Effects of Chronic LHRH Treatment on Brain LHRH Content, Pituitary and Plasma LH and Ovarian Follicular Activity in the Anestrous Ewe

BIOLOGY OF REPRODUCTION 20, 63 3-638 (1979) Effects of Chronic LHRH Treatment on Brain LHRH Content, Pituitary and Plasma LH and Ovarian Follicular Activity in the Anestrous Ewe BRUCE C. AMUNDSON and JON E. WUEATON2 Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108 ABSTRACT Nine mature, anestrous ewes were used in the experiment. Osmotic minipumps (AIza Corp.) containing 1.7 mg synthetic LURU in 170 pl saline were implanted s.c. into 4 sheep. Each ewe received 4 minipumps, implanted 1 week apart. This treatment resulted in a relatively uniform level (54 ± 2 pg/mi, X ± SEM) of LHRH-like immunoreactivity in plasma during the 4 week period. After 4 weeks, the sheep were killed and the stalk-median eminence (SME), hypothalamus proper (HP), preoptic-suprachiasmatic area (P0-SC), a sample of cerebral cortex (C) and the pineal gland (PG) were removed from the brain, homogenized in acidified ethanol and assayed for LI-IRH. Chronic exposure to LHRU was without effect on the contents of LHRH in the SME, HP or P0-SC, which were 50 ± 5, 5.1 ± 0.6 and 4.8 ± 0.8 ng, respectively. LHRU was not detected in C. LHRH treatment did increase the content of LHRH in the PG (treated = 125 ± 24 pg, control = 80 ± 12 pg, P<0.05). These data demonstrate that the midventral brain regions of the anestrous ewe do not take up or retain detectable amounts of exogenous LURU. In contrast, the PG has this ability. Each ewe responded to the initial minipump implantation with a rapid release of LH, reaching peak levels (110 ± 30 ng/mi) 3 h postimplantation, after which LH levels remained above those of control ewes for 24 h. There was also a release of LU following the second week implantation, but compared to the first it was less in magnitude and duration. Basal LU levels were measured during the remainder of the experiment. Continuous administration of LURU for 4 weeks reduced the content of LU in the anterior pituitary by 95% (P<0.0O1). In 3 of the 4 LHRH treated ewes, plasma progesterone levels increased and remained elevated for 12 days, indicative of postovulatory luteal function. Treatment reduced the number of ovarian follicles >2mm in diameter (P<0.01). The initial response of LU to the sustained administration of LHRU appears to be sufficient to induce ovulation and subsequent luteal function. The long term response, however, is not adequate to maintain ovarian follicular development. INTRODUCTION The hypothalamic content of luteinizing hormone releasing hormone (LURH) has been reported to change during the ovine estrous cycle, with highest levels occurring near the time of the preovulatory release of luteinizing hormone (Crighton et al., 1973; Wheaton, 1978). Bioassay of hypothalamic extracts from seasonally anestrous ewes revealed that the LHRH content during this reproductively quiescent period is as high as that found at anytime during the estrous cycle (Jackson et Accepted September 25, 1978. Received July 12, 1978. Scientific Journal Series Paper Number 10,418, of the Minnesota Agricultural Experiment Station, St. Paul. 2To whom requests for reprints should be addressed. al., 1971; Pelletier and Ortavant, 1968). Using radioimmunoassay, however, Wheaton (1978) recently reported that the content of LHRU in the stalk-median eminence (SME) of midseasonally anestrous ewes was only as high as that detected during the early stages of the estrous cycle, significantly less than that during proestrus-estrus. The hypothalamic region of the rat brain has been shown to take up exogenous LURH following a s.c. injection of LHRU (Corbin and Beattie, 1976), after microinjection of [ 2sI1 LHRH into the third ventricle (Recabbaren and Wheaton, 1978) or when incubated with [3H]-LHRH in vitro (Vaala and Knigge, 1974). In the anestrous ewe, on the other hand, infusion of LHRH for 24 h was not found to affect the LU releasing activity of the SME (Chakraborty et at., 1974). The present experiment examined the effects of 4 week treatment with synthetic LURH in anestrous ewes on the 633

