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Effects of small medial preoptic lesions on estrous cycles and receptivity in female rats
Psychoneuroendocrinology,Vol.9, No. 2, pp. 189- 196,1984.
0306-4530/84$3.00 + 0.00 © 1984PergamonPressLtd.
Printed in GreatBritain.
EFFECTS
OF SMALL MEDIAL PREOPTIC LESIONS ON ESTROUS CYCLES AND RECEPTIVITY IN FEMALE RATS* MITZI G. LEEDYt
Department of Physiological Sciences, School of Veterinary Medicine, University of California, Davis, CA 95616, U.S.A.
(Received 6 April 1983; in final form 29 September 1983) SUMMARY The effects of small lesions in the preoptic area (POA) of female rats on estrous cycles, LH responsiveness, and receptivity were studied. Regularly cycling rats received lesions confined to either the periventricular portion or the ventral portion of the rostral preoptic area, or a combination of these two sites. Nine animals showed persistent vaginal cornification (PVC) following lesion placement. All of these had lesions in the periventricular preoptic area. In contrast, five of six animals which showed repeated periods of pseudopregnancy(RPP) had lesions in the most ventral portion of the preoptic area. Lesions which damaged both the periventricular POA and the ventral POA gave mixed results. Following ovariectomy and estrogen and progesterone stimulation, the RPP animals had higher levels of LH 6 hr after the P injection than did the control animals or PVC animals. In three different tests for female sexual behavior--with estrogen alone, estrogen plus progesterone, and low estrogen priming and repetitive sexual stimulation--the PVC animals had consistently lower receptivity than the controls. The RPP animals were more receptive than the controls in all hut the estrogen plus progesterone tests. The decreased levels of receptivity of the PVC animals could he related to a disruption of the LHRH system, while the RPP group responses could reflect a hypersensitivity of remaining neurons to estrogen.
THE effect of lesions in the medial preoptic area (MPOA) on the occurrence of cyclic release of gonadotropins remains unclear. MPOA lesions not involving the suprachiasmatic nucleus have been reported to lead to repeated periods of pseudopregnancy (Clemens et al., 1976; Popolow et al., 1981), to result in the elimination of the LH surge and estrous cycles (Nance et ai., 1977; Terasawa et al., 1979, 1980), or to leave estrous cyclicity unaltered (Gray et al., 1978). These conflicting findings may be due to the extent of the destruction within this region. Smaller lesions restricted to the medial preoptic nucleus, a small periventricular cell group lying caudal to the organum vasculosum of the lamina terminalis (OVLT) resulted in persistent vaginal cornification (PVC) and elimination of the LH surge (Wiegand et ai., 1978, 1982; Terasawa et ai., 1979, 1980). Lesions of the OVLT also have been reported to eliminate vaginal cycles (Piva et al., 1979; Popolow et al., 1981). Likewise, both Clemens et al. (1976) and Popolow et al. (1981) found that small lesions in the ventral periventricular region of the MPOA disrupt estrous cycles and result in PVC. However, Popolow et ai. (1981) reported that more dorsally placed periventricular lesions resulted in repetitive psuedopregnancy, while *This research report was based upon a dissertation submitted to Tulane University in partial fulfillment of the requirements for the Ph.D. degree. The author would like to thank Arnold A. Gerall for his support and guidance throughout the study. This research was supported by grant HD 00867-16 to Arnold A. Gerall. *Present address: Department of Psychology, Ohio Wesleyan University, Delaware, OH 43015, U.S.A. 189
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MITZI G. LEEDY
Clemens et ai. (1976) reported that these lesions did not disrupt estrous cycles. Wiegand et aL (1982) reported that lesions confined to the ventral prechiasmatic (VPC) area and OVLT resulted in irregular cycles with long periods of diestrus, such as that seen with repetitive pseudopregnancy (RPP). The influence of lesions in the MPOA on receptivity in female rats is also unclear. Lesions have been reported to lower the threshold for estrogen's activation of receptivity (Powers & Valenstein, 1972) and to result in higher lordosis quotients following injections of exogenous hormones (Nance et aL, 1977), or to be without effect (Singer, 1968). In contrast, Popolow et ai. (1981) found decreases in receptivity with repeated estradiol benzoate (EB) injections in those animals with rostral MPOA lesions. The present study examined the effects of small lesions within the MPOA on estrous cycles, the LH surge, and receptivity under three types of testing conditions. METHODS
Subjects Female S p r a g u e - Dawley rats were received from Charles River Breeding Lab., Wilmington, MA, when 50 days of age. All animals were housed in a vivarium maintained at 21 °C and on a reversed 14 hr light : 10 hr dark schedule, with the lights coming on at 2100 hr. Food and water were available ad lib. Vaginal smears were taken daily on all animals for approximately two m o n t h s prior to surgery. Those animals showing regular four or five day estrous cycles were randomly assigned to one of the groups having small lesions placed in the ventral preoptic area (VPOA), the periventricular region of the preoptic (PPOA) area, or a combination of these two sites, or receiving a control operation or no operation. Sixty-six animals received lesions, eight animals received control operations and eight were unoperated controls.
