Expression of messenger ribonucleic acids that encode for 3 beta-hydroxysteroid dehydrogenase and cholesterol side-chain cleavage enzyme throughout the luteal phase of the macaque menstrual cycle

To study further the control of the primate corpus luteum, we obtained corpora lutea from cynomolgus macaques at defined stages of the luteal phase and examined steady state mRNA levels in these corpora lutea by Northern analysis for the two major enzymes involved in progesterone biosynthesis, cytochrome P450 cholesterol side-chain cleavage (P450SCC) and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD). mRNAs for both P450SCC and 3 beta HSD were maximal or near maximal shortly after ovulation and luteinization (days 3-5 of the luteal phase). mRNA for P450SCC exhibited a slight, but nonsignificant (P greater than 0.05) decline throughout the remainder of the luteal phase and was undetectable upon luteal regression. Steady state levels of 3 beta HSD mRNA were significantly lower (P less than 0.05) from corpora lutea removed during the midluteal phase (days 7-8 of the luteal phase) than those in newly formed corpora lutea and declined to 10% of early luteal phase values by days 13-15 of the luteal phase. 3 beta HSD mRNA levels fell to nondetectable values upon luteal regression. These results reveal a paradoxical relationship between the steroidogenic activity of the primate corpus luteum in vivo and the steady state levels of the mRNAs that encode for the major enzymes involved in progesterone biosynthesis. Unlike serum progesterone concentrations, which are very low immediately after ovulation and then rise during the midluteal phase, the steady stale levels of P450SCC mRNA and 3 beta HSD appeared to be maximal or near maximal shortly after ovulation and declined throughout the remainder of the luteal phase. These findings are consistent with the notion that luteal lifespan is set at the time of ovulation and luteinization, and the decline in luteal function may be due in part to decay of specialized luteal cell mRNAs with finite half-lives.     

Evidence that human chorionic gonadotropin/luteinizing hormone receptor down-regulation involves decreased levels of receptor messenger ribonucleic acid

Injection of pseudopregnant rats with pharmacological doses of hCG leads to a characteristic decrease in LH/hCG binding by the isolated luteal cells. The steady state levels of LH/hCG receptor mRNA were determined in rat ovaries during hCG-induced down-regulation of the receptor. Northern blots were performed using a 20-mer probe corresponding to a guanine cytosine-rich carboxyl-terminal untranslated region of the LH/hCG receptor cDNA. The hybridization of the probe to LH/hCG receptor mRNA was highly specific, since the probe hybridized only to rat luteal cell RNA fraction, with no signal detected in nontarget tissues. The LH/hCG receptor level was quantitated by [125I]hCG binding to the isolated membrane fractions from the corresponding treatment and control groups. Examination of mRNA levels of the receptor during hCG-induced down-regulation showed a steady decrease from 0-24 h, followed by a gradual increase to control levels from 24-72 h corresponding to days 8-9 of pseudopregnancy. The [125I]hCG-binding activity during down-regulation paralleled the mRNA profile in both the experimental and control groups. Examination of the levels of mRNA for alpha-actin showed no change during this period, suggesting that the loss of LH/hCG receptor mRNA at 24 h was not due to a general loss of mRNA in luteal cells. These results suggest that hCG-induced down-regulation of the LH/hCG receptor in luteal cells involves regulation of the receptors at the message level.     

Alpha and luteinizing hormone beta subunit messenger ribonucleic acids during the rat estrous cycle

Alpha and LH beta subunit mRNAs were measured in pituitaries of 4-day cycling rats during the estrous cycle. A two-fold increase in alpha mRNA occurred between 0800-2000 h on diestrus, but alpha mRNA concentrations were stable during other days of the cycle. LH beta mRNA concentrations were low during estrus and metestrus (11-16 pg cDNA bound/100 micrograms pituitary DNA), but were elevated (27-30 pg) between 0800-2000 h on diestrus. A second increase in LH beta mRNA was observed on the afternoon of proestrus, prior to the onset of the LH surge with maximum values (45 pg) coincident with peak LH secretion. LH beta mRNA concentrations declined rapidly and had fallen to basal values by midnight on proestrus. These data show that alpha and LH beta mRNAs change in a similar manner during metestrus, diestrus and estrus, suggesting coordinate regulation of alpha and LH beta gene expression at these times. During the LH surge, however, LH beta mRNA alone is increased, suggesting that the LH beta gene can be differentially expressed at times when maximum LH secretion is occurring.     

