Dynamics of messenger RNAs encoding inhibin/activin subunits and follistatin in the ovary during the rat estrous cycle

Quantitative changes in ovarian inhibin/activin subunit and follistatin mRNAs during the rat estrous cycle were examined by ribonuclease protection assay using digoxygenin-labeled RNA probes. Levels of ovarian inhibin alpha subunit mRNA remained low throughout estrus, metestrus, and diestrus; abruptly increased on the morning of proestrus; then rapidly decreased when the primary gonadotropin surge occurred. A similar changing pattern was observed in inhibin/activin beta(A) subunit mRNA. On the other hand, inhibin/activin beta(B) subunit mRNA showed a different changing pattern. Levels of beta(B) subunit mRNA remained constant during metestrus and diestrus, abruptly decreased on the afternoon of proestrus, then quickly recovered from the nadir by 1100 h on estrus. Throughout the rat estrous cycle, especially during the periovulatory period, alpha subunit mRNA levels were considerably higher than beta(A) and beta(B) subunit mRNA levels. In addition, changes in plasma concentrations of inhibin A and inhibin B were very similar to that in ovarian beta(A) and beta(B) subunit mRNA levels, respectively, with several-hour delays. These results suggest that levels of beta subunit mRNAs restrict secretion of dimeric inhibins. Levels of follistatin mRNA remained low from the midnight of metestrus to the midnight of diestrus, then increased until initiation of the primary gonadotropin surge. Thereafter, follistatin mRNA decreased, reached the nadir at 0200 h on estrus, then increased abruptly at 1100 h on estrus. Afterward, follistatin mRNA levels remained high until the morning of metestrus. The changing pattern of ovarian follistatin mRNA was similar to, and preceded, the changes in plasma concentrations of progesterone, suggesting that ovarian follistatin may modulate progesterone secretion during the rat estrous cycle.     

Circulating immunoreactive inhibin, gonadotropin, and prolactin levels during pregnancy, lactation, and postweaning estrous cycle in the rat

Serum inhibin levels were measured by heterologous RIA during pregnancy, lactation, and the post-weaning estrous cycle in the rat and correlated with changes in serum FSH and LH and prolactin. Blood was serially collected by cardiac puncture under light ether anesthesia from adult Sprague-Dawley rats on alternate days throughout the experimental period. For the first 8 days of pregnancy, immunoreactive inhibin levels remained high, then gradually decreased to reach a nadir at Day 16, and subsequently rose steeply until parturition. The pattern of serum immunoreactive inhibin levels during early pregnancy does not support a corpus luteum source and the dramatic rise from Day 16 to Day 22 correlates with the recommencement of follicular development in the ovary. Inhibin levels decreased rapidly on the day after birth and were suppressed until Day 8 of lactation, slowly increasing thereafter to reach a plateau from Day 14 until weaning (Day 22.5 of lactation). These changes in inhibin levels positively correlated with LH and FSH and negatively with prolactin, and are consistent with an ovarian source for inhibin associated with the recommencement of follicular development resulting from the diminution of the suckling stimulus. Immediately after weaning, serum immunoreactive inhibin levels showed a 4-day cyclic pattern corresponding to the estrous cycle identified by vaginal smear. Inhibin levels peaked on the day of proestrus, reached a nadir on the day of estrus, and rose slowly during metestrus and diestrus to a new peak at proestrus. Serum FSH levels showed an inverse correlation to inhibin levels consistent with a feedback relationship with inhibin.     

Patterns of gonadotrophin secretion associated with ovulation

A detailed analysis was made of circulating luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin throughout the estrous cycle in rats. Vaginal cytology was recorded daily for 18 or 23 days in 2 groups of adult female rats, 300 and 400 per group. The presence of distended uteri or tubal ova was noted at the time of exsanguination under ether anesthesia. Serum concentrations of LH, FSH, and prolactin were determined by radioimmunoassay. In 404 rats with regular 5-day cycles, all 3 hormones reached maximum concentrations on the afternoon of functional proestrus. During the next 3 days FSH decreased progressively, and LH levels indicated a diurnal fluctuation with minimum concentrations during darkness. Serum prolactin may have b een elevated due to anesthesia and exsanguination and showed a rapid increase which occurred simultaneously with elevated LH and FSH on the afternoon of proestrus. Radioimmunoassay of all 3 hormones indicated that the increases in serum LH and prolactin did not necessarily occur simultaneously. Hypophysectomy and sequential heart puncture of proestrus rats during maximum hormone release showed disappearance rates (half-lives) for LH, FSH, and prolactin of 20, 110, and 13 minutes, respectively.     

