Angiogenesis in the human corpus luteum: changes in expression of angiopoietins in the corpus luteum throughout the menstrual cycle and in early pregnancy

CONTEXT: Blood vessel stabilization is regulated by angiopoietins and important for angiogenesis in the corpus luteum. OBJECTIVE: To study angiogenesis and blood vessel stabilization in the human corpus luteum, changes in expression of angiopoietin (Ang)-1, Ang-2, and their specific receptor, Tie-2, together with the number of blood vessels and pericytes were examined in the corpus luteum throughout the menstrual cycle and in early pregnancy. DESIGN: The number of blood vessels and pericytes was determined by immunohistochemistry for CD34 and alpha-smooth muscle actin, respectively. Ang and Tie-2 expression were examined by immunohistochemistry or RT-PCR. RESULTS: The number of blood vessels increased during the early luteal phase, whereas the number of pericytes was small in the early luteal phase and increased in the midluteal phase, suggesting that angiogenesis is undergoing during the early luteal phase and blood vessels are stabilized in the midluteal phase. Blood vessels and pericytes decreased in number during the late luteal phase. The increased number of both blood vessels and pericytes seen in the corpus luteum of early pregnancy suggests that angiogenesis is undergoing accompanied by blood vessel stabilization. Ang-2 expression with low Ang-1 expression was found during the early luteal phase. Thereafter, increasing Ang-1 expression during the midluteal phase, declining Ang-1 expression with continued Ang-2 expression during the late luteal phase, and relatively high Ang-1 expression in early pregnancy were observed. CONCLUSIONS: The change in Ang expression is closely associated with angiogenesis, blood vessel stabilization, and blood vessel regression during the divergent phases of luteal formation, luteal regression, and luteal rescue by pregnancy.     

Expression and role of the corticotropin-releasing hormone/urocortin-receptor-binding protein system in the primate corpus luteum during the menstrual cycle

CRH/urocortin-receptor-binding protein (CRH/UCN-R-BP) mRNAs are dynamically expressed in the primate ovary during the menstrual cycle. Therefore, studies were designed to localize CRH/UCN-R-BP mRNAs to ovarian cell types, quantitate protein expression during the corpus luteum (CL) lifespan, and investigate the role of this system in the macaque ovary at midcycle. Monkey ovaries were removed during the preovulatory phase and through the luteal phase to localize CRH/UCN-R-BP mRNAs by in situ hybridization and determine their protein levels in CL by Western blotting. Also, vehicle or a CRH receptor antagonist (astressin) was injected into the preovulatory follicle; daily serum samples were analyzed for hormone levels, and ovaries were removed on d 9 of the luteal phase for histological analysis. There was minimal ligand mRNA staining, whereas receptor and CRHBP was detected in the granulosa and theca cells of the preovulatory follicle. However, ligand and receptor mRNA staining was appreciable in luteal cells of the CL during the early luteal phase (ECL) and diminished in the late luteal phase (LCL). CRHBP staining was low in the ECL and increased markedly in the LCL. Ligand and receptor protein expression was also highest during ECL, whereas CRHBP expression was highest at the LCL. Although astressin injection did not prevent follicle rupture, progesterone levels were significantly less by the mid-luteal phase, and estradiol levels never increased above baseline during the CL lifespan. Histological indices of cell degeneration were observed in the astressin-treated CL. Thus, CRH/UCN-R-BP components are expressed in an ovarian cell-specific manner. The expression pattern and results from antagonist injection are consistent with the hypothesis that CRH/UCN-R activation promotes luteal development and/or structure-function in monkeys during the menstrual cycle.     

