The effect of human chorionic gonadotrophin on the expression of progesterone receptors in human luteal cells in vivo and in vitro

The human corpus luteum expresses genomic progesterone receptors (PRs) suggesting that progesterone may have an autocrine or paracrine role in luteal function. We hypothesised that the reduction in luteal PR reported in the late-luteal phase augmented progesterone withdrawal and had a role in luteolysis. We therefore tested the hypothesis that luteal rescue with human chorionic gonadotrophin (hCG) would maintain PR expression. PR was immunolocalised to different cell types in human corpora lutea (n = 35) from different stages of the luteal phase and after luteal rescue with exogenous hCG. There was no change in the staining intensity of theca-lutein cell or stromal cell PR throughout the luteal phase or after luteal rescue. In the late-luteal phase, granulosa-lutein cell PR immunostaining was reduced (P < 0.05) but the trend to reduction was also seen after luteal rescue with hCG (P = 0.055). To further investigate the effect of hCG on granulosa-lutein cell PR expression, an in vitro model system of cultured human luteinised granulosa cells was studied. Cells were cultured for 12-13 days exposed to different patterns of hCG and aminoglutethamide to manipulate progesterone secretion (P < 0.0001). Expression of PR A/B and PR B isoforms was examined by quantitative real-time RT-PCR. PR A/B mRNA was lower (P < 0.05) after 11-13 days of culture than after 7 days of culture. This reduction could not be prevented by hCG in the presence (P < 0.05) or absence (P < 0.05) of stimulated progesterone secretion. The expression of PR B mRNA showed a similar pattern (P = 0.054). Simulated early pregnancy in vivo and hCG treatment of luteinised granulosa cells in vitro did not appear to prevent the down-regulation of PR seen during luteolysis.     

Role of gap junctions in regulation of progesterone secretion by ovine luteal cells in vitro

To evaluate the role of gap junctions in the regulation of progesterone secretion, two experiments were conducted. In Experiment 1, luteal cells obtained on days 5, 10, and 15 were cultured overnight at densities of 50 x 10(3), 100 x 10(3), 300 x 10(3), and 600 x 10(3) cells/dish in medium containing: (1) no treatment (control), (2) LH, or (3) dbcAMP. In Experiment 2, luteal cells from days 5 and 10 of the estrous cycle were transfected with siRNA, which targeted the connexin (Cx) 43 gene. In Experiment 1, progesterone secretion, Cx43 mRNA expression, and the rates of gap junctional intercellular communication (GJIC), were affected by the day of the estrous cycle, cell density, and treatments (LH or dbcAMP). The changes in progesterone secretion were positively correlated with the changes in Cx43 mRNA expression and the rates of GJIC. Cx43 was detected on the luteal cell borders in every culture, and luteal cells expressed 3beta-hydroxysteroid dehydrogenase. In Experiment 2, two Cx43 gene-targeted sequences decreased Cx43 mRNA expression and progesterone production by luteal cells. The changes in Cx43 mRNA expression were positively correlated with changes in progesterone concentration in media. Thus, our data demonstrate a relationship between gap junctions and progesterone secretion that was supported by (1) the positive correlations between progesterone secretion and Cx43 mRNA expression and GJIC of luteal cells and (2) the inhibition of Cx43 mRNA expression by siRNA that resulted in decreased production of progesterone by luteal cells. This suggests that gap junctions may be involved in the regulation of steroidogenesis in the ovine corpus luteum.     