Hypersecretion of follicle-stimulating hormone (FSH) on estrous afternoon in rats treated with RU486 in proestrus

Summary 1. Intact or ovariectomized (OVX) cyclic rats injected or not with RU486 (4 mg/0.2 ml oil) from proestrus onwards were bled at 0800 and 1800h on proestrus, estrus and metestrus. Additional RU486-treated rats were injected with: LHRH antagonist (LHRHa), estradiol benzoate (EB) or bovine follicular fluid (bFF) and sacrified at 1800 h in estrous afternoon. LH and FSH serum levels were determined by RIA.2. RU486-treated intact or OVX rats had decreased preovulatory surges of LH and FSH, abolished secondary secretion of FSH and hypersecretion of FSH in estrous afternoon. The latter was decreased by LHRHa and abolished by EB or bFF. In contrast, EB induced an hypersecretion of LH in RU486-treated rats at 1800h in estrus.3. It can be concluded that in the absence of the proestrous progesterone actions, the absence of the inhibitory effect of the ovary in estrus evoked a LHRH independent secretion of FSH.     

小熊猫繁殖周期血清雌二醇和孕酮含量变化

为研究小熊猫繁殖周期血清雌二醇、孕酮含量变化规律,采用化学发光免疫分析法连续16 次测定了2只成体雌性小熊猫血清雌二醇和孕酮含量变化,历经发情间期、发情期和两次妊娠期;连续9次测定了7只小熊猫妊娠期的孕酮含量变化。结果:(1)发情间期,小熊猫血清雌二醇的水平一直维持在低水平(基础水平),进入发情前期,血清雌二醇水平明显升高,在发情期一直维持高水平,配种后迅速降至基础水平; (2)小熊猫血清孕酮含量在发情间期和发情期均维持在较低水平,直至发情期过后才出现升高,在妊娠期一直维持高水平,峰值出现在5 月;(3)发情的小熊猫不论妊娠与否,在妊娠期内血清孕酮含量均维持在高水平。研究表明:小熊猫血清雌二醇、孕酮含量变化能直接反映其繁殖规律,雌二醇对启动雌性小熊猫季节性繁殖起重要作用;在妊娠期内小熊猫血清孕酮含量升高不能作为判断小熊猫妊娠的标准;雌性小熊猫在妊娠期有假孕现象。     

Mechanism of the stimulatory action of antisteroid RU486 on follicle-stimulating hormone secretion in the cyclic Rat

These experiments explored the mechanism underlying FSH hypersecretion on estrous afternoon in rats injected with RU486 (RU) on proestrus. Four-day cyclic rats were injected with RU at 12:00 h on proestrus (1 or 4 mg/0.2 ml oil; s.c.), and its effects on LH and FSH secretion at 18:30 h on estrus were compared with those of antiprogestagens ZK299 (ZK) (1 or 4 mg/0.2 ml oil; s.c.) and Org31806 (OR) (2 or 8 mg/0.2 ml oil; s.c.). Additionally, rats treated with RU or nembutal (PB) (60 mg/kg; i.p. at 13:00 h on proestrus) were injected with an LHRH antagonist (LHRHa) at 10:00 h on estrus (1 mg/0.2 ml saline; s.c.) or progesterone (P) (7.7, 15.5 or 30.9 mg/0.2 ml oil; s.c.) on proestrus at 10:00 h in RU-injected rats and at 14:00 h in PB-injected rats. Animals were killed by decapitation at 18:30 h on estrus and serum LH and FSH concentrations were determined. Rats treated with 1 or 4 mg of RU or Org or 4 mg of ZK recorded increased serum FSH on estrous afternoon, while 1 mg ZK had no effect. PB increased mainly serum LH levels and, to a lesser extent, FSH levels. P decreased serum FSH concentrations in both RU- and PB-injected rats. LHRHa reversed the effects of PB on FSH secretions, but reduced FSH hypersecretion induced by RU only. These results are interpreted to mean that, in the absence of proestrous afternoon P-inhibitory action of the neural stimulus controlling LHRH release, FSH secretion on estrous afternoon involves two components: one is LHRH dependent while, in contrast to LH secretion, the other is LHRH independent, and only expressed in a low estrogen background.     