Expression of vascular endothelial growth factor and its receptors in the human corpus luteum during the menstrual cycle and in early pregnancy

To investigate the possible role of vascular endothelial growth factor (VEGF) and its receptors in the human corpus luteum (CL), expression of VEGF and its receptors, the fms-like tyrosine kinase and the kinase insert domain-containing region (KDR), was analyzed in the CL during the menstrual cycle and in early pregnancy. Immunohistochemistry revealed that VEGF was localized in luteal cells and both flt-1 and KDR were also localized in luteal cells, in addition to vascular endothelial cells. Messenger RNA (mRNA) expression of VEGF, flt-1, and KDR remained constant in the CL during the luteal phase and was lower in the regression phase. In the pregnant CL, VEGF mRNA expression was higher compared with that in the midluteal phase, and mRNA expression of both flt-1 and KDR was the same as that in the midluteal phase. Western blot analyses revealed that the change in protein expression of VEGF, flt-1, and KDR was similar to that in their mRNA expression. To study the effect of human CG (hCG) on VEGF expression in the CL, corpora lutea obtained from the midluteal phase were incubated with hCG (1 IU/ml) for 6 h. hCG increased the expression of mRNA and protein of VEGF. In conclusion, VEGF and its receptors may play important roles in development and function of the CL, and VEGF may exert a paracrine-autocrine role in regulating luteal function. hCG may act to prolong the life span of the CL by stimulating VEGF expression when pregnancy occurs.     

Opposite effects of ethanol on the activation of adenylyl cyclase in human corpus luteum membranes

The influence of an acute exposure to ethanol on adenylyl cyclase activity in membrane fractions prepared from human corpus luteum was investigated. Ethanol up to a concentration of 5% (v/v) was without effect on basal luteal adenylyl cyclase activity, but markedly potentiated stimulation of NaF and hCG in a dose-dependent manner. In contrast, ethanol progressively inhibited forskolin stimulation at the same range of ethanol concentrations. Maximal NaF and hCG responsiveness of adenylyl cyclase activity was observed at 5% ethanol and reached values 80% and 100% higher than controls without ethanol, respectively. However, at the same ethanol concentration, forskolin-stimulated enzymatic activity was reduced by 40% relative to controls. Equilibrium binding studies involving [¹²⁵I]hCG interaction with luteal membranes in the presence of the concentration of ethanol showing maximal hCG responsiveness indicated that ethanol slightly affected (15% increase) the hCG binding compared to controls, without any appreciable change on the K_d for the hormone. This minor effect of ethanol on gonadotropin binding sites contrasted greatly with the extent at which ethanol maximally potentiated the gonadotropin-stimulated adenylyl cyclase. GTP was found to be less effective than GMP-P(NH)P in sustaining ethanol potentiation, suggesting that ethanol is unlikely to act by inhibiting GTPase activity. These data indicate that the acute effects of ethanol inhibit forskolin-stimulated adenylyl cyclase at concentrations potentiating stimulatory effects of NaF and of hCG, and that the synergistic interaction of ethanol and gonadotropin stimulation of adenylyl cyclase is, at least in part, due to an increase in the functional coupling of the occupied hCG-receptor complex with the components of the enzyme system.     

Luteotropic effects of prostaglandin E2 on the human corpus luteum of the menstrual cycle and early pregnancy

Human corpora lutea (CL) of the menstrual cycle and early pregnancy were excised at operation, cut into pieces, and incubated or superfused in the presence of hCG or prostaglandin (PG) E2. After incubation, the tissue levels of cAMP and the medium concentrations of progesterone (P) were determined, while the concentration of P was analyzed after superfusion. PGE2 stimulated cAMP formation in CL from all phases of the menstrual cycle as well as from early pregnancy and caused an increase in P formation in CL from the early and midluteal phases of the menstrual cycle as well as from early pregnancy. A difference was found in the latency, the lag phase until maximal response, and the duration of response between the effects of PGE2 and hCG on both cAMP and P formation. Thus, the effect of PGE2 started more rapidly and was of shorter duration than that of hCG. The stimulatory effect of PGE2 on CL from early pregnancy was of the same magnitude as that of CL from the menstrual cycle. On the other hand, hCG had less stimulatory effect on cAMP and P formation in CL from early pregnancy compared to CL from the menstrual cycle. We conclude that PGE2 stimulates P and cAMP formation in isolated human CL from all phases of the menstrual cycle as well as in early pregnancy, indicating a luteotropic effect of this PG.     

