. .1. .“.«m "x . 3% flex . .2. ix fixflnfim 3:. 1-7.6 : in?! 9.1.x 15.33.55... 2...: THESIS ;fiiti LIBRARY Michigan State University This is to certify that the dissertation entitled Developmental Toxicities of Polychlorinated Biphenyls on Female Sexual Behavior and Incertohypothalamic Dopaminergic Neurons: Its Implication on the Neural Control of Sexual Behavior of Rats presented by Yu—Wen Chung has been accepted towards fulfillment of the requirements for Ph.D. (kgmnn Zoology-Neuroscience WMU ViesK ’M kW Major pro or WW MSU i: an Affirmative Action/Equal Opportunity Institution 0-12771 PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 11100 W.“ DEVELOP} Sle'AL ‘. Ell? RON! DEVELOPMENTAL TOXICITIES OF POLYCHLORINATED BIPHENYLS ON SEXUAL BEHAVIOR AND INCERTOHYPOTHALAMIC DOPAMINERGIC NEURONS: ITS IMPLICATION ON THE NEURAL CONTROL OF SEXUAL BEHAVIOR OF RATS By Yu-Wen Chung A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology and Neuroscience Program 2000 DEVELOP SEXUAL NEL'RON Polycl hare been my Very little is l two commen dh‘elopmem ms. A1321 ‘. treatment pa and day 10; 72‘ Alllmals Pem tTitatmem W Mod; Tht “50¢de \ C105 (11-7121ij br. ABSTRACT DEVELOPMENTAL TOXICITIES OF POLYCHLORINATED BIPHENYLS ON SEXUAL BEHAVIOR AND INCERTOHYPOTHALAMIC DOPAMINERGIC NEURONS: ITS IMPLICATION ON THE NEURAL CONTROL OF SEXUAL BEHAVIOR OF RATS By Yu-Wen Chung Polychlorinated biphenyls (PCBs) are long-lasting environmental pollutants that have been reported to disrupt development and reproduction in many species. However, very little is known about the effect of PCBs on sexual behavior. I examined the effect of two commercial PCBs, Aroclor 1221 (A1221) and Aroclor 1254 (A1254), on the development of sexual behavior and specific brain dopaminergic neurons in Long-Evans rats. A1221 and A1254 were chosen for their distinct difference in estrogenicity. Three treatment paradigms were used: perinatal treatment on gestation day 14, neonatal day 1 and day 10; neonatal treatment from day 1 to day 7; adult treatment from day 67 to day 72. Animals were tested for sexual behavior in adulthood. Permanent changes were seen in female sexual behavior after developmental treatment with PCB mixtures. Different PCB mixtures appeared to have different critical periods. The change of female sexual behavior in neonatally A1254 treated rats was associated with the disruption of the developing A13 dopaminergic system. Closer examination of the incertohypothalamic dopaminergic systems revealed a dynamic brain asymmetry wherein the presence of some dopaminergic cells was only seen following context-dependent copulatory stimulation. Copulation differentially Induced more cells in the A? Cepu compared to dimorphic in females reee testing sima‘ Intimate th Mal obseh-‘ed n to PCBs. E number in sexually al Neonatal ] in MPG n Tl and p€lVli bfihall‘lor, bti‘Il'een ' behalfigr' induced more detectable tyrosine hydroxylase (TH) immunoreactive (IR) and/or F OS/T H—IR cells in the All and A13 regions. The testing situation, i.e. female-paced or male-paced, also affected the timing of TH-IR and FOS/TH-IR induction. Copulation also induced more detectable FOS-IR cells in the medial preoptic nucleus compared to the noncopulated control. A striking difference in F OS-IR in the sexually dimorphic nucleus of the preoptic area of the hypothalamus (SDN-POA) appeared when females received one ejaculatory series of sexual stimulation from the male under different testing situations. The possible roles of SDN-POA in female reproduction might be to terminate the estrus and female cyclicity for pregnancy. Males were not as susceptible to PCB exposure as female rats since I only observed minor changes in their sexual behavior subsequent to developmental exposure to PCBs. However, I found a strong correlation between male sexual behavior and cell number in the major pelvic ganglia (MPG). The number of the MPG cells showed sexually allomorphism among control females, male copulators and noncopulators. Neonatal PCB treatment resulted in a heterogeneous population of rats with wide spectra in MPG neuronal development. The medial preoptic area, zona incerta, A11 dopaminergic neurons, spinal cord and pelvic ganglia formed a coordinated network involved in the control of sexual behavior. PCB exposure during development of these systems might disrupt the balance between central and peripheral nervous systems and result in the alteration of sexual behavior. To my parents, Hsiu-Ching Chung and Hsiu-Lien Lin and those who helped me navigate through the Nebula of Science For 11 my sincere g of my disser Johnson and l alsr liang-Yo Y years at MS lwould like counting: R Johansen (i Alston-Mil their SUPPO l w llll, {01 lllt’ computer r Years, ESp. me (111an ACKNOWLEDGEMENTS For their advice and influence into learning about the beauty of science, I express my sincere gratitude to my major professor Dr. Lynwood G. Clemens and the members of my dissertation committee, Drs. W Richard Dukelow, Kay E. Holekamp, John 1. Johnson and Antonio A. Nunez. I also thank Drs. Anthony E. Ackerrnan, Gary M. Lange, Kevin Sinchak and Liang-Yo Yang. They taught me experimental techniques I learned during my first three years at MSU. Dr. Jiming F ang is highly appreciated for his technical advice and support. I would like to acknowledge several undergraduate students who helped me with the cell counting: Rose Sydlowski (in the pelvic ganglia), Kimberly Roedel and Jamie A. Johansen (in the medial preoptic area). I also thank other lab associates, Dr. Brenda Alston-Mills, Anne M. Corrigan, Mattew Martin, Kristin Pikul, and Dan Thomhill for their support and company. I would especially like to thank my parents, Hsiu-Ching Chung and Hsiu-Lien Lin, for their encouragement and support. I thank Dr. Kalle Karu, who designed the computer program for recording the behavior tests and has been there for me during these years. Especially warm thanks are extended to many friends who have helped to cheer me during the rough times and to celebrate with me during the happy times. Finally, I thank the faculty and staff in the Department of Zoology and the Neuroscience Program for their support. The work in this dissertation was supported by a NIEHS Superfimd grant (P42ESO491 1-09) and a NSF grant (IBN9728883) to Dr. Lynwood G. Clemens. UflOEH LBTOET. [BTOES‘ CHAPTER Poly. Basn Sexu CHAPTER rAnit FCm.‘ Resu DlSCL LIST OF FIGURES LIST OF TABLES LIST OF SYMBOLS OR ABBREVIATIONS CHAPTER]: INTRODUCTION Polychl Coplanar PCBS Mono-ortho-substituted PCBs Di-ortho-substituted PCBS Coplanar vs. Ortho-substituted PCBS Metabolism of PCBs Toxicities of PCBs Basic Biology of Long-Evans Rats Sexual Differentiation in Rats CHAPTER 2: Animal Female Immunohistochemistry TABLE OF CONTENTS ix xii xiv orinated Biphenyls (PCBS) GENERAL METHODS S Behavior Tests Traditional Lordosis Tests Pacing Tests CHAPTER 3: EFFECTS OF PERINATAL EXPOSURE TO PCBS ON THE DEVELOPMENT OF FEMALE SEXUAL BEHAVIOR AND THE IN CERTOHY POTHALAMIC DOPAMINERGIC NEURON S Introduction Materials and Methods Results Discussion Animals Immunohistochemistry for Caudal Incertohypothalamic Dopaminergic Neurons Statistics Perinatal A1221 treatment affects female sexual behavior. Differential Effect of A1221 and A1254 Aroclor and the Ah and Cholinergic Receptors vi 24 24 25 25 25 26 30 30 32 33 33 34 34 35 35 43 44 FEMALE 5. DOPAMlNl lnlroe Mate Resu Dism CHAPTER REPRODL' Intro Mate Resr Disc CHPArEp SEXTAL 1 A1221."le lnm Mai CHAPTER 4: NEONATAL AND ADULT TOXICITY OF PCB EXPOSURE ON FEMALE SEXUAL BEHAVIOR AND INCERTOHYPOTHALAMIC DOPAMINERGIC NEURON S IN RATS 46 Introduction 46 Materials and Methods 47 Animals 47 Immunohistochemistry for Caudal Incertohypothalamic Dopaminergic Neurons 49 Statistics 49 Results 51 Relation of Neonatal A1221 Treatment to All and A13 Dopaminergic Neurons 51 The Effect of Neonatal A1254 Treatment on All and A13 Dopaminergic Neurons 51 Discussion 57 CHAPTER 5: FEMALE-PACED SEXUAL BEHAVIOR AND ITS LINK TO REPRODUCTION: A TIME COURSE STUDY 62 Introduction 62 Materials and Methods 63 Animals 63 Female Behavior Tests 64 Immunohistochemistry for F08 and TH 65 Statistics 66 Results 67 Time Effect 67 Effect of the Amount of Sexual Stimulation 70 Discussion 73 CHPATER 6: EFFECTS OF PCBS ON MALES AND RELATIONS OF MALE SEXUAL BEHAVIOR TO THE MAJOR PELVIC GANGLIA IN N EONATALLY A1221-TREATED RATS 82 Introduction 82 Materials and Methods 83 Animals 83 Male Behavior Tests 84 Histochemistry for the Major Pelvic Ganglia 84 Statistics 85 Results 86 Discussion 94 vii CHIPI ER 7 Neural Future APPENDIX REFERENC CHAPTER 7: GENERAL DISCUSSION 103 Neural Control of F emale-Paced Sexual Behavior 104 Systems Involved in Female-Paced Sexual Behavior: A Working Hypothesis 104 The Anatomy and the Function of Incertohypothalamic Dopaminergic System in Female-paced Sexual Behavior 107 The Media] Preoptic Nucleus and Female-paced Sexual Behavior m 115 Future Research Directions 116 APPENDIX 118 REFERENCES 137 viii Figure 1. Mr position. Figure 2. Mt Figure 3. Mr Figure 4. PC eateehol-O-n dihydroxixphu deearboxylas hydroxylase. Figure 5. Pa- Figure 6. Pe lordosis freq Figure 7. Fe N) males 2 Figure 8. Pc lhe male afic Ofintrurnissi “gun: 9. Pg Flgure 10.] Figure 12.) [I] Enema] 20 SiOPfS. ..,“_ LIST OF FIGURES Figure 1. Molecular structure of polychlorinated biphenyls. O: ortho, m: meta, p: para position. 2 Figure 2. Molecular structure of polychlorinated dibenzo-p-dioxins. --------------- 5 Figure 3. Molecular structure of polychlorinated dibenzofiirans. 6 Figure 4. Possible mechanisms of PCB effects on dopamine concentration. COMT: catechol-O-methyl transferase; DOPA: L-dihydroxy-phenylalanine; DOPAC: dihydroxyphenylacetic acid; HVA: homovanillic acid; LAAD: L-amino acid decarboxylase; MAO: monoamine oxidase; 3MT: 3-methoxytyramine; TH: tyrosine hydroxylase. 11 Figure 5. Pathways for in situ metabolism of testosterone by neural tissue. -------------- 19 Figure 6. Perinatal A1221 treatment decreased the lordosis quotient (LQ=frequency of lordosis/frequency of 10 mount) of female rats. 36 Figure 7. Perinatal A1221 treatment increased the latencies for female rats to approach (AL) males and return to them afier intromissions (IRL). 37 Figure 8. Perinatal A1221 treatment increased the percentage of times that females leave the male after receiving intromissions (%IL=frequency of intromission leave/frequency of intromission). 38 Figure 9. Perinatal A1254 treatment increased the percent leave following mounts (%ML) or intromissions (%IL). 39 Figure 10. The main effect of asymmetry in A1 1 dopaminergic neurons. -------------- 40 Figure 11. The main effect of asymmetry in A13 dopaminergic neurons. -------------- 41 Figure 12. Neonatal A1254 treatment reduced female lordosis quotient (LQ). ----- 52 Figure 13. Neonatal A1254 treatment shortened the latency for female rats to return to the male after an intromission (IRL). 53 Figure 14. Interaction between neonatal A1254 treatment and the number of TH-IR cells in medial zona incerta (MZI). A1254 decreased the asymmetry as shown by different slopes. 55 ix figure 15.01I the symmetr: FOS-Tll-IR Ct Figure 16. In each bar repre llPlO: male-p EPlh: female- group (p<0.05 dirlereni frorr. Figure 17. Ti. inide each hr: min; llPlO: r FPlh: female- ETOUp - A1254-15mg 200 . . A11(major) A11(minor) No. of TH-IR Cells Figure 10. The main effect of asymmetry in All dopaminergic neurons. 40 2000 ‘ 1800 ' 1600 ‘ 1400 ' 1200 ' No. of TH-IR Cells 1000 ‘ 800 ' 600 Fieure 11.1 2000 -0- Control 1800 ‘ -l:l- A1221-5mg i -A- A1221-15mg % 1600 -<>— A1254-15mg U . E. 1400 ‘ I . £1 1200 ~ ° 4 5 Z 1000 ‘ 800 ' 600 Al3(major) A13(ininor) Figure 11. The main effect of asymmetry in A13 dopaminergic neurons. 41 old female rats with A1221 (20 mg/rat) and reported similar precocious puberty, persistent vaginal estrus, and anovulation. However, only testosterone or antiestrogen decreased the lordosis behavior. It is believed that testosterone has to be aromatized in the brain tissue into estrogen to affect the brain circuits involved in lordosis behavior (McEwen et al., 1977). We did not find any correlation between the lordosis quotient change and the A1 1 or A13 anatomic parameters. Therefore, A1221 may have acted on other systems related to lordosis behavior. Several dopaminergic systems in the hypothalamus and midbrain have been associated with lordosis behavior in female rats, including the substantia nigra (A9), central tegrnental area (A10) (Hemdon, 1976) and the arcuate nucleus (A12) (Ahren et al. , 1971; Lofstrom, 1977). Indeed, PCBS have been shown to decrease dopamine content in the substantia nigra and other hypothalamic nuclei (Seegal et al., 1990, 1991), and it is possible that changes in these dopaminergic systems may be related to the low lordosis quotient in our study. Alternatively, although the whole population of A1 1 or A13 dopaminergic neurons did not show correlations with lordosis behavior, some subpopulation of these neurons may be related to this behavior, e.g., the Al 1 or A13 dopaminergic neurons that were activated during copulation. Other methods of measuring neuronal activities may be a better index than TH-IR cell counts. In the pacing tests, perinatal A1221-treated (5 mg) females took a longer time to approach the male, and returned to the male with longer latencies after intromission than control females. The percentage of times these females left the male following an intromission was also increased. These behavioral alterations may reflect changes in sexual motivation of the female rats and could be the result of increased dopamine level in the nucleus accumbens and the striatum due to the estrogenic effect of A1221 in the critical 42 period of development. Xiao and Becker (1997) found that estrogen implantation in the nucleus accumbens increased dopamine level in this brain region and prolonged the return latencies of female rats receiving intromissions and the implantation into the striatum facilitated the percentage of females leaving males after copulatory events. This pathway appears to be involved in the maintenance but not in the initiation of copulation (the approach latency). On the other hand, since the temporal pattern of female copulation depends heavily on peripheral sensory factors (Erskine, 1989), PCB treatments may have influenced the female’s sensitivity to vaginal and cervical stimulation during copulation. In addition, lesions of the medial preoptic area (MPOA) have been shown to disrupt the temporal pattern of sexual behavior in female rats (Whitney, 1986; Yang & Clemens, 1996). It is plausible that PCBs disrupted development of the MPOA which in some way produced deficits in the temporal pattern of female sexual behavior. Differential Effect of A1221 and A1254 A possible explanation for different effects of Aroclor 1254 and Aroclor 1221 lies in the components of these two PCB mixtures. Aroclor 1221 consists of 40% coplanar and 60% ortho-substituted PCBs (Webb and McCall, 1972; Willis and Addison, 1972). Aroclor 1254, on the other hand, is mainly composed of ortho-substituted PCBS (about 99%; Sissons and Welti, 1971; Webb and McCall, 1972). Comparing the results from A1221 and A1254 treatments, it appears that the lordosis reflex may have been altered by the estrogenic effects of coplanar PCBS since only A1221 decreased the lordosis quotient, while the increase in leaving after a copulatory event may be due to the effect of the ortho- substituted PCBS because both A1221 and A1254 showed changes in these parameters. 