Nerves immunoreactive for the peptides substance P, neurokinin A, calcitonin gene-related peptide or cholecystokinin-octapeptide innervate the uterine cervix in the rat. Nerve terminals are associated with the myometrial and vascular smooth muscle and are distributed throughout the endocervix. These nerves degenerate following neonatal capsaicin treatment indicating that they are small-diameter, unmyelinated, C-type primary afferent nerves. Adult female rats, treated with capsaicin as neonates, exhibit decreased fertility following mating and diminished sensitivity to the induction of pseudopregnancy following copulomimetic electrical stimulation of the cervix. The results also demonstrate that hypothalamo-adenohypophyseal-ovarian interactions, corpus luteum progesterone secretion and uterine sensitivity to progesterone are normal in capsaicin-treated rats. Taken together, these data suggest that the reproductive dysfunction observed in capsaicin-treated rats is due to destruction of the afferent limb of the neuroendocrine copulatory response that facilitates the luteal progesterone secretion necessary to support pregnancy or pseudopregnancy. Thus, it is concluded that the afferent limb of this neuroendocrine response in the rat consists primarily of unmyelinated, peptide-containing, C-type primary afferent nerves.

Maintenance of the luteal phase and progesterone (P4 ) secretion adequate to support nidation and pregnancy or pseudopregnancy in the proestrous rat requires intact innervation of the uterine cervix [1]. Stimulation of the cervix during copulation, or by experimental means, is conveyed via neural pathways to the hypothalamus [2,3] altering the gonadotropin secretion pattern [4,2] and facilitating the secretion of prolactin (Prl), a luteotropin in the rat, by the hypothalamo-hypophyseal axis [5,6,14]. This results in cessation of estrous cyclicity and stimulation of P4 secretion by the corpus luteum [7,4,5], thus completing the neuroendocrine copulation reflex (response) [8]. Our previous results have demonstrated an extensive innervation of the uterine cervix by capsaicin- sensitive primary afferent nerves containing markers for substance P [9], and further, that groups of capsaicintreated (CAP) neonatal rats subsequently have a markedly reduced incidence of pregnancy following confirmed matings [10]. This suggested that the neural limb of the neuroendocrine copulation reflex consisted of primary afferent “c” type nerves.

The purpose of this study was to determine the effects of aging on serotonin (5-HT) pharmacodynamics in rostral hypothalamic tissue of female rats. Monoamine oxidase (MAO) activity was greater in old (19 months old) than in young (3 months old) animals. This difference resulted from a higher Vmax for hypothalamic MAO from old rats, whereas Km's were comparable in both age groups. These enzymatic changes were accompanied by altered uptake and release of [3H]5-HT. Although [3H]5-HT uptake was not different at equilibrium between groups, the equilibrium state was achieved more slowly by hypothalamic tissue from old rats. Basal and potassium-stimulated efflux of [3H]5-HT was significantly greater in old rats compared with young ones and these differences remained during multiple depolarizations. The findings of this study suggest that synaptic levels of hypothalamic 5-HT increase with age. Since pharmacological simulation of this condition in young rats produces physiologic dysfunction, the spontaneous changes that occur in hypothalamic 5-HT pharmacodynamics during aging have the potential to promote senescence.

The purpose of this study was to examine the temporal effects of norepinephrine (NE) stimulation on pineal serotonin (5HT) in vitro. Rat pineal glands were individually incubated in nutrient media for 6 h. After a 2-h preincubation, the glands were exposed to tryptophan (TRYP) or TRYP + norepinephrine (10(-4)M each) for 4 additional h. Media and pineals were then analyzed for 5HT and 5HIAA content. Initial exposure to TRYP increased 5HT levels in the media. Norepinephrine increased media 5HT concentrations further and also caused 5HIAA levels to rise significantly. These findings suggest that 5HT secretion and/or oxidation is an early response to stimulation of the pineal gland by NE. Following prolonged NE exposure, media 5HT declined to control values while 5HIAA remained high. Similarly, intrapineal levels of 5HT fell after prolonged stimulation with NE. Thus, pineal responses to initial and continued stimulation with NE are differentiable as early and late effects upon 5HT. Early effects may include secretion and/or oxidation of 5HT followed later by utilization of 5HT in N-acetylation pathways such as that leading to melatonin synthesis. The finding that 5HT is secreted in response to NE suggests that the indoleamine may be a hormone of the pineal gland. Based upon its known influence on physiologic rhythms, daily secretion of 5HT from the pineal may be an important part of the gland's time-keeping function.