Differential regulation of thyroid hormone receptor messenger ribonucleic acid levels by thyrotropin-releasing hormone

In addition to its well known actions in stimulating TSH and PRL synthesis and secretion, TRH has been shown to decrease the concentration of thyroid hormone receptors (TRs) in GH4C1 cells as measured by nuclear thyroid hormone (T3) binding. In the present study we have investigated the effects of TRH on the levels of mRNA encoding the different forms of TR, TR beta-1, TR beta-2, and TR alpha-1 as well as that of the non-T3-binding variant, c-erbA alpha-2. GH3 cells were incubated with 100 nM TRH in the presence or absence of 1 nM T3 for 48 h, and mRNA levels were determined by Northern blot analysis. Results revealed that there is differential regulation of the individual TRs by TRH at the pretranslational level. The mRNA for the pituitary-specific form of TR, TR beta-2, was down-regulated by 60% by TRH in GH3 cells, while that of its alternative splice product, TR beta-1, was unchanged. A modest change was observed in TR alpha-1 mRNA levels, which were down-regulated by 20%; there was no change in c-erbA alpha-2 mRNA levels. Levels of nuclear T3 binding were assessed under the same conditions, and 100 nM TRH was found to decrease binding by 40% from 0.78 to 0.46 fmol/micrograms DNA. A similar change in nuclear T3 binding was seen after incubation with 1 nM T3. The effect of TRH on the GH mRNA response to T3 was investigated. In the absence of TRH there was a 4-fold induction of GH mRNA after incubation with 1 nM T3. In the presence of 100 nM TRH, no significant induction in GH mRNA by T3 was seen, indicating that T3 responsiveness as well as receptor concentration are diminished by TRH under these conditions.     

Follicle-stimulating hormone beta subunit messenger ribonucleic acid concentrations during the rat estrous cycle

Serum follicle-stimulating hormone (FSH), pituitary FSH content and FSH beta subunit mRNA concentrations were measured at 1 to 3h intervals throughout the 4 day estrous cycle in rats. Serum FSH was stable (range 200-320 ng/ml) apart from the biphasic proestrus surge (5 fold elevation) which was present from 1800h of proestrus through 0800 h on estrus. Basal FSH beta mRNA concentrations from late metestrus through the afternoon of proestrus were 0.10 +/- 0.04 f mol cDNA bound/100 micrograms pituitary DNA. The major increase in FSH beta mRNA began at 2000 h on proestrus, 2 h after the initial rise in serum FSH and peak mRNA concentrations (0.43 +/- 0.08 f mol cDNA bound) occurred at 0200 h on estrus. FSH beta subunit mRNA concentrations were again increased at 2300 h on estrus (peak 0.24 f mol cDNA bound) and remained elevated through 1700 h on metestrus. Pituitary FSH content was transiently increased during metestrus and diestrus, but was elevated at 1000 h through 1900 h on proestrus (peak 5-fold increase). FSH content fell rapidly at 2000h and remained low until 1400 h on estrus when values again rose. These data show that FSH beta mRNA is increased 4-5 fold during the proestrus FSH surge, and a smaller increase occurs on metestrus in the absence of elevated FSH secretion. The increased concentrations of FSH beta mRNA occurred at different times to the previously reported changes in alpha and LH beta mRNAs. Therefore, the data suggest that different mechanisms are involved in the regulation of LH and FSH beta subunit gene expression during the 4-day estrous cycle in rats.     

Follicle-stimulating hormone beta subunit messenger ribonucleic acid concentrations during the rat estrous cycle

