Effect of Testosterone on the Cock Pituitary in vitro Leading to the Release of Gonadotropins

Transcription

1 170 Effect of Testosterone on the Cock Pituitary in vitro Leading to the Release of Gonadotropins Mitsuo KAWASHIMA, Masayuki INAGAMI, Michiharu KAMIYOSHI and Katuhide TANAKA Department of Poultry and Animal Sciences, Gifu University, Kakamigahra-shi, Gifu 504 Testosterone is a major testicular androgen in the fowl1) and has been thought to exercise a negative (inhibitory) feedback control of the pituitary gonadotropin secretion2-10). The site of this testosterone action has been considered to be the hypothalamus7-10), although the action partly at the pituitary level has been suggested5,7). A positive (stimulatory) feedback control of the gonadotropin secretion by testosterone in the male fowl has not been demonstrated. The avian pituitary (adenohypophysis) is divided into two cytologically distinct regions11) which were named "cephalic" and "caudal" lobes12). Between the two lobes, the vascular supply is different13,14), suggesting that the secretion of hormones from the two lobes is under a different control. In immature cockerels, the gonadotropin concentration of the pituitary shows a diurnal rhythm15,16), which appears at an earlier age in the caudal lobe than in the cephalic lobe16). Whether the difference in the age of the appearance of the rhythm is due to a difference in the sensitivity for the feedback action of the testicular androgen on the two lobes or on the hypothalamic nuclei remains unknown. The present study was performed to determine whether testosterone acts on the pituitary itself in respect of the release of gonadotropins and also whether the effect is different between the cephalic and caudal lobe. Materials and Methods Anterior pituitaries were removed from White Leghorn cocks (18-20 months of age; kg BW) at a. m., i. e., at hours in which the pituitary gonadotropin concentration and content are at a relatively high level16,17). In the first series of experiments, the pituitaries were hemisected and placed in separate flasks which contained 2ml Medium 199. Number of the pituitary halves in each flask was 4 (2 pituitary equiv.). min) by using a Warburg apparatus. After the preincubation, the pituitary fragments were transferred to test and control flasks which contained 2ml Medium 199. In the to the medium. The concentration of the steroid was 1 or 10ng per ml. In the control in the same way as in the preincubation. After the incubations, the pituitary fragments Received Feburary 24, 1978.

2 KAWASHIMA et al.: Cock Pituitary GTH Release By Testosterone 171 second and the third series of experiments, only the effect of testosterone (5 and 10ng/ ml) or of synthetic GnRH (Gonadotropin-releasing hormone, or LH-FSH-RH; ng/ml) was examined after dividing the pituitary into cephalic and caudal lobes. Each lobe was hemisected and incubated in the same way, and the media were assayed for the gonadotropic activity. Assays for the gonadotropic activity of the media were performed by using the method of KAMIYOSHI et al.18) which is based on the 32P-uptake by testes of 1-day-old chicks and is a reliable bioassay method for measuring the activity of unseparate gonadotropins (FSH and LH). The dose of the media injected into each chick was 0.8mg pituitary equiv. in the first series of experiments, and 0.4mg pituitary equiv. all assays. The gonadotropic activity was expressed as cpm/mg testis. Student 't' test was used for the significance of difference between means. in conbinations with LH on the 32P-uptake by the chick testis was examined. The dose of the steroids injected into each chick was 0.5 and 5 ng, and the dose of LH (NIH- the 32P-uptake by the chick testis, and that the presence of the steroids did not affect the 32P-uptake response to LH. The effect of synthetic GnRH (250ng/chick) on the chick testicular 32P-uptake was also examined, and the results indicated that synthetic GnRH did not affect the 32P-uptake. Results The media in which the pituitary halves were incubated with testosterone at the concentration of 10ng/ml showed a greater gonadotropic activity than the control ("0" group) in which the other pituitary halves were incubated without testosterone, while the media in which the pituitaries were incubated at a lower concentration (1ng/ ml) of testosterone did not show any significant difference from the control (Table 1). No significant difference was observed between the media in which the pituitaries were When the pituitaries were divided into cephalic and caudal lobes, and then hemisected and incubated with and without testosterone, the media in which the halves of the cephalic lobes were incubated with testosterone showed a greater gonadotropic activity than those in which the other halves were incubated without testosterone (Table 2). However, no difference in the gonadotropic activity was found between the media in which the halves of the caudal lobes were incubated with and without testosterone. The media in which the halves of the cephalic lobes were incubated with GnRH at the concentration of 250 and 500ng/ml showed a greater gonadotropic activity than the media in which the other halves were incubated without GnRH (Table 3). Similar results were also obtained in the incubations of the hemisected caudal lobes.