Follicle-stimulating hormone release on the morning of oestrus was examined by using two different techniques which eliminate LH-releasing hormone (LHRH) stimulation of the pituitary gland. Cyclic female rats were given a potent LHRH antagonist (ALHRH) or were subject to electrolytic lesions of the medial basal hypothalamus (MBH) before or after the pro-oestrous phase of FSH release. Administration of ALHRH at 14.00, 15.30 and 17.00 h or lesioning of the MBH between 11.30 and 13.00 h on pro-oestrus entirely blocked the preovulatory LH surge and both phases of FSH release. Ovulation was abolished in all of these animals. However, when ALHRH was given at 20.30, 22.00 and 23.30 h or lesions of the MBH made between 20.00 and 21.30 h on pro-oestrus after the pro-oestrous FSH and LH surges had occurred, the oestrous phase of FSH release was indistinguishable from that of saline-treated control rats. Ovulation occurred in all of these animals, and the mean number of ova shed was eight/rat. The conclusions are that (1) the pro-oestrous phase of FSH release is dependent upon the hypothalamic hormonal stimulation by LHRH and (2) the oestrous phase of FSH release is entirely independent of direct LHRH stimulation, or any hypothalamic stimulus.

The Ca2+-dependent regulation of the adenylate cyclase activity associated with microsomes isolated from bovine aortic smooth muscle has been studied. Calmodulin content of microsomal membranes employed in these studies was 80 +/- 14 ng/mg as determined by specific radioimmunoassay. In the absence of exogenous calmodulin, Ca2+ concentrations greater than 0.8 microM inhibited adenylate cyclase activity with one-half-maximal inhibition occurring at 2.5 microM Ca2+. In the presence of 5 or 9 microM bovine testis calmodulin, Ca2+ stimulated smooth muscle adenylate cyclase activity with one-half-maximal stimulation occurring at 0.2 microM for both 5 and 9 microM calmodulin. Calmodulin stimulation was observed between 0.1 and 0.8 microM Ca2+. Despite the presence of calmodulin, Ca2+ concentrations greater than 0.8 microM were inhibitory to smooth muscle adenylate cyclase activity. However, calmodulin reduced the sensitivity of the enzyme to inhibition by Ca2+. Trifluoperazine (100 microM) reversed both the calmodulin-dependent stimulation of cyclase activity and the calmodulin-induced decrease in sensitivity to the inhibitory actions of Ca2+. Trifluoperazine alone shifted the curve describing Ca2+ inhibition of cyclase activity to the left. The value of Ca2+ for one-half-maximal inhibition decreased from 2.9 to 1.2 microM. The trifluoperazine-induced shift was reversed by exogenous calmodulin. These data suggest: 1) Ca2+, at physiological concentrations, can stimulate as well as inhibit smooth muscle adenylate cyclase activity; 2) the stimulation of adenylate cyclase activity is mediated by calmodulin; 3) the Ca2+-calmodulin complex reduces the sensitivity of smooth muscle adenylate cyclase to the inhibitory actions of Ca2+; and 4) the level of calmodulin associated with smooth muscle adenylate cyclase may modulate the response (both stimulatory and inhibitory) of the enzyme to Ca2+.

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.

