A single injection of 17β-estradiol facilitates sexual behavior in castrated male mice

Sexual behavior was assessed in castrated adult CD-1 male mice given exogenous steroids under various treatment regimens. Castrated mice maintained on 20 μg testosterone (T) daily for 1 week, but given 250 μg testosterone propionate (TP) on the day of testing showed higher levels of copulatory activity than intact mice or the males receiving an additional dose of 20 μg T on the test day, although plasma testosterone levels were not different at the time of behavioral testing. Castrated males given 50, 125, or 250 μg TP for 1 week including the day of testing showed higher levels of sexual behavior than males receiving the same doses of TP only once, on the test day. A single injection of 17β-estradiol (E₂) completely restored the male copulatory pattern, including ejaculation, in castrated mice under every condition examined. Testosterone and dihydrotestosterone (DHT) were less effective than E₂, as was the combination of E₂ and DHT. The relative efficacy of a single dose of T, DHT, and E₂ plus DHT was dependent upon factors such as the delay between steroid administration and testing, as well as whether or not the castrated mice received androgen replacement prior to testing. Estradiol benzoate (E₂B) was not capable of restoring sexual behavior in castrated mice in this study. The comparison of results obtained with TP, T, E₂, and E₂B suggests that an appreciable, but not necessarily sustained, elevation of E₂ levels in the brain may be critical in the facilitation of male copulatory behavior in mice.     

Steroids and sexual behavior in castrated male rheusus monkeys.

Sexual behavior of long-term castrated rhesus males was not increased by administration of the hydroxylated metabolite of testosterone (T), 17 beta, 19-dihydroxy-5 alpha-androstan-3-one diacetate (19-OH-DHTA) at a dose of 1 mg/kg/day. Simultaneous administration of 19-OH-DHTA and estradiol benzoate (EB) also failed to increase the level of sexual performance, but daily injection (1 mg/kg/day) of testosterone propionate (TP) was very effective in effective in activating sexual behavior.     

Persistent sexual behavior in castrated, recombinant inbred mice.

In contrast to other male mice, many hybrid B6D2F1 males continue to mate after being castrated in adulthood. This persistent sexual behavior in the castrated hybrids presumably is based upon heterozygosity that might occur within individual loci or between different loci. Those possibilities were discriminated by studying BXD recombinant inbred mice. In BXD strains, individual loci are homozygous, but heterozygosity has arisen between separate loci. Males in two of six BXD strains persisted in copulating for 5 mo after being castrated. Thus, it is concluded that retention of masculine sexual behavior in castrated mice is a function of heterozygosity between loci.     

Decline of sexual behavior in castrated male rats: Effects of female precopulatory behavior

From the population of 89 adult sexually inexperienced Wistar male rats 20 animals that initiated copulatory behavior with females exhibiting low intensity of precopulatory behavior (presenting females) were preselected. Prior to castration all 20 males had the same sexual experience: three ejaculatory series in four weekly sessions with females exhibiting high intensity of precopulatory behavior (darting females). Following castration, the decline of copulatory behavior was much slower for the nine males tested with darting females as compared to the 11 males tested with presenting females. Male precopulatory behaviors (anogenital sniffing, touching flanks, etc) outlasted the loss of copulatory behavior and seem to be less dependent on both external and internal determinants. It is concluded that intensive external sexual stimuli can function to compensate, and therefore mask, the subnormal operation of androgen-dependent mechanisms in initiating the copulatory behavior.     

Inhibition of testosterone-induced sexual behavior in the castrated male rat by aromatase blockers

The effect of some aromatase inhibitors (aminoglutethimide, 1,4,6-androstatrien-3, 17-dione, and 4-hydroxy-androstenedione) on testosterone propionate (TP)-induced copulatory behavior was tested in sexually inexperienced castrated male rats. A single injection of 6 mg of TP induced mounting in 48% and ejaculatory pattern in 19% of the rats within 120 hr. Treatment with the aromatase inhibitors (injections every 12 hr for 108 hr) suppressed ejaculation in all but one rat and significantly reduced the number of rats mounting and intromitting. Concurrent administration of estradiol benzoate (EB, 1 or 3 μg every 12 hr) prevented the inhibitory effect of aromatase blockers. No inhibitory effect of the aromatization blockers was observed in rats in which sexual behavior was induced by dihydrotestosterone (1 mg/day) and EB (2.5 μg/day) for 20 days. The results support the concept that aromatization is an essential step for the induction of male sexual behavior by androgen in the rat.     

