Suppression of the proestrus prolactin surge in the rat estrous cycle by urethane anesthesia

Urethane-anesthetized male rats have been used for the analysis of prolactin (PRL)-releasing substances on PRL secretion. However, there are only a few reports investigating the effect of urethane anesthesia on PRL secretion in female rats. In this study, we intended to examine the effects of urethane anesthesia on PRL secretion during proestrus in the rat. Proestrus PRL surge was completely blocked when urethane was administered to rats prior to the critical period of proestrus both at doses of 1.0 g/kg and 1.5 g/kg. Additionally, urethane, at a dose of 1.5 g/kg, was also effective in blocking spontaneous ovulation. An experiment examining pituitary PRL concentration at 1800 h confirmed that urethane (1.0 g/kg) anesthesia prevents the PRL surge from the pituitary. Similarly, urethane anesthesia blocked the LH surge from the pituitary, but LH levels in the urethane-treated group were higher than those in the pentobarbital-treated group.     

A secondary surge of prolactin on the estrus afternoon

It has been described that throughout the estrous cycle of the rat, plasma prolactin (PRL) is basal except on proestrus afternoon when a preovulatory surge occurs. However, there have been controversies about PRL levels on the estrus day. Thus, the aim of this study was to evaluate the existence of a secondary surge of PRL on estrus afternoon and correlate it with plasma estradiol levels. The jugular vein of cycling rats was cannulated at 14:00 h on proestrus and a blood sample was withdrawn at 17:00 h for plasma LH measurement and determination of the preovulatory LH surge occurrence. In order to exclude the regular cycling rats that do not present the gonadotropins preovulatory surge and do not ovulate, only rats showing the LH surge on proestrus were considered in this study. Blood samples were collected hourly during estrus from midnight to 9:00 h (group 1) and from 10:00 to 18:00 h (group 2). In group 1, PRL showed a descending profile from midnight to 9:00 h, whereas the estradiol concentrations were constant. In group 2, a secondary surge of PRL was observed in 20 of 25 (80%) rats and plasma estradiol remained constant, but was higher in animals with the PRL surge. Thus the present data suggest the occurrence of a secondary surge of PRL in the afternoon of estrus that seems to be related to plasma estradiol levels of estrus day, which might exert only a permissive role in this surge generation.     

Prolactin messenger ribonucleic acid concentrations throughout the ovine estrous cycle: assessment relative to prolactin serum and pituitary amounts

Prolactin (PRL) mRNA concentrations were assessed by nucleic acid hybridization assays in pituitaries of ewes representing the defined stages of the ovine estrous cycle. Concomitantly, pituitary and serum PRL concentrations were measured in these ewes using radioimmunoassays. It was observed that PRL serum, pituitary and mRNA concentrations tended to increase near the time of the gonadotropin preovulatory surge, particularly between 24 hrs before behavioral estrus to 5 hours after estrus. However, the changes in PRL mRNA, serum and pituitary concentrations were shown not to be statistically significant. These data suggest that PRL production during the sheep estrous cycle is maintained without dramatic changes in synthesis or secretion.     

Prolactin messenger ribonucleic acid levels, prolactin synthesis, and radioimmunoassayable prolactin during the estrous cycle in the golden Syrian hamster

The purpose of this study was to observe the molecular dynamics of pituitary prolactin (PRL) gene expression during the estrous cycle of the Golden Syrian hamster. PRL messenger ribonucleic acid (mRNA) levels, PRL synthesis (3H-PRL in the incubation media or incubated pituitary after a 3 hr incubation with 3H-leucine), and radioimmunoassayable (RIA) PRL (in the incubation media or incubated pituitary after the 3 hr incubation) were measured in the morning (0930-1100 hr) on each day of the cycle. We observed that all of these PRL indices declined or did not change from Day 2 to Day 3 of the cycle. From Day 3 to Day 4 (proestrus), however, PRL mRNA levels increased 33-38% and media 3H-PRL increased 32-42%, while there were no significant changes in pituitary 3H-PRL, or RIA-PRL in the media or pituitary. From Day 4 to Day 1 (estrus) there was a reciprocal change in the levels of 3H-PRL in the pituitary vs. the media, with the former increasing 37-50% and the latter decreasing 25-32%. Pituitary RIA-PRL also increased 45-64% from Day 4 to Day 1 while media RIA-PRL did not change. These data are consistent with the following hypothesis: On the morning of proestrus (Day 4) in the hamster, PRL mRNA levels are elevated compared to those on Day 3, signaling an increase in PRL synthesis. This newly synthesized PRL is shunted into a "readily releasable" pool on the morning of Day 4 (contributing to the afternoon surge of serum PRL), and into a "preferentially stored" pool by the morning of Day 1 (for release in response to cervical stimulation and use as a luteotrophin to maintain early pregnancy should fertilization occur).     

