Secretion of gonadotrophins change during the luteal phase of the bovine oestrous cycle in the absence of corresponding changes in progesterone or 17β-oestradiol

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SCIENCE Animal Reproduction Science 37 ( 1995 ) 109-119

Secretion of gonadotrophins change during the luteal phase of the bovine oestrous cycle in the absence of corresponding changes in progesterone or 17fl-oestradiol A.S. Cupp, T.T. Stumpf t, F.N. Kojima, L.A. Werth 2, M.W. Wolfe 3, M.S. Roberson 4, R.J. Kittok, J.E. Kinder* Department of Animal Science, University of Nebraska, Lincoln, NE 68583-0908, USA Accepted 12 April 1994

Abstract The hypothesis in the present study was that changes in circulating luteinizing hormone (LH) and follicle stimulating hormone (FSH) would occur during the luteal phase of the oestrous cycle (Days 4-19; Day 0, day of behaviourial oestrus) that were not related to corresponding changes in concentrations of progesterone and 17fl-oestradiol. The stage of the oestrous cycle of cows (n = 18 ) was synchronised to obtain cows that were on alternate days of the cycle. Blood samples were collected every other day at 15 min intervals for 12 h from all cows: Days 4, 6, 8, 10, 12, 14, 16, 18 ( n = 9 ) a n d Days 5, 7, 9, 11, 13, 15, 17, 19 (n = 9 ). Concentrations of LH, FSH, 17fl-oestradiol and progesterone were determined in these samples. Data were compared across days to determine when significant changes occurred in concentrations or patterns of secretion of the gonadotrophins and ovarian steroid hormones during the oestrous cycle. There were significant changes in mean concentrations of FSH in circulation between Days 6 and 12. The most striking changes in secretion of gonadotrophins that could not be explained by changes in gonadal steroids were the fluctuations in amplitude of LH pulses between Days 7 and 12. Amplitude of LH pulses increased between Days 7 and 11 and subsequently decreased between Days 11 and 12 of * Corresponding author: Tel. (402) 472-6438; Fax. (402) 472-6362. Present address: Department of Animal Science, University of Missouri, Columbia, MO 652 l 1, USA. 2 Present address: Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, NE 68583-0908, USA. 3 Present address: Department of Pharmacology,Case Western Reserve University, Cleveland, OH 44106, USA. 4 Present address: Department of Cell Biologyand Anatomy, Oregon Health Sciences University, Portland, OR 97201, USA. 0378-4320/95/$09.50 © 1995 Elsevier Science Publishers B.V. All rights reserved SSDI 0378-4320( 94 )01333-H

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the oestrous cycle. Some changes in gonadotrophin secretion that occurred in the present study can be explained by fluctuations in concentrations of progesterone and 17fl-oestradiol in circulation. Other changes cannot be explained by fluctuations in circulating concentrations of these steroids. We accept our hypothesis because the concomitant changes in mean concentration of FSH between Days 6 and 11 and amplitude of LH pulses between Days 7 and 12 of the bovine oestrous cycle cannot be explained by changes in circulating concentrations of progesterone and 17fl-oestradiol. Keywords:Cattle,endocrinology;Progesterone;Oestradiol;LH; FSH;Oestruscycledynamics

1. Introduction The frequency and amplitude of luteinizing hormone (LH) pulses varied with the day of the oestrous cycle of cows when characteristics of gonadotrophin secretion were evaluated on days 3, 10 or 11 and 18 or 19 (Rahe et al., 1980) and days 4 or 11 (Waiters et al., 1984) of the oestrous cycle (oestrus designated Day 0). Modulation of patterns of LH secretion was attributed to the relative concentration of steroid hormones in circulation on days when blood collection occurred. Varying concentrations of progesterone (Roberson et al., 1989; Kojima et al., 1992) and 17fl-oestradiol (Stumpf et al., 1989; Wolfe et al., 1992; Kinder et al., 1991 ) have differential effects on the pattern of LH secretion. Concentrations of follicle-stimulating hormone (FSH) in circulation are also modulated by concentrations of 17fl-oestradiol in blood plasma of heifers (Wolfe et al., 1989). Recently, we reported that two concentrations of progesterone in circulation also have a differential effect on FSH secretion in cows (Cupp et al., 1992). All of these studies document the well recognized role that the ovarian steroids have in modulation of secretion of the gonadotrophin hormones of bovine females. A thorough evaluation of changes in secretion of the gonadotrophins throughout the luteal phase of the oestrous cycle of the bovine female has not occurred. Therefore, the objective of the present experiment was to determine if changes in secretion of LH and FSH would occur during the luteal phase of the oestrous cycle of bovine females that could not be explained by concomitant changes in concentrations of progesterone and 17fl-oestradiol.

