NIHIHIHII IIHHII 122 120 .THS_ THESXS .- _‘wwm9 -_ “iv II b. . EIBRARY Michigan State Univemay ‘.-'U- 'H‘ - v .— W W "V ww— This is to certify that the thesis entitled Male Sexual Behavior in Deermice (Peromyscus Maniculatus) Following Castration and Hormone Replacement presented by S teven M. Pomerantz has been accepted towards fulfillment of the requirements for M.A. degree in Psychology ’3 // x ./ / ,4 4 ' " - ' 7 :55J’g'm (M2 LA-lyxc' ’ Major professor Date // 415/, 30 0-7639 OVERDUE FINES: . ‘ 25¢ per day per item 1; Kg}. ‘ RETURNING LIBRARY MATERIALS: P g. N 4 In Place in book return to rent charge from circulation rec: ;; flaw”! J ummm MALE SEXUAL BEHAVIOR IN DEERMICE (PEROMYSCUS MANICULATUS) FOLLOWING CASTRATION AND HORMONE REPLACEMENT By Steven M. Pomerantz A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF ARTS Department of Psychology 1980 ABSTRACT MALE SEXUAL BEHAVIOR IN DEERMICE (PERMSCUS MANICUIA‘IUS) POILOWDG CASTRATIW AND WE REPIACEMENI‘ By Steven M. Pomerantz The present study investigated the role of gonadal hormones in mediating male copulatory behavior in male deerndce (Perouyscus neniculatus) . Sexually experienced male deermice were castrated and tested for male sexual behavior. In the weeks following cas- tration male sexual behavior decreased. Ejaculation disappeared first, followed by intromission and, finally, munting. Castrated males who no longer copulated were assigned to one of four treat- ment groups: 200 ug testosterone propionate ('IP); 200 ug dihydro- testosterone propionate CDHTP); 2 ug estradiol benzoate (EB); or sesame oil (OIL). TP and DHTP were both effective in restoring the couplets male sexual behavior pattern. In contrast, EB was effective in inducing ununting and minimally effective in inducing intranissions (vaginal penetration), but did not induce any ejacu- latory responses. These data indicate that in deerndce there is a greater likelihood that testosterone may mediate male sexual behavior through reduction to dihydrotestosterone than through aromatization to estradiol. .I offer many thanks to Dr. Lynwood G. Clemens for his enthusiastic support and advice during this research project. I also wish to thank Dr. Lauren J. Harris, Dr. John I. Johnson, Jr., and Dr. John A. King for their thoughtful comments and suggestions. I also acknowledge the helpful editorial assistance made by Gary Dohanich, Steven ($1th, and Nancy Secor in preparing this manuscript. ' Finally, I affectionately recognize my parents for their encouragement of my academic ambitions . ii TABLE OF CWTENTS LIST OF F IGURES O O O O O O O O O O O O O O O O O O O O O 0 LIST OF WES INTRODUCTION EffectsofCastration . Restorative Effects of Testosterone . . . . . . Biochemistry of Testosterone Metabolism . . . . Effects of Dihydrotestosterone in the Male Rat . Importance of. Aromatization of. Androgens in the Vale Rat Combined Action of Estrogen and Dihydrotestosterone in theMaleRat...................... Comparative Studies of I-brmonal Mediation of. Male Sexual \lU‘leH H (D Behavior....... BEERIMM 1 I O O O O O O O O O O O O O O O O O O O O O O O MMOD O O O O O O O O O O O O O O O O O O O O O O O O O SUbjxts O O O O O O O O O O O I O O O O I O O O 0 O Apmratus O O O O O O O O O O O O O O O O O O O O O O PrOC$ure O O O O O O O O O O O O O O O O I O O O O O masures O O O O O O O O O O O O O O O O O O O O O O mta malWis O O O O O O O O O O O O O O O 0 O O O O REULTS O O O O O O O O O O O O 0 O O O O O O I O O O O EWERIMM 2 O O O 0 O O O O O O O O O O O O O O O O O O O O MEIHOD O O O O O O O O O O O O O O O O O O O O O O O O 0 Subject Assignment and Hormone Treatment . . . . . . Precame O O O O O O O O O I O O O O O O O O O O O O mta malfiis O O O O O O O O O O O O O O O O O O O 0 RESULTS 0 O O O O O O O O O O O O O O O O I O O O O O O DISCLBSIW O O O O O O O O O O O O O O O O O O O O O O O O 0 LIST OF REFERENCES 0 O O O O O O O O O O O O O O O O O O O O 10 15 15 16 16 18 19 24 24 24 25 25 26 30 35 LIST OF FIGURES Figure Page 1 Percentage of. male deemice maintaining momting, intromission and ejaculatory responses on successive we“ EOIIOWim castration O O O O O O I O I O O O O O O 20 Percentage of. castrated male deermice exhibiting mounting intromission and ejaculation on successive weeks after onset of daily hormone treatment with 206 ug 'I'P,200ungP,2ugEBorsesameOIL . . . . . . . . .27 iv Table LIST OF TABLES Page Hormonal Mediation of. Male Sexual Behavior in Mammalian Species Other Than Pattus Norvegicus . . . . . . . . . . 11 Effects of. Castration on Male Oopulatory Performance . . 21 Comparison of. Oopulatory Performance Among Castrated Male Deermice Before and After the Disappearance of fixulation 0 O O O O O O O O O O O O O O O O O O O O O O 23 Effects of. Hormones on Mating Performance of. Castrated Wle memice O O O O O O O O O O O O O O O O I O O O O 029 INTRODUCTION Male sexual behavior is influenced by the endogenous secretion of testicular hormones. Traditionally, a basic methodological approach has been employed men characterizing the role of gonadal hormones in mediating male copulatory behavior. The first step involves removing the testes and observing the effects on the physiology and behavior of the animal. 