Acute versus chronic effects of human chorionic gonadotrophin on relaxin secretion in rhesus monkeys.

Corpora lutea (CL) were removed from rhesus monkeys (N = 26) at 0 h, 9 h, 3 days, 6 days and 10 days during treatment with hCG to simulate blood concentrations of CG during normal pregnancy. Dispersed luteal cells were incubated in vitro at 37 degrees C for 8 h. Immunoreactive relaxin was measured in incubation medium and in cell extract by radioimmunoassay (RIA). Cellular content and release of relaxin into medium increased as simulated early pregnancy progressed. By 3 days, relaxin content had significantly increased (P less than 0.05) and continued to rise throughout simulated early pregnancy. Significant increases in cellular content and release were observed before the time when relaxin has been detected in the peripheral circulation during this treatment regimen. Within group, total relaxin (cells plus medium) was similar before and after incubation (P greater than 0.05). As such, production of relaxin during the 8-h incubation was not evident. In-vitro exposure of the luteal cells to hCG or dbcAMP had no acute effect on cell content or medium concentration of relaxin at any stage of simulated early pregnancy. Since acute effects of hCG and dbcAMP were not evident in vitro, a sustained gonadotrophic influence may be necessary to augment relaxin production/secretion in the primate CL.     

Localization of relaxin in human gestational corpus luteum

Summary Corpora lutea from 12 pregnant women were prepared for immunohistochemical localization of relaxin using a highly specific antiserum. A positive response is given by luteal cells that are diffusely distributed throughout the corpus luteum. These cells do not form a distinctive group in any particular area. A negative response is seen in the adjacent ovarian tissue, and also in nongestational corpora lutea in an early luteal phase.     

Effect of luteinizing hormone and human chorionic gonadotropin on cell populations in the ovine corpus luteum

Two experiments were conducted to examine the effect of treatment with human chorionic gonadotropin (hCG) or ovine luteinizing hormone (LH) on the number and size distribution of steroidogenic luteal cells. In Experiment I, 27 ewes were assigned to one of three groups: 1) hCG (300 IU, i.v.) administered on Days 5 and 7.5 of the estrous cycle (Day 0 = Estrus); 2) LH (120 micrograms, i.v.) administered at 6-h intervals from Days 5 to 10 of the cycle; 3) saline (i.v.) administered as in the LH treatment group. Blood samples were drawn daily from the jugular vein for quantification of progesterone. On Day 10, corpora lutea were collected, decapsulated, weighed, and dissociated into single cell suspensions. Cells were fixed, stained for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity, and the size distribution of 3 beta HSD-positive cells was determined. Treatment with hCG, but not LH, increased (p less than 0.05) concentrations of progesterone in serum and the weight of corpora lutea. Treatment with either hCG of LH increased the proportion of cells greater than 22 micron in diameter and decreased the proportion of cells less than or equal to 22 micron (p less than 0.01). The ratio of small to large luteal cells decreased after treatment with either hCG or LH (p less than 0.05). In Experiment II, 9 ewes were assigned to one of two groups: 1) LH (120 micrograms, i.v.) administered at 6-h intervals from Days 5 to 10 of the estrous cycle, and 2) saline (i.v.) administered as in the LH treatment group.(ABSTRACT TRUNCATED AT 250 WORDS)     

A quantitative study of changes in the human corpus luteum microvasculature during the menstrual cycle

Endothelial cells are the most abundant cell type in the corpus luteum (CL), and changes in blood vessels have been proposed to play a pivotal role in CL regression. We have studied quantitatively the changes in the human granulosa-luteal microvasculature in CL of various ages: young (Days 17-19 of the cycle), mature (Days 20-24), old (Days 25-27), early regressing (follicular phase of the following cycle), and late regressing (luteal phase of the following cycle). Blood vessels were identified by immunohistochemical staining for the endothelial cell marker CD34. Because of the anisotropy of blood vessels, both vertical and transverse sections of the granulosa-lutein layer (GLL) were used to estimate relative (volume, surface, and length densities) and absolute (mean cross-sectional area) vascular variables. Full luteinization from young to mature CL was accompanied by a 61% increase in the mean cross-sectional area of vascular profiles and a 52% increase in the mean volume of granulosa-lutein cells, as an estimator of changes in the volume of the GLL. In old and early regressing CL, there was a progressive increase in relative structural vascular variables, due to the shrinkage of the GLL, whereas the mean cross-sectional area of capillaries showed a 53% decrease from mature to old CL. Finally, in late regressing CL, there was a decrease in most relative structural variables, in spite of the increasingly shrunken GLL. The decrease in the capillary diameter found at the late luteal phase most likely leads to a decreased blood flow, and early changes in blood vessels could initiate and/or accelerate CL regression.     