2. Materials and methods 2.1. Experimental protocol

Thirty-three cows of a composite breed type ( 1/4 Pinzgauer, 1/4 Hereford, 1/ 4 Red Poll, 1/4 Angus) were synchronized (SYNCHRO-MATE-B regimen, Sanoff Animal Health, Overland Park, KS, USA) to a common day of oestrus (oestrus designated Day 0) to obtain cows on alternate days of the oestrous cycle. Of

A.S, Cupp et al. / Animal Reproduction Science 37 (1995) 109-119

i 11

the 33 cows, 18 were selected for the present experiment. Animals were selected on the basis of showing oestrus on 1 of 2 consecutive days. Serial blood samples were collected at 15 min intervals for 12 h every other day of the oestrous cycle from cows using indwelling jugular catheters: Days 4, 6, 8, 10, 12, 14, 16, 18 ( n = 9 ) and Days 5, 7, 9, 11, 13, 15, 17, 19 ( n = 9 ) to examine patterns of LH and FSH in circulation. Blood was allowed to clot at room temperature and was then stored for 24 h at 4°C. Serum was obtained after centrifugation at 1500 Xg for 15 min and stored at - 20 °C until assayed for LH and FSH. Additional blood samples were collected hourly during each 12 h period to evaluate changes in concentrations of 17fl-oestradiol and progesterone in blood. Tubes for collection of blood from which plasma was obtained contained 30% potassium-EDTA (50/11 per tube). Plasma was obtained within 3 h of collection by centrifugation at 1500 X g for 15 min. Samples were stored at - 2 0 °C until assayed for progesterone and 17fl-oestradiol. 2.2. Hormone assays Concentrations of LH in serum samples were determined by radioimmunoassay using duplicate 200/tl samples of blood serum (Adams et al., 1975; Wolfe et al., 1986). Intra- and interassay coefficients of variation were 2.5% and 12.3%, respectively. Rabbit antiserum against ovine FSH (JAD-RAoLH No. 17-6, 7, 9) and highly purified ovine FSH (LER-1976-A2 ) as radiolabeled tracer and standard were used in 200/zl duplicate samples of blood serum in the radioimmunoassay to quantify FSH (Acosta et al., 1983; Wolfe et al., 1989). Intra- and interassay coefficients of variation were 2.6% and 9.6%, respectively. Sensitivity of LH and FSH assays in blood serum was 30 pg m l - 1 and 45 pg m l - 1, respectively. With the radioimmunoassay for progesterone, 50 pl duplicate plasma samples were hexane:benzene extracted and (Roberson et al., 1989) intra- and interassay coefficients of variation were 3.3% and 11.7%, respectively. Concentrations of 17fl-oestradiol were also determined by radioimmunoassay using 200/tl duplicate plasma samples which were ether extracted (Kojima et al., 1992) and the intra- and interassay coefficients were 1.1% and 16.6%, respectively. Sensitivity of progesterone and 17fl-estradiol were 200 pg ml -~ and 0.50 10g ml -~ in blood plasma, respectively. 2. 3. Statistical analyses The pattern of LH in circulation was characterized by determination of mean concentration of LH and frequency and amplitude of LH pulses. Pulsatile characteristics were determined using algorithms (Pulsar software modified for the IBM-PC by J.F. Gitzen and V.D. Ramirez, Urbana, IL, USA). To ensure that all data for the gonadotrophins that were analyzed were from cows in the luteal phase, data from periods early and late in the oestrous cycle when progesterone was at basal levels were deleted from the statistical analyses. The rationale for deleting these data was that the purpose of the study was to

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evaluate variables for gonadotrophins in circulation during the luteal phase of the oestrous cycle. As a result, the numbers of cows included for each day were: Day 4, n=8; Days 5-15, n=9; Day 16, n=8; Day 17, n=6; Day 18, n=6; Day 19, n=4. Data regarding daily hormone means were fit to a linear model containing the fixed effect of day and the random effects of cow and group. Since there was no effect of group, data from all cows were combined for analyses. Variance components were estimated using the REML algorithms of PROC MIXED in SAS. Repeated measures were accounted for by assuming an autocorrelation error structure for residual error which estimates the correlation among repeated measurements on an individual cow. Data were compared across days of the luteal phase to determine when changes in concentration or pattern of secretion of gonadotropin and ovarian steroids occurred (Steel and Torrie, 1980; Statistical Analysis Systems Institute Inc., 1985 ).