'lhe next step is to attempt to comteract the effects of castration through the administration of appropriate gonadal hormones. Finally, the last and most difficult step is to determine the sites and mechanism of action by which testicular hormones are having their facilitative effects. Effects of Castration Castration of males in all mammal ian species that have been sttriied results in a decreased frequency of copulatory behavior; however, the rate and degree to which specific components (i.e. mounting, intromis- sion and ejaculation) of the sexual response decline varies among different species. In some species following gonadectomy the intromis- sion and ejaculation responses are lost simultaneously, while in others intromission is maintained for some time after the animal is no longer ejaculating. In all species that have been studied mounting responses and other precopulatory behaviors (i.e. anogenital sniffing, touching, vocalizing, etc.) are the last components of the male sexual response to decline (reviewed in Larsson, 1979). In addition to castration resulting in deterioration or disappearance of male sexual activity, investigators have also noted other effects on the mating performance of males continuing to copulate. In castrated male rats gonadectomy'produced an increase over preoperative levels in true to first intromission (intromission latency) and tnme from first intromission to ejaculation (ejaculation latency) (Davidson, 1966). Fewer intromissions were required for ejaculation (Davidson, 1966). Also, in both rats and hamsters the frequency of momting (without vaginal insertion) increased for a short time following gonadectomy (Beach and Pauker, 1949; Davidson, 1966). Several morphological changes in the penis of the castrated male parallel his decline in sexual responsiveness. There is a decrease in weight and size of the glans and, also a decrease in number of epithe- lial papillae "penile spines" (Peder, 1971; Phoenix, Gopenhaver, and Brenner, 1976). Whether these morphological changes cause functional disturbances in sexual behavior is not known. However, peripheral atrophy'appears to be related to the deficits seen in copulatory perfor- mance (increase in momts without intromission) . Restorative Effects of Testosterone In all species that have been stLriied, male sexual behavior of castrated males can be restored by testosterone (T) treatment in a dose dependent manner (guinea pig, Grmt and Young, 1953; rat, Beach and Holtz-Tuoker, 1949; Larsson, 1966; hamster, Tiefer and Johnson, 1973; albino mouse, Luttge and Hall, 1973; Champlin, Blight, and M26111, 1963; rabbit, Beyer and Rivaud, 1973; red deer, Lincoln, Guiness, and Snort, 1972; rhesus monkey, Phoenix, Slob, and Soy, 1973) . It has generally been reported that rehabilitative effects of T on copulatory behavior begin to appear after several days of treatment. Mounting behavior returns before intromission and ejaculation. However, in.some long-tenm castrated rats mounting and intromission were restored 24 hr after initial T treatment (Beyer, mbra1i, Naftolin, Larsson, and Perez- Palacios, 1976) and penile reflexes were restored within 8 hr after initial T‘ treatment (Hart, 1980). Many sttriies have investigated whether T can restore sexual behavior of castrated males to intact levels. In mice (Cnamplin et a1., 1963) , guinea pigs (Grmt and Young, 1952; 1953) and hamsters (Beach and Pauker, 1949; Tiefer and Johnson, 1973; Whalen and DeBold, 1974) the mating perfbrmance of castrated males receiving T'was no different from intact males. In rhesus monkeys and rats differences in mating perfor- mance between intact and Tetreated castrated males have not consistently been reported. Same studies report deficits in the Tetreated castrates (Beach, 1944; Beach and Ebltz-Tucker, 1949; Larsson, 1966; Michael and Mfilson, 1974), whereas others report.a complete restoration of sexual behavior to preoperative levels (Peder, 1971; Phoenix et a1., 1973; malen and Luttge, 1971). Biochemistry of Testosterone Metabolism It has been demonstrated in a number of mammalian species with both in vivo and in vitro stuiies that cells in the central nervous system and genital tissues possess specific enzymes which metabolize T to various other steroid hormones. Specifically two major metabolic pathways are: (1) metabolism by aromatase enzymes (aromatization) resulting in the production of estrogen (Callard, Petro, and Ryan, 1978; Dorfman and mgar, 1965; Flores, Naftolin, and Ryan, 1973, Lieberburg and When, 1975; Naftolin, Ryan, Davies, Raddy, Flores, Petro, Kuhn, Write, T‘akooda, and Vblin, 1975; Weisz and Gibbs, 1974) and (2) metabo- lism by Sat-reductase enzymes (Sn-reduction) leading to the production of dihydrotestosterone (DI-IT) and other Sat-reduced androgens (Bruchovsky and Wilson, 1968; Jaffe, 1969; Massa, Justo, and Martini, 1975; Sholiton, Taylor and Lewis, 1974; Sholl, Robinson, and (by, 1975; Whalen and Rezek, 1972) . Currently, it is believed that in mammals II-IT is incapa- ble of being converted to E and vice versa. The potential importance of Q8 metabolism of T is underscored by reports that the extent of aromatization and Sam-reduction varies among different discrete hypothalamic and limbic brain regions (Selmanoff, Brodkin, Weiner, and Siiteri, 1977; Sheridan, 1979). Furthermore, in these same anatomical locations biochemical and autoradiographic stuiies have revealed the existence of specific E and DHT binding macromole- cules, hypothesized receptor sites (Kato and mouchi,1975; Lieberburg and heaven, 1977; Pfaff and Keiner, 1973; Bar and Stumpf, 1977; Zigmond and McEMen, 197D) . Effects of Dihydrotestosterone in the Male Rat mports that DI-IT is more potent than T in promoting cell prolifera- tion and growth of genital tissues (Dorfman and Shipley, 1956; Luttge and malen, 1970) have led to the currently accepted notion that T exerts its stimulatory action on peripheral reproductive tissues through its intracellular Sat-reduction to [RT (Bruchovsky and Wilson, 1968; Gloyna and Wilson, 1969; Mainwaring, 1975) . Fecent support for this hypothesis was reported in CD—1 mice in which in vivo inhibition