Morning Of Diestrus Research Articles

Role of the superior ovarian nerve in the regulation of follicular development and steroidogenesis in the morning of diestrus 1.

Little is known about the role of the superior ovarian nerve (SON) in follicular development during the estrus cycle. The aim of the present study was to analyze the role of neural signals arriving through the SON at the ovaries in the regulation of follicular development and ovarian steroid secretion in diestrus 1 of cyclic rats. Cyclic rats were subjected to left, right, or bilateral SON sectioning or to unilateral or bilateral laparotomy at diestrus 1 at 11:00h. Animals were sacrificed 24h after surgery. Compared to laparotomized animals, unilateral SON sectioning decreased the number of preovulatory follicles, while bilateral SON sectioning resulted in a decreased number of atretic preantral follicles. An important observation was the presence of invaginations in the follicular wall of large antral and preovulatory follicles in animals with denervation. Furthermore, left SON sectioning increased progesterone levels but decreased testosterone levels, which are effects that were not observed in animals that were subjected to right denervation. At 11:00h of diestrus 1, the SON was found to stimulate follicle development, possibly via neural signals, such as noradrenaline and/or vasoactive intestinal peptide, and this stimulation induced the formation of follicle-stimulating hormone receptors. The role of the SON in the regulation of ovarian steroid secretion is asymmetric: the left SON inhibits the regulation of progesterone and stimulates testosterone secretion, and the right nerve does not participate in these processes.

SAT-406 Deletion of Musashi in Gonadotropes Leads to Increased GnRHR Protein Levels and Gonadotrope Dysfunction

Leptin is a critical mediator of metabolic regulation of the hypothalamic-pituitary gonadal (HPG) axis. We have previously shown that leptin is responsible for the optimal expression of GnRHR, a rate-limiting component of the reproductive process. Female mice lacking leptin receptors (Lepr-null) specifically on gonadotropes are sub-fertile. Their reduced GnRHR proteins and normal Gnrhr mRNA levels suggest leptin’s actions on gonadotropes are post-transcriptional. A clue about a candidate translational regulator is seen in our studies showing that Lepr-null gonadotropes have increased expression of Musashi (MSI), which has binding sites within the Gnrhr 3’ UTR. Furthermore, leptin reduced MSI expression specifically in gonadotropes and increased GnRHR expression. We hypothesized that MSI may repress Gnrhr mRNA translation and that leptin alleviates this repression. To determine the effects of MSI on the HPG axis, we developed a gonadotrope-Msi1/2-null mouse line and compared mutant females (MUT) to their littermate controls (CTL) on the morning of diestrus, when the pituitary GnRHR protein levels should reach a peak. The levels of GnRHR proteins (measured by EIA) are significantly increased (1.6X) in the pituitary of the mutant females (CTL: 1.355 ± 0.11 ng/ml vs MUT: 2.202 ± 0.16 ng/ml, p=.0006), with no change in mRNA. The gonadotrope-Msi1/2-null females are subfertile, with litter sizes of 3 ± 0.4 pups, with the first litter at around day 40 and an average of 41 day delay between litters. To understand the downstream effect of the MSI knockout on gonadotropin levels, we measured pituitary and serum LH and FSH protein levels (Luminex EIA) and mRNA (qPCR). Serum FSH levels are decreased by >50% in mutant females (CTL: 0.947 ± 0.14 ng/mL vs MUT: 0.406 ± 0.05 ng/ml, p=.0049), but FSH stores and Fsh mRNAs are unchanged. Additionally, we observed a 2.2X increase in the pituitary LH protein content in mutant females (CTL: 0.328 ± 0.11 ng/ml vs MUT: 0.729 ± 0.09 ng/ml, p=.0174), but no changes in serum LH or Lh mRNA levels. These studies thus show that, as a repressor of GnRHR translation, Musashi can also regulate expression of gonadotropins. In the gonadotrope Lepr-null model, we hypothesized that leptin signals were needed to de-repress Musashi actions and allow GnRHR translation. In contrast, the gonadotrope-MSI-null mice over-express GnRHR, which confirms this role for MSI as a GnRHR regulator. We propose that the increased expression of GnRHR in the gonadotrope MSI-null animals causes the increased LH content, and disrupted FSH secretion, resulting in a higher serum LH:FSH ratio and subfertility. Collectively these data suggest that MSI regulation is necessary for optimal fertility in the adult female mouse. Future studies will determine the impact of loss of MSI on gonadotrope function throughout the estrous cycle.