Surgery Lesions and control operations were performed under Chloropent anesthesia. At the time of the surgery, the animals weighed approximately 2 5 0 - 3 0 0 g. Bilateral electrolytic lesions were made with a 0.33 m m diameter platinum electrode, insulated with Teflon except for approximately 0.3 m m at the tip. Electrode placement for lesions in the V P O A were 0.7 m m anterior to the bregma, 0.3 m m lateral to the superior sagittal sinus and 8.4 m m below the dura. A 0.4 m A direct current was passed for 10 sec through this electrode to a rectal cathode. For lesions of the P P O A region, a direct current was passed two times on each side. The electrode placements were 0.7 m m anterior to the bregma, 0.2 m m lateral to the middle of the superior sagittal sinus, and 7 m m and 7.5 m m below the dura. A 0.4 m A curreht was passed for 5 sec at each of these four electrode sites. For control operations the electrode was lowered into the P P O A , but no current was passed. Following lesion placement or control operations, daily smears were resumed within two days and continued for at least five weeks. L H responsiveness All animals were ovariectomized after a definite pattern of vaginal smears had emerged. Following a two-week recovery period, each animal was injected with 5 ~tg estradiol benzoate (EB) at 0300 hr (6 hr into the light period), followed 48 hr later by an injection of 2 mg progesterone (P). At 0900 hr, 6 hr after the P injection, the animals were anesthetized with ether, and 1 ml of blood was collected from the jugular vein. The plasma was separated and frozen on dry ice. Plasma LH concentrations were determined by radioimmunoassay using the double antibody method. Rat LH (NIAMDD-rat LH-RP-1) was used as the standard. All samples were measured in duplicate in a single assay. Behavior tests One week later, daily injections of 3.3 lag EB were begun and continued for 12 days. Starting 4 hr after lights off on days 1, 3, 5, 7, 10, and 12, all animals were placed with a stud male in a 62 × 25 × 38 cm testing chamber. Behavioral observations were made using dim red illumination, and receptivity was scored on each of 10 mounts as follows: 0 - - n o response to the mount, 1, 2, or 3 for increasing degrees of arch; 0 - - n o darting prior to the mount, or 1 indicating darting; and 0, 1, or 2 for the persistence of the lordosis posture. The receptivity score (RS) was the mean of the ratings of these three components for the 10 mounts. One week after the last EB test, each animal was given injections of 3.3 lag EB on day 1 and 2, and 0.5 mg P on day 4. Four hours after the P injection, receptivity was tested as described for the EB-alone tests. The following
MPOA LESIONS AND REPRODUCTIVE FUNCTION
191
week, this hormone schedule was repeated, and all animals received a second test for receptivity as before. Receptivity in response to repetitive coital stimulation was tested one week later. The animals were primed with injections of 3.3 lag EB 72 and 48 hr prior to testing. Six mating sessions, lasting 15 min each, began at 0, 30, 60, 90, 120 and 150 min. Following each mating session, all animals were allowed to rest for 15 min. Receptivity scores were determined for each session.
Histology Following the final behavior test, all animals were perfused with 10°70 formalin, and 50 pm frozen coronal brain sections were taken. Sections were stained with cresyl violet. Identification o f the lesion sites was determined independently by two judges. The extent o f the lesions was drawn on outlines o f the hypothalamus copied from Konig & Klippel (1974). RESULTS
During the first week following surgery, 11 animals died of unknown causes. Throughout the study, data were taken only from those animals which appeared to be in good health.
Vaginal smears Following surgery, 70~/0 of the animals receiving either the control operation or lesions, regardless of site, entered a period of diestrus that lasted an average of 12 days. After this time, all control operated animals resumed cycling and continued to cycle until ovariectomy. Six animals became repetitively pseudopregnant (RPP) following lesion placement. All of these animals showed several extended periods of diestrus, averaging 12 days each, separated by one or two 4-day cycles. This pattern of vaginal smears continued until ovariectomy. Figures I(A) and (B) show composite drawings of the lesion extents in these animals. Five of these six animals sustained damage to the VPOA area, one of which also had damage in both the VPOA and the PPOA. Nine animals showed persistent vaginal cornification (PVC) following lesion placement. These animals generally displayed at least one 4-day cycle, and some as many as five regular cycles, before entering a state of PVC. All of these animals had damage in the PPOA, and two also had lesions extending into the VPOA. Figures I(C) and (D) show composite drawings of the lesions in these nine animals. L H regulation Analyses of plasma LH levels, following injections of EB and P, were performed on animals divided into three groups: those which showed PVC prior to ovariectomy, those which were RPP, and the pooled control group. Analysis of variance showed a significant difference among these groups, F(2,26) = 18.5, p < 0.001. The control group had a mean of 99 ng/ml (n = 16, S.E.M. ___21.2), the PVC group mean was 58 ng/ml (n = 9, S.E.M. ___ 19.6) and the RPP group mean was 388 ng/ml (n = 6, S.E.M. _+ 84.6). A subsequent Dunnett's test revealed that the RPP group had significantly higher levels of plasma LH than the control, t (2,26) = 7.23, p < 0.005, while no difference was detected between the controls and the PVC group. Receptivity Repeated measures analyses of variance were performed on behavioral data that were grouped on the basis of vaginal smear history. While a significant group difference was
192
MITZI G. LEEDY
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FIG. 1.
