Progesterone production by monkey luteal cell subpopulations at different stages of the menstrual cycle: changes in agonist responsiveness.

Small (less than or equal to 15 microns diameter) and large (greater than 20 microns diam.) luteal cells of the rhesus monkey have been separated by flow cytometry based on light scatter properties. To determine whether the steroidogenic ability and agonist responsiveness of luteal cell subpopulations vary during the life span of the corpus luteum, small and large cells were obtained at early (Days 3-5), mid (Days 7-8), mid-late (Days 11-12), and late (Days 14-15) luteal phase of the cycle. Cells (n = 4 exp./group) were incubated in Ham's F-10 medium + 0.1% BSA for 3 h at 37 degrees C with or without hCG (100 ng/ml), prostaglandin E2 (PGE2; 14 microM), dibutyryl-cAMP (db-cAMP; 5 mM), or pregnenolone (1 microM). Basal progesterone (P) production by large cells was up to 30-fold that by small cells depending on the stage of the cycle. HCG stimulated (p less than 0.05) P secretion by both small (1.8 +/- 0.2-fold) and large (3.7 +/- 0.7-fold) cells in the early luteal phase. HCG responsiveness declined during the luteal lifespan; P production by small cells was not significantly enhanced by hCG by mid luteal phase, whereas that by large cells was stimulated 1.7 +/- 0.2-fold (p less than 0.05) even at late luteal phase. Cell responses to db-cAMP were similar to those for hCG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intraluteal infusions of prostaglandins of the E, D, I, and A series prevent PGF2 alpha-induced, but not spontaneous, luteal regression in rhesus monkeys

A luteotropic role for prostaglandins (PGs) during the luteal phase of the menstrual cycle of rhesus monkeys was suggested by the observation that intraluteal infusion of a PG synthesis inhibitor caused premature luteolysis. This study was designed to identify PGs that promote luteal function in primates. First, the effects of various PGs on progesterone (P) production by macaque luteal cells were examined in vitro. Collagenase-dispersed luteal cells from midluteal phase of the menstrual cycle (Day 6-7 after the estimated surge of LH, n = 3) were incubated with 0-5,000 ng/ml PGE2, PGD, 6 beta PGI1 (a stable analogue of PGI2), PGA2, or PGF2 alpha alone or with hCG (100 ng/ml). PGE2, PGD2, and 6 beta PGI1 alone stimulated (p less than 0.05) P production to a similar extent (2- to 3-fold over basal) as hCG alone, whereas PGA2 and PGF2 alpha alone had no effect on P production. Stimulation (p less than 0.05) of P synthesis by PGE2, PGD2, and 6 beta PGI1 in combination with hCG was similar to that of hCG alone. Whereas PGA2 inhibited gonadotropin-induced P production (p less than 0.05), that in the presence of PGF2 alpha plus hCG tended (p = 0.05) to remain elevated. Second, the effects of various PGs on P production during chronic infusion into the CL were studied in vivo. Saline with or without 0.1% BSA (n = 12), PGE2 (300 ng/h; n = 4), PGD2 (300 ng/h; n = 4), 6 beta PGI1 (500 ng/h; n = 3), PGA2 (300 ng/h; n = 4), or PGF2 alpha (10 ng/h; n = 8) was infused via osmotic minipump beginning at midluteal phase (Days 5-8 after the estimated LH surge) until menses. In addition, the same dose of PGE, PGD, PGI, or PGA was infused in combination with PGF2 alpha (n = 3-4/group) for 7 days. P levels over 5 days preceding treatment were not different among groups. In 5 of 8 monkeys receiving PGF2 alpha alone, P declined to less than 0.5 ng/ml within 72 h after initiation of infusion and was lower (p less than 0.05) than controls. The length of the luteal phase in PGF2 alpha-infused monkeys was shortened (12.3 +/- 0.9 days; mean +/- SEM, n = 8; p less than 0.05) compared to controls (15.8 +/- 0.5). Intraluteal infusion of PGE, PGD, PGI, or PGA alone did not affect patterns of circulating P or luteal phase length.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ovarian and luteal blood flow, and peripheral plasma progesterone levels, in cyclic guinea-pigs