3 172 Japan. Poultry Sci., 15 (4), 1978 Table 1. Gonadotropic activity of media in which hemisected pituitaries of cocks were incubated in vitro with and without testosterone (T), progesterone (P), or 1 Measured by chick testicular 32P-uptake assay. Dose of medium injected into each chick was 0.8mg pituitary equiv. (): No. of assay chicks NS: Not significantly different at 5% level Table 2. Gonadotropic activity of medium in which hemisected cephalic and caudal lobes of the cock pituitary in vitro with and without testosterone for 3hr 1, #, (), NS, **, ## Same as in Table 1, except for the dose injected into each chick which was 0.4mg pituitary equiv.

4 KAWASHIMA et al.: Cock Pituitary GTH Release By Testosterone 173 Table 3. Gonadotropic activity of medium in which hemisected cephalic and caudal lodes of the cock nituitarv in vitro with and without synthetic 1, #, (), NS, **, ## Same as in Table 1, except for the dose injected into each chick which was 0.4mg pituitary equiv. ### Not significantly different from the assay control at 5% level. Discussion The results shown in Table 1 indicate that an in vitro incubation of the cock pituitaries with testosterone causes an increase in the gonadotropic activity of the medium. This seems to imply that the gonadotropin release is stimulated by testosterone, and is contrary to the expectation from the inhibitory feedback action of testosterone on the pituitary gonadotropin secretion reported in the male birds of various species2-10). Whether the increase in the gonadotropic activity of the media is a result from either or both the stimulation of release itself from the pituitary cells or/and the stimulation of gonadotropin biosynthesis which leads to the release remains unknown. The pituitary pic activity of media. The non-effectiveness of progesterone does not accord with the findings reported recently in the male turkey19), in which immunoreactive FSH was released in vitro from isolated pituitary cells by progesterone. This discrepancy may be due to the difference in the method of incubations and of gonadotropin assays used. When the cephalic lobes of the anterior pituitary was incubated with testosterone, the gonadotropic activity of media increased, while in the incubations of the caudal lobes did not cause the increase (Table 2). This indicate a difference in the response to testosterone between the two lobes. Since the concentration of gonadotropins which can be measured by the 39P-uptake assay does not differ between the two lobes of the pituitary of cocks of the same breed at almost the same age under similar feeding conditions16), the concentration of the measurable gonadotropins by the 32P-uptake assay in the two lobes may not be a causative of the difference in the response to testosterone. Although FSH and LH activities are not separable by the 32P-uptake assay, LH is more potent than FSH18), and therefore, a minute increase in LH activity may be more easily detectable than in FSH activity. The difference between the responses of the two lobes