Characterization of the Reproductive Effects of the Vgf-Derived Peptide TLQP-21 in Female Rats: In vivo and in vitro Studies

Background: VGF (non-acronymic), a protein expressed in the hypothalamus and pituitary, is involved in the control of metabolism and body weight homeostasis. Different active peptide fragments are generated from VGF, including TLQP-21. Previous studies of our group reported that this molecule participates also in the regulation of reproductive function in male rats, with predominant stimulatory effects. Methods: We report herein a series of studies on the reproductive effects of TLQP-21 in female rats, as evaluated by a combination of in vivo and in vitro analyses. Results: TLQP-21 modestly increased serum LH levels after systemic administration and directly stimulated pituitary LH and FSH secretion in prepubertal female rats, while acute central injection of TLQP-21 was unable to modify LH secretion at this age. Repeated central administration of TLQP-21 during the pubertal transition (between PND-28 and -35) to female rats fed ad libitum advanced the timing of vaginal opening and increased the percentage of animals with signs of ovulation. Moreover, an analogous treatment slightly enhanced ovarian maturation in pubertal female rats subjected to chronic undernutrition, but was unable to rescue the delay of vaginal opening induced by food deprivation. In addition, TLQP-21 oppositely modified LH secretion in adult female rats depending on the stage of the ovarian cycle: it stimulated LH secretion when injected in the morning of diestrus and decreased the magnitude of the preovulatory LH (but not FSH) surge when injected in the afternoon of proestrus. Conclusions: Our data are the first to document the potential involvement of TLQP-21 in the control of reproductive function in female rats.

Estrus cycle stage modifies the presentation of stress-induced startle suppression in female Sprague–Dawley rats

Tailshock stress causes transient reductions in startle reactivity, associative learning and open field activity in female rats in an ovarian hormone dependent manner. Others have shown estrogen modulation of associative learning by testing across the estrus cycle and pharmacological manipulations. Here we tested whether stress-induced suppression of startle reactivity can be attributed to circulating ovarian hormones. Female rats were tracked across the estrus cycle and subjected to the stressor (2 h periodic tailshock) the morning of diestrus, proestrus, estrus, or metestrus. Startle reactivity was tested 2 h following the cessation of the tailshock. Using a multi-stimulus protocol, we determined there were differences in startle sensitivity and responsivity. Following stressor exposure, estrus females exhibited reduced startle responsivity. In contrast, diestrus females exhibited increased sensitivity to the lowest acoustic stimulus. The results are discussed with respect to ovarian hormone regulation of the immune system and sensory reactivity during and following trauma that may lead to different abnormal behaviors in females in the wake of traumatic stress.

Pituitary Adenylate Cyclase Activating Polypeptide Messenger RNA in the Paraventricular Nucleus and Anterior Pituitary During the Rat Estrous Cycle1

The neuropeptide pituitary adenylate cyclase activating polypeptide (ADCYAP 1, or PACAP) has been demonstrated to enhance gonadotropin-releasing hormone (GnRH)-induced gonadotropin secretion and regulate gonadotropin subunit gene expression in cultures of anterior pituitary cells. In the present study, we used in situ hybridization and real-time polymerase chain reaction to examine the expression of Pacap mRNA within the paraventricular nucleus (PVN) and anterior pituitary throughout the estrous cycle of the rat. Levels of luteinizing hormone in serum and pituitary gonadotropin subunit mRNAs were evaluated and displayed cyclic fluctuations similar to those reported previously. Pacap mRNA expression in the PVN and pituitary varied significantly during the estrous cycle, with the greatest changes occurring on the day of proestrus. Pacap mRNA levels in the PVN declined significantly on the morning of diestrus. During proestrus, PVN Pacap mRNA levels significantly increased 3 h before the gonadotropin surge and then declined. Pituitary expression of Pacap mRNA also varied on the afternoon of proestrus with a moderate decline at the time of the gonadotropin surge and a significant increase later in the evening. Expression of the mRNA species encoding the 288 amino acid form of follistatin increased significantly following the rise in pituitary Pacap mRNA, at the termination of the secondary surge in follicle-stimulating hormone beta (Fshb) gene expression. These results suggest that PACAP is involved in events before and following the gonadotropin surge, perhaps through increased gonadotroph sensitivity to GnRH and suppression of Fshb subunit expression through increased follistatin, as previously observed in vitro.

