The Proestrous Surge of Proactin Enhances Sexua Receptivitiy in the Rat

Transcription

1 BIOLOGY OF REPRODUCTION 32, (1985) The Proestrous Surge of Proactin Enhances Sexua Receptivitiy in the Rat JEFFREY A. WITCHER3 and MARC E. FREEMAN2 Department of Biological Science Florida State University Tallahassee, Florida ABSTRACT The influence of the proestrous surge of prolactin (PrI) on expression of feminine sexual behavior (lordosis) has been investigated. In the first experiment, proestrous rats were treated with a dopamine agonist, bromocriptine (CB-154; 100 g at 1200, 1300, and 1600 h), which blocked the proestrous surge of Pri without affecting the preovulatory surge of luteinizing hormone. Such animals displayed depressed lordosis quotients (LQs) when compared to control animals at 2000 h on proestrus. However, in CB-154-treated animals given ovine Pr! (10 l.u.) at 1400 h on proestrus, LO_s were restored to control levels. Ovariectomized (OVX) rats primed with estradiol benzoate (EB; 2 g for 2 days) produced surges of Prl that were similar in timing to those of proestrous. Once again, CB-154 treatment blocked this PrI surge and significantly depressed the LQ, whereas replacement with ovine PrI returned the LQ to control values. These results suggest that the PrI surge facilitates the expression of lordosis. Ovariectomized/adrenalectomized (ADX) and OVX/sham ADX rats were treated with EB (2 Mg for 2 days) and tested for lordosis on Day 3. Adrenalectomized rats responded with lower levels of lordosis than did sham controls. Administration of progesterone (P4) to ADX rats on Day 3 enhanced the LQ compared to sham ADX values. CB-154 was ineffective in reversing the enhanced LQ, indicating that PrI may be acting through stimulation of adrenal progestins. These data taken together suggest that the proestrous surge of PrI contributes to the normal expression of feminine sexual behavior on proestrus. INTRODUCTION A surge in the secretion of prolactin (Pri) preceding behavioral estrus (Butcher et al., 1974; Smith etal., 1975) presents the possibility that Pr! may influence the expression of feminine sexual behavior. Reports of effects of Pr! administration on sexual behavior include inhibition of receptivity when given in the third ventricle to estrogen + progesterone (P4 )-primed ovariectomized (OVX) rats (Dudley et al., 1982), as well as facilitation of receptivity when applied to the midbrain of estradiol benzoate (EB)- treated OVX rats (Harlan et al., 1983). On the other hand, a surge of Pr! secretion induced by the dopamine antagonist domperidone was found to have no effect on receptivity in EB + Accepted December 6, Received July 20, Supported by NIH grant HO-i 1669 and a Research Career Development award to M.E.F. (NIH grant HD ). 2Correspondence: Dr. Marc E. Freeman, Department of Biological Science, Florida State University, Biology Unit #1, Tallahassee, Florida Present address: Department of Psychology, Univeristy of Pennsylvania, Philadelphia, Pennsylvania P4 -treated rats (Sidersten et a!., 1983a). The aim of this study was to determine whether the proestrous surge of Pri plays a physiologic role in influencing the expression of feminine sexual behavior in the rat. In order to make this determination, the proestrous surge of Prl was suppressed by a dopamine agonist, bromocriptine (CB-154; Cronin et al., 1984). Animal Care and Surgery MATERIALS AND METHODS Sprague-Dawley rats were purchased from Charles River Laboratories (Wilmington, MA). Groups of 3-5 were housed in suspended metal cages under a 12L: 1 2D schedule (lights on at 0600 h) with food and water available ad libirum. Estrous cycles of intact females were monitored daily with vaginal lavage. All surgery was performed under ether anesthesia. Adrenalectomized animals were given 0.9% sodium chloride. Blood Collection Serial blood samples were obtained by atrial cannulae implanted 1 day before collection. Silastic medical grade tubing (Dow-Corning, Midland, MI; catalog number ) was inserted through the external jugular vein while connected to PE-50 tubing (Clay-Adams, Parsippany, NJ) and exteriorized through the dorsal aspect of the neck. Cannulae were filled with heparinized saline (100 lu/mi; Sigma Chemical Co., St. Louis, MO) before and between blood collec- 834