634 AMUNDSON AND WHEATON regional brain content of LHRH, the anterior pituitary content of LU, plasma LU and on ovarian follicular development. MATERIALS AND METHODS Nine 2-year-old Suffolk ewes weighing 67 ± 4 kg ± SD) were used in the experiment. Sheep were penned with vasectomized marker rams and maintained under environmental lighting and temperature. The experiment was conducted in June and none of the ewes had exhibited behavioral estrus for at least 6 weeks. Osmotic minipumps (AIza Corp., Palo Alto, CA) containing LHRH were implanted into 4 ewes. Each of the ewes received a total of 4 minipumps, implanted s.c. under a forelimb 1 week apart. Each minipump contained 1.7 mg synthetic LURH (Abbott) dissolved in 170 p1 saline, an amount calculated to be released from the minipump at a rate of 10 pg/h for 170 h. Jugular blood samples were taken at hourly intervals on the days of implantation and otherwise twice daily. Control ewes were also bled twice daily. Blood was withdrawn into chilled heparinized vacutamer tubes, centrifuged and the plasma stored frozen until assayed for LHRH, LH and progesterone. After 4 weeks, ewes were transported to the abattoir and killed by exsanguination. The brain was removed and the stalk-median eminence (SME), hypothalamus proper (UP), preoptic-suprachiasmatic area (P0-SC), pineal gland (PG), anterior pituitary gland (AP) and a fragment of cortex (C) were excised and immediately frozen. The SME was separated from the AP and adjoining HP. The UP consisted of a block of tissue formed rostrally by a frontal cut made just behind the optic chiasm, caudally by a cut made immediately in front of the mammillary bodies, laterally by sagittal cuts made 3 mm from the opening of the 3rd ventricle and dorsally by a horizontal cut made 4 mm from the base of the HP. The P0-SC tissue block was limited rostro-caudally by a cut made 4 mm rostral to the optic chiasm and the cut forming the rostral border of the HP. Lateral and dorsal walls of the P0-SC were made by extending rostrally the corresponding cuts of the UP. A fragment of C approximately the size of the HP was also removed. Samples of nervous tissues were freeze dried and stored under vacuum until homogenized. Ovaries and uteri were removed, weighed and the number and diameter of ovarian follicles were recorded. The SME, UP, P0-SC area and C were homogenized in 2 ml of 0.2 N acetic acid:ethanol (1:1) and then centrifuged (20,000 X g for 20 mm). Aliquots (5 and 20 p1) of the supernatants were added to phosphate buffered saline (PBS) for LHRU radioimmunoassay. Pineal glands were extracted using the procedure described by Wheaton (1976). Individual glands were homogenized in 1 ml of 2 N acetic acid and centrifuged (20,000 X g for 20 mm). Four ml glacial acetic acid were added to the supernatant, mixed, diluted with H2 0 and freeze dried. Residues were dissolved in PBS, centrifuged (20,000 X g for 20 mm) and aliquots (100 and 200 p1) of the supernatants (adjusted to ph 7.0) were assayed for LURH. Anterior pituitary glands were homogenized in 15 ml PBS, centrifuged (15,000 X g for 30 mm) and the supernatants diluted 1:2,000 for LU assay. Radioimmunoassay of LHRH was conducted according to the protocol of Nett et al. (1973). Synthetic LHRH (Abbott) was used for radioiodination and reference material. Assay sensitivity was approximately 1.5 pg LHRH/tube. Radioimmunoassay of LU was conducted using the procedure reported by Niswender et al. (1969). Results are expressed in terms of NIH-LU-S19. Progesterone was assayed using the method of Garza et al. (1975), with the exception that plasma was twice extracted with 4 ml hexane, instead of the single 10 ml extraction. Assay sensitivity was 25 pg progesterone/tube. 150-60 - 40 - a- E - 10-8- = 6-.5 4-2- 150 - a- 550-50- 00- :i to 5 3 RESULTS Plasma concentrations of LHRH in ewes treated with osmotic minipumps containing LURH are shown in Fig. 1. Within 30 mm after the implantation of the first minipump, there was a significant rise in plasma LHRU levels. Peak levels were detected at 60 mm, after which concentrations declined to levels that remained relatively uniform during the course of the experiment. Endogenous plasma LHRHlike immunoreactivity in the control ewes during the experimental period are also mdi- Res,,,. 06 Ane000000 6*., to U45H In,ptont DAYS FIG. 1. Plasma LU levels and plasma LI-IRH-like immunoreactivity (X ± SEM) in 4 anestrous ewes implanted (4) at weekly intervals with osmotic minipumps containing synthetic LHRU. Progesterone concentrations are from 3 of the 4 treated ewes. The remaining ewe had basal (<0.5 ng/ml) levels of progesterone during the experimental period. The mean ± 95% confidence intervals for hormone levels in control anestrous ewes (n = 5) are indicated by the stippled areas.