of 5 -reductase activity with the compomd 4-androsten—3—one—l7 -carboxyl ic acid (17 C) effectively blocked the stimulatory effects of T on seminal vesicle and penis growth, but did not interfere with BET-stimulated growth of these tissues (Luttge, Jasper, Gray, and Sheets, 1977) . Since DHT is considered to be the active metabolite of T in peri— pheral sex tissues, a logical extension of this concept was to determine whether DHT was also the active metabolite of T in C58 structures mediating male sexual behavior. McDonald and co-workers (1978) reported that Dt-IT propionate (DHTP) administered at a dosage of 125 ug/day for 8 days to sexually inexperienced rats that had been castrated six weeks previously failed to induce copulatory behavior. wales given similar treatment with testosterone propionate (TP) exhibited copulatory behavior equivalent to intact controls. Many other studies have replicated this finding for DHT and other SK-reduced androgens (Beyer, Larsson, Perez-Palacios, and Nbrali, 1973; Peder, 1971; Peder, Naftolin, and Ryan, 1974; Johnston, Grmwell, Benson, Kandel, and Petro, 1975; Larsson, Sodersten, and Beyer, 1973; Larsson, Sodersten, Beyer, Morali, and Perez-Palacios, 1976; Luttge, Hall, Wallis, and Campbell,1975) but have gone further in showing that the castrates could not.maintain sexual behavior when hormone replacement was initiated immediately following castration (Parrott, 1974, 1976; Whalen and Luthge, 1971; Yahr and Gehrling, 1978). In contrast to these findings several studies reported that under certain conditions DHT can stimulate sexual behavior in.some castrated male rats. Although.not so effective as T, DHT implanted into the preoptic area of the brain did activate male sexual behavior in some long-team castrated males (Johnston and Davidson, 1972). Whalen and Luttge (1971) found that when DHT (800 ug/day) was.administered immedi- ately following castration it was not effective in maintaining sexual behavior, but when administered to long-term castrated males, DHT was effective in restoring male copulation. Similarly, in two other studies ejaculatory responses were restored in at least 50% of long-team cas— trated males receiving either 500 ug or 1 mg daily DHT treatment (Paup, Mennin, and Gorski, 1975; Sodersten, 1975) and 33% of long-term.cas- trates receiving 200 ug/day DHTP treatment (Baum and‘Vreebung, 1976). Several points are noteworthy in reviewing these studies. First, in maintenance paradigms DHT(P) has always been fomd to be ineffective in promoting male sexual behavior. By contrast, in reinstatement para- digms, IHNP) , when administered at a sufficiently high dosage over an extended period of thme, was quite effective, although not as potent as T, in restoring the complete male copulatory pattern. DHT also has been found to be'more potent in the free alcohol state than in its esterified state (DHTP) . Thus, it appears that DI-IT is not the major active metabolite of T mediating male sexual behavior in the rat. However, [HT can exert a stimulatory action in the CNS and periphery which facilitates male sexual behavior and growth of genital tissues. Importance of Aromatization of Androgens in the Male Rat thtil recently, as a result of the large number of stuiies finding that Ste-reduced androgens failed to promote male copulatory behavior in the rat, the focus of research has been on determining whether or not aromatization of androgens to estrogen was necessary for an androgen to centrally mediate male sexual behavior (McDonald et a1., 1970) . This hypothesis was supported by findings that T, other aroma- tizable androgens and B were effective in fully stimulating male sexual behavior in castrated rats (Ball, 1937, 1939; Beach, 1942; Beyer et a1., 1973; mvidson, 1969; Johnston et a1., 1975; Morali, Larsson, Perez- Palacios, and Beyer, 1974; Parrott, 1974, 1975, 1976, Sodersten, 1973; malen and Dittge, 1971). A more definitive test of whether T must be aromatized to E: for the indiction of male copulation in the rat was provided by experiments which blocked either the aromatization of T to E or the E: receptor itself. Christensen and Clemens (1975) demonstrated that intrapreoptic administration of the aromatization inhibitor, 1,4,6—androstatriene— 3,17-dione (ATD) , blocked T-stimulated male sexual behavior in castrated male rats. However, sexual behavior was restored in male rats receiving intrapreoptic administration of E alone or in combination with ATD. Similarly, other reports indicated that mating behavior is inhibited in castrated male rats receiving systemic injections of both T and an aromatization blocker, but males treated with E and DHT in addition to the aromatization blocker exhibited normal copulatory behavior (Beyer et a1., 1976; Morali, Larsson, and Beyer, 1977). Etrther support for the aromatization hypothesis came from studies finding that administering compomds which block the E receptor inhibited T—stimulated male sexual behavior (Luttge, 1975; Beyer et a1., 1976). Although it might be concluded from these studies that male sexual behavior can be accounted for solely by the action of estrogenic metabo- lites of T, other evidence questions the validity of this conclusion. Ebr example, systemic treatment with E stimulated the full copulatory pattern only when administered at extremely high dosages and over a long period of time (Davidson, 1969; Sodersten, 1973). Moreover, adrenalec- tomy blocked ejaculations in E-treated rats; therefore, suggesting the possibility that androgens of adrenal origin may be partially responsi- ble for the facilitative effects of B on copulation (Gorzalka, Rezek, and malen, 1975) . Combined Action of