Qualitative and Quantitative Changes in Plasma Luteinizing Hormone (LH) under Stimulation by Intravenous Infusion of Synthetic Luteinizing Hormone-Releasing Hormone (LHRH) in Japanese Monkeys (Macaca fuscata) as Assessed by Electrofo-

Using 5 adult Japanese monkeys (Macaca fuscata) of both sexes, the qualitative and quantitative changes in plasma LH caused by LHRH administration were examined before and after castration. Five hundred μg synthetic LHRH was infused for 2hrs and blood samples were obtained every 15min for 5hrs under ketamine hydrochloride anesthesia. Plasma LH levels were determined by an in vitro bioassay using the mouse testis interstitial cell preparation. The LHRH infusion induced a biphasic release of LH in intact animals of both sexes and in castrated females but not in castrated males. The biphasic pattern of LH release was characterized by an initial acute rise followed by a plateau or a fall and the consecutive second rise after a long latent period. Aliquots of plasma samples obtained during 30-90 (corresponding to the initial rise) and 120-210min (corresponding to the second rise) after the initiation of LHRH infusion were pooled separately (plasma pools I and II, respectively) and subjected to isoelectrofocusing (IEF) fractionation. The IEF fractionation was performed in ampholine-sucrose gradient, pH range 3.5-10, under a constant electrical power supply (3W, 700V) for 48hrs at 4°C. After the completion of the IEF fractionation the LH activity in each fraction was measured by the in vitro bioassay. The IEF fractionation separated plasma LH into 6 distinct species in terms of pI values. When the IEF profiles of plasma pools I and II in intact animals of both sexes were compared, the increase and the decrease in acidic to neutral LH and alkaline LH, respectively, were consistent and significant in plasma pool II. Changes of the same kind were further exaggerated when the IEF profiles in castrated animals were compared with those in non-castrated ones. The acidic LH may represent the LH species newly synthesized and not well processed. The LH species possessing pI values of 7.5-8.5 is likely to be the mature type of LH exhibiting high biological potency. The LH species migrating in the high alkaline region seems to be the LH of over-maturation. Luteinizing hormone (LH) is not a hormone of homology but consists of heterogenous molecular species. LH species of various molecular sizes were demonstrated by gel chromatography (Peckham and Knobil, 1976a, b; Liu and Jackson, 1981). Pulsechase labeling techniques revealed that the big LH might represent the precursor form of native LH (Liu and Jackson, 1981). Isoelectrofocusing fractionation separated LH into multiple LH species in terms of pI values, possessing their own biological and immunological properties (Robertson and Diczfalusy, 1977; Wakabayashi, 1977; Strollo et al., 1981). It is generally accepted that LH is released Recevied January 5, 1983 102 HAMADA et al. Endocrinol. Japon. February 1983 in a biphasic pattern in response to a constant stimulation by synthetic luteinizing hormone-releasing hormone (LHRH); i. e. the initial acute rise followed by a plateau or a fall, and the consecutive second rise appearing after a long latent peiod (Bremnar and Paulsen, 1974; Koninckx et al., 1976; Wang et al., 1976; Hoff et al., 1977; Hudson et al., 1981). The so-called two pool theory was proposed in order to explain this biphasic release of the hormone. The first rise was attributed to the release of releasable LH which might be stored in secretory granules situated closely to the cell membrane. In contrast to this, the long latent period prior to the second rise may indicate that some time-consuming processes, such as biosynthesis of new LH molecules or modification of newly synthesized LH, are involved in the LH cells for the second LH release (De Koning et al., 1976; Hoff et al., 1977). If this is the case, the difference in the ratio of each LH species could be expected in the circulating LH during the first and second rises following LHRH stimulation. The present study was undertaken to confirm