BRAIN, PITUITARY AND OVARIAN RESPONSE TO CHRONIC LHRH 635 cated in Fig. 1. Levels of endogenous LHRHlike activity were significantly less than the levels of plasma LHRH detected after the administration of exogenous LHRH (split-plot analysis, df= 1,7). Four week treatment with LHRH was without effect on the LHRU contents of the SME, HP or P0-SC area (Table 1). LHRH was not detected in extracts of C from either group of sheep. Pineal glands from LHRH treated sheep did contain more LHRH than the glands from control sheep. Each ewe responded to the initial implantation of the minipump with a rapid release of LH (Fig. 1). Peak concentrations reached 110 ± 30 ng/ml (X ± SEM) and occurred 3 h postimplantation. LU levels were still elevated a day later, but by the 2nd day, concentrations were not significantly different from those of control ewes. Implantation of the second minipump also resulted in a rapid release of LH, which was, however, less in magnitude and duration than that which followed the implantation of the first minipump. Regardless of implantation of new minipumps during the 3rd and 4th weeks, plasma LH levels remained in the range of control concentrations for the remainder of the experiment. At the conclusion of the experiment, chronic exposure to elevated levels of plasma LHRH had reduced the AP content of LH by 95% (Table 2). AP weights were similar in the treated and control ewes. Basal concentrations of plasma progesterone were less than 0.3 ng/mt in all ewes (Fig. 1). In 2 of the 4 LHRH treated ewes, progesterone levels increased 4 days after the initial minipump implantation. In another ewe, it increased 4 days after the second implantation. Progesterone concentrations remained elevated in the 3 ewes for an average of 12 days. Basal progesterone levels were measured throughout the 4 week period in the other treated sheep. Uterine and ovarian weights were similar in the treated and control sheep but there was a significant reduction in the number of ovarian follicles greater than 2 mm in diameter in the sheep given LHRU (Table 3). None of the ewes was detected in estrus during the experimental period. DISCUSSION Weekly implantation of osmotic minipumps containing LURH resulted in a relatively uniform level of LHRU in plasma during the treatment period. Based upon the following equation for the determination of the secretion rate of an endocrine gland: X = A X VD X C x T, where A = 0.693 - t 1/2 (exogenous LHRH in anestrous sheep, t 1/2 = 6.7 mm (Nett et at., 1973), VD = distribution volume (assume 4 1), C = concentration of hormone in plasma (54 pg LHRH/ml), T = time conversion (60 mm/h), the calculated rate (X) at which LURH entered the systemic plasma was 1.3 pg/h, substantially less than the 10 pg/h theoretically released from the minipump. Apparently, a considerable amount of LHRH released from the minipump at the site of subcutaneous implantation is degraded or does not diffuse into the systemic circulation. In the C term of the equation, control levels of plasma LHRHlike immunoreactivity were not subtracted from the concentrations of LHRH in the treated sheep because exogenous LHRH has been previously shown to be quantitatively recovered from ovine plasma (Nett et at., 1973). Although endogenous LURU-tike immunoreactivity has been reported to exist in ovine plasma by a number of other investigators using different antisera (Nett et at., 1974; Jeffcoate et al., 1974; Kerdelhu et al., 1973), TABLE 1. Regional brain content ( ± SEM) of LHRH in anestrous ewes treated for 4 weeks with osmotic minipumps containing synthetic LURH and in control anestrous ewes. LHRH Tissue Treated Control Stalk-median eminence 43 ± 3 ng 50 ± 5 ng Hypothalamus proper 5.1 ± 0.9 ng 5.1 ± 0.6 ng Preoptic-suprachiasmatic area 3.2 ± 0.3 ng 4.8 ± 0.8 ng Pineal gland 125 ± 24 pg 80 ± 12 pg ap<0o5 t test.

