Department of Obstetrics and Gynecology, School of Medicine, Chiba University, Chiba

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1 Endocrinol. Japon. 1971, 18 (6), 477 `485 Radioimmunoassays for Rat Follicle Stimulating and Luteinizing Hormones KATSUYOSHI SEKI, MITSUNORI SEKI, TERUFUMI YOSHIHARA AND HIDEYASU MAEDA Department of Obstetrics and Gynecology, School of Medicine, Chiba University, Chiba Synopsis Radioimmunoassays for rat FSH and LH have been studied taking advantage of rat FSH and LH radioimmunoassay kits supplied by the NIAMD. Separation of free and antibody bound labeled hormone was accomplished by the double antibody method with pre-precipitation technique. Although rat GH and prolactin did not react in the FSH assay, LH crossreacted slightly to a negligible extent. Hypophysectomized rat serum inhibited the reaction between labeled rat FSH and anti-rat FSH serum. To eliminate the nonspecific interference by serum, hypophysectomized rat serum was added to the diluent for reference preparations in the FSH assay. Rat GH and prolactin did not react in the LH assay. However, FSH crossreacted in the LH assay to a greater degree than LH in the FSH assay. This indicates that LH concentrations may be overestimated when samples with extraordinary large amounts of FSH are measured. Hypophysectomized rat serum did not influence the LH assay. The FSH activity of pituitary extracts determined by bioassay and that by radioimmunoassay agreed well. And there was also a good correlation between LH values obtained both by bioassay and by radioimmunoassay. Although a marked decrease in pituitary FSH and LH levels was found between proestrus and estrus, serum FSH and LH levels were elevated only slightly at 2:00 PM of the proestrus day. Serum and pituitary FSH and LH levels increased after ovariectomy. Recent development of radioimmunoassays for rat follicle stimulating hormone (FSH) and luteinizing hormone (LH) has made it possible to measure FSH and LH in sera obtained from individual rats as well as in sera obtained sequentially from the same rat (Monroe et al., 1968; Daane and Parlow, 1971). Radioimmunoassays for rat FSH and LH have been studied taking advantage of rat FSH and LH radioimmunoassay kits kindly supplied by the National Institute of Arthritis and Metabolic Diseases (NIAMD), Rat Pituitary Distribution Program. Some characteristics of the radioimmunoassays were examined and FSH and LH levels in rats under known physiological processes Received for publication August 24, were determined by the radioimmunoassays to validate the assays. Materials and Methods Bioassay Pituitary extracts were bioassayed for FSH concentration by the ovarian augmentation method of Steelman and Pohley (1953). LH was estimated by the ovarian ascorbic acid depletion method of Parlow (OAAD) (1961). Hormones and antisera Hormones and antisera were obtained from the NIAMD. NIAMD-Rat FSH-I-1 was used for iodination in the FSH assay. It has an FSH activity 100 ~ NIH-FSH-S1 and an LH activity less than ~ NIH-LH-S1. NIAMD-Rat LH-I-1, having an LH activity 1.0 ~NIH-LH-S1 and an FSH activity less

