The distribution of gonadotropin-releasing hormone (GnRH) was studied in the brain of adult female rats with three immunocytochemical techniques using antisera to unconjugated synthetic GnRH and to GnRH conjugated with limpet hemocyanin. GnRH was found in nervous tissue surrounding blood vessels of the organum vasculosum of the lamina terminalis. In the median eminence it occurred in nervous tissue associated primarily with the tuberoinfundibular sulci throughout their extent. Cephalic to the pars tuberalis GnRH often spread across the median eminence from sulcus to sulcus. Caudally, with widening of the median eminence, GnRH occurred dorsal to the tuberoinfundibular sulci, and especially in the external lamina medial to the sulci. A broad median zone of the median eminence was rather free of GnRH. GnRH was most concentrated in the region of continuity between the dorsolateral walls of the infundibulum and floor of the third ventricle where the tuberoinfundibular sulci are deep. Caudal to the infundibulum GnRH was disposed in a flat zone through the cephalic portion of the floor of the mammillary recess. In the median eminence GnRH appeared to be located in axons that terminated there. The amount of demonstrable GnRH varied significantly from rat to rat. The distributions of GnRH as revealed by use of antisera to unconjugated and conjugated GnRH were essentially the same. The apparent order of sensitivity of the immunocytochemical methods was: the peroxidase-antiperoxidase (PAP) (Sternberger et al.) procedure greater than the immunoglobulin-enzyme bridge (Mason et al.) procedure smaller than the conjugated antibody (Nakane and Pierce) procedure.
s y n t h e t i c l u t e i n i z i n g hormone-releasing hormone (LRH), LRH was l o c a l i z e d i n t h e p e r i p h e r a l r e g i o n o f t h e median eminence i n t h e mouse and r a t , and more g e n e r a l l y i n t h e median eminence o f t h e guinea p i g . t u d i e s i n d i c a t e s t h a t l u t e i n i z i n g hormone-releasing hormone (LRH) i s elabor a t e d i n t h e hypothalamus, t r a n s p o r t e d t o t h e median eminence i n axons, andt h e r e secreted i n t o t h e primary plexus o f t h e hypothalamo-hypophyseal vascular system. G r e a t l y needed a t t h e moment i s a c l e a r demonstration o f what nerve c e l l s e l a b o r a t e LRH and where t h i s hormone i s released i n t o t h e blood stream. Recently Leonardell i e t a1 . ( ' 7 3 ) used immunofl uorescence t o d e s c r i b e t h e l o c a li z a t i o n o f LRH i n t h e median eminence o f t h e mouse and guinea p i g . Subsequently,they ( B a r r y e t a l . , ' 7 3 ) r e p o r t e d t h e l o c a l i z a t i o n o f LRH i n nerve c e l l bodies s c a t t e r e d through t h e a n t e r i o r hypothalamus from t h e p r e o p t i c and septa1 areas t o t h e caudal p o r t i o n of t h e t u b e r cinereum.The o b j e c t i v e o f our p r e l i m i n a r y paper i s t o r e p o r t t h e l o c a l i z a t i o n o f LRH i n hypothalami o f r a t , mouse and guinea p i g u t i l i z i n g immunohistochemistry.MATERIALS AND METHODS Four female r a t s o f t h e Sprague-Dawley s t r a i n , one female and one male o f s t r a i n 129 SvSl mouse, and one female guinea p i g were 129
When GnRH is radioiodinated by the chloramine-T method, two immunoreactive labeled species are formed at pH 6.5 with a chloramine-T: GnRH molar ratio of 11:1, whereas four bands (I, IIa, IIb, and III) are separated by polyacrylamide gel electrophoresis when the hormone is iodinated at pH 7.5 in a system containing a 97:1 molar ratio of chloramine-T:GnRH. Because they were more stable and were more immunoreactive than the other products, band I and band IIa from the latter system were used separately as tracers with Niswender antiserum R-42 in radioimmunoassays for GnRH. The standard curves of each tracer are distinct: when analyzed after log-logit transformation, the band I curve had a mean slope of -3.31 +/- 0.2 (SE) and a 50% B/Bt level of 9 +/- 0.8 pg (n=8) of synthetic GnRH, whereas the band IIa standard curve had a slope of -2.30 +/- 0.6 and a 50% B/Bt value of 20 +/- 0.9 pg (n=11). The sensitivity of both assays is approximately 2.0 pg. Gn RH concentrations in plasma and serum samples assayed with band I were consistently greater than those assayed with band IIa. Normal adult male plasmas assayed with band I measured 21 +/- 0.9 pg/ml, whereas band IIa values were 8 +/- 0.4 pg/ml. No difference between plasma and serum was detected, nor was there any difference among adult men, adult women, prepubertal children, hypogonadal patients, or hypopituitary patients with either assay. Plasma GnRH concentrations were also similar in jugular and vena cava samples from intact and castrated male rats. Because many of the samples were at or below the sensitivity of the band IIa assay, they were concentrated after extraction with either methanol or acid-ethanol. However, endogenous immunoreactive GnRH could not be concentrated by these extraction procedures. As measured in the band IIa assay, hypothalamic extracts from control adult male rats contained 3.1 +/- 0.4 ng while hypothalami from castrated rats contained 1.4 +/- 0.1 ng. Similar but slightly lower values were obtained with band I. In contrast, the GnRH content of pineal glands from intact and castrated male rats was similar (approximately 150 pg) when determined in either assay. These studies emphasize that: 1) the characteristics of the radioiodinated hormone can influence the quantitation of GnRH; and 2) endogenous plasma concentrations of GnRH are much lower than previously reported.
A series of des-His2 octa- and nonapeptide analogues of luliberin (luteinizing hormone-releasing hormone) with modifications in the 1 and 6 positions, and in some instances the 10 position, has been prepared. Some of these analogues are potent inhibitors of luliberin in vitro and in vivo. The use of ultraviolet absorption measurements for evaluating peptides containing tyrosine and tryptophan is described. An efficient synthesis of O-methyl-d-tyrosine is reported.
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