2 PROESTROUS PRL SURGE AND LORDOSIS 835 tions. Blood was collected at 1000, 1200, 1400, 1600, 1800, and 2100 h, with replacement of equivalent volumes of saline. Blood was centrifuged at 1000 X g for 10 mm at room temperature and plasma was stored at -20#{176}C until assayed for Prl and luteinizing hormone (LH). Cannulated animals were housed individually in Plexiglas cages. Treatments Steroid hormones (Sigma) were solubiized in cottonseed oil and administered intramuscularly in a volume of 0.1 ml cottonseed oil. Bromocriptine (CB-154; Sandoz Labs, East Hanover, NJ) was initially solubiized in absolute ethanol and diluted with water to a suspension containing 100 Mg/0.2 ml of 10% ethanol (EtOH). Aliquots (one hundred Mg) of CB- 154 were given intraperitoneally at 1200, 1400, and 1600 h. Ovine prolactin (NIADDK o-prl-16; 30.5 lu/mg) was solubiized in 50% polyvinylpyrrolidone (PVP; Eastman-Kodak Co., Rochester, NY) in 0.9% saline at a concentration of 10 IU/0.1 ml and administered Hormone subcutaneously. Assays Determinations of PrI and LI-I in plasma were accomplished using double antibody radioimmunoassays. Precipitating antibody (goat antirabbit gammaglobulin; Antibodies Inc., Davis, CA) was used in both Pri and LH assays. PrI assays were performed using NIADDK rat Prl-RP-1 (11 lu/mg) as the standard, and NIADDK antirat Prl-S-8 (1:3125) was used as first antibody. Rat PrI (NIADDK rat Prl-l-5; 30 lu/mg) was iodinated by the lactoperoxidase method (Miyachi et al., 1972). Assays were incubated for 24 h at 37#{176}C between additions of hormone antibody, iodinated hormone, and precipitating antibody. Precipitates were separated by centrifugation 48 h after addition of precipitating antibody. Luteinizing hormone assays were performed using NIADDK rat LH-RP-1 (0.03 X NIH LH-S-1) as the standard and NIADDK antirat LII- S-7 (1:5000) as the first antibody. Rat LH (NIADDK rat LH-I-6; 1 X NIH LH-S-1) was iodinated using the chloramine-t method (Greenwood et al., 1963). Assay incubation conditions were the same as above with the exception that the temperature was 4#{176}C. Behavioral Testing Behavioral tests were conducted in rectangular testing arenas (approximate dimensions 60 X 32 X 40 cm) in the darkened colony room. Observations were made under illumination of a 40-watt red bulb. Sexually experienced males were allowed to acclimate in the arenas 5-10 mm before introduction of test females. A test was considered complete when the female had been mounted 10 times. Females were observed for the incidence of lordosis behavior (ventral arching of the spinal column), an indication of receptivity. Lordosis behavior was quantified as a lordosis quotient (LOJ: LQnumber of lordoses displayed/number of mounts experienced X 100. Behavioral tests and blood sampling were performed on separate groups of animals. Ovariectomized (OVX) rats were pretested for competency in displaying lordosis behavior in response to exogenous estradiol benzoate (EB) and P4 before inclusion in an experimental group. Females were pretested after administration of 2 Mg EB 54 and 34 h before behavioral testing. Approximately 5 h before testing 500 Mg P4 were given. Attainment of an LQ of 80 or greater during the pretest was a requirement for inclusion in further studies. Animals were allowed 6 days after pretesting before introduction to experiments. No pretests were given to intact animals. Experiments Experiment 1. Proestrous rats were assigned to one of three treatment groups. Two groups received CII- 154 (100 pg in 10% EtOH at 1200, 1400, and 1600 h) with one receiving 50% PVP/0.9% saline and the other opri (10 IU at 1400 h) in 50% PVP/0.9% saline. A third group received injections of both vehicles. All groups were tested for display of lordosis at 2000 h proestrus (lights off at 1800 h). Blood was collected from cannulated animals receiving the same treatment. Experiment 2. Ovariectomized rats received 2 Mg EB at 54 and 30 h before behavioral testing. On the day of testing, two groups received CB-154 (100Mg at 1200, 1400, and 1600 h). One of these received 50% PVP/0.9% saline, and the other received opri as previously described. The third group received only vehicle injections. Animals were tested for the display of lordosis behavior