The lack of a physiologic effect of the stress-induced decrease of the proestrous prolactin surge in the rat

Experiments were performed to determine whether the restraint stress-induced decrease in the proestrous prolactin (PRL) surge blocked luteolysis of the corpora lutea (CL), affected ovulation, or prevented the induction of pregnancy/pseudopregnancy in the next cycle. In all experiments rats were either stressed on proestrus and estrus, administered daily sc injections of 1 mg/day of 2-Br-alpha-ergocryptine (CB-154) for 4 days starting on diestrus II or not treated. In one experiment animals were sampled on the afternoon of proestrus to determine the effect of restraint stress on plasma PRL values and sacrificed on the morning of proestrus in the next cycle. Ovaries were removed, weighed, fixed and examined for number of CL. Restraint stress resulted in a significant increase in ovarian weight when compared to controls; CB-154 resulted in significant increases in ovarian weight when compared to stress and control animals. However, only CB-154-treated animals had a significant increase in the number of CL when compared to controls. In another experiment, animals were sacrificed on estrus of the next cycle and the oviducts examined for the number of ova. There were no differences among groups. In the final experiment, animals were placed with males of proven fertility on proestrus of the next cycle and examined for evidence of sperm in the vaginal lavage and/or vaginal plugs. CB-154 prevented the induction of pregnancy or pseudopregnancy due to a carry over effect of the drug on PRL surges. Restraint stress had no significant effect on the induction of pregnancy or pseudopregnancy. We conclude that there is no physiological significance to the stress-induced decrease of the proestrous PRL surge with respect to ovarian function or fertility.     

The influence of anesthetics on the estrogen-induced afternoon prolactin surge. Althesin does not block the surge

The effect of continuous anesthesia produced by ether, ketamine, urethane and althesin on the afternoon prolactin (PRL) surge was examined using aortic catheters in ovariectomized, estrogentreated rats. Ether and ketamine (ip or ia) completely abolished the diurnal PRL surge. Urethane (ip) from two sources and administered at two doses, which produced surgical anesthesia, initially suppressed the PRL surge, however, PRL levels rose slightly in the early evening. The effect of althesin (ip) on the afternoon PRL surge was variable; in some animals the surge was similar to that seen in unanesthetized controls, while in others the surge was reduced or absent. The variable effect of althesin (ip) seemed to be a result of fluctuations in the depth of anesthesia since ia infusion of althesin at a constant rate did not suppress the afternoon PRL surge.     

Inhibin activity and secretion of gonadotropin during the period of follicular maturation

To evaluate the relative contributions of the ovarian inhibin and estradiol-17 beta (E) on the regulation of FSH secretion, inhibin and E in ovarian venous plasma (OVP) and FSH and LH in peripheral plasma were simultaneously measured using superovulating rats with special reference to follicular maturation. By the transplantation of a pituitary gland from adult male rats under the kidney capsule between 1100 and 1200 hr on diestrus-1 in cyclic rats, superovulation was successfully induced on the morning of the next estrus without any additional treatment with human chorionic gonadotropin (hCG). The number of maturing follicles capable of ovulating in response to hCG significantly increased at 12 hours after the grafting as compared with sham-operated controls and further increases occurred until the afternoon of proestrus. In the superovulating rat, first and second surges of FSH were completely blocked and an LH surge was also partially suppressed during the periovulatory period when surges of FSH and LH were normally observed in controls. Contents of FSH as well as LH in the animal's own pituitary gland were suppressed significantly after the grafting as compared with controls. A marked increase in inhibin activity in OVP of rats with a pituitary transplant occurred concomitantly with an increase in the number of follicles capable of ovulating whereas E levels in OVP did not so. Inhibin activity in OVP at each point was much higher in the pituitary grafted rats than in controls but this was not true for E levels. These results suggest that ovarian inhibin derived from the maturing follicles rather than E may be a primary factor for regulation of FSH secretion, and high levels of endogenous inhibin can suppress synthesis of LH as well as FSH in the pituitary gland of the female rat.     

Inhibin secretion during the rat estrous cycle: relationships to FSH secretion and FSH beta subunit mRNA concentrations

Serum inhibin and FSH and FSH beta subunit mRNA levels were measured at 3h intervals throughout the 4 day estrous cycle in female rats and hourly between 1000 and 2400 h of proestrus. On proestrus, serum inhibin concentrations fell during the late morning-early afternoon, then increased transiently during the late afternoon gonadotropin surges. Inhibin levels decreased during the late evening of proestrus, coincident with the FSH surge-related rise in FSH beta mRNA levels. Serum inhibin remained relatively stable during estrus and early metestrus, but rose during the late evening of metestrus and remained elevated until early diestrus. FSH beta mRNA levels were elevated on late estrus and early metestrus and declined during the evening of metestrus as serum inhibin levels increased. These data show that concentrations of serum inhibin change during the estrous cycle and that a general inverse relationship exists between serum inhibin and FSH levels and FSH beta mRNA concentrations in the pituitary. This suggests that inhibin may inhibit FSH beta gene expression and FSH secretion during the 4 day cycle in female rats.     