Serum follicle-stimulating hormone (FSH), pituitary FSH content and FSH beta subunit mRNA concentrations were measured at 1 to 3h intervals throughout the 4 day estrous cycle in rats. Serum FSH was stable (range 200-320 ng/ml) apart from the biphasic proestrus surge (5 fold elevation) which was present from 1800 h of proestrus through 0800 h on estrus. Basal FSH beta mRNA concentrations from late metestrus through the afternoon of proestrus were 0.10 +/- 0.04 f mol cDNA bound/100 micrograms pituitary DNA. The major increase in FSH beta mRNA began at 2000 h on proestrus, 2 h after the initial rise in serum FSH and peak mRNA concentrations (0.43 +/- 0.08 f mol cDNA bound) occurred at 0200 h on estrus. FSH beta subunit mRNA concentrations were again increased at 2300 h on estrus (peak 0.24 f mol cDNA bound) and remained elevated through 1700 h on metestrus. Pituitary FSH content was transiently increased during metestrus and diestrus, but was elevated at 1000 h through 1900 h on proestrus (peak 5-fold increase). FSH content fell rapidly at 2000 h and remained low until 1400 h on estrus when values again rose. These data show that FSH beta mRNA is increased 4-5 fold during the proestrus FSH surge, and a smaller increase occurs on metestrus in the absence of elevated FSH secretion. The increased concentrations of FSH beta mRNA occurred at different times to the previously reported changes in alpha and LH beta mRNAs. Therefore, the data suggest that different mechanisms are involved in the regulation of LH and FSH beta subunit gene expression during the 4-day estrous cycle in rats.     

Follicular development during the luteal phase of the human menstrual cycle

The aims of the present studies were to determine the number, size range, health, and steroidogenic activities of antral follicles in normal human ovaries during the luteal phase of the menstrual cycle. Steroidogenic activity was assessed from the levels of androstenedione, testosterone, and estradiol in follicular fluid and the levels of extant and FSH-stimulable aromatase activity and FSH-stimulable progestin synthesis in the granulosa cells. Data for luteal phase ovaries were compared to those obtained for ovaries from the late follicular phase. On average, 94% (range, 70-100%) of the luteal phase follicles (greater than or equal to 1 mm diameter) were atretic as assessed by oocyte viability and granulosa cell number. The largest healthy follicles during the mid- to late luteal phase were 4-4.5 mm in diameter; these contained high levels of aromatizable androgen (500-2000 ng/ml), low levels of estradiol (less than 10 ng/ml), and granulosa cells with an extant level of aromatase activity 200 times lower than that in a preovulatory follicle. Based on these biochemical criteria, healthy (luteal phase) follicles were not distinguishable from atretic follicles. Granulosa cells from the luteal phase follicles were responsive to FSH with respect to progesterone and estradiol biosynthetic activity; the aromatase system in the cells from the mid- to late luteal phase follicles was significantly more responsive to FSH than that in cells from late follicular or early luteal phase follicles (P less than 0.05). These data suggest that the number of healthy luteal phase follicles (greater than or equal to 1 mm diameter) available for subsequent preovulatory development is limited.     

The roles of estradiol and progesterone in decreasing luteinizing hormone pulse frequency in the luteal phase of the menstrual cycle

During the luteal phase of the menstrual cycle, plasma progesterone (P) and estradiol (E2) concentrations are elevated, and LH (and by inference GnRH) pulse frequency is slow. In contrast, LH pulse frequency increases during the early follicular phase when plasma E2 and P are lower. To examine the mechanism(s) responsible for the slower GnRH pulse frequency in the luteal phase, we maintained plasma P, E2, or both at midluteal concentrations from the midluteal phase to the time of the next early follicular phase and measured the effects on LH secretion. Thirteen normal women with regular menstrual cycles were studied during two or three cycles. Blood was obtained every 10 min during 10-h studies. Control cycle luteal and early follicular studies were followed by a second control study in the luteal phase of the treatment cycle. P (six women), E2 (seven women), or both (five women) then were given twice daily by im injection for 6-12 days until the day corresponding to the early follicular study of the control cycle (EF + P, EF + E2, or EF + E2 + P). A final study was performed 1 week after the injections were discontinued (F). LH pulse frequency was low in the midluteal phase [3.2 +/- 0.2 (+/- SE) pulses/10 h] and increased by the early follicular phase (8.0 +/- 0.8 pulses/10 h) in the control cycles. The increase in LH pulse frequency was not significantly inhibited by administration of P (6.7 +/- 0.7 pulses/10 h; EF + P). However, during both E2 alone and E2 + P, LH pulse frequency remained low (EF + E2, 3.6 +/- 0.8; EF + E2 + P, 2.0 +/- 0.7 pulses/10 h). The mean plasma FSH concentrations paralleled changes in LH pulse frequency, increasing from the luteal to the early follicular phase in the control cycles and during P injections and remaining low during E2 and E2 + P injections. We conclude that continued exposure to P alone does not maintain GnRH pulse frequency at midluteal phase values and that any effect of P requires the presence of E2. As E2 alone maintained lower LH pulse frequency, E2 may act directly to decrease the pulsatile GnRH secretion or it may potentiate the effects of low (less than 3.2 nmol/L) P concentrations.     

