CHANGES IN SERUM LEVELS OF LH AND FSH IN PROGESTERONE.TREATED OVARIECTOMIZED EWES FOLLOWING A SINGLE INJECTION OF ESTROGEN

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1 CHANGES N SERUM LEVELS OF LH AND FSH N PROGESTERONE.TREATED OVARECTOMZED EWES FOLLOWNG A SNGLE NJECTON OF ESTROGEN B. E. HOWLAND, W. M. PALMER'?, andl. M. SANFORD' Departments of roral Biology and zanimal Science, University of Manitoba, Winnipeg, Man. R3 E 0W3. Received Aug. 1977, accepted 7 Nov Howr-rNo, B. 8., Perruen, W. M. rno SlNnono, L. M Changes in serum levels of LH and FSH in progesterone-treated ovariectomized ewes following a single injection of estrogen. Can. J. Anim. Sci. 58: 15-. The effect of dose and duration of progesterone treatment on estrogen-induced LH and FSH release was studied in ovariectomized ewes. n each of two years, groups of ewes were injected with 5 or l0 mg progesterone daily for 4 or 10 days before an injection of mg estradiol-17b. Experiments were conducted in early July (year 1) or mid-augus t (year ). Jugular blood was collected immediately before and hourly from 14 to 30-h after estrogen injection. Serum hormone levels were determined by radioimmunoassay. Combined data from the two years showed that an LH release occurred in four of five ewes treated with 5 mg progesterone for either 4 or l0 days, five of five ewes treated with l0 mg progesterone for 4 days but only one of five ewes treated with 10 mg progesterone for l0 days. The onset of estrus and the rise in LH both tended to occur earlier after estrogen treatment in year than in year l, suggesting an effect of season. Only one ewe showed a simultaneous release of LH and FSH; in the remainder, the levels of FSH declined after estrogen injection and remained depressed throughout the sampling period. Mean FSH levbls tended to be higher in ewes receiving l0 mg progesterone than in ewes receiving 5 mg progesterone in year, but the reverse tended to occur in year (progesterone dose x year, P <.05). The results suggest that a 10-day treatment with a moderate dose of progesterone can reduce the incidence of LH release induced by a high dose of estrogen and that the estrogen-induced LH release in progesterone-treated ovariectomized ewes is usually not accompanied by a release of FSH. On a 6tudi6 sur des brebis ovariectomis6es les effets de la dose et de la dur6e d'un traitement h la progest6rone sur 1a d6charge oestrog6nique de LH et de FSH. Pendant ans, des groupes de brebis ont regu par injection 5 ou 10 mg de progesterone par jour pour 4 ou l0jours avant une injection d'estradiol-17. Les essais ont 6t6 r6alis6s au d6but dejuillet (1"" ann6e) ou en d6but d'ao0t ("). Des pr6lbvements de sang jugulaire ont 6t6 faits imm6diatement avant et ensuite toutes les heures de l4 A 30 h aprds 'injection de 'oestrogbne. Les teneurs hormonales s6riques ont 6t6 d6termin6es par dpreuve de radio-immunit6. Les r6sultats r6unis des deux ann6es montrent que la lib6ration de LH s'est produite chez 4 des 5 brebis trait6es h 4 mg de progest6rone pendant 4 ou 10 jours, chez toutes les cinq brebis recevant l0 mg pendant 4 jours, mais chez seulement 1 des 5 brebis recevant cette dose pendant 10 jours. Le d6clenchement de l'oestrus et la mont6e du taux de LH se sont manifest6s plus vite aprbs le traitement oestrogbne dans la " ann6e que dans la 1"", ce qui laisse soupgonner un effet de saison. Une brebis seulement a enregistr6 une d6charge simultan6e de LH et de FSH. Chez les autres, les valeurs FSH ont baiss6 aprbs l'injection d'oestrogbne et sont demeur6es faibles durant toute la p6riode de pr6lbvement. La deuxidme ann6e, les taux moyens de FSH 6taient plus 6lev6s chez les brebis trait6es h l0 mg qu'h 5 mg de progest6rone, mais 'inverse semble s'otre produit dans la premidre ann6e (interaction significative ir P < 0.05 dose de Can. J. Anim. Sci.5t: 15- (Mar. l97t) 15