EBtrogen and Dihydrotestosterone in the Male Rat Recent studies have addressed the possibility that B may stimulate male mating behavior by acting synergistically with either peripherally- or intracerebrally-acting androgens. Castrated male rats exhibited momting, intromission and ejaculation following treatment with sub— threshold dosages of E: plus DHT (Baum and Vreeburg, 1973; Peder et a1., 1974; Larsson et a1., 1973) . Neither of these hormones when given alone stimulated intromission or ejaculation, but some of the E-treated males exhibited mounting. In addition, Davis and Barfield (1979) reported that a higher percentage of castrated male rats administered E in the medial anterior hypothalamic-preoptic area in conjunction with system— ically administered DHT exhibited the full copulatory'pattern than males receiving intracerebral E or DHT treatment alone. On the basis of these studies it was proposed that the neural mechanisms controlling copulatory behavior are mediated by the estrogenic metabolites of T, whereas Sukreduced metabolites of T facilitate sensory inputs important for copulation by acting peripherally to sthmulate the sex organs. This concept of a non—neural site of action for DHT was challenged in a study by Iodder and Baum (1977) . Penile factors contributing to sexual performance mere reduced or eliminated by bilaterally transecting the pudendal nerve. This operation, in addition to reducing the occurrence of penile erections, eliminated intromissions and ejaculations. How- ever, pudendectomized and castrated males given DHT + E had a higher momting frequency than males given T? or 3 alone. It was suggested from these data that both DHT and E act synergistically in the CNS to control male copulation. The spinal cord appears to be one likely site of action for DHT. Radioactive labelled GIT, but not B, has been shown to accumulate in ventral horn motor neuron nuclei in the lumbar spinal cord (Breedlove and Arnold, 1979; Sar and Stumpf, 1977b). This finding correlates well with reports that either DHTP or TP, but not B, activated sexual reflex- es in spinally transected castrated male rats (Hart, 1979) . These 10 results further supported the hypothesis that both aromatized and 5&- reduced metabolites of T have important CNS effects mediating male copulatory behavior in the rat. Comparative Studies of I-brmonal Mediation of Male Sexual Behavior Other mammalian species have not been so extensively stuiied as the rat; however, evidence has accumulated concerning the role played by T's metabolites in activating male sexual behavior. Table 1 provides a brief review of the stuiies carried out to date. [HT(P) was effective in stimulating copulation in rabbits (Agmo and Sodersten, 1975; Beyer and Rivaud, 1973) , hamsters (DeBold and Clemens, 1978; Whalen and DeBold, 1974) , Swiss Webster mice (Luttge and Hall, 1973) , CD-1 mice in maintenance paradigms only (Wallis and Luttge, 1975) , guinea pigs (Alsum and Soy, 1974) and rhesus monkeys (Phoenix, 1974) , but was ineffective in sheep (Parrott, 1978) . In addition to rats, E promoted copulatory behavior in CD-1 mice (Wallis and Luttge, 1975) , Swiss Webster mice (Edwards and Burge, 1971) , red deer (Fletcher, 1978), hamsters (DeBold and Clemens, 1978; Noble and Alsum, 1975) and sheep (Parrott, 1978) , but was without effect in rabbits (Beyer, de la Tbrre, Larsson, and Perez- Palacios,il975) , guinea pigs (Alsum and Goy, 1974) and rhesus monkeys Phoenix, 1978) . In rabbits (Agmo and Sodersten, 1975) , sheep (D'Occhio and Brooks, 1976), hamsters (DeBold and Clemens, 1978) and CD-1 mice (Wallis and Luttge, 1975) synergistic actions of E and our have been reported . Table l. l-brmonal Mediation Of Male Sexual Behavior In Mammalian Species Other Than Rattus Norveg icus Species Guinea Pig (Cavia porcellus) Hamster (Mesocricetus auratus) Conclusions DHTP was as effective as TP in restoring sexual behavior. EB has no restorative effects (Alsum and Soy, 1974) . DHTP activated complete copulatory pattern, but was less effective than T? over long-term administration. EB restored momting behavior, but not intromission or ejaculation. Best results produced with EB acting syner- gistically with [HT (Christensen et 31., 1973; 068an and Clemens, 1978; Noble and Alsum, 1975; Payne and Ben— net, 1976; Whalen and DeBold, 1974). Table 1 (Cont) Species Albino muse (Mas musculus) Rabbit (Orycytolagus cuniculus) .12 Conclusions (1) CD-l strain: EB maintained and restored copulatory behavior, but was less effective than TP. GIT was less potent than BB in maintaining sex behavior and was unable to restore copulation in long-term castrated males. EB + DHT more potent than EB or DHT alone (Luttge and Hall, 1973; Wallis and Luttge, 1975). (2) Swiss Webster: [HT was as potent as T? in stimulating the complete copulatory pattern. EB restored momting behavior only (Edwards and Burge, 1971; Luttge et a1., 1974). DHT was less potent than T in acti- vating copulation. EB has no stimu- lative effects. Best results pro- duced with EB acting synergistically with DHT (Agmo and Sodersten, 1975; Beyer and Rivaud, 1973; Beyer et a1., 1975) . Table 1 (cont) Species Red Deer (Cervus elaphus) Rhesus ankey (Macaca mul atta) Sheep (Ovis aries) l3 Cbnclusions EB was more potent than TP in main- taining copulatory behavior. DHT not tested (Fletcher , 1978) . DHTP restored complete mating pat— tern, but was less potent than TP. EB has no restorative effects. 19- hydroxytestosterone (aromatizable an- drogen) did not induce copulation (Phoenix, 1974, 1976). EB was less effective than TP in ac- tivating sexual behavior. [HT has no stimulative effects. DHT + EB more potent than EB alone (D'Occhio and Brooks, 1976; Parrott, 1978). 