Effects of luteinizing hormone and human chorionic gonadotropin on corpus luteum cells in a spheroid cell culture system

The human corpus luteum (CL) is a highly vascularized, temporarily active endocrine gland and consists mainly of granulosa cells (GCs), theca cells (TCs), and endothelial cells (ECs). Its cyclic growth and development takes place under the influence of gonadotropic hormones. If pregnancy does occur, human chorionic gonadotropin (hCG) takes over the function of luteinizing hormone (LH) and, in contrast to LH, extends the functional life span of the CL. In this study, we investigated the effects of hCG and LH in a spheroidal cell culture model of CL development. Our data indicate that GCs secrete factors under the control of hCG that increase sprout formation of EC-spheroids. We demonstrate that the most prominent of these factors is VEGF-A. Furthermore, we found that both LH and hCG decrease sprout formation of GC-spheroids. After forming EC-GC coculture spheroids and consequently bringing GCs and ECs in close contact, sprouting increased under the influence of hCG, however not under LH. These experiments provide evidence for an hCG dependent functional switch in the GCs after coming in contact with ECs. Moreover, it demonstrates the considerably different effects of hCG and LH on GCs although their signaling is transmitted via the same receptor.     

Detection of relaxin by immunohistochemistry in the corpus luteum during lactation

The occurrence of relaxin in corpora lutea (CL) throughout lactation was studied in rats and pigs using the avidin-biotin immunoperoxidase procedure and homologous antisera to purified relaxins. In the rat, both CL from the previous pregnancy (CLp) and CL formed after postpartum ovulation, termed CL of lactation (CLL), were studied. In the rat, relaxin was localized only in cells of the CLp in early lactation, and immunostaining declined with advancing lactation. In late lactation (Days 16-20), immunoreactive relaxin first appeared in cells of the CLL, although the intensity was less relative to that observed in the CLp in early lactation. Cells of the CLp were sensitive to the effects of exogenous prostaglandins (PG) as shown by a loss of relaxin immunostaining at both 12 and 48 h after a PGF2 alpha challenge. In the sow, the CLp showed highest immunostaining in early lactation with a gradual reduction as lactation progressed, such that by Day 20 lactation, immunostaining was lost. These localization studies show that immunoreactive relaxin is present in the CL during lactation. Low levels of relaxin localized in the CLL of late lactation in the rat probably represents newly formed hormone, whereas the immunostaining in the CLp of the pig and rat appears to be residual relaxin and an indicator of the degeneration of the CLp with advancing lactation.     

The secretory patterns of relaxin and human chorionic gonadotropin in human pregnancy

Relaxin and human chorionic gonadotropin (hCG) were simultaneously determined in the same serum samples obtained from pregnant women. Although the secretory pattern of relaxin, in general, appeared to parallel that of hCG during human pregnancy, several discrepancies were discerned in the secretory patterns of the two hormones. The mean hCG concentration significantly differed between weeks 4-7 and 8-11 of pregnancy, but the mean relaxin concentration did not. The mean relaxin concentration began to decrease at weeks 16-19 whereas that of hCG did so at weeks 12-15. The mean relaxin concentration at weeks 4-7 was significantly higher than that at weeks 24-27, though there was no significant difference between the mean hCG concentrations in the two periods. These differences in the secretory pattern of relaxin from that of hCG indicate that relaxin secretion in pregnancy is not determined only by the circulating level of hCG. The responsiveness of the corpus luteum of pregnancy to hCG stimulation of relaxin secretion may vary as a function of the age of the corpus luteum, and this may partially account for the differences between the secretory pattern of relaxin and that of hCG observed in the present study.     