Ovarian and luteal blood flow rates were studied using radioactive microspheres in guinea-pigs between Day 6 of the oestrous cycle and Day 1 of the following cycle. Peripheral plasma progesterone levels were measured by radioimmunoassay on the same days of the oestrous cycle. Ovarian blood flow was greatest between Days 9 and 12 and had fallen by Day 16 both in absolute (ml . min-1) and relative (ml.min-1.g-1) terms. Luteal weight and blood flow were also greatest between Days 9 and 12 and had fallen sharply by Day 16. The highest mean (+/- s.d.) luteal flows measured were 0.10 +/- 0.04 ml.min-1 per corpus luteum, and 24.26 +/- 9.3 ml.min-1.g-1 luteal tissue on Day 10 of the cycle. Mean peripheral plasma progesterone levels reached a maximum of 3.66 +/- 1.1 ng/ml at Day 12 of the cycle and fell thereafter, reaching 0.74 +/- 0.5 ng/ml by Day 1 of the following cycle. Plasma progesterone levels declined significantly between Days 12 and 14 of the cycle, whereas no significant drop in luteal blood flow was demonstrable until after Day 14. These data do not support the idea that declining luteal blood flow is an initiating mechanism in luteal regression in the guinea-pig.     

Prostaglandin F2 alpha, oxytocin and progesterone secretion by bovine luteal cells at several stages of luteal development: effects of oxytocin, luteinizing hormone, prostaglandin F2 alpha and estradiol-17 beta

Bovine luteal cells from Days 4, 8, 14 and 18 of the estrous cycle were incubated for 2 h (1 x 10(5) cells/ml) in serum-free media with one or a combination of treatments [control (no hormone), prostaglandin F2 alpha (PGF), oxytocin (OT), estradiol-17 beta (E) or luteinizing hormone (LH)]. Luteal cell conditioned media were then assayed by RIA for progesterone (P), PGF, and OT. Basal secretion of PGF on Days 4, 8, 14 and 18 was 173.8 +/- 66.2, 111.1 +/- 37.8, 57.7 +/- 15.4 and 124.3 +/- 29.9 pg/ml, respectively. Basal release of OT and P was greater on Day 4 (P less than 0.01) than on Day 8, 14 and 18 (OT: 17.5 +/- 2.6 versus 5.6 +/- 0.7, 6.0 +/- 1.4 and 3.1 +/- 0.4 pg/ml; P: 138.9 +/- 19.5 versus 23.2 +/- 7.5, 35.4 +/- 6.5 and 43.6 +/- 8.1 ng/ml, respectively). Oxytocin increased (P less than 0.01) PGF release by luteal cells compared with control cultures irrespective of day of estrous cycle. Estradiol-17 beta stimulated (P less than 0.05) PGF secretion on Days 8, 14 and 18, and LH increased (P less than 0.01) PGF production only on Day 14. Prostaglandin F2 alpha, E and LH had no effect on OT release by luteal cells from any day. Luteinizing hormone alone or in combination with PGF, OT or E increased (P less than 0.01) P secretion by cells from Days 8, 14 and 18. However on Day 8, a combination of PGF + OT and PGF + E decreased (P less than 0.05) LH-stimulated P secretion. These data demonstrate that OT stimulates PGF secretion by bovine luteal cells in vitro. In addition, LH and E also stimulate PGF release but effects may vary with stage of estrous cycle.     

Effect of rate of change of luteinizing hormone concentration on in-vitro progesterone secretion within rat corpora lutea during differentiation.

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

Peripheral inhibin levels in relation to climatic variations and stage of estrous cycle in buffalo (Bubalus bubalis )

The present study investigated the peripheral plasma inhibin levels in relation to 1) the stage of estrous cycle and the effect of climatic variations. Blood samples were collected from cyclic buffalo (n=5) once daily for 32 consecutive days during the tropical hot humid (summer) and cold (winter) seasons. Estrus was recorded by parading a vasectomized bull as well as by plasma progesterone determination. In the winter season, peripheral inhibin concentrations which were lowest (0.35 +/- 0.02 ng/ml) during the mid-luteal phase of estrous cycle (Day 6 to Day 14, Day 0 = day of estrus) increased significantly (P < 0.02) to 0.47 +/- 0.04 ng/ml during the late luteal phase (Day -4 to Day -2) and then further to 0.52 +/- 0.03 ng/ml (P< 0.02) during the periestrus phase (Day -1 to Day 1). Inhibin concentrations then decreased significantly (P < 0.02) to 0.40 +/- 0.03 ng/ml during the early luteal phase (Day 2 to Day 5). In the summer season the differences in peripheral inhibin concentrations among different phases of estrous cycle were found to be nonsignificant. A comparison of the circulating inhibin concentrations between the two seasons indicated that inhibin concentrations were significantly higher in the late luteal phase (P < 0.01) and periestrus phase (P < 0.05) during the winter season compared with corresponding periods during the summer season. The present study suggests that peripheral inhibin concentrations change in the estrous cycle during cooler breeding season and that environmental heat stress can cause a reduction in peripheral inhibin concentrations.     