Influence of progesterone on sexual behavior in castrated male rhesus monkeys treated with testosterone

The effect of progesterone (P) on sexual behavior was tested in five long-term castrated rhesus monkeys treated simultaneously with testosterone (T) and P. A control group consisting of three long-term castrates was treated only with T. Plasma levels of T and P were measured at weekly intervals, and behavioral data were collected for each animal twice a week throughout the study. Progesterone has been reported to inhibit sexual behavior in male guinea pigs and mice, but the levels of behavior in our two groups of monkeys did not differ from each other over 8 weeks of treatment and testing.     

Oxytocin in the medial preoptic area facilitates male sexual behavior in the rat

Oxytocin (OT) is a versatile neuropeptide that is involved in a variety of mammalian behaviors, and its role in reproductive function and behavior has been well established. The majority of pharmacological studies of the effects of OT on male sexual behavior have focused on the paraventricular nucleus (PVN), ventral tegmental area (VTA), hippocampus, and amygdala. Less attention has been given to the medial preoptic area (MPOA), a major integrative site for male sexual behavior. The present study investigated the effects of intra-MPOA administration of OT and (d(CH2)51, Tyr(Me)2, Thr4, Orn8, Tyr-NH29)-vasotocin, an OT antagonist (OTA), on copulation in the male rat. The relationship between OT receptor (OTR) binding levels in the MPOA and sexual efficiency was also explored. Microinjection of OT into the MPOA facilitated copulation in sexually experienced male rats, whereas similar injections of an OTA inhibited certain aspects of copulation but had no significant effect on locomotor activity in an open field. Contrary to expectation, sexually efficient males had lower levels of OTR binding in the rostral MPOA compared to inefficient animals. The present data suggest that OT activity in the MPOA is not necessary for the expression of male sexual behavior but is sufficient to facilitate copulatory behaviors and improve sexual efficiency in sexually experienced male rats. These data also suggest that OTR activity in the MPOA stimulates anogenital investigation, facilitates the initiation of copulation, and plays a role in the sensitization effect of the first ejaculation on subsequent ejaculations.     

Effects of diverse androgens on the sexual behavior and morphology of castrated male quail

Castrated male Japanese quail were injected for 15 days with 1 mg/day of testosterone propionate (TP), testosterone (T), androstenedione (AE), androsterone (AO), 5α-dihydrotestosterone benzoate (5α-DHTB), or 5β-dihydrotestosterone (5β-DHT), or with oil. Copulation was activated to a significant extent only by TP and T. Strutting was activated only by TP, T, and AE. Proctodeal (foam) glands were well-developed in birds injected with TP, T, AE, or 5α-DHTB. Additional data were obtained following implantation of pellets of crystalline T, AE, AO, or 5α-DHT. T pellets activated copulation, but AO and 5α-DHT pellets did not. Effects of AE require further study. These results suggest that conversion of androgen to estrogen is necessary for the activation of copulation in the male quail.     

The influence of androgenic and estrogenic hormones on sexual behavior in castrated adult male pigs

The objectives of these studies were to evaluate the influence of testosterone propionate (TP), estradiol cypionate (EC), dihydrotestosterone propionate (DHTP), EC + TP, EC + DHTP, and TP + DHTP on traits of masculine sexual behavior in castrated adult male pigs of different breeds. Masculine sexual behavior was restored and maintained by TP, whereas EC initially activated sexual behavior, including copulation and ejaculation, but was unable to sustain copulatory behavior for the 8- to 18-week periods that were evaluated. Treatment with DHTP was ineffective for stimulation of sexual behavior; thus, it is suggested that testosterone promotes some aspects of masculine sexual behavior in male pigs via aromatization to estrogen, but both androgen and estrogen are required for maintenance of the full complement of masculine sexual behavior traits.     