43 A1221, 5 mg, had effects on the female’s response latencies (Figure 7) but the dose of 15 mg A1221 did not. Since estrogen interacts with the dopaminergic system (Mani er al. 1994), and since coplanar and ortho-substituted PCBs have opposite effects on dopamine levels (Seegal et al. 1990, 1997), the differential effect in the A1221 5 mg and 15 mg groups may be attributed to the antagonism between two types of PCBs. Interestingly, the dose response curve of commercial PCBS is sometimes biphasic (Chishti et al., 1996). The biphasic dose response may also result from the antagonism between the coplanar and ortho-substituted PCBS. Aroclor and the Ah and Cholinergic Receptors It is well known that coplanar PCBs can mimic the structure of TCDD as described in Chapter 1, and therefore bind to the Ah receptor. However, there are two structural requirements for induction of Ah receptor activity: the presence of at least two adjacent halogen atoms in the lateral positions of each benzene ring (positions 3,4,3’,4’) and the absence of halogen atoms adjacent to the biphenyl bridge (position 2,6,2’,6’) (Poland & Glover, 1977). None of the coplanar components in A1221 fulfill these requirements. In addition to changes in brain dopamine function, postnatal day 10 exposure to individual congeners (including ortho-substituted congeners and 3,4,3’,4’-TCB) resulted in long-term changes in behavior and regional brain muscarinic and nicotinic receptor densities (Eriksson et al., 1991; Eriksson & Fredriksson, 1996). The persistent changes in Cholinergic receptor function may alter the normal dopaminergic/Cholinergic balance, resulting in altered brain function (Consolo et al., 1992; Marchi et al., 1992; Di Chiara & Morelli, 1993). In summary, permanent changes were seen in feminine sexual behavior after perinatal treatment with the PCB mixture, A1221 or A1254. The lordosis reflex may be altered by the estrogenic effects of the coplanar PCBS, while the increase in leaving after a copulatory event may be due to the effect of the ortho-substituted PCBS. 45 CHAPTER 4: NEONATAL AND ADULT TOXICITY OF PCB EXPOSURE ON FEMALE SEXUAL BEHAVIOR AND IN CERTOHYPOTHALAMIC DOPAMINERGIC NEURONS IN RATS Introduction In the previous study we found that perinatal PCB treatments produced deficits in female sexual behavior. However, the timing of PCB treatment seems to be a crucial factor in PCB toxicity (Seegal, 1992; Brouwer et al., 1995). In contrast to the decreases in dopamine concentrations observed following adult PCB exposure, perinatal exposure to Aroclor 1016 significantly increased brain dopamine concentrations (Seegal, 1992). In addition, coplanar PCBS only showed toxic effects during development. Adult coplanar PCB treatment did not produce deficits in treated animals. On the contrary, ortho- substituted PCBS are active during both development and adulthood (Brouwer et al., 1995). Furthermore, the process of sexual differentiation and the physiological environment are different at various developmental stages (Maclusky & Naftolin, 1981). Hypothalamic dopaminergic neurons start to display sex differences in dopamine metabolism at birth (Reisert & Pilgrim, 1991) and around puberty (Davis et al., 1996). Therefore, it is important that we also examine the neonatal and adult effects of PCBs. In this chapter, we compare the effects of neonatal treatment with two commercial PCB mixtures, A1221 (compounds with estrogenic activity) and A1254 (non-estrogenic compounds), on the development of female sexual behavior and incertohypothalmic dopaminergic neurons as well as the effect of these compounds when administered to adult female rats. The dose was chosen as 1/3 of the perinatal treatment to mimic the day 1 injection used in the perinatal study. To ensure the delivery of PCBS, I used daily 46 injection for one week. In this experiment I also used F OS immunoreactivity to firrther refine our assessment of the function of incertohypothalamic dopaminergic neurons during copulation. Materials and Methods Neonatal PCB exposure from day 1 to day 7 was used to determine whether PCB exposure would affect adult sexual behavior. To ascertain whether the effects were specific to development, I also exposed adult rats from day 67 to day 72. Since PCBs accumulate in body fat, owing to their high lipophilicity and low biodegradation rate, the actual exposure can extend beyond the time of injection. Animals Neonatal Treatment Long-Evans rats were randomly paired and placed with the male over night. Pregnant female rats were then housed alone. After parturition, lactating rats were treated daily with sesame oil or a PCB solution (Fisher Scientific, Pittsburgh, PA) from day 1 (parturition day) to day 7. In the A1254 experiment, 10 mg or 20 mg A1254 were dissolved in 14 ml sesame oil (Sigma Chemical Co., St. Louis, MO) as the working solution (0.71 mg/ml and 1.43 mg/ml, respectively). In the A1221 experiment, 12.5 mg or 25 mg A1221 were dissolved in 17.5 ml sesame oil as the working solution (about 0.71 mg/ml and 1.43 mg/ml, respectively). Lactating rats were given daily intraperitoneal (ip) injections of sesame oil or the PCB working solution (0.5 ml/rat; daily dosage about 0.357 mg and 0.714 mg, respectively) at 0900 hr (2 hr before lights off) thus providing 47 offspring with lactational exposure to the PCBS at a total dosage of 0 mg (sesame oil group, control), 2.5 mg (about 7 mg/Kg) or 5 mg (about 14 mg/Kg) A1254 (or A1221). [Note: The actual dosage transferred to the pups was estimated to be 7 pg (1.2 mg/Kg) and 14 pg (2.3 mg/Kg), respectively (Takagi et al., 1986).] Pups born on the same day were randomly assigned to the dam and the number of pups per dam was reduced to eight at parturition. On the same day, at least one darn was randomly assigned to each treatment (control, 2.5 mg PCBs and 5 mg PCBS). Pups were weaned on day 28. Males and females were housed in separate cages after weaning. At 60 days of age, female offspring were ovariectomized and treated with hormones for behavior tests as described in Chapter 2. Adult Treatment In order to separate developmental PCB effects from concurrent residual effects of stored PCBS, I examined the behavior of females treated with PCBS as adults. Sixty sexually mature (60-day-old) female rats were ovariectomized and treated with hormones as described in Chapter 2. After four behavior tests, they were randomly divided into five groups [control, 2.5 mg (8 mg/Kg) A1221, 5mg (16 mg/Kg) A1221, 2.5 mg (8 mg/Kg) A1254, 5 mg (16 mg/Kg) A1254]. To prepare the PCB working solution, 30 mg or 60 mg A1254 (or A1221) was dissolved in 8.4 ml sesame oil (3.57 mg/ml and 7.14 mg/ml, respectively). The control group received 0.1 ml sesame oil (ip injection at 0900 hr) per day for a week. The other four groups received 0.1 ml of the PCB working solution for a week [total dosage 2.5 mg (daily dosage 0.357 mg, about 1 mg/Kg) or 5 mg (daily dosage 0.714 mg, about 2.4 mg/Kg) A1254 (or A1221), ip injection at 0900 hr]. Four hours after the last PCB treatment, rats were tested for female sexual behavior (1300 hr, 2 48 hr after lights off). Behavioral analyses were based on data from the last two tests for the pre- and post-treatment comparisons. Immunohistochemistry for Caudal Incertohypothalamic Dopaminergic Neurons Female rats were randomly selected (at least one from each assigned litter in the neonatal treatment groups) for immunohistochemical processing and sacrificed one hour after the introduction of the female into the testing arena in the last behavior test. Since there was no difference in the behavior of adult treatment groups, immunohistochemistry was not performed in the adult experiment. In the other groups, brains were sectioned coronally at 25 pm with a cryostat. Frozen sections were collected and every fourth section was processed for F08 and TH immunohistochemistry as described in Chapter 2. The primary antibody (c-FOS oncoprotein mouse monoclonal antibody, 1:40, Novocastra Laboratories Ltd., UK/Vector Laboratories, CA) and normal goat serum (Vector Laboratories, diluted with TBS/0.3% Triton X-100 according to the manufacturer's directions) were used. The biotinylated secondary antibody was goat-anti-mouse IgM (Vector Laboratories, diluted with TBS/0.3% Triton X-100 according to the manufacturer's directions). The primary antibody for TH was monoclonal mouse-anti-TH (1 : 10,000, Sigma Chemical Co., St. Louis, MO) with normal horse serum and the secondary antibody was horse-anti-mouse IgG (Vector Laboratories). Statistics Neonatal Treatments The mean of each behavioral parameter in each test was automatically calculated 49 by the computer program. The average data of four behavior tests were then used for statistical analyses. Since there was no difference between assigned litters in each treatment group, pooled data for treatment groups were used for further statistical analyses. Approach latency, mount return latency, intromission return latency and postejaculatory refractory period were analyzed by one-way ANOVA. Fisher’s LSD post hoc test was performed between the control and PCB-treated group when there was a significant difference among treatments (p<0.05). Percentages of mount leave, intromission leave, mount stay and intromission stay were analyzed by Kruskal-Wallis Test. LQ, the percentage of times that females showed a lordosis response in 10 mounts (standard test) or more (pacing test), was also analyzed by Kruskal-Wallis Test. The post hoc tests were performed between the control and PCB-treated group when there was a significant difference between treatments (p<0.05). The number of TH-IR neurons in the All or A13 region was analyzed by both one-way ANOVA and Kruskal-Wallis Test. To compare the measurements of two sides of All or A13, two-way ANOVA (two sides as repeated measurements) was used. I used the number of TH-IR neurons (or FOS/TH-IR neurons) on the major side divided by the number of TH-IR neurons (or F OS/T H-IR neurons) on the minor side as an index of asymmetry. The asymmetric indices were analyzed by Kruskal-Wallis Test. Adult Treatment Data from the fourth and the fifth behavior tests were treated as repeated measurements (pre- and post- treatments). Two-way ANOVA was used to analyze female approach latency, mount return latency, intromission return latency and postejaculatory refractory period. Percentages of mount leave, intromission leave, mount 50 stay, intromission stay and LO were analyzed by Friedman’s test. Results There was no adverse effect on the general health as measured by body weight (data not shown) of the exposed animals in the neonatal or adult PCB treatment groups. At adulthood, experimental females showed regular estrous cycles as indicated by daily vaginal smears for ten days before ovariectomy. Neonatal treatment with A1254 significantly reduced the lordosis quotient (Figure 12), and shortened the latency for female rats to return to the male after an intromission (Figure 13). Other measurements of female sexual behavior were not affected. Neither neonatal treatment with A1221 nor adult treatment with either PCB compounds affected female sexual behavior. Relation of Neonatal A1221 Treatment to All and A13 Dopaminergic Neurons Neonatal A1221 treatment had no effect on the number of A1 1 or A13 TH-IR neurons across treatment groups. Since groups did not differ the data were pooled. Analysis revealed an asymmetry in both the A11 and A13 TH-IR cell groups with one side of brain (major side) containing more TH-IR cells than the other (minor) (p<0.001 and p<0.005, respectively). The FOS/TH-IR A13 neurons also showed significant asymmetry (p=0.005) while the F OS/T H-IR A1 1 neurons did not. The Effect of Neonatal A1254 Treatment on All and A13 Dopaminergic Neurons There was no difference in the number of A1 1 or A13 TH-IR neurons between 51 [:1 Control (n=15) A1254 2.5mg (n=15) A1254 5mg(n=15) 100 - 90 - r 80 1 70 .. .e u 60 - *1: T LQ (%) 40 l 20; 10- LQ (Nohpaced) LQ (Paced) Figure 12. Neonatal A1254 treatment reduced female lordosis quotient (LQ). 52 35 30 ‘ I 25 ‘ 20 ' IRL (sec) - * 15 - T * . n=15 r 10 " i "—15 n=15 Control A1254‘2.5mg A1254 5mg Figure 13. Neonatal A1254 treatment shortened the latency for female rats to return to the male after an intromission (IRL). 53 neonatal A1254 treatment groups. However, there was a significant interaction between neonatal A1254 treatments and the number of All or A13 TH-IR neurons on the two sides of the brain (p<0.05 and p<0.005, respectively). In control females, one side of the All and A13 region had more TH-IR neurons (major) than the other (minor) (p<0.0001 and p<0.05, respectively). A1254 treatment decreased the asymmetry of A13 TH-IR neurons in A1254-treated females (Figure 14, showing different slopes). The FOS/TH-IR A13 neurons also showed the pattern of asymmetry (p<0.05), and the interaction between A1254 treatment and the number of A13 F OS/T H-IR neurons was also significant (p<0.005). The FOS/TH-IR A11 neurons also showed the pattern of asymmetry (p<0.001), but no interaction was found between A1254 treatment and the number of A1 1 F OS/T H-IR neurons. Since there was a significant interaction between neonatal A1254 treatment and the asymmetry of FOS/TH-IR A13 neurons, we examined the possible correlation between the asymmetry of F OS/T H-IR A13 neurons and the behavioral changes in the intromission return latency and the lordosis quotient. To represent the asymmetry of F OS/T H-IR A13 cells, we used the number of cells in the major side divided by the number of cells in the minor side as the asymmetric index. Correlation analyses revealed that the asymmetric index of F OS/T H-IR A13 cells was significantly (F0577, p<0.05) correlated with the intromission return latency but not the lordosis quotient. A linear regression analysis showed a significant linear relation between intromission return latency and the asymmetric index of FOS/TH-IR A13 neurons (r=0.577, p<0.05, Figure 15). 54 1400 ' -0— Control (n=4) 1200 j -El— A1254 2.5mg (n=4) .4. - 1000 . A1254 5mg (n-4) 800 ' l 600 4 No. of TH-IR Cells 400 ‘ 200 " 0 . . A13 (major) A13 (minor) Figure 14. Interaction between neonatal A1254 treatment and the number of TH-IR cells in the A13 region. A1254 decreased the asymmetry as shown by different slopes. 55 90 : Y=-3.71+12.445*X;R"2=.333 0 80 - r=0.577,p<0.05 70- 60: 50': O 40: 30': 0 20-0 0 o 10‘ IRL (sec) Asymmetric Index Figure 15. There is a significant correlation between the intromission return latency and the asymmetric index of FOS/TH-IR cells in the zona incerta. Asymmetric index = No. of FOS/TH-IR cells (major) / No. of FOS/TH-IR cells (minor). 