Induction of relaxin secretion in rhesus monkeys by human chorionic gonadotropin: dependence on the age of the corpus luteum of the menstrual cycle

Peripheral concentrations of immunoreactive relaxin are undetectable in primates during the nonfertile menstrual cycle, but become measurable during the interval when chorionic gonadotropin (CG) rises in early pregnancy. The objectives of the current study were to determine if exogenous CG, administered in a dosage regimen which invoked patterns and concentrations resembling those of early pregnancy, would induce relaxin secretion in nonpregnant rhesus monkeys, and whether the induction was dependent on the age of the corpus luteum (CL) at the onset of treatment. Female rhesus monkeys received twice-daily i.m. injections of increasing doses of human CG (hCG) for 10 days beginning in the early (n = 4), mid (n = 6) or late (n = 4) luteal phase of the menstrual cycle [5.3 +/- 0.3, 8.3 +/- 0.5, and 12.0 +/- 0.4 days after the midcycle luteinizing hormone (LH) surge, respectively; means +/- SEM]. Whereas immunoreactive relaxin was nondetectable in the luteal phase of posttreatment cycles, detectable levels of relaxin were observed in 2 of 4, 5 of 6, and 3 of 4 monkeys during hCG treatment in the early, mid and late luteal phase, respectively. Although CG treatment rapidly enhance progesterone levels, the appearance of relaxin was deferred; relaxin was first detectable 9.0 +/- 1.0 and 4.7 +/- 1.9 days after the onset of CG treatment at early and late luteal phases. Patterns of relaxin concentrations differed among groups (P less than 0.05, ANOVA; split plot design) and relaxin levels were lowest (P less than 0.01) in monkeys treated during the early luteal phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin F in reproductive tissues collected during the luteal phase of the estrous cycle and at parturition in Virginia opossums (Didelphis virginiana )

Prostaglandin F(2alpha) (PGF(2alpha)) plays a role in the regression of the corpus luteum (CL) in a number of placental mammals. However, the mechanism of luteal regression has not been extensively studied in marsupials. The objectives of this study were to characterize changes in concentrations of PGF(2alpha) within utero-ovarian (UO) tissue/venous plasma during the luteal phase of the estrous cycle in Virginia opossums, to correlate these changes with those of plasma progesterone (P(4)), and to characterize the peripheral pattern of 13,14-dihydro-15-keto-PGF(2alpha) (PGFM) in parturient opossums. Ovaries, uteri, UO venous plasma and peripheral plasma were collected on Days 5, 9 and 12 after induced ovulation (n = 3 to 4 opossums/group). In addition, concentrations of PGFM were measured in peripheral plasma collected from two opossums during late gestation (Days 7,9,11 and 12) and at parturition (Day 13). Concentrations of P(4), PGFM and PGF(2alpha) in tissue homogenates and plasma samples were estimated by radioimmunoassay. In nonpregnant opossums, peripheral P(4) levels were highest on Day 5 (38.8 +/- 11.1 ng/ml, x +/- SEM) declined on Day 9 (22.6 +/- 7.4 ng/ml), and were at basal levels by Day 12 (2.4 +/- 0.7 ng/ml). Endometrial concentrations of PGF(2alpha) increased (P = 0.056) from Day 5 (15.7 +/- 4.1 ng/g) to Day 9 (92.1 +/- 61.0 ng/g) and were maintained to Day 12 (97.2 +/- 25.7 ng/g). Prostaglandin F(2alpha) concentrations in UO plasma increased (P < 0.01) from Day 5 (143.1 +/- 32.7 pg/ml) to Day 12 (333.0 +/- 32.4 pg/ml). Prostaglandin F(2alpha) concentrations in ovarian tissue followed a similar pattern and were correlated with UO concentrations (r = 0.708, P < 0.05). In pregnant opossums, the highest levels of peripheral PGFM were recorded in the peripartum period, when luteal regression would also be expected to occur. The negative temporal relationship between peripheral concentrations of P(4) and concentrations of PGF(2alpha) in UO tissue/venous plasma observed in this preliminary study is consistent with the notion that PGF(2alpha) from the ovary and/or uterus may play a role in CL regression in the opossum.     

