Estrogen — Induced release of luteinizing hormone in prepubertal and postpubertal heifers

THERIOGENOLOGY ESTROGEN

- INDUCED RELEASE OF LUTEINIZING HORMONE PREPUBERTAL AND POSTPUBERTAL HEIFERS

K. K. Schillo(a),

D. J. Dierschke

IN

and E. R. Hauser

Department of Meat a Animal Science and Wisconsin Regional Primate Research Center University of Wisconsin, Madison, WI 53706(C) Received

for

November 16, Accepted: February 10, Abstract

publication:

1982 1983

This experiment was designed to determine the age at which estradiol-176 (E2) first induces a preovulatory-like surge of luteinizing hormone (LH) in prepubertal heifers. Responses of prepubertal animals 3 to 4 and 5 to 6 months of age were compared with those of postpubertal heifers that received 25 mg prostaglandin F2a at 0800 hr on day 15 of the estrous cycle. E2 (500119) induced surges of LH in 5 to 6 months of age and l/5 heifers 3 to 4 months of age, 3/3 heifers 5/5 postpubertal heifers. Duration of response and interval between injection and peak of the response were longer in postpubertal E2 heifers than in those 5 to 6 months old (PtO.lO). Peak response and total amount of LH released were greater in animals 5 to 6 months old (P
(a)Trainee of the Endocrinology-Reproductive Physiology Program. Present address: Department of Veterinary Bioscience, University of Illinois, Urbana, IL 61801. (b)Reprint requests and correspondence: Dr. E. R. Hauser, Meat and Animal Science Dept., 1675 Observatory Drive, University of Wisconsin, Madison, WI 53706. (c)Supported by the College of Agriculture and Life Sciences, Meat and Animal Science, (Paper number 7g5), Grant No. RR-00167 from the National Institute of Health, and Grant No. 5T32HD07007-03 awarded by the National Institutes of Child Health and Human Development, DHEW. Publication No. 22-028 .from the Wisconsin Regional Primate Research Center. The authors thank Ors. L. Reichert, G. Niswender and the Endocrine Study Section, National Institutes of Health, for donating luteinizing hormone used for iodination, luteinizing hormone used for standard and luteinizing hormone antiserum. We also thank Dr. R. B. Staigmiller for donating progesterone antiserum and tyrosine methyl ester of progesterone used for iodination. We acknowledge Mr. E. Hoch, Mr. P. Bringle, Mr. J. Kane, and Mr. Tim Old for their excellent technical assistance and Ms. Julie Busby and Ms. Sue Kading for typing this manuscript.

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THERIOGENOLOGY Introduction Estradiol-17ti (E2) was shown to facilitate release of luteinizing hormone (LH) in ovariectomized cows by Short et al. (1) and in ovariectomized heifers by Beck and Convey (2). EncKtrations of LH increased to maximum levels between 12 and 22 hr following E2 injection. The responses resembled preovulatory surges normally induced by endogenous estrogen secreted by growing follicles in cycling females. Development of this positive feedback system may be an important Therefore, we conducted an experiment to step leading to puberty. determine the age at which E2 first induces a preovulatory-like surge Other objectives were to compare of LH in prepubertal heifers. responses of prepubertal heifers with those of sexually mature animals, and to determine if an induced surge of LH would result in ovulation. At the time our experiment was initiated, Staigmiller et _* al (3) reported that E2 stimulated LH release in heifers at 5 months of age, but not at 3 months of age. It is important to compare our results with those of Staigmiller -et al. (3) because of differences between breeds, management conditions and experimental designs. Materials and Methods Twenty-four Angus x Holstein heifers were purchased from Wisconsin farmers at different times of the year when they were one month of age. They were housed in a shed open to the south with access to an exercise paddock, and were fed milk replacer until they were able to consume a ration of chopped hay and grain (67% TDN) ad libitum. The experimental trials were conducted between April and Max 1980,hen animals were 3 to 4, 5 to 6 and 11 to 14 months of age. Heifers of this type usually attain puberty at 6 to 10 months of age. Heifers were assigned randomly to one of two treatments (Table 1); single injections of 5OOpg E (d) in 1 ml corn oil, or vehicle, were Since one administered IM at 1900 hr on day 1 of the experiment. objective was to compare responses in prepubertal heifers with those of postpubertal heifers, it was necessary to assess the effect of E2 in sexually mature heifers that would respond to E2. Because of the negative feedback action of progesterone extant during the luteal phase, heifers are sensitive to Ep only during the follicular phase (4). The following adult model was used to ensure that E2 was administered during the responsive phase. Heifers which were 11 to 14 months of age, and had exhibited at least two estrous cycles of 19 to 22 days, were injected IM with 25 mg prostaglandin F2a (PGF2a)(e) at 0800 hr on day 15 of the estrous cycle. This treatment has been shown to cause luteal regression within 8 to 12 hr (5,6). PGF2a was followed by E2 or vehicle at 2000 hr. Prepubertal heifers did not receive PGF20. (d)Sigma Chemical Co., St. Louis, MO., Lot No. 19C-0519. (e)LutalyseP, Upjohn Co., Kalamazoo, MI.