Increases in the basal secretion rate of follicle-stimulating hormone (FSH) accompany age-associated changes in serum FSH levels on estrus

This laboratory has recently reported that by 5-6 months of age, alterations in the secretion and production of follicle-stimulating hormone (FSH) occur in virgin female rats which precedes the age-related disruption of estrous cycles and attenuation of preovulatory gonadotropin surges. Specifically, circulating immunoreactive FSH levels are higher on estrus in rats 5 months and older compared to levels measured in 2- to 3-month-old rats. Therefore, the present study was conducted to explore a possible mechanism for this age-related increase in FSH levels. At 1400 hr on proestrus, estrus and diestrus-1, groups (n = 6-12 rats/group) of 3- and 7-month-old, cyclic rats were decapitated, trunk blood was collected, and anterior pituitary glands were bisected and placed in incubation flasks containing 1 ml media (medium 199). Following a 30-min preincubation period, hemipituitary fragments were incubated for an additional 2 hr. Media and serum FSH levels were quantified by RIA. Levels of FSH were twofold higher in the serum of 7-month-old rats than 3-month-old rats on estrus. Similarly, the basal secretion rate (BSR) of FSH (expressed as ng FSH/ml/2 hr) was significantly (P less than 0.05) higher from incubated hemipituitary fragments of 7-month-old estrous rats than from fragments obtained from younger estrous rats (7 month: 1637 ng/ml/2 hr vs 3 months: 1253 ng/ml/2 hr). Neither the serum FSH levels nor the BSR of FSH differed between age groups on proestrus or diestrus-1. These results show that age-associated increases in circulating FSH levels on estrus may be attributed to an enhanced basal secretion of FSH from the pituitary gland.     

Circulating radioimmunoassayable inhibin during periods of transient follicle-stimulating hormone rise: secondary surge and unilateral ovariectomy

We have measured changes in circulating immunoreactive (ir-) inhibin in male and female rats using an RIA with an antiserum raised against porcine inhibin alpha (1-26)-Gly-Tyr. The same synthetic peptide was used for standards and for the preparation of tracer. Serum ir-inhibin levels were significantly higher in intact female than in intact male rats (p less than 0.001). Immunoreactive inhibin was significantly reduced in both sexes 24 h after bilateral gonadectomy (p less than 0.0001). Unilateral ovariectomy (ULO) of female rats on metestrus caused a transient decrease in serum inhibin 8 h after surgery, but levels were not significantly different from those of sham-operated controls at later times after surgery. Increases in serum FSH and LH were observed for 8-18 h after ULO. Serum ir-inhibin levels were also measured on the early morning of estrus during the secondary FSH surge. At this time, ir-inhibin levels were low, while FSH levels were high and LH levels were low. These results show that serum ir-inhibin levels in rats are decreased at times when serum FSH levels are high.     

Effects of thymulin and GnRH on the release of gonadotropins by in vitro pituitary cells obtained from rats in each day of estrous cycle

The effects of thymulin and GnRH on FSH and LH release were studied in suspension cultures of anterior pituitary cells from female adult rats sacrificed on each day of the estrous cycle. The spontaneous release of gonadotropins by pituitaries, as well as their response to GnRH or thymulin addition, fluctuated during the estrous cycle. Adding thymulin to pituitary cells from rats in diestrus 1 increased the concentration of FSH; while in cells from rats in estrus, FSH level decreased. Thymulin had a stimulatory effect on the basal concentration of LH during most days of the estrous cycle. Adding GnRH increased FSH release in cells from rats in diestrus 1, diestrus 2, or proestrus, and resulted in higher LH levels in cells obtained from rats in all days of the estrous cycle. Compared to the GnRH treatment, the simultaneous addition of thymulin and GnRH to cells from rats in diestrus 1, diestrus 2, or proestrus resulted in lower FSH concentrations. Similar results were observed in the LH release by cells from rats in diestrus 1, while in cells from rats in proestrus or estrus, LH concentrations increased. A directly proportional relation between progesterone serum levels and the effects of thymulin on FSH release was observed. These data suggest that thymulin plays a dual role in the release of gonadotropins, and that its effects depend on the hormonal status of the donor's pituitary.     

