram/kg/day) on the day of oestrus and for 3 days thereafter to cyclic and mated goats, significantly (P less than 0.001) inhibited progesterone and prostaglandin secretion and reduced conception rate. Co-administration of PGI-2 (200 micrograms/day) with OXA resulted in a steady increase of progesterone and establishment of pregnancy, but co-administration of PGF-2 alpha (175 micrograms/day) with OXA had no effect. It is suggested that oxytocin is required for early development of the corpus luteum and such effects may be mediated via PGI-2 production.

Corpora lutea (CL) were obtained from immature rats primed with pregnant mares' serum gonadotrophin followed by human chorionic gonadotrophin (hCG). Two days after hCG, CL were isolated, placed in perifusion culture and exposed to control medium or specific pulses of luteinizing hormone (LH). In Expt 1, a frequency of 1 pulse LH/h (amplitude 500 pg/ml, duration 40 min, 30 ng/min) increased progesterone secretion compared with control values (P less than 0.05). In Expt 2, LH rate was held constant and the amplitude and duration of a single LH pulse varied; 250 and 500 ng LH/ml initially stimulated progesterone secretion equivalently, but increasing the duration of the LH pulse prolonged high progesterone secretion. These observations suggest that at less than or equal to 500 ng LH/ml, once a stimulatory amplitude is obtained, higher amplitudes do not further increase progesterone secretion, while increasing pulse duration further enhances progesterone secretion. In Expt 3, the LH pulse amplitude was 250 ng/ml and the rate set at 0, 5 or 30 ng LH/min; only 30 ng LH/min resulted in sustained stimulation of progesterone (P less than 0.05). Taken together, these data demonstrate that the characteristics which determine whether an LH pulse will be stimulatory include not only amplitude and duration but also the rate at which an amplitude is obtained.

Immature rats were injected with pregnant mares' serum gonadotrophin followed by human chorionic gonadotrophin (hCG). Ovaries were removed 0, 2, 5 or 8 days after hCG and either prepared for morphometric analysis or perifused with 0, 5 or 30 ng luteinizing hormone (LH)/min. In a second study, ovaries were removed on Day 2 or 8 and perifused with 0.1 mg 8-br-cyclic adenosine 5'-phosphate/ml (8-br-cAMP). On Day 0, the granulosa cells of the preovulatory follicles were small (53 +/- 0.5 microns2) with a cytoplasmic to nuclear (Cy:Nu) ratio less than or equal to 1.5. By Day 2, corpora lutea (CL) were present and composed of 95% small luteal cells (diameter less than 125 microns2, Cy:Nu greater than or equal to 3.0) and 5% large luteal cells (diameter greater than 125 microns2, Cy:Nu ratio greater than or equal to 3.0). The percentage of large luteal cells increased to 36 +/- 7% by Day 5, suggesting that they are derived from a select population of small luteal cells. Basal progesterone secretion increased from 38 +/- 5 on Day 0 to 1010 +/- 48 pg/mg/ml on Day 8. The rate of 5 ng LH/min stimulated progesterone secretion on Days 0, 2 and 8; 30 ng LH/min stimulated progesterone secretion on Days 0, 2 and 8, but not on Day 5; 8-br-cAMP stimulated progesterone secretion on both Days 2 and 8. These data demonstrate that once granulosa cells are induced to luteinize they lose their capacity to secrete progesterone in response to 5 ng LH/min and do not regain their responsiveness to LH rate until they completely differentiate. The loss of this LH responsiveness appears to be due to an inability to stimulate sufficient intracellular cAMP concentrations, since cAMP stimulates progesterone secretion on both Days 2 and 8.

Ewes were treated with exogenous follicle-stimulating hormone (FSH) and oestrus was synchronized using either a dual prostaglandin F-2 alpha (PGF-2 alpha) injection regimen or pessaries impregnated with medroxy progesterone acetate (MAP). Natural cycling ewes served as controls. After oestrus or AI (Day 0), corpora lutea (CL) were enucleated surgically from the left and right ovaries on Days 3 and 6, respectively. The incidence of premature luteolysis was related (P less than 0.05) to PGF-2 alpha treatment and occurred in 7 of 8 ewes compared with 0 of 4 controls and 1 of 8 MAP-exposed females. Sheep with regressing CL had lower circulating and intraluteal progesterone concentrations and fewer total and small dissociated luteal cells on Day 3 than gonadotrophin-treated counterparts with normal CL. Progesterone concentration in the serum and luteal tissue was higher (P less than 0.05) in gonadotrophin-treated ewes with normal CL than in the controls; but luteinizing hormone (LH) receptors/cell were not different on Days 3 and 6. There were no apparent differences in the temporal patterns of circulating oestradiol-17 beta, FSH and LH. High progesterone in gonadotrophin-treated ewes with normal CL coincided with an increase in total luteal mass and numbers of cells, which were primarily reflected in more small luteal cells than in control ewes. Gonadotrophin-treated ewes with regressing CL on Day 3 tended (P less than 0.10) to have fewer small luteal cells and fewer (P less than 0.05) low-affinity PGF-2 alpha binding sites than sheep with normal CL. By Day 6, luteal integrity and cell viability was absent in ewes with prematurely regressed CL. These data demonstrate that (i) the incidence of premature luteal regression is highly correlated with the use of PGF-2 alpha; (ii) this abnormal luteal tissue is functionally competent for 2-3 days after ovulation, but deteriorates rapidly thereafter and (iii) luteal-dysfunctioning ewes experience a reduction in numbers of small luteal cells without a significant change in luteal mass by Day 3 and, overall, have fewer low-affinity PGF-2 alpha binding sites.

