Localization of estrogen and androgen receptors in male reproductive tissues of mice and rats

Using immunohistochemical methods, we studied the cell-type- and species-specific expressions of estrogen receptor (ER) isoforms (ER alpha and ER beta) and androgen receptors (ARs) in the male reproductive tract and accessory sex glands of mature mice and rats. ER alpha and ER beta showed cell-type- and species-specific distributions, respectively. In contrast, AR was localized in the epithelial and stroma cells of all tissues examined in this study, in both species. In mice, the epithelial cells of the ductuli efferentes showed a strong ER alpha-immunoreaction, and those of the caput epididymis, coagulating glands, and prostate also exhibited a positive reaction. Stroma cells, except in the ductuli efferentes, showed a positive ER alpha-immunostaining. In rats, ER alpha was detected in very few cell types: the epithelial cells of the ductuli efferentes showed a strong reaction, and the stroma cells of the ampullary and urethral glands exhibited a weak reaction. ER beta was localized in the epithelial cells of the prostate in mice, while the reaction was faint or negative in both the epithelial and stroma cells of other tissues. In rats, the ER beta-immunoreaction was strongest in the epithelial cells of the ventral prostate. The epithelial cells of the corpus and cauda epididymis, ductus deferens, and urethral glands, and the stroma cells of the urethral glands were also positively ER beta-immunostained. Almost the same AR distribution pattern was observed in both species. In particular, strong AR-immunostaining was present in the epithelial cells of the caput and corpus epididymis, seminal vesicle, and ventral prostate. These results indicate that species and tissues differences should be taken into careful consideration in assessing the physiological and pharmacological effects of sex steroids (particularly estrogens) on the reproductive tissues of male rodents.     

Roles of estrogen receptors alpha and beta in differentiation of mouse sexual behavior

Sex differences in brain and behavior are ubiquitous in sexually reproducing species. Developmental differences in circulating concentrations of gonadal steroids underlie many sexual dimorphisms. During the late embryonic and early perinatal periods, the testes produce androgens, thus, male brains are exposed to testosterone, and in situ testosterone is aromatized to estradiol. In contrast, females are not exposed to high concentrations of testosterone or estradiol until puberty. In many species, neural sex differences and sexually dimorphic behaviors in adults are initiated primarily by estradiol exposure during early development. In brain, estradiol activates two independent processes: masculinization of neural circuits and networks that are essential for expression of male-typical adult behaviors, and defeminization, the loss of the ability to display adult female-typical behaviors. Here, data for the roles of each of the known estrogen receptors (estrogen receptor alpha and estrogen receptor beta) in these two processes are reviewed. Based on work done primarily in knockout mouse models, separate roles for the two estrogen receptors are suggested. Estrogen receptor alpha is primarily involved in masculinization, while estrogen receptor beta has a major role in defeminization of sexual behaviors. In sum, estradiol can have selective effects on distinct behavioral processes via selective interactions with its two receptors, estrogen receptor alpha and estrogen receptor beta.     

Sexual behavior of the naive male mouse as affected by the presence of a male and a female performing mating behavior.

Sexually naive male mice allowed to observe conspecifics engaged in copulation later exhibited a preference (p<0.01) for the odor of estrous females over that of diestrous females. They also mounted estrous females more frequently than diestrous females (p<0.001). Naive males observing noncopulating pairs or isolated females exhibited a preference (p<0.01) for the odor of diestrous females over that of estrous females but showed no difference in mounting behavior in the presence of estrous versus diestrous females.     

Localization of estrogen receptors and estrogen receptor-mRNA in female mouse thymus.

The present study was performed to demonstrate estrogen receptor (ER) and ER-mRNA in female mouse thymus. The results are as follows: (i) thymic tissue contains ER in both reticuloepithelial(RE)- and T-cell fractions, the ER level being three-fold higher in the former fraction than in the latter; and (ii) thymic tissue contains ER-mRNA at 6.2 kb, a large amount of which was localized in the RE cells and less in the T cells. From these results it is suggested that estrogen (E) mediates some immune function of the mouse thymus through its receptor within RE cells and/or T cells.     