Secretory dynamics of bioactive and immunoactive LH during the oestrous cycle of the sheep

Blood samples were collected every 15 min for 6 h during the follicular (1 day before oestrus), and early (Days +1 to +3), mid- (Days +4 to +8), and full (Days +9 to +14) luteal phases of the oestrous cycle. Serum concentrations of immunoactive LH were measured by radioimmunoassay. The biological activity of serum LH was determined by an in-vitro bioassay that uses LH-induced testosterone production from mouse interstitial cells as an endpoint. Only ovine and bovine LH and hCG had appreciable activity in this bioassay. The temporal pattern of secretion of bioactive LH paralleled the secretory pattern of immunoactive LH at all stages of the ovine oestrous cycle. However, the secretory pattern itself varied regularly through the oestrous cycle. The frequency of secretory excursions of LH was highest during the follicular phase (6.2 +/- 0.9 pulses/6 h) and was progressively reduced through the luteal phase (1.1 +/- 0.1 pulses/6 h during full luteal phase). Conversely, amplitude of secretory excursions of immunoactive LH was low during the follicular phase (0.79 +/- 0.08 ng/ml) and significantly (P less than 0.05) increased during the mid- and full luteal phases (1.49 +/- 0.10 and 2.37 +/- 0.20 ng/ml, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics and regulation of the ovarian cycle in vervet monkeys (Chlorocebus aethiops)

This study was designed to evaluate the timecourse of ovarian and pituitary endocrine events throughout the menstrual cycle in the vervet monkey, and whether circulating luteinizing hormone (LH) or the uterus regulates the functional lifespan of the vervet corpus luteum. Daily saphenous blood samples were collected from adult females (1) during spontaneous menstrual cycles (n = 7), and (2) during cycles in which a gonadotropin-releasing hormone antagonist (acyline) was administered for 3 days at midluteal phase (n = 3), and (3) for 30 days following recovery from hysterectomy (n = 4). Estradiol (E) and progesterone (P) levels were assayed using electrochemoluminescent assays. Gonadotropin levels were measured by radioimmunoassay using reagents developed for the assay of follicle-stimulating hormone and LH in macaques. Spontaneous cycles exhibited a midcycle E rise (476+/-49 pg/ml), engendering an LH surge, 12+/-1 days after onset of menses, followed by a luteal phase with peak P levels of 4.7+/-0.9 ng/ml. Histologic evaluation of the ovaries at late follicular phase or early luteal phase revealed the presence of a single, large Graafian follicle or developing corpus luteum, respectively. Acyline treatment caused a significant (P<0.05) decline in P levels (2.9+/-0.5 vs 0.5+/-0.3 ng/ml, 0 vs 48 h post-treatment) and premature menstruation compared with untreated controls (P<0.05). Hysterectomy had no apparent effect on the monthly pattern or levels of circulating E or P. Thus, the characteristics and regulation of the ovarian cycle in vervets appear similar to those in women and macaques, with cyclicity dependent on pituitary gonadotropin hormones and independent of a uterine luteolytic factor.     

Pattern of ovarian progesterone secretion during the luteal phase of the ovine estrous cycle

Pulsatile secretion of progesterone has been observed during the late luteal phase of the menstrual cycle in the rhesus monkey and human. As the luteal phase progresses in each of these species, there is a pattern of decreased frequency and increased amplitude of progesterone pulses. The present study was designed to determine the pattern of progesterone secretion during the late luteal phase (Days 10-16) of the normal ovine estrous cycle. Five unanesthetized ewes, each bearing an indwelling cannula in the utero-ovarian vein, were bled every 15 min from 0800 h on Day 10 through 0800 h on Day 16 of the estrous cycle. With the computer program PULSAR, it was determined that progesterone secretion was episodic, with pulsations observed on all days. Analysis of variance was used to determine differences in frequency, amplitude, and interpeak interval (IPI) of progesterone pulses among ewes and days. The ewes averaged 8.0 +/- 0.63 pulses of progesterone per 24 h. Mean frequency of pulses was not different between days but showed differences between ewes. Mean amplitude of progesterone pulses was 7.0 +/- 0.27 ng/ml, with no differences observed either between days or between ewes. Mean IPI was 197 +/- 7.1 min, and, like frequency, the IPI was not different between days, but varied between ewes. No consistent temporal relationship was found between progesterone pulses and luteinizing hormone (LH), as determined by bioassay and radioimmunoassay, on Day 14 of the cycle in one ewe. The results indicate that progesterone secretion is episodic during the luteal phase of the ovine estrous cycle and is independent of LH pulses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of ovarian tissue reduction on the menstrual cycle: persistent normalcy after near-total oophorectomy

