FOLLOWING OVARIECTOMY AND ESTRADIOL INJECTIONld,

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1 THE EFFECT OF PHOTOPERIOD ON SERUM CONCENTRATIONS OF LUTEINIZING AND FOLLICLE STIMULATING HORMONES IN PREPUBERTAL )ieifers FOLLOWING OVARIECTOMY AND ESTRADIOL INJECTIONld, P. J. Hansen, 1. A. Kamwanja and E. R. Hauser Dept. of Meat and Animal Science University of Wisconsin Madison, WI Received for Publication: April 27, 1982 Accepted: August 24, 1982 Abstract Elevlen heifers, between 63 and 197 days of age, were exposed to 18 hr lisht/sday (L) or natural photoperiods (N), beginning October 19, Thi?y were ovariectomized 8 weeks later. LH concentrations after ovariectomy were not affected by photoperiod, but the rate of increase of FSH after ovariectomy was greater (P<O.lO) for group L than for group N. Three weelcs after ovariectomy, heifers were injected, IV, with 0.1 vg/kg estradiol17s. LH concentrations initially decreased after injection. This was followed by a series of pulses larger than those prior to injection. FSH concentrations declined after injection and remained low throughout the sampling period. The net response of LH concentrations to estradiol (mean postinjection concentration minus mean preinjection concentration) was greater (P=O.O5) for group L (4.7 * 0.49 ng/ml) than for group N (2.9 * 0.37 ng/ml). Photoperiod did not affect the net response of FSH concentrations to estradiol. We concluded that exposing prepubertal heifers to 18 hr light/day during the winter resulted in a greater rate of increase of FSH after ovariectomy and greater estrogeninduced LH release. Because the response of LH to estradiol17g differed from the response of FSH, these hormones may be regulated differently. INTRODUCTION In northern latitudes, age at puberty in heifers is influenced by season. Menge et al. (1) and Roy et al. (2) observed that springborn heifers reached@!%rty earlier th=gifers born in autumn. Grass et al. (3) observed that the dates of puberty attainment for heifers bofti in ztumn and fed a low plane of nutrition were bimodally distributed. Some animals became pubertal in summer and early autumn; few reached puberty in late autumn and winter and many reached puberty the following spring or summer. In a study in which heifers were reared in environmental chambers programmed to simulate conditions of temperature and photoperiod (ajmeat and Animal Science Paper No Supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, NRS Award 5T32HD and USAIDUSDA. The authors thank Drs. R. Stauffacher and K. Schillo, E. Hoch, P. Bringle and J. Kane for their technical assistance, Drs. L. Reichert, D. Bolt and G. Niswender and the National Pituitary Agency for providing materials used in RIA and S. Kading and J. Busby for typing this manuscript. NOVEMBER 1982 VOL. 18 NO

2 and photoperiod characteristic of Wisconsin (4), those reared from six months of age to puberty under conditions of spring, summer and early autumn became pubertal at younger ages than those reared under conditions of autumn, winter and early spring. At least part of the influence of season on age at puberty was due to photoperiod, since exposure to supplemental photoperiod during winter hastened the onset of puberty (5,6). Seasonal or photic influences on reproduction in other species have been found to be partially mediated by changes in the ability of gonadal steriods to inhibit (7,8,9,10,11) or promote (12,13) the secretion of gonadotropins. The mechanisms by which photoperiod influences sexual maturity in cattle have not been investigated. The objectives of this experiment were to determine whether photoperiod alters the effect of ovariectomy and estradioll7g (E2) injection on serum concentrations of luteinizing hormone (LH) and follicle stimulating hormone (FSH). Intravenous injection of E2 at 0.1 pg/kg body weight has been shown to cause an initial decrease in LH concentrations, followed by an increase above preinjection concentrations (14). A similar response of FSH to an intramuscular injection of E2 has been observed (15). During prepubertal development in cattle, estrogen becomes less effective in inhibiting LH secretion (14) and more effective in stimulating LH secretion (16,17). It was hypothesized that during the winter the magnitude of suppression of LH and FSH following an injection of E2 at 0.1 kg/kg body weight would be less and the subsequent increases in concentration would be greater for heifers exposed to 18 hr light/day than for heifers exposed to natural photoperiods. MATERIALS AND METHODS Eleven heifers, ranging in age from 63 to 197 days and sired by Angus bulls and out of dams of various breeds, were randomly assigned within age group (6385, , days) to one of two treatments. Beginning October 19, 1979 (week 0) heifers were exposed to either 18 hr light/day (L, n=6) or natural photoperiods (N, n=5) for 11 weeks. They were fed a diet of alfalfabrome hay, ad libitum, and grain (about 3 kg/head/day). The two groups were housg%@%separate sheds open to the south to a drylot. The animals were confined to the sheds from 1530 hr to 0900 hr each day and allowed access to shed and drylot at other times. Temperatures in the two sheds were similar. Twentytwo fluorescent lamps of 40 watts each in the sheds of the L group were on from 0300 to 2100 each day. Weekly blood samples (10 ml) were taken via jugular venipuncture during weeks 1 to 7 and daily samples were taken during weeks 7 to 9. At week 8, animals were ovariectomized via high lumbar incision using an ecraseur to sever the ovarian attachments. One heifer in group N died after surgery and data collected after ovariectomy from one heifer in group L was excluded because she was incompletely ovariectomized. At week 11 (January 4), each animal was tethered and an indwelling cannula was placed in one jugular vein. Ten ml blood samples were taken via cannula from 0900 to 1200 hr at 30min intervals. After the 552 NOVEMBER 1982 VOL. 18 NO. 5