Implants containing vehicle or oestradiol-17 beta (10 mg) were placed into pairs of corpora lutea (CL) with and without prostaglandin F-2 alpha (PGF-2 alpha) (100 micrograms) on Day 11 and CL were collected on Day 19, in cyclic gilts (Exp. 1). The results demonstrated that CL implanted with PGF-2 alpha with or without oestradiol-17 beta had a markedly lower (P less than 0.01) weight (mg) and progesterone concentration (ng/mg) than CL with vehicle-or oestradiol-17 beta-implanted or unimplanted CL, which were similar (149 and 7.2 vs. 304 and 49.6, respectively). In Exp. 2, CL implanted with vehicle, oestradiol-17 beta or PGE-2 remained fully functional until Day 19, whereas CL implanted with oestradiol-17 beta +/- PGF-2 alpha and PGE-2 + PGF-2 alpha exhibited lower (P less than 0.05) weight and progesterone concentrations; CL implanted with PGE-2 + PGF-2 alpha were heavier (P less than 0.05) and tended (P less than 0.10) to have greater progesterone concentrations than CL implanted with oestradiol-17 beta + PGF-2 alpha. In Exp. 3, a dose-dependent (P less than 0.05) effect of PGE-2 on preventing regression induced by PGF-2 alpha was observed on Day 19. These data demonstrate a direct effect of PGE-2, but not of oestradiol-17 beta in protecting the CL against luteolysis induced by PGF-2 alpha.

The function of oxytocin receptors in the corpus luteum of pregnant ewes was investigated by infusing saline or oxytocin (100 ng/min) into the utero-ovarian artery of pregnant ewes (62 +/- 5 days, n = 12). During a 4-h infusion, plasma oxytocin (OT) concentration increased to 268 +/- 80 pg OT/ml in the OT-infused group and remained unchanged at 2.5 +/- 1.5 pg OT/ml in the saline-infused group. Progesterone concentration in jugular venous plasma (17 +/- 9 ng/ml) rapidly decreased during oxytocin infusion to 59 +/- 10% and 26 +/- 9% of control at 1.5 and 2 h, respectively; the utero-ovarian venous concentration of 64 +/- 38 ng/ml decreased by a similar magnitude during oxytocin infusion. Electron microscopy of corpora lutea, removed at the end of the experiments, showed no indication of luteolytic changes following oxytocin infusion. It was concluded that oxytocin markedly and rapidly reduces progesterone secretion in pregnant ewes.

In Experiment 1, an osmotic minipump containing oxytocin was implanted s.c. in ewes for 12 days beginning on Day 10 of the oestrous cycle, producing approximately 100 pg oxytocin/ml in the plasma. Two days after the start of infusion, all ewes were injected with 100 micrograms cloprostenol and placed with a fertile ram. At slaughter 22 days later, 9 (75%) of the 12 control (saline-infused) ewes were pregnant compared with 1 (11%) of the 9 ewes infused with oxytocin. In the control group, midcycle plasma concentrations of oxytocin were significantly higher in nonpregnant than in pregnant ewes. In Experiment 2, an infertile ram was used throughout to avoid any possible effects of pregnancy and oxytocin infusions were given at different stages of the oestrous cycle. Otherwise the protocol was similar to that in Exp. 1. Oxytocin infusion during luteolysis and the early follicular phase had no effect on the subsequent progesterone secretion pattern, but infusions beginning the day before cloprostenol-induced luteolysis and lasting for 7 or 12 days and infusions beginning on the day of oestrus for 4 days all delayed the subsequent rise in plasma progesterone by approximately 3-4 days. In these animals, the cycle tended to be longer. It was concluded that an appropriate oxytocin secretion pattern may be necessary for the establishment of pregnancy in ewes and that a high circulating oxytocin concentration during the early luteal phase delays the development of the young corpus luteum.