14 Among the many different species of muroid rodents research con- cerned with hormonal detenminants of male copulatory behavior has been conducted exclusively on the laboratory rat (Rattus norvegicus) , labora- tory mouse (Mus musculus) and golden hamster (mesocricetus auratus) . This lack of diversity points to a need to extend the number of species being stuiied so that general principles of hormonal factors regulating male sexual behavior can be derived. Dewsbury (1979) has recently reported normative data on male copulatory behavior in deermice (Peromyscus maniculatus bairdi) . The present stuiy sought to characterize the role played by gonadal hormones in mediating male reproductive behavior in this cricetid species. Specifically, the pattern of decline of male mating behavior and perfor— mance was examined following castration. Secondly, in castrated male deermice T, DHT, and E were compared for their effectiveness in stimu- lating male copulation. All results were discussed .in relationship to similar experiments performed using other mammalian species. In this manner the comparative base for mderstanding hormone-behavior inter- actions could be further expanded. MERIMENT 1 The purpose of this experiment was to assess the effects of castra- tion on male sexual behavior in P,m. bairdi. METHOD Subjects . The srbjects in this experiment were 45 male P.r_m_. bairdi bred in the laboratory from stock originally trapped in East Lansing, Michigan. All were at least 120 days old at the beginning of behavioral testing. Males were selected on the basis of having achieved to ejaculations within a test on at least two out of three copulatory behavior tests, one of which being their third behavioral test. Precastration data were collected from these selection tests. Chce the ejaculation criterion was achieved the males were castrated mder betofane (Pitman-Moore,Inc.) anesthesia and housed individually in plastic cages, 48 X 27 X 13 cm. Wood shavings were used for bedding and cotton Nestlets (Ancare Oorp.) were provided as nesting material. Deermice were maintained on a reverse day-night cycle of 16 hr light and 8 hr dark with lights off at 1030. Food and water were available at all times. 16 Apparatus. TeSts for copulatory behavior were condmted in the home cages of the males. Behavioral data were recorded on an Esterline-Angus Event Recorder. Procedure. To induce sexual receptivity, stimulus females were injected with 60 ug estradiol benzoate (EB) approximately 72 hr before testing and 600 ug progesterone approximately 6 hr before testing. msts were begun with the introduction of the female into the male's home cage 4 hr after the lights went out. The room in which the tests were conducted mas illuminated with a 25 watt red bulb. Male copulatory behavior in 3.51. bairdi is comprised of momts (with thrusting), intromissions (with vaginal insertion), and ejacula— tions. The male is capable of achieving multiple ejaculations within a given time period. The temporal pattern of the behavior is such that each copulatory series is comprised of several intromissions and termi- nates with an ejaculation. Ebllowing ejaculation there is a period of sexual quiescence (post ejaculatory interval) before the next copulatory series is initiated. A mating test was terminated when one of the following criteria had been satisfied: (1) 30 min after the start of the test with no intromission; (2) no ejaculation within 20 min of the first intromission of a copulatory series; (3) no intromission within 15 min after completion of the first copulatory series; (4) completion of two copulatory series. Both before and after castration males were tested once a meek. Following castration, if the males failed to exhibit any sexual response on three consecutive copulatory behavior tests they were .17 no longer tested and automatically scored as sexually unresponsive for the remainder of the experiment. Measures. During each behavioral test the following copulatorylmeasures were recorded: momt latency (ML)- time in seconds from introduction of the female to the first mount or intromission, whichever came first; intro- mission latency (IL)- time in seconds from introduction of the female to the first intromission; ejaculation latency (EL)- time in seconds from first intromission of a copulatory series to an ejaculation; intromis~ sion frequency (IF)- number of intromissions in a copulatory series; momt frequency (MF)- number of momts in a copulatory series; mean interintromission interval (MIII)- mean interval in seconds between intromissions in a copulatory series including the interval between the last intromission and ejaculation. For the intact.males, intromissions were followed by'a brief period of sexual inactivity (grooming, exploration, digging), whereas a mount was followed aLmost immediately by'another mount attempt until an intromission was achieved. In contrast, there were many instances in castrated deenmice in which a mount or cluster of mounts and intromissions was followed by'a brief period of sexual inactivity not distinguishably'different from that seen after intromissions in intact males. Sachs and Barfield (1970) employed the team “mount bouts” to identify a similar clustering of mounts and intromissions observed in rats. After castration we recorded mount bout frequency (MBF)- number of momt bouts in a copulatory series; and mean intermount bout interval (MIMBI)- mean interval in seconds between momt 18 bouts including the interval separating the last momt bout and ejacula- tion. The abbreviation for a measure followed by a hyphen signifies the copulatory series to which the