We investigated the role of the adrenal glands in the maintenance of serum testosterone concentrations during the 4-day estrous cycle in the rat. Animals were untreated, adrenalectomized (ADX), or sham-ADX. The operations did not alter with the length of the estrous cycle. Three to five estrous cycles after surgery, we decapitated rats for the collection of trunk blood and measured the FSH, LH, and testosterone in the serum by radioimmunoassays. Serum FSH and LH concentrations were similar in the three groups of rats during the estrous cycle except at 1000 hr of estrus when serum FSH levels were lower in the untreated rats than in the other two groups. In untreated rats, serum testosterone concentrations were lowest during estrus and highest after the onset of the preovulatory surges of FSH and LH in serum during the afternoon of proestrus. No significant differences in serum testosterone concentrations were observed between groups except that levels in the ADX rats were lower than levels in the untreated rats at 1000 and 1800 hr of diestrous day 1, and levels in the ADX rats were lower than levels in the sham-ADX rats at 0600 hr of estrus. Serum testosterone levels were undetectable in rats ovariectomized and adrenalectomized for 2 weeks. The results indicate that the adrenal glands contribute little to the maintenance of serum testosterone levels during the rat 4-day estrous cycle and that the rise in serum testosterone concentration from estrus to the afternoon of proestrus is likely the result of ovarian secretions.

We investigated 1) if the administration of porcine follicular fluid (p FF) on proestrus could block the entire periovulatory gonadotropin secretion in the 4-day cyclic rat, 2) if the maintenance of elevated serum 17β-estradiol (E2) concentrations during proestrus and estrus has any influence on periovulatory FSH release, either alone or in combination with pFF, and 3) if the administration of pFF to proestrous rats interferes with LHRH-induced gonadotropin release. We collected blood through a jugular cannula at 4-h intervals from 1400 h on proestrus to 1000 h on estrus and measured the plasma for LH and FSH by RIA. Experimental protocols included injecting pFF at low (0.1 ml) or high (0.5 ml) doses, early (at 1000, 1300, and 1530 h on P), or late (at 1800, 2100, and 2400 h on P), or at all six times, or implanting sc an E2 Silastic capsule at 1300 h on P. We injected the pFF through the cannula, except for the 1000 h injection which was ip. Either dose ofpFF (early) completely blocked the proestrous increase in plasma FSH observed in normal and pig serum-injected controls, but plasma FSH was only partially suppressed during the morning of estrus during the time a secondary elevation in plasma FSH was observed in controls. Plasma FSH did not rise during estrus when the high dose of pFF was administered late or at all times. If the low dose of pFF was not injected at 1000 h despite its injection at the other five times, a small elevation in plasma FSH was observed throughout proestrus and estrus, and a proestrous LH surge occurred. Interestingly, the LH surge was blocked in most rats which received either dose of pFF if the injection at 1000 h on proestrus was included. E2 had no effect on the periovulatory gonadotropin surges, and it did not prevent the small rise in plasma FSH during estrus in rats given the low dose of pFF early. Additional rats were ovariectomized and administered phenobarbital on the early afternoon of proestrus. In these rats, plasma LH remained low, and plasma FSH rose by 2200 h on P to levels which were similar to those observed during the secondary rise in plasma FSH during estrus in controls. The high dose of pFF administered six times completely blocked, and the low dose of pFF administered six times suppressed, the elevations in plasma FSH in the phenobarbital-treated ovariectomized rats, and E2 did not suppress plasma FSH further in phenobarbital-treated ovarectomized rats given the low doses of pFF. Constant rate iv infusion of LHRH at 50 ng/h from 1400–1700 h on proestrus did not elevate plasma FSH in intact rats given the high dose of pFF early, but did cause a partial rise in plasma LH during proestrus compared to values observed in phenobarbital-blocked, LHRH-infused controls. The results suggest that 1) decreasing inhibin titers may be involved in the selective release of pituitary FSH during estrus, 2) decreasing E2 titers play no major role in the selective release of pituitary FSH during estrus, either alone or in association with decreasing inhibin titers, 3) pFF can block the preovulatory LH surge when administered on the morning of proestrus, and 4) the action of pFF to completely suppress proestrous but not necessarily estrous gonadotropin secretion is probably exerted at the anterior pituitary gland to suppress LHRH-induced gonadotropin release.