MPOA LESIONS AND REPRODUCTIVEFUNCTION
193
found in the six behavior tests given during EB treatment (F = 3.84; 2,28 d.f.; p < 0.05), subsequent Dunnett's tests comparing the control group to the PVC and R P P groups failed to reach significance. However, Fig. 2 shows that the R P P group had higher mean receptivity levels than the controls on most days, while the PVC animals were considerably lower than either the control or R P P animals. As seen in Fig. 3, the R P P and control animals showed high levels of receptivity during the EB and P tests, while the PVC animals were not as receptive. Again, the difference among groups was significant (F = 4.62; 2,27 d.f.; p < 0.05). No differences in receptivity levels were found between the two behavior tests.
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FIG. 2. Mean receptivity scores for the control, RPP, and PVC groups for six tests given during daily EB treatment.
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FIG. 3. Mean receptivity scores for the control, RPP, and PVC groups on two tests given following treatment with EB and P. FIG. 1. Semidiagrammatic reconstruction, adapted from Wiegand & Terasawa (1982), of lesions in the sagittal (left) and coronal (right) planes. RPP animals (A,B); PVC animals (C,D). Abbreviations: AA, anterior hypothalamus; AC, anterior commissure; ARC, arcuate nucleus; DA, dorsal hypothalamic area; DB, diagonal band of Broca; DM, dorsomedial nucleus; f, fornix; L, Lamina terminalis; LP, lateral preoptic area; MP, medial preoptic area; MPN, medial preoptic nucleus; OC, optic chiasm; PA, paraventricular nucleus; SCN, suprachiasmatic nucleus; ST, bed nucleus of stria terminalis; VM, ventromedial nucleus.
194
MITZ] G. LEEDY
Using repeated measures analyses of variance for the repetitive coital stimulation tests, the PVC animals again showed the lowest mean receptivity. The interaction between groups and tests was significant: F = 2.17; 10,130 d.f.; p < 0.05, and a significant group difference was found, F = 3.43; 2,26 d.f.; p < 0.05. Figure 4 shows that the RPP animals had considerably higher receptivity levels than the control animals at all times. Both the RPP and control animals showed increases in receptivity over time. However, the PVC animals showed lower levels, and only slight improvement over trials with the male. 2.4 O U
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FIG. 4. Mean receptivity scores for the control, RPP, and PVC groups for the repetitive coital stimulation test.
DISCUSSION
While many investigators have studied the effects of MPOA lesions on vaginal cycles, the results are not clear. PVC has generally been found only when the ventral portion of the MPOA or portions of the suprachiasmatic area are damaged (Barraclough et al., 1964; Bishop et ai., 1972; Clemens et aL, 1976). However, Kimura & Kawakami (1978) have postulated that the periventricular portion of the MPOA-suprachiasmatic system is necessary for the functional integration of ovulation. In the present study, damage to the area just lateral to the walls of the third ventricle, while not always disrupting cycles, did result in PVC in nine animals. These lesions did not infringe on the suprachiasmatic area. In support of this, Gray et ai. (1978) found that six animals with lesions sparing the most medial portion of the MPOA responded to injections of EB and P with an LH surge, while lesions that destroyed the periventricular portion eliminated this surge. More recently, Wiegand & Terasawa (1978, 1982) have reported that lesions restricted to the periventricular preoptic area resulted in PVC. Their lesions also eliminated the LH surge in response to EB injections and EB plus P. These findings may be related to disruption of the LHRH system. Kozlowski & Hostetter (1978) have found cell bodies containing LHRH in the MPOA just lateral to the ventricle. Barry (1979) suggests that the preopticoterminal LHRH tract, with cell bodies in the OVLT, POA, and anterior hypothalamus, has axons that not only terminate in the OVLT but also cross between the ependymal cells of the ventricular wall. Lesions resulting in PVC in the present study always destroyed the ventricular walls and were
M P O A LESIONS AND REPRODUCTIVE FUNCTION
195