56 Discussion Neonatal treatment with the nonestrogenic PCB mixture, A1254, significantly altered female sexual behavior and the organization of dopaminergic neurons in the A13 region of the brain. However, neonatal treatment with the estrogenic PCBs, A1221, had no detectable effect either on female sexual behavior or on dopaminergic neurons in All and A13 regions. Comparing these results with those from the previous chapter, it is clear that the estrogenic component of PCBs has the potential to alter development of lordosis and pacing behaviors but such effects appear restricted to prenatal development. On the other hand, the nonestrogenic component of PCBs may disrupt sexual differentiation in a manner quite different from the estrogenic PCBs and the critical period for this action occurs during neonatal development. Since both estrogenic and nonestrogenic PCBs reduced lordosis behavior but had opposite effects on pacing behavior, it appears that these two behaviors are controlled by different developmental mechanisms. In addition, there may be at least two phases for the differentiation of brain systems involved in lordosis behavior: the initial prenatal phase may be influenced by estrogen whereas the second phase may be related to the development of d0paminergic systems. Furthermore, PCB toxicity is region-specific with A13 being more susceptible to PCB treatment than A1 1. Although the reduction of LQ in A1254-treated animals was not related to the brain regions examined in this study, the short intromission return latency did appear to be associated with a reduction in putative dopamine asymmetry in the A13 region. This brain area sends fibers rostrally to several nuclei in the hypothalamus involved in the control of sexual behavior (Lindvall & Bjorklund, 1983), e.g., the medial preoptic area 57 (MPOA). Incertohypothalamic dopaminergic neurons (including A11, A13 and A14) provide the major dopaminergic inputs to the MPOA region (Lindvall & Bjorklund, 1983; Simerly & Swanson, 1986), and lesions of the MPOA have been shown to disrupt the temporal pattern of sexual behavior in female rats (Whitney, 1986; Yang & Clemens, 1996). The A13 region also receives return projections from the MPOA (Swanson et al., 1987). Asymmetry of dopaminergic systems in the brain is well documented (Glick et al., 1982; Caligiuri et al., 1989; Afonso et al., 1993). In the striatum dOpamine laterality has been correlated with turning behavior in females that showed a strong turning preference towards one side while a similar correlation was not seen in males (Dark et al., 1984). Asymmetry in neostriatal and accumbens dopamine metabolism has been associated with variations in locomotion (Speciale et al., 1986) and hippocampal dopamine concentration has been related to lateralization of T-maze choice in rats (Diaz- Palarea et al., 1987). The laterality of the A13 shown in the present study provides an example of how asymmetrical brain organization may be related to behavior involving approach and avoidance components (e. g. female-paced sexual behavior). The zona incerta (including A13 dopaminergic neurons) is involved in the initiation of locomotor (Milner & Mogenson, 1988) and orienting movements (Kim et al., 1992). Electrical stimulation of the zona incerta in cats may lead to escape responses (Kaelber, 1979). The initiation of female-paced sexual behavior falls into the category of orienting movement since it has a desired direction (towards the male). The escape of the female from the male after a mount may result from the nociception provoked by body contact, and may parallel the escape response in cats. Thus, the two sides of the A13 may 58 play separate roles in approaching or leaving the male. Perhaps the activation of one side of the A13 is related to approach behavior while the activity of the other side favors the avoidance component of the same behavior. It is intriguing to consider the possibility that when both sides of the A13 region are active during copulation, the asymmetry of neuronal activities between the two sides may be associated with the interval between the approach and the avoidance components (return latency). In subsequent work, we have found that this asymmetry in F OS/T H-IR is a dynamic phenomenon associated with recent experience (next chapter). In view of this, caution is warranted concerning interpretation since it is not clear whether the asymmetry is a functional aspect of the system that mediates sexual behavior, or whether the behavior results in the emergence of the asymmetrical F OS/T H immunoreactivity. While the role of A11 dopaminergic neurons in female sexual behavior is unclear, anatomical distribution of the terminal fields for these cells suggest that they may have an inhibitory effect on mating-induced noxious input to the spinal cord and mediate an analgesic effect during copulation (Jensen & Smith, 1982; Jensen & Yaksh, 1984). The A1 1 cell group provides spinal dopaminergic projections (Lindvall & Bjorklund, 1983), which are involved in autonomic regulatory processes (Smith & Devito, 1984; Skagerberg & Lindvall, 1985) and the inhibition of noxious input to the spinal cord (Fields, 1978; Jensen & Smith, 1982; Jensen & Yaksh, 1984). Mechanical or copulatory stimulation of the vagina and cervix of the rat produces a variety of behavioral effects (Komisaruk, 1978) including facilitation of lordosis (Rodriguez-Sierra et al., 1975) and selective inhibition of responses to noxious stimulation (Crowley et al., 1976; Komisaruk et al., 1976; Komisaruk & Wallman, 1977; Gomora et al., 1994). Both the pelvic and 59 hypogastric nerves play a critical role in conveying genital sensory activity relevant to vaginocervical stimulation-evoked responses during mating (Cunningham etal., 1991; Cueva-Rolon etal., 1996). Bilateral transection of both the pelvic and hypogastric nerves not only eliminates the analgesic effects of vaginocervical stimulation (VCS) (Gomora et al., 1994) but also reduces the intromission return latency (Erskine, 1992). Coincidentally, the A1 1 dopaminergic fibers in the spinal cord are located at sites where the pelvic and hypogastric nerves enter the spinal cord (Lindvall & Bjorklund, 1983) and where the neural circuitry mediating VCS-induced analgesia is found (Watkins et al., 1984). Therefore, A11 dopaminergic neurons could play an indirect role in regulating the time females stay with the male and the lordosis intensity through some form of A1 1 analgesic effect. On the other hand, other brain areas, e. g. A13, might dictate the motor components directly. In addition to possible central effects of A1254, there is a possibility of the peripheral effects. PCBS have been reported to alter peripheral nerve conduction velocity (Murai & Kuroiwa, 1971). Such effects could result in altered sensitivity to sexual stimulation. Severing the pelvic nerve decreased female return latencies in female pacing tests (Erskine, 1992), a result similar to the effect of neonatal A1254 treatment. It is possible that PCBS altered the pelvic nerve processes and/or decreased pelvic sensitivity of the female resulting in the shortening of the return latencies in our neonatal experiments. Adult PCB treatment had no effect on female sexual behavior, suggesting that effects seen as a result of neonatal exposure represent developmental influences of PCBS, not concurrent effects. Seegal suggested that the prolonged suppression of brain 60 dopamine concentrations following PCB exposure was due to biochemical alterations induced by the initial exposure to PCBs, rather than to continual, low-level exposure via fat stores (Seegal et al., 1992, 1994). Our adult study further supported the idea that changes in sexual behavior were developmental effects, not the result of residual PCBS. Moreover, the dose needed to disrupt sexual behavior was much lower in neonates than in adults. In summary, permanent changes were seen in feminine sexual behavior after neonatal treatment with the PCB mixture, A1254, but not afier treatment with the estrogenic PCBS, A1221. These behavioral changes were associated with the disruption of developing A13 dopaminergic system. The asymmetry of A13 may provide a good model for studying motor control of female-paced sexual behavior. The A1 1 dopaminergic system may indirectly modulate this behavior. These studies suggest that PCBS could disrupt the development of A13 dopaminergic systems and thereby reduce the reproductive success of exposed animals. 61 CHAPTER 5: FEMALE-PACED SEXUAL BEHAVIOR AND ITS LINK TO REPRODUCTION: A TIME COURSE STUDY Introduction When placed in a two-compartment chamber which allows free entrance and exit from a compartment containing a sexually active male, estrous female rats spontaneously regulate or pace the timing of the copulatory stimulation received from males (Bermant, 1961; Peirce & Nuttall, 1961; Krieger et al., 1976; Erskine, 1985). Female-paced sexual behavior appears to regulate the timing of vaginocervical stimulation and plays an important role in ensuring the activation of the neuroendocrine reflex responsible for prolongation of ovarian corpora luteal function for pregnancy (Erskine, 1989). My previous studies showed that the distribution of FOS immunoreactive A13 dopaminergic neurons on two sides of the brain might be important for female-paced sexual behavior (Figure 14). However, the correlation did not clarify whether the change in the asymmetry of A13 dopaminergic activity was the cause of behavioral disruption or vice versa. Further, since these data were collected from animals that had been treated neonatally, I decided to investigate more closely the relationship of copulatory behavior and changes in brain activity using normal females. Since female-paced sexual behavior is dynamic in its nature, I predict that the neuronal activity in the A13 region would also be dynamic during copulation if A13 dopaminergic neurons are involved in female-paced sexual behavior. Thus, F OS irmnunoreactivity (an index for neuronal activity as described in Chapter 2) in the A13 region should show differences among females that have copulated and those that have not. 62 A time-course study of F OS immunoreactivity in the A13 region should also show dynamic changes during the course of female-paced copulation. In addition, a comparison of female- paced and male-paced females should reveal differences in this brain area as well as in efferent A13 target sites. Since neurons of the incertohypothalamic system (including A11, A13, and A14) provide dopaminergic input to the medial preoptic area of the hypothalamus (MPOA) (Lindvall & Bjorklund, 1983; Simerly & Swanson, 1986), and since MPOA lesions have been shown to disrupt the temporal pattern of female-paced sexual behavior in rats (Whitney, 1986; Yang & Clemens, 1996), we propose that the MPOA is a potential efferent of the A13 region that is related to female-paced sexual behavior. We would then predict that F OS immunoreactivity would differ between female-paced and male-paced animals and also vary over time in the A13 and MPOA regions. In this chapter, we compared the A1 1, A13 and the medial preoptic nucleus of the MPOA (MPON) from normal female rats tested in female-paced and male-paced paradigms for different time periods (10 min, 30 min, 1 hr) as well as with different amounts of vaginocervical stimulation (1E, 2E, 3E and 4B). A group of noncopulated females served as the baseline control. Materials and Methods Animals Sixty-day-old Long-Evans female rats were ovariectomized and given hormone treatments for behavior tests as described in Chapter 2. 63 Female Behavior Tests We divided 42 rats into seven time-course groups (6 rats/group). The control group received no sexual stimulation and stayed in the home cage. F P—10, FP-30 and FP-lh groups were placed with a male for 10 min, 30 min and 1 hr respectively under female-paced condition. MP-lO, MP-30 and MP-lh groups remained with a male for 10 min, 30 min and 1 hr under male-paced condition. Females were returned to their home cage after the behavior test except the FP-lh and MP-lh groups which were immediately sacrificed. One hour after the first sexual encounter, all the other females were also deeply anesthetized with sodium pentobarbital (75 mg/Kg) and sacrificed for brain tissue processing. Thus, the time from the beginning of sexual stimulation was constant. Four latencies were measured in the male-pacing test: mount latency, intromission latency, ejaculation latency, and postejaculatory interval. Mount latency was the time interval measured from the introduction of the female to the first mount or intromission performed by the male. Similarly, intromission latency was the time interval measured fi'om the introduction of the female to the first intromission performed by the male. Ejaculation latency, however, was the time interval from the first intromission to the following ejaculation performed by the male. Postejaculatory interval was the time interval measured from the first ejaculation to the following intromission. The frequencies of different copulatory events (mount frequency and intromission frequency) were also recorded. Inter-intromission interval, the average interval between intromissions, and intromission rate, the rate of intromissions per minute, were analyzed as well. In the female-paced paradigm, other parameters were used together with the above 64 measures as described in Chapter 2. Immunohistochemistry for F08 and TH Tyrosine hydroxylase immunoreactivity (TH-IR) was used to identify dopaminergic neurons in the A11 and A13 regions of the brain. We also double labelled the brain sections with F OS immunohistochemistry to identify mating-activated dopaminergic neurons. F OS- IR of the medial preoptic nucleus of the hypothalamus (MPON) was also analyzed. Female rats were randomly selected for immunohistochemical processing and sacrificed 1 hr after the first sexual encounter (the introduction of female into the testing arena). The procedure for FOS/TH immunohistochemistry was described in Chapter 2. Brains were sectioned coronally at 25 pm with a cryostat. Frozen sections were collected and every fourth section was processed for F08 and TH immunohistochemistry. At least one brain from each group was processed simultaneously. (Note: All the sections for MPON were double stained with F OS/T H except the ones for the thionine staining.) The primary antibody (Ab-5 c-FOS rabbit polyclonal antisera, 1:10,000, Oncogene Research Products, MA) and normal goat serum (Vector Laboratories, diluted with TBS/0.3% Triton X-100 according to the manufacturer’s directions) were used. The biotinylated secondary antibody was goat-anti-rabbit IgG (Vector Laboratories, diluted with TBS/0.3% Triton X-100 according to the manufacturer's directions). After the PCS immunohistochemistry, sections were incubated in 5% dimethyl sulfoxide for 30 min and rinsed three times with TBS. They then underwent the immunohistochemistry with the primary antibody for TH (monoclonal mouse-anti-TH, 1:5,000, Sigma Chemical Co., St. Louis, MO) with normal horse serum (Vector Laboratories, diluted with TBS/0.3% Triton X-100 according to the manufacturer's 65 directions) and the same steps as described in Chapter 2. TH-[R and FOS/TH-IR perikarya from both sides of the sections were quantified in the A11 and A13 regions using camera lucida. Systematic section method was used for cell counting, and the estimated neuronal population was calculated by the product of sample count and the period of the systematic sample (Konigsmark, 1970). F OS-IR in the MPON was quantified on a Macintosh computer using the public domain NIH Image program (developed at the US. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). Brain sections fiom each animal were selected to match 5 plates from the Atlas of the rat brain (Paxinos & Watson, 1986) at the level from plate 18 (Bregrna: -0.26 mm, Interaural: 8.74 mm) to plate 22 (B: -0.92 mm, 1: 8.08 mm). The images were taken at 100x maginification. The plates were magnified to the appropriate size to match the computer printouts of the image. The plate was then superimposed on the computer printouts and F OS-IR nuclei were counted inside the MPON region. All the slides were number-coded and the person responsible for counting was not aware of the experimental design. Statistics The mean of each behavioral parameter in each test was automatically calculated by the computer program. The average data of four behavior tests were then used for statistical analyses. Mount latency, intromission latency, ejaculation latency and postejaculatory interval were analyzed by one-way ANOVA. Fisher’s LSD post hoc test was performed when there was a significant difference among groups (p<0.05). Mount frequency, intromission frequency, inter-intromission interval and intromission rate were analyzed 66 by the Kruskal-Wallis Test. A post hoc test was performed when there was a significant difference among groups (p<0.05). Approach latency, mount return latency, intromission return latency and postej aculatory refractory period were analyzed by one-way AN OVA. Fisher’s LSD post hoc test was performed between different time course groups when there was a significant difference among treatments (p<0.05). Percentages of mount leave, introrrrission leave, mount stay and intromission stay were analyzed by the Kruskal-Wallis Test. LQ, the percentage of times that females showed a lordosis response in 10 mounts (standard test) or more (pacing test), was also analyzed by the Kruskal-Wallis Test. The post hoc tests were performed between different groups when there was a significant difference among treatments (p<0.05). The number of TH-IR neurons in the All or A13 region was analyzed by both one-way AN OVA and Kruskal-Wallis Test. The number of FOS-IR cells in the MPON region was analyzed by both one-way AN OVA and Kruskal-Wallis Test. To compare the measurements of two sides of A1 1, A13 or MPON, two-way ANOVA (two sides as repeated measurements) was used. Results Time Effect Copulation increased the total number of TH-IR (p<0.005, Figure 16) and FOS/TH- IR (p<0.0005, Figure17) cells in the A13 region. The induction of A13 TH-IR or FOS/I‘H- IR cells was asymmetric (p<0.0001, p<0.001, respectively) on two sides of the brain. Copulation for 10 min increased the number of A1 3 TH-IR cells under the female- paced condition, but not in the male-paced paradigm. An increase in TH-IR cell number was only seen in the male-paced females that copulated for 30 min and 1 hr. The number of TH- 67 3500 $33 3000 - 1500-4 2000 - 1500 - No. ofTH-IR Celt 1000 ~ d {o {o a I. sit (a re Copuht'nn T'm (min) Figure 16. Tina corme of tyrosine hydroxylase imrmnm'eactivity (TH-IR) in the A13 regal. FP: female-paced; MP: tulle-paced; *: sipfificantly different from the noncopulated group (0 min), p<0.05; **: siglificantly different from the mopulnted group, p<0.005; ”*: siglifionnfly different from the rumoplflnted Mp, p<0.0001). 