2 SEKI et al. Endocrinol. Japon. December 1971 than 0.04 ~NIH-FSH-S1, was used for iodination in the LH assay. NIAMD-Rat FSH-RP-1 with a biological potency 2.1 ~NIH-FSH-S1 was employed for the reference preparation of the FSH assay and NIAMD-Rat LH-RP-1 with a biological potency 0.03 ~NIH-LH-S1 for the reference preparation of the LH assay. Highly purified NIAMD-Rat GH-I-1 and NIAMD-Rat prolactin-i-1 were used to test the specificity of the assays. NIAMD-Anti-Rat FSH-Serum-1 (Anti-RFSH) and NIAMD-Anti-Rat LH-Serum-1 (Anti-RLH) were used. Precipitating antiserum Anti-rabbit gamma globulin serum (Anti-RGG) was obtained from Calbiochem. Hypophysectomized rat serum Hypophysectomized rat serum was a gift from Dr. Nakanowatari, Teikoku Hormone Mfg. Co. Ltd. It was obtained by exsanguinating adult female rats of Wistar-strain ten days after hypophysectomy. Animal samples Animals used in this study were adult female Sprague-Dawley rats weighing approximately 200gr each. They were kept in a 14-hr light (6:00AM-8:00 PM) and 10-hr dark environment. Individual blood samples were obtained from the carotid artery under light ether anesthesia. The blood samples were allowed to clot at room temperature and centrifuged. The serum was stored at-20 Ž until assayed. Pituitary glands were collected and weighed immediately after killing the rats. The pituitaries were reaction was stopped by the addition of 125ƒÊg (25ƒÊl) of sodium metabisulfite. The entire reaction mixture was applied to a Sephadex G-75 column equilibrated with PBS. One column eluates were collected and counted for radioactivity. Assay Provedure The assay method is based on a double antibody radioimmunoassay with pre-precipitation technique similar to that described by Hales and Randle (1963) for insulin. Antisera and normal rabbit serum (NRS) were diluted with PBS-0.025M EDTA. Labeled hormones were diluted with PBS-0.1% BSA. And reference preparations and samples were diluted with PBS-1% BSA. Anti-RFSH was used in a dilution of 1:300 and Anti-RLH in a dilution of 1:10,000. Anti- RGG was used in a dilution of 1:20 and NRS in a dilution of 1:100. Equal volumes of Anti-RFSH or Anti-RLH and Anti-RGG were preincubated together for 2 days at 4 Ž.Algquots of 0.2ml of the preincubated antisera were added to the tubes containing 0.1ml (approximately 10,000cpm) of labeled hormone, 0.1ml of NRS and 0.2ml of sample or standard. After the tubes were incubated for further 5 or 6 days, they were centrifuged and the supernatant carefully aspirated. The precipitate containing the antibodybound hormone was counted in an automatic gamma counter. The standard curve was obtained by expressing the radioactivity recovered from reactions with known amounts of unlabeled hormone as a percentage of the initial radioactivity and by plotting this value against the logarithm of the concentration of unlabeled hormone. placed in a large excess volume of acetone in the refrigerator. The acetone was changed 24hr later. Results After another day or longer, the glands were dried at room temperature and stored in a desiccator at Radioiodination Specific activities of labeled rat FSH to fine powder in a mortar, homogenized in distilled water and diluted with 0.01M phosphate-buffered (125I-RFSH) and rat LH (125I-RLH) were in 0.15M NaCl with 1% bovine serum albumin (PBS- 1% BSA) just prior to assay. Radioiodination Purified Rat FSH-I-1 and LH-I-1 were iodinated at room temperature by a modification of the chloramine-t method of Greenwood et al. (1963). All reagents were dissolved in PBS, ph 7.6. Five ƒêg (50ƒÊl) of Rat FSH-I-1 or LH-I-1 and 30ƒÊl of 0.5M sodium phosphate buffer, ph7.6 were added to approximately 0.6mCi aliquots of carrier and thiosulfate free sodium (1251) iodide in NaOH (Iso-Serve, Inc(, U.S.A.) dispensed at the bottom of a small glass tube. Then 100ƒÊg (20ƒÊl) of chloramine-t was added and the reaction mixture was agitated for one minute. The the range from 70 to 150ƒÊc/ƒÊg. Radioimmunoassay for rat FSH Purified Rat FSH-I-1, Rat FSH-RP-1 and a crude pituitary extract inhibited the reaction between 125I-RFSH and Anti-RFSH essentially in the same way (Fig. 1). To test the specificity of the FSH assay, the effect of rat pituitary hormones available to us was examined. Purified Rat GH-I-1 and prolactin- I-1 even at the levels as high as 1ƒÊg did not react in the FSH assay. However, purified Rat LH-I-1 slightly inhibited the reaction

3 RADIOIMMUNOASSAYS FOR RAT FSH AND LH Fig. 1. Dose-response curves for purified rat J SH (NIAMD-Rat FSH-I-1), reference preparation of rat FSH (NIAMD-Rat FSH-RP-1), a crude pituitary extract, purified rat LH (NIAMD-Rat LH-I-1), purified rat prolactin (NIAMD-Rat Prolactin-I-1) and purified rat GH (NIAMD-Rat GH-I-1) in the FSH assay. between 125I-RFSH and Anti-RFSH. The degree of crossreaction with LH was less than 1% on a weight basis. When hypophysectomized rat serum was added to the FSH assay, binding of 125I- RFSH to Anti-RFSH was inhibited significantly compared with tubes containing only PBS-1% BSA. For this reason, hypophysectomized rat serum was added to the diluent for reference preparations and the serum concentration of the standard solution was made equal to that of samples. In this instance, the dose-response curve of serum from an ovariectomized rat was identical with the standard curve in hypophysectomized rat serum. Figure 2 illustrates these points. Accuracy of measurement was determined by adding known amounts of FSH-RP-1 to serum. Mean recovery was 92.7%. This means that the presence of serum does not Fig. 2. Standard curves in the FSH assay. Note the difference between the standard curve in PBS-1% BSA and that in hypophysectomized rat serum (1:2). Also is shown a dose-response curve of an ovariectomized rat serum. interfere with the accuracy of measurements. The sensitivity of the FSH assay was 100ng FSH-RP-1/ml of serum. When a sample was measured on four seperate occasions the between-assay coefficient of variation was 10.8%. Radioimmunoassay for rat LH In the LH assay, highly purified Rat LH-I-1. Rat LH-RP-1 and a crude pituitary extract gave similar dose-response curves in shape and slope (Fig. 3). When highly purified Rat FSH-I-1 was added to the LH assay, binding of 125I-RLH to Anti-RLH was slightly inhibited, resulting in a shallow dose-responsc curve very different from the standard curve The degree of crossreaction with FSH in the LH assay was 6% on a weight basis. Purified

