Effect of Estradiol on Secretion of Luteinizing. Hormone during the Follicular. Phase of the Bovine Estrous Cyclel. T. T. STUMPF,. M. L. DAY,3. M. W. WOLFE,.
BIOLOGY
OF REPRODUCTION
Effect of Estradiol
T. T. STUMPF,
40, 91-97
(1989)
on Secretion of Luteinizing Hormone during the Follicular Phase of the Bovine Estrous Cyclel M. L. DAY,3 P. L. WOLFE,
M. W. WOLFE, R. J. KITFOK,
Department University Lincoln,
A. C. CLUTrER,4 and J. E. KINDER2
of Animal
J. A. STOTTS,
Science
of Nebraska-Lincoln
68583-0908
Nebraska
ABSTRACT Mean concentrations of luteinizing hormone (LII) increase during the follicular phase of the estrous cycle in cows. The working hypotheses in the present study were (1) that increasing concentrations of l73-estradiol (E2) during the follicular phase of the estrous cycle cause an increase in mean concentration of LII by increasing amplitude of pulses of LII, and (2) that increasing E2 concentrations during this stage of the estrous cycle decrease frequency of pulses of LII in bovine females. Day of estrus was synchronized in seventeen mature cows. Treatments were initiated on Day 16 of the experimental estrous cycle (Day 0 = estrus). At Hour 0 (on Day 16), 4 cows were lutectomized. Lutectomy of these cows (EE; n = 4) allowed for endogenous secretion of E2. The remaining cows were ovariectomized at Hour 0 and were assigned to one of three E2 treatments: luteal phase E2 (LE, n = 5), increasing then decreasing E2 (DE, n = 5), and no E2 (NE, n = 3). Cows in the group that received LE were administered one E2 implant at Hour 0, which provided low circulating concentrations of E2 similar to those observed during the luteal phase of the estrous cycle. Cows in the group that received DE were administered one E2 implant at Hour 0, and additional implants were administered at 8-h intervals through Hour 40; then, two implants were removed at Hours 48 and 56, and one implant was removed at Hour 64. This protocol mimicked the normal preovulatory rise and fall of circulating concentrations of E2. Blood samples were collected (at 12-mm intervals) from Hour 2 through Hour 80. The response of mean concentration of LII over time was quadratic for cows receiving the DE, LE, and NE treatments. Linear and quadratic responses in the amplitudes of pulses of LII over time were determined in the cows receiving the DE, LE, and NE treatments. Amplitudes of pulses of LH increased at a greater rate through Hour 40 in the cows receiving the DE treatment (time of increasing concentrations of E2 in DE treatment) as compared to those receiving either the LE or NE treatments. The overall quadratic response for frequency of pulses of LII over time was signifIcant for all treatments, with no signcant interactions between treatments. From these results we conclude that the amplitude of pulses of LII increases at a greater rate due to increasing concentrations of E2 during the follicular phase of the bovine estrous cycle and that frequency of pulses of LII is not influenced by E2 during this stage of the estrous cycle.
INTRODUCTION
(Rahe et at., 1980). Declining levels of progesterone at luteolysis result in an increase in frequency of pulsatile release of LH in cows (heland and Roche, 1982; Imakawa et at., 1986) and in ewes (Goodman and Karsch, 1980). During the follicular phase of the bovine estrous cycle after the decline in circulating concentrations of progesterone, there is a linear increase in mean concentration of LH prior to the preovulatory surge of LH (Chenault et a!., 1975; Imakawa et a!., 1986). In addition, after the demise of the corpus luteum, there is an increase in circulating concentrations of 1 7-estradiol (E2) in cows (Schallenberger et al., 1984) and ewes (Karsch et a!., 1979). Imakawa et at. (1986) reported this rise in circulating concentrations of E2 was parallel to the increases in mean concentrations of LH observed
hormone (LH) is secreted in a pulsatile manner during the bovine estrous cycle with the pattern of secretion dependent on stage of the estrous cycle Luteinizing
Accepted March 21. 1989. Received December 1, 1988. tThe data from this study were presented in pail at the 78th American Society of Animal Science Annual Meeting. Kansas State University, Manhattan, KS. This research was supported by USDA CRG 86-01108 is published as paper no. 8805, Journal Series Nebraska Agricultural Research Division. 2Repnnt requests. 3Present address: Department of Animal Science, The Ohio State University. Columbus, OH 43210-1095. 4Present address: Department of Animal Science, Oklahoma State University, Stillwater, OK 74074.