generally restricted to a small area just lateral to the ventricle. Thus, these lesions probably destroyed L H R H processes which may be important for the L H surge. In the present study, apparently neither the control animals nor the PVC animals exhibited an L H surge following ovariectomy and treatment with EB and P. The doses of hormones given (5 gg EB; 2 mg P) have been reported to elicit a surge in normal rats (Samson & McCann, 1979), although higher doses are commonly used for this purpose (Taleisnik et aL, 1978; Wiegand et al., 1982). Since only one blood sample was taken, the surge may have occurred earlier or later in the other groups, but was not detected. From the present study, it is not known if the PVC animals had elevated thresholds for elicitation of the surge. However, the RPP animals had a reduced threshold in comparison to the control animals, since these RPP animals reliably showed an LH surge. This could be due to an increased responsiveness to estrogen following incomplete destruction of an estrogen-sensitive neural area (Nance et al., 1977). Confusion exists in the findings of the effects of preoptic area lesions on female receptivity. Lesions here have been reported to result in either no change in sexual behavior (Singer, 1968; Gray et al., 1978) or an increase in receptivity (Powers & Valenstein, 1972; Nance et ai., 1977). Recently, Popolow et al. (1981) found decreased levels of receptivity in female rats with lesions restricted to the periventricular portion of the rostral POA. In the present study the PVC animals, all of which sustained P P O A damage, showed lower levels of sexual behavior than the controls. There may be a common cause for the lowered receptivity and for the ovarian dysfunction. An interruption in the L H R H system also may be involved in the behavioral deficits. Numerous studies have shown that L H R H can facilitate female sexual behavior (Pfaff, 1973; Moss & McCann, 1973, 1975; Moss & Foreman, 1976; Foreman & Moss, 1977, 1979). Also, Kozlowski & Hostetter (1978) have found that antiserum to L H R H can decrease receptivity in ovariectomized animals injected with EB and P. It is possible that the P P O A lesions, associated with PVC in the present study, destroyed part of the L H R H system involved in sexual receptivity. Further evidence for this comes from the repetitive coital stimulation tests. Foreman & Moss (1977) found that receptivity increased sooner and reached higher levels in those animals which received exogenous L H R H in addition to a low priming dose of estrogen. In the present study, the PVC animals showed almost no increases in receptivity over time in this paradigm. This result may be due to inadequate levels o f endogenous L H R H preventing the increase in receptivity which occurred in the control and R P P animals. The finding that the R P P animals had higher levels of receptivity than the control animals in the behavior tests with EB alone is consistent with the results of the L H surge data. Powers & Valenstein (1972) also found that animals with MPOA lesions had lower thresholds to estrogen for the activation of receptivity. This lower threshold may be due to the small size o f the lesions. Nance et aL (1977) proposed that incomplete destruction of the estrogen-sensitive neurons may lead to hypersensitivity in the remaining neurons. The involvement of the POA in ovarian function, gonadotropin secretion, and receptivity needs to be further explored. It is likely that destruction of large areas of the MPOA yields conflicting results, since multiple areas which may serve differing and even opposing functions are destroyed. Small lesions, when accurately placed, may give more information on these functions.
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MITZl G. LEEDY REFERENCES
BARRACLOUGH, C. A., YRARRAZAVAL, S. & HUTTON, R. (1964) A possible hypothalamic site of action of progesterone in the facilitation of ovulation in the rat. Endocrinology 75,838 - 845. BARRY,J. K. (1979) Immunohistochemistry of luteinizing hormone-releasing hormone-producing neurons of the vertebrates. Int. Rev. Cytol. 60, 1 7 9 - 2 2 1 . BISHOP, W., FAWCETT, C. P., KRULICH, L. & MCCANN, S. M. (1972) Acute and chronic effects of hypothalamic lesions on the release of FSH, LH, and prolactin in intact and castrated rats. Endocrinology 91,643 - 6 5 5 . CLEMENS,J. A., SMALSTIG,E. B. & SAWYER,B. D. 0976) Studies on the role of the preoptic area in the control of reproductive function in the rat. Endocrinology 99, 728 - 735. FOREMAN,M. M. & MOSS, R. L. (1977) Effects of subcutaneous injection and intrahypothalamic infusion of releasing hormones upon lordotic response to repetitive coital stimulation. Horm. Behav. 8, 2 1 9 - 2 3 4 . FOREMAN, M. M. & Moss, R. L. (1979) Roles o f gonadotropins and releasing hormones in hypothalamic control of lordotic behavior in ovariectomized estrogen-primed rats. J. comp. Physiol. Psychol. 