(Received 9th January 1974)

RELEASE OF LH AND FSH IN THE NORMAL INTACT RAM BY SYNTHETIC LH-RF AND THE EFFECT OF PRETREATMENT WITH TESTOSTERONE PROPIONATE C. R. N. HOPKINSON, H. C. PANT and R. J. FITZPATRICK Department of Veterinary Clinical Studies, University of Liverpool, Leahurst, Neston, The Wirral L64 7TE (Received 9th January 1974) It has been shown that the synthetic decapeptide LH-RF (Matsuo, Baba, Nair, Arimura & Schally, 1971) can cause release of LH in the ram (Arimura, Debeljuk, Matsuo & Schally, 1972). The present study was designed to investigate the effects of graded doses oflh-rf on plasma levels of LH and FSH and the effect of testosterone propionate (TP) pretreatment on pituitary responsiveness. Five sexually active rams were used\p=m-\threeclun Forest and two Welsh Mountain\p=m-\which were maintained indoors away from ewes. The experiments were conducted between March and August, outside the main breeding season. The samples collected from the jugular vein, using both venepuncture and catheterization, were quickly centrifuged and stored at \m=-\20\s=deg\c.collections were made every 5 min for at least 20 min before the rapid intrajugular injection of LH-RF and then for a further 45 min. Samples were also taken 60 and 90 min after injection and, when 50 \g=m\g LH-RF was administered, the period of frequent sampling was extended. All experiments began at approximately 14.00 hours and at least 3 days were allowed to elapse between experiments. The TP (Koch Light, Colnebrook, Bucks) was administered by intramuscular injection in arachis oil in a 50-mg dose at 09.00 hours and again at 18.00 hours on the first 2 days followed by 100 mg at 09.00 hours on the 3rd day. The res ponse of rams pretreated with TP to a dose of LH-RF was compared with the response to the same dose of LH-RF (7 days earlier) in the same animal pre treated with vehicle only. Plasma LH was assayed by a double antibody method similar in procedure to that of Niswender, Reichert, Midgley & Nalbandov (1969). An equine anti-bovine LH, preabsorbed with bovine TSH (Snook, Saatman & Hansel, 1971) was used at a final dilution of 1 : 200,000. Ovine LH (LER 10 56) was used for iodination by the chloramine-t method (Greenwood, Hunter & Glover, 1963). A rabbit anti-equine y-globulin serum was prepared and used at a final dilution of 1 : 100 and NIH LH SI3 was used as the standard. The * Present address : Abteilung Andrologie, Hautklinik, Universität Düsseldorf, 4 Düsseldorf, Moorenstrasse 5, West Germany. 135

C. R. N. 136 Hopkinson et al. assay system did not cross-react with LH-RF and the cross-reaction with ovine FSH was not greater than the stated contamination with LH. There remained a 25% cross-reaction with ovine TSH but, as pointed out by many previous authors, this is probably due to contamination of the TSH by immunologically active LH. The method was able to detect 0-32 ng/ml (95 % ct/min of the buffer control tubes) and the within-assay variation was 9-9%, 5-0% and 3-6% for the tubes containing less than 0-5 ng, 0-5 to 1-0 ng and more than 1-0 ng, respectively. The between assay variation was 10%. All samples were assayed Text-fig. 1. The effect of different doses of LH-RF on LH levels in a ram (upper section) :, 125 ng; A, 250 ng;, 500 ng;, 1-25 ßg;, 5 ßg;, 10 ßg; O, 50 ßg. The lower section shows the basal levels (saline only injected) in two rams sampled in mid-june. Similar levels were found in late February. in triplicate and, whenever the effect of TP pretreatment was being investigated, the appropriate control samples were included in the same assay. Ovine FSH was measured essentially as described by Hopkinson & Pant (1973). Highly purified rat FSH (NIAMD rat FSH 1-1) rather than human FSH was iodinated and this obviated the need for prior absorption with LH and TSH. The standard used was NIH FSH S9. The within-assay coefficient of variation was 8-3%. Text-figures 1 and 3 illustrate the typically stable levels of LH and FSH. It was concluded that basal levels were stable enough to make studies involving

LH and FSH release in intact Text-fig. 2. dose range. Log dose versus response (ALH) 137 rams for three rams, 125 ng Text-fig. 3. The effect of 50 ßg LH-RF on serum FSH levels in four The effect of saline alone in two of the rams (, ) is also shown. to 10 ßg being the (,, ) rams,