91
STUMPF
92
during the follicular phase of the estrous cycle in intact heifers. During this time in the ovine estrous cycle, increasing concentrations of E2 diminish the magnitude of the increase in tonic secretion of LH initiated by progesterone withdrawal (Goodman et a!., 1980). This results in a decrease in amplitude of pulses of LH, with frequency of pulses of LH remaining similar to that of ovariectomized ewes that do not receive E2 (Goodman and Karsch, 1980). In contrast to these data, increased concentrations of E2 administered to ovariectomized cows via an implant for 30 days caused an increase in mean concentration of LH and amplitude of pulses of LH, but decreased the frequency of pulses of LH (Kinder et a!., 1983; Imakawa et at., 1986). The purpose of the present study was to determine the effects of increasing concentrations of E2 during the follicular phase of the bovine estrous cycle on secretion of LH prior to the preovulatory surge of LH. Our working hypotheses were that (1) increasing concentrations of E2 during the follicular phase of the bovine estrous cycle would increase mean concentration of LH by increasing amplitude of pulses of LH, and (2) increasing concentrations of E2 during this stage of the estrous cycle would suppress the frequency in which pulses of LH were released from the anterior pituitary. MATERIALS
Experimental
AND
METHODS
Design
The estrous
of 17 mature beef cows 3-9 yr of to the same day of the estrous cycle utilizing SYNCRO-MATE-B (CEVA Laboratories, Inc., Overland Park, KS). The cows were randomly assigned to one of four treatments initiated on Day 16 (Day 0 = estrus) of the experimental estrous cycle. At Hour 0, 4 cows were lutectomized via high lumbar laparotomy. The purpose of lutectomy was to allow for endogenous secretion of E2 (EE, n = 4). The remaining cows were ovariectomized at Hour 0 and received one of three E2 replacement treatments: luteal phase E2 (LE, n = 5), increasing then decreasing E2 (DE, n = 5) and no E2 (NE, n = 3). Estradiol was administered via s.c. polydimethylsioxane implants (3.35 mm i.d. x 4.65 mm o.d. x 13.5 cm; Dow-Coming, Midland, MI) filled with 17-estradiol (Sigma Chemical Company, St. Louis, MO). caudal to the shoulder blade. Cows in the group that received LE were administered one E2 implant at Hour 0. This implant was to age
were
cycles
synchronized
ET AL. provide circulating concentrations of E2 similar to those observed during the luteal phase of the estrous cycle. The cows in the group that received DE were administered one E2 implant at Hour 0, and additional E2 implants were administered at Hours 8, 16, 24, 32, and 40 of the study. Two implants were then removed at Hours 48 and 56 and one implant was removed at Hour 64. This treatment protocol was designed to mimic the preovulatory rise and fall of circulating concentrations of E2 after luteolysis. l’his implant regimen was established by a preliminary study (T. T. Stumpf, unpublished data) in which the desired increase and decrease in concentrations of E2 was based on follicular phase concentrations of E2 subsequent to induced luteolysis (Zalesky et a!., 1985). All implants of E2 used in the treatment protocol were administered s.c. to one of two ovanectomized cows 6 days prior to initiation of the study. Implants containing E2 were removed from these cows immediately prior to time of implantation of the cows used in the present experiment. The implants of E2 were placed in ovariectomized cows before initiation of the study, so any E2 that collected on the surface of the implants would be cleared by metabolic processes. This prevented a transient rise in E2 following implantation of the experimental cows. At Hour 0 in cows receiving NE and at Hours 8-56 in cows receiving NE, LE, and EE, s.c. sham polydimethylsioxane implants (3.35 mm i.d. x 4.65 mm o.d. x 13.5 cm) were administered