Ovarian Control of Prolactin Secretion During Late Pregnancy in the Rat1 ROBERT S. BRIDGES2 AND B. D. GOLDMAN Department of Biobehavioral Sciences, The University of Connecticut, Storrs, Connecticut 06268 ABSTRACT. The involvement of the ovaries during late pregnancy in the rat upon serum prolactin was investigated. Ovariectomy on day 17 or day 21 of pregnancy prevented the dramatic rise of prolactin found in sham-ovariectomized animals between days 21 and 23 of pregnancy. While animals ovariectoinized on day 17 of pregnancy failed to carry

viable fetuses beyond day 19 of pregnancy, rats ovariectomized on day 21 of pregnancy had viable pups in utero on day 23 of pregnancy. These data suggest that the rise in serum prolactin during late pregnancy is stimulated by ovarian factors. (Endocrinology 97: 496,1975)

ERUM estrogen and prolactin levels are high S at approximately the same time during the estrous cycle (1-4) and during pregnancy in the

the females had mated. The presence of sperm in the smear was designated as day 1 of pregnancy. Pregnant animals were then housed separately in plastic cages (22 x 43 x 15 cm). In Experiment 1, pregnant rats were either ovariectomized under ether anesthesia on day 17 of pregnancy and then bled by decapitation on day 19, 21, or 23 of pregnancy, or were sham-ovariectomized (sham-ovx) on day 17 of pregnancy and then bled at the same times as the ovariectomized animals. An additional group of pregnant rats was decapitated on day 17 of pregnancy. All bleedings were performed between 10:00 and 11:00 except on day 23 when decapitation occurred between 8:00 and 9:00. Following decapitation, the uteri of all animals were examined to validate pregnancy and to determine whether live pups were present. In a second experiment pregnant females were assigned to one of three groups. Group 1 females were ovariectomized under ether anesthesia on day 21 of pregnancy between 12:00 and 14:00. Group 2 females were sham-ovariectomized at the same time. Both of these groups were bled by decapitation between 8:00 and 9:00 on day 23 of pregnancy. In Group 3, animals were bled by decapitation between 8:00 and 9:00 on day 21 of pregnancy. Following sacrifice the uteri were examined as described in Experiment 1.

rat (1,2,5). Ovariectomy (ovx) will suppress serum prolactin levels in virgin rats, while estrogen will elevate prolactin levels in the same animals (6). Apparently, the rise in estrogen during the estrous cycle and pregnancy stimulates the release of pituitary prolactin resulting in elevated serum prolactin concentrations. Contrary to this hypothesis, recent reports (7,8) have suggested that the rise in serum prolactin during late pregnancy in the rat is either independent of ovarian factors (7) or under an inhibitory ovarian influence (8). However, in these studies blood was collected by heart puncture while the animal was lightly anesthesized (7) or by heart puncture while the animal was conscious (8). The method of blood collection can greatly affect the level of serum prolactin. Ether anesthesia, for example, will result in a rapid elevation of serum prolactin (3). The present study reexamines the role of the ovaries in the increased titer of prolactin found during late pregnancy in the rat.

Materials and Methods Animal preparations. Purdue-Wistar rats from our colony were maintained on 13:11 h light:dark schedule (lights on 7:00) with food and water available ad libitum. Between 90 and 120 days of age females were housed with adult male rats. Vaginal smears were made daily (10:00) to detennine whether Received January 27, 1975. 1 Aided in part by NIH Grant HD 05481 awarded to B. D. Goldman. 2 Address reprint requests to Robert S. Bridges.

Hormone assay. Bloods were refrigerated at 4 C following decapitation and were then centrifuged at 2000 rpm for 20 min. Sera were stored at -20 C until they were assayed for prolactin content. All sera within each experiment were assayed simultaneously; however, the sera from the two experiments were analyzed in separate assays. Prolactin content was determined by radioimmunoassay and all values are expressed in terms of the NIAMDD Rat Prolactin RP-1 Reference Preparation (11 lU/mg). All serum samples were run in duplicate and the mean for each animal was used in statistical calculations. Data were analyzed using the t for planned comparisons and the Fisher Exact Probability test. The sensitivity of the prolactin assay was approximately 0.5 ng.

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NOTES AND COMMENTS Results Ovx day 17 of pregnancy: prolactin and fetal viability. Ovariectomy of rats on day 17 of pregnancy prevented the rise in serum prolactin that occurred between days 21 and 23 in sham-ovx animals (Table 1). Prolactin levels remained low in the ovariectomized animals bled on days 19, 21, and 23 of pregnancy. In contrast, prolactin levels of sham-ovariectomized rats gradually rose from day 17 to day 21 and then increased dramatically between days 21 and 23 of pregnancy (t = 20.50, P < 0.001). A comparison of serum levels between sham-ovx and ovx animals showed that serum prolactin values were higher in sham-ovx females on both days 21 (t = 2.60, P < 0.05) and 23 (t = 13.75, P < 0.001) of pregnancy. Ovariectomy resulted in decreased fetal survival. Significantly more pregnant ovariectomized animals failed to carry live fetuses in utero on either days 21 (Fisher Exact Probability, P < 0.001) or 23 (Fisher Exact Probability, P < 0.001). Since failure to maintain viable fetuses was associated with the suppression of prolactin levels after ovariectomy on day 17 of pregnancy, an attempt to dissociate these factors was made in the second experiment. Ovx day 21 of pregnancy: prolactin and fetal viability. Serum prolactin levels were elevated in sham-ovx animals on day 23 of pregnancy,