636 AMUNDSON AND WHEATON TABLE 2. Anterior pituitary content of LH and weight (X ± SEM) in anestrous ewes treated for 4 weeks with synthetic LURH and in control anestrous ewes. - LHRH Anterior pituitary Treated Control LU content (pg/gland) 104 ± 8 1911 ± 248 Dry weight (mg) 210 ± 35 230 ± 21 test. the source of the activity has been questioned (Nett and Adams, 1977;Jonas et al., 1975). Exposure to circulating LURH for 4 weeks did not elevate the contents of LHRH in the SME, HP, P0-SC area or C. Likewise, Chakraborty et al. (1974) reported that the infusion of 2.3 pg LHRH/h for 24 h into anestrous ewes had no effect on hypothalamic LU releasing activity. These data indicate that the midventral brain regions of the anestrous ewe either do not take up or retain detectable amounts of exogenous LHRH. Further research is needed to determine if this response is also typical of ewes during the breeding season. Pineal glands of treated sheep, on the other hand, did contain more LHRH than those of control animals. LHRH has been identified in the ovine PG using immunohistochemistry (Kozlowski and Zimmerman, 1974). White et al. (1974) reported that large quantities of LHRH are present in extracts of sheep PGs. Wheaton and Harsdorf (1977) also have demonstrated the existence of LHRH in the ovine PG using radioimmunoassay and bioassay, although the amounts detected were much less than those described by White et at. (1974). The PG of the rat has been shown to take up exogenous LHRH (Dupont et al., 1974; Redding and Schally, 1973). Implantation of the initial osmotic minipump resulted in a rapid discharge of pituitary LH. After 24 h, LH levels had decreased, but still remained above control levels. Implantation of a second minipump also produced a release of LH, but it was smaller in magnitude and duration. Imptantations of new minipumps during the 3rd and 4th weeks did not elicit significant elevations in plasma LU levels. In other investigations, infusions of LHRH have led to only a short lived release of LH. Infusion of LHRH for 24 h into anestrous ewes produced peak plasma LH levels by 3 h after which LH had returned to preinfusion levels by 16 h (Chakraborty et at., 1974). Infusion of LHRH into ovariectomized sheep resulted in peak plasma LU levels by 1.5 h and a return to preinfusion levels by 12 h (Piper et at., 1973). The tack of a sustained release of LH in response to the continuous administration of LHRH appears to be due, at least in part, to the reduction of LU in the AP. Chakraborty et at. (1974) observed a 75% reduction in the AP content of LH after a 24 h infusion. At the termination of the present study, LH stores in the AP had been reduced by 95%. Also, a decrease in the responsiveness of the ovine gonadotroph to the frequent injection of TABLE 3. Uterine and ovarian weight (X ± SEM) and the total number of ovarian follicles greater than 2 mm in diameter in anestrous ewes (n = 4) treated with synthetic LHRH for 4 weeks and in control anestrous ewes (n = 5). LHRH Treated Control Uterine weight (g) 25 ± 4 27 ± 2 Ovarian weight (g) 1.8 ± 0.2 2.5 ± 0.4 Number of ovarian follicles >2 mm ia 11 Mean follicular diameter (mm) 2 4.8 ap<ool Chi-square.

BRAIN, PITUITARY AND OVARIAN RESPONSE TO CHRONIC LHRH 637 LHRH has been demonstrated, without a coincident reduction in the AP content of LU (Rippel et a!., 1974). This so called down regulation may contribute to the limited duration of LH release. Three of the 4 anestrous ewes treated with LHRH exhibited plasma progesterone profiles which resembled those occurring during the tuteal phase of the estrous cycle. Ovulation has been reported to occur in more than half the anestrous ewes treated with a single injection of synthetic LHRH (Haresign et at., 1975; Crighton et at., 1975). Nonetheless, in most of the ewes that ovulated, postovutatory plasma progesterone levels remained basal. A single injection of the LHRH analog, D-Leu6 des G1yNH2 #{176}-LHRH ethylamide, which releases more LU than does LHRH in the