Differential Gonadotropin-Releasing Hormone (GnRH) and GnRH Receptor Messenger Ribonucleic Acid Expression Patterns in Different Tissues of the Female Rat across the Estrous Cycle

GnRH, the main regulator of reproduction, is produced in a variety of tissues outside of the hypothalamus, its main site of synthesis and release. We aimed to determine whether GnRH produced in the female rat pituitary and ovaries is involved in the processes leading to ovulation. We studied the expression patterns of GnRH and GnRH receptor (GnRH-R) in the same animals throughout the estrous cycle using real-time PCR. Hypothalamic levels of GnRH mRNA were highest at 1700 h on proestrus, preceding the preovulatory LH surge. No significant changes in the level of hypothalamic GnRH-R mRNA were detected, although fluctuations during the day of proestrus are evident. High pituitary GnRH mRNA was detected during the day of estrus, in the morning of diestrus 1, and at noon on proestrus. Pituitary GnRH-R displayed a similar pattern of expression, except on estrus, when its mRNA levels declined. Ovarian GnRH mRNA levels increased in the morning of diestrus 1 and early afternoon of proestrus. Here, too, GnRH-R displayed a somewhat similar pattern of expression to that of its ligand. To the best of our knowledge, this is the first demonstration of a GnRH expression pattern in the pituitary and ovary of any species. The different timings of the GnRH peaks in the three tissues imply differential tissue-specific regulation. We believe that the GnRH produced in the anterior pituitary and ovary could play a physiological role in the preparation of these organs for the midcycle gonadotropin surge and ovulation, respectively, possibly via local GnRH-gonadotropin axes.

GABA release in the medial preoptic area of cyclic female rats

GABA is a potent regulator of gonadotropin-releasing hormone neurons in the hypothalamus. To determine the profile of GABA release in the medial preoptic area where the gonadotropin surge generator resides, an in vivo microdialysis study was performed in cyclic female rats. The microdialysis samples were collected and sequential blood samples (150 μl each) were also obtained, at 1-h intervals. During estrus and diestrus 1, GABA release in the medial preoptic area was relatively low. A small increase in the GABA release began in the afternoon of diestrus 1 and attained its peak in the morning of diestrus 2, but declined in the afternoon of that day. The GABA release markedly increased from late in the night of diestrus 2 through the morning of proestrus, when it attained its peak, and thereafter it declined sharply until the critical period of proestrus. A distinct preovulatory luteinizing hormone surge was observed in the afternoon of proestrus in all proestrous rats. From these results we suggest that the preovulatory elevation of the GABA release from the night through to the morning of proestrus, followed by a sharp decline, is closely associated with the onset of the preovulatory luteinizing hormone surge in cyclic female rats. The present study is the first to report the 4-day profile of GABA release in the medial preoptic area during the estrous cycle.

Effect of ovarian steroids on hypothalamic norepinephrine-induced adenylate cyclase activity

The adenylate cyclase activity induced by norepinephrine (10(-8)-10(-9) M) was studied in hypothalamic particulate fractions from female rats. The effect of estradiol on this activity was studied in rats that were ovariectomized in diestrus 1, injected with estradiol benzoate (50 micrograms/kg body weight), and killed 48 h later. The effect of progesterone was studied in fractions from female cycling rats injected in the morning of diestrus 2 with progesterone (2 mg/rat); these animals were killed 30 or 48 h after the steroid injection. The blockade of norepinephrine-induced adenylate cyclase activity by alpha- and beta-blocking agents (10(-8)-10(-9) M) was also evaluated. The enzymatic activity was determined by monitoring the capacity to produce cAMP from ATP at saturated levels; cAMP was assayed by radioimmunoassay. At 48 h after administration, estradiol benzoate increased the norepinephrine-induced adenylate cyclase activity in the hypothalamus. This effect was not changed by the presence of phenoxybenzamine, an alpha-adrenoceptor blocker, but was greatly reduced by propranolol, the beta-adrenoreceptor blocker. In contrast, the progesterone, at 30 and 48 h after injection, decreased the hypothalamic adenylate cyclase activity, and this effect was preferentially antagonized in the presence of phenoxybenzamine rather than propranolol. These results suggest that estrogen and progesterone act selectively on one or another type of adrenergic receptor at the hypothalamic level.

