Effect of Neonatal Diethylstilbestrol Exposure on Luteinizing Hormone ...

BIOLOGY OF REPRODUCTION 48, 828-832 (1993)

Effect of Neonatal Diethylstilbestrol Exposure on Luteinizing Hormone Secretion following Ketamine Anesthesia and Gonadotropin-Releasing Hormone in Castrated Postpubertal Rats KENNETH A. FABER' and CLAUDE L. HUGHES, JR. Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology Duke University Medical Center, Durham, North Carolina 27710 ABSTRACT Neonatal diethylstilbestrol (DES) exposure diminishes pituitary responsiveness to GnRH in castrated postpubertal rats 4 h after placement of intra-atrial cannulae under ketamine anesthesia. Ketamine anesthesia has been reported to blunt the preovulatory LH surge in female rats and to decrease baseline LH levels in males immediately following administration, although its effect lasted less than 2 h. It is not known what impact ketamine has on baseline LH levels or on GnRH-stimulated LH release in estrogen-exposed castrated rats. This study was designed to determine 1) whether the changes in GnRH-induced LH secretion in DES-exposed castrated rats are partially attributable to a direct effect of ketamine; 2) the gender-specific effects of ketamine on LH secretion; and 3) the DES-dependent effects of ketamine on LH secretion. In this experiment we ascertained the effect of ketamine anesthesia for right heart cannulation on LH levels in male and female 42-day-old castrated rats that were exposed to either corn oil or DES (0.1 jpg/day) on Days 1 through 10 of life. LH secretion in DES-exposed animals increased, in contrast to that in control animals, immediately (< 10 min) after anesthesia and cannulation. Mean LH levels in DES-exposed females were lower than in controls before GnRH administration and were blunted following GnRH. While ketamine and catheterization were associated with decreased LH titer in males, the extent of that decrease was not dependent on exposure to DES. Both sexes and exposure groups achieved baseline catheter LH values by 4 h. This experiment demonstrates that 1) the pituitary response to ketamine anesthesia and catheterization depends on gender and neonatal DES exposure and 2) the previously reported decreases in GnRH-induced LH secretion due to neonatal treatment with DES cannot be attributed to ketamine-induced suppression of basal gonadotropin secretion.

INTRODUCTION Neonatal estrogen exposure in female rats changes sexual behavior, physiology, and central nervous system anatomy [1-4]. These changes reflect masculinization of the normal pattern of female sexual differentiation. We have reported that neonatal injection of DES (0.1 ig or 1 Vtg), the plant estrogen genistein, o,p'-DDT, or the mycoestrogen zearalenone decreases GnRH-stimulated LH release in pubertal castrated females [5,6]. The animals were castrated in order to isolate the developmental effects of neonatal sex-steroid exposure from those during the pubertal period. One implication of those studies is that the phenomena associated with natural exposures to environmental estrogens may be explained by decreases in pituitary sensitivity to GnRH. In the present studies, intra-atrial catheters were placed under anesthesia with ketamine hydrochloride (100 mg/ kg) 4 h before GnRH stimulation. Blood sampling was done when the animals were awake and moving freely in their cages. Ketamine is known to block spontaneous GnRH release and to decrease peripheral LH levels in adult rats [710], but the effect has been reported to last less than 2 h [9, 10]. It is not known whether this property of the ketamine effect on LH secretion is altered in castrated rats ex-

posed neonatally to estrogen. If estrogen-exposed females were more sensitive to the effects of ketamine than control females, either in the degree of LH suppression or the length of time needed for recovery from anesthesia, then differences in GnRH-stimulated LH release as seen in the previous studies might have been partially due to ketamineinduced pituitary suppression. This study tests the hypothesis that ketamine has exaggerated effects on LH secretion in castrated, estrogen-exposed rats, and that this anesthesia-associated phenomenon explains the decrease in pituitary sensitivity to exogenous GnRH seen in previous studies. MATERIALS AND METHODS Pregnant rats of the Charles River CD strain (Raleigh, NC) were purchased at 15-17 days gestation and maintained in air-conditioned quarters with Purina Laboratory Chow (Ralston-Purina, St. Louis, MO) and water available ad libitum. The lighting schedule was 14L:10D, with lights-on from 0500 to 1900 h EST. Day of delivery was defined as Day 1 if delivery was observed to have occurred before 1200 h. Pups were treated from Days 1-10 with 0.05 ml injections (s.c.) of either corn oil or 0.1 RIg DES dissolved in corn oil. All pups within a litter received the same treatment. On Day 21 of life, the animals were castrated under ketamine anesthesia (100 mg/kg) and weaned. Six siblings were housed in each plexiglass cage. On Day 42, the pups

Accepted November 19, 1992. Received August 3, 1992. 'Correspondence: Kenneth Faber, M.D., Campus Box B198, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262.

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KETAMINE EFFECTS ON LH SECRETION log LH (ngm) 2.6 -

Legend (12)

nsa

--

2.1 1.6 -

(13)

(18)

ns~~~~~

-A Control Male

*

DES Male

+

DES Female

(8)

*

1.1-

(12)

Control Female

P (12)

0.6 (12) 0.1 (1S \.