at 2000 h. Blood was collected from a group of similarly treated animals. Experiment 3. Ovariectomized animals were treated with 2 Mg EB for 2 days and adrenalectomized (ADX) or sham ADX the second day. Sham adrenalectomy included visualizing and handling but not removal of the adrenal glands. The following day animals were tested for display of lordosis at 2000 h. Five days later all OVXIADX animals received 2 days of EB priming as before. On the next day all animals received 500Mg P4 at 1430 h. In addition the ADX rats were given CB-154 treatment (100 Mg) at 1200, 1400, and 1600 h or 10% EtOH vehicle injections. All animals were tested for display of lordosis at 2000 h. Statistical A nalyses Kruskal-Wallis one-way analysis of variance and Mann-Whitney U tests were used to assess differences in blood hormone levels (Siegel, 1956). Lordosis behavior was analyzed by parametric analysis of variance followed by least significant difference tests where appropriate (Fryer, 1966). Experiment I RESULTS The proestrous surge of Pr! was blocked in rats treated with CB-154 (P<0.02) when compared to controls (Fig. 1). The same treatment failed to affect the proestrous surge of LH (Fig. 1). Proestrous rats treated with CB-154 displayed significantly attenuated LQj (P<0.05) when compared to controls (Fig. 2). In addition CB-1 54-treated rats given exogenous oprl presented LQs significantly greater than animals treated with CB-154 alone (P<O.05) and equivalent to those of proestrous controls.

3 836 WITCHER AND FREEMAN Experiment 2 Ovariectomized rats treated with EB for 2 days displayed surge-like Prl secretion on the afternoon of the third day (Fig. 3). Treatment with CB-154 on the third day eliminated surge-like secretion of Pr! (P<0.02). In addition, CB-154 treatment lowered LH titers (P<0.02), but did not eliminate the expected LH surge. CB-1 54-treated rats displayed significantly lower levels of lordosis (P<0.05) than did control rats (Fig. 4). Again, as in intact rats, exogenous oprl reversed the inhibitory effect of CB- 154 on expression of lordosis (P<0.05). Experiment 3 Ovariectomized rats treated with 2 pg EB for 200 Pr days and adrenalectomized the second day of EB treatment (Fig. 5, left panel) exhibited less lordosis behavior than sham ADX rats (P<0.05) at 2000 h on Day 3. Both ADX and sham ADX rats primed with 2 pg EB for 2 days displayed high levels of lordosis behavior 4.5 h following 500 pg P4 treatment (Fig. 5, right panel). Equivalent high lordosis behavior was observed in response to P4 regardless of whether ADX animals received CB-154 or not. DISCUSSION Results of the present study indicate that the proestrous surge of Prl influences the subsequent expression of feminine sexual behavior on proestrus. The data suggest that the influence of Pr! is exerted through the adrenal glands, possibly mediated by progesterone release. Others have found various effects of Pr! on lordosis behavior. These effects range from inhibition (Dudley et a!., 1982) to facilitation (Harlan et al., 1983) to no effect (S#{246}dersten et a!., 1983a). Hyperprolactinemia chronically induced by pituitary transplants beneath the kidney capsu!e suppressed lordosis behavior in estrogen + P4 -primed intact rats (Dudley et a!., 1982). These same authors also found that an PROESTRUS LH I- z!i 80.f -J 6C 20 rh 4 K OO rio pvp ce-l54 PVP CB-l54 o-prl TIME ON PROESTRUS TREAFMENT FIG. 1. A comparison of the pattern of plasma Pr! and LU levels on the afternoon/evening of proestrus. The closed circles represent mean values of animals given CB-154 (n=10) and the open circles represent mean values of animals receiving 10% EtOH (n=9) injections. Injections of CB-154 (100 Mg) were given at 1200, 1400, and 1600 h on proestrus. The vertical line through each circle represents the standard error. P<0.02 vs. EtOH. FIG. 2. A comparison of lordosis quotients of proestrous rats at 2000 h given one of three treatments: 1) EtOH plus PVP (n=13); 2) CB-154 plus PVP (n=15); or 3) CB-154 plus opri (n=i3). CB-154 treatment regimens were the same as previously described. Ovine PrI (10 lu) was given (s.c.) in PVP at 1400 h proestrus. Each bar represents mean lordosis quotient with standard error represented by vertical line. P<0.05 vs. EtOH/PVP and CB-154/0PRL.