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THERIOGENOLOGY

Table

1.

Experimental

Number Age (months)

Design

of Heifers

of heifers

Y estradiol-17e

3-4 5-6 11-14

and Body Weights

5

Corn

4 3 4

z

oil

Body weight (kg, mean * S.E.)

105.9 178.8 329.1

*

9.3 6.2 * 12.4 l

Blood s m les were obtained via indwelling jugular cannulae (60 cm penetration)tfP at 60-min intervals between 1600 hr on day 1 of the experiment and 2300 hr on day 2. Additional samples were obtained by venipuncture at 1600 hr on days 2 through 6, 9, 15, 18 and 21 postinjection. Blood was allowed to clot at 2O'C for 2 hours and at 4OC for an additional 24 hr. Serum recovered after centrifugation was stored at -2O'C until analyzed by radioimmunoassay (RIA). Serum concentrations of LH were determined in all samples collected on days 1 while progesterone concentrations were determined in the and 2, pretreatment samples taken at 1600 hr and in all samples collected between days 2 and 21 postinjection. Serum concentrations of LH were determined by the RIA developed by for our laboratory by Carter et al. y;;wender e al. (7) as described NIH-LH-Bm was used as standard (range = 0.25 to 20 ng). Init dilution of first antibody (GDN B225) was 1:40,000. Serum was assayed Withinand between-assay coefficients in duplicate 200 ~1 aliquants. of variation were 7 and 13X, respectively. Serum concentrations of progesterone were determined by the RIA Initial dilution of first antibody described by Staigmiller et al. (9). The range 3 zandards was 64 to 11,200 pg. Withinand was l:lO,OOO. between-assay coefficients of variation for pooled sera from pregnant Serum aliquants of 500 ~1 were extracted cows were 18 and 6% (n=3). with 5 ml glass distilled hexane and reconstituted with 500 ~1 buffer (0.01 M phosphate buffer with 0.9% NaCl and 0.1% gelation, pH 7.1) prior Progesterone concentrations were determined in duplicate 200 ~1 to RIA. reconstituted extract. Procedural loss of aliquants of 3H-progesterone averaged 30%. The response to E2 concentrations following by 2 standard deviations ng/ml (4,lO).

(f)Bard

I Cath,

Murray

was considered a preovulatory-like surge if LH E2 injection exceeded the preinjection levels for 6 hrs and reached a peak of at least 10

Hill,

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729

THERIOCENOLOGY Chi-square analysis was used to detect differences in number of animals exhibiting surges of LH. Characteristics of responses in the two older groups of heifers were compared in the following manner. Wilcoxon's two sample test (11) was used to determine the effect of age on sum of LH concentrations comprising the response (total LH) and on Durations of responses and maximal concentrations during responses. intervals between E2 injections and maximal responses were compared by determine Correlation coefficients were calculated to t tests. activity associations between traits. Luteal was identified by palpation of a corpus luteum per rectum, and by serum concentrations of progesterone which exceeded i-iigiiiil-% days 2 through 9 during the post-treatment sampling period.

days. their

Postpubertal and 5 Three to 4 month small size.

to 6 month-old heifers old heifers could not

were palpated every 7 be palpated because of

fitted to Second degree polynomials were progesterone concentrations in postpubertal heifers receiving E2 or corn oil. Regression coefficients and intercepts were then compared by t tests to identify differences in patterns of progesterone. Results Luteinizing

Hormone

Treatment and surges of LH (Table but the corn oil, increased with age.

age influenced the number of animals exhibiting No surges were observed in animals treated with 2). number of Ep-treated animals demonstrating surges These responses are shown in Figures 1 through 4. Table

2.

Responses

Number Age (months)

3-4 5-6 11-14 Level of significance for age effectsb

Corn

to Treatment

of heifers exhibiting an LH surge oil

o/4 o/3 o/4

P>O.25

Estradiol-17a

115 313 515

Level of significance for treatment effects

P>O.25 P
P=O.lO

aDetermined by concentrations of LH which exceeded preinjection concentrations by 2 s.d. for at least 6 hr and reached a peak at least 10 ng/ml. bChi-square analysis.