2 16 canadan JouRNAL of ANMAL scrence progest6rone-ann6e). La conclusion qui se d6gage des travaux est qu'un traitement mod6r6 de progest6rone pendant l0 jours peut r6duire les cas de d6charge de LH provoqu6e par une forte dose d'oestrogbne et que la lib6ration de LH sous commande oestrog6nique chez les brebis ovariectomis6es trait6es d la progest6rone ne s'accompagne pas habituellement d'une d6charge de FSH. Treatment with estrogen can induce a release of LH in anestrous (Goding et al. 1969; Beck and Reeves 1973), ovariectomized (Radford et al. 1969; Scaramuzzi et a. l97l; Howland and Palmer 1973) and cyclic ewes (Bolt et al. l97l; Howland et al. l91l). n cyclic ewes, a relatively high dose of estrogen is required to induce a release of LH which is small compared with the normal preovulatory LH release, and treatment is effective early in the cycle but not at mid-cycle (Bolt et al. l97l; Howland et al. 1971). The relative ineffectiveness of estrogen in inducing LH release in the cyclic ewe may be due to the inhibitory action of progesterone. At mid-cycle, the serum progesterone levels are high and have been elevated for a number of days. hogesterone given along with a low dose of estrogen effectively blocks the estrogeninduced release of LH in ovariectomized ewes (Scaramuzzi et ai. l91l). However, when a high dose of estrogen (one that is effective in cyclic ewes) is administered to ovariectomized ewes, progesterone injected at 0, 4,8 or 4 h before the estrogen does not block the LH release (Yuthasastrakosol et al. 1974). Thus the effectiveness of progesterone in blocking an estrogeninduced LH release may depend on duration of exposure to endogenous or exogenous progesterone, as well as the circulating blood level. The present study was conducted to determine the effects of dose of progesterone and duration of treatment on estrogen-induced LH and FSH release in ovariectomized ewes. MATERALS AND METHODS n each of two years, groups of two or three ewes, which had been ovariectomized for at least 1 mo, were injected with 5 or l0 mg progesterone daily for either 4 or l0 days prior to an injection of mg estradiol-17b. n year l, four Finnish Landrace, two Suffolk and four crossbred ewes were employed. n year, five Finnish Landrace and five crossbred ewes were used. Ewes were assigned to treatment groups at random. n year l, the estrogen was injected on 4 July and in year on 0 Aug. Both steroids were dissolved in peanut oil and injected intramuscularly. Progesterone was injected at 0800 h each day including the day after estrogen treatment. The estrogen was injected at 800 h on the 4th or loth day of progesterone treatment. Jugular blood was collected in evacuated tubes immediately before and hourly from 14 to 30 h after estrogen injection. Rams were kept with the ewes and observations for estrus were made during the period of frequent blood sampling. Blood samples were chilled immediately on ice and centrifuged within 36 h to recover serum. Serum was stored at -0"C until assayed. Serum levels of LH were determined by radioimmunoassay (Niswender et al. 1969) using methodology previously described (Howland 197). Antlbody #573 was used at an initial dilution of 1:100,000. Concentrations of LH are expressed in terms of NH-LH-S14. The serum levels of FSH were measured by a multi-species double antibody radioimmunoassay (Sanford et al. 1976). Concentrations of FSH are expressed in terms of NH-FSH-S6. The concentration of progesterone in serum was determined in year only by assaying serum collected at 14, and 30 h after estrogen injection. These time periods corresponded to 0, 8 and 16 h after progesterone injection. Progesterone was measured by radioimmunoassay as described by Yuthasastrakosol etal. (1974).