3 1200 bleeding, estradiol175(b) (0.1 pg/kg body weight dissolved in 85:15 propylene glycol:o.9% saline at a concentration of 3.35 pg/mlf was injected into the jugular vein contralateral to the one cannulated. Body weights (X SE) on January 4 were kg (L) and 180 f 41 kg (N). Blood samples were taken at 15min intervals from 1200 to 1300 hr, at 30 min intervals from 1300 to 2400 hr and at hourly intervals from 2400 to 0900 hr. Hormone Assays Serum LH and estradiol concentrations were assayed as described by Schillo et al. (14). Limits of sensitivity for LH and estradiol were 0.9 ng/mra% 6.5 pg/ml, respectively. Intra and interassay coefficients of variations (CVl for LH assays were 12.6% and 18.5%. All estradiol samples were assayed in one assay and intraassay CV was less than 5%. Serum samples were not chromatographed and therefore concentrations are expressed as estrogen rather than as estradiol. Serum FSH was measured in a radioimmunossay developed by Bolt (18). Validation procedures and modifications for use in our laboratory are as follows. Five pg of USDAFSHBP3 (approximately 70 times potency of NIHFSHSl) was radioiodinated as described by Bolt (19) except that 1 mci 1125, 25 wg chloraminet dissolved in 5 v M P04, ph 7.5 and 100 ug sodium metabisulfite disolved in 20 ~1 0.1 M P04, ph 7.5 were used. Radioactive FSH was diluted with 0.05 M phosphate buffered saline, ph 7.0 with 0.1% gelatin (PBSgel) to approximately 30,000 cpm/loo ~1 for use in the assay. Antiserum to the beta subunit of bovine FSH (USDA 50122) was used in the assay at an initial dilution of 1:12,500 or 1:15,000 with a 1:300 dilution of normal rabbit serum in 0.05 M PBS containing 18.6 g/l of ethylenediamine tetraacetic acid, disodium salt (PBSEDTA). Either 400 ~1 of serum (assayed in duplicate) or 100 ~1 of standard (USDAFSHBP3) were added to 12 x 75 mm tubes, adding PBSgel so that final volume was 500 pl. Two hundred ~1 of antiserum and 100 ~1 of trace were added to all tubes and then the tubes were incubated at room temperature for 48 hr. Sheep antirabbit gamma globulin (200 ~1, 1:30 dilutiondin PBSEDTA) was then added and allowed to rea$t for 24 hr at 4 C. The reaction was then stopped with 2 ml cold (4 C) 0.01 M PBS, ph 7.5. Bound radioactive FSH was separated from free FSH by centrifugation. Afterward, supernatants were poured off and tubes counted in a gamma counter. Average binding of radioactive hormone to antiserum when no standard or serum was present (Bo) was 12.7%. Standard ranged from 0.1 to 15 ng USDAFSHBP3/tube. When 2, 4 or 6 ng standard was added to serum, the correlation between added and recovered hormone was Inhibition of binding by adding varying amounts of bull and ovariectomized/adrenalectomized cow serum pools was parallel to inhibition of binding by varying amount of standard. Limits of sensitivity (g@ of Bo) ranged from 0.16 to 0.27 ng/tube. All samples below the limit of sensitivity were given the value of the mean limit of sensitivity (0.20 ng/tube) for statistical analysis. Mean (bisigma Chemical Co., St. Louis, MO. NOVEMBER 1982 VOL. 18 NO