A Mathematical Model for the Actions of Activin,Inhibin, and Follistatin on Pituitary Gonadotrophs

The timed secretion of the luteinizing hormone (LH) and follicle stimulating hormone (FSH) from pituitary gonadotrophs during the estrous cycle is crucial for normal reproductive functioning. The release of LH and FSH is stimulated by gonadotropin releasing hormone (GnRH) secreted by hypothalamic GnRH neurons. It is controlled by the frequency of the GnRH signal that varies during the estrous cycle. Curiously, the secretion of LH and FSH is differentially regulated by the frequency of GnRH pulses. LH secretion increases as the frequency increases within a physiological range, and FSH secretion shows a biphasic response, with a peak at a lower frequency. There is considerable experimental evidence that one key factor in these differential responses is the autocrine/paracrine actions of the pituitary polypeptides activin and follistatin. Based on these data, we develop a mathematical model that incorporates the dynamics of these polypeptides. We show that a model that incorporates the actions of activin and follistatin is sufficient to generate the differential responses of LH and FSH secretion to changes in the frequency of GnRH pulses. In addition, it shows that the actions of these polypeptides, along with the ovarian polypeptide inhibin and the estrogen-mediated variations in the frequency of GnRH pulses, are sufficient to account for the time courses of LH and FSH plasma levels during the rat estrous cycle. That is, a single peak of LH on the afternoon of proestrus and a double peak of FSH on proestrus and early estrus. We also use the model to identify which regulation pathways are indispensable for the differential regulation of LH and FSH and their time courses during the estrous cycle. We conclude that the actions of activin, inhibin, and follistatin are consistent with LH/FSH secretion patterns, and likely complement other factors in the production of the characteristic secretion patterns in female rats.     

Effects of complete hypothalamic deafferentation on the estrous phase of follicle-stimulating hormone release in the cyclic rat

We investigated whether neural afferents to the medial basal hypothalamus play an acute role in the estrous phase of FSH release in the 4-day cyclic rat. A cannula was inserted into the right atrium of the heart under brief ether anesthesia during the early afternoon of proestrus for subsequent blood collections and injection of LHRH. In some of the rats, the medial basal hypothalamus was surgically isolated from the rest of the brain with a small knife under brief ether anesthesia between 2000 h and 2130 h of proestrus. Control groups consisted of naive rats which were not treated during the night of proestrus and sham-operated animals in which the knife was lowered to the corpus callosum between 2000 h and 2130 h or proestrus. Rats were bled at 2200 h of proestrus and at 0200 h, 0600 h and 1000 h of estrus for radioimmunoassay of plasma FSH and LH. The plasma FSH levels in all 3 groups between 2200 h of proestrus and 1000 h of estrus were elevated above levels observed in other cannulated rats bled to the onset of the proestrous phase of FSH release at 1400 h of proestrus. There were no statistically significant differences in plasma FSH or LH concentrations at any of the time periods between the 3 groups of serially bled rats. The deafferentation procedure did not appear to impair the pituitary gland's ability to secret gonadotrophins as injection of 50 ng of LHRH after the bleeding at 1000 h of estrus caused substantial elevations in plasma FSH and LH concentrations which were not different between the 3 groups. The results suggest that neural afferents to the medial basal hypothalamus play no acute role in the estrous phase of FSH release in the cyclic rat.     