These experiments were designed to evaluate whether removal of approximately 95% visible ovarian tissue would interrupt the short- or long-term regulation of cyclic ovarian function. On cycle Days 2 4 (onset of menses = Day 1), the entire left ovary and approximately 90% of the right ovary were removed from three cycling cynomolgus monkeys. After approximately 95% ovariectomy, there was an acute elevation of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which lasted 11 +/- 2 days. A midcycle-like gonadotropin surge occurred 20 +/- 3 days following approximately 95% ovariectomy; the next menses occurred 19 +/- 1 days later. Follicular phase patterns of estradiol preceded the midcycle gonadotropin surge, and luteal phase progesterone levels indicated subsequent ovulation. Two of three monkeys resumed normal menstrual cyclicity in the following cycle with follicular phase, luteal phase, and menstrual cycle lengths similar to pretreatment levels. Histological examination of the ovarian remnant removed on Day 21 of the next cycle revealed a morphologically normal corpus luteum and many small follicles. A second group of 6 rhesus monkeys also underwent approximately 95% ovariectomy for long-term evaluation of menstrual cyclicity; typical 28-day menstrual cycle patterns were observed in 4 of the 6 monkeys for 5 mo, with 2 of these 3 animals maintaining regular menstrual cycles for 1 yr. In summary, our data suggest that normal ovarian function, i.e. recruitment, selection, and dominance of the ovulatory follicle, ovulation, and subsequent corpus luteum function, is maintained with only approximately 5% of functional ovarian tissue remaining.(ABSTRACT TRUNCATED AT 250 WORDS)     

The synchrony of prostaglandin-induced estrus in cows was reduced by pretreatment with hCG

The induction of optimal synchrony of estrus in cows requires synchronization of luteolysis and of the waves of follicular growth (follicular waves). The aim of this study was to determine whether hormonal treatments aimed at synchronizing follicular waves improved the synchrony of prostaglandin (PG)-induced estrus. In Experiment 1, cows were treated on Day 5 of the estrous cycle with saline in Group 1 (n = 25; 16 ml, i.v., 12 h apart), with hCG in Group 2 (n = 27; 3000 IU, i.v.), or with hCG and bovine follicular fluid (bFF) in Group 3 (n = 21; 16 ml, i.v., 12 h apart). On Day 12, all cows were treated with prostaglandin (PG; 500 micrograms cloprostenol, i.m.). In Experiment 2, cows were treated on Day 5 of the estrous cycle with saline (3 ml, i.m.) in Group 1 (n = 22) or with hCG (3000 IU, i.v.) in Group 2 (n = 20) and Group 3 (n = 22). On Day 12, the cows were treated with PG (500 micrograms in Groups 1 and 2; 1000 micrograms in Group 3). Blood samples for progesterone (P4) determination were collected on Day 12 (Experiment 1) or on Days 12 and 14 (Experiment 2). Cows were fitted with heat mount detectors and observed twice a day for signs of estrus. Four cows in Experiment 1 (1 cow each from Groups 1 and 2; 2 cows from Group 3) had plasma P4 concentrations below 1 ng/ml on Day 12 and were excluded from the analyses. In Experiment 1, cows treated with hCG or hCG + bFF had a more variable (P = 0.0007, P = 0.0005) day of occurrence of and a longer interval to estrus (5.9 +/- 0.7 d, P = 0.003 and 6.2 +/- 0.8 d, P = 0.005) than saline-treated cows (3.4 +/- 0.4 d). The plasma P4 concentrations on Day 12 were higher (P < 0.0001) in hCG- and in hCG + bFF-treated cows than in saline-treated cows (9.4 +/- 0.75 and 8.5 +/- 0.75 vs 4.1 +/- 0.27 ng/ml), but there was no correlation (P > 0.05) between plasma P4 concentrations and the interval to estrus. In Experiment 2, cows treated with hCG/500PG and hCG/1000PG had a more variable (P = 0.0007, P = 0.002) day of occurrence of and a longer interval to estrus (4.2 +/- 0.4 d, P = 0.04; 4.1 +/- 0.4 d, P = 0.03) than saline/500PG-treated cows (3.2 +/- 0.1 d). The concentrations of plasma P4 on Days 12 and 14 of both hCG/500PG- and hCG/1000PG-treated cows were higher (P < 0.05) than in saline/500PG-treated cows (7.3 +/- 0.64, 0.7 +/- 0.08 and 7.7 +/- 0.49, 0.7 +/- 0.06 vs 5.3 +/- 0.37, 0.5 +/- 0.03 ng/ml). The concentrations of plasma P4 on Days 12 or 14 and the interval to estrus were not correlated (P > 0.05) in any treatment group. The concentrations of plasma P4 on Days 12 and 14 of hCG/500PG- or hCG/1000PG-treated cows were correlated (r = 0.65, P < 0.05; r = 0.50, P < 0.05). This study indicated that treatment of cows with hCG on Day 5 of the estrous cycle reduced the synchrony of PG-induced estrus and that this reduction was not due to the failure of luteal regression.     