3. Results 3.1. Characteristics o f L H secretion

Days during the oestrous cycle when significant changes in characteristics of LH secretion occurred are described in Table 1. Mean concentration of LH increased on Days 16, 17 and 19 of the oestrous cycle with the lowest concentration of LH occurring on Day 12. Frequency of LH pulses was variable throughout the oestrous cycle with the lowest frequency of LH pulses occurring on Days 7 and 9-13. The greatest pulse frequency occurred on Days 16 and 19 of the oestrous cycle. The amplitude of LH pulses was greater on Days 8-11 and then on Days 16, 17 and 19 than on other days of the estrous cycle. Data for secretory profiles of LH for individual cows are depicted in Figs. 1-4. 3.2. Characteristics of F S H secretion

Days during the oestrous cycle when changes in characteristics of FSH secretion occurred are described in Table 1. No detectable pulsatile pattern of FSH secretion was observed. Mean concentrations of FSH were greater on Day 5 and on Days 7-11 when compared with either Day 6 or Days 12-14 of the oestrous cycle. There were further increases in concentrations of FSH on Days 17 and 19 when compared with Days 18 and 16. Increased mean concentrations of FSH were detected in 12 of 18 cows between Days 8 and 11 of the oestrous cycle. Figs. 1, 2, 3 or 4 depict examples where FSH was increased on either Days 8, 9 or 10. This slight but consistent increase in FSH for individual cows was only detected for one period of serial blood collection in most cows and by the following period of collection FSH had returned to concentrations detected earlier in the oestrous cycle. Fig. 1 depicts data from one cow where FSH was increased during two periods of serial blood collection. Five of the 12 cows that had increased FSH

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between Days 8 and 11 had subsequent increases in FSH during Days 12-17 of the oestrous cycle (Fig. 1, example of this pattern).

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3.3. Concentrations of steroid hormones Data for concentrations of 17fl-oestradiol and progesterone are included in Table 1. Mean concentrations of 17fl-oestradiol decreased between Days 4 and 13

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A.S. Cupp et al. / Animal Reproduction Science 37 (1995) 109--119

of the oestrous cycle and then increased from Days 13 through 19. Concentrations of 17fl-estradiol were greater on Days 4 and 5 of the oestrous cycle with the greatest concentration occurring on Day 19. Concentrations of progesterone increased between Days 4 and 15 of the oestrous cycle with the lowest concentrations being detected on both Day 4 and 19. Concentrations of progesterone decreased from Days 17 through 19. Data for progesterone and 17fl-oestradiol of individual cows are depicted in Figs. 1-4. To determine if changes in the ratio of 17fl-estradiol to progesterone occurred during the luteal phase of the oestrous cycle, a ratio was calculated and daily values are included in Table 1. The ratio of 17fl-oestradiol to progesterone was greater and declined gradually in value from Day 4 though Day 13. This, we suspect, was due to increasing concentrations of progesterone being produced by a developing corpus luteum. The ratio then increased gradually from Days 15 through 19 of the oestrous cycle. This change in ratio of 17fl-oestradiol to progesterone presumably occurred as a result of the demise of the corpus luteum and increasing concentrations of 17fl-oestradiol being produced by the ovulatory follicle.