4 IHERIOGENOLOGY intraassay and interassay CV were 8.3% and 13.1%, respectively; Statistical Analysis Mean preovariectoniy concentrations of LH and FSH were calculated by averaging the values of samples from week 1 to week 7 and mean postovariectomy concentrations were calculated as the average of values from weeks 8 to 9. LH and FSH levels from 1 to 7 days after ovariectomy were regressed on time after ovariectomy within animal and the slope of the regression line was designated the rate of hormone increase. Data from blood samples collected on the day of E2 injection were divided into preinjection and postinjection periods and mean hormone concentrations were calculated for both periods. The net response to E2 was defined as mean postinjection concentration minus mean preinjection concentration. Even though the response of LH to E2 was bimodal, postinjection data were not divided into suppression and increased release periods because of the difficulty in objectively defining when these two phases of LH response occurred. Treatment differences for all traits were tested for significance by Wilcoxon's two sample test (20). RESULTS AND DISCUSSION Photoperiod did not affect concentrations of LH or FSH at any sampling time prior to ovariectomy. Mean concentrations prior to ovariectomy (weeks 1 to 7) were ng/ml (L) vs ng/ml (N) for LH and 0.52 * 0.02 ng/ml (L) vs 0.52 KO3 ng/ml (N) for FSH. Concentrations of LH and FSH after ovariectomy are presented in figure 1. Levels of both hormones rose in all animals. Neither mean concentrations of LH from 1 to 7 days after ovariectomy [3.9 f 1.24 ng/ml (L) vs 4.1 * 0.35 ng/ml (N)] nor mean rate of increase in LH LO.53 * 0.m ng/ml/day (L) vs 0.64 * 0.13 ng/ml/day (NJ1 were affected (P>O.lO) by photoperiod. TliQgh not significant, mean concentrations of FSH from 1 to 7 days after ovariectomy tended to be higher for L (1.7 * 0.3% ng/ml) than for N heifers ( ng/ml). The mean rate of rise in FSH from 1 to 7 days after ovariectomy (ng/ml/day) was greater (P<O.lO) for L ( ) than for N ( ) heifers. The difference in the rate of increase of FSH concentrations after ovariectomy could have resulted from differences in circulating inhibitors or their rate of clearance, the number or affinity for these inhibitors in the hypothalamus and pituitary or the rate of synthesis and release of FSH. Since the rate of increase of LH concentrations was not affected by photoperiod,"control of LH levels must differ from that of FSH. Effect of Estradiol Administration Photoperiod did not affect (P>O.lO) mean concentrations of LH C5.4 * 0.67 ng/ml (L) vs 5.5 * 0.12 ng/ml (N)l or FSH ng/ml (L) vs 4.0 * 0.3% ng/tiir (Nil prior to Ep administration. Preinjection lgels of estrogen were generally nondetectable and reached maximum concentrations at 15 to 30 min after injection, except for one animal 554 NOVEMBER 1982 VOL. 18 NO. 5

5 that peaked at 45 min. Photoperiod did not influence (P>O.lO) mean estrogen concentration from 15 min to 1 hr after injection [17.1 i 4.36 pg/ml (L) vs 13.4 * 2.70 pg/ml (N)] nor peak estrogen concentration c32.2 * 7.m pg/ml (L) z 25.7 * 9.57 pg/ml (N)] I I Cl TIME RELATIVE TO OVRRIECTOMY (DAYS1 Figure 1. Effect of photoperiod on LH and FSH concentrations before and after ovariectomy (day 0). Representative patterns of LH concentrations after E2 injection are presented in figure 2. LH concentrations initially declined after injection in three of five heifers exposed to L and in three of four NOVEMBER 1982 VOL. 18 NO

6 Figure 2. Representative patterns of LH and FSH concentrations after injection of estradiol17s (0.1 wg/kg body weight). Heifers exposed to 18 hr light/day are indicated by L while heifers exposed to natural winter photoperiods are indicated by N. Note the absence of initial decrease of LH concentrations after estradiol injection in heifer No. 1. heifers exposed to N, and concentrations remained low for approximately 0.5 to 2.5 hrs. Suppression was followed by a series of pulses greater in magnitude than preinjection pulses. Animals in which suppression did not occur had similar large pulses. This E2induced LH release was not characteristic of the preovulatory surge (21) as increases in LH concentration were followed by declines to levels similar to those before the increase. Similar responses of LH concentrations to an intravenous injection of E2 have been observed in heifers (141, sheep (22) and monkeys (23). Intramuscular injection of 500 Hg E2 elicited preovulatorylike surges of LH in heifers (17) indicating that the type 556 NOVEMBER 1982 VOL. 18 NO. 5