Expression of Bcl-2 and Bax in the human corpus luteum during the menstrual cycle and in early pregnancy: regulation by human chorionic gonadotropin

To investigate the relationship between apoptosis and the Bcl-2/ Bax system in the human corpus luteum (CL), the frequency of apoptosis and expression of Bcl-2 and Bax were examined in the CL during the menstrual cycle and in early pregnancy. In situ analysis of DNA fragmentation showed that the number of apoptotic cells was much greater in the regressing CL than that in the midluteal phase CL, whereas there were almost no apoptotic cells in the CL of early pregnancy. Immunohistochemistry revealed that Bcl-2 expression was observed in the luteal cells in the midluteal phase and early pregnancy, but not in the regressing CL. In contrast, Bax immunostaining was observed in the regressing CL, but not in the midluteal phase and early pregnancy. bcl-2 messenger ribonucleic acid (mRNA) levels in the CL during the menstrual cycle were highest in the midluteal phase and lowest in the regressing CL. In the CL of early pregnancy, bcl-2 mRNA levels were significantly higher than those in the midluteal phase. In contrast, bax mRNA levels were highest in the regressing CL and remarkably low in the CL of early pregnancy. Western blot analyses revealed that Bcl-2 expression was significantly lower in the regressing CL than in the midluteal phase and early pregnancy, and that Bax expression was, in contrast, significantly higher in the regressing CL than in the midluteal phase and was remarkably low in the CL of early pregnancy. When corpora lutea of the midluteal phase were incubated with hCG, hCG significantly increased the mRNA and protein levels of Bcl-2 and significantly decreased those of Bax. In conclusion, Bcl-2 and Bax may play important roles in the regulation of the life span of the human CL by controlling the rate of apoptosis. hCG may act to prolong the life span of the CL by increasing Bcl-2 expression and decreasing Bax expression when pregnancy occurs.     

Adenylate cyclase in the primate corpus luteum during chorionic gonadotropin treatment simulating early pregnancy: homologous versus heterologous desensitization

Stimulation of the primate corpus luteum by endogenous CG in early pregnancy or by exogenous CG in simulated conditions is transient despite continued exposure to this luteotropic hormone. The transitory response to CG is not due to the down-regulation of gonadotropin receptors. The current studies were designed to determine if the transient response involves a postreceptor lesion at the membrane level, i.e. the loss of CG receptor activation of adenylate cyclase. Nonpregnant female rhesus monkeys received increasing doses of hCG for up to 10 days beginning near the typical time of implantation (9 days post-LH surge) to simulate early pregnancy. Corpora lutea were removed at specific intervals after the onset of hCG treatment, luteal homogenates were prepared, and adenylate cyclase activity was assessed by the conversion of [alpha-32P]ATP to [32P] cAMP. Basal activity of adenylate cyclase was unchanged throughout the in vivo hCG treatment interval. Nonhormonal activators, such as forskolin (100 microM) and 5'-guanylylimidodiphosphate (50 microM) stimulated (P less than 0.05) adenylate cyclase to a similar extent (greater than 10-fold the control level) throughout hCG treatment. On day 0, both gonadotropins (hCG and human LH; 250 nM) and prostaglandins (PGE2 and PGI2; 500 nM) stimulated cAMP production (approximately 3-fold the control level; P less than 0.05). The responses of adenylate cyclase to PGE2 and PGI2 did not diminish throughout the in vivo hCG treatment. In contrast, exposure to hCG for 3 days reduced the sensitivity of adenylate cyclase to gonadotropin. Moreover, adenylate cyclase in luteal tissue after 6-10 days of treatment was insensitive to hCG. The loss of gonadotropin sensitivity of adenylate cyclase by 6 days of hCG treatment correlated with the decline in circulating progesterone levels. These results demonstrate that 1) the gonadotropin-responsive adenylate cyclase of the macaque corpus luteum is also stimulated by paracrine factors, notably PGs of the E and I series; and 2) CG exposure stimulating early pregnancy conditions leads to homologous, not heterologous, desensitization of the adenylate cyclase system. We hypothesize that homologous desensitization of the adenylate cyclase system is an important mechanism leading to the transient response of the primate corpus luteum to CG in early pregnancy.     