Effects of intraventricular injection of delta receptor antagonist ICI 154, 129 on the secretion of luteinizing hormone and prolactin in the proestrous rat.

In order to ascertain the role of delta receptors in the control of gonadotropin secretion, a preferential delta receptor antagonist ICI 143,129 was microinjected into the third ventricle through chronically implanted cannulae and the effects on the serum concentration of luteinizing hormone (LH) and prolactin (PRL) were determined in female rats in proestrus. When the injection was given at 1030 h, ICI 154,129 (50 micrograms) exerted no significant effects on either LH or PRL. However, in the rat given a microinjection of ICI 154,129 at 1300 h, an afternoon rise in LH occurred in advance and was of greater magnitude, with the peak time more than 1 h earlier and the peak amplitude approximately 100% greater than that in the control rat, respectively. The injection also suppressed the PRL rise during the plateau phase. The results indicate that delta receptors are involved in the mediation of the inhibitory influence of endogenous opioids on the surge of LH in proestrus, and that delta receptors mediate the facilitatory influence of opioids on the PRL surge during the plateau phase.     

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.     

Suppression of the prolactin surges in the pseudopregnant rat by urethane anesthesia

In pseudopregnancy of the rat prolactin (PRL) is released in the form of twice daily surges (nocturnal and diurnal surges). An attempt was made to examine the effects of urethane anesthesia on PRL surges during pseudopregnancy of the rat. In a preliminary study, using the continuous blood sampling method, the nocturnal PRL surge was completely blocked when urethane (1.0g/kg BW) was administered at 0:00 hr. Urethane (1.0g/kg BW) was injected at 0:00 or 12:00 hr, and serum and pituitary PRL concentrations were measured at 6:00 or 18:00 hr, respectively, to study the effects of urethane on nocturnal or diurnal PRL surges. There were no serum PRL surges during either the nocturnal or diurnal periods following urethane injection. The experiment examining pituitary PRL concentration at 6:00 or 18:00 hr confirmed that urethane (1.0g/kg) anesthesia suppressed the release of PRL surge from the pituitary.     

Heme oxygenase in the rat anterior pituitary: immunohistochemical localization and possible role in gonadotropin and prolactin secretion

The objectives of this study were to determine if heme oxygenase (HO), which catalyzes the degradation of heme and the formation of carbon monoxide (CO), is localized in the rat anterior pituitary and, if so, to determine if hemin (a substrate for HO) or chromium mesoporphyrin (CrMP) (an inhibitor of HO), alter pituitary gonadotropin and prolactin secretion. For localization of HO, sections of anterior pituitaries obtained from mature Holtzman Sprague-Dawley rats in different stages of the estrous cycle were immunostained for two of the HO isoforms, HO-1 and HO-2. The immunostaining for the inducible HO isoform (HO-1) was limited to discrete populations of pituitary cells, whereas the constitutive isoform (HO-2) had a more widespread distribution. The afternoon surge of leutinizing hormone (LH) in the plasma of ovariectomized, estradiol-treated rats was advanced by 2 hr after 7 days of treatment with CrMP (4 micro M/kg), and this effect was reversed when hemin (30 micro M/kg) was co-administered with CrMP. The afternoon follicle-stimulating hormone (FSH) surge was not affected by either treatment. In contrast, the afternoon prolactin (PRL) surge was completely blocked or delayed by CrMP treatment, and this effect was not reversed by hemin. In vitro perifusion of pituitary explants with CrMP also significantly reduced PRL release compared with secretion from untreated explants. In vitro gonadotropin-releasing hormone (GnRH)-stimulated FSH secretion was significantly increased from pituitary explants of ovariectomized, estradiol-treated rats treated in vivo with hemin but was unaffected by CrMP treatment, whereas GnRH-stimulated LH release was not affected by hemin but was increased by CrMP treatment. In conclusion, this study demonstrates that HO exists in the rat anterior pituitary gland, and that a substrate and an inhibitor of this enzyme alter the secretion of gonadotropins and PRL.     