4 SEKI et al. Endocrinol. Japon. December 1971 Fig. 3. Dose-response curves for purified rat LH (NIAMD-Rat LH-I-1), reference preparation of rat LH (NIAMD-Rat LH-RP-1), a crude pituitary extract, purified rat FSH (NIAMD-Rat FSH-I-1), purified rat prolactin (NIAMD-Rat Prolactin-I-1) and purified rat GH (NIAMD-Rat GH-I-1) in the LH assay. Rat GH-I-1 and prolactin-i-1 did not change the percentage of binding of 125I-RLH to Anti-RLH. In contrast to the FSH assay, no detectable LH was found in sera from hypophysectomized rats and the dose-reresponse curve of serum from an ovariectomized rat was parallel to the standard curve in PBS-1% BSA in the LH assay (Fig. 4). The sensitivity of the assay was 15ng Rat LH-RP-1/ml of serum. The coefficient of variation was 12.7% when a sample was assayed on four seperate occasions. The mean recovery of Rat LH-RP-1 added to serum was 92.7%. Comparison of bioassay and radioimmunoassay Pituitary extracts previously bioassayed for Fig. 4. Standard curves in the LH assay. Note the identity between the standard curve in PBS-1% BSA and that in hypophysectomized rat serum (1:2). Also is shown a dose-response curve of an ovariectomized rat serum demonstrating the identity to these. FSH by the ovarian augmentation method were radioimmunoassayed for FSH and LH. The result is shown in Table 1. The FSH potency estimated by bioassay was very close to that obtained by radioimmunoassay. The ratio of biologic to immunologic FSH potency ranged from 0.63 to Furthermore, pituitary extracts bioassayed for LH by OAAD were radioimmunoassayed for FSH and LH. As shown in Table 2, there was also a good correlation between LH values obtained by bioassay and radioimmunoassay. The ratio of biologic to immunologic LH potency fluctuated from to 0.85 to 1.71.

5 RADIOIMMUNOASSAYS FOR RAT FSH AND LH Table 1. Correlation of bioassay and radioimmunoassay potency estimates for rat FSH FSH and LH relative potencies are expressed in terms of NIH-FSH-S1 and NIH-LH-S1. *NIAMD-Rat FSH-RP-1 was used as the standard for FSH-RIA potencies. **NIH-FSH-S3 was used as the standard for ovar. aug. bioassay potencies. *** NIAMD-Rat LH-RP-1 was used as the standard for LH-RIA potencies. +LH content by RIA as Đg/pituitary FSH content by RIA as Đg/pituitary. #FSH content by ovar. aug. bioassay as Đg/pituitary FSH content by RIA as Đg/pituitary. Table 2. Correlation of bioassay and radioimmunoassay potency estimates for rat LH FSH and LH relative potencies are expressed in terms of NIH-FSH-Sl and NIH-LH-S1. * NIAMD-Rat LH-RP-1 was used as the standard for LH-RIA potencies. ** NIH-LH-S10 was used as the standard for OAAD potencies. *** NIAMD-Rat FSH-RP-1 was used as the standard for FSH-RIA potencies. +LH content by RIA as Đg/pituitary FSH content by RIA as Đg/pituitary. #LH content by OAAD as Đg/pituitary LH content by RIA as Đg/pituitary.