Adenylate cyclase in the corpus luteum of the rhesus monkey. III. Changes in basal and gonadotropin-sensitive activities during the luteal phase of the menstrual cycle

The activity of adenylate cyclase was examined in corpora lutea (CL) obtained from rhesus monkeys at specific stages in the luteal phase of the menstrual cycle [3-5, 6-8, 9-12, 13-15, and 16 days (menses) after the midcycle LH surge]. The conversion of [alpha-32P]ATP to [32P]cAMP was used to monitor adenylate cyclase activity. cAMP production in luteal homogenates was assessed in the absence (basal activity) and presence of maximum stimulatory doses of forskolin (100 microM), 5'-guanylylimidodiphosphate [GMP-P(NH)P; 50 microM], GTP (50 microM), and GTP plus increasing doses of hLH and hCG. Basal activity was low in the early luteal phase (days 3-5; mean +/- SE, 1.2 +/- 0.2 pmol cAMP/mg protein X min), increased (P less than 0.05) by the midluteal phase (days 6-8 and 9-12, 2.1 +/- 0.4 and 2.0 +/- 0.3 pmol/mg X min, respectively), and then declined (P less than 0.05) during the late luteal phase (days 13-15 and 16-menses, 1.6 +/- 0.3 and 1.2 +/- 0.5 pmol/mg X min, respectively). Activity stimulated by GTP and GMP-P(NH)P [e.g. GMP-P(NH)P approximately 12 times basal level] followed the same pattern as basal activity during the luteal phase. In contrast, cAMP production in the presence of forskolin did not change significantly throughout the luteal phase. In the midluteal phase (days 6-8 and 9-12; n = 12), hCG and human LH (hLH) stimulated adenylate cyclase in a similar dose-dependent manner. Maximal stimulation of cAMP production by hCG was about 10% greater (P less than 0.05) than that by hLH; the activation constant was 12.3 nM for hCG and 28.3 nM for hLH. The maximal response to hLH and hCG as well as the sensitivity of adenylate cyclase to activation by hLH were greater (P less than 0.05) in the midluteal phase than in the early or late luteal phase. Decreased basal, gonadotropin-stimulated, and guanine nucleotide-stimulated cAMP production and diminished sensitivity of adenylate cyclase to hLH correlated with a decline (P less than 0.05) in circulating progesterone and luteal weight during the late luteal phase. Thus, the adenylate cyclase system of the rhesus monkey CL undergoes significant changes during the luteal phase which are associated with the development and regression of the CL of the menstrual cycle. Mechanisms that modulate gonadotropin and nucleotide activation of adenylate cyclase without interfering directly with the catalytic unit are implicated in the changes that accompany luteolysis.     

Human corpus luteum: luteinizing hormone and chorionic gonadotropin receptors during the menstrual cycle