Effects of arachidonate metabolism inhibitors on basal and human chorionic gonadotropin-stimulated progesterone secretion by rat corpus luteum cells in vitro

Arachidonic acid (AA) and its metabolites mediate many physiological processes including reproduction and endocrinology. The current study investigated effects of several inhibitors of AA cascade on steroidogenesis by rat corpus luteum cells in vitro. Dispersed luteal cells prepared from rat corpus luteum on day 6 of pseudopregnancy secreted progesterone (P4) in time-dependent and human chorinonic gonadotropin (hCG)-dependent fashion. Arachidonyl trifluoromethyl ketone, a preferential inhibitor of the type IVA phospholipase A(2) (PLA(2)-IVA), stimulated basal P4 secretion and had no influence on hCG-stimulated steroidogenesis. A novel and more specific inhibitor pyrrophenone inhibited hCG-induced P4 secretion. A cyclooxygenase inhibitor indomethacin did not affect basal secretion but inhibited hCG-stimulated secretion. Nordihydroguaiaretic acid tended to decrease basal P4 secretion and completely inhibited hCG-stimulated secretion. Obtained results suggest that AA metabolic cascade, which is triggered at least in part by PLA(2)-IVA activity, is potentially implicated in hCG-stimulated P4 secretory response in the rat corpus luteum.     

Ultrasonographic assessment of the endometrium in rhesus monkeys during the normal menstrual cycle

This study was undertaken to determine whether cyclical changes in the endometrium of the rhesus monkey could be observed by using ultrasound. Three indices of endometrial size were examined: the antero-posterior (or ventro-dorsal), longitudinal, and transverse diameters. Changes in the ultrasonic reflectivity of the endometrium were also assessed. We have attempted to correlate these endometrial parameters with the hormonal status of the animal. Ultrasonography was performed for an average of 12 consecutive days during 19 menstrual cycles. All ultrasonic recordings were normalized to the day of the estradiol (E2) peak (Day 0). We found that the reflectivity of the endometrium was dependent on the stage of the cycle: during the follicular phase, the endometrium appeared less echogenic (darker) compared to the myometrium; in the luteal phase, the endometrium was more echogenic (lighter). During the follicular phase (Days -9 to 0), there was a linear increase in the antero-posterior (p less than 0.001), longitudinal (p less than 0.05), and transverse (p less than 0.001) diameters. In the luteal phase (Days 1-15), no significant changes were observed in these diameters. An estimated endometrial volume (EEV) was obtained by the product of the antero-posterior, longitudinal, and transverse diameters. Each animal observed during the follicular phase (n = 14) exhibited a peak in the EEV, which correlated with the day of the E2 peak (p less than 0.01). From this study, we conclude that the sonographic appearance of the endometrium of the rhesus monkey reflects the cyclical changes that occur during the menstrual cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessment of circadian rhythms throughout the menstrual cycle of female rhesus monkeys

Reproductive cyclicity has a significant influence on the regulation of circadian rhythms in rodents. Studies have suggested that there are changes in body temperature rhythms between the follicular and luteal phases in human females. This study examined the effects of menstrual cyclicity on physiological and behavioral circadian rhythms in female rhesus monkeys (Macaca mulatta), an acknowledged biomedical model. Seven unrestrained subjects were implanted with a biotelemetry transmitter to measure body temperature and heart rate and an accelerometer was used to measure physical activity. Water was available ad libitum and drinking was measured via an electronic circuit attached to a water lixit. A video-based task system, the Psychomotor Test System, provided environmental enrichment and delivered a pelletized diet. Mean, phase, and amplitude of each rhythm were calculated. Estrogen and progesterone conjugates were assayed and quantified from daily urine samples to identify follicular and luteal phases of the menstrual cycle. Average circadian variables were then compared between these phases. Heart rate was significantly (P< or =0.05) delayed in the luteal phase. Albeit non-significant, analysis showed a trend toward decreased circadian amplitude of body temperature in the luteal phase.     

Effects of chorionic gonadotropin on peripheral monocytes vary with the phases of the menstrual cycle

The qualitative and quantitative effects of physiological concentrations of chorionic gonadotropin (CG) on monocytes of women vary with the phases of the menstrual cycle. During the follicular phase, the hormone inhibits phagocytosis; stimulates the secretion of interleukin (IL) 6 (IL-6), interferon α (IFN-α), and the protein component of apolipoprotein A1 (APO-A1); and activates myeloperoxidase (MPO). During the luteal phase, CG stimulates phagocytosis and APO-A1 secretion, inhibits MPO, and does not shift the levels of IL-6 and INF-α. Regardless of the menstrual phase, the hormone does not modify the release of elastase or the production of granulocytic colony-stimulating growth factor, IL-1α, and tumor necrosis factor α.     