Regulation of steroidogenic acute regulatory protein and luteinizing hormone receptor messenger ribonucleic acid in hen granulosa cells.

The regulation of steroidogenic acute regulatory protein (StAR) in vitro by gonadotropins was investigated in granulosa cells from prehierarchal and preovulatory hen follicles. Basal levels of StAR messenger RNA (mRNA) in undifferentiated granulosa cells from prehierarchal (6- to 8-mm) follicles were consistently low, but detectable, and were significantly increased by treatment with 8-bromo-cAMP and FSH (but not LH) within 3-6 h of culture. After 20 h of culture, 8-bromo-cAMP, FSH, and LH each increased StAR mRNA levels above those in control cultured cells, and the delayed response to LH treatment was associated with increased levels of LH receptor (LH-R) mRNA. On the other hand, inhibition of mitogen-activated protein (MAP) kinase signaling, using the MAP kinase kinase inhibitors U0126 and PD98059, in the presence of FSH further increased StAR mRNA and protein levels, LH-R mRNA levels, and progesterone synthesis compared with those in cells cultured with FSH alone. The highest basal expression of StAR mRNA during follicle development was found in granulosa from the largest (F1) preovulatory follicle, with comparatively lower levels in granulosa from less mature (F2 plus F3) preovulatory follicles. Treatment with LH rapidly increased StAR mRNA and protein (but not LH-R mRNA) expression in cultures of F1 granulosa and in combined F2 plus F3 granulosa within 3 h, although the magnitude of stimulation was greater in F2 plus F3 granulosa. Compared with results from granulosa cells from prehierarchal follicles cultured for 20 h, inhibition of MAP kinase signaling in the presence of LH for 1 h failed to further enhance levels of StAR or LH-R expression or progesterone production in F2 plus F3 follicle granulosa compared with the effect of LH treatment alone. These results demonstrate that StAR expression in the hen ovary is up-regulated by gonadotropins at least in part via cAMP signaling. The ability of MAP kinase kinase inhibitors to potentiate gonadotropin-induced StAR and LH-R expression plus progesterone synthesis in prehierarchal follicle granulosa cells in vitro suggests that inhibition of paracrine or autocrine factor-mediated MAP kinase signaling in vivo may be a prerequisite for the full potentiation of granulosa cell steroidogenesis that occurs after recruitment into the preovulatory hierarchy. Finally, these results fail to support a role for MAP kinase signaling in acutely modulating LH-mediated StAR expression or progesterone production in hierarchal follicles, such as occurs during the preovulatory surge of progesterone.     

Regulation of luteinizing hormone receptor messenger ribonucleic acid levels by gonadotropins, growth factors, and gonadotropin-releasing hormone in cultured rat granulosa cells

The induction of LH receptors in granulosa cells is prerequisite for ovarian follicles to ovulate and form corpora lutea. Earlier studies have demonstrated the modulatory role of gonadotropins, growth factors, and GnRH on ovarian LH receptor content. We have now analyzed the influences of gonadotropins (FSH, LH, and PRL), several growth factors, and GnRH on LH receptor mRNA levels in cultured granulosa cells. Cells were obtained from immature estrogen-treated rats and cultured in medium containing FSH with or without growth factors or GnRH for 48 h. Some cells were also treated with FSH for 48 h, followed by treatment with FSH, LH, or PRL for another 2 days. Cellular total RNA was extracted, and blot hybridization with 32P-labeled LH receptor cRNA or 28S ribosomal RNA cDNA probes was performed. Treatment of granulosa cells with FSH increased the levels of five species of LH receptor mRNAs in a dose- and time-dependent manner. In FSH-primed cells, LH receptor mRNA levels were maintained by FSH, LH, and PRL. In contrast, treatment of cells with basic fibroblast growth factor or epidermal growth factor suppressed FSH induction of LH receptor mRNA in a dose-dependent manner, whereas treatment with insulin-like growth factor-I had no effect. In addition, GnRH suppressed FSH-stimulated LH receptor mRNA levels in a dose-dependent manner; the effects of GnRH could be counteracted by coincubation with a GnRH antagonist, suggesting mediation by specific GnRH-binding sites. These studies demonstrated that the observed stimulatory effects of gonadotropins (FSH, LH, and PRL) and the inhibitory effects of growth factors (epidermal growth factor and basic fibroblast growth factor) and GnRH on LH receptor content are correlated to their regulation of LH receptor mRNA levels. The granulosa cell culture system should provide a useful model for studying LH receptor gene regulation.     