To characterize and determine the concentration of LH/hCG receptors in human corpora lutea of the menstrual cycle, we measured occupied and unoccupied receptors and determined the association (Ka) and dissociation (Kd) constants individually in 23 corpora lutea (CL) and 4 corpora albicantia obtained at the time of tubal ligation from 25 normal cycling women. We found no [125I]hCG binding in any of the corpora albicantia. Scatchard plot analysis for each CL revealed a linear binding plot indicative of a single set of LH/hCG receptors. The mean concentration of unoccupied receptors was 36 +/- 10 (+/- SE) fmol/mg protein in the early luteal phase (days 15-19; n = 5), 64 +/- 11 fmol/mg protein in the midluteal phase (days 20-25; n = 13), and 42 +/- 19 fmol/mg protein in the late luteal phase (days 26-30; n = 5). The concentrations of occupied receptors were 56 +/- 8, 46 +/- 6, and 54 +/- 12 fmol/mg protein in the early, mid-, and late luteal phases, respectively. Total (occupied plus unoccupied) receptor concentrations reached maximum levels of 110 +/- 11 fmol/mg protein in the midluteal phase. Ka increased progressively from 12 +/- 4 X 10(9) mol/L-1 in the early luteal phase to 19 +/- 7 X 10(9) and 21 +/- 8 X 10(9) mol/L-1 in the mid- and late luteal phases. We conclude that in normal CL, 1) total and unoccupied LH/hCG receptor levels parallel progesterone secretion; 2) changes in the binding affinity may be important in sustaining and/or rescuing the CL; and 3) loss of LH/hCG receptors is probably related to luteolysis.     

The corpus luteum of the primate menstrual cycle is capable of recovering from a transient withdrawal of pituitary gonadotropin support

To further our understanding of the role of pituitary gonadotropin secretion in the control of corpus luteum function during the primate menstrual cycle, we have used an experimental model which enables us to directly control pituitary gonadotropin secretion throughout the luteal phase. Specifically, we have asked whether cessation of progesterone secretion, or functional luteolysis, resulting from a 3-day withdrawal of gonadotropin support, culminates in an irreversible loss of luteal responsiveness to further gonadotropic stimulation; and do the effects of gonadotropin deprivation vary with the age of the corpus luteum? Endogenous gonadotropin secretion was abolished in seven adult rhesus monkeys by placing radiofrequency lesions in the arcuate nucleus region of the medial basal hypothalamus. Endogenous gonadotropin secretion and ovulatory menstrual cycles were then restored by chronic pulsatile infusion of GnRH (1 pulse/h). Control luteal phases supported by this GnRH regimen exhibited typical plasma progesterone patterns and ranged from 14-17 days in length. In experimental cycles, endogenous gonadotropin secretion was interrupted for a 3-day period during the early (days 2-5), mid (days 8-11), or late (days 13-16) stages of the luteal phase. During the GnRH deprivation period, bioassayable and immunoreactive serum LH was undetectable. The disappearance of circulating LH was followed by a rapid fall in plasma progesterone levels regardless of the stage of the luteal phase. The restoration of gonadotropin secretion resulted in a resumption of progesterone secretion when the gonadotropin deprivation period was imposed during the early or midluteal phase. In each instance, the resumption of progesterone secretion continued for a period of time which effectively completed the typical 14- to 17-day functional lifespan of the corpus luteum of the menstrual cycle. Thus, the luteal phase was neither shortened nor lengthened by a 3-day interruption of luteal function resulting from withdrawal of gonadotropic support. When gonadotropin secretion was interrupted during the late luteal phase (days 13-16), restoration of gonadotropin secretion on day 16 did not result in resumption of progesterone secretion. Our findings confirm our earlier demonstration that progesterone secretion during the luteal phase of the non-fertile menstrual cycle is dependent on pituitary gonadotropic support.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of reduced luteinizing hormone concentrations on corpus luteum function during the menstrual cycle of rhesus monkeys

To further define the relationship between plasma LH concentrations and progesterone secretion by the primate corpus luteum, we examined luteal function in rhesus monkeys in response to reduced LH concentrations during the luteal phase of the menstrual cycle. Five anovulatory rhesus monkeys received a pulsatile infusion of synthetic GnRH (6 micrograms/pulse; one pulse per h, iv) to restore menstrual cyclicity. During the early luteal phase (4-5 days after ovulation), the amount of GnRH administered per pulse was reduced to 1/250th or 1/750th of the standard GnRH infusion regimen. Plasma LH concentrations, determined by bioassay, were reduced by approximately 50% during cycles maintained by reduced GnRH concentrations compared with the standard GnRH dosage. Serum progesterone concentrations were maintained for 5-6 days after GnRH reduction and declined thereafter, and premature menstruations were observed in four of seven cycles maintained by the 1/250th GnRH reduction and four of six cycles maintained with the 1/750th GnRH reduction. These results are consistent with the hypothesis that luteal regression during the nonfertile menstrual cycles of primates is due primarily to an alteration in luteal cell responsiveness to LH, rather than a reduction in the gonadotropic drive to the corpus luteum per se. When plasma LH concentrations were reduced during the early luteal phase to values below those found during the onset of luteal regression in control cycles, luteal function was maintained for 5-6 days. However, as the luteal phase progressed, the reduced LH concentrations were unable to sustain progesterone secretion, and premature menses occurred in some, but not all, animals.     