The effects of exogenous rat LH or FSH on the release of endogenous FSH in the cyclic rat have been investigated. Rats were administered phenobarbitone to block the spontaneous increases in gonadotrophins in plasma during pro-oestrus and oestrus and then cannulated through the jugular vein or cannulated and hypophysectomized during the late morning or early afternoon of pro-oestrus. Comparison of patterns of plasma FSH in hypophysectomized and intact rats after i.v. injection of 0·5 μg FSH at 17.00 h suggested that exogenous FSH stimulated the release of endogenous FSH in less than 5 h. Intravenous LH (2 μg at 16.00 and at 18.00 h) raised the level of FSH in plasma between 2 and 6 h after the first injection of LH. Both gonadotrophins stimulated FSH release by the pituitary gland during the morning of oestrus. Comparison of patterns of plasma FSH in hypophysectomized and intact rats after i.v. injection of 0·25 or 0·05 μg FSH at 14.00 h suggested that the latency between FSH injection and stimulation of some FSH release by the pituitary gland is as short as 2 h. Intravenous LH (3,4 or 9 μg) at 14.00 h did not increase the level of FSH in plasma within 2 h and was only minimally effective in raising the level within 4 h. Intravenous LH (2 μg at 16.00 and at 18.00 h) on the afternoon of dioestrus day 2 was nearly as effective in increasing the levels of FSH in plasma as it was when administered to pro-oestrous rats. This procedure did not raise the plasma levels of FSH in rats used on dioestrus day 1. The results suggest that in the phenobarbitone-blocked, pro-oestrous rat (1) a small increase (less than that observed spontaneously) in plasma rat FSH during pro-oestrus is effective in stimulating FSH release by the pituitary gland, (2) an increase in plasma rat FSH can exert positive feedback on its own secretion within 2 h and (3) a large increase in plasma rat LH is not very effective in increasing the plasma level of FSH over a period of 4 h. The results also suggest that the spontaneous increase in plasma levels of FSH and, to a lesser extent, of LH is involved in causing the selective phase of FSH release which occurs during late pro-oestrus and the morning of oestrus, and that LH and FSH act differently, but not necessarily by way of a different mechanism, to stimulate release of FSH by the pituitary gland.

Experiments were conducted in vivo to investigate further if the control of the pulsatile plasma LH phenomenon in ovariectomized (OVX) rats is located in the brain or in the adenohypophysis. Luteinizing hormone releasing hormone (LHRH) was infused at a constant rate (2--100 ng/h) through an indwelling venous cannula in unanesthetized, unrestrained OVX rats. Blood samples were collected at 5-min intervals through a second venous cannula prior to and during LHRH infusion for subsequent radioimmunoassay of plasma LH. LHRH infusion at 12.5, 50 and 100 ng/h did not interfere with the magnitude or the periodicity of LH pulses in plasma but the range within which plasma LH fluctuated was elevated. Phenobarbital (75 mg/kg BW; i.p.) blocked the pulsatile plasma LH and maintained the plasma LH nearly constant at reduced levels. Pulse i.v. injections of LHRH but not constant rate i.v. infusions restored pulsatile LH patterns in phenobarbital-treated OVX rats. The results are consistent with the view that pulsatile LHRH release is responsible for the pulsatile nature of plasma LH in OVX rats. The results do not support the concepts of a short-loop feedback of LH or an ultra-short-loop feedback of LHRH on LH secretion at least on an acute basis.

