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903 Changes in preproenkephalin (PPE) mRNA levels in the ventromedial hypothalamus (VMH) during the rat estrous cycle
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DEVELOPMENTAL CHANGE OF NEURONAL AROMATASE EXPRESSION IN THE SEPTAL, PREOPI'IC, STRIAL AND AMYGDALOID REGIONS OF THE RAT. Koh Shinoda, Mamoru Na~ano and Hiroshi Sasaki, Igpt. o f Anatomv_ ]], Kinki U n i v e r s i t y S c h o o l o f M e d i c i n e , 377-2, O h n o - H i g a s h i , O s a k a s a v a m a , Osaka 589, Japan. Using highly specifc anti-aromatase (P-450arom) antiserum, which was produced against the antigen purified through immunoaffinity cohimn coupled with a monoclonal antibody monospecific to P-450arom, regional and subcellular Iocalizafions of neuronal aromatase were iwanunohistochemically evaluated in developing rat septal, preoptie, strial and ,'unygdaloid regions from late prenatal to infantile stages. Aromatase-inuurmoreacfive (AROM-I) neurons were present in several distinct regions, which could largcly be classified into three groups. The first, in which weak-to-moderate immunostaining occurs only in limited pre- or perinatal days around birth (EI6-P2), included the anterior medial preoptie nucleus, the periventricular preoptic nucleus, neurons associated with the strial part of the preoptic area and the rostta'alportion of the medial preoptie nucleus. The second is a striking AROM-I cell-~oup found in the "medial prcoptico-amygdaloid neuronal arc (mPO-AM)", which extends from the lateral periphery of the medial preoptie nucleus to the principal nucleus of the bed nucleus of the stria terminalis and continues to the posterodorsal part of the medial amygdaloid nucleus. These AROX M nettrons appeared by El6, reaching a peak in number and staining intensity between E18-P2 and substantially diminishing to weak staining after the perinatal stage (but still remained stained w~nkly). After P14, a third ~ o u p of ARO•I-I neurons emerged in the intermediate part of the lateral septal nucleus, the oval nucleus of the bed nucleus of the stria terminalis and the central mnygdaloid nucleus. The mPO-AM appeared to serve as a major aromatization center in developing rat brains, while aromatization enzyme immunoreacfivity increased in the third Voup (especially in the central amygdaloid nucleus) after the late infantile stage. The distribution and developmental patterns were basically similar in males and females, suggesting that the transiently prominent aromatase immunoreactivity is not induced by males .p.p.p.p~fie androgen surges occurring around birth. Inununo-dectron microscopic analysis showed that subncuronal aromatase was predominantly localized on the nuclear membrane and endoplasmic retieulum. The former location appeared appropriate for the efficient conversion of androgen into estrogen just prior to binding to the nuclear receptors.
903
CHANGES IN PREPROENKEPHALIN (PPE) m R N A L E V E L S IN THE VENTROMEDIAL HYPOTHALAMUS (VMH) DURING THE RAT ESTROUS CYCLE TOSHIYA FUNABASHI, PHILIP J. BROOKS, STEVEN P. KLEOPOULOS, CHARLES V. MOBBS, FUKUKO K I M U R ~ A N D DONALD W. PFAFF Lab. of Neurobiol. Behav., The Rockefeller Univ., 1230 York Ave., New York, NY 10021, USA and !Dept. of Physiol., Yokohama City Univ. Sch. of Med., 3-9 Fukuura, Kanazawa-Ku, Yokohama 236, Japan To understand the relationship between female ratreproductive systems and PPE neurons under physiological conditions , we exam/ned changes in PPE mRNA levels in the VMH during the rat estrous cycle by means of northern blotting and in situ hybridization histochemistry (ISHH). In the nortnern blotting studies, the level of PPE mRNA in the VMH was significantly increased by noon of proestrus compared to that on the morning and stayed high until the day of metestrus, and returned toward low levels on the day of diestrus. In addition, we found that ~APDH mRNA levels were significantly higher while calcineurin mRNA levels were significantly lower, on the day of proestrus. ISHH experiment revealed that the level of PPE mRNA on the afternoon of proestrus was about two hold higher than that on the afternoon of diestrus, indicating that these changes in PPE m R N A l e v e l s were specific in the VMH and not due to variation depending on phase of circadian rhythm. The present study provides additional informations regarding possible implications of the PPE gene expression in female reproductive systems, since changes in PPE mRNA levels may be associated with estrogen and progesterone concentrations during the estrous cycle.
904
LOCALIZATION OF PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE (PACAP) IN THE HYPOTHALAMUS-PfTUITARY SYSTEM. AN IMMUNOELECTRON M I C R O S C O P I C STUDY. YAHE S H I O T A N I , S H I G E H A R U KIMURA AND C H I Z U K O YANAIHARA*, Dept.of N e u r o a n a t . , B i o m e d i c a l Res. Center and Dept.o~ Pharmacy ~, Osaka Univ. Med. School, 2-2, Yamadaoka, Suita, Osaka 565 Japan The localization of PACAP in the h y p o t h a l a m u s - p i t u i t a r y system in rats was examined i m m u n o e l e c t r o n m i c r o s c o p i c a l l y using an a n t i s e r u m to synthetic PACAP 1-38 (R0831). PACAP-immunopositive reaction was observed in the supraoptic nucleus(SO), paraventricuiar magnocel±ular nucleus(PV), internal as well as external layer of the median eminence, posterior(PP) and intermediate(IP) lobe, but not anterior lobe, of the p i t u i t a r y gland. In SO, PV and IP, DAB reaction products were distributed diffusely in the cytoplasmic matrix, frequently attaching to the membrane of rER. In PP, reaction products were o b s e r v e d among the secretory granules, but sometimes on them.