4. Discussion

In the present study, there were dynamic changes in patterns of LH and FSH in circulation during the luteal phase of the oestrous cycle in cows. Some of these changes can be explained by the changing concentrations of progesterone and 17fl-oestradiol. Progesterone influences the frequency of LH pulses in ewes (Goodman and Karsch, 1980; Goodman et al., 1981 ) and cows (Stumpf et al., 1993). The increased frequency of LH pulses late in the luteal phase of the oestrous cycles in the present study presumably resulted from the corresponding declining concentrations of progesterone. Frequency of LH pulses is greater when concentrations of progesterone are lower in cows (Roberson et al., 1989; Cupp et al., 1992; Kojima et al., 1992). Amplitude of LH pulses has been reported to be greater during the luteal phase compared with other parts of the oestrous cycle of cattle (Rahe et al., 1980). Fluctuations in amplitude of LH pulses occurred during the luteal phase of the oestrous cycle of cows in the present study. We suspect that fluctuations in the amplitude of LH pulses between Days 16 and 19 of the oestrous cycle resulted from declining progesterone. Changes in amplitude of LH pulses between Days 7 and 12 are the most significant finding in the present study. Progesterone has been implicated as the primary modulator of amplitude of LH pulses; however, changes in concentrations of progesterone during this portion of the oestrous cycle cannot explain the changes in amplitude of LH pulses (Table 1 ). The increase and subsequent decrease in amplitude of LH pulses between Days 7 and 12 may be related to dynamics in the development and/or demise of dominant ovarian follicles that occurs during this portion of the oestrous cycle of the cow (Sirois and Fortune, 1988; Ginther et al., 1989). The altered hormonal milieu that results from ovarian follicle atresia and/or development between Days 7 and 12 might lead to subtle changes in

A.S. Cupp et al. / Animal Reproduction Science 3 7 (1995) 109-119

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hormones in circulation which enhance the amplitude of LH pulses. Increased amplitude of LH pulses between Days 7 and 12 of the oestrous cycle indicates that LH may be involved in development of dominant ovarian follicles because LH support is necessary for follicles to produce the androgenic precursors for 17fl-oestradiol (Fortune and Quirk, 1988). The increased LH support during Days 7 and 12 may have a role in the establishment of dominant estrogen active follicles during this portion of the oestrous cycle. It is also possible that the developing corpus luteum is producing factors other than progesterone which affect the amplitude of LH pulses at this time during the oestrous cycle (AI-Gubory et al., 1992 ). Corpora lutea of cattle contain high concentrations of oxytocin (Wathes and Swann, 1982). To date, there is only evidence which supports a role for secretion of oxytocin during luteal regression, and not during the early portion of the oestrous cycle. The greater mean concentration of LH during the latter part of the luteal phase probably results from the increase in frequency of LH pulses. The enhanced LH during Days 16-19 of the oestrous cycle would allow for initiation of development of the dominant follicle into the ovulatory follicle. An enhancement of amplitude of LH pulses in cattle continues after luteal regression and results from the increasing 17fl-oestradiol during the follicular phase of the oestrous cycle (Stumpfet al., 1989). Concentrations of FSH did not vary within cows throughout each serial blood collection period. There was no evidence of pulsatile secretion of FSH in circulation in individual cows across days of the oestrous cycle which is in contrast with data that have been previously reported (Adams et al., 1992). However, there were subtle increases in mean concentration of FSH between Days 7 and 11 in a majority of the cows in the present study. These increases may relate to changes in FSH that have been reported to be associated with recruitment of ovarian follicles in cattle (Adams et al., 1992 ). Some of the cows that had altered profiles of FSH and LH secretion between Days 7 and 12 had similar alterations in the pattern of FSH secretion later in the luteal phase. These might be cows that had three waves of ovarian follicular growth during their oestrous cycle if the changes in FSH detected in the present study are associated with changes in follicular dynamics. We suspect that the changes in FSH and amplitude of LH pulses that were detected in 12 of 18 cows between Days 7 and 12 of the oestrous cycle may have occurred in more cows. We may have failed to detect changed patterns of these hormones in some cows owing to the regimen of blood collection used. Serial blood collections were conducted on alternate days of each cow's luteal phase. Therefore, cows not exhibiting changes in secretion of LH and FSH between Days 7 and 12 of the oestrous cycle may have had altered patterns of secretion of LH and FSH on days when serial blood collection did not occur.

5. Conclusion The present study comprises a comprehensive evaluation of LH and FSH secretion during the luteal phase of the oestrous cycle of the cow. Predictable changes

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in LH and FSH occurred as a result of fluctuations in progesterone and 17fl-oestradiol during the luteal phase of the oestrous cycle. In addition, there are changes in secretion of LH and FSH that cannot be explained by fluctuations in progesterone and 17fl-oestradiol. Therefore, we accept our hypothesis that there are changes in FSH and LH secretion during the luteal phase of the oestrous cycle that cannot be attributed to corresponding changes in progesterone or 17fl-oestradiol. We propose that future studies focus on intensive evaluations in changes of LH and FSH during times of development or atresia of dominant ovarian follicles in bovine females.