68 450 400 - 350 4 300 ~ 250 1 zoo 7 No. of FOSlTH-IR Celt 150 - 100 - so I I I I I I I 0 10 20 30 40 50 60 '10 Copulat'nn Time (min) Figure 17. Time course of FOSITH inununomasfivity (IR) in the A13 region. FP: female-paced; MP: shale-paced; a: significantly diflennnt from the FP-10 group, p<0.05; *: significantly different from the noncopulated group (0 min), p<0.005; **: significantly different from the noncopulated group, p<0.0005; ***: significanflydiflerent from the noncopulated goup, p<0.000 l. 69 IR cells in the female-paced 30 min group was low, but an increase in TH-IR cells was seen in the female-paced 1 hr group (Figure 16). Copulation for 10 min increased the number of A 1 3 F OS/T H-IR cells in female- paced rats, but not in the male-paced paradigm. The induction of FOS/TH-IR cell number was not seen in either condition at 30 min. The increase in F OS/T H-IR cells occurred in both female-paced and male—paced 1 hr groups (Figure 17). Copulation did not increase the total number of TH-IR cells in the A1 1 region. However, the increase in F OS/TH-IR cells was seen in the male-paced females copulated after 1 hr (p<0.001, Figure 18) and the induction was also asymmetric (p<0.001). Copulation increased the number of FOS-IR cells in the MPON as measured by the sum of five plates (p<0.01, Figure 19). The increase in F OS-IR cells was restricted in plate 20 (p<0.005) and plate 21 (p<0.05) and the induction was significant in both female-paced and male-paced females that copulated for 1 hr (Figure 19). Effect of the Amount of Sexual Stimulation To control for the amount of sexual stimulation that the female received within the same time period under a different testing paradigm, I also analyzed data using ejaculation as a unit for sexual stimulation. Copulation increased the number of TH-IR cells at the major side of the A13 region (p<0.05). The induction of A13 TH-IR cells was asymmetric (p<0.0001) with respect to the two sides of the brain. More sexual stimulation did not induce more TH immunoreactivity after one ejaculatory series since there was no difference among different ejaculatory series. Copulation increased the number of F OS-IR cells in the MPON as measured by the 70 800 ~ .[ —o— man 700 - —o— mun 600 u 500 . 400 . 300 . No. of F OSfI'H-IR (Tel 200 - 100 - r r I 0 10 20 3; 40 50 60 70 Copulatinn Time (min) Figure 18. Time course of FOSJTH inunnmoreadivity in the All region. FP: female-pawl; MP: male-paced; *: significantly difiement from the noncopulated group (0 min), p<0.005). 71 2200 2000 - 1000 - 1600 - 1400 - 1200 a No. of FOS-IR Cells 1000 - 000 ‘ ‘00 I I I I l I T 0 10 20 30 40 50 60 70 Copulatinn Time (min) Figure 19. Time course of HS immunoreactinity in the medialpmeopti: nucleus of the hypothalamus (MPON). FP: female-paced; MP: male-paced. *: significantly difiement from noncopulated g'oup (0 min), p<0.05; **: significantly different finnm noncopulated goup (0 min), p<0.005; a: significantly difl‘ennt fium MP- lh group, p<0.05. 72 sum of five plates (p<0.01, Figure 20). The increase in FOS-IR cells was seen only in plate 20 (p<0.05), 21 (p<0.05) and 22 (p<0.005) of both l-hr groups (Figure 20). There was no FOS—IR asymmetry between two sides of the brain in the MPON. A striking finding showed that a dense core of FOS-IR cells in the sexually dimorphic nucleus of the preoptic area (SDN-POA) occurred in the FP-lE group but not in the MP-lE group (Figure 21). After copulation for 2 ejaculatory series, both female-paced and male-paced females showed this dense core of F OS-IR cells in the SDN-POA (Figure 21). There was no difference between the male behavioral parameters in different testing situations (data not shown). Discussion I was able to replicate our previous finding of A11 and A13 TH-IR asymmetry following copulation in normal female rats. The asymmetry may be a dynamic phenomenon associated with recent sexual experience since copulation for various lengths of time induced differential changes in TH immunoreactivity. In addition, the TH-IR asymmetry in these two brain regions may be fundamentally different since copulation for various time periods changed the total number of detectable TH-IR cells in the A13 but not in the A11 region. Moreover, difl‘erent testing situations also affected the timing of TH-IR induction. Copulation also induced F OS/T H-IR in both A1 1 and A13 regions. However, the timing of induction seemed to be different for the two brain areas. In the A11 region, copulation induced more detectable F OS/T H-IR cells only in the 1 hr male-paced group but not in the other groups. As for the A13 region, the number of detectable FOS/TH-IR cells 73 2400 2200 - mi 1800 - 1000 - 1400 - 1200 q No. of FOS-IR Cells 1000 - 800 - 600 . u u . u NC 13 2B 3B 43 Copulat'nn Stimulatinn Figure 20. FOS irmnunorcactivity in the MPON region. FP: female-paced; MP: male-paced; E: ejaculatory series; *: significanty diflerent from the noncopulated group (0 min), p<0.05; **: significantly diflerent from the noncopulated group, p<0.005). 74 Figure 21. FOS immunoreactivity in the sexual dimorphic nucleus of the preoptic area of the hypothalamus under various sexual stimulation. A, B: noncopulated; C: male-paced 1 ejaculatory series; D: female-paced 1 ejaculatory series; E: male-paced 2 ejaculatory series; F: female-paced 2 ejaculatory series. 75 dramatically increased in the F P-lO females. While a similar increase was not seen in the FP-3O females, the number did increase in FP-lh females. In the male-paced females, the induction of F OS/TH-IR did not show significant difference until the l-hr test period. F emale-paced mating seemed to maximize the induction of F OS/TH-IR cells in the A13 region in a short period of time (10 min) while male-paced mating required a longer period (1 hr). It is unclear from these data whether there was a decline of F OS/TH-IR cells in the FP-3O females or whether the increase seen at 10 min was followed by a decline. The induction of F OS-IR cells in the MPON seemed to follow the same pattern of the induction of FOS/TH-IR cells as seen in the A13 region with a little delay in time. Considering the reciprocal connections between MPON and A13, it is quite reasonable that these two regions should share the same pattern of neuronal activity during copulation. A13 activity may be the trigger of the induction of F OS—[R in the MPON. Pfaus et al. (1996) found that artificial vaginocervical stimulation (VCS) produced a threshold pattern of F OS activation in the MPOA. The artificial VCS was designed to stimulate an intromission. In the rostral MPOA, generally moderate and steady numbers of FOS-IR cells were induced between 1 and 20 VCSs, but a sharp rise in F OS-IR cells occurred following 30-50 VCSs. In the caudal MPOA, VCS appeared to induce three different thresholds of F OS expression, the first increased from 1 to 10 VCSs, a second increase from 10 to 40 VCSs, and a third increase at 50 VCSs. To consider the factor of VCS through the time course, we also examined the FOS induction using ejaculation as a unit since the amount of VCSs female received in l ejaculatory series (E) in our behavior test is close to 10 VCSs in the artificial treatment in Pfaus’s study (1996). Perhaps the most striking finding in this Chapter is that the SDN-POA is activated at 76 1B in the female—paced but not in the male-paced females. Although the SDN-POA was characterized over 20 years ago (Gorski et al., 1978), no true function has been assigned to this sexually dimorphic nucleus. Considering the timing of SDN-POA activation in the female-paced and male-paced females and its anatomical connections (Simerly & Swanson, 1986, 1988), I propose that the function of SDN-POA in female rats may be related to the termination of estrus, stimulation of the prolactin release for pregnancy and the inhibition of LHRH release for normal cycling (Figure 22). In rats, one of the behavioral consequences of mating is an abbreviation of the period of estrus (Hardy & DeBold, 1972; Erskine, 1989). Although large numbers of temporally uncontrolled intromissions can induce estrus abbreviation (Erskine & Marcus, 1981), many fewer are sufficient if the female is allowed to pace coital contacts with males (Erskine & Baum, 1982; Erskine, 1985). Estrus abbreviation may occur afier 10 intromissions in the female-paced situation (Erskine,1985), but has been demonstrated under male-paced condition only after 25 intromissions (Erskine & Marcus, 1981) or after ad lib mating has occurred for 40 min (Lodder & Zeilmaker, 1976). The stimulation needed for the induction of SDN-POA F OS-IR cells in our female-pacing test (1E) and male-pacing test (2E) is quite compatible with 10 intromissions mentioned in Erskine’s female-paced (1985) and male- paced (25 intromissions, 1981) paradigms. Therefore, considering the timing and the stimulation for the induction of SDN-POA FOS-IR, it is likely that this area might be related to the termination of estrus in female rats. Indeed, a significant increase in dopamine concentration in the preoptic-anterior hypothalamic area of the sexually refiactory females was observed (Clark et al., 1986). The termination of estrus (sexually refractory) may be due to the increase dopamine transmission from the incertohypothalamic system (including A13) 77 MPON(SDN) GABA GABA + - - DA A12 (Arc) LHRH Neurons 4— A13 (21) <— All + DA DA DA LHRH + ' + Spinal Cord DA * PRL Release LH Release . + Analgesra Pregnancy Estrus Cycle Female-Paced Sexual Behavior Figure 22. Hypothetical diagram for the possible functions of the sexually dimorphic nucleus of the preoptic area (SDN). DA: dopamine; GABA: gama- aminobutyric acid; LH: luteinizing hormone; LHRH: luteinizing hormone- releasing hormone; MPON: medial preoptic area; PRL: prolactin; ZI: zona incerta. 78 to the medial preoptic area (including SDN-POA). F emale—paced sexual behavior can regulate the vaginocervical stimulation (V CS) and play an important role in ensuring the activation of the neuroendocrine reflex responsible for prolongation of ovarian corpora luteal fimction (Erskine, 1989). In a variety of species, copulation with intromission or manual VCS results in the release of prolactin to sustain luteal function (Terkel & Sawyer, 197 8) which prepares the uterus for implantation of the embryos. The induction of this progestational state is critical for the establishment and maintenance of pregnancy, and is positively correlated with the number of female—paced intromissions received by the female (Adler et al., 1970; Komisaruk & Steinman, 1986). It has been shown that the frequency of intromissions received by the female rat during estrus is a critical determinant of whether the ovarian corpora lutea secrete the quantities of progesterone necessary to maintain pregnancy (Adler, 1969, 1983; Chester & Zucker, 1970). VCS activates a neuroendocrine reflex are which initiates a pattern of twice-daily surges of the prolactin secretion; these nocturnal and diurnal prolactin surges are necessary for the prolonged luteal progesterone secretion of pregnancy and pseudopregrrancy (Gunnet & Freeman, 1983). Intromissions received by females during female-paced mating induce pregnancy/pseudopregnancy more readily than do male-paced intromissions (Erskine et al. , 1989). Female-paced as opposed to male-paced mating appears to enhance the effectiveness of each intromission in inducing these neuroendocrine changes (Erskine, 1989). The differences between female-paced and male-paced females in the behavioral and neuroendocrine responses to coital stimulation may result from differences in intromission duration (Erskine et al., 1989) or ejaculation duration (Fang et al., 1998) by males (longer in the female-paced situation). 79 The effect of the SDN-POA in stimulating the prolactin release might be through the inhibition of the other POA region (Clemens etal., 1971, 1976; Wiegand et al., 1980; Gunnet & Freeman, 1983, 1985) or the dopaminergic neurons in the arcuate nucleus (Moore, 1987) by its GABAergic neurotransmission or due to the release of a prolactin releasing factor, thyrotropin-releasing hormone (Tashjian et al., 1971). The SDN contains thyrotropin- releasing hormone neurons (Simerly et al., 1986) and GABAergic neurons (Sagrillo & Selrnanoff, 1997). The POA is known to tonically inhibit the expression of the nightly surge of prolactin during pseudopregnancy following cervical stimulation. Lesions of the medial preoptic area trigger the release of nocturnal prolactin surges and induce repetitive, spontaneous pseudopregnancy (Clemens et al., 1976; Wiegand et al., 1980; Gunnet & Freeman, 1983, 1985). Conversely, electrochemical stimulation of the preoptic area reduces prolactin secretion perhaps by increasing the inhibitory action of this brain site (Clemens et al., 1971). It has been proposed that the medial preoptic area may contain neurons which exert a chronic inhibitory effect on the prolactin surge (Gunnet & Freeman, 1983). Bilateral implantation of muscimol—mannitol mixture into the medial preoptic/anterior hypothalamic area increased blood prolactin level (Lamberts et al., 1983; Bach et al., 1992). SDN-POA neurons might inhibit the effect of other POA neurons and stimulate the release of prolactin through their GABAergic terminals. Indeed, the axons of the GABAergic neurons in the medial preoptic area often ramified into an apparent network of local collaterals, possibly forming local circuits in this region (Hoffman et al., 1994). Female-paced sexual behavior seems to maximize the induction of F OS-IR in the medial preoptic area including the SDN-POA. The induction of FOS-IR in the SDN-POA may be related to the termination of estrus, the release of prolactin for pregnancy and the 80 interruption of regular cycling of LH. The relation between A13 dopaminergic neurons and the medial preoptic area may serve as the link between sexual behavior and the neuroendocrine systems that mediate implantation and the onset of pregnancy. 