or removed according to the treatment protocol for cows receiving DE. Indwelling jugular catheters were inserted and cows were placed in stanchions one day prior to initiation of treatments. All cows were acclimated to stanchions prior to the study. Hay was provided ad libitum throughout the 80-h period of blood collection and water was routinely provided. Blood samples were serially collected (5 ml at 12mm intervals) from Hour 2 through Hour 80 for determination of concentrations of LH. An additional sample (10 ml) was collected immediately before surgery (Hour 0) and at hourly intervals from Hour 2 through Hour 80 for progesterone and E2 analyses. The 5-mi blood samples were allowed to coagulate at room temperature and were then stored at 4#{176}C. The 10-mi blood samples were immediately stored at 4#{176}C. All samples were centrifuged within 48 h of collection, and serum was decanted, frozen, and stored at -20#{176}C until assayed.
ESTRADIOL Hormone
MODULATION
Analysis
Serum concentrations of progesterone were quantified from aliquots of samples collected at Hour 0 and Hours 2 through 10 for each cow by the radioimmunoassay validated by Anthony et al. (1981). Intra- and interassay coefficients of variation for progesterone assays were 2.5 and 14.5%, respectively, and the average sensitivity was 0.11 ng/ml of serum. Serum concentrations of LH were determined in all samples (5 ml) obtained during the period of serial blood collection by a double antibody radioimmunoassay (Golter et a!., 1973; Garcia-Winder et at., 1986), using rabbit antiserum against bovine LH (JJR-RABLH #5), highly purified iodinated ovine LII (LER-1056-C2) as labeled hormone, and NIH-LH-B7 as standard. All samples collected during the experiment for an individual cow were analyzed in a single assay. The assays were performed in pairs such that cows of different treatments were represented in each assay. Intra- and interassay coefficients of variation for LH assays were 3.6 and 13.7%, respectively, and the average sensitivity was 80 pg/mI of serum. Serum concentrations of E2 were measured in pools of the hourly samples collected for each cow (Hours 2-4; then Hours 5-8, 9-12, 77-80) with the radioimmunoassay validated by D’Occhio et at. (1982). Each assay contained the sample collected at Hour 0 and all of the pooled samples for an individual cow. Assays were performed in triplicate with cows of different treatments represented in each assay. Intra- and interassay coefficients of variation for E2 assays were 1.7 and 11.7%, respectively, and the average sensitivity was 1.5 pg/ml of serum. Mean concentration of LH and E2, amplitude of pulses of LH, and frequency of pulses of LH were calculated within 4-h periods (to characterize changes over time), with the exception that Period 1 contained data from only Hour 2 through Hour 4. Mean concentration of LH per period was calculated as the average concentration of the samples collected during that period for an individual cow. Pulses of LH were identified and amplitudes of pulses of LH were calculated by the methods of Goodman and Karsch (1980). Frequency of pulses of LH for Period I (Hours 2-4) was determined as the total number of pulses detected divided by 0.75, thereby representing a 4-h period. Frequency of pulses of LH per 4-h period (Periods 2 through 17) was determined as the total number of pulses detected during that particular period for an individual cow. Ampli.
.
.
,
OF LH
93
tude of pulses of LH per period was calculated as the average amplitude of all pulses detected during that particular period. Mean concentration of E2 per period was derived by determining concentration of E2 in the pooled hourly samples for an individual cow. Any data for LH collected during or subsequent to a preovulatory-like surge of LH for a particular cow were not included in the analyses of the data.