but remained low in ovx females (Table 1). Prolactin increased between days 21 and 23 in sham-ovx females (t = 4.38, P < 0.001), but remained at day 21 levels on day 23 in ovx rats. A comparison between sham-ovx and ovx animals on day 23 of pregnancy indicates that serum prolactin levels were significantly higher in sham-ovx females (t = 7.36, P < 0.001). In contrast to animals ovariectomized on day 17 of pregnancy (Experiment 1), rats ovariectomized on day 21 of pregnancy were found to have viable fetuses in utero following bleeding on day 23. Each of the 13 animals in this latter group had four or more live fetuses in utero following decapitation. These live pups were able to survive when fostered to lactating rats. Discussion The data demonstrate that the rise in serum prolactin occurring during late pregnancy in the rat is under ovarian influence. Ovariectomy prevented the increase in the serum levels of prolactin on days 21 and 23 of pregnancy and on day 23 of pregnancy in the two experiments, respectively. This finding is consistent with data that suggest that the rise in ovarian estrogen occurring prior to parturition may stimulate an increase in serum prolactin levels (4,6,9). The findings of this study are in disagreement with those previously reported (7,8). We found no increase in serum prolactin following ovariectomy during late pregnancy, while

TABLE 1. Serum prolactin levels and fetal viability following ovariectomy during late pregnancy in the rat

Experiment 1

2

% N with 1 or more live pups

N

Pregnancy day bled

8

17

9.33 ± 0.64

100

Sham-ovx Day 17

12 8 7

19 21 23

12.02 ± 0.84 15.65 ± 2.35 139.58 ± 5.90**

100 100 100

Ovx day 17

11 8 8

19 21 23

10.73 ± 0.76 9.26 ± 0.72*** 9.69 ± 1.09* 34.20 ± 6.24

Treatment Intact

Intact

5

21

Sham-ovx Day 21

11

23

Ovx day 21

13

23

Prolactinf ng/ml ± SEM

151.45 ± 17.50**** 29.92 ± 3.66*

82 0* 0* 100 100 100

* Compared to sham-ovx controls, P < 0.001. ** Compared to day 21 sham-ovx animals, P < 0.001. *** Compared to day 21 sham-ovx animals, P < 0.05. **** Compared to day 21 intact animals, P < 0.001. f Variation in prolactin values between experiments was due to interassay variability.

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498

Endo

NOTES AND COMMENTS

Simpson et al. (7) and Vermouth and Deis (8) reported increased titers of prolactin in ovariectomized pregnant rats. Simpson et al. (7) reported that the rise in prolactin occurring during late pregnancy was independent of ovarian control. They bled animals only until day 22 when a moderate rise in prolactin occurred. In the present report, the dramatic rise of prolactin levels occurred on day 23 of pregnancy. It is possible that if Simpson et al. (7) had measured prolactin levels on day 23, they also would have found that ovariectomy inhibited the rise in this hormone. The method of blood collection employed by Simpson et al. (7) also might have masked the inhibitory effect of ovariectomy on prolactin release. In their study blood was collected via heart puncture while animals were under ether anesthesia. Since ether is known to stimulate prolactin secretion (3), the rise in prolactin found in both ovx and sham-ovx animals (7) might have been a function of the ether stress. If animals had been decapitated, it is likely that these investigators would have found lower prolactin levels in the ovx pregnant rats and differences in prolactin levels between ovx and sham-ovx animals during late pregnancy. Vermouth and Deis (8) bled pregnant rats via heart puncture while the animals were conscious. These workers reported an increase in serum prolactin levels following ovariectomy and proposed that prolactin secretion was inhibited by ovarian progesterone, which is high in intact animals through day 21 of pregnancy (10,11). However, we found no similar increase

1975

Vol 97 i No 2

in prolactin following ovariectomy. Perhaps, bleeding rats by heart puncture while the rats are conscious might also trigger a "stress" response of prolactin. The present study does not exclude the possibility that extra-ovarian sources may stimulate prolactin release during late pregnancy in the rat, but it appears that the dramatic increase in serum prolactin that occurs between days 21 and 23 of pregnancy is due primarily to ovarian stimulation. References 1. Yoshinaga, K., R. A. Hawkins, and J. F. Stocker, Endocrinology 85: 103, 1969. 2. Amenomori, Y., C. L. Chen, and J. Meites, Endocrinology 86: 506, 1970. 3. Neill, J. D., Endocrinology 87: 1192, 1970. 4. Smith, M. S., M. E. Freeman, and J. D. Neill, Endocrinology 96: 219, 1975. 5. Linkie, D. M., and G. D. Niswender, Endocrinology 90: 532, 1972. 6. Chen, C. L., and J. Meites, Endocrinology 86: 503, 1970. 7. Simpson, A. A., M. H. W. Simpson, and P. N. KulkarniJ Endocrinol 57: 425, 1973. 8. Vermouth, N. T., and R. P. D e i s J Endocrinol 63: 13, 1974. 9. Neill, J. D., In Greep, R. O., E. B. Astwood, E. Knobil, W. H. Sawyer, S. R. Geiger (eds.), Handbook of Physiology, Section 7—Endocrinology, vol. IV, Part 2, Baltimore, 1974, p. 469. 10. Pepe, G. J., and I. Rothchild, Endocrinology 95: 275, 1974. 11. Grota, L. J., and K. B. Eik-Nes J Reprod Fertil 13: 83, 1967.

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Ovarian control of prolactin secretion during late pregnancy in the rat.

The involvement of the ovaries during late pregnancy in the rat upon serum prolactin was investigated. Ovariectomy on day 17 or day 21 of pregnancy pr...
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