Delayed effect of low progesterone concentrations on bovine uterine PGF(2alpha) secretion in the subsequent oestrous cycle

Low progesterone concentrations during the bovine oestrous cycle induce enhanced responsiveness to oxytocin challenge late in the luteal phase of the same cycle. The delayed effect of low progesterone concentrations during one oestrous cycle on uterine PGF(2alpha) secretion after oxytocin challenge on day 15 or 16 of the subsequent cycle was studied by measuring the concentrations of the major PGF(2alpha) metabolite (13,14-dihydro-15-keto PGF(2alpha); PGFM) in plasma. Two experiments were conducted, differing in the type of progesterone treatment and in the shape of the low progesterone concentration curves. In Expt 1, progesterone supplementation with intravaginal progesterone inserts, with or without an active corpus luteum, was used to obtain high, or low and constant plasma progesterone concentrations, respectively. In Expt 2, untreated cows, representing high progesterone treatment, were compared with cows that had low but increasing plasma progesterone concentrations that were achieved by manipulating endogenous progesterone secretion of the corpus luteum. Neither experiment revealed any differences in plasma progesterone concentrations between the high and low progesterone groups in the subsequent oestrous cycle. In both experiments, both groups had similar basal concentrations of PGFM on day 15 (Expt 1) or 16 (Expt 2) of the subsequent oestrous cycle, 18 days after progesterone treatments had ended. In both experiments, the increases in PGFM concentrations in the low progesterone groups after an oxytocin challenge were markedly higher than in the high progesterone groups. These results indicate that low progesterone concentrations during an oestrous cycle have a delayed stimulatory effect on uterine responsiveness to oxytocin during the late luteal phase of the subsequent cycle. This resulting increase in PGF(2alpha) secretion may interfere with luteal maintenance during the early stages of pregnancy.     

Effect of beta-carotene and vitamin A on progesterone production by bovine luteal cells

The relationship between concentrations of plasma vitamin A and c-carotene and corpora lutea was studied using 52 Holstein cows. Bovine luteinizing hormone was added to incubation tubes in doses of 0, 10, or 100 ng/ml. Regression of progesterone secretion by luteal cells in vitro on plasma beta-carotene was positive and significant for corpora lutea collected during the winter months when plasma beta-carotene was low. The two were unrelated during the summer months when beta-carotene was higher. Similar regressions for in vitro progesterone production and vitamin A were not significant in either season. These results suggest that in vivo beta-carotene status is related to bovine luteal function in vitro.     

Corpora lutea induced by gonadotrophin-releasing hormone treatment of anoestrous Welsh Mountain ewes: reduced sensitivity to luteinizing hormone in vivo and to chorionic gonadotrophin in vitro

Seasonally anoestrous Welsh Mountain ewes received 250 ng gonadotrophin-releasing hormone (GnRH) every 2 h, with (Group 1; n=13) or without (Group 2; n=14) progesterone priming for 48 h. Fourteen control ewes (Group 3) were studied during the luteal phase in the breeding season. Animals in Group 4 (n=12) received progesterone priming followed by 250 ng GnRH at increasing frequency for 72 h, while ewes in Group 5 (n=13) were given three bolus injections of 30 microg GnRH at 90-min intervals. All treatment regimens induced ovulation. However, only corpora lutea (CL) from ewes in Group 3 (breeding season) or Group 4 exhibited normal luteal function. Luteal luteinizing hormone (LH) receptor levels were significantly higher on day 12 than day 4, and CL from groups with adequate CL (3 and 4) had significantly higher (125)I-human chorionic gonadotrophin (hCG)-binding levels than the three groups with inadequate CL on day 12. LH-binding affinity was unchanged. Exogenous ovine LH (10 microg) in vivo on days 3 or 11 after ovulation induced a pulse of progesterone in ewes with adequate CL: however, ewes in Groups 1, 2 and 5 showed no significant response. Basal progesterone secretion in vitro was significantly greater on day 4 than on day 12. Maximal steroidogenic responses of adequate and inadequate CL to hCG and to dibutyryl cyclic-3',5'-AMP were similar at both stages of the luteal phase. However, the EC50 for hCG on days 4 and 12 was 10-fold lower for groups with an adequate CL (0.1 IU hCG/ml) than for inadequate-CL groups (1 IU hCG/ml; P <0.05). Thus, in addition to the well-characterized premature sensitivity of GnRH-induced inadequate CL to endometrial luteolysin, we have shown (1) a marked decrease in total number of cells in the CL, a profound reduction in vascular surface area, and a decrease in mean large luteal cell volume (with no change in large luteal cell numbers), (2) decreased luteal LH receptor and progesterone content compared with adequate CL and (3) that CL that were becoming, or were destined to become, inadequate failed to respond to ovine LH in vivo and were 10-fold less sensitive to hCG in terms of luteal progesterone secretion in vitro.     