Transitory increases of luteinizing hormone and follicle-stimulating hormone following luteectomy in hemiovariectomized primates significantly increase progesterone secretion in vitro by the subsequent corpus luteum

Ten chronically hemiovariectomized cynomolgus and rhesus monkeys were luteectomized 5.5 +/- 0.3 days after the midcycle luteinizing hormone (LH) and follicle-stimulating hormone (FSH) surge in two consecutive cycles. The corpus luteum (CL) was removed, weighed, dispersed with collagenase and the luteal cells counted. Luteal cells (50,000/ml) were incubated in Ham's F10 medium for 3 h at 37 degrees C either in the presence or absence of 100 ng/ml human chorionic gonadotropin (hCG). Daily blood samples were taken from the monkeys throughout the study for determination of LH, FSH, estradiol (E2) and progesterone levels. Within 5 days following each luteectomy (LX), all monkeys responded with a significant increase in FSH and LH (P less than 0.05). Ovulatory LH/FSH surges occurred 14.4 +/- 0.5 days after the first LX. Hormonal profiles of serum progesterone prior to the first and second LX, CL weight and number of luteal cells/CL were similar (P greater than 0.05). However, luteal cells obtained at the second LX produced more progesterone (P less than 0.05) in vitro under basal and hCG-stimulated conditions than cells from the first LX. The areas under the LH and FSH curves following the first LX were highly correlated (P less than 0.05) with the in vitro progesterone production following the second LX. Thus, the monkeys with the largest areas under the LH and FSH curves subsequently had the highest in vitro progesterone production.     

The role of the adenylyl cyclase system in the regulation of corpus luteum function in the human and in nonhuman primates

We have reviewed the properties of luteinizing hormone/human chorionic gonadotropic (LH/hCG)-sensitive adenylyl cyclase (AC) of human corpus luteum (CL) and its regulation by several hormones and nonhormonal activators. We have also described the changes in enzyme activity in membrane preparations of human and cynomolgus monkey CL obtained at various stages of the menstrual cycle and pregnancy. The data have been analyzed with respect to the functional status of the luteal tissue and to the species differences among primate CL. In the menstrual cycle, luteal AC responsiveness to LH/hCG was detectable during the midluteal phase, but not during the late luteal phase or in the follicular phase of the following cycle. In addition, nonhormonal stimulation was high in CL obtained during the midluteal and late luteal phases, but declined drastically by the follicular phase of the next cycle. In early pregnancy, the enzyme was unresponsive to LH/hCG stimulation, yet its sensitivity to nonhormonal stimulation was similar, if not identical, to that of midluteal phase CL. Functional activity was also evident at the end of pregnancy. These results demonstrate that expression of AC activity in primate luteal membrane changes significantly with varying hormonal status under physiologic conditions. It is concluded that the AC system in luteal membranes is an effective model to study the mechanisms that regulate function and life span of the human and nonhuman primate CL.     