3 HOWLAND ET AL.-LH RELEASE N EWES t7 Table 1. Effects of progesterone dosage, heatment duration and year on peak serum LH concentration, time of peak LH and mean FSH concentration after a single injection of estradiol-l7b Dose (mgl Treatment duration (days) Year No. ewes Peak LH Time of Meant FSH conc. peak LH conc. (ng/ml) (h) (ng/ml) l0 l0 10 fthe value for each ewe is the mean of 8 observations. *Values are means for numbers of ewes indicated. Where applicable, the data were analyzed by analysis of variance. RESULTS The serum LH profiles for individual ewes after estrogen treatment are shown in Fig. 1. A ewe was presumed to have responded to estrogen if a serum LH value, during the period h after estrogen treatment, exceeded 0 nglml and was greater than the pre-estrogen value. Using these criteria, an LH release occurred in four of five ewes treated with 5 mg progesterone for either 4 or 10 days, five of five ewes treated with 10 mg progesterone for 4 days and one of five ewes treated with l0 mg progesterone for 10 days. Multiphasic LH releases were noted in several animals in year 1 but this pattern ofrelease was not observed inyear. Source Peak LH conc ' '7 The highest LH value between 14 and 30 h after estrogen for each ewe was considered the peak LH concentration. Means for peak LH concentrations and time of peak LH are shown in Table l The interaction of progesterone dose'with days of treatment for peak LH concentration approached significance atlhe 5Vo level (Table ). This interaction is consistent with the observation that four of five ewes treated with 10 mg progesterone for l0 days failed to release LH in response to estrogen. The timing of the peak LH concentration relative to estrogen injection tended to differ between years (Tables and ). The rise in serum LH in ewes that responded to estrogen occurred earlier in year than in year 1 (Fig. ). Table. Mean squares forpeak LH concentration, time ofpeak LH and mean FSH concentration Progesterone (P) Days of treatment (D) Year (Y) PD PY DY PDY Error *P <.05, *x*p <.1 t 84 1,184 8s4 t 56Ra*x Time of peak LH ** Mean FSH conc.,16 4l 36,068*** 5,5r9 88,866* 10,36 3,'763 9,83'7

4 18 CANADAN JOURNAL OF ANMAL SCENCE The change in serum FSH concentration after estrogen injection is shown in Fig. 3. With the exception of one ewe, serum FSH concentrations decreased during the initial 14 h after estrogen and then declined at a much slower rate during the remainder of the sampling period. An analysis of variance of mean FSH values for each ewe (mean of all samples collected) revealed an interaction (P <.05) of progesterone dose with year (Tables and ). This interaction was the result of higher FSH values in ewes receiving l0 mg progesterone than in ewes receiving 5 mg progesterone in year, while the reverse tended to occur in year 1. The treatment differences in mean FSH concentration were not due to differences in -c \or C:') ro6r c Bo response to estrogen. The same interaction was significant when pre-estrogen FSH values were analysed (data not shown). At present we can offer no explanation for this interaction. One ewe receiving l0 mg progesterone for 10 days responded to estrogen with a release of LH. n contrast to the other ewes that released LH, this one had an accompanying FSH release (Fig. a). The mean (-+- SE) serum progesterone concentrations at 0,8 and 16 h after progesterone injection were , and.17!.03 ng/ml, respectively, for ewes injected with 5 mg progesterone and.85 -f.04, f.14 and r-.10 ng/ml, respectively, for ewes injected with Yeor Yeor i t\- ot4t6t doys lo doys Hours Affer Estrogen Fig. la. Serum profiles of LH for individual ewes after injection of mg estradiol-178 following treatment with 5 mg progesterone daily for 4 or l0 days in each of two years (F, Finnish Landrace; S, Suffolk: C. crossbred).