Disruption of Estrous Cyclicity Following Administration of a Luteinizing Hormone-Releasing Hormone Antagonist to the Preoptic Area of the Rat1

Experiments were designed to test the hypothesis that LHRH receptors in the preoptic area (POA) are of physiological importance for maintaining estrous cyclicity in the rat. Bilateral cannulae were implanted just dorsal to the POA. Estrous cycles were monitored daily by vaginal smears. Antide, a long-acting LHRH antagonist, was infused bilaterally (2.5 micrograms/side) in the POA or the hypothalamus on the mornings of diestrus I and II. As controls, at separate times, rats also received similar infusions of either vehicle (1:1 water:propylene glycol) or a bombesin antagonist (#B0650; Sigma, St. Louis, MO). Collection of daily vaginal smears continued, and the number of days from the first infusion to the next day of estrus that preceded a normal cycle was recorded. After infusion of Antide into the POA, rats demonstrated varying durations of interrupted cycles ranging from 11 days to more than 100 days. These periods of disruption were characterized by either long periods of diestrus, long periods of estrus, or an extended period of diestrus followed by an extended period of estrus. After infusion of Antide into the dorsomedial, ventromedial, or anterior hypothalamic areas, rats had either a 4- or 5-day estrous cycle and continued to cycle normally. Likewise, infusions into the septum had no effect. Infusion of vehicle or bombesin antagonist into any of the hypothalamic or POA sites tested also resulted in no interruptions in the cyclic activity of the rats. Therefore, it appears that functional LHRH receptors in the POA are necessary to drive the normal estrous cycle.

Effects of Acute Estradiol on 5-Hydroxytryptamine and Dopamine Receptor Subtype mRNA Expression in Female Rat Brain

The aim of the present study was to determine the effect of estradiol-17β, in its positive feedback mode for the release of prolactin and luteinizing hormone (LH), on gene expression of 5-hydroxytryptamine (5-HT) and dopamine receptors. Gene expression was determined by measurement of the levels of receptor mRNA by in situ hybridization in brain sections from adult female rats. The animals were ovariectomized under halothane anesthesia on the morning of diestrus, given a sc injection of either estradiol benzoate (EB) or oil (vehicle), and killed between 1600-1700 h of the next day, presumptive proestrus. The injection of EB in this preparation is known to stimulate a massive surge of LH and prolactin in the late afternoon of presumptive proestrus. The injection of EB produced a significant increase in silver grain densities representing 5-HT 2 receptor mRNA in the dorsal raphe nucleus (DRN) and a significant reduction in these grain densities in the medial septum and diagonal band of Broca. The number of cells expressing 5-HT 2 receptor mRNA was increased by 290% in the DRN and decreased by 25% in the medial preoptic area. Estradiol had no effect on silver grain densities, or numbers or percentages of cells expressing 5-HT 2 receptor mRNA in any of the other nine brain regions or choroid plexus studied, and had no effect on the levels of 5-HT 1A, 5-HT 1C, D 1, or D 2 receptor mRNA in any region studied. These data suggest that the 5-HT 2 receptor is a key receptor involved in mediating the positive feedback stimulation by estradiol-17β of LH and prolactin release. Since estradiol increases 5-HT levels in the DRN, the data raise the intriguing possibility that estrogen positive feedback on LH and prolactin release is mediated by a feed-forward 5-HT drive which results in increased 5-HT release at DRN-derived nerve terminals on hypothalamic neurons which control LH and prolactin release.