Lactation reduces prolactin levels in reproductively experienced female rats

Long-term alterations in prolactin (PRL) secretion following reproductive experience have been demonstrated in both women and female rats. In the rat, these changes include decreased PRL secretion in response to a dopamine antagonist challenge following ovariectomy, decreased post-coital diurnal and nocturnal prolactin surges in multigravid versus primigravid females, as well as decreased suckling-induced prolactin release in multiparous versus primiparous females. To date, there have been no studies examining PRL secretion following reproductive experience in cycling female rats. Studies in women, however, have demonstrated a reduction in basal PRL secretion during the menstrual cycle. The purpose of the present work was to determine whether similar changes occur in the rat during the estrous cycle and to what extent lactation is involved in these effects. In addition to examining PRL, potential parity-induced changes in estradiol secretion were also studied. The findings revealed a significant decrease in PRL levels during the afternoon of proestrus, which was only observed in primiparous females that had lactated. Significant differences in estradiol secretion were not detected following reproductive experience. Thus, a reduction in the PRL surge on the afternoon of proestrus is a consequence of reproductive experience that requires both pregnancy and lactation.     

Pituitary adenylate cyclase activating polypeptide messenger RNA in the paraventricular nucleus and anterior pituitary during the rat estrous cycle

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.     

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.     

gamma-Aminobutyric acid activity in the olfactory bulb of the rat during the sexual cycle and response to olfactory stimuli.

Glutamic acid decarboxylase activity in the main and accessory olfactory bulbs throughout the sexual cycle of the rat was studied. The effect of male pheromonal secretion on enzyme activity during proestrus and estrus day was also tested. The enzyme activity showed circadian rhythm during the estrous cycle. This rhythm was disrupted during diestrus-2 afternoon in the main bulb and came back during proestrus afternoon. A different pattern of enzyme activity was present in the accessory bulb, since the circadian rhythm was altered during proestrus morning, returning during estrus afternoon. Male odor exposition did not change enzyme profile activity during proestrus day and during estrus morning in the main bulb. In contrast, in the accessory bulb the olfactory stimuli induced opposite changes to that found in rats from the vivarium during proestrus. Comparison of enzyme activity in olfactory stimuli-deprived rats with that of pheromone-stimulated rats during proestrus showed that male odor exposure specifically affects accessory bulb enzyme activity. It is concluded that the changes of the olfactory bulb GABAergic system during proestrus and estrus day, or that evoked by odor stimuli, demonstrate the discriminative response of this system between the accessory olfactory bulb and the main olfactory bulb.     

Differences in the stress response of prolactin in young and aged female rats

The drawing of blood by orbital sinus puncture (OSP) under ether anesthesia is known to produce a marked increase in serum prolactin (PRL levels in young cycling female rats. The effect of this stressful procedure on PRL release was compared in young and aged female rats. Nonstressed PRL levels were obtained from blood drawn by decapitation. Whereas OSP with a one-minute ether exposure induced a marked increase in PRL levels in young rats on all days of the estrus cycle, older cycling female rats on the day of diestrus -1 and aged rats exhibiting prolonged diestrus (PD) showed virtually no increase above nonstressed levels. However, increasing the ether exposure time to five minutes did produce a rise in PRL levels. Old cycling female rats on the day of estrus and aged rats exhibiting constant estrus (CE) did show a PRL increase comparable to that seen in young animals. Ovariectomy (OVX) completely abolished the stress response seen in aged CE rats. The response, though markedly decreased, was still present in young ovariectomized rats. These experiments show that the stress-induced rise in PRL promoted by OSP under either anesthesia is markedly diminished in aged rats exhibiting a diestrus state. The attenuated response seen in these rats is believed due to factors characteristic of the diestrous state of aging.     

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.     

A change in basal FSH release accompanies the onset of the second or selective phase of increased serum FSH in the cyclic rat

Experiments were undertaken to investigate if changes occur at the level of the anterior pituitary gland to result in selective follicle-stimulating hormone (FSH) release during late proestrus in the cyclic rat. At 1200 h proestrus, prior to the preovulatory luteinizing hormone (LH) surge in serum and the accompanying first phase of FSH release, serum LH and FSH concentrations were low. At 2400 h proestrus, after the LH surge and shortly after the onset of the second or selective phase of FSH release, serum LH was low, serum FSH was elevated about 4-fold, pituitary LH concentration was decreased about one-half and pituitary FSH concentration was not significantly decreased. During a two hour $\text{in}$$\text{vitro}$ incubation, pituitaries collected at 2400 h released nearly two-thirds less LH and 2.5 times more FSH than did pituitaries collected at 1200 h. Addition of luteinizing hormone releasing hormone (LHRH) to the incubations caused increased pituitary LH and FSH release. However, the LH and FSH increments due to LHRH in the 2400 h pituitaries were not different from those in the 1200 h pituitaries. The results indicate that a change occurs in the rat anterior pituitary gland during the period of the LH surge and first phase of FSH release which results in a selective increase in the basal FSH secretory rate. It is suggested that this change is primarily responsible for the selective increase in serum FSH which occurs during the second phase of FSH release.