Temporal changes in reproductive hormones and conceptus-endometrial interactions during embryonic diapause and reactivation of the blastocyst in European roe deer (Capreolus capreolus)

The roe deer blastocyst is in diapause between August and December, after which time it expands and elongates rapidly before implantation. Blood samples were taken from 30 animals to define temporal changes in reproductively important hormones to investigate the physiological cues present at embryo reactivation. In 15 of these animals, changes in uterine and conceptus protein synthesis and secretion, and luteal progesterone release during diapause and reactivation, were assessed after culture of these tissues in vitro. Oestradiol concentrations remained low during diapause (1.07 +/- 0.4 pg ml(-1)) and expansion (1.2 +/- 0.4 pg ml(-1)) but increased by 30 times at trophoblast elongation (49.17 +/- 0.37 pg ml(-1)). Prolactin remained at basal concentrations (4.69 +/- 0.86 ng ml(-1)) and increased after implantation (12.34 +/- 2.71 ng ml(-1)). Peripheral progesterone concentrations and luteal progesterone release remained constant throughout diapause, reactivation and implantation (peripheral progesterone: 3.82 +/- 1.97 ng ml(-1); luteal progesterone: 6.72 +/- 0.81 ng mg(-1) protein). Incorporation of a radiolabel into conceptus secretory proteins increased by four times at expansion compared with diapause, whereas incorporation into endometrial secretions remained constant. At elongation, incorporation into endometrial secretions increased two times and conceptus secretions increased 32 times. Two-dimensional electrophoresis and fluorography showed that the profile of endometrial secretory proteins was constant until implantation when qualitative changes were evident. Although a role for an endocrine maternal trigger of reactivation from diapause cannot be dismissed, these data provide no supporting evidence and indicate that the conceptus itself may drive reactivation.     

Hypothyroidism prolongs corpus luteum function in the pregnant rat

It has been shown that hypothyroidism in the rat produces a prolongation of pregnancy associated with a delay in the fall of circulating progesterone (P4) at term. The aim of the present work is to determine whether the delayed P4 decline in hypothyroid mother rats is due to a retarded induction of P4 degradation to 20alphaOH P4 or to a stimulation of its synthesis, and to investigate the possible mechanisms that may underlie the altered luteal function. We determined by RIA the circulating profile of the hormones (TSH, PRL, LH, P4, PGF2alpha, and PGE2) involved in luteal regulation at the end of pregnancy and, by semiquantitative RT-PCR, the expression of factors involved in P4 synthesis (CytP450scc, StAR, 3betaHSD, PRLR) and metabolism (20alphaHSD, PGF2alphaR, iNOS and COX2). Our results show that the delay in P4 decline and parturition is the resultant of retarded luteal regression, caused by a combination of decreases in luteolytic factors, mainly luteal PGF2alpha, iNOS mRNA expression and also circulating LH, and increased synthesis or action of luteotrophic factors, such as luteal and circulating PGE2 and circulating PRL. All these changes may be direct causes of the decreased 20alphaHSD mRNA and protein (measured by western blot analysis) expression, which in the presence of unchanged expression of the factors involved in P4 synthesis results in elevated luteal and circulating P4 that prolonged pregnancy and also may favor longer survival of the corpus luteum.     