DEVELOPMENTAL CHANGE OF NEURONAL AROMATASE EXPRESSION IN THE SEPTAL, PREOPI'IC, STRIAL AND AMYGDALOID REGIONS OF THE RAT. Koh Shinoda, Mamoru Na~ano and Hiroshi Sasaki, Igpt. o f Anatomv_ ]], Kinki U n i v e r s i t y S c h o o l o f M e d i c i n e , 377-2, O h n o - H i g a s h i , O s a k a s a v a m a , Osaka 589, Japan. Using highly specifc anti-aromatase (P-450arom) antiserum, which was produced against the antigen purified through immunoaffinity cohimn coupled with a monoclonal antibody monospecific to P-450arom, regional and subcellular Iocalizafions of neuronal aromatase were iwanunohistochemically evaluated in developing rat septal, preoptie, strial and ,'unygdaloid regions from late prenatal to infantile stages. Aromatase-inuurmoreacfive (AROM-I) neurons were present in several distinct regions, which could largcly be classified into three groups. The first, in which weak-to-moderate immunostaining occurs only in limited pre- or perinatal days around birth (EI6-P2), included the anterior medial preoptie nucleus, the periventricular preoptic nucleus, neurons associated with the strial part of the preoptic area and the rostta'alportion of the medial preoptie nucleus. The second is a striking AROM-I cell-~oup found in the "medial prcoptico-amygdaloid neuronal arc (mPO-AM)", which extends from the lateral periphery of the medial preoptie nucleus to the principal nucleus of the bed nucleus of the stria terminalis and continues to the posterodorsal part of the medial amygdaloid nucleus. These AROX M nettrons appeared by El6, reaching a peak in number and staining intensity between E18-P2 and substantially diminishing to weak staining after the perinatal stage (but still remained stained w~nkly). After P14, a third ~ o u p of ARO•I-I neurons emerged in the intermediate part of the lateral septal nucleus, the oval nucleus of the bed nucleus of the stria terminalis and the central mnygdaloid nucleus. The mPO-AM appeared to serve as a major aromatization center in developing rat brains, while aromatization enzyme immunoreacfivity increased in the third Voup (especially in the central amygdaloid nucleus) after the late infantile stage. The distribution and developmental patterns were basically similar in males and females, suggesting that the transiently prominent aromatase immunoreactivity is not induced by males .p.p.p.p~fie androgen surges occurring around birth. Inununo-dectron microscopic analysis showed that subncuronal aromatase was predominantly localized on the nuclear membrane and endoplasmic retieulum. The former location appeared appropriate for the efficient conversion of androgen into estrogen just prior to binding to the nuclear receptors.
903
CHANGES IN PREPROENKEPHALIN (PPE) m R N A L E V E L S IN THE VENTROMEDIAL HYPOTHALAMUS (VMH) DURING THE RAT ESTROUS CYCLE TOSHIYA FUNABASHI, PHILIP J. BROOKS, STEVEN P. KLEOPOULOS, CHARLES V. MOBBS, FUKUKO K I M U R ~ A N D DONALD W. PFAFF Lab. of Neurobiol. Behav., The Rockefeller Univ., 1230 York Ave., New York, NY 10021, USA and !Dept. of Physiol., Yokohama City Univ. Sch. of Med., 3-9 Fukuura, Kanazawa-Ku, Yokohama 236, Japan To understand the relationship between female ratreproductive systems and PPE neurons under physiological conditions , we exam/ned changes in PPE mRNA levels in the VMH during the rat estrous cycle by means of northern blotting and in situ hybridization histochemistry (ISHH). In the nortnern blotting studies, the level of PPE mRNA in the VMH was significantly increased by noon of proestrus compared to that on the morning and stayed high until the day of metestrus, and returned toward low levels on the day of diestrus. In addition, we found that ~APDH mRNA levels were significantly higher while calcineurin mRNA levels were significantly lower, on the day of proestrus. ISHH experiment revealed that the level of PPE mRNA on the afternoon of proestrus was about two hold higher than that on the afternoon of diestrus, indicating that these changes in PPE m R N A l e v e l s were specific in the VMH and not due to variation depending on phase of circadian rhythm. The present study provides additional informations regarding possible implications of the PPE gene expression in female reproductive systems, since changes in PPE mRNA levels may be associated with estrogen and progesterone concentrations during the estrous cycle.
904
LOCALIZATION OF PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE (PACAP) IN THE HYPOTHALAMUS-PfTUITARY SYSTEM. AN IMMUNOELECTRON M I C R O S C O P I C STUDY. YAHE S H I O T A N I , S H I G E H A R U KIMURA AND C H I Z U K O YANAIHARA*, Dept.of N e u r o a n a t . , B i o m e d i c a l Res. Center and Dept.o~ Pharmacy ~, Osaka Univ. Med. School, 2-2, Yamadaoka, Suita, Osaka 565 Japan The localization of PACAP in the h y p o t h a l a m u s - p i t u i t a r y system in rats was examined i m m u n o e l e c t r o n m i c r o s c o p i c a l l y using an a n t i s e r u m to synthetic PACAP 1-38 (R0831). PACAP-immunopositive reaction was observed in the supraoptic nucleus(SO), paraventricuiar magnocel±ular nucleus(PV), internal as well as external layer of the median eminence, posterior(PP) and intermediate(IP) lobe, but not anterior lobe, of the p i t u i t a r y gland. In SO, PV and IP, DAB reaction products were distributed diffusely in the cytoplasmic matrix, frequently attaching to the membrane of rER. In PP, reaction products were o b s e r v e d among the secretory granules, but sometimes on them.