93, 556 - 565. GRAY, G. D., SODERSTEN, P., TALLENTIRE,D. & DAVIDSON, J. M. (1978) Effects of lesions in various structures of the suprachiasmatic-preoptic region on LH regulation and sexual behavior in female rats. Neuroendocrinology 25, 174 - 19 I. KIMURA, F. & KAWAKAMI, M. (1978) Reanalysis of the preoptic afferents and efferents involved in the surge of LH, FSH, and prolactin release in the proestrous rat. Neuroendocrinology 27, 74 - 85. KONIG, J. F. R. & KLIPPEL, E. R. A. (1974) The Rat Brain: A Stereotaxic Atlas o f the Forebrain and Lower Parts o f the Brainstem. Robert E. Drieger, New York. KOZLOWSKI, G. P. & HOSTETTER, G. (1978) Cellular and subcellular localization and behavior effects of gonadotropin-releasing hormone (Gn RH) in the rat. In Brain-Endocrine Interaction IlL Neural Hormones and Reproduction, Scott, D. E., Kozlowski, G. P. and Weindl, A. (Eds.), pp. 1 3 8 - 1 5 3 . S. Karger, New York. Moss, R. L. & MCCANN, S. M. (1973) Induction of mating behavior in rats by luteinizing hormone-releasing factor. Science 181, 1 7 7 - 179. Moss, R. L. & MCCANN, S. M. (1975) Action of luteinizing h o r m o n e releasing factor (LRF) in the initiation of lordosis behavior in the estrone-primed ovariectomized female rat. Neuroendocrinology 17, 3 0 9 - 318. Moss, R. L. & FOREMAN, M. M. (1976) Potentiation of lordosis behavior by intrahypothalamic infusion of synthetic luteinizing hormone-releasing hormone. Neuroendocrinology 20, 1 7 6 - 181. NANCE, D. M., CHRISTENSEN, L. W., SHRYNE, J. E. & GORSKI, R. A. (1977) Modification in gonadotropin control and reproductive behavior in the female rat by hypothalamic and preoptic lesions. Brain Res. Bull. 2 , 3 0 7 - 312. PFAFF, D. W. (1973) Luteinizing hormone-releasing factor potentiates lordosis behavior in hypophysectomized ovariectomized female rats. Science 182, 1148 - 1149. pIVA, F., BORRELL, J., LIMONTA, P., GAVAZZI, G. & MARIINI, L. (1979) Role of the amygdala and the organum vasculosum lamina terminalis in the control of ovarian function in the female rat. J. Steroid Biochem. I!, 1 0 0 7 - 1014. POPOLOW, H. P., KING, J. C. & GERALL, A. A. (1981) Rostral medial preoptic area lesions influence on female estrous processes and L H R H distribution. Physiol. Behav. 27, 8 5 5 - 861. POWERS, B. & VALENSTEIN, E. S. (1972) Sexual receptivity: facilitation by medial preoptic lesions in female rats. Science 175, 1003 - 1005. SAMSON, W. K. & MCCANN, S. M. (1979) Effects of lesions in the organum vasculosum lamina terminalis on the hypothalamic distribution of luteinizing hormone-releasing hormone and gonadotropin secretion in the ovariectomized rat. Endocrinology 1 0 5 , 9 3 9 - 946. StNGER, J. J. (1968) Hypothalamic control of male and female sexual behavior in female rats. J. comp. Physiol. Psychol. 6 6 , 7 3 8 - 742. TALEISNIK, S., CALIGARIS, L., BELTRAMINO, C. & CACERES, A. (1978) Stimulatory and inhibitory effects of ovarian steroids on g o n a d o t r o p h i n secretion in ovariectomized rats after anterior hypothalamic deafferentation. J. Endocr. 77, I I - 2 3 . TERASAWA,E., WIEGAND,S. J., RUBENS, L. V. & BRIDSON, W. E. (1979) Further studies of lesions of the medial preoptic nucleus (MPN) on the control of luteinizing hormone (LH) release in female rats. Presented at the Ninth Annual Meeting of the Society for Neuroscience, Atlanta, GA. TERASAWA, E., WIEGAND, S. J. & BRIDSON, W. E. (1980) A role for medial preoptic nucleus on afternoon of proestrus in female rats. Am. J. Physiol. 234, E 5 3 3 - E539. WIEGAND, S. J., TERASAWA, E. & BRIDSON, W. E. (1978) Persistent estrus and blockade of progesteroneinduced LH release follows lesions which do not damage the suprachiasmatic nucleus. Endocrinology 102, 1645 - 1648. WIEGAND, S . . l . & TERASAWA, E. (1982) Discrete lesions reveal functional heterogeneity of suprachiasmatic structures in regulation of gonadotropin secretion in the female r a t Neuroendocrinology 34, 395 - 404.
0306-4530/84$3.00 + 0.00 © 1984PergamonPressLtd.
Printed in GreatBritain.
EFFECTS
OF SMALL MEDIAL PREOPTIC LESIONS ON ESTROUS CYCLES AND RECEPTIVITY IN FEMALE RATS* MITZI G. LEEDYt
Department of Physiological Sciences, School of Veterinary Medicine, University of California, Davis, CA 95616, U.S.A.
(Received 6 April 1983; in final form 29 September 1983) SUMMARY The effects of small lesions in the preoptic area (POA) of female rats on estrous cycles, LH responsiveness, and receptivity were studied. Regularly cycling rats received lesions confined to either the periventricular portion or the ventral portion of the rostral preoptic area, or a combination of these two sites. Nine animals showed persistent vaginal cornification (PVC) following lesion placement. All of these had lesions in the periventricular preoptic area. In contrast, five of six animals which showed repeated periods of pseudopregnancy(RPP) had lesions in the most ventral portion of the preoptic area. Lesions which damaged both the periventricular POA and the ventral POA gave mixed results. Following ovariectomy and estrogen and progesterone stimulation, the RPP animals had higher levels of LH 6 hr after the P injection than did the control animals or PVC animals. In three different tests for female sexual behavior--with estrogen alone, estrogen plus progesterone, and low estrogen priming and repetitive sexual stimulation--the PVC animals had consistently lower receptivity than the controls. The RPP animals were more receptive than the controls in all hut the estrogen plus progesterone tests. The decreased levels of receptivity of the PVC animals could he related to a disruption of the LHRH system, while the RPP group responses could reflect a hypersensitivity of remaining neurons to estrogen.