α1 adrenergic regulation of estrogen-induced increases in luteinizing hormone-releasing hormone mRNA levels and release

Prazosin, an α 1 adrenergic antagonist, was used to examine the relationship between adrenergic inputs and the stimulatory effects of estrogen on LHRH mRNA and release. Bilateral cannulae were implanted just dorsal to the preoptic area (POA). Estrous cycles were monitored daily by vaginal smears. On the morning of diestrus, each rat was ovariectomized and assigned to one of three treatment groups: Control — injected with sesame oil ( n = 5); Surge — injected with estradiol benzoate (EB, 10 μg) to produce an LH surge ( n = 5); or, Surge + Prazosin — injected with EB and a prazosin-filled inner cannula was put into the POA ( n = 6). Between 4–6 pm of the following day, rats were anesthetized, decapitated, trunk blood collected, and brains were stored in liquid nitrogen. In situ hybridization was performed using a 32P end-labelled 59-mer complementary to LHRH mRNA. Reduced silver grains, proportional to LHRH mRNA content, were quantified. Treatment with estrogen alone resulted in an LH surge and a 50% increase ( P < 0.05) in numbers of cells expressing LHRH. This estrogen-induced increase and the LH surge were completely blocked ( P < 0.01) by prazosin. Prazosin also decreased ( P < 0.01) the median number of grains per cell from 81 (Surge) to 65 grains per cell (Surge + Prazosin). When the number of grains in LHRH-expressing neurons were totalled, EB increased ( P < 0.05) LHRH gene expression by 53%, and local administration of prazosin completely blocked ( P < 0.01) this increase. Since, coincident with estradiol stimulating the LH surge, it increased potential LHRH synthetic capacity (as measured by LHRH mRNA) and since administration of prazosin into POA blocked the ability of estrogen to do so, it appears that an endogenous α 1 ligand, probably norepinephrine, is a mediator of stimulatory effects of estrogen on LHRH biosynthesis as well as release.

Effects of immobilization stress on tuberoinfundibular dopaminergic (TIDA) neuronal activity and prolactin levels in lactating and non-lactating female rats

The effects of immobilization stress on the prolactin secretory response and on the activity of the tuberoinfundibular dopaminergic (TIDA) neurons were determined in intact, virgin female rats on the morning of diestrus or proestrus and in post-partum, lactating female rats. The virgin females exhibited a significant increase in circulating levels of prolactin which was evident by 1 minute and persisted during the immobilization (5 minutes). In contrast, the prolactin secretory response in lactating females was significantly attenuated compared to non-lactating animals. The activity of the TIDA neurons was not altered by the 5 minutes of stress. Even after 30 minutes of immobilization, TIDA neuronal activity was not affected in either the lactating or cycling females. These data suggest that the cycling female rat is capable of a prolactin secretory response to the stressor without inhibition of TIDA neuronal activity. It seems likely that prolactin releasing factors mediate this response. In contrast, stress did not produce a similar prolactin increase during lactation. It seems likely that, during lactation, the pituitary is not sensitive to releasing factors unless the TIDA neurons are inhibited. There appear to be differences in the sensitivity of the pituitary depending on the physiological state of the model employed.

The effect of transient dopamine antagonism on thyrotropin-releasing hormone-induced prolactin release in female rats during the estrous cycle

Prolactin (PRL) release induced by TRH was examined on each day of the estrous cycle in female rats in which pituitary dopamine (DA) receptors were blocked pharmacologically. The objective was to determine if an interaction exists between hypothalamic inhibitory and releasing hormones with regard to prolactin (PRL) secretion. Domperidone (0.01 mg/rat i.v.) followed 5 minutes later by the administration of the DA agonist 2-Br-α-ergocryptine meleate (CB-154, 0.5 mg/rat i.v.) were used to produce a transient (< 1 hr) dopamine blockade. One hour later, thyrotropin-releasing hormone (TRH, 1.0 μg/rat i.v.) was given to stimulate PRL release. On the morning of proestrus, TRH released a significantly greater quantity of PRL into the plasma after DA antagonism compared to control animals which did not receive the dopamine antagonist. Dopamine antagonism also enhanced the effectiveness of TRH on the mornings of estrus and metestrus. The response on estrus was significantly greater than the response on proestrus. However by the morning of diestrus, TRH-“releasable” PRL was greatly diminished. Our results suggest that DA antagonism is able to shift differing quantities of PRL into a TRH “releasable” pool on several days of the estrous cycle and that the control of this mechanism is acute.