81 CHAPTER 6. EFFECTS OF PCBS ON MALES AND THE RELATION OF MALE SEXUAL BEHAVIOR TO THE MAJOR PELVIC GANGLIA IN N EONATALLY A1221-TREATED RATS Introduction I have discussed the effects of PCBS on the development of female sexual behavior and brain dopaminergic systems in the previous chapters since my studies were primarily focused on females. However, I also examined sexual behavior of the male litterrnates. Since I had a skewed sex ratio in the animal colony, I did not have a complete data set for all the experiments. While I did not observe striking PCB effects, the analysis did reveal an intriguing relation between male sexual behavior and measures of the major pelvic ganglion. In this chapter, I summarize the male behavioral data from the perinatal and neonatal PCB treatment groups. Since PCBS did not have strong effects on male sexual behavior, I did not undertake the studies of the brain dopaminergic systems in these subjects. In addition, I only examined the pelvic ganglia of the neonatally A1221- treated rats since the technique was not developed and available to use with prior treatment groups. The pelvic plexus comprises a major pelvic ganglion and some satellite ganglia lying on either side of the dorsolateral lobes of the prostate in male rats. In female rats, these bilateral ganglia (no satellite ganglia) were located at the uterine cervix-vagina junction and were called paracervical or pelvic ganglia. For the convenience of discussion, I refer to the male major pelvic ganglia and female pelvic ganglia as the MPG. The MPG is a mixed autonomic ganglion composed of both parasympathetic and sympathetic elements (Dail et al., 1975; Dail and Evans, 1978), which innervate 82 androgen sensitive accessory organs of male rats as well as the pelvic viscera in both male and female rats (Langworthy, 1965; Purington et al., 1973; Quinlan et al., 1989). The MPG receives innervation from the spinal cord at L6 and S1 levels via the pelvic nerve and from the thoracolumbar outflow via the hypogastric nerve (Quinlan et al., 1989) Nissl-stained or tyrosine hydroxylase-immunoreactive (TH-IR) neurons in the MPG display a strong sexual dimorphism. There are about 2.5 times more neurons in the male than in the female MPG (Greenwood et al., 1985). Many TH neurons are found in the male MPG, but very few in the female MPG (Vera and Nadelhaft, 1992). The cavernous (penile) nerve from the MPG, which courses along the urethra and innervates the corpus cavernosum, is essential for penile erection in male rats (Quinlan et al., 1989; Sachs and Liu, 1991). Pelvic and pudendal nerves from the MPG, on the other hand, are important sensory pathways for female sexual behaviors (Erskine, 1992). Here I report the behavioral data of the effect of neonatal A1221 treatment on sexual differentiation of the MPG in rats. Materials and Methods Animals PCB treatments were described in Chapter 3 (perinatal) and Chapter 4 (neonatal). Female offspring from PCB-treated female rats were tested as described previously. Male offspring were tested intact at 90 days of age. They were placed with sexual experienced female rats over night at the testing arena two weeks before real tests (day 76). One more pretest was observed on day 83. Males were then tested for sexual behavior once per week for four weeks. Untreated rats of the same strain and age were purchased from the 83 breeder (Harlan Spraque Dawley, Inc., Indianapolis, IN) and tested with the same protocol. Male Behavior Test To test for male behavior, a sexually receptive female was placed with a male rat in a single-compartment testing chamber (56x44x49 cm). Male behaviors (mount, intromission, and ejaculation) were then recorded by a computer with a event recording program. Copulatory behavior of the male rats was characterized by a series of mounts, with or without vaginal insertion, then eventually accumulated in ejaculation. Intromission patterns (mount with vaginal insertion) could be distinguished behaviorally from mounts without penetration by the presence of a deep thrust and a springing dismount. An ejaculation occurred after several intromissions and followed by a refractory period. Histochemistry for the Major Pelvic Ganglia Rats were anesthetized with sodium pentobarbital (75 mg/Kg). The abdominal wall was opened and the MPG were removed and fixed with 4% paraforrnaldehyde in 0.1M phosphate buffer (pH 7.4) over night at 4°C. The MPG was then dehydrated through an ethanol series (50%, 70%, 90%, 100%, 30 min, 2 times each) and finally transferred into ethanol/xylene (1:1, 30 min) then 100% xylene (30 min, 2 times). Afier incubating with xylene/paraffin (1 :1) two times (30 min each) at 60°C, the MPG was infiltrated with pure paraffin at 60°C (1 hr, 2 times) and embedded into a paraffin cube. The MPG was sectioned at 30 pm with a microtome. Paraffin ribbons were mounted onto gelatin-coated slides, stained with thionine as described in Chapter 2, and covered with a 84 coverslip in DPX mounting media (Electron Microscopy Sciences). Neurons were quantified on every tenth section in one side of the MPG using camera lucida. Slides were number-coded and the person responsible for counting was not aware of the experimental design. Statistics The analyses of female behavioral data were described in previous chapters. To assess the temporal pattern of male sexual behavior, four latencies were measured: mount latency, intromission latency, ejaculation latency, and postejaculatory interval. Mount latency was the time interval measured from the introduction of the female to the first mount or intromission performed by the male. Similarly, intromission latency was the time interval measured from the introduction of the female to the first intromission performed by the male. Ejaculation latency, however, was the time interval from the first intromission to the following ejaculation performed by the male. Postejaculatory interval was the time interval measured from the first ejaculation to the following intromission. The mean of each behavioral parameter in each test was calculated by the computer program. The mean of four behavior tests were then used to calculate the mean for each litter and for further statistical analyses using StatView (Abacus Concepts, Inc., Berkeley, CA). Mount, intromission and ejaculation latencies and postejaculatory intervals were analyzed by one-way ANOVA. The frequencies of different copulatory events (mount frequency and intromission frequency) were also recorded and analyzed by the Kruskal-Wallis Test. The hit rate, defining as the intromission frequency divided by the total number of mounts and intromission, was also calculated and analyzed by the Kruskal-Wallis Test. Interintromission interval, the average interval between 85 intromission rate, the rate of intromissions per min, were analyzed by the Kruskal-Wallis Test as well. Since the sample size for perinatal A1221 treatment groups was not sufficient for statistical analyses, data were discarded. In order to understand why male noncopulators had fewer cells in the MPG, I examined the relation between the number of cells in the MPG and male sexual behavior. Since there was no difference among treatment groups, I pooled the data of PCB-treated rats and did the correlation analyses using MPG cell number as the independent variable and the behavioral parameters as dependent variables. The number of neurons in the MPG was analyzed by both one-way ANOVA and the Kruskal-Wallis Test. Fisher’s LSD post hoc test was performed when there was a significant difference among treatments (p<0.05). Correlation analysis was performed between behavior parameters (dependent variables) and the number of neurons in the MPG (independent variable). Linear regression analysis was followed if significant correlations were found (p<0.05). F tests were performed between PCB-treated and purchased rats in both sexes. Results There was no difference in male sexual behavior or the cell number of the MPG among neonatal A1221 treatment groups. However, there was a sex difference in the number of neurons in the MPG with control males having more cells than control females. Within control males, the number of cells in the MPG was significantly different between copulators and noncopulators (Figure 23). The cell counts of non-copulators’ MPG were significantly fewer than those of the control male copulators but significantly greater than those of control females. 86 14000 ~ I 12000 - 0 an 10000 - 2 0 * 5 8000 - I .5 1 n: E 6000 4 U a: ‘- 1 ‘3 4000 4 =4 * O I z . 2000 . n=6 r 0 . . . Copulator Noncopulator Female 1 | Male Figure 23. Sexual allomorphism in the number of cells in the major pelvic ganglia (MPG). 87 The mount frequencies of PCB-treated male rats were significantly correlated with the number of neurons in the MPG (correlation coefficient = 0.77, p<0.01). Linear regression analysis showed a negative linear relationship between male mount frequencies and the number of neurons in the MPG (p<0.01, Figure 24). The ejaculation latencies of PCB-treated male rats were significantly correlated with the number of neurons in the MPG (correlation coefficient = 0.79, p<0.01). Linear regression analysis showed a negative linear relationship between male ejaculation latencies and the number of neurons in the MPG (p<0.01, Figure 25). The interintromission intervals of PCB-treated male rats were significantly correlated with the number of neurons in the MPG (correlation coefficient = 0.82, p<0.01). Linear regression analysis showed a negative linear relationship between male interintromission intervals and the number of neurons in the MPG (p<0.01, Figure 26). The intromission rates of PCB-treated males were significantly correlated with the number of neurons in the MPG (correlation coefficient = 0.73, p<0.05). Linear regression analysis showed a positive linear relationship between male intromission rates and the number of neurons in the MPG (p<0.05, Figure 27). The hit rates of PCB-treated males were significantly correlated with the number of neurons in the MPG (correlation coefficient = 0.81, p<0.01). Linear regression analysis showed a positive linear relationship between male hit rate and the number of neurons in the MPG (p<0.01, Figure 28). We also examined the MPG of newly purchased non-treated rats to confirm these correlations between male sexual behavior and the neuron number of the MPG. However, we did not find any significant correlations in the non-PCB treated rats. Further analyses revealed that the distribution of cell numbers in the PCB-treated group 88 _ O 45 . Y = 52.87 - .003 * x 40 j r= 0.77, p<0.01 35 1 Mount Frequency 11000 0 V r ' l 7000 9000 13000 15000 17000 No. of Cells in the MPG Figure 24. Linear relation between male mount frequency and the number of cells in the major pelvic ganglia (MPG). Correlation coefficient = 0.77, p < 0.01. 89 . 1000 j 0 Y = 1239.466 - .064 * X 900 - r = 0.79, p<0.01 Ejaculation Latency (sec) 100 . . . . . . 7000 9000 11000 13000 I ' I j I ' I I fiI 15000 17000 No. of Cells in the MPG Figure 25. Linear relation between male ejaculation latency and the number of cells in the major pelvic ganglia (MPG). Correlation coefficient = 0.79, p < 0.01. 90 100 . o 1': 90 j Y = 115.297 - .006 * X it: 80 - I' = 0.82, p<0.01 «at . E 70 ‘. ~53 60 - g .. g 50 ‘ E 40 ~ § 30 4 5 20 . 10...,.....,.,.,.,.,. 7000 9000 11000 13000 15000 17000 No. of Cells in the MPG Figure 26. Linear relation between male interintromission interval and the number of cells in the major pelvic ganglia (MPG). Correlation coefficient =03Lp<00L 91 3.25 0 Y = -.242 + 1.813E-4 * X A 3 ‘ r=0.73,p<0.05 o 75 2.75 - E 2.5 J 8 g 2.25 . E 2 . g 1.75 . E 1.5 - 1.25 1 1.,.,.,.,.,.,..fi1.,. 7000 9000 11000 13000 15000 17000 No. of Cells in the MPG Figure 27. Linear relation between male intromission rate and the number of cells in the major pelvic ganglia (MPG). Correlation coefficient = 0.73, p < 0.05. 92 Y = -.07 + 4.868E-5 * x 0 r = 0.81, p<0.01 .64 0 g .5“ m .. E .4“ .3“ .2“ .O .1 yrrl'r l'r—rl l 7000 9000 11000 13000 15000 17000 No. of Cells in the MPG Figure 28. Linear relation between male hit rate and the number of cells in the major pelvic ganglia (MPG). Hit rate = intromission frequency (IF) divided by the sum of mount frequency (MF) and IF [IF/(MF+IF)]. Correlation coefficient = 0.81, p<00L 93 and the untreated group was very different. The F test showed that these two groups in both sexes had significantly different variances (p<0.0001). The non-PCB treated rats were more homogeneous than the PCB-treated rats (Figure 29), although the mean number of neurons in the MPG did not differ for the two groups. Discussion I was able to reproduce the sex difference in the cell number of the MPG (Greenwood et al., 1985) in the control rats. Moreover, I found that the number of neurons in the MPG was significantly different between copulators and non-copulators within control males. In addition, MPG cell number of PCB-treated rats had significantly greater variance than that of the untreated rats. One possible explanation for the difference in the MPG was the various somatosensory inputs from their mothers during development. It is well known that lactating rats spend a great deal of time licking the genital area of their male pups which ensures the proper development of male sexual behavior (Moore & Morelli, 1979). Mother rats displayed significantly more anogenital licking towards male pups as opposed to female pups over the first 18 postpartum days (Moore & Morelli, 1979; Richmond & Sachs, 1984). Administering testosterone neonatally to female pups increased the amount of anogenital licking received from mothers (Moore, 1982). Additional experiments suggested that testosterone stimulates the production of a chemosensory cue by the preputial glands. This preputial pheromone is excreted in the urine, and is preferentially ingested by mothers (Moore, 1981; Moore & Samonte, 1986). Depriving rat pups of normal levels of maternal anogenital licking by making mothers anosmic (Moore, 1984) or by applying perfume to pups’ anogenital region (Birke & 94 No. of Anrmak' l2 l0 - I 5000 7000 fi T I sooo nooo moo rsooo moo No. of Celb inure MPG Figure 29. Distribution of the MPG cell number. MPG: major pelvic ganglia;PCB: animals fem neonatal A1221 experiment 95 Sadler, 1987) led to deficits in later masculine cepulatory behavior of both male and female offspring. These behavioral deficits were correlated with a significant reduction in the number of motor neurons in the sexually dimorphic spinal nucleus of the bulbocavemosus muscle in male rats whose mothers were made anosmic over the first 14 postpartum days (Moore et al., 1992). It is also possible that the deficits of male sexual behavior are also correlated with a significant reduction in the number of neurons in the MPG. Males deprived of maternal anogenital licking later displayed significantly longer ejaculation latencies and longer interintromission intervals than controls (Moore, 1984). We found that longer ejaculation latency and longer interintromission intervals are correlated with fewer neurons in the MPG of PCB-treated animals. It is possible that maternal anogenital licking is correlated with the development of the MPG. Possibly noncopulators do not receive enough anogenital licking thus resulting in under- development of the MPG. Since reproduction is not essential to animal survival, it is likely that the neurons in the MPG of the noncopulators were largely those innervating the colon or bladder while those neurons innervating the penis for erectile mechanisms may well be reduced. More somatosensory inputs by neonatal maternal anogenital licking may induce more neuronal growth and/or prevent the neuronal death in the MPG during the development, hence the more neurons in the MPG and more neurons innervating the penis resulting in better performance in male sexual behavior. However, further investigation would be needed to draw such conclusions. The possible explanation for the large variance in PCB-treated rats is that PCBs disrupt the maternal recognition of male rats by disrupting testosterone metabolism (Derr and Dekker, 1979) in the male pups or by masking the maternal recognition of their 96 urine. Alternatively, PCBs per se may disrupt the development of the MPG. It is known that tyrosine hydroxylase (TH), DOPA decarboxylase, and choline acetyltransferase (CAT) activities in the MPG are significantly reduced by postnatal castration on day 10- 11. Testosterone and dihydrotestosterone replacement reversed all developmental enzyme- activity deficits (Melvin & Hamill, 1987 & 1989). In castrated animals, estrogen therapy reversed the deficits in CAT activity, but was ineffective in reversing the alterations in TH activity (Melvin & Hamill, 1989). Neonatal Aroclor 1221 treatment may disrupt the balance of gonadal hormones and cause the variation in the cell number of the MPG. Another alternative is that the PCB affected the mothers’ behavior directly. I found correlations among five measures of male sexual behavior and the neuron nrunber in the MPG. The most important correlation is probably the relation between the hit rate and the MPG cell count. That result indicates that all the correlations we found may be due to the ability to achieve penile erection. Rats with fewer MPG neurons had more unsuccessful attempts (mounts), copulated slower and took a longer time to achieve ejaculation than rats with more MPG neurons. A brief review of the physiology of penile erection may provide insight into those correlations. Three autonomic centers participate in the innervation of the penis (Figure 30). The sacral cord gives rise to preganglionic parasympathetic fibers that travel in the pelvic nerve to innervate ganglion cells in the pelvic plexus. This is the major pathway responsible for penile vasodilation. Although the mechanism of action is uncertain, the neurotransmitters of the postganglionic neurons are acetylcholine, vasoactive intestinal peptide (VIP) and nitric oxide (NO) (Burnett et al., 1992; Dail, 1993). An alternate pathway for penile vasodilation is centered in the lumbar cord. This sympathetic pathway reaches penile neurons in the pelvic plexus via the hypogastric nerve. Evidence suggests 97 Spinal Cord Hypogastric Nerve T11-12 L1-2 — — — — —l Pudendal Nerve Ach/VIP/NO I (vasodilation) Sympathetic l l I i | L M 6 . l Pelvic Ganglion Pelvrc Nerve | Parasympathetic 51 l . I Cavernous | Nerve NE/NPY ' (Constriction) I ' Ach/VIP/NO l (vasodilation) V V lschiocavernosi and Corpus Bulbospongiosus Cavernosum Muscles l Penile Erection Figure 30. Neural control of penile erection. Ach: acetylcholine; L: lumbar; NE: noradrenaline; NPY: neuropeptide Y; NO: nitric oxide; S: sacral; T: thoracic; VIP: vasoactive intestinal polypeptide. 