Pituitary alpha-subunit messenger ribonucleic acid remains elevated during the latter stages of the preovulatory luteinizing hormone surge

In this study, pituitary alpha-subunit mRNA amounts were assessed during the latter stages of the preovulatory LH surge in the normal cycling ewe. The times chosen were 15 and 25 h after the onset of behavioral estrus, designated E+15 and E+25. These mRNA values were correlated with pituitary and serum LH concentrations to assess the possible role of subunit synthesis during these times of the cycle, and then compared to values obtained from hybridizations of RNAs from animals in other groups representing defined stages of the cycle. One of these groups included animals during the midluteal phase (day 12), whose amounts were assigned a value of 1. It was observed that the amounts of alpha-subunit mRNA in E+15 and E+25 groups, as measured by translations and cDNA hybridizations, were approximately 10-fold higher than the day 12 values and were roughly similar to those observed in a group examined 5 h after estrus. Thus, it is evident that the alpha mRNA levels are not only elevated during the LH surge, but also remain high at a time when both serum and pituitary LH concentrations have fallen dramatically, suggesting the involvement of changing mRNA amounts in the feedback systems associated with the maintenance of pituitary and serum gonadotropin amounts.     

Nocturnal slowing of pulsatile luteinizing hormone secretion in women during the follicular phase of the menstrual cycle

The gonadotropin secretion pattern in normal reproductive age women (n = 5) was evaluated for the presence of a circadian rhythm. The women volunteered for a series of 24-h admissions in different phases of their menstrual cycles (early follicular, late follicular, and midluteal). Plasma LH and FSH levels were determined by RIA in blood samples drawn through indwelling venous catheters at 20-min intervals throughout a normal 24-h sleep-wake cycle. The gonadotropin secretory pattern was subjected to cosine analysis for identifying rhythmicity. The LH interpulse interval fluctuated with a significant 24-h rhythm during the early follicular phase in four of the five women. The maximum interpulse intervals occurred during the early morning between 0100 and 0500 h (mean, 0250 h), with a corresponding increase in LH pulse amplitude occurring within the same time interval (mean, 0320 h). We found no consistent 24-h rhythms in overall mean plasma LH levels during any phase of the menstrual cycle, nor did we find a significant rhythmicity in either LH interpulse interval or LH pulse amplitude during the late follicular or luteal phase. These results demonstrate that the LH pulse-generating system is frequency modulated on a circadian basis during the early follicular phase of the human menstrual cycle.     

Pulsatile secretion of luteinizing hormone during the menstrual cycle of rhesus macaques

The secretion of LH, PRL, and cortisol was investigated in 4 sexually mature female rhesus macaques with cardiac catheters protected by tethers. Based on endocrine parameters, all 4 of the animals ovulated within 2 months from the time they were tethered, and regular menstrual cycles of 24-34 days were observed. The catheters remained patent for 6-12 months without reposition or repair. Plasma levels of 2 stress-labile hormones, PRL and cortisol, showed diurnal fluctuations comparable to those observed in untethered animals. The frequency of LH secretory episodes was determined by measuring bioactive LH in blood samples collected at 10-min intervals in the follicular phase and at 15-min intervals in the luteal phase of the menstrual cycle. In 10 trials during the follicular phase, we estimated that an average of between 14 and 15 LH pulses occurred every 12 h. The interpulse interval ranged between 20-80 min and averaged 50 min. No change in pulse frequency was observed across the follicular phase. The number of LH pulses decreased after ovulation, and by the end of the luteal phase, the interpulse interval was 4-6 h. One example during the preovulatory LH surge revealed the high frequency, high amplitude nature of LH secretion at that time. Our experience indicates that tethered animals with cardiac catheters show no hormonal indications of stress and represent the best available model for studies requiring frequent and prolonged access to the vascular system. Our data suggest that peripheral LH fluctuations in rhesus monkeys, as in other mammals, are pulsatile, and the frequency of these pulsatile episodes changes with different phases of the menstrual cycle, presumedly in response to varying stimuli to the pituitary from the brain.