4 PROESTROUS PRL SURGE AND LORDOSIS 837 acutely induced elevation of central levels of Pr! (by administration of Pr! into the third ventricle) suppressed lordosis behavior in estrogen + P4-primed OVX rats. A pharmacologically induced hyperprolactinemia using a dopamine antagonist, domperidone, resu!ted in no effect of lordotic expression in EB + P4-primed OVX rats (Slidersten et al., 1983a). However, when Pr! was administered in the midbrain in nonreceptive EB-primed OVX rats, a facilitation of lordosis was noted (Harlan et al., 1983). The apparent incongruent results of the above studies may be a reflection of the experimental models used. The first two studies (Dudley et al., 1982;Sbderstenetal., 1983a) utilized highly receptive rats in which it might have been difficult to elicit a further facilitation of feminine sexual behavior by Prl administration. Harlan et al. (1983) used an animal model in which an enhancement of lordosis behavior was observed only following treatment. Perhaps Pr! may exert a biphasic effect on expression of!ordosis I- z IJ 0 -J OVX (2ug EB/DAY) X 2 TAThENT FIG. 4. A comparison of lordosis quotients of OVX rats treated with EB (2 Mg) for 2 days and tested on Day 3 at 2000 h. Animals were given one of three treatments: 1) EtOH plus PVP (n=15); 2) CB-154 plus PVP (n17); or 3) CB-154 plus opri (n=13). CB-154 and oprl treatments were the same as described previously. Each bar represents the mean lordosis quotient with standard error represented by the vertical line., P<0.05 vs. EtOH/PVP. UI TIME ON DAY TiFEE FIG. 3. A comparison of the pattern of plasma PrI and LU levels on the third afternoon/evening following 2 days of EB (2 Mg) treatment. The closed circles represent mean values for animals treated with CB-1 54 (n=6). The open circles represent mean values for animals treated with EtOH (n#{176}6).cb-154 treatments were the same as described previously. The vertical lines through the circles represent the standard errors., P<0.02 vs. EtOH. FIG. 5. A comparison of lordosis quotients from OVX rats that were ADX (n=9) or sham ADX (n-9). Left panel illustrates lordosis quotients following 2 days of EB (2 pg) treatment and testing at 2000 h on Day 3. The right panel illustrates lordosis quotients of the same rats 1 wk later after 2 days of EB plus P4 (500 pg) on Day 3 at 2000 h. Sham ADX rats (n=9) were given EtOH injections and ADX rats were given EtOH (n=5) or CB-154 (n=4) injections. Each bar represents mean lordosis quotient with standard error represented by vertical line., P<0.05 vs. sham ADX.