98 that this alternate pathway (the supra-sacral erector outflow) is also cholinergic-, VIP- ergic and/or possibly NO-ergic (Alrn et al.., 1995 ; Domoto & Tsumori, 1994). Vasoconstriction in the penis is mediated by noradrenergic-neuropeptide Y fibers of sympathetic chain origin. Noradrenergic fibers probably reach the penis via the pudendal nerve. The pudendal nerve, a somatic nerve, innervates skeletal muscle at the base of the penis. Once autonomic mechanisms have increased blood flow to the penis, a further rise in pressure in erectile tissue may be obtained by contraction of the ischiocavernosi and bulbospongiosus muscles (Dail, 1993). Therefore, stimulation of the pudendal nerve or sympathetic trunk resulted in constriction and retraction of the penis of the cat and rabbit (Sjostrand & Klinge, 1979; Langley & Anderson, 1985), whereas the erectile response could be produced by electrical stimulation of either the pelvic or cavernous nerve (Quinlan et al., 1989). One possible mechanism involved in the correlation between male sexual behavior and the MPG is that with more MPG neurons, the number of VIP/NO neurons in the MPG was increased resulting in a greater release of VIP/NO during copulation, hence faster and better penile erection. VIP and NO are potent vessel dilators that can facilitate penile erection via vascular mechanisms in the penis (Andersson & Wagner, 1995). In my most recent study, female rats treated with testosterone propriate neonatally had more neurons in the MPG, and the increase in the MPG neurons could be accounted for an increase in VIP neurons. It is possible that male rats with more MPG neurons may have more VIP/NO neurons as well, but further investigation is needed to confirm that. I would expect an even higher correlation between VIP/NO neuron number and male sexual behavior. 99 One important message from the correlations is that local circuits in the MPG may be sufficient to produce penile erection because the interintromission intervals, intromission rates and hit rates are highly correlated with the neuron number in the MPG. Indeed, the somatic component in the erectile response was not essential because there was no difference between curarized and control rats. Other evidence has shown that testosterone enhances the erectile response of cavernous nerve stimulation and acts peripherally to the spinal cord, but not in the central nervous system. These results all support that a local circuit peripheral to the spinal cord is sufficient and androgen also acts on the local circuit around the MPG. The sites of testosterone action appear to be situated on neurons rather than on penile erectile tissue, and proerectile postganglionic parasympathetic neurons in the MPG seem to be the likely target sites for gonadal steroids (Giuliano et al., 1993). However, the efferent control from the central nervous system also inhibits the efficiencies of penile erection because pelvic nerve transection increases intromission rate in male rats (Lodder & Zeilmaker, 1976a). Electric stimulation of the sympathetic preganglionic or postganglionic fibers innervating the genital organs can produce ejaculation without erection (Langley & Anderson, 1985). Tracing studies in cats, rats, dogs, and monkeys have shown that the preganglionic neurons for the MPG are located in the interrnediolateral nucleus of the spinal cord and send dendritic projections into areas of laminae V-VII and the dorsal commissure that receive afferent input from the penis. Thus the afferent and efferent components of the reflex pathway are in close proximity. In rats, penile afferents also project into the ventral horn and appear to make contacts with the soma and dendrites of motor neurons. These connections could be involved in the somatic reflex mechanisms 100 involved in copulation and in reflex responses such as the bulbocavemous reflex (Andersson & Wagner, 1995). According to the results from PCB-treated male rats, as well as previous reports (Giuliano et al., 1993; Andersson & Wagner, 1995), the MPG seemed to play an important role in penile erection in addition to other basic autonomic functions. Female rats were not expected to see similar results because they lacked the erectile tissues. Since the pelvic plexus innervates the female cervix, vagina and uterine horns, it is more important for the pelvic nerve to carry the afferents responsible for the induction of pseudopregnancy or for all phases of pregnancy, from activation of corpora luteal through and including normal parturition (Carlson & DeFeo, 1965). In this sense, we would expect the intromission return latency or the postej aculatory refractory period of female rats to be more likely related to the number of preganglionic neurons receiving the afferent information. An interesting finding (Berkley et al., 1990) from pelvic nerve recording showed that the rat detected the vaginal stimulus at very low levels of distension by brief orienting movements toward the stimulus at its onset. They made escape responses only when distension levels were great enough to exceed vaginal compliance. This is very similar to the responses of female rats in the female-paced sexual behavior. Females escape from males afler receiving vaginal cervical stimuli (intromissions or ejaculations), and the return latency is longer after greater stimulation (Erskine, 1992). In fact, our recent experiment, showed that the VIP neuron number, not the total cell number, in the MPG is highly correlated to female intromission return latency and postejaculatory refractory period (Fang et al., 1998). I found a significant difference in the variance analysis of the MPG neurons between PCB-treated animals and those not treated with PCBS. It would seem that during 101 normal development, sex hormones (estrogen or testosterone) act as the developmental restraint to create a homogeneous population with the MPG neuron number of most animals located within a normal range. PCBS, by disrupting sex hormone metabolism or maternal care during development resulted in a heterogeneous population. The increased variance may explain why I was able to find strong correlations between male sexual behavior and the cell counts of the MPG. 102 CHAPTER 7: GENERAL DISCUSSION Permanent changes were seen in female sexual behavior after developmental treatment with the PCB mixtures, A1221 or A1254. Perinatal A1221 treatment lowered female receptivity and increased the latency for the female to approach the male during copulation and increased avoidance of the male by the female. Perinatal A1254 treatment, on the other hand, only increased the avoidance component of female-paced sexual behavior. Neonatal A1254 treatment, however, decreased female receptivity as well as the latency for the female to approach the male after receiving an intromission; neonatal A1221 treatment had no effect on these measures. Thus, different PCB mixtures appeared to act during different critical periods in development. The estrogenic A1221 seemed to act prenatally while the nonestrogenic A1254 was disruptive only neonatally. Some of the changes in female sexual behavior were associated with the disruption of the developing A13 dopaminergic system. Examination of the incertohypothalamic dopaminergic systems revealed a functional brain asymmetry wherein the presence of some dopaminergic cells was only seen following copulatory stimulation. The brain appeared to restructure itself in relation to the environmental context. When the brain was stimulated during copulation, more incertohypothalamic dopaminergic neurons could be recruited to process information and to perform the task. Further analysis in normal females showed the emergence of neuronal activity in the sexually dimorphic nucleus of the preoptic area (SDN-POA) in sexually active females. This activity coincided with the sensory and temporal factors associated with the release of prolactin and the induction of the progestational state of pregnancy. When 103 given the opportunity to pace her copulation, the female was able to maximize the effect of copulatory stimulation and thereby facilitate the onset of activity in the SDN-POA. PCBS also disrupted the development of the major pelvic ganglion (MPG) in the male. Analysis of these changes revealed strong correlations between several measures of male sexual behavior and cell number in the MPG. In the normal male these relations appear more elusive due to the restricted variance in cell number. Neural Control of Female-Paced Sexual Behavior Based on the findings reported in the previous chapters, I have formulated a hypothesis concerning the events that bring about the female’s timing of copulation (Figure 31). This hypothesis, while preliminary, is summarized below and is followed by a review of existing anatomical and functional studies that lend support to this concept. Systems Involved in Female-Paced Sexual Behavior: A Working Hypothesis The incertohypothalamic dopaminergic systems are seen as areas that integrate motor and sensory, as well as limbic information, and play important roles in the coordination of neuroendocrine processes with motor function in female-paced sexual behavior. Such a functional system might act in the following manner. When the female first encounters the male, visual, auditory, and olfactory information are relayed to the zona incerta and the medial preoptic area. The decision to approach or avoid may reflect processes in the medial preoptic area. The zona incerta, with its extensive connections to the brain areas involved in motor functions, enables the female to perform the behavior of approaching or avoiding the male. Sensory information from the first mount or 104 A d't £3533 _> Medial preoptic area stimulations ‘ A12 44> ''''' I I \ x d— Olfactory stimulation I Pituitary I ‘ Motor systems I Zona Incerta I PRL l . Ovary | t . Progesterone , All I I Pregnancy V I Brain Stem —————— _ I -’ Spinal cord H Pelvic ganglion I\¢ Female-paced Cervix, Vagina Sexual Behavior T Vaginocervical stimulation Figure 31. Hypothetical diagram for systems involved in female- paced sexual behavior. PRL: prolactin; SDN: sexually dimorphic nucleus of the preoptic area. 105 intromission the female receives from the male brings about increased activity in A1 1 dopaminergic neurons which are associated with modulation of potentially noxious somatic sensation associated with copulation, e. g. vaginal distention beyond compliance (Berkeley et al., 1988). The notion that the MPOA is involved in the decision to approach the male is strengthened by the finding that MPOA has been implicated in the initiation of goal-oriented or motivated behaviors associated with reproduction (Swanson & Mogenson, 1981). Neurons in the MPOA may influence locomotor activity through a direct pathway via caudal regions of the zona incerta, and then continuing on to the pedunculopontine nucleus (a major component of the mesencephalic locomotor region) (Swanson et al., 1987). Lesions of the MPOA have been shown to disrupt the temporal pattern of sexual behavior (Whitney, 1986; Yang & Clemens, 1996) in female rats. It seems clear that a number of goal-oriented behaviors, including female-paced sexual behavior, can be influenced by manipulation of the MPOA (Swanson et al., 1987). The number of intromissions and their timing can be directly related to activity in the SDN-POA. When ejaculation occurs, 1 suggest that the sexually dimorphic nucleus of the preoptic area initiates the processes that terminate the estrus, stop the cyclic release of gonadotropin and facilitate prolactin release. PCB treatment during development may disrupt the balance among these systems in a number of ways. While this hypothesis is probably far too simple, in the following section anatomical, physiological and behavioral data will be used to support the main elements of this concept and indicate areas of weakness. 106 The Anatomy and the Function of Incertohypothalamic Dopaminergic System in Female-paced Sexual Behavior The incertohypothalamic system is the link between the spinal cord and the medial preoptic area in the hypothesis. It (Figure 32) was first described by Bjorklund et al. in 1975. On the basis of fiber distribution, a caudal and a rostral part can be discriminated: the caudal part extends from the area of the dopamine-containing cell bodies in the caudal thalamus, the posterior hypothalamic area and the medial zona incerta (the All and A13 cell groups) into the dorsal part of the dorsomedial nucleus and the dorsal and anterior hypothalamic areas; the rostral part extends from the area of the rostral periventricular dopaminergic cell system (the A14 cell group) into the medial preoptic area and the periventricular and suprachiasmatic preoptic nuclei. The system also extends into the most caudal portion of the lateral septa] nucleus. The projection areas of these neurons signify an involvement in the control of pituitary hormone secretion (ijrklund et al., 1975). These anatomical data support but do not necessarily prove our hypothesis about the function in gonadotropin and prolactin release. A11 Dopaminergic Neurons The A1 1 dopaminergic neurons are the link between the spinal cord and the zona incerta, and they may modulate the analgesia that occurs during female-paced copulation in our hypothesis. This cell group is known to be the major source of fibers running in the periventricular catecholamine projection system, which innervates, e.g., medial and midline thalamus and several hypothalamic nuclei (Lindvall & Bjorklund, 1974). These cell bodies are located in the dorsal and posterior hypothalamus, zona incerta, and caudal 107 Figure 32. Locations of the cell bodies and terminals of the incertohypothalamic dopaminergic system are schematically depicted on coronal sections. AC: anterior commissure; ah: anterior hypothalamic area; All, 13, 14: dopaminergic cell groups; dh: dorsal hypothalamic area; dm: dorsomedial nucleus; F: fornix; MT: mammilothalamic tract; 0C: optic chiasm; ph: posterior hypothalamus; pom: medial preoptic area; PPN: preoptic periventricular nucleus; pv: paraventricular nucleus; st: bed nucleus of stria terminalis (from Bjorklund et al., 1975) 108 thalamus (Figure 33). The A11 fiber system is a component of the dorsal longitudinal fasciculus of Schfitz, which is a bidirectional tract interconnecting the lower brain stem and spinal cord with the periaqueductal gray and hypothalamic and thalamic areas. The A11 dopaminergic projections in the spinal cord are located in the dorsal gray of the cervical, thoracic, lumbar, and sacral cord, and in the intermediate gray of the thoracic cord. In the dorsal born, the dopaminergic fibers are most abundant in the lateral parts of the superficial layers and in the adjoining reticular nucleus. The highest density of the dopaminergic fibers is found in the intermediolateral cell column and in the area surrounding the central canal at thoracic and upper lumbar levels. This dopaminergic innervation is not uniform along the intermediolateral column but forms clusters or patches of varicose fibers corresponding to the clustering of preganglionic sympathetic neurons along the intermediolateral column (Skagerberg et al., 1982; Lindvall & Bjorklund, 1983), which indicates important functions for this system involving sympathoexcitatory function (Bemthal & Koss, 1979; Gebber & Snyder, 1979; Simon & Schramm, 1983; Smith & Devito, 1984; Skagerberg & Lindvall, 1985) important for sexual behavior and sperm transport (Carlson & DeFeo, 1965; Erskine, 1992; Traurig & Papka, 1993). The concentration of dopaminergic terminals in the dorsal horn suggests a modulatory role for the diencephalospinal A11 dopaminergic system in the processing of sensory information, such as pain (Lindvall & Bjorklund, 1983). Available evidence from studies with various nociceptive tests suggests that spinal dopaminergic mechanisms have an inhibitory effect on noxious input to the spinal cord (Jensen & Smith, 1982, 1983; Jensen & Yaksh, 1984). In fact, stimulation-produced analgesia has been elicited 109 Figure 33. The location of All cell bodies and their spinal projections (from Skagerberg et al., 1982). 