Variations in concentration of oxytocin and vasopressin in the paraventricular nucleus of the hypothalamus during the estrous cycle in rats

Oxytocin (OT) and arginine-8-vasopressin (AVP) were measured by radioimmunoassay in micropunched hypothalamic neurosecretory nuclei of estrous cycling female Sprague-Dawley rats. In the paraventricular nucleus (PVN): the concentration (pg/microgram protein) of OT was significantly higher in rats in diestrus than during proestrus, estrus, or metestrus, while the concentration during metestrus was significantly greater than in proestrus and estrus; the concentration of AVP was significantly lower in animals in estrus than during the other three stages; because the paraventricular OT levels dropped before proestrus, the AVP/OT ratio was significantly greater in animals in proestrus than in diestrus, metestrus, and estrus. In the supraoptic nucleus (SON) a similar trend was noted: the concentration of OT was highest during diestrus, and AVP was lowest during estrus, though neither was significantly different from other stages. Because the OT and AVP cycles in the SON were asynchronous, the ratio of AVP to OT was significantly higher in proestrus than in metestrus or diestrus and significantly greater in estrus than during diestrus. In contrast to these two areas, peptide concentrations did not vary significantly across the estrous cycle in other sites of nonapeptide synthesis, i.e. the anterior commissural nucleus (ACN) and the suprachiasmatic nuclei (SCN).     

Prolactin secretion and its response to stress during the estrous cycle of the rats

Serum and pituitary prolactin (PRL) concentrations were measured during the estrous cycle of the rat with particular attention to the afternoons of the days of proestrus and estrus. Homogenizing machines, a Polytron and Sonifier, were used to extract PRL from the pituitary gland. The effects of ether anesthesia and restraint were also examined on the afternoons of both proestrus and estrus. The occurrence of a surge in PRL secretion during proestrus was confirmed with a peak at 1500 h, and this was accompanied by a decline in pituitary PRL content. A relatively high level of serum PRL was observed in the afternoon of estrus, during which time pituitary PRL content increased progressively. Ether anesthesia had no effect on the proestrus PRL surge, while restraint enhanced it. On the afternoon of estrus, restraint completely suppressed the rise in serum PRL, but ether anesthesia failed to suppress it completely. From these results, the following conclusions were drawn: 1) the PRL surge on the afternoon of proestrus occurs without synthesis of the hormone in the pituitary; 2) PRL secretion on the afternoon of estrus is accompanied by its synthesis in the gland; 3) the PRL response is distinct for each type of stress applied; and 4) PRL secretion is thus regulated by different mechanisms in proestrus and estrus.     

Ovarian steroidogenic enzyme activities during the rat estrous cycle

We have correlated the concentrations of serum LH, estradiol and progesterone with the activities of 2 ovarian steroid biosynthetic enzymes during the rat estrous cycle. Ovarian 3 β-hydroxysteroid dehydrogenase isomerase (3-βHSD) activity decreased from 29 ± 6 nmol/mg protein/ min (mean ± SEM) in diestrus, to 7 ± 0.4 nmol/mg protein/min in late proestrus (p < 0.005), and subsequently increased to 36 ± 9 nmol/mg protein/min in metestrus (p < 0.01). Ovarian 17-hydroxylase (17-OH) activity decreased from early to late proestrus (3.3 ± 0.2 vs 2.2 ± 0.2 nmol/mg protein/min, p <0.0025), and subsequently increased to 3.9 ± 0.2 in metestrus (p<0.001). Serum LH, estradiol and progesterone peaked during proestrus, and reached a nadir during estrus. We conclude that the activities of 3-βHSD and 17-OH in the rat ovary vary markedly during the estrous cycle. These changes may underlie the pattern of steroid secretion characteristic of this process.     

Prolactin receptors in the rat mammary gland. Change during the estrous cycle

Ovine prolactin (o-PRL) binding to mammary gland membranes was studied during the estrous cycle in the rat. Groups of rats were decapitated throughout the 4-day estrous cycle at 10 h00 on the days of diestrus I, diestrus II and estrus and at 10 h00, 12 h00, 16 h00 during the day of proestrus. Daily vaginal smears were taken to determine the stage of the estrous cycle which was also controlled by PRL and LH serum levels. Prolactin receptors were quantified in the 100 000 g pellet. For one Scatchard analysis, mammary gland membranes from 5 animals were pooled. Results given are the mean of 4 or 5 pools. Results obtained showed that the apparent affinity constant (KA) remained unchanged during the days of diestrus II and at all the times studied of proestrus and showed a slight but significant decrease on the days of estrus and diestrus I (or metestrus). The binding capacity did not vary from the day of diestrus II to the proestrus 16h00 (11.3 +/- 2.8 fmoles/mg protein) but sharply increased on the day of estrus (190.4 +/- 35.9 fmoles/mg protein). Binding capacity remained elevated on the day of diestrus I. This increase of PRL receptors on the day of estrous would appear to be an important step in preparing mammary gland for pregnancy and lactation.     