Effects of different gonadotropin pulse frequencies on corpus luteum function during the menstrual cycle of rhesus monkeys

In the nonfertile menstrual cycle, the frequency of episodic LH secretion declines from approximately 1 pulse/h in the early luteal phase to 1 pulse/4-8 h in the mid- to late luteal phase, but the relevance of this phenomenon to the initiation of functional luteal regression is not completely understood. We investigated whether a reduction in LH pulse frequency causes a decline in luteal progesterone production by experimentally reducing LH pulse frequency during the early luteal phase, and measured the effects on the subsequent plasma progesterone pattern and the onset of luteal regression. Rhesus monkeys were rendered anovulatory by placing radiofrequency lesions in the arcuate region of the medial basal hypothalamus or surgically transecting the hypothalamic-pituitary stalk. Endogenous gonadotropin secretion and ovulatory menstrual cycles were restored by pulsatile infusion of synthetic GnRH at a frequency of 1 pulse/h. Commencing on days 3-6 of the luteal phase, GnRH frequency was changed to either 1 pulse/8 h (four animals) or 1 pulse/24 h (four animals), or maintained at the standard 1 pulse/h frequency (four animals). Luteal phases of 13- to 17-day duration were observed in all animals kept on the 1 pulse/h frequency and in three of four animals in which the frequency was changed to 1 pulse/8 h on day 3 of the luteal phase. Daily midluteal phase (days 5-10) plasma progesterone levels observed in response to the 1 pulse/h and 1 pulse/8 h infusion regimens were similar (mean +/- SE, 4.1 +/- 0.4 vs. 3.2 +/- 0.4 ng/ml; P greater than 0.1). In contrast, short luteal phases were observed in all animals after the LH pulse frequency was reduced to 1 pulse/24 h. Comparison of plasma LH responses to a representative GnRH pulse of each GnRH infusion regimen revealed that the maximal LH levels attained in response to 1 pulse/8 h (47.5 +/- 11.5 ng/ml) were significantly greater (P less than 0.05) than the maximal LH levels attained in response to 1 pulse/h (30.5 +/- 3.2 ng/ml) or 1 pulse/24 h (27.2 +/- 5.0 ng/ml). Progesterone levels remained elevated for 140-200 min after the LH pulse resulting from the 1 pulse/8 h infusion regimen. In response to the 1 pulse/24 h infusion regimen, plasma progesterone levels remained elevated for 60 min after the LH pulse.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in biochemical markers of osteoblastic activity during the menstrual cycle