5 174 Japan. Poultry Sci., 15 (4), 1978 to testosterone might be a result from a difference in the release of FSH and LH between the two lobes, i. e., LH is released from the cepnalic lobe while FSH is released from the caudal lobe in response to testosterone. However, if this were the case, this is inconsistent with the reported findings that the cephalic lobe contains FSH while the caudal lobe contains LH20). It may be likely then to consider that the cephalic lobe is merely more susceptible to testosterone than the caudal lobe to release either or both FSH and LH. In contrast to the results from the incubations with testosterone, the results from the incubations with GnRH showed that the gonadotropic activity of the media increased when the caudal lobes were incubated as well as when the cephalic lobes were incubated (Table 3). This indicates that the caudal lobes are capable to respond to GnRH as well as the cephalic lobes. It is obscure whether the difference between the responses to testosterone and to GnRH found in the caudal lobe incubations is a result from a difference in the intensity or/and nature of actions in the mechanism causing the release of either or both gonadotropins in vitro. The results obtained in the present study suggest that testosterone acts on the pituitary and leads to the release of gonadotropins, and that this stimulatory action for the release of gonadotropins is more pronounced in the cephalic lobe than in the caudal lobe within the anterior pituitary of cocks. However, the mechanism by which testosterone leads to the release of gonadotropins in vitro needs further investigations. Summary assayed for the gonadotropic activity by using a bioassay method based on 32P-uptake by chick testes. The media in which the pituitary halves were incubated with testosterone (10ng/ml) showed a greater gonadotropic activity than those in which the other pituitary halves were incubated without testosterone. Incubations with progesterone When pituitaries were divided into cephalic and caudal lobes, and each lobe was hemisected and incubated in the same way, the incubation of the cephalic lobes with testosterone (5 and 10ng/ml) resulted in the increase in the gonadotropic activity of the media, while the incubation of the caudal lobes did not result in the increase. Synthetic GnRH (250 and 500ng/ml) caused the increase in the gonadotropic activity of the media in both incubations of the cephalic lobes and of the caudal lobes. The results suggest that testosterone acts on the pituitary and leads to the release of gonadotropins, and that the responsiveness of the pituitary to testosterone differs between the cephalic and caudal lobes. Acknowledgements We are indebted to Dr. Hideichi ASANO, Teikoku Zouki Pharm Co. Ltd, for the supply of the steroid hormones, and to NIAMD, NIH, U.S.A., for the gift of NIH-LH-S

6 18 and synthetic GnRH. KAWASHIMA et al.: Cock Pituitary GTH Release By Testosterone 175 This work was supported by a research grant from the Ministry of Education (No ). References 1) LAKE P. E, and B. J. A. FURR: Physiology and Biochemistry of the Domestic Fowl, vol. 3 (Bell, D. J. & B. M. Freeman, eds.), p. 1469, Academic Press, London, New York, ) LOFTS, B.: Gen. Comp. Endocrinol. 2, 258, ) UEMURA, H.: Endocrinol. Japon. 11, 185, ) KOBAYASHI, H. and D.S. FARNER: Gen. Comp. Endocrinol. 6, 443, ) STETSON, M.H.: Gen. Comp. Endocrinol. 19, 37, ) DAVIES, D.T., L.P. GOULDEN, B.K. FOLIETT and N.L. BROWN: Gen. Comp. Endocrinol. 30, 477, ) WILSON, E.F.: Aspects of Neuroendocrinology (Bargman, W. & B. Sharrer, eds.), p. 274, Springer-Verlag, Berlin, ) CUSICK, E.K, and F.E. WILSON: Gen. Comp. Endocrinol. 19, 441, ) WADA, M.: Z. Zellforsch. Mikrosk. Anat. 124, 507, ) FOLLETT, B.K.: Breeding Biology of Birds (Farner, D.S., ed.), p. 209, National Academy of Sciences, Washington, D.C., ) WINGSTRAND, K.G.: The Pituitary Gland (Harris, G.W. & B. T. Donovan, eds.), vol. 1, p. 58, Butterworths, London, ) RAHN, H. and B.T. PAINTER: Anat. Rec. 79, 297, ) VITUMS, A., S. MIKAMI, A. OKSCHE and D.S. FARNER: Z. Zellforsch. Mikrosk. Anat. 64, 541, ) DOMINIC, C.J.: Proc. 1st International Symp. Avian Endocrinology (Calcutta), p. 23, ) HASHIGUCHI, M., M. KAMIYOSHI and K. TANAKA: Japan. Poultry Sci. 14, 199, ) KAMIYOSHI, M., M. YOSHIHARA and K. TANAKA: Jap. J. Zootech. Sci. 48, 424, ) KAMIYOSHI, M., M. HASHIGUCHI and K. TANAKA: Japan. Poultry Sci. 14, 202, ) KAMIYOSHI, M., K. TANAKA and Y. TANABE: Endocrinology 91, 385, ) GODDEN, P.M.M., M.R. LUCK and C.G. SCANES: Acta Endocrinol. 85, 713, ) BRASCH, M. and T.B. BETZ: Gen. Comp. Endocrinol. 16, 241, 1971.

7 176 Japan, Poultry Sci., 15 (4), 1978