730

of

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100 423

3-4MOS

‘B,+yycyyq,, 2

0

2 4

6 8

10121416

,, ,j,, 1820222426

HOURS

Figure 1. Serum concentrations of LH in samples obtained at 60 min intervals in the only 3 to 4 month-old heifer exhibiting a surge of LH. Estradiol-178 (E2) was injected at time = 0.

-2

0

2 4

6

8 10 12 14 16 182022242628 HOURS

Figure 2. Serum concentrations of LH (mean f s.e.) in samples obtained at 60 min intervals from 3 to 4 month-old heifers receiving corn oil (x; n=4) or estradiol-176 (0; n=4) at time = 0. The latter results exclude the data illustrated in Figure 1.

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rHERIOGENOLOGY

HOURS

Figure 3. Concentrations of LH (mean l s.e.) in samples obtained at 60 min intervals from 5 to 6 month-old heifers receiving corn oil (x; n=3) or 500 vg estradiol-17s (0; n=3) at time = 0. The latter results include data only from those animals exhibiting a surge of LH.

1 l-14

LIE -2

MO 5.

,,,,,,,,,, 0

2 4

6

, 8 iQ121416182022a426 HOURS

Figure 4. Serum concentrations of LH (mean * s-e.) in samples obtained at 60 min intervals from 11 to 14 month-old heifers receiving corn oil (x; n=4) or 500 pg estradiol-176 (0; n=5) at time = 0. These heifers were treated on day 15 of the estrous cycle, 12 hr after an intramuscular injection of 25 mg prostaglandin F_~u.

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THERIOGENOLOGY Although four of five heifers, 3 to 4 months old, did not exhibit LH surges after E2, concentrations of LH appeared to be lower and less variable than in control animals between 1 and 8 hr postinjection. This was apparently due to the negative feedback action of E2 which was previously characterized in prepubertal heifers (12). Several individuals in this group showed a slight rise in LH at 12 to 18 hr postinjection, but these were too small and too brief to be identified as surges by our criteria. The 3 to 4 month-old heifer that exhibited an LH surge after E2 had the heaviest body weight in this group. Maximal concentrations of LH in heifers 5 to 6 months old were higher than those in postpubertal animals (108 * 29.6 vs 38.3 * 14.6 Total LH was also greater in heifers 5 K 6 months old nglml; PO.25). The combined correlation coefficient for all heifers exhibiting surges was nonsignificant (r=-0.37; P>O.25). Progesterone Although one heifer 3 to 4 months old and 3 heifers 5 to 6 months old exhibited LH surges, only the youngest animal had elevated concentrations of progesterone indicative of luteal activity (Figure 6). The elevation was neither as great in magnitude nor as long in duration as that of postpubertal heifers (Figure 5). Four of five postpubertal heifers receiving E2 showed elevated concentrations of progesterone, and corpora lutea were detected by palpation. Number of days between PGF2a and estrus was 2.8 * 0.5. The animal that did not have elevated concentrations of progesterone exhibited estrus 3 days after PGF2a, but no corpus luteum was palpated. Elevated concentrations of progesterone were also present in 3 of 4 heifers that received PGF2a and corn oil. All of these animals exhibited estrus 2 days after PGF2a and had corpora lutea following treatment. The remaining heifer did not exhibit estrus and no corpus luteum was detected.

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0

23456

9

Serum concentrations of progesterone Figure 5. (mean * s.e.) from II to 14 month-old heifers treated with corn oil (upper panel) or 500 pg estradiol-178 (E2, lower panel) on day 0. Treatments were given 12 hr after an intramuscular injection of PGFza on day 15 of the estrous cycle.

Serum concentrations of progesterone Figure 6. the only 3 to 4 month-old heifer exhibiting animal received This surge of LH. 500 estradiol-176 on day 0.

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in a 119

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THERIOGENOLOGY heifers were not Patterns of progesterone in postpubertal influenced by treatment. Second degree polynomials describing patterns between days 2 and 21 were similar for the two treatment groups (P>O.25). were Preinjection concentrations of progesterone inversely proportional to total LH (i-=-0.95; P
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THERIOGENOLOGY follicle size and number of palpable follicles increased in heifers prior to puberty (16,18). Furthermore, treatment with pregnant mare's serum gonadotropin and human chorionic gonadotropin induced ovulation in This evidence suggests that the heifers 2 to 5 months of age (19). and/or amount of gonadotropin secretion at this stage of pattern stimulate follicle growth to development were not adequate to preovulatory stage.