Effects of progesterone receptor blockers on human granulosa-luteal cell culture secretion of progesterone, estradiol, and relaxin

We have developed culture methods for human luteinizing granulosa cells (GLC) that support the timely and dynamic secretion of estrogen (estradiol-17beta; E(2)), progesterone (P(4)), and relaxin (Rlx) in patterns that mimic serum hormone concentrations during the luteal phase of the menstrual cycle. Additional hCG, to simulate rescue of the corpus luteum, prevented the normal decline in GLC hormone production. To test the importance of the P(4) receptor in P(4) production, GLC were treated in vitro with two P(4) receptor antagonists. Human GLC received one of two hCG support protocols: a Baseline group simulating the normal luteal phase or a Rescue group simulating early pregnancy. Baseline and Rescue groups were treated with either RU-486 or HRP2000 either early or late in the cell culture period. The effects of treatments or control on ovarian steroid and peptide hormone production were determined (significant difference was P < 0.05). In the Rescue group, late treatment resulted in an immediate and dramatic decline in E(2), P(4), and Rlx secretion to nearly nondetectable levels within 1 day after treatment, and hormones remained depressed for the remaining 10 days of culture. In contrast, early treatment resulted in a decline in steroid hormone secretion that returned to control levels within 5 days of cessation of treatment, and Rlx secretion was delayed for approximately 5 days more than in controls. The data support the hypothesis that P(4) may be a required autocrine factor, not only for its own production but also for the maintenance of full endocrine function of the corpus luteum.     

In. vitro evaluation of corpus luteum function of cycling and pregnant rhesus monkeys: Progesterone production by dispersed luteal cells

Corpus luteum function in the cycling and the pregnant rhesus monkey (Macaca mulatta) was evaluated through short term $\text{in vitro}$ studies of progesterone production by suspensions of collagenase-dispersed luteal cells in the presence and absence of exogenous gonadotropin (human chortonic gonadotropin, HCG). Cells from mid-luteal phase of the menstrual cycle secreted progesterone, as measured by accumulation of this hormone in the incubation medium, and responded to the addition of 100 ng HCG/ml with a marked increase in progesterone secretion above basal level ($\text{63.7 ± 13.1}\text{versus}\text{24.7 ± 5.5}\text{ng progesterone}\text{/}\text{ml}\text{/5 × 10}{}^4\text{cells}\text{/ 3}\text{hr}\text{,}\text{X}\text{± S.E.,}\text{n}\text{= 6;}\text{p}\text{< 0.05}$). However, luteal cells from early pregnancy (23–26 days after fertilization) secreted significantly less progesterone than cells of the non-fertile menstrual cycle (3.6 ± 2.4 versus 24.7 ± 5.5 ng/ml/5 × 10⁴ cells/3 hr, n = 3; p < 0.05) and did not respond to HCG with enhanced secretion. By mid-pregnancy (108–118 days gestation) luteal cells exhibited partially renewed function, and near the time of parturition (163–166 days gestation) basal and HCG-stimulated progesterone secretion (30.2 ± 5.6 and 63.0 ± 13.0 ng/ml/5 × 10⁴ cells/3 hr, respectively; n = 3) was equivalent to that of cells from the luteal phase of the non-fertile menstrual cycle. The data suggest that following a period around the fourth week of gestation, when steroidogenic activity is markedly diminished, the corpus luteum of pregnancy progressively reacquires its functional capacity and at term exhibits gonadotropin-sensitive steroidogenesis similar to that of the corpus luteum of the menstrual cycle.     

In-vitro and in-vivo responsiveness of the corpus luteum of the mare to gonadotrophin stimulation

Dispersed horse luteal cells were used to evaluate the ability of horse LH, hCG and PMSG to stimulate progesterone secretion in vitro. Morphological characterization of these cells before gonadotrophin stimulation indicated the presence of two populations of cells based on cell diameters. In luteal cells incubated as suspended cells, horse LH and hCG stimulated (P less than or equal to 0.05) progesterone production at all levels of treatment. Stimulation of progesterone secretion by hCG was greater (P less than or equal to 0.05) than by horse LH over the range of concentrations utilized. When mares (N = 7) received an intramuscular injection of 1000 i.u. hCG on Days 3, 4 and 5 after the end of oestrus, there was an increase (P less than or equal to 0.05), in peripheral progesterone concentrations beginning on Day 7 and continuing until Day 14 compared with controls (N = 7). Peripheral progesterone concentrations continued to be elevated in hCG-treated mares for Days 15-30 after oestrus in those mares that conceived. Although treatment with hCG increased progesterone concentrations, it had no influence on anterior pituitary release of LH as measured by frequency and amplitude of LH discharge. We conclude that the mare corpus luteum is responsive to gonadotrophins in vitro and that exogenous hCG can enhance serum progesterone concentrations throughout the oestrous cycle and early pregnancy.     