110 from part of the area of the All cell group (; Rhodes & Liebeskind, 1978; F leetwood— Walker eta1., 1988). Together with the pharmacological data, it has been suggested that the diencephalospinal A1 1 dopaminergic neurons might constitute an endogenous pain inhibitory system (Skagerberg & Lindvall, 1985), which may be important to sustain female receptivity during copulation. Zona Incerta and A13 Dopaminergic Neurons The A13 dopaminergic neurons within the zona incerta are the integration center of motor, sensory and limbic information in the hypothesis. The zona incerta can be subdivided into a ventral zone (ventral sector of pars caudalis and pars ventralis) and a dorsal zone (pars dorsalis). The somesthetic (somatosensory cortex, trigeminal complex, and dorsal column nuclei), collicular, and cerebellar projections terminate at the ventral zone. Limbic input (cingulate cortex), on the other hand, is directed into a dorsal zone. The contingent of somatosensory afferents is relatively large and there is a high degree of overlapping between the different somatosensory terminal fields within the ventral zona incerta. This suggests a participation of zona incerta in the treatment of somesthetic information and/or in the transmission of noxious stimuli (Roger & Cadusseau, 1985; Shammah-Lagnado et al., 1985) during copulation. The efferent connections of the zona incerta terminate mainly in an ipsilateral system of descending and ascending fibers distributed to reticular structures of the brain stem, precerebellar nuclei, the middle and deep layers of the superior colliculus, the pretectum, perioculomotor, the parvocellular portion of the red nucleus, the central gray substance, the nucleus tegmenti dorsalis lateralis, the ventral horn of the cervical spinal lll cord, non-specific thalamic nuclei, basal ganglia, hypothalamic structures and a subpallidal district of the substantia innominata (Ricardo, 1981). Taken together, the zona incerta is the main tecto-subthalamic target regions and it also projects to the superior colliculus (Watanabe & Kawana, 1982; Araki et al., 1984; Kim et al., 1992; Kuenzle, 1996) and ipsilateral cortical fields, including sensory, limbic and association areas, with more incertal neurons projecting to the SI/MI cortices than. to any other cortical area. The pathway orients toward the visual or frontal cortex and the projections possibly involved in limbic and circadian mechanisms, and may be important during copulation. For example, based on the input-output organization of the zona incerta, this nucleus may act as an important integrative center linked to the reticular activating system in the midbrain (MacKenzie et al., 1984), and involved in motor functions including drinking, visual discrimination, locomotion, muscular response to morphine and posture (Sinnamon, 1984; Mogenson & Wu, 1986; Nakanishi et al., 1992). In addition, it is also important in the control of nociception (Wardas et al., 1987). Therefore, the zona incerta may be involved in the motor component of female-paced sexual behavior. Electrical stimulation of the zona incerta leads to motor responses (Kaelber & Smith, 1979). Microelectrode recording in the monkey disclosed the existence of zona incertal units that respond to passive limb manipulations and to visual stimuli, are activated when the animal reaches for objects of interest and fire during the performance of a visuomotor tracking task (Crutcher et al., 1980). Other evidence suggests that the zona incerta might be a link in pathways involved in the transfer of visual information to motor circuits (Legg, 1979). Considering the anatomical afferents 112 of zona incerta, the information about limb movement might reach the zona incerta via the projections from the dorsal column nuclei, whereas visual input could conceivably be conveyed to zona incerta by way of pathways originating from the ventral lateral geniculate nucleus, the visual areas of the cerebral cortex and the pretectum. In the context of motor integration, zona incerta receives substantial projections from the primary motor and retrosplenial cortices, cerebellum as well as apparently minor projections from other motor-related structures, such as the entopeduncular and red nuclei. Considering zona incertal efferents, they can be traced to several motor-related structures, such as precerebellar nuclei, the nucleus tegmenti pedunculopontinus pars compacta, the ventral horn of the spinal cord, many brain stem territories that send fibers to the spinal cord, both segments of the globus pallidus and the thalamic nuclei whose efferents reach the motor cortex; even a sparse direct projection from zona incerta to the primary motor cortex has been suggested (Jackson & Crossman, 1981; Ricardo, 1981; Roger & Cadusseau, 1985; Shammah—Lagnado et al., 1985). Zona incerta may be involved in the escape component of female-paced sexual behavior. Electrical stimulation of zona incerta in cats leads to typical escape responses (Kaelber & Smith, 1979); moreover, zona incertal units in the cat can be driven by noxious stimuli over wide body regions, whereas monkeys will work to terminate electrical stimulation of zona incerta (Berkley, 1980). These functional observations suggest the involvement of the zona incerta in nociceptive behavior which is, in part, important during copulation. Indeed, several structures which serve as sources of zona incertal afferents have been implicated in pain mechanisms. These structures include the periaqueductal gray substance, the trigeminal sensory complex, the nucleus reticularis 113 gigantocellularis, the nucleus raphe magnus and dorsalis, the pretectum, the superior colliculus and the reticular nucleus of the thalamus (Shammah-Lagnado et al., 1985). The zona incerta may also be involved in the motivational component of female- paced sexual behavior. Apparently, the diffusely organized projection from zona incerta to the cerebral cortex and spinal cord may play a role in the general arousal associated with a variety of motivated behaviors, such as self-stimulation and sexual behavior (K6hler et al., 1984). Indeed, afferent projections to points of self-stimulation in the medial prefrontal cortex of the rat include the zona incerta (Vives et al., 1983). Subthalamic lesions centered on the caudal zona incerta just dorsal to the subthalamic nucleus eliminated sexual behavior in males (Maillard & Edwards, 1988). Subthalamic lesions produced no obvious impairment in locomotion, posture, limb use, muscle tone or sensory motor orientation. Even so, the fact that electrical stimulation of the subthalamus elicits coordinated stepping suggests that the region is linked with systems directly concerned with movement and locomotion. These links could be particularly important in the process by which sexual motivation is translated into sexual behavior (Maillard & Edwards, 1988). In addition, the zona incerta may be involved in the initiation of female- paced sexual behavior since it is involved in the initiation of locomotor activity (Milner & Mogenson, 1988) and the generation of orienting movements (Kim et al., 1992). A13 Dopaminergic neurons with perikarya (Dahlstrom & Fuxe, 1964) located in the rostral portion of the medial zona incerta have been implicated in a variety of functions including the regulation of gonadotropin secretion (Wilkes et al., 1979; MacKenzie et al., 1984; James et al., 1987; Sanghera et al., 1991). Indeed, the extensive projections of A13 dopaminergic neurons were identified in hypothalamic areas 114 important for gonadotropin secretion (Wagner et al., 1995). The Medial Preoptic Nucleus and Female-paced Sexual Behavior The medial preoptic nucleus acts as a coordinating unit for controlling sexual behavior and reproduction in the hypothesis. It plays an important neuroendocrine role since it has extensive reciprocal connections to almost all parts of the periventricular zone of the hypothalamus, including the anteroventral periventricular nucleus, anterior part of the periventricular, paraventricular and arcuate nuclei, and contains receptors for estrogen, progesterone and testosterone. Its involvement in the autonomic mechanisms is indicated by the connections to dorsal and lateral parvicellular parts of the paraventricular nucleus, the parabrachial nucleus, and the nucleus of the solitary tract. Other connections of the medial preoptic nucleus suggest participation in the initiation of specific motivated behaviors. For example, connections to the ventromedial and dorsomedial hypothalamic nuclei may be related to the control of reproductive and ingestive behaviors, respectively. The execution of these behaviors may involve activation of somatomotor regions via connections to the substantia innominata, zona incerta, ventral tegmental area, and pedunculopontine nucleus. Connections to other regions that project directly to the spinal cord, such as the periaqueductal gray, the laterodorsal tegmental nucleus, certain medullary raphe nuclei and the magnocellular reticular nucleus may also be involved in modulating somatic and/or automonic reflexes. Finally, the medial preoptic nucleus may influence a wide variety of physiological mechanisms and behaviors (including female sexual behavior) through its massive connections to the ventral part of the lateral septal nucleus, the bed nucleus of the stria 115 terminalis, the lateral hypothalamic area, the supramammillary nucleus, and the ventral tegmental area, all of which have extensive connections with regions along the medial forebrain bundle (Simerly & Swanson, 1988). Future Research Directions To better understand the mechanisms of PCB toxicity in the dopaminergic neurons during development, pure PCB congeners should be used. The comparison between pure coplanar and ortho-substituted PCBS is especially important since they may have diverse effects. The antagonistic or synergistic effects between coplanar and ortho- substituted PCBS are of equal significance. To determine the critical period, prenatal, neonatal, adult as well as perinatal treatments are equally important since the same compound may have different effects in each developmental stage because of different sets of molecules and different cellular environment triggered in each developmental step may result in various outcomes. The effect of PCBs on A14 dopaminergic neurons should be examined because of their vicinity to the MPOA and possible sexual dimorphism of these dopaminergic neurons. A12 dopaminergic neurons should be examined as well because of its involvement in the control of pituitary function. The effect of PCBs on A9 and A10 dopaminergic systems and their relation to sexual behavior are also worth tackling. The efferent areas of A9 and A10, the striatum and nucleus accumbens, are especially important since they are involved in female paced sexual behavior and the effect of PCBS on these sites may be related to the behavioral changes. The ontogeny of the incertohypothalamic dopaminergic system is virtually unknown. Its involvement in the sexual differentiation and the control of sexual behavior 116 needs further investigation. The sex difference in the susceptibility of this system to PCBS is also interesting. The sex difference in the induction of TH-IR in the incertohypothalamic dopaminergic system by copulation is an exciting finding that calls for further investigation. Finally, the suggestion that the SDN is a dynamic, context- dependent nucleus needs further examination and testing. 117 APPENDIX 118 Table 1. The effect of perinatal Aroclor 1221 (A1221) treatment on female sexual behavior and All or A13 dopaminergic neurons. Control A1221 A1221 (sesame oil) (5 mg) (15 mg) Approach Latency 759410.983 22.7l9i7.149b 3.7930568“ (sec) (n=8) (n=8) (n=6) Mount Return Latency 12.375i2.568 15.363i1.959 8.067:1:0.772 (sec) (n=8) (n=8) (n=6) Intromission Return 18.950i2.670 36.938i8.772a 11.133zt1.602c Latency (sec) (n=8) (n=8) (n=6) Postejaculatory 58.350:t13.900 85.238i13.014 61.983i9.88l Refractory Period (sec) (n=8) (n=8) (n=6) Percentage of Mount 36.676i4.824 3562816615 27.736i5.34l Leave (n=8) (n=8) (n=6) Percentage of 5504017129 73.104dz2.754a 49.466zt4.554° Intromission Leave (n=8) (n=8) (n=6) Lordosis Quotient 83. 12515083 4812518014" 4416711535? (n=8) (n=8) (n=6) A1 1 TH-IR Neurons 1020.6i101.0 (n=5) 1274.0:t91.l 876.0i105.0 (total) Q1=3) (n=5) All TH-IR Neurons 545.4i52.2 686.0:t16.5 46441580 (major) (n=5) (n=3) (n=5) A1 1 TH-IR Neurons 475.2i52.6 588.0:h74.8 411.6:t48.9 (minor) (n=5) (n=3) (n=5) A13 TH-IR Neurons 2898.0002t4l9.006 2321.000i435.087 2409.600:l:l99.082 (total) (n=5) (n=3) (n=5) A13 TH-IR Neurons 1609.200dz233.566 13020001211544 1302.600rh132.308 (major) (n=5) (n=3) (n=5) A13 TH-IR Neurons 1288.800i189.011 1019.000i224.007 1107.000i76.703 (minor) (n=5) (n=3) (n=5) Data were represented as meaniSE 3: significantly different from control (p<0.05) b: significantly different from control (p<0.01) c: significantly different from 5 mg group (p<0.01) TH-IR: tyrosine hydroxylase immunoreactive 119 Table 2. The effect of perinatal Aroclor 1254 (A1254) treatment on female sexual behavior and A1 1 or A13 dopaminergic neurons. Control A1254 A1254 (sesame oil) (5 mg) (15 mg) Approach Latency 7.5942t0.983 6.583i0.823 7.3332t1.794 (sec) (n=8) (n=6) (n=5) Mount Return 12.375i2.568 11.350i2.010 9.880dz2.805 Latency (sec) (n=8) (n=6) (n=5) Intromission Return 18.950i2.670 27.433i4.167 17.000i3.044 Latency (sec) (n=8) (n=6) (n=5) Postejaculatory 58.350:I:I3.900 74.883d:11.858 80.000i11.312 Refractory Period (n=8) (n=6) (n=5) (sec) Percentage of Mount 36.676i4.824 57.250i3.880al 55.763i8.437a Leave (n=8) (n=6) (n=5) Percentage of 55.0402t7.129 77.873dz4.771b 77.701zt4.990b Intromission Leave (n=8) (n=6) jn=5) Lordosis Quotient 83.125d:5.083 78.333i6.412 92.000i2.550 01:8) (F61 (n=5) A1 1 TH-IR Neurons 1020.600:!:101.049 N.D. 835.500i252.813 (total) (n=5) (n=4) A1 1 TH-IR Neurons 545400152207 N.D. 451.500i121.814 (major) (n=5) (n=4) A11 TH-IR Neurons 475.200zt52.604 N.D. 411.600i48.886 (minor) (n=5) (n=lD A13 TH-IR Neurons 2898.000i419.006 N.D. 2488.500i560.727 (total) (n=5) (n=4) A13 TH-IR Neurons 1609.200i233.566 N.D. 1353.750i330.262 (major) (n=5) (n=4) A13 TH-IR Neurons 1288.800i189.01 1 ND. 1134.750i241.344 (minor) (n=5) (n=4) Data were represented as meaniSE. N.D.: not determined a: significantly different from control (p<0.05) b: significantly different from control (p<0.01) TH-IR: tyrosine hydroxylase immunoreactive 120 Table 3. The effect of neonatal Aroclor 1254 (A1254) treatment on female sexual behavior. Control (sesame oil) A1254 (2.5 mg) A1254 (5 mg) Approach Latency 7.754i0.933 960012.968 5.445i0.885 (sec) (n=1 5) (n= 1 5) (n=] 5) Mount Return 9.882i0.960 11.591il.441 12.273zt3.359 Latency (sec) (n=15) (n=15) (n=15) Intromission Return 24.729i5.557 14.469i1.655* 12.863i1.014* Latency (sec) (n=15) (n=15) (n=15) Postej aculatory 52.643:t6.822 43.896i4.676 41 .6352t4.447 Refractory Period (n=15) (n=15) (n=15) (sec) Percentage of 39.353i4.157 40.643i4.584 32.480:t3.920 Mount Leave (n= 1 5) (n= 1 5) (n=15) Percentage of 60.647i4.157 59357224584 67520113920 Mount Stay (n=15) (n=15) (n=15) Percentage of 61.178i4.669 51.8952t4.03l 64.127:t4.422 Interintromission (n=15) (n=15) (n=15) Leave Percentage of 38.822i4.669 48.105i4.031 35.873zt4.422 Interintromission (n=15) (n=15) (n=15) Stay Lordosis Quotient 85.779i2.444 40.223i4.730* 56.444i8.122* (Nonpacing) (n=15) (n=15) (n=15) Lordosis Quotient 84.757i3.652 52.684i5.657** 60.383i5.630** (Pacing) (n=15) (n=15) (n=15) Data were represented as meaniSE. * significantly different from control (p<0.05) ** significantly different from control (p<0.001) 121 Table 4. The effect of neonatal Aroclor 1221 (A1221) treatment on female sexual behavior. Control (sesame oil) A1221 (2.5 mg) A1221 (5 mg) Approach Latency 8.690i1.373 7.474zi:O.857 7.679:t0.776 (sec) (n=14) (n=13) (n=14) Mount Return 11.697i1.788 21.288i9.641 7.606zt1.070 Latency (sec) (n=14) (n=13) (n=14) Intromission Return 18.912i2.503 16.802zt1.581 13.470:1:2.191 Latency (sec) (n=14) (n=13) (n=14) Postej aculatory 43. 