Comparing subpopulations of plasma progesterone using cluster analyses

The production of progesterone during the luteal phase of the estrous cycle regulates early embryonic development, uterine function, and luteal function. Plasma progesterone concentrations were measured during a spontaneous estrous cycle in lactating Holstein-Friesian cows of New Zealand or North American ancestry, fed either fresh pasture or a total mixed ration. Subpopulations of profiles were identified using 2 approaches: 1) shape-based clustering and 2) trait-based clustering. Subpopulations of profiles were compared using key progesterone profile components, which described the early and late luteal phase of the estrous cycle. The application of resistant nonlinear smoothing functions across raw progesterone profiles resulted in a high degree of agreement between the raw data and smoothed estimates (R² = 0.93). Both clustering techniques resulted in the identification of 3 similar subpopulations of progesterone profiles. The distribution of animals in these defined subpopulations and the association with cow strain and diet were examined. The early luteal phase was similar for the shape-based categories, but differed in the trait-based analyses. Differences in the late luteal phase and the onset of functional luteal regression in the different clusters were evident for both the shape- and trait-based analyses. The distribution of animals across the subpopulations identified by shape-based clustering was not associated with either cow strain or diet. However, within the 3 subpopulations identified using trait-based clustering, cow strain affected the shape of the progesterone profile. Isolation of 3 subpopulations within a small data set showed that otherwise normal progesterone profiles exhibit significant variation. The differences in the luteal phase of these subpopulations, in particular progesterone early in the luteal phase and the early onset of functional luteal regression, may provide an insight into factors associated with subfertility.     

Direct in vivo effects of leptin on ovarian steroidogenesis in sheep

Leptin, the metabolic fat hormone, has been shown to have effects on reproduction in mice and to modulate steroid production by cultured ovarian somatic cells in a number of species. However, a direct role of leptin on normal ovarian function in vivo has not been shown. In this paper the effect of passive immunisation against leptin (experiment 1; 20 ml antiserum or non-immune plasma i.v.; n = 6/treatment) and direct ovarian infusion of leptin (experiment 2; 0, 2 or 20 mug recombinant ovine leptin; n = 4/treatment) during the early follicular phase was investigated in sheep with ovarian autotransplants, which allow recovery of ovarian venous blood and regular non-invasive scanning of the ovary. Passive immunisation against leptin resulted in an acute increase (P < 0.05) in ovarian oestradiol secretion but had no effect on gonadotrophin concentrations, ovulation or subsequent luteal function. Conversely, direct ovarian arterial infusion of the low dose of leptin resulted in an acute decline (P < 0.05) in ovarian oestradiol secretion whereas the high dose, which resulted in supra-physiological leptin concentrations, had no effect on oestradiol production compared with the controls. Neither dose of leptin had any effect on gonadotrophin concentrations or ovulation but both doses resulted in an increase (P < 0.05) in progesterone concentrations over the subsequent luteal phase. In conclusion, together these data provide strong in vivo evidence that leptin can modulate ovarian steroidogenesis directly and acutely in ruminants and suggest that leptin is an alternate regulatory system whereby nutritional status can regulate reproductive activity.     

Association between energy balance and luteal function in lactating dairy cows

Our objective was to determine the relationship between energy balance and secretion of progesterone in lactating dairy cows. Eight primiparous and 24 multiparous lactating Holstein cows were studied from parturition to 100 d postpartum or conception. Cows calved normally and remained healthy throughout the study. All cows were fed ad libitum a total mixed diet formulated to satisfy requirements for maintenance and lactation. Intake of feed and production of milk per cow were measured twice daily. Body weight was determined weekly. Daily energy balance was determined by subtracting energy required for maintenance and lactation from intake of energy. Concentrations of progesterone were determined in milk sampled every 3rd d. For at least 4 successive d postpartum, 81% of cows were in negative energy balance. Variation in energy balance was explained largely by intake of energy. Duration of luteal phases was not associated with energy balance. Energy balance within 9 d postpartum was correlated positively with concentration of progesterone within second and third postpartum luteal phase. Postpartum interval to nadir and magnitude of nadir of energy balance interacted to reduce progesterone within second and third postpartum estrous cycles. Thus, in lactating cows, secretion of progesterone is reduced by spontaneous caloric deficit and is modulated by timing and magnitude of maximal caloric deficit. Spontaneous caloric deficit is a potential source of infertility in lactating dairy cows.     