Differential effect of testosterone and dihydrotestosterone on the sexual behavior of prepuberally castrated male rabbits

The effect of testosterone propionate (Tp) and dihydrotestosterone propionate (DHTp) at doses of 1, 3 and 9 mg daily for 30 days on the copulatory behavior of prepuberally castrated male New Zealand white rabbits was studied. Tp was significantly more effective than DHTp in eliciting copulatory behavior at each dose level tested. Three milligrams Tp was the minimal dose required to elicit mounting consistently. DHTp at the high dose level (9 mg) only elicited sexual activity comparable to that observed with the low dose of Tp (1 mg). The results suggest that T does not need to be reduced to DHT to stimulate sexual behavior in the male rabbit.     

Effect of some antiestrogens and aromatase inhibitors on androgen induced sexual behavior in castrated male rats

The effect of antiestrogens (MER-25, ICI-46474, and cis-clomiphene) and aromatase inhibitors (5-α-androstanedione, metopirone, and aminoglutethimide) on androgen induced copulatory behavior was tested in sexually inexperienced castrated male tats. Daily injections of 1 mg testosterone (T) for 21 days induced sexual activity in most subjects (61% mounting). Daily pretreatment with MER-25 or cis-clomiphene at three dose levels did not block the behavioral response to T. ICI-46474 at the high dose level (1 mg/kg) elicited a significant depressory effect on the sexual behavior of the T treated castrated rats. A single injection of 6 mg testosterone propionate (TP) induced mounting behavior in 56% of the tested rats within 120 hr. Treatment with metopirone or 5 α-androstanedione (injections every 12 hr for 96 hr) did not inhibit the response to TP. By contrast, aminoglutethimide (5 or 15 mg every 12 hr for 96 hr) abolished the behavioral response to androgen.     

Plasma testosterone and sexual behavior following intracerebral implantation of testosterone propionate in the castrated male rat

Interpretation of behavioral and other effects of intracranial steroid implants depends on knowledge of the rate of release of the implanted hormones into the general circulation. Testosterone propionate implants (200 μg, pellets) in the median eminence and pituitary were found to result in circulating levels of testosterone (T) twice as high as those in the anterior hypothalamus-preoptic area (AHPOA), posterior hypothalamus (PH), and cortex (Ctx). Implants in all cranial areas examined resulted in plasma T levels in the lower range of circulating T found in normal rats for the first 24 hr postoperatively, decreasing thereafter and reaching very low levels by the end of 2 weeks. There were no significant differences in the plasma T levels in rats with implants in the AHPOA, PH, and Ctx, but AHPOA implants were slightly more effective in restoration of sexual behavior than PH implants, and both of these implants were considerably more effective than those in the cortex. There was no apparent correlation between behavioral responses and peripheral levels of T. The major conclusion of this study was that the effects of hypothalamic implants of T on male sexual behavior cannot be explained by the presence of T in the peripheral circulation.     

Steroid-independent male sexual behavior in B6D2F2 male mice

It is well established that male sexual behavior (MSB) is regulated by gonadal steroids; however, individual differences in MSB, independent of gonadal steroids, are prevalent across a wide range of species, and further investigation is necessary to advance our understanding of steroid-independent MSB. Studies utilizing B6D2F1 hybrid male mice in which a significant proportion retain MSB after long-term orchidectomy, identified as steroid-independent-maters (SI-maters), have begun to unravel the genetic underpinnings of steroid-independent MSB. A recent study demonstrated that steroid-independent MSB is a heritable behavioral phenotype that is mainly passed down from B6D2F1 hybrid SI-maters when crossed with C57BL6J female mice. To begin to uncover whether the strain of the dam plays a role in the inheritance of steroid-independent MSB, B6D2F1 hybrid females were crossed with B6D2F1 hybrid males. While the present study confirms the finding that steroid-independent MSB is a heritable behavioral phenotype and that SI-mater sires are more likely to pass down some components of MSB than SI-non-maters to their offspring, it also reveals that the B6D2F2 male offspring that were identified as SI-maters that displayed the full repertoire of steroid-independent MSB had the same probability of being sired from either a B6D2F1 SI-mater or SI-non-mater. These results, in conjunction with previous findings, indicate that the specific chromosomal loci pattern that codes for steroid-independent MSB in the B6D2F2 male offspring may result regardless of whether the father was a SI-mater or SI-non-mater, and that the maternal strain may be an important factor in the inheritance of steroid-independent MSB.     