7 of response of LH after Ep depends on amount and site of administration. As shown in figure 2, both the suppression and subsequent increase in LH concentrations after Ep varied between heifers within treatments in time of initiation and magnitude. Rather than trying to subjectively divide the postinjection period into periods of suppression and stimulation, the effect of photoperiod on net response of LH to E2 was determined by comparing mean postinjection concentrations minus mean preinjection concentrations. Thus defined, the net response of LH concentrations to Ep was greater (P=O.O5) for group L (4.7 * 0.49 nglml) than for group N (2.9 f 0.37 ng/ml 1. This effect of photoperiod was probably due to differences in Epinduced stimulation of LH release, since the length and magnitude of suppression was small relative to the E2induced release. Differences in suppression between groups L and N would have to be extreme to cause the observed difference in net response. The onset of puberty may be preceded by development of positive feedback mechanisms, since the ability of estradiol to induce preovulatorylike LH surges increases with age (16,171. Long photoperiods may hasten the onset of puberty, in part, by increasing the rate of development of positive feedback actions of estrogen on LH release. Our data are consistent with this hypothesis and findings that estrogen induced greater release of LH in cows during summer than during winter (13). Concentrations of FSH increased from 0900 to 1200 hr in seven of nine animals (figure 2). After E2 injection, levels declined in all animals and became lower than concentrations at injection 1.5 to 3 hrs after injection. Thereafter, concentrations of FSH remained low. Larger doses of E2, injected subcutaneously, caused an increase in FSH concentrations (1.51, suggesting that the nature of the response of FSH to E2, like LH, is dependent on dose or method of administration. Net response of FSH concentrations to E2 was not affected (P>O.lO) by photoperiod LO.83 * 0.29 ng/ml (L) ng/ml (N)]. Patterns of FSH concentrations after estradiol injection and the effect of photoperiod on these patterns differed from those of LH, indicating differential control of these two hormones. Estradiol may have affected these two hormones in separate ways by either affecting release of different hypothalamic regulatory peptides or by directly affecting pituitary cell release of the hormones. In vitro, estrogen caused an increase in release of LH from pituitary zlmut had no effect on release of FSH (24). In conclusion, photoperiod affected rate of rise of FSH after ovariectomy and release of LH after estradiolinjection. Greater release of LH after estradiol in heifers exposed to 18 hr light/day as compared to heifers exposed to natural photoperiods suggests that photoperiod may affect age at puberty by altering the rate of developmental changes in positive feedback actions of estradiol. Because LH patterns after estradiol injection differed from patterns of FSH, these two hormones may be regulated differently. NOVEMBER 1982 VOL. 18 NO

8 REFERENCES Menge, A. C., Mares, S. E., Tyler, W. J. and Casida, L. E. Some factors affecting age at puberty and the first 90 days of lactation in Holstein heifers, J. Dairy Sci. 43: (1960). Roy, J. H. B., Gillies, C. M., Perfitt, M. W. and Stobo, I. J. F. Effect of season of the year and phase of the moon on puberty and on the occurrence of oestrus and conception in dairy heifers reared on high planes of nutrition, Anim. Prod. 31:1326 (1980). Grass, J. A., Hansen, P. J., Rutledge, J. J. and tlauser, E. R. Genotype x environmental interactions on reproductive traits of bovine females I. Age at puberty as influenced by genotype, dietary regimen and season, J. Anim. Sci. 55:in press (1982). Hansen, P. J., Schillo, K. K., Kamwanja, L. A., Hauser, E. R. and Dierschke, D. J. The influence of season on sexual development in the bovine female: Ovarian growth and body weight as related to the onset of puberty, pp in Dynamics of Ovarian Function. N. B. Schwartz and M. A. HunzickerDunn (eds.), Raven Press, New York (1981). Peters, R. R. and Tucker, H. A. Prolactin and growth hormone responses to photoperiod, Endocrinology 103: (1978). Hansen, P. J. and Hauser, E. R. The effect of photoperiod on the onset of puberty in heifers, Biol. Reprod. 26:lllA, abstract (1982). Pelletier, J. and Ortavant, R. Photoperiodic control of Ltl release in the ram: II. Lightandrogens interaction, Acta Endocrinologica 78: (1975). Tamarkin, L, Hutchinson, J. S. and Goldman, B. D. Regulation of serum gonadotropins by photoperiod and testicular hormone in the Syrian hamster, Endocrinology 99: (1976). Legan, S. J., Karsch, F. J. and Foster, D. L. The endocrine control of seasonal reproductive function in the ewe: A marked change in response to the negative feedback action of estradiol on luteinizing hormone secretion, Endocrinology 101: (1977). Legan, S. J. and Karsch, F. J. Photoperiodic control of seasonal breeding in ewes: Modulation of the negative feedback action of estradiol, Biol. Reprod. 23: (1980). Webster, G. F/i. and Haresign, W. The hormonal control of the seasonalitv of breedina in the ewe. Anim. Prod. 32:372, abstract (1981). Land, R. B., Wheeler, A. G. and Cart, the oestrogen induced LH discharge of Landrace and Scottish Blackface ewes, Biophys. 16: (1976). W. R. Seasonal variation in ovariectomized Finnish Ann. Biol. Anim. Biochem. 558 NOVEMBER 1982 VOL. 18 NO. 5

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