measure refers (i.e. MF-2) . Data Analysis. Comparisons of the percentage of males no longer exhibiting ejacu— lation, intromission or mounting behavior on each week following castra— tion were made using a Chi-square test. In order to further analyze the behavioral data, each male for each of the copulatory measures was assigned a score equal to the mean of his precastration behavior tests in which ejaculation occurred and another score equal to the mean of his postcastration behavior tests in which ejaculation occurred. Within- srbject comparisons of copulatory performance among ejaculating males before and after castration were conducted by using a matched-pairs T-test. Finally, data from castrated males were collected so that one set of data was composed of the males' first behavior test following castration in which ejaculation occurred, the second set of data was composed of the males' last behavior test following castration in which they ejaculated, and the final data set was composed of the males' first behavior test in which they failed to ejaculate, but in which intromis- sions were still being achieved. Che-way analysis of. variance was conducted on these sets of data. when the assumption of homogeneity of variance was violated log transformations were performed. Significant F ratios were followed by the Student-Newman-Keuls procedure (Winer, 1962) . 19 RESULTS The percentage of males.maintaining ejaculation, intromission and mounting behavior in successive weeks following castration is illus- trated in Figure 1. By the fourth week after gonadectomy 80% of the males failed to ejaculate. Further analysis revealed that the number of males that had lost the ejaculatory response by weeks 4 and 5 was significantly'greater than the number in which intromission had disap- peared (Week 4, X2(1)=4.20, p<.02; Week 5, x2(1)=3.72, p<.05). Also, the number of males who were no longer exhibiting intromission by weeks 3 and 5 was significantly greater than the number of males who were no longer exhibiting mounting behavior (Week 3, x2(1)=3.o6, p<.05; Week 5, x2(1)=3.96, p<.05) . It should be noted that failure to exhibit copula- tory behavior one week did not necessarily'mean that the male would fail to exhibit behavior in the following week. In Table 2 the copulatory perfonmance of males achieving ejacula- tion following castration is compared with their precastration perfor- mance. Time measures increased significantly following gonadectomy (ML, IL, EL, M111, and MIMBI), as did MF. After castration IF-l and IF—2 were significantly less than before castration, whereas MBF-l and MBF-z were no different from precastration levels. In order to further assess the behavioral changes which occurred after castration, comparisons of copulatory performance were made between castrated males' first and last test with ejaculation and their X 0? MAlES MAINIAINING BEHAVIOR 20 l O O 90 p a o ' 5,”. '.. ‘o-oooooooo‘ M o U N T I N G .\ "5...... t—' 'N T R o M I s S I o N 70 '3‘ . .-.-... EJACULATION .\ ', \n 6 O ‘. ‘l .‘u \' v ”'3. so % '3 \.‘ 5‘. .\ ".3. 4o \ . xx .\ “a 3 O \.\ \. t \. '3‘ 2° 3 \ \- o \ ‘. I '\.‘ \q ". ........ . ....... o ..‘.‘ \Cm—‘f ......... ~ ‘.‘ .90-.-" .-.. \ ' ......... ‘ .~' -0..- 0-0-I. \. ..... o . . “Q.-"...-.-. .m. WEEKS AFTER CASTRATION Figure 1. Percentage of male deermice maintaining mounting, intro- mission and ejaculatory responses on successive weeks following castration. 21 a Table 2. Effects of Castration on Male Copulatory Performance Measure Pre-Castration Post-Castration pb N ML (sec) 529 i 39 '747 i 57 <0.001 33 IL (sec) 545 t 41 789 s 58 <0.001 33 MF-l 2.1 i .5 9.3 r 2.0 <0.001 33 IF-l 12.3 t 1.0 9.6 r .8 €0.05 33 MB-l 12.8 i 1.0 11.9 s .8 ns 33 EL-l (sec) 354 i 20 430 t 29 <0.05 33 MIII-l (sec) 31.3 i 2.1 60.5 s 8.9 <0.001 33 MLMBI-l (sec) 30.0 r 2.0 41.7 r 4.3 <0.001 33 PEI (sec) 400 t 17 419 s 15 ns 25 MF-Z 1.5 i .4 4.2 r 1.5 <0.05 20 IF-Z 11.1 s .7 8.8 r 1.0 <0.05 20 MB—Z 11.1 s .7 9.8 s 1.0 ns 20 BL (sec) 141 r 9 182 t 17 <0.05 20 MIII-Z (sec) 12.2 t .8 22.1 r 1.9 <0.001 20 MEMBI-Z (sec) 12.0 i .9 18.1 i 1.6 <0.001 20 Note. ML- Mount latency; IL- Intromission latency; MFa Mount frequency; IF- Intromission frequency; MB= Mount bout frequency; EL- Ejaculation latency; MIII= Mean interintromission interval; MIMBI- Mean intermount bout interval; PEI- Post ejaculatory interval an scores a o y or an ma 3 w 1c ejacu ate gMe ( sax) n1 f i 1 h' h ' 1 d Two-tailed matched-pair T-test 22 first test following the loss of ejaculation in which intromissions occurred. Results are presented in Table 3. In castrated males' first test after losing ejaculation MIMBI was significantly longer than both the first and last test with ejaculation. Also, MIII of males who were no longer ejaculating was significantly longer than the castrated males' first test with ejaculation. However, MIII in the last test before the ejaculatory response disappeared did not differ from either of the other groups. In both the last test with ejaculation and the first test in which ejaculation did not occur MF was significantly increased over the first test with ejaculation. There were no differences between the groups in IF, MBF'or IL. Also, there was no difference in ELfibetween the two groups of ejaculating males. 23 Ace. v mes Ho. v ms AmeHv mouoom amuse no we «a . z ----- Hv ..... oe n nee on n nee Aoooo as m v H H.s o.~e n e.eo m.~H h o.ee e.o e ~.ne Aooov one: m v ~.H eea.oe m.~a n o.me o.e n e.ne o.m n m.nm Aooov Haze: ..... av m.~ e H.~H e.H e m.~u H.H n o.- as: ..... me.e o.H n o.k e.e n H.o H.H n m.HH eH m.~ v H eeo.n m.o n e.e~ o.m e o.me e.~ e m.