anestrous ewe (Rippel et at., 1975), also induces ovulation, but in contrast to LHRH, in analog treated ewes there is a postovutatory rise in serum progesterone levels (Frandle et at., 1977). The release of LH which followed the implantation of the first osmotic minipump was not greater in magnitude than that which follows a single injection of LHRH, but it was longer in duration. These data indicate that the extended exposure of granulosa cells to elevated levels of LH is necessary fo the formation of a functional corpus luteum. Plasma LU levels in the ewe that did not reflect a rise in plasma progesterone levels appeared to be similar to the others. This and the results from other aforementioned investigations, indicate that for unknown reasons, some anestrous ewes do not ovulate or form a functioqal corpus luteum in response to the release of LU induced by treatment with LHRH. There was a significant reduction in the number of ovarian follicles greater than 2 mm in diameter in the anestrous sheep treated with LHRH for 4 weeks. Plasma LH levels in the control ewes were observed to fluctuate with pulses reaching up to 13 ng/ml. Similar fluctuations have previously been reported to occur in the anestrous ewe (Scaramuzzi and Baird, 1977; Yuthasastrakosal et at., 1975). Although plasma LH levels in the LHRH-treated sheep were within the range of control levels, they did not reveal the same degree of fluctuation. It is possible that these periodic pulses of circulating LH are necessary for follicular growth. Also, plasma levels of FSH may have been reduced by treatment to such an extent that follicutar development was minimized. ACKNOWLEDGMENTS Appreciation is expressed to Dr. T. M. Nets (Colorado State University) for providing LHRU antiserum (#42), to Dr. G. D. Niswender (Colorado State University) for ovine LU antiserum (#15) and to Dr. L. E. Reichert (Emory University) for highly purified ovine LU used for radioiodination. NIH-LH-S19 was supplied through the NIAMDD Pituitary Hormone Program. The authors wish to thank Abbott Laboratories for supplying synthetic LURH. REFERENCES Chakraborty, P. K., Adams, T. E., Tarnavsky, G. K. and Reeves, J. J. (1974). Serum and pituitary LU concentrations in ewes infused with LH-RH/ FSH-RH. J. An. Sci. 39, 1150-1157. Corbin, A. and Beattie, C. W. (1976). Effect of luteinizing hormone-releasing hormone (LHRH) and an LHRH antagonist on hypothalamic and plasma LHRH of hypophysectomized rats. Endocrinology 98, 247-2 50. Crighton, D. B., Foster, J. P., Haresign, W. and Scott, S. A. (1975). Plasma LU and progesterone levels after single or multiple injections of synthetic LU-RH in anestrous ewes and comparison with levels during the oestrous cycle. J. Reprod. Fert. 44, 121-124. Crighton, D. B., Hartley, B. M. and Hamming, G. E. (1973). Changes in the luteinizing hormone releasing activity of the hypothalamus, and in pituitary gland and plasma luteinizing hormone during the oestrous cycle of the sheep. J. Endocrinol. 58, 377-385. Dupont, A., Labrie, F., Pelletier, G., Puviani, R., Coy, D. H., Coy, E. J. and Schally, A. V. (1974). Organic distribution of radioactivity and disappearance of radioactivity from plasma after administration of 3U-luteinizing hormone-releasing hormone to mice and rats. Neuroendocrinology 16, 65-73. Frandle, K. A., Kinder, J. E., Coy, D. U., Schally, A. V., Reeves, J. J. and Estergreen, V. L (1977). Plasma progestins in anestrous ewes treated with D-Leu Des GIyNH2-LHRH Ethylamide. J. An. Sci. 46, 486-491. Garza, R., Burns, B., Manlimos, F. and Abraham, C. (1975). Radioimmunoassay of plasma progesterone without chromatography. Anal. Let. 8, 225-23 2. 1-laresign, W., Foster, J. P., I-Iaynes, N. B., Crighton, D. B. and Lamming, G. E. (1975). Progesterone levels following treatment of seasonally anoestrous ewes with synthetic LU-releasing hormone. J. Reprod. Fert. 43, 269-279. Jackson, C. L., Roche, J. F., Foster, D. L. and Dzuik, P. J. (1971). Luteinizing hormone releasing activity in the hypothalamus of anestrous and cyclic ewes. Biol. Reprod. 5, 5-12. Jeffcoate, S. L., Fraser, U. M., Holland, D. T. and Gunn, A. (1974). Radioimmunoassay of luteinizing hormone-releasing hormone (LU-RH) in serum from man, sheep and rat. Acta Endocrinol. 75, 625-635. Jonas, H. A., Burger, U. G., Cumming, I. A., Findlay, J. K. and Dekretser, D. M. (1975). Radioim-