Effects of dihydrotestosterone and estradiol benzoate pretreatment upon testosterone-induced sexual behavior in the castrated male rat

Three groups of inexperienced castrated male rats were treated daily for 15 days with oil, estradiol benzoate (1 μg), or dihydrotestosterone (1 mg), and thereafter injected daily with testosterone (1 mg) for 21 days. Sexual behavior was tested every third day after the start of the pretreatment until day 36. Estradiol benzoate or dihydrotestosterone failed to elicit sexual behavior. Pretreatment with dihydrotestosterone, but not estradiol benzoate, significantly shortened the intervals to initiation of mounting and intromission in response to testosterone. The results suggest that fully developed genitals (penis and/or sexual accessories) facilitate initiation of copulatory behavior in response to testosterone administration.     

Temporary inhibition by progesterone of sexual behavior in intact male mice

Administration of 0.50 mg or 1.00 mg progesterone (P) daily resulted in suppression of portions of the male sexual repertoire. Mice treated with 0.25 mg P daily behaved like control subjects. Sexual behavior of mice treated with the higher P doses resembled that of controls three weeks after cessation of P treatment. No lasting effects of P on total body weight or on weights of testes or seminal vesicles were detected.     

Raloxifen prevents bone loss in castrated male mice

Raloxifen is a selective estrogen receptor modulator which prevents bone loss in ovariectomized female mice in a fashion similar to estrogens. Since testosterone-deficient male mice also lose bone mass, we were interested in testing the effects of raloxifen on bones in intact and castrated male mice. Bone density was significantly reduced in castrated animals (1.36+/-0.04 g/ml) as compared to intact animals (1.42+/-0.03 g/ml) (p<0.01). When castrated mice with extraordinarily low concentrations of testosterone and with reduced weight of seminal vesicles were treated with raloxifen, the changes in bone density and bone minerals resulting from castration (1.36+/-0.04 g/ml) were entirely prevented (1.40+/-0.01 g/ml). Cortical bone was lost in orchidectomized mice, and this decrease in cortical thickness of the femur was prevented by raloxifen administration. Raloxifen in a dose used in humans for treatment of osteoporosis decreased the weight of seminal vesicles, an organ which is highly sensitive to the androgenic effect, decreased the concentration of testosterone (12.5+/-2.8 micromol/l) (p<0.01) but not to the same level as in the case of castrated animals (0.6+/-0.3 micromol/l), and did not have any effect on bone density or mineral content in intact mice. The results of the present study may thus be interpreted as supporting the hypothesis that raloxifen is an effective agent against the deleterious effects of castration-induced osteopenia in male mice and also support the hypothesis that estrogens may have physiological skeletal effects in male mice.     

Restoration of male sexual behavior by adult exogenous estrogens in male aromatase knockout mice

We previously found that male aromatase knockout (ArKO) mice that carry a targeted mutation in exons 1 and 2 of the CYP19 gene and as a result cannot aromatize androgen to estrogen show impaired sexual behavior in adulthood. To determine whether this impairment was due to a lack of activation of sexual behavior by estradiol, we studied here male coital behavior as well as olfactory investigation of sexually relevant odors in male ArKO mice following adult treatment with estradiol benzoate (EB) or dihydrotestosterone propionate (DHTP). Again, we found that gonadally intact ArKO males show pronounced behavioral deficits affecting their male coital behavior as well as their olfactory investigation of volatile body odors but not that of soiled bedding. Deficits in male coital behavior were largely corrected following adult treatment with EB and the androgen DHTP, suggesting that estradiol has prominent activational effects on this behavior. By contrast, adult treatment with EB to either castrated or gonadally intact ArKO males did not stimulate olfactory investigation of volatile body odors, suggesting that this impairment may result from a lack of proper organization of this behavior during ontogeny due to the chronic lack of estrogens. In conclusion, the present studies suggest that the behavioral deficits in sexual behavior in male ArKO mice result predominantly from a lack of activation of the behavior by estrogens. This is in contrast with earlier pharmacological studies performed on rats and ferrets that have suggested strong organizational effects of estradiol on male sexual behavior.     