5 838 WITCHER AND FREEMAN behavior, as has been suggested for P4 (Morin, 1979). Acute effects of Pr! treatment may be inhibition (Dudley et a!., 1982) or facilitation (Harlan et al., 1983) of lordosis behavior on a site-dependent basis, whereas chronic peripheral hyperprolactinemia may require a substantial period of time to exert its inhibitory effects (S#{246}dersten et a!., 1983b). The present design pharmacologically suppressed expected endogenous Pr! surges using a dopamine agonist, CB Animals treated with CB-154 (intact or OVX treated with EB) displayed decreased lordosis behavior at 2000 h. Since this decrement of lordosis was overcome in CB-154-treated animals by exogenous replacement with oprl, a Pr! influence on lordosis behavior is suggested, as opposed to a drug effect of CB Ovariectomized animals primed with 2 pg EB for 2 days displayed a moderate level of receptivity (LQ60). Receptivity could be theoretically inhibited or enhanced from this submaximal level. Thus, the observed decrement in lordosis behavior following CB-1 54 treatment indicates that a suppressed level of Prl secretion, correlated with depressed receptivity, may be a direct result of inhibition of the hormone that plays a physiologically significant modulatory role in receptivity. Direct central effects of Pr! on the expression of sexua! behavior have been reported (Dudley et a!., 1982; Harlan et a!., 1983). However, PrI could also exert its facilitatory effects on!ordosis behavior by indirectly stimulating adrenal progestin release (S#{246}dersten et al., 1983a). Therefore, it became of interest to resolve whether the present Pr! effect was of central or peripheral mediation. Estradiol benzoate-primed OVX rats displayed reductions in!ordosis behavior following CB-154 treatment, eliminating the ovary as a required structure. Ovariectomized rats primed with EB showed enhanced lordotic response following stimulation of adrenal progesterone release by adrenocorticotropic hormone (ACTH; Feder and Ruf, 1969; Resko, 1969). Pro!actin has been shown to have greater efficacy at stimulating release of adrenal progestins than ACTH (Piva et al., 1973). Indeed, OVX/ADX rats, when primed for 2 days with 2 pg EB, displayed suppressed levels of lordosis behavior when compared to OVX/sham ADX rats (Fig. 5). Ovariectomized rats when treated with EB for 2 days respond with a Pr! surge on the afternoon of the third day. Estradiol benzoate treatment also stimulates a normal Pr! surge in OVX/ADX animals (Mann et a!., 1976) without exciting progesterone secretion. Since no differences in receptivity were noted in EB + P4 -primed OVX/ADX rats regardless of CB-154 treatment, a pituitary Pr! surge may not be the direct agent of action for the expression of lordosis. The adrenal gland may contribute to the proestrous surge of P4 and Pr! may stimulate adrenal P4 secretion on proestrus. Indeed, pentobarbital administration on the afternoon of proestrus blocks the surge of LH (Barraclough et a!., 1971; Freeman et al., 1976), Pr! (Wuttke et al., 1971), and P4 (Barradough et al., 1971; Freeman et al., 1976). A!- though the full proestrous surge of P4 can be stimulated by LH in pentobarbita!-blocked rats, substitution of PrI alone can cause a small but significant elevation of peripheral plasma concentrations of P4 (Barraclough et al., 1971). This small elevation may be a reflection of an adrenal contribution to the proestrous surge of P4 as well as the timing of mating behavior in the intact rat (Nequin and Schwartz, 1971). These data further suggest that the adrenal contribution is PrI dependent. Other investigations have pointed to the adrenal glands as important in timing the proestrous surge of LH (Nequin and Schwartz, 1971; Lawton, 1972). However, these studies do not eliminate an adrenal-independent modulatory role for Prl acting centrally. Indeed, as stated above, the OVX or OVX/ADX rat stimulated with EB + P4 is maximally receptive (LQ100) compared to proestrous (LQ80) or OVX EB-treated rats (LQ60). Under conditions of maximal stimulation by ovarian steroids, inhibition of Pr! secretion with CB-1 54 may not affect the LQ and therefore the modulatory role for PrI may not be apparent. Thus, Pr! can still be acting centrally and/or peripherally through stimulation of adrenal progesterone secretion to influence lordosis. REFERENCES Barraclough, C. A., Collu, R., Massa, R. and Martini, L. (1971). Temporal interrelationship between plasma LH, ovarian secretion rates and peripheral plasma progestin concentrations in the rat: effects of Nembutal and exogenous gonadotropins. Endocrinology 88: Butcher, R. L., Collins, W. E. and Fugo, N. W. (1974). Plasma concentrations of LH, FSH, prolactin, progesterone, and estradiol-1 7 throughout the 4-day estrous cycle of the rat. Endocrinology 94: Cronin, M. J., Evans, W. S. and Thorner, M. 0. (1984). One minute of bromocriptine irreversibly inhibits

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