Effect of estradiol and tamoxifen on GnRH self-priming in perifused rat adenohypophysial cells.

Columns of enzymatically dispersed rat pituitary cells have been used to examine the effects of estradiol and the estrogen receptor antagonist, tamoxifen, on the responsiveness of the gonadotropes to repeated challenges with GnRH. Synthetic GnRH stimulated readily the release of LH from the pituitary cells. When challenged repeatedly with the same submaximal dose (8 nmol/l) of the releasing hormone, cells from animals in diestrus 2 or pro-estrus, like those from juvenile rats, showed a marked progressive increase in their sensitivity, but those from rats in estrus or diestrus 1 did not. Adenohypophysial cells removed from adult rats ovariectomized 14 days previously also failed to exhibit sensitization even when estradiol-17 beta (1 and 10 nmol/l) was included in the perifusion fluid. However, those from ovariectomized rats treated with appropriate doses of the steroid (1.5 microgram/day sc for 14 days or 5 micrograms sc 22 h before decapitation) did. The capacity of pituitary cells from intact rats to exhibit priming on the anticipated day of pro-estrus was unaffected by the administration of the estrogen receptor antagonist, tamoxifen (700 micrograms/100 g sc on the morning of diestrus 1 and 24 h later). Similarly, addition of tamoxifen (1 mumol/l) to the incubation medium failed to influence the capacity of pituitary cells from juvenile rats to exhibit priming in vitro. The results support the concept that the ability of GnRH to 'prime' the gonadotropes is dependent on estradiol and indicate that the exposure of the hypothalamo-pituitary complex to critical levels of estradiol prior to autopsy is necessary for the gonadotropes to retain their capacity to exhibit priming in vitro.

Analysis of estradiol-independent and -dependent endogenous opioid peptide suppression of pulsatile LH release between the mornings of diestrus 2 and proestrus in the rat estrous cycle

The aim of this study was to analyze possible estradiol (E 2)-independent and -dependent endogenous opioid peptide (EOP) suppression of pulsatile LH release between the mornings of diestrus 2 (D2) and proestrus by examining the LH response to naloxone infusions in the presence or absence of proestrous levels of E 2. Pulsatile LH secretion remained unchanged between D2 and proestrus but mean blood LH levels, pulse amplitude and frequency increased within 24 hr following ovariectomy on D2. This increase was due in large part to the loss of E 2 negative feedback, since restoration of physiological proestrous E 2 levels returned LH pulse frequency to proestrous a.m. levels and greatly reduced pulse amplitude. In ovariectomized rats lacking E 2 negative feedback, continuous infusion of the EOP receptor antagonist naloxone (0.5 and 2 mg/kg/hr) caused a further increase in pulse amplitude and frequency. This naloxone-induced increment in pulsatile LH release was exerted via centrally located EOP receptors since naloxone did not alter pituitary responsiveness to LHRH, and its stimulatory action on pulsatile release was diminished by simultaneous infusion with morphine. Naloxone also increased pulsatile LH release in E 2-treated animals. The naloxone-induced increments in LH pulse amplitude were the same in the presence or absence of E 2 negative feedback. Moreover, the increments in amplitude produced by naloxone in E 2-treated rats were significantly less than those resulting from the combination of ovariectomy plus naloxone infusion in empty capsule-implanted rats. These data indicated that naloxone infusion in E 2-implanted animals blocked an E 2-independent EOP suppression of this parameter of pulsatile release. In contrast, with both naloxone doses tested, there was a trend for naloxone-induced increments in pulse frequency to be larger in ovariectomized rats previously implanted with E 2 rather than empty capsules. Although these differences were not statistically significant, the naloxone-induced increments in pulse frequency in E 2-treated rats (unlike those in pulse amplitude) were large enough so that they were also significantly different from the overall increases in pulse frequency caused by the combination of ovarian steroid removal coupled with blockade of EOP receptors during naloxone infusion in empty capsule-implanted animals. This raises the possibility that naloxone infusions in E 2-treated rats compromised both an E 2-independent EOP suppression of pulse frequency and a small, E 2-dependent component as well. Overall, the data demonstrate that between the mornings of D2 and proestrus, EOPs act independent of E 2 to suppress both LH pulse amplitude and frequency, and the negative feedback of E 2 on LH pulse amplitude is not mediated by EOPs whose receptors are antagonized by naloxone. The data also suggest the possibility that E 2 suppression of pulse frequency may be mediated, at least in part, by EOPs.