6 SEKI et al. Endocrinol. Japon. December 1971 Table 3. Anterior pituitary FSH and LH contents in relation to the stages of the estrous cycle * Mean + standard error LH FSH Fig. 5. Concentrations of LH and FSH in rat sera during the estrous cycle, expressed in terms of NIAMD-Rat LH-RP-1 and NIAMD-Rat FSH-RP- 1/ml. D = diestrus, PE = proestrus, E = estrus and M = metestrus. FSH and LH levels during the estrous cycle Vaginal smears were taken for two consecutive cycles prior to sacrifice. Rats were killed at 2:00PM. Pituitary FSH content was the highest at diestrus and it decreased between proestrus and estrus (Table 3). Serum FSH levels elevated slightly at proestrus and detectable levels of serum FSH were also found at other stages of cycle (Fig. 5). A marked decrease in pituitary LH content was found to occur between proestrus and estrus (Table 3) and slightly elevated levels of serum LH were found at proestrus (Fig. 5). Effect of ovariectorny on FSH and LH levels Female adult rats weighing approximately 200 gr each were bilaterally ovariectomized without regard to the stage of the estrous cycle. The rats were killed 1, 2, 4 and 8 weeks after ovariectomy and pituitary and serum FSH and LH levels were determined. The result is shown in Figure 6. Serum and pituitary FSH increased markedly 1 week after ovariectomy and continued to increase through the following weeks. However, the increase of serum and pituitary LH 1 week after ovariectomy was not so marked as that of FSH. Serum and pituitary LH attained their maximum levels at 4 weeks and serum LH decreased slightly at 8 weeks. Discussion Monroe et al. (1968) were the first to report

7 RADIOIMMUNOASSAYS FOR RAT FSH AND LH Fig. 6. The pituitary and serum FSH-I (closed circles, circles, Đg or ng NIAMD-Rat LH-RP-1) levels at various intervals after ovariectomy. radioimmunoassay for rat LH utilizing rat LH for iodination and immunization. Niswender et al. (1968) developed more sensitive radioimmunoassay for rat LH using ovine LH for both immunization and iodination. Radioimmunoassay for rat FSH has also appeared and been applied for estimations of FSH in sera from rats under some physiological and pathological conditions (Yamamoto et al., 1970; Swerdloff et al., 1971; Daane and Parlow, 1971). Radioimmunoassays for rat FSH and LH presented here are more sensitive than any existing bioassays and the assay for LH is as sensitive as the radioimmunoassay for rat LH using ovine LH described by Niswender et al. (1968). The values of FSH and LH determined both by radioimmunoassay and by bioassay for pituitary extracts corresponded well. Monroe et al. (1968) and Niswender et al. (1968) reported a good correlation between bioassay and radioimmunoassay estimates of rat LH concentrations for pituitary extracts. Daane and Parlow (1971) reported that both results of FSH-radioimmunoassay and bioassay of rat serum samples and pituitaries were in excellent agreement. The values of LH in sera and serum extracts as estimated by radioimmunoassay, however, were reported to be considerably lower than the values obtained by bioassay (Monroe et al., 1968; Bogdanove et al., 1971). The nonspecific interference by serum observed in the FSH assay was also reported by Swerdloff et al. (1971). It was eliminated by adding hypophysectomized rat serum to the diluent for reference preparations and keeping the serum concentration of each tube constant throughout the assay. Interference by gamma-globulin (Hales and Randle, 1963) or by complement (Morgan et al., 1964) was shown in the double antibody radioimmunoassay for insulin. The former was eliminated by pre-precipitating the insulin antibody with second antibody and the latter by the addition of EDTA. Since the interference by serum in the FSH assay could not be eliminated by pre-precipitation technique and by the addition of EDTA, it does not seem to be due to gamma-globulin or complement, but seems to be due to reaction between Anti-RFSH and some components in rat serum. LH crossreacted slightly in the FSH assay, but the degree of crossreaction was not large. Thus, LH apparently does not influence the FSH assay. However, the degree of crossreaction with FSH in the LH assay was 6 on a weight basis. This crossreaction with FSH may result in a slight overestimation of LH when samples with maximum amounts of FSH are measured. On the whole, the present observations on pituitary and serum FSH and LH levels dur-