Acknowledgments We thank Robin Hopper for her patience in the preparation of this manuscript; Karl Moline, Jeff Bergman and Bob Broweleit for management of experimental animals; Ken Pearson and Georgette Caddy for assistance with hormone analyses; Tom Rathje for assistance in analysing the data. We also thank each of the following for materials that allowed us to complete our study: Dr. Jerry Reeves, LH antisera; Dr. Leo Reichert, Jr., purified LH and FSH; Dr. Jim Dias, FSH antisera; Dr. Norman Mason, 17fl-oestradiol antisera; Dr. H. Edward Grotjan, Jr., Four Fit program for 17p-oestradiol analysis. This paper is published as Paper Number 10110, Journal Series, Nebraska Agricultural Research Division. This research was supported by appropriated funds from the State of Nebraska and the USDA CRGO (90-37240-5714).

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Goodman, R.L. and Karsch, F.J., 1980. Pulsatile secretion of luteinizing hormone: Differential suppression of ovarian steroids. Endocrinology, 107:1286-1290. Goodman, R.L., Bittman, E.L., Foster, D.L. and Karsch, F.J., 1981. The endocrine basis of the synergistic suppression ofluteinizing hormone by estradiol and progesterone. Endocrinology, 109:14141417. Kinder, J.E., Garcia-Winder, M., Imakawa, K., Day, M.L., Zalesky, D.D., D'Occhio, M.L., Stumpf, T.T., Kittok, R.J. and Schanbacker, B.D., 1991. Circulating concentrations of 17 fl-estradiol influence pattern of LH in circulation of cows. Domestic Anita. Endocrinol., 8: 463-469. Kojima, N., Stumpf, T.T., Cupp, A.S., Werth, L.A., Roberson, M.S., Wolfe, M.W., Kittok, R.J. and Kinder, J.E., 1992. Exogenous progesterone and progestins used in estrous synchrony regimens do not mimic the corpus luteum in regulation of LH and 17,8- estradiol in circulation of cows. Biol. Reprod., 47:1009-1017. Rahe, C.H., Owens, R.E., Fleeger, J.L., Newton, H.J. and Harms, P.J., 1980. Pattern of plasma luteinizing hormone in the cyclic cow: Dependence upon period of the estrous cycle. Endocrinology, 107:498-503. Roberson, M.S., Wolfe, M.W., Stumpf, T.T., Kittok, R.J. and Kinder, J.E., 1989. Luteinizing hormone secretion and corpus luteum function in cows receiving two levels of progesterone. Biol. Reprod., 41:997-1003. Sirois, J. and Fortune, J.E., 1988. Ovarian follicular dynamics during the estrous cycle in heifers monitored by real-time ultrasonography. Biol. Reprod., 39:308-317. Statistical Analysis Systems, 1985. Users Guide: Statistics. SAS Inst. Inc., Cary, NC. Steel, R.D.G. and Torrie, J.H., 1980. Principles and Procedures of Statistics: A Biometrical Approach, 2nd edn. McGraw-Hill, New York. Stumpf, T.T., Day, M.L., Clutter, A.C., Stotts, J.A., Wolfe, P.L., Kittok, R.J. and Kinder, J.E., 1989. Effect of estradiol on secretion of luteinizing hormone during the follicular phase of the bovine estrous cycle. Biol. Reprod., 41:91-97. Stumpf, T.T., Roberson, M.S., Wolfe, M.W., Hamernik, D.L., Kittok, R.J. and Kinder, J.E., 1993. Progesterone, 17ff-estradiol, and opioid neuropeptides modulate pattern of luteinizing hormone in circulation of the cow. Biol. Reprod., 49:1096-1101. Waiters, D.L., Schams, D. and Schallenberger, E., 1984. Pulsatile secretion of gonadotrophins, ovarian steroids and ovarian oxytocin during the luteal phase of the oestrous cycle of the cow. J. Reprod. Fertil., 71:479-491. Wathes, D.C. and Swann, R.W., 1982. Is oxytocin an ovarian hormone? Nature, 297:225-227. Wolfe, M.W., Roberson, M.S., Stumpf, T.T., Kittok, R.J. and Kinder, J.E., 1992. Circulating concentrations and pattern of luteinizing hormone and follicle stimulating hormone in circulation are changed by circulating concentration of 17 ,8-estradiol in the bovine male and female. J. Anim. Sci., 70: 248-253.