In conclusion, the crucial physiological event triggering onset of puberty in heifers probably involves stimulation of ovarian growth the positive feedback rather than development of system. One possibility is that gonadotropin secretion increases because of the decreased responsiveness of the hypothalamo-pituitary axis to negative feedback action of E2 (12).

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THERIOGENOLOGY References

1.

Short, R. E., Howland, B. E., Randel, R. D., Christness, Bellows R. A. Induced LH release in spayed cows, J. (1973). -37:551-557

2.

Beck, T. W. and Convey E. M. Estradiol hormone concentrations in the bovine, J. (1977).

3.

Staigmiller, R. B., Short, R. E. and Bellows, R. A. Induction of LH surges with 17s estradiol in prepubertal beef heifers: An age dependent response, Theriogenology (1979a). -11:453-459

4.

Hansel, W. and Echternkamp, S. domestic animals, Am. Zoologist.

5.

Carter, M. L. Control of ovarian function during the estrous cycle and early postpartum period in the beef cow, Ph.D. Thesis, University of Wisconsin, (1980) p,p. 69-77.

6.

Weston, P. G. and Hixon, J. E. F2cl administration on in vitro corpora lutea, Biol. Rep%d.59-268 -

7.

Niswender, G. D., Reichert, L. E., A. V. Radioimmunoassays for bovine Endocrinology (1969). -84:1166-1173

8.

Carter, M. L., Dierschke, D. J., Rutledge, J. J. Effect of gonadotropin-releasing hormone and pituitary-ovarian reproductive function and postpartum cows, J. Anim. Sci. -51:903-910 (1980).

9.

Staigmiller, R. B., Short, R. E., Effect of nutrition on response to (1979). -48:1182-1190

D. S. and Anim. Sci.

control of luteinizing Anim. Sci -45:1096-1101

E. Control of ovarian 12:225-243 (1972). -

function

in

Effects of -in vivo prostaglandin progesterone synthesis by bovine (1980). Midgley, A. R. and Nalbandov, and ovine luteinizing hormone,

Bellows, R. FSH in beef

and Hauser, E. R. calf removal on performance in

A. and Carr, T. B. cows, J. Anim. Sci.

10.

Rahe, C. H., Owens, R. E., Fleeger, J. L., Newton, H. J. and Harms, P. G. Pattern of plasma luteinizing hormone in cyclic cows: Dependence upon period of the .cycle, Endocrinology -107:498-503 (1980).

11.

Steel, R. G. D. and Torrie, J. Statistics, McGraw-Hill Book pp. 542-543.

12.

Principles H. Co., Inc.,

and

New

Procedures of York, (1980)

Schillo, K. K., Dierschke, D. J. and Hauser, E. R. Regulation of LH secretion in prepubertal heifers: Increased threshold to negative feedback action of estradiol, 3. Anim. Sci. -54:325-336 (1982).

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THERIOGENOLOGY 13.

Estradiol treatment and Swanson, L. V. and McCarthy, S. K. luteinizing hormone (LH) response of prepubertal Holstein heifers, Biol. Reprod. -18:475-480 (1978).

14.

Beck, T. W., Smith, V. G., Seguin, B. E. and Convey, E. M. Bovine LH, GH and prolactin following chronic implantation of serum, steroids and subsequent ovariectomy, J. Anim. ovarian Sci. (1976). -42:461-468

15.

Estrus and LH Rajamahendran, R., Lague, P. C. and Baker, R. D. ovariectomized heifers following vaginal devices release in containing ovarian steriods, J. Anim. Sci. -49:554-559 (1979).

16.

Hafs, H. 0. and Morrow, Swanson, L. V., and prolaction, characteristics serum LH, glucocorticoids from first estrus to breeding heifers, J. Anim. Sci. -34:284-293 (1972).

17.

Schillo, K. K., Dierschke, D. J. and Hauser, E. R. month of birth and age on patterns of LH secretion Theriogenology 18:593-598 (1982). heifers. -

18.

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 puberty. In: Dynamics of Ovarian Function. (N. B. Schwartz and Hunzicker-Dunn, eds.) Raven Press, New York. (1981), pp. M. 239-244.

19.

Spilman, C. H., Seidel, G. E., Larson, L. L., Vukman, G. R. Progesterone, 208-hydroxypregn-4-en-3-one Foote, R. H. luteinizing hormone levels in superovulated prepuberal postpuberal cattle, Biol. Reprod. 2:116-124 (1973).

738

Ovarian D. A. progesterone and size in Holstein

Influences of in prepubertal

and and and

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