Serum levels of osteocalcin [OC; bone Gla protein (BGP)] and bone alkaline phosphatase (B-AP) are both correlated to osteoblastic activity, which may be regulated by several hormones, including estrogen, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], and PTH. Estrogen shows reproducible variations during the menstrual cycle, while available data on variations in serum 1,25-(OH)2D3 and serum immunoreactive PTH show midcyclic increases or no changes. In the present study we evaluated osteoblastic activity by measuring serum OC and B-AP during the menstrual cycle in eight healthy women, aged 20-47 yr. The cycles were synchronized by LH peaks, and follicular and luteal periods were normalized by lengths. Repeated measures analysis of variance showed that serum OC varied significantly (P less than 0.05), with highest levels during the luteal period. Although the same pattern was seen for serum B-AP, the variation just failed to reach significance (P less than 0.10), but the mean level was significantly higher during the luteal than during the follicular period (P less than 0.05). Gonadotropins and ovarian sex hormones showed significant variations. There were no significant changes in serum vitamin D-binding protein, serum total and free 1,25-(OH)2D3 index, or serum immunoreactive PTH-(1-84), but serum levels of somatomedin-C showed a significant variation, with the highest level during the luteal period (P less than 0.05). Blood levels and urinary excretion of minerals exhibited no significant variations. Cross-correlation studies between OC and estradiol showed the highest correlation coefficient, when OC was lagged about 7 days after estradiol (r = 0.69; P less than 0.05). Moreover, a high correlation was found between OC and somatomedin-C when matched at concurrent time points (r = 0.76; P less than 0.01). No significant correlations were found between the other calcium-regulating hormones and OC when matched at concurrent time points. In conclusion, we found a significant effect of the menstrual cycle on the serum levels of two osteoblastic bone markers, OC and B-AP. The changes indicated that osteoblastic activity is higher during the luteal period. However, whether the changes are caused by direct or indirect effects of the fluctuations in calciotropic hormones is still unresolved.     

Identification and quantitative determination of steroids in bovine corpus luteum during oestrous cycle and pregnancy.

Neutral steroids in bovine corpus luteum were isolated by liquid-gel chromatography on hydrophobic Sephadex, and were analyzed by computerized gas chromatography-mass spectrometry. The presence of progesterone and 20beta-hydroxy-4-pregnen-3-one was confirmed. In addition, 3beta-hydroxy-5-pregnen-20-one, 5-pregnene-3beta,20beta-diol, 3beta-hydroxy-5alpha-pregnan-20-one and 5alpha-pregnane-3beta,20beta-diol were fully identified, and 3-hydroxy-4-pregnen-20-one, 4 pregnene-3,20-diol, 22-hydroxycholesterol and 20,22-dihydroxycholesterol were partially characterized. Steroid sulphates were not detected. Quantification of the six fully identified steroids was based on peak areas in specific fragment ion current chromatograms constructed by the computer. During the 4th-19th day of the oestrous cycle the steroid concentrations varied as follows: progesterone 6.0-36.7 mug/g wet luteal weight, 20beta-hydroxy-4-pregnen-3-one 0.8-5.5 mug/g, 3beta-hydroxy-5-pregnen-20-one 1.0-7.1mug/g, 5-pregnene-3beta,20beta-diol smaller than 0.2-0.9 mug/g, 3beta-hydroxy-5alpha-pregnan-20-one 1.7-8.6 mug/g, and 5alpha-pregnane-3beta,20beta-diol smaller than 0.2-1.2 mug/g. The concentrations of progesterone and 3beta-hydroxy-5-pregnen-20-one seemed to vary in parallel and were low during days 11-17. During this period the concentrations of 5-pregnene-3beta,20beta-diol and 5alpha-pregnane-3beta,20beta-diol were highest as was the relative contribution of all three 20beta-hydroxysteroids to the total amount of steroids. The relative amount of 3beta-hydroxy-5alpha-pregnan-20-one seemed to be highest during days 4-6. The total steroid concentration in corpora lutea taken in early pregnancy (75-105 days) was 18-47 mug/g. In the period 75-90 days, progesterone constituted only 35-42% of the total steroids, 3beta-hydroxy-5alpha-pregnan-20-one as much as 23-40% and the total 20beta-hydroxysteroids 18-30%. The total steroid concentration in corpora lutea taken in midterm and late pregnancy was 21-77 mug/g. In this period progesterone was by far the predominant steroid and constituted about 80-90% of the total steroids in corpora lutea taken between days 150 and 240. Possible correlations between luteal growth, steroid oxidoreductases and steroid concentrations are discussed.     

