No abstract

Immunocytochemical identification of nerve terminals reacting specifically with anti-LHRH antisera has recently been reported in various species, like the guinea pig (1) and the duck (2), employing a double antibody labeling technique, with the second antibody bound to fluorescein isothiocyanate (3). However, visualization of such terminals has not yet been described in the rat; on the other hand, a double labeling with a second, peroxidase bound, antibody does not seem to have been successfully applied to that material as yet. In the present paper, we report identification of fibers containing an antigen reacting with anti-LHRH in the rat median eminence, using both fluorescein and peroxidase labels.Methods. The antibodies were obtained from rabbits injected with synthetic LHRH (Hoffman-LaRoche, Basle) coupled to guinea pig gamma globulin (4), according to the technique described by Avramkas ( 5 ) . In a radioimmunoassay system (6), the titer of the antiserum is approximately 1/100,000; it can detect as little as 1-5 pg of LHRH. Its specificity was tested against oxytocin, lysin-vasopressin and TRH, as well as against LH, FSH and Prolactin; it does not cross react with these hormones at doses up to 500 pg. With this antibody, endogenous levels of LHRH were determined in the rat hypothalamus; values ranged between 1 and 3 ng/hypothalamus of normal male rats. Injected to cyclic female rats (200 pl ip at 1200 noon of the day of proestrus), this antiserum inhibited the preovulatory surge of LH and FSH and blocked ovulation (7).Male rats were perfused by aortal cannulation with various fixatives (200 ml total volume) under light chloral hydrate anesthesia. The fixa-tives used were either Bouin-Hollande without acetic acid (4% formalin, 4% picric acid, 2.5% copper acetate and 10% of a saturated solution of Hg sublimate), 4% paraformaldehyde in saline solution, or picroformol. After dissection, the hypothalamus was postfixed for 4 hr at 4". The tissues were dehydrated in graded butanol series followed by a mixture of paraffin and butanol and embedded in paraffin. Five micrometers serial sections were rehydrated, washed in water overnight, immersed in 0.01 A4 saline phosphate buffer solution (PBS) for 30 min and incubated with the antiserum against LHRH at various dilutions for 90 min. Adjacent sections were incubated for the same time with (a) normal rabbit serum, (b) serum of a rabbit immunized against guinea pig gamma globulins, (c) anti-LHRH serum absorbed previously with an excess of LHRH by overnight incubation at 4" of 100 p1 of pure serum with 20 pl of a 3. lob4 M solution of synthetic LHRH, (d) anti-LH serum (8), (e) anti-LHRH serum incubated overnight with a cytoplasmic fraction from cerebral cortex and subsequently centrifuged ( 17,OOOg for 30 min)in an attempt to eliminate background staining. Some sections were treated for 5 min with 0.5 M HCl, or with 1% trypsin in PBS, prior to incubation with the antisera. After thorough washing in PBS, a second incubation with either peroxidase-or fluorescein-labeled sh...

We have previously reported that GnRH stimulates the synthesis of both the alpha- and beta-polypeptide chains of LH. In the present study, in order to investigate the mechanisms involved in the GnRH regulation of LH subunit synthesis, we have explored the effects of cAMP and a phorbol ester [12-O-tetradecanoyl phorbol 13-acetate (TPA)] using anterior pituitary cells in primary culture incubated in the presence of [35S]methionine. The radioactivity incorporated into alpha and LH beta immunologically related polypeptides was measured after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the labeled material immunoextracted from cells and media with specific antisera. The cAMP analog 8-Br-cAMP (at concentrations 0.25-2 mM), the cholera toxin (6-60 nM), and forskolin (10-100 microM) induced, like GnRH, an increase in the [35S]methionine incorporation into alpha- and LH beta-subunits. On the other hand, TPA (50-100 nM) also enhanced the synthesis of LH subunits. After a 5-h incubation in the presence of GnRH, 8-Br-cAMP, and TPA in different combinations, no cumulative effect was observed. These results demonstrate that intracellular cAMP and TPA are potent activators of both alpha- and LH beta-polypeptide chain synthesis, suggesting that cAMP as well as diacylglycerols may act as intracellular mediators of the GnRH effect on LH subunit synthesis.

A single ip injection (0.2 ml) of a rabbit antiserum to LHRH was given at noon on proestrus (day 0) to 4-day cycling rats. A blockade of the preovulatory surge of LH and FSH was observed, as well as inhibition of ovulation; the prolactin (PRL) surge was unaffected. Four days later, palsma LH and FSH were still low and hypoprolactinemia appeared, while the estrous cycle was unaffected. Then, after a transient diestrous period, vaginal smears indicated a pattern of persistent estrus for at least 240 days starting from day 8 after treatment. Hypothalamic LHRH remained low throughout this period, with the exception of a transient rise around day 36; hypothalamic serotonin and dopamine were unchanged. A peak of FSH in serum appeared on day 28 and an elevation of serum LH on day 36; this latter rise was concomitant with the increase of hypothalmic LHRH and with ovarian luteinization. Except for this short period, the ovaries showed a picture of persistent estrus, with large, cystic follicles. During the whole experimental period, basal serum FSH levels were higher than those of LH. Pituitary FSH and LH contents remained in the range usually found during the normal estrous cycle. Starting from the 8th day after treatment, a marked hyperprolactinemia appeared. Serum estradiol and progesterone were assayed on days 10 and 40 after injection; no significant increase in either steroid was observed on day 10, but on day 40 the levels of estradiol increased to values similar to those of progestrus, while progesterone showed only a small, but significant, increase. Thus, a single injection of LHRH antiserum (AS) in a normal cycling rat provoked a long-term alteration in hypothalamic function. The results so far obtained suggest that the long-lasting hyperprolactinemia induced by this treatment, might be due to an abnormal ovarian secretion.