Neurosteroids stimulate G protein-coupled sigma receptors in mouse brain synaptic membrane

Dehydroepiandrosterone, its sulfate (DHEAS) and pregnenolone sulfate, representative neurosteroids as well as (+)-pentazocine concentration-dependently stimulated the [35S]GTPgammaS binding in synaptic membranes of mouse prefrontal cortex. These stimulations were blocked by NE-100, a sigma-receptor antagonist, and by progesterone, another type of neurosteroid. The DHEAS-induced stimulation was blocked by the pertussis toxin (PTX)-treatment, and completely recovered by reconstitution of PTX-treated membranes with recombinant G(i1), but not with G(oA). DHEAS also stimulated the [35S]GTPgammaS binding in the coronal sections of mouse brain in NE-100- or progesterone-reversible manner. These findings suggest that some neurosteroids may act on metabotropic sigma receptors, and this study may be the first to show the coupling of neurosteroid binding site and G(i).     

Hypothalamic nuclear estrogen receptors and lordosis behavior in hamsters

These experiments examined the effects of a high and a low dose of estradiol benzoate (EB) in enhancing lordosis behavior and correlated these effects with the retention of hypothalamic nuclear estrogen receptors (NER) in female hamsters. Ten micrograms EB was significantly more effective in facilitating sexual receptivity in hamsters when it was followed 36 or 48 hr later by 0.5 mg progesterone (P), but not when P was given 24 hr after EB. Low levels of behavioral responses were observed in animals that received P at 24, 36 or 48 hr after 2 micrograms EB. Correspondingly, although the hypothalamic NER levels were equally elevated 24 hr after either a low or a high dose of EB, these receptor concentrations remained high at 36 and 48 hr post EB, only in those animals that received the high estrogen dose. The results of these experiments suggest that the long-term retention of NERs (which is maintained by a single high EB dose) may play an important role in the enhancement of sexual receptivity in hamsters.     

A diallel analysis of male mouse sex behavior

Seventeen sex behavior variables resulting from 538 matings by 135 males of A/J, C57BL/6J, and DBA/2J inbred mouse strains and their reciprocal hybrids were analyzed using the diallel cross, fixed-effects model for maternal effects. Proportions of total genetic variance ranged from 0.09 to 0.62. Proportions of variance due to common genic contributions of the paternal lines ranged from 0.00 to 0.33. Proportions of variance due to inter-and intra-allelic interactions ranged from 0.00 to 0.29. Only one variable showed maternal effects.Supported by USPHS grant MH 13885.     

Maternal behavior in male rats: Critical times for the suppressive action of androgens

To determine whether the first day of life is the only period during infancy when removal of the testes permanently alters maternal behavior in rats, male Sprague-Dawley rats were castrated on either Day 1, 5, 10, 25 or 70 of life and were tested as adults for maternal behavior following testosterone replacement. Males castrated on Day 1, 5 or 10 responded significantly sooner to pups and spent longer times exhibiting maternal behavior than did males castrated on Day 25 or later. However removal of the testes on the first day of life was maximally effective. Treatment with testosterone neither inhibited nor facilitated maternal behavior. Thus there is no one critical period in infancy for differentiation of the neural substrate for maternal behavior, although removal of androgen at birth is maximally effective for the onset of maternal behavior in the adult.     

Enhancement by estrogen treatment of -bungarotoxin binding in fetal mouse amygdala cells reaggregated in vitro

Dissociated amygdala cells from 17-day-old mouse fetuses were cultivated in a flask for 7 days to form aggregates. In the aggregates there developed typical synaptic structures and [125I]alpha-bungarotoxin binding capacity. An addition of estradiol (100 ng/ml) in the culture medium caused a significant increase in the toxin binding capacity. From these results it is concluded that estradiol enhances alpha-bungarotoxin binding capacity in the amygdala by exerting direct effects on the developing amygdala cells.     

Immunoneutralization of gonadotropin-releasing hormone type-III suppresses male reproductive behavior of cichlids

To investigate the roles of gonadotropin-releasing hormone (GnRH) types in reproductive behaviors, antisera against GnRH1, GnRH2 and GnRH3 were stereotaxically administered into the intracerebroventricular region to neutralize the three native GnRH types in the brain of male tilapia Oreochromis niloticus. Reproductive behaviors (nest-building and aggressive behaviors), and morphological changes of the three GnRH systems were investigated by immunocytochemistry. GnRH1, GnRH2 and GnRH3 immunoreactive fibers were significantly decreased following injections of GnRH antisera indicating successful neutralization of their respective endogenous GnRH peptides. GnRH1- and GnRH2-immunoneutralization did not inhibit reproductive behaviors but GnRH3-immunoneutralization significantly decreased nest-building ability (Saline: 26.5 ± 3.7%/day versus GnRH3: 6.1 ± 2.9%/day, P < 0.001), nest size (Saline: 0.67 ± 0.09 points versus GnRH3: 0.10 ± 0.05 points, P < 0.0002) and aggressive behavior (Saline: 2.34 ± 0.19 points versus GnRH3 1.06 ± 0.12 points, P < 0.0001). These observations provide evidence that GnRH3 is a potent neuromodulator of reproductive behaviors in male tilapia.     