Effects of estrogen on serum leptin levels and leptin mRNA expression in adipose tissue in rats

OBJECTIVE: In this study, we examined changes in serum leptin levels during the estrus cycle and the role of estrogen in these changes. METHODS: We measured serum leptin levels during normal estrus cycles in intact rats and estradiol-17beta (E2)-induced artificial estrus cycles in ovariectomized rats. RESULTS: Serum leptin levels increased 1.6-fold from 4.2 +/- 0.2 ng/ml during diestrus stage 2 to 6.7 +/- 0.9 ng/ml during proestrus stage during the 4-day estrus cycle. During the E2-induced estrus cycle, serum leptin levels increased 2.3-fold from 2.3 +/- 0.1 ng/ml at estrus to 5.4 +/- 1.2 ng/ml at proestrus. E2 also increased serum leptin concentrations and leptin mRNA expression in adipose tissue of immature rats. DISCUSSION: These findings suggest that increased serum leptin induced by estrogen during proestrus may trigger the preovulatory release of luteinizing hormone. Furthermore, our findings indicate that estrogen has a positive effect on leptin production in adipose tissue.     

Amygdala activation by corticosterone alters visceral and somatic pain in cycling female rats

Irritable bowel syndrome (IBS) is often seen in women, and symptom severity is known to vary over the menstrual cycle. In addition, activation of the hypothalamic-pituitary-adrenal (HPA) axis enhances symptomology and patients with IBS have increased activation of the amygdala, a brain region known to facilitate HPA output. However, little is known about the effects of amygdala activation during different stages of the menstrual cycle. We therefore investigated the effects of amygdala activation on somatic and visceral pain perception over the rat estrous cycle. Female Wistar rats were implanted with either corticosterone (Cort) or cholesterol as a control onto the dorsal margin of the central amygdala. Visceral sensitivity was quantified by recording the visceromotor response (VMR) to colorectal distension (CRD) and somatic sensitivity was assessed via the Von Frey test. In cholesterol controls, both visceral and somatic sensitivity varied over the estrous cycle. Rats in proestrus/estrus responded to CRD with an increased VMR compared with rats in metestrus/diestrus. Somatic sensitivity followed a similar pattern with enhanced sensitivity during proestrus/estrus compared with metestrus/diestrus. Elevated amygdala Cort induced visceral hypersensitivity during metestrus/diestrus but had no effect during proestrus/estrus. In contrast, elevated amygdala Cort increased somatic sensitivity during both metestrus/diestrus and proestrus/estrous. These results suggests that amygdala activation by Cort eliminates spontaneously occurring differences in visceral and somatic pain perception, which could explain the lowered pain thresholds and higher incidence of somatic pain observed in women with IBS.     

Puberty and ovulatory release of gonadotropins in spontaneously hypertensive rats

The age at vaginal opening, estrous cyclicity, serum levels of luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL) on the day of proestrus, and number of ova and ovarian weight as measured on the day of estrus in spontaneously hypertensive (SH) and genetically matched normotensive Wistar Kyoto (WKY) female rats were compared. In SH rats, there was a significant delay in the vaginal opening, but the regular 4-day estrous cycle followed afterwards. No significant changes were observed in the afternoon increase in serum LH, FSH and PRL on the day of proestrus in SH and WKY rats, although the basal levels of LH and PRL in the morning (11:00 h) were lower in SH rats than in WKY rats. The mean number of ova in SH rats was also less than in WKY rats, whereas the ovarian relative weight was similar in both species of rats. It can be said that SH rats undergo certain, but not critical, endocrine and/or neuroendocrine changes related to reproduction.