Changes in available gonadotropin receptors in the corpus luteum of the rhesus monkey during simulated early pregnancy

Stimulation of the primate corpus luteum (CL) by endogenous CG in early pregnancy or by exogenous human CG (hCG) in simulated early pregnancy, is transient, despite continued exposure to rising concentrations of CG. The objective of this study was to determine if the transitory response of the CL to CG is related to changes in gonadotropin receptors. Numbers and affinities of available LH/CG binding sites were characterized in the CL of rhesus monkeys (n = 27) during prolonged CG exposure in simulated early pregnancy, and the temporal relationship between changes in receptor parameters and in luteal function was examined. Administration of hCG increased progesterone concentrations above pretreatment levels within 9 h (2.2 +/- 0.8 vs. 7.6 +/- 1.1 ng/ml, mean +/- SE, P less than 0.05); the relative increase (345% of control) in serum progesterone was more profound than that of available hCG binding sites (135%, P greater than 0.05) or luteal weight (128%, P greater than 0.05) over this interval. Receptor affinities for hCG remained comparable to pretreatment values (Kd = 1.1 +/- 0.2 X 10(-10) M) throughout this 9-h period. A significant diminution in available hCG receptors occurred between 9 h (12.7 +/- 2.1 fmol/mg tissue) and 3 days (7.4 +/- 1.5 fmol/mg tissue) of hCG treatment (P less than 0.05). The loss of available CG receptors preceded a significant decline in serum progesterone concentrations. Serum progesterone decreased by 6 days (4.0 +/- 0.6 ng/ml, P less than 0.05) of hCG treatment, as did receptor affinity for hCG (Kd = 4.7 +/- 0.9 X 10(-10) M, P less than 0.05). Numbers and affinities of available receptors for hCG and serum progesterone concentrations fell before any decrease in luteal weight. Binding capacities and receptor affinities for human LH were comparable to those for hCG throughout simulated early pregnancy. In conclusion, the population of available LH/CG receptors in the macaque CL is maintained, or perhaps modestly increased, amidst dramatic stimulation of luteal function during early CG exposure. The subsequent diminution of number and affinity of available LH/CG receptors during prolonged exposure to CG in early pregnancy may compromise CL function and thus participate in the establishment of the transient nature of the luteal response to CG.     

The expression of angiotensin and endothelin system members in bovine corpus luteum during estrous cycle and pregnancy

The aim of this study was to determine the changing profiles of the mRNA expression of members of angiotensin and endothelin system in bovine corpus luteum (CL) from different stages of the estrous cycle and pregnancy. Corpora lutea were accordingly assigned to the following stages; d 1–2, 3–4, 5–7, 8–12, 13–18, >18 (after regression) of estrous cycle and of early and late pregnancy (<4 and >4 mo). The block RT-PCR analysis of CL showed a significantly higher angiotensin converting enzyme (ACE) mRNA expression during mid and late luteal phases as well as after regression, but lower levels during pregnancy. Full quantitative real-time RT-PCR (LightCycler) confirmed this pattern of ACE mRNA expression. The angiotensin receptor type 1 (AT1R) mRNA expression was relatively stable throughout the periods examined. In contrast, AT2R mRNA temporarily decreased on d 8–12, followed by an increase to the highest levels during late luteal phase, and it remained at high levels during regression and pregnancy. Concentration of angiotensin II (Ang II) peptide in luteal tissue was highest after ovulation (d 1–2), decreased afterward, increased again during late luteal phase, and decreased to lower levels during regression and pregnancy. The mRNA expression and peptide concentration of endothelin 1 (ET-1) was high after ovulation followed by a decrease during mid and late luteal phases and increased again to the highest level after regression. The endothelin receptor type B (ETR-B) mRNA expression increased during late luteal phase and further after regression. In contrast, ETR-A and endothelin converting enzyme 1 (ECE-1) mRNA expression were relatively constant during all stages examined. In conclusion, the regulatory changes of both angiotensin and endothelin family members during early luteal phase and again during late luteal phase suggest a possible modulatory role of these vasoactive peptide families for bovine CL formation and regression.