5 HOWLAND ET AL.-LH RELEASE N EWES t9 10 mg progesterone. t is assumed that these values are representative of the daily progesterone profiles over the duration of treatment. ln year l, seven of ten ewes came into estrus 17-8 h after estrogen injection. n year, eight of ten ewes were observed in estrus 14- l8 after estrogen injection. There was no indication that dose or duration of progesterone treatment affected the incidence or time of estrus. DSCUSSON This study was designed to determine whether the serum level of progesterone, the duration of exposure of the hypothalamic-pituitary complex to proges- L c.) c J 40 r to. Yeor terone, or both, are responsible for the reported difference in the ability of estrogen to induce LH release at days 3 or 4 compared with days 10 or 1l of the cycle (Bolt et al. l97l; Howland et al. l91l). Ovariectomized animals were used as a model to avoid effects of endogenous gonadal steroids and the duration ofprogesterone treatment was 4 or 10 days in order to simulate the duration of progesterone exposure corresponding to days 4 or 10 of the estrous cycle. The low and high doses of progesterone were anticipated to achieve serum levels of progesterone similar to those observed at days 4 or l0 of the cycle, respectively. Although l0 mg progesterone for 10 days appeared to interfere with the 4 doys lo doys i"\r Yeor \,' t\ ti ic,jr. ffi o1416ra046'450 Hours After Estrogen Fig. lb. Serum profiles of LH for individual ewes after injection of mg estradiol-17b following treatment with l0 mg progesterone daily for 4 or 10 days in each of two years (F, Finnish Landrace; S, Suffolk; C, crossbred).

6 0 CANADAN JOURNAL OF ANMAL SCENCE =60 E o50 5 r40 J lltt.r o 14 ',",1. o1,",",?,,""1*'" a 30 f ig.. Mean (+ SE) serum levels of LH in ewes with an estrogen-induced release of LH in year (O-O) and year (O O). Means represent eight and six animals in year and, respectively. estrogen-induced LH release, the maximum serum levels achieved (8 h after treatment) were considerably lower than the values measured in ewes on day l0 of the estrous cycle (Yuthasastrakosol et al. 1975). However, this daily dose of progesterone is known to be effective in preventing estrus (Dutt and Casida 1948) and presumably is effective in inhibiting gonadotropin release. The results of the present study suggest that treatment with daily injections of 10 mg progesterone for l0 days can reduce the incidence of the estrogen-induced LH release. The previous observation that 10 mg or even 30 mg of progesterone injected prior to mg estradiol-l7b failed to block LH release (Yuthasastrakosol et al. l9l4) can be explained by insufficient duration of treatment. t appears that high blood levels of progesterone per se do not block the positive effect of a large dose (1 mg) of estradiol-176 but somewhat lower blood levels of progesterone over a 1O-day period can result in a blocking effect. Scaramuzzi et al. (1971) reported that estrogen and progesterone given simultaneously to ovariectomized ewes results in no LH r a L o1416 a Hours Afler Estrogen Fig. 3. Mean (+ SE) serum levels of FSH in ewes following estrogen injection. Each value is based on 0 observations. release. However, the ewes had been treated for an ll-day period with progesterone prior to the combined steroid treatment and it is possible that the progesterone injected with the estrogen insured the continuation of the previously established refractory state. This study was not designed to determine seasonal differences in response to estrogen. However, the differences between years, in timing of LH peaks and estrus cannot be ignored. Both estrus and the rise in LH tended to occur earlier after estrogen treatment in year (mid-august) than year 50?oo 530 )?a aat Hours Afler Eslrogen :oo l AA Fig. 4. Serum profiles of LH (O-O) and FSH (O-O) in an ovariectomized ewe after treatment with 10 mg progesterone for l0 days before a single injection of mg estradiol- 17B. r00