143:1:7 .4 l 6 62.077zt6.855 45.63 1i4.792 Refractory Period (n=14) (n=13) (n=14) (sec) Percentage of 30.947i7.583 50.031i6.858 29.181i5.577 Mount Leave (n=14) (n=13) (n=14) Percentage of 6905317583 49.969i6.858 70.819:1:5.577 Mount Stay (n=14) (n=13) (n=14) Percentage of 52.712zt6.091 67.882i6.757 62.976zt5.207 Interintromission (n=14) (n=13) (n=14) Leave Percentage of 47.288i6.091 32.118i6.757 37.024i5.207 Interintromission (n=14) (n=13) (n=14) Stay Lordosis Quotient 81.429i5.108 91.795i2.155 84.286i3.346 (Nonpacing) (n=14) (n=13) (n=14) Lordosis Quotient 74.048i3.697 80.976i2.588 76.976d:3.439 (Pacing) (n=14) (n=13) (n=14) Data were represented as meaniSE. 122 Table 5-1. The effect of adult polychlorinated biphenyl treatment on female sexual behavior. Test Control Aroclor 1254 Aroclor 1254 (sesame oil) (2.5 mg) (5 mg) Approach Latency Pre 383310.548 558311.819 441710.543 (sec) (n=12) (n=12) (n=12) Post 475010.986 355610.818 508311.300 (n=12) (n=9) (n=12) Mount Return Pre 629111.401 1050011734 455010.673 Latency (sec) (n=1 1) (n=10) (n=10) Post 695511.013 981413.394 906411.453 (n=1 1) (n=7) (n=1 1) Intromission Return Pre 720810.768 12.71712.449 10.31711.727 Latency (sec) (n=12) (n=12) (n=12) Post 13.91714.709 23.70014951 1643313097 (n=12) (n=9) (n=12) Postejaculatory Pre 2491716543 3408318670 3483316292 Refractory Period (n=12) (n=12) (n=12) (sec) Post 77917122586 (n=12) 54889114690 71000110392 (n=9) (n=12) Percentage of Mount Pre 3128414781 4687518777 44.70119.401 Leave (n=12) (n=12) (n=12) Post 4859218828 42200115049 5019017940 (n=12) (n=7) (n=12) Percentage of Pre 62.6 1 518.3 85 6346915493 5189916598 Interintromission (n=12) (n=12) (n=12) Leave Post 71 26717246 72.61016. 140 82.12416.103 (n=12) (n=9) (n=12) Lordosis Quotient Pre 7083317732 8000017177 74.16717.120 (Nonpacing) (n=12) (n=12) (n=12) Post 8750014286 7916714994 8083316088 (n=12) (n=12) (n=12) Lordosis Quotient Pre 8208315641 8436412909 79.27313.628 (Pacing) (n=12) (n=1 1) (n=] 1) Post 8325014085 8709116231 8218213858 (n=12) (n=ll) (n=ll) Data were represented as mean1S.E. 123 Table 5-2. The effect of adult polychlorinated biphenyl treatment on female sexual behavior (continued). Control Aroclor 1221 Aroclor 1221 @esame oil) (2.5 mg) (5 mg) Approach Latency (sec) Pre 383310.548 616711.392 583311.272 Q1=12) (n=12) (n=12) Post 475010.986 458311.033 408310.633 (n=12) (n=12) (n=12) Mount Return Latency Pre 629111.401 15.70014992 690911.112 (sec) (n=] 1) (n=10) (n=1 1) Post 6.95 511 .0 1 3 962711.636 770911.258 (n=11) (n=11) (n=11) Intromission Return Pre 720810.768 2100816764 1029112539 Latency (sec) (n=12) (n=12) (n=11) Post 13.91714709 1608314914 1358312990 (n=12) (n=12) (n=12) Postejaculatory Pre 2491716543 3000019823 3241715562 Refractory Period (sec) (n=12) (n=12) (n=12) Post 77.917122586 52273112857 47250111058 (n= 1 2) (n=1 1) (n=12) Percentage of Mount Pre 3128414781 44789110365 39.41717.309 Leave (n=12) (n=1 1) (n=12) Post 4859218828 56717110023 49.47815.120 (n=12) (n=12) (n=1 1) Percentage of Pre 62.615183 85 5549217680 5442519300 Interintromission Leave (n=12) (n=12) (n=12) Post 71.26717246 786201555 5 7050216954 (n=12) (n=12) (n=12) Lordosis Quotient Pre 7083317732 7666715551 7916716450 (Nonpacing) (n=12) (n=12) (n=12) Post 8750014286 7500016455 8000016629 Q=Q (n=12) (n=12) Lordosis Quotient Pre 8208315641 8200013238 7158314458 (Pacing) (n=12L (n=12) (n=12) Post 8325014085 8358315610 7850015479 (n=12) (n=12) (n=12) Data were represented as mean1S.E. 124 Table 6. The effect of neonatal Aroclor 1221 (A1221) treatment on All and A13 dopaminergic neurons. Control (sesame oil) A1221 (2.5 mg) A1221 (5 mg) A1 1 TH-IR Neurons 9031138818 10864159351 8613331256794 00ml) (n=4) (n=5) 01:30 All TH-IR Neurons 483000169902 596800147521 4613331138974 (major) (n=4) (n=5) (n=3) All TH-IR Neurons 435000157093 489600117463 4000001118006 (minor) (n=4) (n=5) (n=3) All Asymmetric 1.11010.044 1.21910.081 l.l4310.041 Indexl (n=4) (n=5) (n=3) A1 1 FOS/TH-IR 97000128862 82400116570 178667152679 Neurons (major) @=4) (n=5) (n=3) All FOS/TH-IR 81.000115610 105600126126 162667168521 Neurons (minor) (n=4) (n=5) (n=3) All Asymmetric 1.16510.170 099810.264 140310.489 Index 2 (n=4) (n=5) (n=3) Ratio of All 1918213283 1475413910 45327114044 FOS/TH-IR (major) (n=4) (n=5) (n=3) Ratio of All 1826511653 21.28014958 42353118383 F OS/T H-IR (minor) (n=4) (n=5) (n=3) A13 TH-IR Neurons 18550001324433 17904001168236 14746671485443 (total) (n=4) (n=5) (n=3) A13 TH-IR Neurons 10750001180742 12144001163387 8866671266827 (major) (n=4) (n=5) (n=3) A13 TH-IR Neurons 7800001158114 576000172078 5880001223762 (minor) Q1=4) (n=5L (n=3) A13 Asymmetric 1.41510.l42 2.21810339 1.69110357 Indexl (n=4) (n=5) (n=3) A13 F OS/T H-IR 275000148232 230400121858 188000170086 Neurons (major) (n=4) (n=5) (n=3) A13 FOS/TH-IR 188000151562 123200117591 182667149817 Neurons (minor) (n=4) (n=5) (n=3) A13 Asymmetric 163810.241 194010.158 107710.258 Index 2 (n=4) (n=5L (n=3) Ratio of A13 2569312052 1991012505 2038011388 F OS/TH-IR (major) (n=4) (n=5) (n=3) Ratio ofAl3 2303512465 2132811576 3381315747 FOS/TH-IR (minor) (n=4) (n=5) (n=3) Data were represented as mean1S.E. Asymmetric Index 1 = TH-IR Neurons (Major) / TH-IR Neurons (Minor) Asymmetric Index 2 = [F OS/T H-IR Neurons (Major)] / [F OS/T H-IR Neurons (Minor)] TH-IR: tyrosine hydroxylase immunoreactive Table 7. The effect of neonatal Aroclor 1254 (A1254) treatment on All and A13 dopaminergic neurons. Control (sesame oil) A1254 (2.5 mg) A1254 (5 mg) A11 TH-IR Neurons 6090001129207 6880001115703 6432001153745 (Total) (n=4) (n=5) (n=5) A1 1 TH-IR Neurons 38900016191 1* 389600158468 358400179162 (Major) (n=4) (n=5) (n=5L A11 TH-IR Neurons 220000167469 298400161013 284800175274 (Minor) (n=4) 4n=5) (n=5) A11 Asymmetric 211110.443 1.43210.217 167310.459 Index 1 (n=4) (n=5) (n=5) A11 FOS/TH-IR 58000113216“ 60000123152 61600112998 Neurons (Major) (n=4) (n=5) (n=5) A1 IFOS/TH-IR 26000111372 46400113775 41600110852 Neurons (Minor) (n=4) (n=5) (n=5) A11 Asymmetric 208110.504 209411.000 165210.208 Index 2 (n=4) (n=5) (n=5) Ratio ofAll 1460011257 1673217856 17.24611635 FOS/TH-IR (Major) E4) (n=5) (n=5) Ratio of All 1446518881 1409113693 1674412092 F OS/T H-IR (Minor) (n=4) (n=5) (n=5) A13 TH-IR Neurons 12140001200749 10960001212119 12580001362018 (Total) (n=4) (n=4) (n=4) A13 TH-IR Neurons 9750001227569“ 641000182662 7210001203098 (Major) (n=4) (nfl 01:3 A13 TH-IR Neurons 239000132099 4550001130705 5430001158420 (Minor) (n=4) (n=4) (n=4) A13 Asymmetric 468711.741 200410.724 136310.113 Index 1 (n=4) (n=4) (n=4) A13 FOS/TH-IR 256000128752“ 81000120290 251000167278 Neurons (Maj or) (n=4) (n=4) (n=4) A13 FOS/TH-IR 81 .00016. 191 74000142411 138000136679 Neurons (Minor) (n=4) (n=4) (n=4) A13 Asymmetric 325110.508 180710.510 184510.135 Index 2 (n=4) (n=4) (n=4) Ratio of A13 2860214197 1418215056 35.13812872 FOS/TH-IR (Major) (n=4) (n=4) (n=4) Ratio of A13 3485012783 23823112409 25.93312206 F OS/T H-IR (Minor) (n=4) (n=4) (n=4) Data were represented as mean1S.E. Asymmetric Index 1 = TH-IR Neurons (Major) / TH-IR Neurons (Minor) Asymmetric Index 2 = [F OS/T H—IR Neurons (Major)] / [FOS/TH-IR Neurons (Minor)] The major side is significantly different from the minor side (p<0.05) TH-IR: tyrosine hydroxylase immunoreactive 126 Table 8. Time course of A13 F OS and tyrosine hydroxylase immunoreactivities. Treatment No. of TH-IR No. of TH-IR No. of FOS/TH- No. of F OS/T H- cells (major) cells (minor) IR cells (major) IR cells (minor) Control 453 1 42 408 1 47 52 1 9 59 1 14 (n=4) FP-lO min 977 1 17ac 8281 40° 155 1 132‘ 149 1 42" (FD FP-3O min 7391101bc 6411108be 109111” 95116bc (n=6) FP-l h 116711333 105011348 1731303 1711258| (n=5) MP-lO 832 1 42c 696 1 37c 85 1 35bcd 65 1 9bed min n=3) MP-3O 899 1 120ac 744 1 148° 103 1 8be 105 1 9bc min Q=6) MP-l h 14211247a 12871216a 158117“ 175122a (n=4) Data were represented as mean1S.E. a: significantly different from the control (p<0.05) b: significantly different from F P-l h (p<0.05) c: significantly different from MP-l h (p<0.05) d: significantly different from F P-lO min (p<0.05) FP: F emale-paced MP: Male-paced TH-IR: tyrosine hydroxylase immunoreactive 127 Table 9. Time course of A11 F OS and tyrosine hydroxylase immunoreactivities. Treatment No. of TH-IR No. of TH-IR No. of F OS/T H- No. of FOS/TH- cells (major) cells (minor) IR cells (major) IR cells (minor) Control 575 1 45 441 1 50 118 1 20 98 1 28 (n=4) FP-lO min 689 1 19 645 1 20 172 1 10° 149 1 24 (11:3) FP-30 min 527 1 30 469 1 15 a 73 1 15‘” 63 1 20‘” (n=6) FP-l h 716149 616194 2381393 206153 111:5) MP-lO 684145 637129 101117‘” 84117° min 61=3) MP-3O 563 1 50 456 1 73 69 1 16‘” 43 1 13‘” min (n=6) MP-l h 677 1 106 567 1 77 334 1 96a 284 1 873 (11:4) Data were represented as mean1S.E. a: significantly different from the control (p<0.05) ": significantly different from FP-lh (p<0.05) °: significantly different from MP-lh (p<0.05) d: significantly different from FP-10 min (p<0.05) F P: Female-paced MP: Male-paced TH-IR: tyrosine hydroxylase immunoreactive 128 Table 10. Time course of F OS immonoreactive cells in the medial preoptic nucleus. Treatment Plate 20 Plate 21 Sum of Plate 18 to Plate 22 Control 74 1 2 311 1 25 758 1 85 QI=3) FP-lO min 204 1 65° 421 1 97° 1144 1 242° (n=5) FP-3O min 254 1 68ad 681 1 133ad 1717 1 301ad (n=3) FP-l h 267 1 37a 471 1 52 1369 1 108 (n=8) MP-lO 116 1 12‘” 353 1 57° 928 1128‘” min (n=6) MP-30 196 1 52° 468 1 54 1410 1 307 min (n=3) MP-l h 323 1131‘ 686183” 18151133“ (n=8) Data were represented as mean1S.E. 8: significantly different from the control (p<0.05) ": significantly different from FP-l h (p<0.05) °: significantly different from MP-l h (p<0.05) d: significantly different from MP-lO min (p<0.05) FP: F emale-paced MP: Male-paced 129 Table 11. All FOS and tyrosine hydroxylase immunoreactivities under various sexual stimulation. Treatment No. of TH-IR No. of TH-IR No. of F OS/T H- No. of FOS/TH- cells (major) cells (minor) IR cells (major) IR cells (minor) Control 5751 45 441 1 50 118 1 20 98 1 28 (n=4) FP-lE 689119“ 645120 172110 149124 (n=3) FP-2E 621191b 5391100 1641101 1531118 (n=3) FP-3E 539146" 516155 136178 111163 (n=3) FP-4E 595 1 61b 557 1 52 128 1 37 129 1 39 (n=3) MP-lE 684145" 637129 101117 84117 (n=3) MP-ZE 6291 382‘ 564 1 23 52 1 13 41 1 16 (n=3) MP-3E 535151b 4191110 123110 79122 (n=3) MP-4E 6151123a 4131113 2241107 162193 (n=4) Data were represented as mean1$.E. °: significantly different from the control (p<0.05) b: significantly different from MP-4E (p<0.05) E: ejaculatory series FP: F emale-paced MP: Male-paced TH-IR: tyrosine hydroxylase immunoreactive 130 Table 12. A13 F OS and tyrosine hydroxylase immunoreactivities under various sexual stimulation. Treatment No. of TH-IR No. of TH-IR No. of F OS/T H- No. of F OS/T H- cells (major) cells (minor) IR cells (major) IR cells (minor) Control 453 1 42 408 1 47 52 1 9 59 1 14 (11:4) FP-lE 977117 828140 155113 149142 QI=3J FP-2E 8471195 7521221 11917 112122 (n=3) FP-3E 8771217 7691214 127131 91124 (n=3) FP-4E 892 1 255 804 1 223 159 1 59 167 1 55 (11:3) MP-lE 832 1 42b 696 1 37 85 1 35 65 1 9 (n=3) MP-2E 10631176zal 96712172' 11716 12115 (n=3) MP-3E 640 1 22" 504 1 98" 120 1 37 133 1 45 (11:3) MP-4E 135312033 111312213 141122 139123 01:41 Data were represented as mean18.E. 3: significantly different from the control (p<0.05) b: significantly different from MP-4E (p<0.05) E: ejaculatory series F P: Female-paced MP: Male-paced TH-IR: tyrosine hydroxylase immunoreactive 131 1r Table 13. FOS-immunoreactive cells in the medial preoptic nucleus under various sexual stimulation. Treatment Plate 20 Plate 21 Plate 22 Sum of Plate 18 to Plate 22 Control 74 1 2 311 1 25 282 1 42 758 1 85 (n=3) FP-lE 204 1 65 421 1 97"c 309 1 64” 1144 1 242‘” (n=51 FP—ZE 228 1 47 402 1 78” 350 1 135” 1237 1 152” (n=4) FP-3E 287 1 101"d 544 1 61 258 1 25‘” 1456 1 204 $3) FP-4E 320 1 4"d 714 1 120"1 618 1 86a 1948 1 204a (n=2 MP-IE 116112"c 353157"c 240151"c 9281128bc (n=6) MP-2E 176 1 33 468 1 54 297 1 63” 1185 1 90"c (n=3) MP-3E 255 1 16"d 559 1 58 352 1 70” 1608 1 288ad (n=3) MP-4E 3111211! 72711513 6741923 19681206" (n=4) Data were represented as mean1S.E. 2': significantly different from the control (p<0.05) b: significantly different from F P-4E (p<0.05) °: significantly different from MP-4E (p<0.05) d: significantly different from MP-IE (p<0.05) E: ejaculatory series F P: Female-paced MP: Male-paced 132 Table 14. The effect of perinatal Aroclor 1254 (A1254) treatment on male sexual behavior. Control A1254 A1254 (sesame oil) (5 mg) (15 mg) Mount Latency (sec) 1183613675 1088012868 1583815492 (n=14) (n=5) (n=8) Intromission Latency 2266415572 1978013428 53.963120.445 (sec) (n=14) (n=5) (n=8) Ejaculation Latency 435421148348 560360179972 5129251109679 (sec) (n=14) (n=5) (n=8) Postejaculatory 322200115059 338720118857 359850121460 Interval (sec) (n=14) (n=5) (n=8) Mount Frequency 1395711538 1236011836 1468813605 (n=14) (n=5) (n=8) Intromission 13.97111 . 121 1216010851 1357511073 F requengy (n=14) (n=5) (n=8) Inter-intromission 3177112496 4960015022" 3907516717 Interval (sec) (n=14) (n=5) (n=8) Intromission Rate 207310.144 134710.081“ 202610.365 (min'l) (n=14) (n=5) (n=8) Data were represented as mean1S.E. significantly different from control (p<0.05) 133 Table 15. The effect of neonatal Aroclor 1254 (A1254) treatment on male sexual behavior. Control A1254 A1254 (sesame 011) (2.5 mg) (5 mg) Mount Latency (sec) 976011.806“ 950011.867” 17.43813.838 (n=32) (n=32) (n=32) Intromission Latency (sec) 45333117440 28031111530 3275019389 (n=32) (n=32) @=32) Ejaculation Latency (sec) 493021138194 489771151251 401750130274 Q1=32L (n=32) (n=32) Postejaculatory Interval (sec) 388250122003 365667120683 30245019116 (n=12) (n=12) (n=2(D Mount Frequency 1 1.87511 .1 13 1559412590 862510.962 (n=3 2) (n=3 2) (n=32) Intromission Frequency 976011.806 950011.867 17.43813.838 (n=32) (n=32) (n=32) Inter-intromission Interval 1225010687 1235710891 1091710802 (sec) Q=28) (n=28) (n=12) Intromission Rate (min‘) 198610.164 225410.301 169810.147 (n=24) (n=20) (n=12) Data were represented as mean18.E. * significantly different from A1254 5 mg group (p<0.05) 134 Table 16. There is no effect of neonatal Aroclor 1221 (A1221) treatment on male sexual behavior. Control A122] A122] Untreated (sesame oil) (2.5 mg) (5 mg) Mount Latency (sec) 17.0163 10.4116 9.9113 3.810.] (n=12) (n=12) (n=12) (n=21) Intromission Latency 55.11220 19.7139 46.81215 15.1146 (see) (n=12) (n=12) (n=12) (n=21) Ejaculation Latency 542.71 109.4 41351666 578311360 480.41667 (sec) (n=12) (n=12) (n=11) (n=21) Postejaculatory Interval 37831252 328.4111.9 374.0 131.2 355.2111.4 (sec) (n=12) (n=12) (n=11) (n=21) Mount Frequency 15913.0 12.8135 16.1144 11.4120 (n=12) (n=12) (n=11) (n=2 1) Intromission Frequency 11.111.1 11210.9 12410.8 10.7106 (n=12) (n=12) (n=11) (n=21) Inter-intromission Interval 48.7182 38.5167 48. 1113.4 43.8145 (see) (n=12) (n=12) (n=11) (n=21) Intromission Rate (min'l) 1610.2 2210.3 1.9102 1.710.] (n=12) (n=12) (n=] 1) (n=21) Hit Rate 0.4100 0.510.] 0510.0 0610.0 (n=12) Jn=12) (n=11) (n=21) Data were represented as mean1S.E. Note: The data of untreated rats are included but cannot be used to compare with A1221- treated rats because of different breeding condition and homogeneity (F test p<0.0001). 135 Table 17. There is no effect of neonatal Aroclor 1221 (A1221) treatment on the cell number of the major pelvic ganglia. Control A122] (2.5 mg) A1221 (5 mg) Untreated (sesame oil) Male 1078331940 130386111024 120840115466 11430.51 616.9 (n=6) (n=7) (n=5) Q1=2 1) Female 3251713045 6244011791.] 3708.01 442.3 2938311862 (n=6) (n=5) (n=5) (n=8) Data were represented as mean1S.E. Note: The data of untreated rats are included but cannot be used to compare with A1221- treated rats because of different homogeneity (F test p<0.0001). l3 6 REFERENCES 137 REFERENCES Adkins-Regan, E; Mansukhani, V; Thompson, R; Yang, Sharlene: Organizational actions of sex hormones on sexual partner preference. Brain Res. Bull. 44(4): 497-502, 1997. 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