The effects of chronic treatment with norgestomet on follicular dynamics, corpus luteum growth and function as well as the temporal relationships among body temperature, oestrous behaviour, the luteinizing hormone (LH) surge and ovulation following implant removal were studied in 16 Holstein heifers. Oestrous cycles of the heifers were initially synchronized using 2 injections of prostaglandin F-2 alpha (PGF-2 alpha) 12 days apart. The heifers were then implanted with a norgestomet ear implant for 9 days, beginning either at the middle of the synchronized cycle (dioestrus) or at the end of the synchronized cycle (pro-oestrus). Follicular dynamics, corpus luteum growth and regression, and plasma progesterone were not affected by norgestomet treatment at dioestrus. The dominant follicle present at the time of norgestomet implantation in the pro-oestrus group was maintained during the 9-day implant period of 6 of 8 heifers and ovulated after implant removal. Time from implant removal to onset of standing oestrus and time to LH peak following implant removal were highly correlated with the time of ovulation (r = 0.92 and 0.96, respectively). Onset of standing oestrus and the LH peak and the onset of standing oestrus and peak vaginal and rectal temperatures were also highly correlated (r = 0.96, 0.82 and 0.81, respectively). It is concluded that any decrease in pregnancy rates following treatment with norgestomet is not due to asynchrony among oestrus, the LH surge and ovulation.

Clomiphene citrate (2 mg/kg body wt) given on the day of mating can block or interrupt pregnancy in guinea-pigs. Corpus luteum function, uterine histology, implantation and embryo development were studied in clomiphene-treated and control animals on Days 5, 9 and 20 of pregnancy. Following treatment, only 25% of the females were regularly pregnant, presenting large and healthy foetuses. The other females examined showed either pregnancy with embryos undergoing resorption or no sign of pregnancy. In these females, corpus luteum size was reduced, progesterone concentrations were very low and the endometrial glands and the epithelium were often altered. It is concluded that clomiphene causes a reduction in fertility by altering the uterus and, by directly or indirectly inducing luteolysis, causes later pregnancy loss.

Oxytocin was administered to Dorset and Shropshire ewes in one experiment and to Dorset ewes in a further 4 experiments. In Exp. 1, concentrations of plasma progesterone and lengths of the oestrous cycle in ewes given oxytocin subcutaneously twice a day on Days 0-3, 2-5, 4-7, 6-9, 8-11, 10-13, 12-15 or 14-17 were similar to those of control ewes. In Exp. 2, intraluteal infusions of oxytocin from Day 2 to Day 9 after oestrus had no effect on concentration of progesterone, weight of CL collected on Day 9 or length of the oestrous cycle. In Exp. 3, intraluteal infusions of oxytocin on Days 10-15 after oestrus had no effect on weight of CL collected on Day 15. In Exp. 4, s.c. injections of oxytocin on Days 3-6 after oestrus had no effect on weight of CL collected on Day 9, concentrations of progesterone or length of the oestrous cycle. In Exp. 5, s.c. injections of oxytocin twice a day did not affect the maintenance and outcome of pregnancy in lactating and nonlactating ewes. Exogenous oxytocin, therefore, does not appear to affect luteal function at any stage of the ovine oestrous cycle although oxytocin has been reported by others to alter ovine CL function.

Ovine granulosa cells respond, through transcriptional mechanisms, to preovulatory concentrations of gonadotrophins by secreting an Mr 30,000 polypeptide. To determine the stages of the oestrous cycle at which this polypeptide is secreted, corpora lutea were collected on Days 3, 7, 10, 13 and 16 (Day 0 = oestrus; n = 4 per group), cut into 1-mm slices, and incubated for 6-7 h with [35S]methionine. Radiolabelled polypeptides of intra- and extra-cellular origin were separated by polyacrylamide gel electrophoresis and quantitated by densitometry. The Mr 30,000 polypeptide was secreted at all stages of the luteal phase tested (Days 3-16), and represented approximately 24% of the total labelled polypeptide present in the medium; polypeptides of approximate Mr 14,000, 25,000 and 46,000 accounted for most of the other secreted proteins. Neither pituitary hormones (LH, FSH, prolactin) nor cholera toxin (chosen to activate adenylate cyclase) affected the rate of production of Mr 30,000 polypeptide, indicating that, once secretion has been initiated in the granulosa cells, it is not readily modulated by hormonal intervention after luteinization. Incubation of luteinized granulosa cells with tunicamycin (inhibits N-linked glycosylation reactions) showed that the secreted polypeptide consists of a heavily glycosylated amino acid backbone of approximately Mr 20,000. Western blot analysis established further that the polypeptide was not an inhibin subunit. However, NH2-terminal amino acid sequencing of the first 25 amino acids revealed a 68% sequence identity between the secreted polypeptide (Mr 30,000) and a human tissue inhibitor of metalloproteinases.