Isolation and characterization of cell subpopulations from the monkey corpus luteum of the menstrual cycle

This study was designed to test the hypothesis that the corpus luteum of primate species consists of cell subpopulations that differ in physical characteristics, function, and regulation by endocrine and paracrine factors. The corpus luteum (n = 25) was removed from rhesus monkeys at the mid-luteal phase of the menstrual cycle (Days 7-8 after the surge of luteinizing hormone, LH) and enzymatically dispersed. Freshly dispersed cells were analyzed and sorted on the basis of their forward and 90 degrees light scatter (FLS and 90LS, respectively) properties using an EPICS C flow cytometer. Freshly dispersed and sorted cells were fixed, stained histochemically for the presence of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and measured to determine their diameters. Freshly dispersed (MIX) and sorted cells from corpora lutea during the early (Days 4-5 after the LH surge; n = 4) and mid-luteal phases of the cycle were incubated in vitro and steroid production was assessed. The size distribution of dispersed cells revealed four peaks that corresponded to small (10-15 microns in diameter) 3 beta-HSD-negative, and small, medium (16-20 microns), and large (greater than 20 microns) 3 beta-HSD-positive cells. Analysis of dispersed cells for FLS and 90LS demonstrated two continua (C alpha and C beta). C alpha contained single cells and cell clusters; 99.7 +/- 0.3% (n = 3) of the cells were less than or equal to 15 microns in diameter and 96.7 +/- 0.3% were 3 beta-HSD-negative. C alpha cells produced low levels of progesterone (0.2 +/- 0.1 ng/ml per 5 x 10(4) cells; n = 3) in vitro under basal conditions. C beta consisted of single cells from 10 microns to 40 microns in diameter and contained the lipid-filled and 3 beta-HSD-positive cells. Two regions (R1 and R3) of C beta were defined and their cells separated. In R1, 96 +/- 2% (n = 3) of the cells had diameters of less than or equal to 15 microns, whereas 82 +/- 4% (n = 3) of those in R3 were greater than or equal to 20 microns. Basal progesterone production by R3 cells from early luteal phase of the cycle was 12 times greater than that by R1 cells (n = 3 per group).(ABSTRACT TRUNCATED AT 400 WORDS)     

Gonadotropin and steroid regulation of matrix metalloproteinases and their endogenous tissue inhibitors in the developed corpus luteum of the rhesus monkey during the menstrual cycle

The factors regulating the dynamic expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in the primate corpus luteum (CL) during the menstrual cycle are unknown. We hypothesized that LH or progesterone (P) regulate interstitial-collagenase (MMP-1), the gelatinases (MMP-2 and -9), TIMP-1, and TIMP-2 in the CL. Hormone ablation/replacement was performed in rhesus monkeys on Days 9-11 of the luteal phase in five treatment groups (n = 4/group): control (no treatment), antide (GnRH antagonist), antide + LH; antide + LH + trilostane (TRL; 3beta-hydroxysteroid dehydrogenase inhibitor), and antide + LH + TRL + R5020 (nonmetabolizable progestin). On Day 12, the CL was removed and the RNA and protein isolated for real-time polymerase chain reaction and immunoassays, respectively. The MMP-1 mRNA increased 20-fold with antide, whereas LH replacement maintained MMP-1 mRNA at control levels. Likewise, TRL increased MMP-1 mRNA 54-fold, and R5020 prevented this effect. Immunodetectable MMP-1 protein also increased with antide or TRL; these increases were abated with LH or R5020. Gelatinase mRNA and/or protein levels increased with antide (e.g., 3-fold, MMP-2 mRNA), and LH replacement reduced protein levels (e.g., 11-fold, MMP-2). The TRL increased MMP-9, but not MMP-2, expression; however, R5020 replacement had no effect on mRNA or protein levels. The LH treatment increased TIMP-1 and -2 mRNA and TIMP-1 protein expression compared to controls and antide groups, whereas R5020 enhanced only immunodetectable TIMP-1. These data strongly suggest that LH suppresses MMP-1 in the primate CL via P and that it also suppresses gelatinases, either at the mRNA (MMP-2) or protein (MMP-2 and -9) levels, perhaps in part via steroids, including P. In contrast, LH promotes TIMP expression, perhaps via steroids, including P.     