THE effect of lesions in the medial preoptic area (MPOA) on the occurrence of cyclic release of gonadotropins remains unclear. MPOA lesions not involving the suprachiasmatic nucleus have been reported to lead to repeated periods of pseudopregnancy (Clemens et al., 1976; Popolow et al., 1981), to result in the elimination of the LH surge and estrous cycles (Nance et ai., 1977; Terasawa et al., 1979, 1980), or to leave estrous cyclicity unaltered (Gray et al., 1978). These conflicting findings may be due to the extent of the destruction within this region. Smaller lesions restricted to the medial preoptic nucleus, a small periventricular cell group lying caudal to the organum vasculosum of the lamina terminalis (OVLT) resulted in persistent vaginal cornification (PVC) and elimination of the LH surge (Wiegand et ai., 1978, 1982; Terasawa et ai., 1979, 1980). Lesions of the OVLT also have been reported to eliminate vaginal cycles (Piva et al., 1979; Popolow et al., 1981). Likewise, both Clemens et al. (1976) and Popolow et al. (1981) found that small lesions in the ventral periventricular region of the MPOA disrupt estrous cycles and result in PVC. However, Popolow et ai. (1981) reported that more dorsally placed periventricular lesions resulted in repetitive psuedopregnancy, while *This research report was based upon a dissertation submitted to Tulane University in partial fulfillment of the requirements for the Ph.D. degree. The author would like to thank Arnold A. Gerall for his support and guidance throughout the study. This research was supported by grant HD 00867-16 to Arnold A. Gerall. *Present address: Department of Psychology, Ohio Wesleyan University, Delaware, OH 43015, U.S.A. 189
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MITZI G. LEEDY
Clemens et ai. (1976) reported that these lesions did not disrupt estrous cycles. Wiegand et aL (1982) reported that lesions confined to the ventral prechiasmatic (VPC) area and OVLT resulted in irregular cycles with long periods of diestrus, such as that seen with repetitive pseudopregnancy (RPP). The influence of lesions in the MPOA on receptivity in female rats is also unclear. Lesions have been reported to lower the threshold for estrogen's activation of receptivity (Powers & Valenstein, 1972) and to result in higher lordosis quotients following injections of exogenous hormones (Nance et aL, 1977), or to be without effect (Singer, 1968). In contrast, Popolow et ai. (1981) found decreases in receptivity with repeated estradiol benzoate (EB) injections in those animals with rostral MPOA lesions. The present study examined the effects of small lesions within the MPOA on estrous cycles, the LH surge, and receptivity under three types of testing conditions. METHODS
Subjects Female S p r a g u e - Dawley rats were received from Charles River Breeding Lab., Wilmington, MA, when 50 days of age. All animals were housed in a vivarium maintained at 21 °C and on a reversed 14 hr light : 10 hr dark schedule, with the lights coming on at 2100 hr. Food and water were available ad lib. Vaginal smears were taken daily on all animals for approximately two m o n t h s prior to surgery. Those animals showing regular four or five day estrous cycles were randomly assigned to one of the groups having small lesions placed in the ventral preoptic area (VPOA), the periventricular region of the preoptic (PPOA) area, or a combination of these two sites, or receiving a control operation or no operation. Sixty-six animals received lesions, eight animals received control operations and eight were unoperated controls.
Surgery Lesions and control operations were performed under Chloropent anesthesia. At the time of the surgery, the animals weighed approximately 2 5 0 - 3 0 0 g. Bilateral electrolytic lesions were made with a 0.33 m m diameter platinum electrode, insulated with Teflon except for approximately 0.3 m m at the tip. Electrode placement for lesions in the V P O A were 0.7 m m anterior to the bregma, 0.3 m m lateral to the superior sagittal sinus and 8.4 m m below the dura. A 0.4 m A direct current was passed for 10 sec through this electrode to a rectal cathode. For lesions of the P P O A region, a direct current was passed two times on each side. The electrode placements were 0.7 m m anterior to the bregma, 0.2 m m lateral to the middle of the superior sagittal sinus, and 7 m m and 7.5 m m below the dura. A 0.4 m A curreht was passed for 5 sec at each of these four electrode sites. For control operations the electrode was lowered into the P P O A , but no current was passed. Following lesion placement or control operations, daily smears were resumed within two days and continued for at least five weeks. L H responsiveness All animals were ovariectomized after a definite pattern of vaginal smears had emerged. Following a two-week recovery period, each animal was injected with 5 ~tg estradiol benzoate (EB) at 0300 hr (6 hr into the light period), followed 48 hr later by an injection of 2 mg progesterone (P). At 0900 hr, 6 hr after the P injection, the animals were anesthetized with ether, and 1 ml of blood was collected from the jugular vein. The plasma was separated and frozen on dry ice. Plasma LH concentrations were determined by radioimmunoassay using the double antibody method. Rat LH (NIAMDD-rat LH-RP-1) was used as the standard. All samples were measured in duplicate in a single assay. Behavior tests One week later, daily injections of 3.3 lag EB were begun and continued for 12 days. Starting 4 hr after lights off on days 1, 3, 5, 7, 10, and 12, all animals were placed with a stud male in a 62 × 25 × 38 cm testing chamber. Behavioral observations were made using dim red illumination, and receptivity was scored on each of 10 mounts as follows: 0 - - n o response to the mount, 1, 2, or 3 for increasing degrees of arch; 0 - - n o darting prior to the mount, or 1 indicating darting; and 0, 1, or 2 for the persistence of the lordosis posture. The receptivity score (RS) was the mean of the ratings of these three components for the 10 mounts. One week after the last EB test, each animal was given injections of 3.3 lag EB on day 1 and 2, and 0.5 mg P on day 4. Four hours after the P injection, receptivity was tested as described for the EB-alone tests. The following
MPOA LESIONS AND REPRODUCTIVE FUNCTION
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week, this hormone schedule was repeated, and all animals received a second test for receptivity as before. Receptivity in response to repetitive coital stimulation was tested one week later. The animals were primed with injections of 3.3 lag EB 72 and 48 hr prior to testing. Six mating sessions, lasting 15 min each, began at 0, 30, 60, 90, 120 and 150 min. Following each mating session, all animals were allowed to rest for 15 min. Receptivity scores were determined for each session.