Decap

Ketamlne/Cateter

FIG. 1. Log-transformed serum LH levels (NIDDK-rLH-RP3; ng/ml + SEM) at decapitation or immediately following ketamine (100 mg/kg) and right heart catheterization in male and female castrated postpubertal rats exposed to either corn oil or DES (0.1 i.g/day, Days 1-10). * p < 0.0001, same-sex controls vs. DES; ct,p < 0.001, female controls vs. DES; 3,p < 0.0001, decapitation vs.ketamine/catheter; ns= not significant. (n) = Number of animals in each group.

in each litter were either decapitated at 0800 h or injected with ketamine (100 mg/kg) for the purpose of right heart catheterization. Time of ketamine injection was considered t = 0 and surgery did not begin until the animals were clearly asleep and not responsive to tail pinching. A blood sample (0.3 cc) was drawn from the catheter immediately upon insertion (less than 10 min after ketamine injection) and at 10 min, 15 min, 30 min, 1 h, 2 h, 3 h, and 4 h after ketamine injection. Blood volume was replaced with an equal volume of 10 U/ml heparinized saline each time. The animals received GnRH (Factrel, Lot #3880235; Ayerst Laboratories, New York, NY) dissolved in saline (50 ng/kg; 1 ml/kg) i.v. 4 h after cannulation. Blood samples were collected via the catheter immediately before (t = 4 h) and 5, 10, 15, and 30 min after injection of GnRH. Blood samples were allowed to clot at room temperature and were then centrifuged at approximately 1500 x g for 10 min. Serum was then aspirated and frozen at -20°C for later RIA. Serum LH concentrations were measured by double-antibody RIA with a rat LH RIA kit supplied by NIDDKD and the National Hormone and Pituitary Program (University of Maryland School of Medicine, Baltimore, MD). Second antibody (sheep, anti-rabbit) was graciously supplied by Lee Tyrey, Ph.D. (Duke University Medical Center, Durham, NC). Aliquots of serum (50 ll or less) were assayed for LH in duplicate and the means were expressed in terms of NIDDKD-rLH-RP3. Intra- and interassay coefficients of variation for the measurement of LH in three serum pools were 4.8% and 8.2%, respectively. The assay sensitivity was 0.48 ng/ml of serum. The log-transformed LH values were used to assure homogeneity of the variance. Basal and stimulated LH were

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compared with univariate and multivariate analysis of variance (ANOVA). When LH was less than the assay sensitivity, the Kruskal-Wallis test was used. Statistical significance was taken at ot = 0.05. Differences in LH within subjects was determined with least squared means testing with appropriate adjustments of a via the Bonferroni correction. RESULTS The influence of decapitation without ketamine versus that of ketamine anesthesia and cannulation in control and DES-exposed castrated postpubertal rats is shown in Figure 1. Neonatal DES exposure was associated with decreased LH in DES-exposed animals at decapitation (no ketamine) compared to same-sex controls. LH in DES-treated rats of both sexes increased immediately following catheterization while there was no change in LH in control animals. LH Secretion in Females Serum LH levels following ketamine and GnRH in castrated postpubertal female rats are presented in Figure 2. We used repeated measures analysis of variance to compare the change in LH from the first catheter sample to each subsequent sample during the 4-h interval before GnRH stimulation. The mean LH in females decreased from the first catheter sample only at 30 min, and that decrease was greater in the DES-exposed group. The LH titer was significantly higher in control rats than in DES rats at each sampling. There were no differences in LH level due to time in either exposure group, and there was no interaction of time and neonatal exposure agent. There was no trend towards the linear or concave in either control or DES groups. In contrast to unstimulated LH secretion, LH levels following GnRH at t = 4 h depended on exposure agent, time after GnRH, and an interaction of time and agent (Fig. 2). Diminished GnRH-induced LH secretion in DES-exposed females is illustrated by several factors: 1) decreased LH levels compared to those in control animals 5, 10, 15, and 30 min following GnRH; 2) decreased net LH secretion at 5 min compared to that in control animals; 3) decreased maximum LH; and 4) reduced area under the LH stimulation curve. LH Secretion in Males LH levels after ketamine and in response to GnRH in male postpubertal castrated rats are presented in Figure 3. LH levels at 10, 15, 30, 60, and 120 min were significantly lower than the LH titer in the first catheter sample; however, neonatal exposure agent did not influence the degree of the difference between LH levels in the first sample and those in later samples. This implies that anesthesia/catheterization, and not DES exposure, was responsible for decreases in LH titers in males. When corrected for multiple comparisons, there were no differences in LH levels be-

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FABER AND HUGHES

log LH (ng/ml)

log LI (ng/ml)

2.4-

z2-

2.2-

Legend

S

2-

i-

1.8-

Legend

1.8-

Control

1.6-

-U- DES

1.4-

1.6

-

Control

·-

DES

-

Control

1.2

1.41.2-

1-

1-

4tchc

0.80.6i

0.8-

0.60.4-

0.4*

*

*

30 " lhr ' 2hr

3'hr

4hr

*

0.2

*

0.2-

·

·

0

0

0

//

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