Action of androgen and estrone implants on sexual behavior and reproductive organs of castrated male rabbits

Sexually mature but inexperienced male rabbits were castrated, immediately implanted with either testosterone (T), estrone (E₁), dihydrotestosterone (DHT), T + E₁, or DHT + E₁, and tested for male sexual behavior. Other castrates were not implanted, and testing was either begun immediately (Ca-I) or delayed for 4 weeks (Ca-D). Intact males served as controls (C). Latency to mount a teaser female and to ejaculate into an artificial vagina was tested twice in a morning three times per week for 8 weeks. Then, animals were sacrificed, and reproductive organs were measured. The Ca-I group responded slowly to sexual training and ceased nearly all sexual activity by 8 weeks, whereas the Ca-D males seldom displayed interest in the teaser female. Intact controls and the T and T + E₁, groups all responded to the teaser and mounted and ejaculated within a few seconds. DHT and E₁, individually maintained the sexual activity of castrates equivalent to that of C for 4–5 weeks, but the time required to mount and, particularly, to ejaculate increased thereafter. The results with DHT + E₁ were equivocal in that castrates with this hormone combination sustained sexual activity equivalent to that of the controls for 7 weeks, but one animal in particular became sexually inactive the last week of the experiment. Penis weight was at least partially maintained in all implanted castrates. Accessory sex gland weight was smallest in the DHT group and was significantly increased in the T + E₁ and DHT + E₁ groups. The largest ejaculates of fluid were obtained in the group receiving E₁ alone. These results may be interpreted to indicate a role of both androgen and estrogen centrally and peripherally in the rabbit.     

Synergistic actions of estrogen and androgen on the sexual behavior of the castrated male rabbit.

Daily injections of 2.5 mg dihydrotestosterone (DHT) for 30 days induced sexual behavior in 19% of piepuberally and 62% of postpuberally castrated New Zealand white male rabbits. Combined treatment of 2.5 mgm DHT plus 5 μgm of estradiol benzoate (EB) activated sexual behavior in 100 and 85% of prepuberally and postpuberally castrated rabbits respectively. Moreover, subjects (Ss) receiving DHT + EB displayed sexual activity in a significantly higher percentage of tests and presented a higher frequency of mounts and intromissions than those Ss receiving only DHT. The results demonstrate that estrogen synergizes with androgen (DHT) to stimulate sexual behavior in the male rabbit.     

Impaired male sexual behavior in activin receptor type II knockout mice

Integration of multiple hormonal and neuronal signaling pathways in the medial preoptic area (mPOA) is required for elicitation of male sexual behavior in most vertebrates. Perturbation of nitric oxide synthase (NOS) activity in the mPOA causes significant defects in male sexual behavior. Although activins and their signaling components are highly expressed throughout the brain, including the mPOA, their functional significance in the central nervous system (CNS) is unknown. Here, we demonstrate a neurophysiologic role for activin signaling in male reproductive behavior. Adult activin receptor type II null (Acvr2-/-) male mice display multiple reproductive behavioral deficits, including delayed initiation of copulation, reduced mount, and intromission frequencies, and increased mount, intromission, and ejaculation latencies. These behavioral defects in the adult mice are independent of gonadotropin-releasing hormone (GnRH) homeostasis or mating-induced changes in luteinizing hormone (LH) and testosterone levels. The impairment in behavior can be correlated to the nitric oxide content in the CNS because Acvr2-/- males have decreased NOS activity in the mPOA but not the rest of the hypothalamus or cortex. Olfactory acuity tests confirmed that Acvr2-/- mice have no defects in general odor or pheromone recognition. In addition, motor functions are not impaired and the mutants demonstrate normal neuromuscular coordination and balance. Furthermore, the penile histology in mutant mice appears normal, with no significant differences in the expression of penile differentiation marker genes compared with controls, suggesting the observed behavioral phenotypes are not due to structural defects in the penis. Our studies identify a previously unrecognized role of activin signaling in male sexual behavior and suggest that activins and/or related family members are upstream regulators of NOS activity within the mPOA of the forebrain.