8 SEKI et al. Endocrinol. Javon. December 1971 ing the estrous cycle and after ovariectomy appear to agree with earlier studies made by bioassay. Schwartz and Bartosik (1962) observed a drop in pituitary LH potency between proestrus and estrus. Ramirez and McCann (1964) showed that plasma LH rose to a peak on the afternoon of proestrus. These observations were confirmed by Anderson and Mcshan (1966). The present FSH and LH levels to ovariectomy, however, has been studied by only a few workers (Labhsetwar, 1969). Purves and Griesbach (1955) found that the pituitary content of FSH rose during the first two weeks after ovariectomy and was then followed by a rise in LH. Although this study was not quantitative, it is in accordance with the present finding that the rise of the pituitary content of FSH, but not of LH, was marked one between proestrus and estrus is similar to the majority of reports. Caligaris et al.(1967), week after ovariectomy. While a gradual increase in serum LH after ovariectomy was using the mouse ovarian augmentation recently reported using radioimmunoassay method of Brown (1955), showed a drop in pituitary FSH concentration on the afternoon of proestrus, but they could not find detectable levels of FSH in plasma on the afternoon of (Gay et al., 1969), it appears to be for the first time that changes in serum FSH levels at various lengths of time after ovariectomy were studied by radioimmunoassay. proestrus. McClintock and Schwartz (1968), using the ovarian augmentation method of Steelman and Pohley (1953), showed a decrease in pituitary FSH content with a concomitant Acknowledgements increase in plasma FSH on the The authors are grateful to the National Institute afternoon of proestrus. The conflicting of Arthritis and Metabolic Diseases for providing the observations on plasma FSH levels on the rat FSH and LH radioimmunoassay kits, and to Dr. afternoon of proestrus may be ascribed to Nakanowatari, Teikoku Hormone Mfg. Co. Ltd., for the lack of sensitivity of the bioassays used. hypophysectomized rat serum. Kamioka (1970), using the most sensitive Igarashi-McCann's FSH bioassay (Igarashi and McCann, 1964), showed that plasma FSH rose to a peak on the afternoon of proestrus. Recently, Monroe et al.(1969) and Daane and Parlow (1971) measured serum FSH and LH levels throughout the estrous cycle by radioimmunoassay. They showed that serum FSH and LH levels rose on the afternoon of proestrus and that the maximal elevation of serum FSH persisted until the morning of estrus, while serum LH levels were low after the midnight of proestrus. However, we failed to detect such marked elevations of serum FSH and LH at 2:00PM on proestrus day. Such different results can be explained by the difference in frequency between our blood collection and others'. An increase in pituitary and serum FSH and LH levels after ovariectomy is well established (Parlow, 1964). The time course of the response in References Anderson, R. R. and W. H. Mcshan (1966). Endocrinology 78, 976. Bogdanove, E. M., N. B. Schwartz, L. E. Reichert, Jr., and A. R. Midgley, Jr.(1971). Ibid. 88, 644. Brown, P.S.(1955). J, Endocrinol. 13, 59. Caligaris, L., J. J. Astrada, and S. Taleisnik (1967). Endocrinology 81, Daane, A. T. and A. F. Parlow (1971). Ibid. 88, 653. Gay, V. L. and A. R. Midgley, Jr.(1969). Ibid. 84, Greenwood, F. C., W. M. Hunter, and J. S. Glover (1963). Biochem. J. 89, 114. Hales, C. N. and P. J. Randle (1963). Ibid. 88, 137. Igarashi, M. and S. M. McCann (1964).

9 RADIOIMMUNOASSAYS FOR RAT FSH AND LH Endocrinology 74, 440. Kamioka, J. (1970). Acta. Obst. et Gynaec. Jap. 17, 168. Labhsetwar, A. P. (1969). J. Reprod. Fert. 18, 531. Mclintock, J. A. and N. B. Schwartz (1968). Endocrinology 83, 433. Monroe, S. E., A. F. Parlow, and A. R. Midgley, Jr. (1968). Ibid. 83, Monroe, S. E., R. W. Rebar, V. L. Gay, and A. R. Midgley, Jr. (1969). Ibid. 85, 720. Morgan, C. R., R. L. Sorenson, and A. Lazarow (1964). Diabetes 13, 579. Niswender, G. D., A. R. Midgley, Jr., S. E. Monroe, and L. E. Reichert, Jr. (1968). Proc. Soc. Exp. Biol. Med. 128, 807. Parlow, A. F. Human Pituitary Gonadotropins, (edited by A. Albert). Charles C Thomas, Springfield, Illinois, p.301 (1961). Parlow, A. F. (1964). Endocrinology 74, 489. Purves, H. D. and W. E. Griesbach (1955). Ibid. 56, 374. Ramirez, V. D. and S. M. McCann (1964). Ibid. 74, 814. Schwartz, N. B. and D. Bartosik (1962). Ibid. 71, 756. Steelman, S. L. and F. M. Pohley (1953). Ibid. 53, 604. Swerdloff, R. S., P.C. Walsh, H. S. Jacobs, and W. D. Odell (1971). Ibid. 88, 120. Yamamoto, M., N. D. Diebel, and E. M. Bogdanove (1970). Ibid. 87, 798.