7 HOWLAND ET AL.-LH RELEASE N EWES 1 1 (early July). A shorter interval between estrogen treatment and estrus was reported by Fletcher and Lindsay (l9ll) for ovariectomized ewes treated during the breeding season as compared with the non-breeding season. n year l, a number of multiphasic LH releases were noted. This unusual release pattern could be characteristic of individual ewes studied in year or could represent a seasonal effect in progesterone-treated ovariectomized ewes. n a subsequent experiment (unpublished) estrogen-treated ovariectomized ewes that had not received progesterone failed to show multiphasic releases of LH in either June or July. Although breed differences in responsiveness to estrogen have been reported (Land et al. 1976), the observed differences between progesterone treatment groups and between years in the present study cannot be readily explained as breed effects. Of the four ewes that failed to respond to estrogen after treatment with 10 mg progesterone for 10 days, two were Finnish Landrace ewes and two were crossbreds. Finnish Landrace ewes might be expected to be less responsive to estrogen than ewes of less prolific breeds (Land et al. 1916), however, rhe only ewe that responded to estrogen in the above mentioned group was a Finnish Landrace. A difference in the mean LH profile between years (Fig. ) occurred in spite of the fact that Finnish Landrace ewes were represented in approximately equal numbers in each year. n all but one ewe, serum FSH concentrations declined within the first 14 h after estrogen injection and remained suppressed for the remainder of the sampling period. A simultaneous rise and fall in FSH and LH was observed in the remaining ewe. This response was similar to the simultaneous release of both gonadotropins that occurs in anestrous ewes following estrogen administration (Jonas et al. l9l3; Reeves et al. 1974; Pant and Ward 1974) or treatment with GnRH (Reeves et al. 197; Jonas et al. 1973). The preovulatory release of LH in cyclic ewes is also accompanied by a release of FSH (Salamonsen et al. 1973). The surge of both gonadotropins occurring after estrogen treatment in anestrous ewes or preceding ovulation in cyclic ewes could be due to a release of GnRH from the hypothalamus. Recent work by Coppings and Malven (197O indicates that an alternative explanation, i.e. facilitation of pituitary response to GnRH by estrogen, cannot account for the release of LH following estrogen treatment. The lack of a simultaneous release of both gonadotropins in most ewes in the present study suggests that GnRH released from the hypothalamus was more effective in bringing about LH rather than FSH release. This could be related to the amount of GnRH released or the relative responsiveness of LH- and FSH-secreting pituitary cells. Whether either of these possibilities is related to the ovariectomized state or progesterone treatment remains to be determined. ACKNOWLEDGMENTS This study was supported by grants A 648, A 651 and A 0107 from the National Research Council of Canada. The authors thank Drs. G. D. Niswender, Colorado State University, and L. E. Reicher, Jr., Emory University and the Endocrlne Study Section, NH, for providing radioimmunoassay materials. We are grateful to Dr. G. D. Phillips for ovariectomizing the ewes, to Dr. P. Yuthasastrakosol for assaying progesterone and to D. B. Beaton for other technical assrstance. BECK, T. W. and REEVES, J. J Serum luteinizing hormone (LH) in ewes treated with various dosages of 17B-estradiol at three stages of the anestrous season. J. Anim. Sci. 36: BOLT, D. J., KELLEY, H. E. and HAWK, H. W Release of LH by estradiol in cycling ewes. Biol. Reprod. 4: COPPNGS, R. J. and MALVEN, P. V Biphasic effect of estradiol on mechanisms regulating LH release in overiectomized sheep. Neuroendocrin olo gy l: DUTT, R. H. and CASDA, L. E Alteration of the estrual cycle in sheep by use of progesterone and its effect upon subsequent