Changes in prostaglandin secretion by the regressing bovine corpus luteum

Secretion of prostaglandins (PGs) by the regressing corpus luteum (CL) was investigated in the cow. Six cows were implanted with microcapillary dialysis membranes of a microdialysis system (MDS) into the CL during Days 8-9 (Day 0 = estrus), and a prostaglandin (PG) F2alpha analogue (Estrumate) was injected intramuscularly (i.m.) to induce luteolysis. Acute increases in intraluteal release of PGF2alpha and PGE2 were observed during the first 4 h, followed by decreases over the next 8 h. Intraluteal release of both PGs gradually increased again during the period 48-72 h. Concentrations of PGF2alpha in ovarian venous plasma (OVP) were 4-13 times higher than those of jugular venous plasma (JVP) (P < 0.001) during the period of the experiment, and increased from 24 h after treatment with Estrumate (P < 0.05). Cyclooxygenase (COX)-2 mRNA expression increased (P < 0.05) at 2 and 24 h after treatment with Estrumate. The results indicated that local release of PGF2alpha and PGE2, and COX-2 mRNA expression were increased by Estrumate in the regressing CL at the later stages of luteolysis. Thus, luteal secretion of PGs may be involved in the local mechanism for structural rather than functional luteolysis.     

Effect of inhibition of estrogen synthesis during the luteal phase on function of the corpus luteum in rhesus monkeys

It has been hypothesized that estrogen synthesized by the corpus luteum initiates luteal regression during the nonfertile menstrual cycle in primates. To study the role of endogenous estrogens in functional regression of the monkey corpus luteum, we administered the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) to rhesus monkeys during the luteal phase of the menstrual cycle. Twice-daily oral administration of ATD suppressed systemic and intraluteal estrogen levels by 80-90%. The midluteal phase rise in estradiol concentrations that occurs in rhesus monkeys was completely abolished by ATD treatment. Despite suppression of estrogen synthesis during the luteal phase, mean menstrual cycle length and length of the luteal phase were not different than in control monkeys treated with vehicle only. Progesterone levels were lower in the ATD-treated group on the second and third day of treatment, but did not differ from control levels during the remainder of the cycle. These data suggest that elevated estrogen synthesis during the luteal phase of the menstrual cycle is not a prerequisite for spontaneous luteolysis in rhesus monkeys.     

Regulation of progesterone secretion in human syncytiotrophoblast in culture by human chorionic gonadotropin.

In the present investigation we sought to define the specific sites in the pathway of placental progesterone biosynthesis that underlie the action of human chorionic gonadotropin (hCG). When the cells were challenged with dibutryl cAMP (dbcAMP), forskolin or isobutylmethylxanthine, they produced significantly higher amounts of progesterone which in the presence of the hCG antibody was reduced to the level of the control set of cells. Trophoblast cells cultured in serum free medium with 25-hydroxycholesterol (25-OHC) produced increased amounts of progesterone. In the presence of hCG antibody at a concentration which neutralized the secreted hCG, the steroid production was completely blocked, even when the 25-OHC was added to the medium. Also, direct quantitation of the cytochrome P450 SCC enzyme in the absence of hCG indicated a significant decrease. The exogenous addition of low density lipoproteins (LDL) increased the progesterone secretion by the trophoblast cells in culture. Neutralization of hCG by the antibodies, however, drastically reduced the LDL induced progesterone secretion, which was restored by the addition of dbcAMP to the medium. Based on these findings, we suggest a stimulatory effect of hCG on normal trophoblast cells at the level of LDL utilization and cytochrome P450 SCC enzyme. Since dbcAMP could mimic these actions of hCG, the data suggest a possible autocrine/paracrine role of hCG on the trophoblast cells. An additive effect of hCG and cAMP on progesterone secretion observed in our studies, indicate that apart from hCG, adenylate cyclase activity may also be regulated by other factors.