“ e: ..... He.a «NH n moo so n one one e one . SH oeooeooeeoo Hoses Ame A~v Ass I>HecH uccowuficwem m haco macemmfieouucm umoa umoq umoh uuufim owsmmoz ezmmes zooeessoaem ezmmmeo zooeeoeoaee couuoasumfim mo oocnwooeemmen can nouu< can uuomom ooesuooa can: toumuummo wcoe< oocosuouuom huoumfioeoo we comaumasoo .m mance EXPERIMENT 2 The first experiment established that the expression of male copulatory behavior in male deermice depends upon the secretion of gonadal hormones. The purpose of Experiment 2 was to investigate the role of T and two of its major metabolites, a and an, in the mediation of male copulatory behavior in P.r_m_. bairdi. Castrated males were treated with these hormones to determine whether sexual behavior could be reinstated . METHOD Subject Assignment and Hormone Treatment. The Subjects were 32 gag. M from Dcperiment l. Males were matched according to the number of postcastration tests required to reach the criterion of three successive weeks without exhibiting any sexual behavior and assigned to one of four hormone treatment groups. Beginning on the morning after their third unsuccessful mating test in Elucperiment 1, males received daily subcutaneous hormone injections for six weeks. The treatments used were: 200 ug testosterone propionate (TP); 200 ug SK-dihydrotestosterone propionate (DHTP); 2 ug estradiol benzoate (EB); or sesame oil (OIL). lbrmones were dissolved in .02 cc sesame oil. The 200 ug dosage of T? was selected on the basis of preliminary data which indicated that 100 ug TP was ineffective in 24 25 restoring sexual behavior, 200 ug TP was highly effective, and 400 ug TP increased mortality. In the course of the stlriy one MP and one EB animal died. Procedure. The males were tested once a week for sexual behavior during hormone therapy. Testing procedures, behavioral measures recorded and preparation of sthmulus females were the same as those employed in EXperiment 1. The males were sacrificed and weighed after the sixth behavioral test. The seminal vesicles and ventral prostate gland were removed, cleaned of all adipose tissue and weighed on a Mettler balance to the the nearest mg. Data Analysis. All behavioral data and histological data were analyzed with Mann-Whitney U tests . 26 RESULTS The percentage of males displaying momts, intromissions and ejaculations for each of the six sexual behavior tests is illustrated in Figure 2. T? and HTTP were equally effective in restoring momts, intromissions and ejaculations. The percentage of EB-treated males exhibiting mounting was intermediate between the androgen treatment groups and the OIL controls. In the EB group, animals displaying momting on a given test did not necessarily continue to show momting on subsequent tests. However, six out of seven EB-treated males displayed mounting on at least one test. EB was minimally effective in restoring intromission and totally ineffective in activating ejaculatory behavior. Table 4 presents data from tests in which copulation occurred. TP-treated males took significantly longer to initiate copulation than HITP males. Ch all other copulatory measures, the two groups did not differ. Whereas TP and EB males did not differ in ML or IL, males receiving EB exhibited significantly more mounts and fewer intromissions than T? males. EB males tended to cluster their mounts into momt bouts and a mean mount bout frequency of 9.4 was obtained. There were no differences in body weight among the different treatment groups, with the mean body weight for TP, HITP, EB and OIL males being 18.8, 19.2, 18.2 and 18.1 g respectively. TP and HTTP males did not differ in mean combined seminal vesicle-ventral prostate weight Figure 2. 27 100 ’TP ......... goon-uncaugncnncncnog.4. OH I? “"0'. 30 O ‘0 60 ‘I' .~..°.--I’U-°-0-O-° E8 1 MOUN I. 40 ' . ,r‘ 20 ,I '\ f 100 2 I? '30 60 X INIRO. °-.--- .-.-.0.5 o 40 20 j" -" 100 80 '._----____..‘./ \ 60 E JAC. \ \ q I 4O 20 ‘ ‘ ‘ .- . I o . . ‘ EBOL I 2 3 4 5 6 WEEKS OF HORMONE TREAI’MENI Percentage of castrated male deermice exhibiting mounting, intromission and ejaculation on successive weeks after onset of daily treatment with 200 ug TP, 200 ug DHTP, 2 ug EB or sesame OIL. 28 (237 and 274 mg respectively) , but both were significantly heavier (p<.lwl) than the EB and OIL males (20 and 24 mg respectively). 29 Table 4. Effects of Hormones on Mating Performance of Castrated Male Deermice Measure TPa DHTPa EBb - 74 Tests with M/I/E (2)0 81/81/69 83/81/75 43/19/0 ML (sec) 564 i 115 301 i 38* 751 i 169 IL (sec) 576 t 122 309 t 39* 928 t 215 MF~1 2.6 i 1.1 3.6 i 1.3 41.4 t 8.6** IF-l 19.0 t 3.5 15.0 i 2.5 3.5 : l.6** EL-l ' 489 t 54 378 t 70 --- PEI (sec) 421 i 23 440 t 53 --- MF-Z 1.2 t .4 .6 t .2 --- IF-Z 12.9 r 1.7 11.5 t 1.5 --- EL-2 162 t 16 152 2 30 --- aMean scores (tSEM) only for animals which ejaculated Mean scores (iSEM) only for positive responding animals c . . Percentage of tests on which mounts, intromissions, or ejaculations occurred ' *P<0.05 vs TP **P<0.01 vs T? DISCUSSION The removal of gonadal honmones by castration resulted in a decline in male sexual behavior of g.r_n_. bairdi with ejaculation disappearing first, then intromission, and, finally, mounting. The complete pattern of male sexual behavior (mounting, intromision, and ejaculation) was restored by the administration of either TP or its SK-reduced metabo— lite, DHTP. In contrast, EB restored mounting behavior, but was only minimally effective in restoring intromission and did not induce any ejaculatory responses. TP and DHTP were both equally effective in restoring male copulatory behavior in castrated deermice. Both groups of males responded shnilarly on all measures of copulatory performance except for DHTP males having a shorter latency to