8 CANADAN JOURNAL OF ANMAL SCENCE ovulation and fertility. Endocrinology 43: 08- t7. FLETCHER,. C. and LNDSAY, D. R Effect of oestrogen on oestrous behavior and its variation with season in the ewe. J. Endocrinol. 50: GODNG, J, R., CATT, K. J., BROWN, J. M., KALTENBACH, C. C., CUMMNG,. A. and MOLE, B. J Radioimmunossay for ovine luteinizing hormone. Secretion of luteinizing hormone during estrus and following estrogen administration in the sheep. Endocrinology 85: t33-t4. HOWLAND, B. E., AKBAR, A. M. and STORMSHAK. F Serum LH levels and luteal weight in ewes following a single injection of estradiol. Biol. Reprod. 5: 5-9. HOWLAND, B. E Effect of restricted feed intake on LH levels in female rats. J. Anim. Sci. 34: HOWLAND, B. E. and PALMER, W. M Estrogen-induced changes in serum luteinizing hormone levels in ovariectomized ewes. Can. J. Anim. Sci. 53: JONAS, H. E., SALAMONSEN, L. A., BURGER, H. G., CHAMLEY, W. A., CUM- MNG,. A., FNDLAY, J. K. and GODNG, i. R Release of FSH after administration of gonadotropin-releasing hormone or estradiol to the anestrous ewe. Endocrinology 9: 86-86s. LAND, R. B., WHEELER, A. G. and CARR, W. R Seasonal variation in the oestrogen induced LH discharge of ovariectomized Finnish Landrace and Scottish Blackface ewes. Ann. Biol. Anim. Biochim. Biophys. 16: NSWENDER, G. D., RECHERT, L. E., JN., MDGLEY, A. R., Jr.., and NALBANDOV, A. V Radioimmunoassay for bovine and ovine luteinizing hormone. Endocrinology 84: l166-1r73. PANT, H. C. and WARD, W. R Effect of intravenous infusion of oestradiol-178. with and without prior progesterone treatment, on the plasma luteinizing hormone and folliclestimulating hormone concentrations in anoestrous ewes. J. Endocrinol. 61: V-V (Abstr.). RADFORD, H. M., WHEATLEY,. S. ANd WALLACE, A. L. C The effects of oestradiol benzoate and progesterone on secretion of luteinizing hormone in the ovariectomized ewe. J. Endocrinol. 44: REEVES, J. J., ARMURA, A., SCHALLY, A. V., KRAGT, C. L., BECK, T. W. ANd CASEY, J. M Effects of synthetic luteinizing hormone-releasing hormone/follicle stimulating hormone-releasing hormone (LH- RH/FSH-RH) on serum LH, serum FSH and ovulation in anestrous ewes. J. Anim. Sci. 35: REEVES, J. J., BECK, T. W. and NETT, T. M Serum FSH in anestrous ewes treated with 17 -estradiol. J. Anim. Sci. 38: SALAMONSEN, L. A., JONAS, H. A., BURGER. H. G., BUCKMASTER, J, M., CHAMLEY. W. A., CUMMNG,. A., FNDLAY. J. K. and GODNG, J. R A heterologous radioimmunoassay for folliclestimulating hormone: Application to measurement of FSH in the ovine estrous cycle and in several other species including man. Endocrinology 93: SANFORD, L. M., FAMAN, C., HOW- LAND. B. E. and PALMER, W. M The profile of follicle-stimulating hormone secretion in the ram. Can. J. Anim. Sci. 56: SCARAMUZZ, R. J., TLLSON, S. A., THORNEYCROFT,. H. and CALDWELL, B. V Action of exogenous progesterone and estrogen on behaviorial estrus and luteinizing hormone levels in the ovariectomized ewe. Endocrinology E8: YUTHASASTRAKOSOL, P., HOWLAND, B. E.. SMARAKS, S. and PALMER, W. M Estrogen-induced LH release in progesterone-treated ovariectomized ewes. Can. J. Anim. Sci.54: YUTHASASTRAKOSOL, P., PALMER, W. M. and HOWLAND, B. E Luteinizing hormone, oestrogen and progesterone levels in peripheral serum of anoestrous and cyclic ewes as determined by radioimmunoassay. J. Reprod. Fertil. 43:57-65.

9 This article has been cited by: 1. D.L. Hamernik, T.M. Nett Measurement of the amount of mrna for gonadotropins during an estradiol-induced preovulatory-like surge of LH and FSH in ovariectomized ewes. Domestic Animal Endocrinology 5:, [Crossref]. L.N. Kanchev, B.M. Stankov, H. Dobson Early pituitary follicle stimulating hormone response to gonadotrophin releasing hormone in ewes. Theriogenology 8:4, [Crossref]