638 AMUNDSON AND WUEATON munoassay for luteinizing hormone-releasing hormone (LURH): Its application to the measurement of LHRH in ovine and human plasma. Endocrinology 96, 384-393. Kerdelhu, B., Jutisz, M., Gillessen, D. and Snider, R. 0. (1973). Obtention of antisera against a hypothalamic decapeptide (luteinizing hormone/follicle stimulating hormone releasing hormone) which stimulates the release of pituitary gonadotrophins and development of its radioimmunoassay. Biochimica Biophysica Acta 297, 540-548. Kozlowski, C. P. and Zimmerman, E. A. (1974). Localization of gonadotropin-releasing hormone (Gn-RU) in sheep and mouse brain. Anat. Rec. 178, 396 Abstr. Nett, T. M. and Adams, T. E. (1977). Further studies on the radioimmunoassay of gonadotropinreleasing hormone: Effect of radioiodination, antiserum and unextracted serum on levels of immunoreactivity in serum. Endocrinology 101, 113 5-1144. Nett, T. M., Akbar, A. M. and Niswender, C. D. (1974). Serum levels of luteinizing hormone and gonadotropin-releasing hormone in cycling, castrated and anestrous ewes. Endocrinology 94, 7 13-718. Nett, T. M., Akbar, A. M., Niswender, G. D., Hedlund, M. T. and White, W. F. (1973). A radioimmunoassay for gonadotropin-releasing hormone (Gn- RH) in serum. J. Clin. Endocr. Metab. 36, 880-885. Niswender, C. D., Reichert, L. E., Jr., Midgley, A. R., Jr., and Nalbandov, A. V. (1969). Radioimmunoassay for bovine and ovine luteinizing hormone. Endocrinology 84, 1166-1173. Pelletier, J., and Ortavant, R. (1968). Influence de Ia dur#{233}e quotidienne d #{233}clairement sur l activit hypothalamique LRF du Belier. Compt. Rend. 266, D1604-1606. Piper, E. H., Perkins, J. U., Tugwell, D. R. and Vaught, W. G. (1973). Effect of LRF infusions on LH secretion patterns. J. An. Sci. 37, 324. Abstr. Recabarren, S. E. and Wheaton, J. (1978). Estradiol potentiation of hypothalamic uptake of LU-RH from the CSF. Neuroendocrinology 27, 1-8. Redding, T. W. and Schally, A. V. (1973). The distribution, half-life and excretion of tritiated luteinizing hormone-releasing hormone (LU-RH) in rats. Life Sci. 12, 23-32. Rippel, R. H., Johnson, E. S. and White, W. F. (1974). Effect of consecutive injections of synthetic gonadotropin releasing hormone on LU release in the anestrous and ovariectomized ewe. J. An. Sci. 39, 907-914. Rippel, R. H., Johnson, E. S., White, W. F., Fujino, M., Fukuda, T. and Kobayashi, 5. (1975). Ovulation and gonadotropin-releasing activity of (D-leu, des gly-nh2 0, pro-ethylamide)-gn-rh, Proc. Soc. Exp. Biol. Med. 148, 1193-1197. Scaramuzzi, R. J. and Baird, D. T. (1977). Pulsatile release of luteinizing hormone and the secretion of ovarian steroids in sheep during anestrus. Endocrinology 101, 1801-1806. Vaala, S. S. and Knigge, K. M. (1974). Transport capacity of the median eminence; in vitro uptake of 3H-LRF. Neuroendocrinology 15, 147-157. Wheaton, J. E. (1976). LH-RH immunological activity in ovine pineal glands. J. An. Sci. 43, 310 Abstr. Wheaton, J. E. (1978). Regional brain content of LHRH in sheep during the estrous cycle, seasonal anestrus and after ovariectomy. Endocrinology, In Press. Wheaton, J. E. and Uarsdorf, S. E. (1977). Biological and immunological LU-RH activity in ovine pineal glands. Fed. Proc. 36, 279 Abstr. White, W. F., Hedlund, M. T., Weber, C. F., Rippel, R. H., Johnson, E. F. and Wilber, J. R. (1974). Pineal gland: a supplemental source of hypothalamic-releasing hormones. Endocrinology 94, 1422-1426. Yuthasastrakosol, P., Palmer, W. M. and Howland, B. F. (1975). Luteinizing hormone, oestrogen and progesterone levels in peripheral serum of ewes as determined by radioimmunoassay. J. Reprod. Fert. 43, 57-65.