Morphometric analysis of cell types in the ovine corpus luteum throughout the estrous cycle

The cellular composition of ovine corpora lutea obtained during the early (Day 4), mid (Days 8 and 12), and late (Day 16) stages of the estrous cycle was determined by morphometric analysis. Individual corpora lutea were collected via midventral laparotomy from a total of 19 ewes. A center slice from each corpus luteum was processed for electron microscopy and subsequent morphometric analysis of the numbers and sizes of steroidogenic and nonsteroidogenic cells. Luteal weight progressively increased throughout the estrous cycle (p less than 0.05). Corpora lutea collected on Day 16 were assigned to one of two subgroups on the basis of gross appearance and weight: nonregressed (NR, 542 +/- 25 mg) or regressed (R, 260 +/- 2 mg). There were no significant changes in the proportion of the corpus luteum occupied by small luteal cells (19 +/- 2%) or large luteal cells (36 +/- 1%) throughout the estrous cycle. The total number of steroidogenic cells per corpus luteum increased from 21.8 +/- 3.7 (X 10(6)) on Day 4 to 61.7 +/- 5.4 (X 10(6)) on Day 8 (p less than 0.05) and remained elevated thereafter. The number of small luteal cells was 10.0 +/- 2.7 (X 10(6)), 39.7 +/- 1.4 (X 10(6)), 46.1 +/- 5.8 (X 10(6)), 49.0 +/- 13.7 (X 10(6)), and 29.9 +/- 8.6 (X 10(6)) on Days 4, 8, 12, 16 (NR), and 16 (R), respectively (p less than 0.05, Day 4 vs. Days 8, 12, 16 NR). In contrast, the number of large luteal cells was 11.8 +/- 1.5 (X 10(6)) on Day 4 and did not vary significantly during the remainder of the estrous cycle. The numbers of nonsteroidogenic cell types increased (p less than 0.05) from Day 4 to Day 16 (NR) but were decreased in regressed corpora lutea (Day 16 R). Regression was characterized by a 50% decrease (p less than 0.05) in the total number of cells per corpus luteum from 243 +/- 57 ( X 10(6)) on Day 16 (NR) to 125 +/- 14 ( X 10(6)) on Day 16 (R) (p less than 0.05). Small luteal cells remained constant in volume throughout the entire estrous cycle (2520 +/- 270 microns 3), whereas large luteal cells increased in size from 5300 +/- 800 microns 3 on Day 4 to 16,900 +/- 3300 microns 3 on Day 16 (NR) (p less than 0.05). In summary, small luteal cells increased in number but not size throughout the estrous cycle, whereas large luteal cells increased in size but not number.     

Changes in gonadotrophin secretion and ovarian antral follicular activity in seasonally breeding sheep throughout the year

Overall, significantly more antral follicles greater than or equal to 1 mm diameter were present in Romney ewes during anoestrus than in the breeding season (anoestrus, 35 +/- 3 (mean +/- s.e.m.) follicles per ewe, 23 sheep; Day 9-10 of oestrous cycle, 24 +/- 1 follicles per ewe, 22 sheep; P less than 0.01), although the mean numbers of preovulatory-sized follicles (greater than or equal to 5 mm diam.) were similar (anoestrus, 1.3 +/- 0.2 per ewe; oestrous cycle, 1.0 +/- 0.1 per ewe). The ability of ovarian follicles to synthesize oestradiol did not differ between anoestrus and the breeding season as assessed from the levels of extant aromatase enzyme activity in granulosa cells and steroid concentrations in follicular fluid. Although the mean plasma concentration of LH did not differ between anoestrus and the luteal phase of the breeding season, the pattern of LH secretion differed markedly; on Day 9-10 of the oestrous cycle there were significantly more (P less than 0.001) high-amplitude LH peaks (i.e. greater than or equal to 1 ng/ml) in plasma and significantly fewer (P less than 0.001) low amplitude peaks (less than 1 ng/ml) than in anoestrous ewes. Moreover, the mean concentrations of FSH and prolactin were significantly lower during the luteal phase of the cycle than during anoestrus (FSH, P less than 0.05, prolactin, P less than 0.001). It is concluded that, in Romney ewes, the levels of antral follicular activity change throughout the year in synchrony with the circannual patterns of prolactin and day-length. Also, these data support the notion that anovulation during seasonal anoestrus is due to a reduced frequency of high-amplitude LH discharges from the pituitary gland.