Histology Following the final behavior test, all animals were perfused with 10°70 formalin, and 50 pm frozen coronal brain sections were taken. Sections were stained with cresyl violet. Identification o f the lesion sites was determined independently by two judges. The extent o f the lesions was drawn on outlines o f the hypothalamus copied from Konig & Klippel (1974). RESULTS
During the first week following surgery, 11 animals died of unknown causes. Throughout the study, data were taken only from those animals which appeared to be in good health.
Vaginal smears Following surgery, 70~/0 of the animals receiving either the control operation or lesions, regardless of site, entered a period of diestrus that lasted an average of 12 days. After this time, all control operated animals resumed cycling and continued to cycle until ovariectomy. Six animals became repetitively pseudopregnant (RPP) following lesion placement. All of these animals showed several extended periods of diestrus, averaging 12 days each, separated by one or two 4-day cycles. This pattern of vaginal smears continued until ovariectomy. Figures I(A) and (B) show composite drawings of the lesion extents in these animals. Five of these six animals sustained damage to the VPOA area, one of which also had damage in both the VPOA and the PPOA. Nine animals showed persistent vaginal cornification (PVC) following lesion placement. These animals generally displayed at least one 4-day cycle, and some as many as five regular cycles, before entering a state of PVC. All of these animals had damage in the PPOA, and two also had lesions extending into the VPOA. Figures I(C) and (D) show composite drawings of the lesions in these nine animals. L H regulation Analyses of plasma LH levels, following injections of EB and P, were performed on animals divided into three groups: those which showed PVC prior to ovariectomy, those which were RPP, and the pooled control group. Analysis of variance showed a significant difference among these groups, F(2,26) = 18.5, p < 0.001. The control group had a mean of 99 ng/ml (n = 16, S.E.M. ___21.2), the PVC group mean was 58 ng/ml (n = 9, S.E.M. ___ 19.6) and the RPP group mean was 388 ng/ml (n = 6, S.E.M. _+ 84.6). A subsequent Dunnett's test revealed that the RPP group had significantly higher levels of plasma LH than the control, t (2,26) = 7.23, p < 0.005, while no difference was detected between the controls and the PVC group. Receptivity Repeated measures analyses of variance were performed on behavioral data that were grouped on the basis of vaginal smear history. While a significant group difference was
192
MITZI G. LEEDY
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MPOA LESIONS AND REPRODUCTIVEFUNCTION
193
found in the six behavior tests given during EB treatment (F = 3.84; 2,28 d.f.; p < 0.05), subsequent Dunnett's tests comparing the control group to the PVC and R P P groups failed to reach significance. However, Fig. 2 shows that the R P P group had higher mean receptivity levels than the controls on most days, while the PVC animals were considerably lower than either the control or R P P animals. As seen in Fig. 3, the R P P and control animals showed high levels of receptivity during the EB and P tests, while the PVC animals were not as receptive. Again, the difference among groups was significant (F = 4.62; 2,27 d.f.; p < 0.05). No differences in receptivity levels were found between the two behavior tests.
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FIG. 2. Mean receptivity scores for the control, RPP, and PVC groups for six tests given during daily EB treatment.
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FIG. 3. Mean receptivity scores for the control, RPP, and PVC groups on two tests given following treatment with EB and P. FIG. 1. Semidiagrammatic reconstruction, adapted from Wiegand & Terasawa (1982), of lesions in the sagittal (left) and coronal (right) planes. RPP animals (A,B); PVC animals (C,D). Abbreviations: AA, anterior hypothalamus; AC, anterior commissure; ARC, arcuate nucleus; DA, dorsal hypothalamic area; DB, diagonal band of Broca; DM, dorsomedial nucleus; f, fornix; L, Lamina terminalis; LP, lateral preoptic area; MP, medial preoptic area; MPN, medial preoptic nucleus; OC, optic chiasm; PA, paraventricular nucleus; SCN, suprachiasmatic nucleus; ST, bed nucleus of stria terminalis; VM, ventromedial nucleus.