Statistical
Analysis
Regression analyses were carried out using a splitplot design with cow as the whole plot and period of sample collection (continuous variable) as the split plot (Steele and Torrie, 1980; Draper and Smith, 1981). The overall effect of treatment was tested with cowwithin-treatment as the error term. To test significant treatment by period of sample collection interactions, within-animal regressions of hormone characteristics of interest (mean concentration of LH, amplitude of pulses of LH, frequency of pulses of LH) on period of sample collection were fitted. The resulting within-animal regression parameters were analyzed by analysis of variance according to Allen et a! (1983) and Sanders (1978). Specific treatment means of regression parameters were compared using f-tests.
RESULTS
Mean concentration of serum progesterone over all treatments had declined to 1 ng/ml by Hour 10. Lutectomy of the cows in the EE group did not elicit the endogenous secretion of E2 expected for cows in this treatment. It was expected that the profile for E2 would be similar to the preovulatory rise and fall of circulating concentrations of E2 after induced luteolysis in intact cows (Zalesky et at., 1985). Instead, there was a gradual decline in concentrations of E2 in serum over time in the cows in the EE group (Fig. 1). This effect was also demonstrated in that only 2 of 4 cows receiving the EE treatment displayed a preovulatory surge of LH (mean time of LII surge 43.5 ± 6.4 h). Desired concentrations of E2 in circulation were obtained in cows receiving the DE, LE, and NE treatments (Fig. 1). Concentrations of E2 in serum increased through Period 10 (Hours 37-40) and then decreased through the conclusion of the study in the cows receiving the DE treatment. This was reflected with 4 of 5 cows receiving the DE treatment displaying a preovulatory surge of LH (mean time of LH surge = 47.7 ± 2.0 h). Serum =
STUMPF
94
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19
PERIOD
65-6873-76
HOURS
FIG. 1. Regression of mean concentration of estradiol (Ez) over time by treatment. Serum concentrations of E2 were measured in pools of hourly samples collected for each cow (see MateriaLs and Methods for details). Mean concentration of E2 was calculated within 4-h periods (except Period 1 contained data from only Hour 2 through Hour 4) for an individual cow. Treatments: EE = endogenous E2 secretion; DE = increasing then decreasing E2 replacement; LE = luteal phase E2 replacement; NE = no E2 replacement.
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PERIOD
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FIG. 2. Regression of mean concentration of luteinizing hormone (LII) over time by treatment. Serum concentrations of LH were measured in all samples collected for each cow. Mean concentration of LH was calculated within 4-h periods for an individual cow (except Period I contained data from only Hour 2 through Hour 4; see Materials and Methods for details). Treatments: DE = increasing then decreasing estmdiol (E2) replacement; LE = lutcal phase E2 replacement; NE = no E2 replacement.
5
7
9
17-20
25-28
33-36
ii 41-44
13
15
49-52
57-60
17
PERIOD
65-68H0URS
FIG. 3. Regression of amplitude of pulses of luteinizing hormone (LII) over time by treatment. Mean amplitude of LH pulses was calculated within 4-h periods for an individual cow (except Period I contained data from only Hour 2 through Hour 4, see Materials and Methods for details). Treatments: DE = increasing then decreasing estradiol (E2) replacement; LE = luteal phase E2 replacement; NE = no E2 replacement.
There
concentrations of E2 in the cows receiving the LE treatment initially declined, until the time when the implant of E2 administered at Hour 0 was able to maintain low circulating concentrations of E2. Serum concentrations of E2 in the cows receiving the NE treatment paralleled those of the cows receiving the LE treatment, but the concentrations were lower throughout the study. No cows receiving the LE or NE treatments displayed a preovulatory surge of LH.
3 9-12
was no significant change over time for mean of LH or amplitudes of pulses of LH in cows from the EE treatment group (data not shown). A significant quadratic response (p