Steroidogenic responses of pig corpora lutea to insulin-like growth factor I (IGF-I) throughout the oestrous cycle

This study was designed to investigate the roles of insulin-like growth factor I (IGF-I), IGF-type I receptor (IGF-IR) and IGF-binding proteins (IGFBPs) in regulating progesterone secretion by pig corpora lutea during the oestrous cycle, and the signal transduction pathways involved in mediating the steroidogenic actions of IGF-I. Corpora lutea were collected on days 4, 7, 10, 13 and 15 or 16 of the oestrous cycle, enzyme dissociated and the luteal cells were cultured for 24 h in Medium 199 with IGF-I (0-100 ng ml(-1)), long R(3)-IGF-I (0-100 ng ml(-1)), anti-IGF-I (Sm 1.2B; 0-10 microg ml(-1)), anti-IGF-IR (alphaIR3; 0-2 microg ml(-1)), or IGF-I signal transduction pathway inhibitors (phosphatidylinositol (PI)-3-kinase: 100 nmol Wortmannin l(-1) and 10 micromol LY 294002 l(-1); MAP kinase: 50 micromol PD 98059 l(-1)) to investigate their effects on IGF-I (100 ng ml(-1)) stimulated progesterone secretion. Pig luteal cells displayed dose-dependent responses to IGF-I and long R(3)-IGF-I on days 4 and 7 of the oestrous cycle, but not on days 10-16. There was no difference in the ED(50) or V(max) (maximal response) values between IGF-I and long R(3)-IGF-I. Neither anti-IGF-I nor anti-IGF-IR had significant effects on progesterone secretion, at any dose or day. Wortmannin and LY 294002 blocked IGF-I stimulated progesterone secretion, but PD 98059 was without effect. Finally, IGF-I (6 microg) infused into the ovary on day 7 in vivo significantly increased progesterone secretion within 45 min of infusion. The conclusions of this study are: (1) IGF-I has steroidogenic actions only on 'young' (days 4-7) pig corpora lutea; (2) endogenous IGF-I and IGFBP are insufficient to modulate progesterone secretion in vitro; and (3) the steroidogenic actions of IGF-I are mediated via PI-3-kinase.     

Potential role for peroxisome proliferator-activated receptor gamma in regulating luteal lifespan in the rat

Peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to stimulate progesterone production by bovine luteal cells. We previously reported higher expression of PPARgamma in old compared with new luteal tissue in the rat. The following studies were conducted to determine the role of PPARgamma in rat corpora lutea (CL) and test the hypothesis that PPARgamma plays a role in the metabolism of progesterone and/or luteal lifespan. Ovaries were removed from naturally cycling rats throughout the estrous cycle, and pseudopregnant rats. mRNA for PPARgamma and P450 side-chain cleavage (SCC) was localized in luteal tissue by in situ hybridization, and protein corresponding to PPARgamma and macrophages identified by immunohistochemistry. Luteal tissue was cultured with agonists (ciglitazone, prostaglandin J2) or an antagonist (GW-9662) of PPARgamma. Progesterone was measured in media by RIA and levels of mRNA for 20alpha-hydroxysteriod dehydrogenase (HSD) and bcl-2 were measured in luteal tissue after culture by RT-PCR. An inverse relationship existed between the expression of mRNA for SCC and PPARgamma. There was no effect of PPARgamma agonists or the antagonist on luteal progesterone production in vitro, or levels of mRNA for 20alpha-HSD. PPARgamma protein was localized to the nuclei of luteal cells and did not correspond with the presence of macrophages. In new CL, ciglitazone decreased mRNA for bcl-2 on proestrus, estrus, and metestrus. Interestingly, GW-9662 also decreased mRNA for bcl-2 on proestrus and diestrus in old and new CL, and on metestrus in new CL. These data indicate that PPARgamma is not a major player in luteal progesterone production or metabolism but may be involved in regulating luteal lifespan.     

The human corpus luteum: which cells have progesterone receptors?