138 C. R. N. Hopkinson et al. small doses of LH-RF meaningful and this was borne out by the low and stable pre-injection levels. The effects of various doses of LH-RF on plasma LH in a ram are shown in Text-fig. 1. Doses of LH-RF ranging from 125 ng to 50 µg were used. All rams responded to the lowest dose (P<0-01). There was, however, great variation in the sensitivity (ALH/ng LH-RF) of the rams to LH-RF which could not be related to breed or body weight. It can be seen that apart from increasing the maximal plasma LH level, an increased dose of LH-RF also increased the duration of LH release. The latter effect was predominant for the higher doses and with 50-^g doses of LH-RF, levels were still rising at the end of the 2^-hr collection period in three rams. With doses up to 10 µg, the responses were increase had a correlation proportional to the log dose (the average percentage coefficient of 0-988, P<0-01) as shown in Text-fig. 2. The average of the first five post-injection levels was used to compute the increase in plasma LH since the secretion showed evidence of pulsatility. These results are basically similar to those reported in men (Kastin & co-authors, 1971). After the injections of 10 µg and 50 ^g LH-RF, FSH increased (P<0-05) in one out of five and three out of four rams, respectively (Text-fig. 3). For these LH-RF doses, however, the maximal FSH increase observed was 81% of basal and the mean was 45 % whereas the comparable increases in LH were 700% to 1000% respectively. The FSH response was also delayed compared with the LH response. Thus, LH-RF can cause release of FSH in the ram, but not as effectively as it causes release of LH. These results also demonstrate that LH-RF can cause release of LH alone under conditions in which the pituitary is evidently capable of releasing FSH, since a stable detectable level of the latter was maintained. It seems most unlikely that the FSH necessary to maintain this basal level could be released by this decapeptide. Two other explanations (not mutually exclusive) seem more plausible : firstly, that the pituitary maintains its basal rates of FSH secre tion without releasing factor stimulation and, secondly, that an additional releasing factor is involved which is more effective at releasing FSH than this decapeptide. From the dose-response studies, it was decided to use 5-^g and 10-^g doses to study the effect of TP pretreatment on LH release. Four rams were used and it was found that the pretreatment significantly reduced the response (P<0-05) when the results were analysed by factorial analysis of variance (Snedecor & Cochran, 1956). The degree to which the response was inhibited varied be tween individual animals from 4-5 % to 52 %, with a mean of 25 % and S.D. of 18-2%, and was not related to breed or body weight. Relatively low doses of LH-RF were used since the responses to these doses appear similar to the spontaneous surges of LH reported by Bolt (1971) which were inhibited by exogenous testosterone. There was no significant interaction between LH-RF dose level and inhibition. No significant difference in basal levels of LH were detected though it is not possible to say that no effect was present since the assay was relatively imprecise when quantifying low levels of LH. Inhibition by testosterone of LH-RF-induced LH release has also been reported in rats (Debeljuk, Arimura & Schally, 1972) and men (von zur Mühlen & Kobberling,

LH and FSH release in intact rams 139 1973). These results demonstrate that a large dose of exogenous TP can inhibit the sensitivity of the pituitary to LH-RF. Whether endogenous levels of testos terone are sufficient to have the same effect, i.e. whether the results are physio logical or pharmacological, is unknown. Unfortunately, testosterone assays were not available. Schally, Redding & Arimura (1973) have shown that high doses of sex steroids, including testosterone, can inhibit the release in vitro of LH and FSH induced by LH-RF from rat pituitaries. Sar & Stumpf (1973) have shown that male rat pituitaries take up and concentrate tritiated testos terone. The relative importance of sex steroids acting at the pituitary and hypothalamic levels in the control of gonadotrophin release in the normal intact ram awaits further investigation. The author wishes to thank Dr R. B. Snook for equine anti-bovine LH, Dr J. C. Hendrick for anti-ovine FSH, Dr H. Gregory and Dr J. Gormley of Imperial Chemical Industries for LH-RF, Dr L. E. Reichert, Jr, for highly purified ovine LH and the NIAMD, Bethesda, Maryland, for FSH and LH standards and highly purified rat FSH. The technical assistance of Mr J. Wickham and Mr M. Chapman is acknowledged. The Wellcome Trust is thanked for financial support. REFERENCES Arimura,., Debeljuk, L., Matsuo, H. & Schally, A. V. (1972) Release of luteinizing hormone by synthetic LH-releasing hormone in the ewe and ram. Proc. Soc. exp. Biol. Med. 139, 851. Bolt, D. J. (1971) Changes in the concentration of luteinizing hormone in plasma of rams following administration of oestradiol, progesterone or testosterone. J. Reprod. Fert. 24, 435. Debeljuk, L., Arimura, A. & Schally, A. V. (1972) Effect of testosterone and estradiol on the LH and FSH release induced by LH-releasing hormone (LH-RH) in intact male rats. Endocrinology, 90, 1578. Greenwood, F. C, Hunter, W. M. & Glover, J. S. (1963) The preparation of I131-labelled human growth hormone of high specific activity. Biochem. J. 89, 114. Hopkinson, C. R. N. & Pant,. C. (1973) Heterologous radioimmunoassay of circulating ovine FSH in normal ewes. J. Physiol., Lond. 231, 52p. Kastin, A. J., Schally, A. V., Gual, C, Midgley, A. R., Miller, M. C. & Cabeza, M. (1971) Dose-response relationship of luteinizing hormone to luteinizing hormone-releasing hormone in man. J. clin. Invest. 50, 1551. Matsuo, H., Baba, Y., Nair, R. M. G., Arimura, A. & Schally, A. V. (1971) Structure of the porcine LH and FSH releasing hormone. I. The proposed amino acid sequence. Biochem. biophys. Res. Commun. 43, 1334. Niswender, G. D., Reichert, L. E., Jr, Midgley, A. R. & Nalbandov, A. V. (1969) Radioimmuno assay for bovine and ovine luteinizing hormone. Endocrinology, 84, 1166. Sar, M. & Stumpf, W. E. (1973) Cellular and subcellular localization of radioactivity in the rat pituit ary after injection of l,2-3h-testosterone using dry-autoradiography. Endocrinology, 92, 631. Schally, A. V., Redding, T. W. & Arimura, A. (1973) Effect of sex steroids on pituitary responses to LH- and FSH-releasing hormone in vitro. Endocrinology, 93, 893. Snedecor, G. W. & Cochran, W. G. (1956) Statistical Methods. Iowa State University Press, Ames, Iowa. Snook, R. B., Saatman, R. R. & Hansel, W. (1971) Serum progesterone and luteinizing hormone levels during the bovine estrous cycle. Endocrinology, 88, 678. von zur Mühlen,. & Köbberling, J. (1973) Effect of testosterone on the LH and FSH release induced by LH-releasing factor (LRF) in normal men. Hormone Metab. Res. 5, 266.