The Effect of Transient Dopamine Antagonism on Thyrotropin-Releasing Hormone-Induced Prolactin Release in Ovariectomized Rats Treated with Estradiol and/or Progesterone*

PRL release was studied in ovariectomized (OVX) rats pretreated with estradiol benzoate (EB), progesterone (P), or a combination of both steroids using a protocol that was selected to mimic ovarian steroid changes that have been observed during the female rat 4-day estrous cycle and early pregnancy. On the morning of the experiment, the animals received injections of either the dopamine (DA) antagonist domperidone (0.01 mg/rat iv) or vehicle (acetic acid in saline). Five minutes later, all animals received injections of the DA agonist 2-bromo-alpha-ergocryptine (CB-154; 0.5 mg/rat, iv) followed 60 min later by the administration of TRH (1.0 microgram/rat, iv). Plasma obtained from blood samples taken during the experiment was assayed for PRL by RIA. In OVX or P-treated OVX rats, a transient blockade of DA by domperidone did not alter the sensitivity of the pituitary to TRH administration, as measured by an increase in plasma PRL. However, such an effect of DA blockade was induced by 2 days of EB treatment and was maintained and amplified by P administration after EB injections. We conclude that enhancement of the PRL-releasing effect of TRH by DA antagonism, a mechanism we previously observed in female rats during midlactation, proestrus, estrus, and metestrus using the present drug protocol, can be induced by estrogen and maintained by P. Further, our data suggest that the previously observed loss of this secretory mechanism on the morning of diestrus may be due to the decrease in plasma P that takes place between metestrus and diestrus.

Changes in plasma progesterone, estradiol, follicle-stimulating hormone and luteinizing hormone during diestrus and ovulation in rats with 5-day estrous cycles: effect of antibody against progesterone.

Progesterone secretion remained significantly higher during diestrus in the 5-day cyclic rat than in the 4-day cyclic animal. Injection of a sufficient amount of antiprogesterone serum (APS) at 2300 h on metestrus in a 5-day cycle advances ovulation and completion of the cycle by 1 day in the majority of animals (75 and 80%, respectively). Progesterone (250 micrograms) administered with APS eliminated the effect of the antiserum. Within 2 h after administration of APS, levels of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) elevated significantly, while a significant elevation of plasma estradiol above the control value followed as late as 36 h after the treatment. None of the 5-day cyclic rats treated with APS showed ovulatory increases of FSH and LH at 1700 h on the second day of diestrus, although 3 of the 4 animals receiving the same treatment ovulated by 1100 h on the following day. The onset of ovulatory release of gonadotropins might have been delayed for several hours in these animals. These results indicate that recurrence of the 5-day cycle is due to an elevated progesterone secretion on the morning of diestrus, and suggest that a prolongation of luteal progesterone secretion in an estrous cycle suppresses gonadotropin secretion. Rather than directly blocking the estrogen triggering of ovulatory LH surge, the prolonged secretion of luteal progesterone may delay the estrogen secretion itself, which decreases the threshold of the neural and/or hypophyseal structures for ovulatory LH release.

Interaction between estradiol and a nonsteroidal factor in porcine follicular fluid in regulating LH pulse amplitude between the mornings of diestrus 2 and proestrus in the rat.