Studies comparing the regressing corpus luteum with the rescued corpus luteum have demonstrated that human chorionic gonadotrophin (hCG) has effects on cell types that do not express hCG receptors. As progesterone synthesis is hCG dependent and the corpus luteum has been shown to express genomic progesterone receptors, progesterone is a candidate molecule for these paracrine effects. This study aimed to define the cellular localisation of progesterone receptors in the human corpus luteum using dual-staining immunohistochemistry for genomic progesterone receptors and specific cellular markers. Well-characterised corpora lutea (n = 12) from different stages of the luteal phase were studied. The same distribution was observed in all corpora lutea examined. The steroidogenic cells (3beta-hydroxysteroid dehydrogenase positive) and both theca-lutein (17alpha-hydroxylase positive) and granulosa-lutein (aromatase positive) express progesterone receptors, as do stromal fibroblasts (vimentin positive, fibroblast antigen positive). Vascular endothelial cells (CD31 positive), pericytes (alpha-smooth muscle actin positive), macrophages (CD68 positive) and fibroblasts within the central clot do not express nuclear progesterone receptors. Progesterone is a candidate messenger molecule for the effects of hCG on the matrix metalloproteinase-producing stromal fibroblasts. Some of the effects of hCG on steroidogenic cells may be mediated by progesterone, but its effects on blood vessels and macrophages require alternate paracrine signalling mechanisms. In addition, there appears to be at least two fibroblast populations in the corpus luteum.     

Infrequent milk progesterone measurements in daughters enable bull selection for cow fertility

The interval from calving to first luteal activity (CLA) has been suggested as an unbiased and, therefore, preferable measure for selection on female fertility in dairy cattle. However, measurement of this interval for individual cows is not feasible for reasons of cost and labor associated with the necessary frequent (milk) progesterone measurements. The objective of this study was to test the hypothesis that mean sire progesterone profiles based on individual progesterone measurements of daughters at 3- to 6-wk intervals have prospects as a measure for female fertility when selecting sires in a progeny testing scheme. In this study, progesterone concentrations were measured in milk samples collected at routinely performed milk recordings during the first 100 d of lactation of daughters of 20 test bulls. It is demonstrated that a) mean progesterone profiles can be used to calculate the earliest stage of lactation at which at least 50% of the daughters of a test bull has a milk progesterone level >3 ng/mL (indicating luteal activity) and that b) this stage, at which 50% of the daughters of a bull have an active corpus luteum (CLA50%), varies largely between test bulls. We conclude that selecting sires based on daughter CLA50% may improve female fertility.     

Relationships among body condition score,milk constituents,and postpartum luteal function in Norwegian dairy cows

Relationships among body condition score (BCS), milk constituents, and resumption of postpartum luteal function were studied in 162 lactations of first- and second-parity Norwegian dairy cows. Milk components included acetone, lactose, fat, protein, urea, and ratios of fat to protein and fat to lactose. Milk progesterone concentrations were used to determine intervals from calving to first luteal response (> 5 ng/ml). Intervals to first luteal response were divided into categories of early (< or = 24 d) or late (> 24 d) responses. Higher BCS were observed during wk 4 and 5 postpartum among both first- and second-parity cows with early compared with delayed luteal responses. Second-parity cows with early onset of luteal function also had higher BCS from wk 6 through 12, whereas first-parity cows with early onset of luteal function had higher BCS from wk 13 through 15. Higher acetone levels from wk 2 through 4 postpartum were associated with late luteal response in second-parity cows. Greater milk lactose content during wk 1, 2, 3, 6, 7, and 8 postpartum and higher fat fractions during wk 4 postpartum were related to early luteal response in second-parity cows. Relationships between milk constituents and onset of luteal function were less evident and occurred later postpartum among first-parity cows than among second-parity cows. Measures of weekly milk composites obtained during the early postpartum period and BCS were closely associated with postpartum resumption of luteal function. Acetone and lactose values in milk from the first 4 wk postpartum predicted postpartum luteal function in second-parity cows at a sensitivity of 0.84 and specificity of 0.86.     