194
MITZ] G. LEEDY
Using repeated measures analyses of variance for the repetitive coital stimulation tests, the PVC animals again showed the lowest mean receptivity. The interaction between groups and tests was significant: F = 2.17; 10,130 d.f.; p < 0.05, and a significant group difference was found, F = 3.43; 2,26 d.f.; p < 0.05. Figure 4 shows that the RPP animals had considerably higher receptivity levels than the control animals at all times. Both the RPP and control animals showed increases in receptivity over time. However, the PVC animals showed lower levels, and only slight improvement over trials with the male. 2.4 O U
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FIG. 4. Mean receptivity scores for the control, RPP, and PVC groups for the repetitive coital stimulation test.
DISCUSSION
While many investigators have studied the effects of MPOA lesions on vaginal cycles, the results are not clear. PVC has generally been found only when the ventral portion of the MPOA or portions of the suprachiasmatic area are damaged (Barraclough et al., 1964; Bishop et ai., 1972; Clemens et aL, 1976). However, Kimura & Kawakami (1978) have postulated that the periventricular portion of the MPOA-suprachiasmatic system is necessary for the functional integration of ovulation. In the present study, damage to the area just lateral to the walls of the third ventricle, while not always disrupting cycles, did result in PVC in nine animals. These lesions did not infringe on the suprachiasmatic area. In support of this, Gray et ai. (1978) found that six animals with lesions sparing the most medial portion of the MPOA responded to injections of EB and P with an LH surge, while lesions that destroyed the periventricular portion eliminated this surge. More recently, Wiegand & Terasawa (1978, 1982) have reported that lesions restricted to the periventricular preoptic area resulted in PVC. Their lesions also eliminated the LH surge in response to EB injections and EB plus P. These findings may be related to disruption of the LHRH system. Kozlowski & Hostetter (1978) have found cell bodies containing LHRH in the MPOA just lateral to the ventricle. Barry (1979) suggests that the preopticoterminal LHRH tract, with cell bodies in the OVLT, POA, and anterior hypothalamus, has axons that not only terminate in the OVLT but also cross between the ependymal cells of the ventricular wall. Lesions resulting in PVC in the present study always destroyed the ventricular walls and were
M P O A LESIONS AND REPRODUCTIVE FUNCTION
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generally restricted to a small area just lateral to the ventricle. Thus, these lesions probably destroyed L H R H processes which may be important for the L H surge. In the present study, apparently neither the control animals nor the PVC animals exhibited an L H surge following ovariectomy and treatment with EB and P. The doses of hormones given (5 gg EB; 2 mg P) have been reported to elicit a surge in normal rats (Samson & McCann, 1979), although higher doses are commonly used for this purpose (Taleisnik et aL, 1978; Wiegand et al., 1982). Since only one blood sample was taken, the surge may have occurred earlier or later in the other groups, but was not detected. From the present study, it is not known if the PVC animals had elevated thresholds for elicitation of the surge. However, the RPP animals had a reduced threshold in comparison to the control animals, since these RPP animals reliably showed an LH surge. This could be due to an increased responsiveness to estrogen following incomplete destruction of an estrogen-sensitive neural area (Nance et al., 1977). Confusion exists in the findings of the effects of preoptic area lesions on female receptivity. Lesions here have been reported to result in either no change in sexual behavior (Singer, 1968; Gray et al., 1978) or an increase in receptivity (Powers & Valenstein, 1972; Nance et ai., 1977). Recently, Popolow et al. (1981) found decreased levels of receptivity in female rats with lesions restricted to the periventricular portion of the rostral POA. In the present study the PVC animals, all of which sustained P P O A damage, showed lower levels of sexual behavior than the controls. There may be a common cause for the lowered receptivity and for the ovarian dysfunction. An interruption in the L H R H system also may be involved in the behavioral deficits. Numerous studies have shown that L H R H can facilitate female sexual behavior (Pfaff, 1973; Moss & McCann, 1973, 1975; Moss & Foreman, 1976; Foreman & Moss, 1977, 1979). Also, Kozlowski & Hostetter (1978) have found that antiserum to L H R H can decrease receptivity in ovariectomized animals injected with EB and P. It is possible that the P P O A lesions, associated with PVC in the present study, destroyed part of the L H R H system involved in sexual receptivity. Further evidence for this comes from the repetitive coital stimulation tests. Foreman & Moss (1977) found that receptivity increased sooner and reached higher levels in those animals which received exogenous L H R H in addition to a low priming dose of estrogen. In the present study, the PVC animals showed almost no increases in receptivity over time in this paradigm. This result may be due to inadequate levels o f endogenous L H R H preventing the increase in receptivity which occurred in the control and R P P animals. The finding that the R P P animals had higher levels of receptivity than the control animals in the behavior tests with EB alone is consistent with the results of the L H surge data. Powers & Valenstein (1972) also found that animals with MPOA lesions had lower thresholds to estrogen for the activation of receptivity. This lower threshold may be due to the small size o f the lesions. Nance et aL (1977) proposed that incomplete destruction of the estrogen-sensitive neurons may lead to hypersensitivity in the remaining neurons. The involvement of the POA in ovarian function, gonadotropin secretion, and receptivity needs to be further explored. It is likely that destruction of large areas of the MPOA yields conflicting results, since multiple areas which may serve differing and even opposing functions are destroyed. Small lesions, when accurately placed, may give more information on these functions.
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MITZl G. LEEDY REFERENCES
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