We investigated the importance of anterior afferents to the medial basal hypothalamus (MBH) on the increases in plasma FSH during the periovulatory period in the 4-day cyclic rat. We served the anterior connections to the MBH either at 1200 h on proestrus (before the time of onset of the normal spontaneous LH surge in plasma and the associated first phase of FSH release) or near the end of the LH surge and first phase of FSH release at 2000 h on proestrus (before the onset of the second or selective phase of FSH release). Analyses of FSH and LH in blood collected through indwelling atrial catheters or from the trunk after decapitation showed that anterior deafferentation of the MBH at 1200 h on proestrus blocked the proestrous LH surge, the elevations in plasma FSH during proestrus and estrus, and ovulation. In contrast, when brain surgery was delayed until 2000 h on proestrus, the second phase of FSH release and ovulation occurred. In rats with retrochiasmatic transections made at 1200 h, a constant rate iv infusion of LHRH from 1500-1800 h on proestrus restored the LH surge, both phases of increased plasma FSH, and ovulation. The results suggest that 1) the prevolutory LH surge and the first phase of FSH release are dependent on rostral afferents to the MBH which result in hypothalamic LHRH release and 2) the role of rostral afferents to the MBH in the second phase of FSH release is solely to result in hypothalamic LHRH release during proestrus.

Although a gonadal regulation of hypophysial gonadotrophin secretion is established, the nature of this feedback is not fully known. In certain pathological conditions (1) or after selective destruction of the germinal epithelium (2), FSH levels are elevated whereas LH levels are not. These observations suggest that the secretion of FSH is under the control of a factor other than that controlling LH secretion (2). Recent evidence in the female (3) also supports the concept of the dissociation of secretion of FSH and LH.

The effect of bovine testicular extracts (bTE) on plasma FSH levels in both castrate and X-irradiated male rats has been examined. Biological activity was demonstrated in 34-day-old, orchidectomized rats with doses ranging from 50 to 250 mg. Testicular weights of X-irradiated rats 49 days after X irradiation were decreased by approximately 67%, whereas the ventral prostate weights were unaffected. Plasma FSH levels (383 ng/ml) increased significantly as a result of X irradiation (873 ng/ml). Plasma LH (51 ng/ml) also rose as a result of X irradiation (93 ng/ml), suggesting that there was some interstitial cell damage or some Sertoli cell damage. Two hundred and fifty milligrams of bTE administered in 4% gelatin given once daily for 3 days suppressed the elevated plasma FSH, but was without effect on plasma LH. Castration and a further 3 days of treatment resulted in a significant postcas-tration suppression in plasma FSH (control = 1942 ng/ml; bTE = 1283 ng/ml), and resulted in a suppression of LH as well. This LH response (control = 453 ng/ml; bTE = 285 ng/ml) is probably due to the large dose of bTE employed. A bovine kidney extract (bKE) was totally ineffective in suppressing FSH or LH when given at a dose of 250 mg/ rat/day in 4% gelatin. These data lend further support to the existence of inhibin, contained in bTE, and to its role as a regulator of FSH in the male. The probable site of origin of inhibin is the Sertoli cell.

The interaction of exogenously administered gonadotropin- releasing hormone (GnRH) and inhibin-containing gonadal preparations on the pituitary in intact male rats has been studied. The inhibin preparations were crude bovine testicular extracts (bTE) or porcine follicular fluid (pFF). Both preparations were extracted with ether and/or charcoal to remove steroids. The biological activity of both the bTE and the pFF was established in the 34-day-old acutely castrated male rat. Two experimental approaches were used. In series A, the inhibin preparation was administered first, and 6 h later, a single iv injection of GnRH (125 or 500 ng) was given. In series B, GnRH was infused continuously for 8 h, but after 2 h, the inhibin preparation was administered (sc). In series A, both bTE and pFF completely suppressed the 125-ng GnRH-induced rise in plasma FSH; however, neither was effective when 500 ng GnRH were administered. Plasma LH levels were unaffected by bTE or pFF. In series B, neither the bTE nor the pFF was effective in preventing the 22 ng/min GnRH-induced rise of either FSH or LH. However, when GnRH was infused at the rate of 11 ng/min, both the bTE and the pFF suppressed plasma FSH levels. LH levels were unaffected. Thus, both the bTE and pFF exert similar sex-independent inhibitory effects on the pituitary to control FSH secretion selectively. The data are most simply interpreted as evidence that GnRH and inhibin reversibly interact at the level of the pituitary.