Progesterone concentration in the marsupial Sminthopsis macroura: relationship with the conceptus, uterine glandular regeneration and body weight

Close examination of hormonal profiles and uterine morphology in the marsupial reproductive cycle highlights significant differences between pregnant and non-pregnant cycles. In the polyovular dasyurid marsupial Sminthopsis macroura, we identified changes associated with gestation by comparing ovarian and plasma progesterone concentrations, uterine weights, uterine epithelial mitoses, body weights and gestation lengths between pregnant and non-pregnant luteal phases. The plasma progesterone profile of S. macroura was biphasic, peaking during unilaminar blastocyst expansion and on the day of implantation. Periods of rapid embryonic development were associated with increasing plasma progesterone concentrations and animal body weight. For the first time in a polyovular marsupial, we identified 1) a correlation between ovarian progesterone concentration and conceptus number during the luteal phase just prior to implantation (total ovarian progesterone), indicating a conceptus influence on progesterone concentration; 2) a pulse of uterine epithelial mitotic activity at the time of implantation and 3) increased mitotic activity in pregnant animals during unilaminar blastocyst formation compared with non-pregnant animals. Gestation length was reduced by up to 15%, due to the loss of, or reduction in, the four-cell arrest and more rapid definitive blastocyst expansion. This is the first time a conceptus influence on gestation length has been identified in a dasyurid. This study provides further evidence for the modification of the luteal phase by pregnancy in S. macroura.     

Prostaglandin F2alpha increases endothelial nitric oxide synthase in the periphery of the bovine corpus luteum: the possible regulation of blood flow at an early stage of luteolysis

Prostaglandin F(2)(alpha) (PGF(2)(alpha)) released from the uterus causes alterations in luteal blood flow, reduces progesterone secretion, and induces luteolysis in the bovine corpus luteum (CL). We have recently discovered that luteal blood flow in the periphery of the mature CL acutely increases coincidently with pulsatile increases in a metabolite of PGF(2)(alpha) (PGFM). In this study, we characterized changes in regional luteal blood flow together with regional alterations in endothelial nitric oxide synthase (eNOS) expression during spontaneous luteolysis and in response to PGF(2)(alpha). Smooth muscle actin-positive blood vessels larger than 20 microm were observed mainly in the periphery of mature CL. PGF(2)(alpha) receptor was localized to luteal cells and large blood vessels in the periphery of mid-CL. PGF(2)(alpha) acutely stimulated eNOS expression in the periphery but not in the center of mature CL. Injection of the NO donor S-nitroso-N-acetylpenicillamine into CL induced an acute increase in luteal blood flow and shortened the estrous cycle. In contrast, injection of the NOS inhibitor l-NAME into CL completely suppressed the acute increase in luteal blood flow induced by PGF(2)(alpha) and delayed the onset of luteolysis. In conclusion, PGF(2)(alpha) has a site-restricted action depending on not only luteal phase but also the region in the CL. PGF(2)(alpha) stimulates eNOS expression, vasodilation of blood vessels, and increased luteal blood flow in periphery of mature CL. Furthermore, the increased blood flow is mediated by NO, suggesting that the acute increase in peripheral blood flow to CL is one of the first physiological indicators of NO action in response to PGF(2)(alpha).     

Cell death induced by serum deprivation in luteal cells involves the intrinsic pathway of apoptosis