The object of this study was to examine the effect of porcine follicular fluid (PFF) alone or in combination with estradiol (E2) on pulsatile LH release during the interval between the mornings of diestrus 2 (D2) and proestrus in the rat. Steroids were removed from PFF by charcoal extraction. Preliminary studies indicated that 1 ml PFF given intraperitoneally suppressed FSH secretion for up to 15 h, with an onset of action between 3 and 4 h and maximal suppression between 6 and 9 h. In subsequent experiments, six groups of animals were bled continuously for 3 h between 07.30 and 10.30 h at a rate of 50 microliter whole blood/5 min: group 1 was bled on D2; group 2 was sham ovariectomized on D2 (08.30-09.30 h), immediately implanted with an empty capsule, given saline at 12.00 and 24.00 h, and bled on proestrous AM; groups 3-6 were ovariectomized on D2, implanted with an empty or E2 capsule, given 1 ml saline or PFF at 12.00 and 24.00 h, and bled 24 h following ovariectomy (OVX). Between D2 and proestrus plasma E2 levels increased, and there was no change in any parameter of pulsatile LH release. However, OVX on D2 reduced plasma E2 levels and increased mean blood LH levels above proestrous values due to increases in LH pulse amplitude and frequency. Restoration of physiological proestrous levels of E2 reduced the increase in mean blood LH levels, by lowering pulse frequency to proestrous values and by greatly reducing pulse amplitude. However, LH pulse amplitude and mean blood LH levels were still higher than values on proestrus. PFF alone produced no alteration in any parameter of pulsatile LH release compared with saline-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Ovarian steroid regulation of pulsatile luteinizing hormone release during the interval between the mornings of diestrus 2 and proestrus in the rat.

The object of this study was to determine the influence of ovarian steroids on pulsatile LH release in the interval between the mornings of diestrus 2 (D2) and proestrus in the rat. Four groups of rats were bled continuously for 3 h between 09.30-12.30 h at a rate of 75 microliters whole blood/6 min: bled on D2; sham ovariectomy (OVX) on D2 and bled on proestrus; OVX on D2, implanted with empty or oil-filled capsules, and bled 24 h later; and OVX on D2, implanted with estradiol (E2) capsules, and bled 24 h later. Between D2 and proestrus, plasma E2 levels increased from 13 +/- 1 to 42 +/- 9 pg/ml, and progesterone levels decreased from 27 +/- 3 to 13 +/- 2 ng/ml, the latter reflecting the decline of the corpus luteum early on D2. Between D2 and proestrus there was no change in mean blood LH levels, LH pulse amplitude, or pulse frequency. However OVX on D2 increased mean blood LH levels 2.5-fold over values on proestrus due to a 3.5-fold elevation in LH pulse amplitude and an 80% increase in pulse frequency. E2 levels fell in these rats to 8 +/- 1 pg/ml. Restoration of physiological proestrous levels of E2 (46 +/- 5 pg/ml) significantly reduced the increase in mean blood LH levels by lowering pulse frequency to proestrous values, and by causing a 50% reduction in pulse amplitude. However, LH pulse amplitude and therefore mean blood LH levels were still higher than values on proestrus.(ABSTRACT TRUNCATED AT 250 WORDS)

Does corpus luteum function autonomously during estrous cycle in the rat? A possible involvement of LH and prolactin

This study examined the role of LH and prolactin in the control of corpus luteum function during 4-day cycles in the rat. Bromocriptine (BRC) treatment was performed on proestrus or/and estrus morning that means before or after the preovulatory release of LH. This caused complete blood prolactin depression from the time of injection until diestrus 1 afternoon. This decrease in blood prolactin concentration was associated with a rise in the tonic level of LH secretion in those females which received BRC as soon as on proestrus. We first observed that injection on the morning of proestrus of doses of BRC capable of blunting prolactin secretion on proestrus afternoon did not significantly impair the preovulatory release of LH and did not prevent ovulation occurring during the following night. The life span of the corpora lutea edified from ovarian follicles rupturing before or under BRC administration did not exceed that of those formed under physiological circumstances since 4-day cycles culminating in ovulation constantly took place in all the treated animals whatever the time of BRC injection. To determine the pattern of luteal activity in the absence of prolactin secretion, we measured blood progesterone concentration from estrus until late diestrus in female rats injected with BRC on proestrus and/or estrus at 1100h. The initiation of the function of corpus luteum on estrus and the achievement of its full activity on diestrus 1 did not appear to be affected by BRC. By contrast the level of blood progesterone declined more rapidly on the morning of diestrus 2 in BRC-treated than in control females. The capacity for autonomous progesterone secretion by corpus luteum of the cycle was discussed in the light of previous and present observations.