initiate copulation than TP males. The disappearance of ejaculation before intromission observed in deermice following castration has also been reported to occur in guinea phgs (Grunt and Toung, 1953) and rhesus monkeys (Phoenix et a1., 1973). In male rats some investigators have f6und that intromissions were maintained after ejaculatory responses had disappeared (Beach and Holtz-Tucker, 1949; Stone, 1939), although others have reported that ejaculations and intromissions disappeared simultaneously (Davidson, 1966; Larsson, 1966). In addition to deermice, mounting response have been reported to continue after intromissions have disappeared in rats 30 31 (Davidson, 1966) , hamsters (Beach and Pauker, 1949), guinea pigs (Grunt and Yomg, 1953), rabbits (Stone, 1932), dogs (Beach, 1970; Hart, 1968), cats (Rosenblatt and Aronson, 1958) and rhesus monkeys (Phoenix et 81., 1973) . The results of the hormone replacement study are similar to those reported for guinea pigs which also repond equally well to TP or DHTP treatment (Alsum and Coy, 1974) . In all other species in which DHT(P) activated the complete male sex behavior pattern, it was not fomd to be as potent as T(P): hamsters (whalen and DeBold, 1974); Swiss Webster mice (Luttge and Hall, 1973); rabbits (Agmo and Sodersten; 1975; Beyer and Rivaud, 1973); and rhesus monkeys (Phoenix, 1974). Moreover, the observation that DHTP was at least as potent as TP in stimulating seminal vesicles and ventral prostate gland agrees with studies on rats (Whalen and Luttge, 1971) , two strains of mice (Luttge and Hall, 1973) and hamsters (Payne and Bennett, 1976) . In the present stLdy EB stimulated a high frequency of momting behavior in six out of seven animals. Higher doses of BB resulted in the animals becoming very lethargic (Pomerantz, unpublished observa- tions) ; therefore, it would appear unlikely that intromission and ejacu— lation of castrated male deermice were not facilitated due to inadequate levels of E. Among other species in which DBT(P) promoted sexual behavior, EB failed to activate sexual behavior in guinea pigs (Alsum and Gay, 1974) , rhesus monkeys (Phoenix, 1978) and rabbits (Beyer et a1., 1975). However, hamsters (DeBold and Clemens, 1978; Noble and Alsum, 1975) and Swiss Webster mice (Edwards and Burge, 1971) responded to BB in a fashion similar to deermice (i.e. exhibiting a high 32 frequency of mounting and only rarely intromitting) . The failure of EB to restore intromissions or ejaculation in these species may be related to the inability of EB to stimulate genital structures. However, this is in contrast to the activation of the complete copulatory pattern by BB in castrated rats (Sodersten, 1973) and deer (Fletcher, 1978). Additionally, BB-induced momting behavior may depend on adrenal secre— tion. In castrated male rats being treated with EB, adrenalectomy prevented ejaculations; however, adrenalectomized males continued to momt (Gorzalka et a1., 1975). Species differences or similarities in the ability of T's metabo- lites, DHT and B, to induce male sexual behavior may reflect a varying degree of reliance on T metabolism by Sx-reduction and aromatization respectively. It is noteworthy that castrated males in two cricetid rodent species, deermice and hamsters, responded similarly to E3 administration, exhibiting mounting and, infrequently, intromission. In both species DHT(P) administration stimulated the full copulatory pattern; however, DHTP was a more potent activator of male sexual behavior in deermice than in hamsters. It remains to be determined whether metabolism of T to DHT (SS-reduction) or to E (aromatization) is necessary for T activation of male copulator behavior. Castration had a pronounced effect on the timing of male copulatory behavior in male deenmice. This influence may have affected other components of copulatory performance. In comparison to precastration levels, castrated deermice exhibited a substantial increase in interintromission and intermount bout intervals, and a concomitant decrease in the number of intromissions preceeding ejaculation. This 33 rise in MIII and MIMBI observed in deermice fbllowing castration may'be related to their ability to attain an ejaculation with fewer intromis- sions than were necessary for ejaculation before castration. Castrated rhesus monkeys also exhibited an increase in interintromission interval and a decrease in the number of intromissions preceeding ejaculation when compared to precastration levels (Michael and Wilson, 1974). After castration male rats achieved an ejaculation in fewer intromissions than before castration, but no increase in interintromission interval was observed (Davidson, 1966; Larsson, 1966). However, in intact male rats when the interintromission interval was lengthened beyond nonmal lhmits, males needed fewer intromissions to achieve ejaculation (Bermant, 1964; Hard and Iarsson, 1970). The interval separating intromissions and/or mount bouts also appeared to be a factor in the disappearance of the ejaculatory response among castrated male deermice who were still displaying intromissions. Their inability to achieve ejaculation could arise frqm their increase in interintromission and intermount bout intervals beyond a lhmit necessary to achieve an ejaculation. Although not directly analagous, in intact rats ejaculations were prevented by experimentally enforcing interintromission intervals of greater than 19 minutes (Larsson, 1968). In summary, castration of male deermice resulted in a decline in male sexual behavior with ejaculation disappearing first, followed by intromission and, finally, mounting. 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