The corpus luteum is a transient endocrine gland specializing in the production of progesterone. The regression of the corpus luteum involves an abrupt decline in its capacity for producing progesterone followed by its structural involution, which is associated with apoptosis of the luteal cells. An in vitro experimental approach is needed to study the molecular mechanisms underlying hormonal regulation of luteal cell death under defined experimental conditions. In this study, we investigated simian virus-40-transformed luteal cells to determine whether they can be driven to apoptosis and, if so, to define the intracellular pathway involved. Luteal cells were cultured in the presence or absence of fetal bovine serum for 24 or 48 h. Under serum starvation conditions, the luteal cells underwent growth arrest accompanied by cell death as evaluated by dye exclusion, and confirmed by two-color fluorescence cell viability/cytotoxicity assay. We next studied whether serum starvation-induced death of luteal cells occurred by apoptosis. Morphologic features of apoptosis were observed in cells stained with hematoxylin after being subjected to serum starvation for 48 h. The apoptotic nature was further confirmed by in situ 3'-end labeling and fragmentation of genomic DNA. Apoptosis of serum-deprived luteal cells was dependent upon caspase activation. Serum starvation induced cleavage of poly (ADP-ribose) polymerase (PARP), suggesting that caspase-3 had been activated under the stress of withdrawal of growth factors. This was confirmed by cleavage of full-length procaspase-3. Finally, the fact that serum starvation promoted the cleavage of full-length procaspase-9 and the decrease in the expression of endogenous Bid, a BH-3-only proapoptotic protein of the Bcl-2 family, indicates that the intrinsic (i.e., mitochondrial) pathway of apoptosis was activated. In summary, we have characterized an in vitro experimental model of luteal cell death that can be utilized to evaluate the role of hormones in apoptosis of luteal cells under defined culture conditions, and to study the mechanism of luteal regression.     

Administration of vascular endothelial growth factor Trap during the 'post-angiogenic' period of the luteal phase causes rapid functional luteolysis and selective endothelial cell death in the marmoset

The intense angiogenesis characteristic of early corpus luteum development is dependent upon vascular endothelial growth factor (VEGF) as inhibitors of VEGF administered at the peri-ovulatory period suppress endothelial cell proliferation and progesterone secretion. We now report that administration of VEGF Trap, a soluble decoy receptor-based inhibitor, at the mid- or the late luteal phase in the marmoset results in a rapid decline in plasma progesterone. Since vascularisation of the corpus luteum is largely complete by the mid-luteal phase, it suggested that this functional luteolysis involved mechanisms other than inhibition of angiogenesis. A second experiment investigated the role of VEGF in maintaining the integrity of the luteal vasculature and hormone-producing cells. VEGF Trap was administered to marmosets in the mid-luteal phase and ovaries were obtained 1, 2, 4 or 8 days later for localisation of activated caspase-3 staining in the corpus luteum and compared with those obtained 2, 4 and 8 days after administration of control protein. The number of cells with activated caspase-3 staining was significantly increased after administration of VEGF Trap. Dual staining of activated caspase-3 with the endothelial cell marker CD31 showed that at 1 day post-treatment, more than 90% caspase-3-stained cells were vascular endothelium, prior to detection of an increasing incidence in death of hormone-producing cells on days 2 and 4. Staining with CD31 showed that the endothelial cell area was decreased after treatment. By 8 days after treatment, corpora lutea had regressed to varying degrees, while all control corpora lutea remained healthy. These results show that VEGF inhibition in the mid- or the late luteal phase induces functional luteolysis in the marmoset that is associated with premature and selective death of endothelial cells.     

Use of concentrations of progesterone and estradiol-17 beta in milk in monitoring postpartum ovarian function in dairy cows

Data from artificial insemination, rectal palpation, and hormone assays were used to characterize postpartum reproductive activity in 54 dairy cows. Progesterone and estradiol-17 beta were measured in milk samples collected for 120 d (Trial 1) or 65 d (Trial 2). Progesterone was higher and estradiol was lower in milk than in serum. Values for both hormones in milk were highly correlated with those in serum. Most cows (64%) had short first luteal phases (less than or equal to 12 d). First rise (28 d) in progesterone was later (33.4 vs. 24.9 d) for cows having short rather than normal (greater than 12 d) luteal phases. Cows were classified as having a short luteal phase followed by a normal luteal phase or as having normal luteal phases for the first two estrous cycles. Estradiol for the 6 d prior to each luteal phase was higher preceding the second phase than the short phase or those preceding both phases of cows with normal phases. Follicular function prior to ovulation, as measured by estradiol, was not responsible for short-lived corpora lutea. Concentrations of progesterone in milk in the late luteal phase prior to insemination were related to fertility.