Haplotype analysis of the estrogen receptor 1 gene in male genital and reproductive abnormalities

BACKGROUND: We have recently suggested that homozygosity for a specific 'AGATA' haplotype within a approximately 50 kb linkage disequilibrium (LD) block of the gene for estrogen receptor alpha (ESR1) may raise the susceptibility to cryptorchidism by enhancing estrogenic effects of environmental endocrine disruptors (EEDs). METHODS: Haplotype analysis of ESR1 was performed in 328 Japanese subjects, i.e. 70 patients with micropenis (MP), 43 patients with hypospadias (HS), 80 patients with spermatogenic failure (SF) and 135 control males. Genotyping was performed by the 5' nuclease assay. RESULTS: The LD block was identified in each of the patient groups and in the control males. The frequency of homozygotes for the specific 'AGATA' haplotype was markedly higher in the HS patients [P = 0.0000033, odds ratio [OR] = 11.26] and slightly higher in the MP patients (P = 0.034, OR = 3.64) than in the control males, and the 'AGATA' haplotype was strongly associated with HS (P = 0.0000022, OR = 11.26) and weakly associated with MP (P = 0.040, OR = 3.64) in a recessive mode. There was no significant difference between the SF patients and the control males. CONCLUSIONS: Our results support the hypothesis that homozygosity for the specific ESR1 'AGATA' haplotype may increase the susceptibility to the development of male genital abnormalities in response to estrogenic EEDs.     

Electrophysiological and ultrasonic correlates of reproductive behavior in the male rat

The purpose of the present study was to examine the electrophysiological correlates of copulatory behavior in the rat and correlate brain electroencephalographic (EEG) activity with the ultrasonic vocalizations that have been shown to play a major role in the integration of mating behavior. Ultrasonic vocalizations of male rats with chronically implanted hippocampal and cortical electrodes were monitored during mating with a female rat. Hippocampal theta rhythms were significantly correlated with high activity, mounting, intromissions, and preejaculatory excitatory behavior and were also significantly associated with 50-kHz short ultrasonic vocalizations. Postmount or postintromission behaviors (grooming, exploration) were closely correlated with an absence of ultrasonic vocalizations and the onset of irregular low-amplitude hippocampal EEG recordings. Long 22-kHz vocalizations occurred in all tests during the postejaculatory refractory period. Shorter 22-kHz vocalizations occurred during mating and were associated with unsuccessful intromissions or mounting attempts. Postejaculatory long 22-kHz vocalizations were significantly associated with irregular high-amplitude hippocampal EEG tracings, and preejaculatory short 22-kHz vocalizations were also accompanied by sleep-like irregular high-amplitude hippocampal EEG tracings with cortical spindling. The correlation of ultrasonic vocalizations with electroencephalographic and behavioral manifestations of central arousal and inhibitory processes suggests that these vocalizations are indicators of the sexual arousal of the mating pair.     

Sexual behavior, reproductive physiology and sperm competition in male mammals

Sperm competition involves competition between the gametes of two or more males of a species for fertilization of a given set of ova. Sperm competition is widespread among mammals, as in many other groups of vertebrates. Effects of sexual selection, via sperm competition, upon the evolution of reproductive physiology and behavior are much better understood in invertebrates (and especially in insects) than is the case for mammals. However, if the reproductive organs of male mammals are viewed as an integrated system for production and delivery of spermatozoa (and accessory glandular secretions) to females, then it is logical to assume that sperm competition might influence the evolution of all parts of the system, as well as associated physiological mechanisms (e.g., testicular endocrinology) and behavior (e.g., copulatory patterns). Here we analyze and review relationships between mating systems, relative testes sizes and sperm morphology, phallic morphology, circulating testosterone levels and sexual behavior in male mammals.     

Interaction of androgens and estrogens in the control of sexual behavior in male Japanese quail

A series of 4 experiments was performed to study the relative contribution of androgens and estrogens in the activation of sexual behavior in castrated male quail. The synthetic androgen methyltrienolone (R 1881) which is not metabolized in androgen target tissues activated sexual behavior in castrated birds and at the dose level of 0.5-1 mg/day/animal had the same potency as testosterone (T). However R 1881 was much more active than T in the induction of cloacal gland growth and activation of crowing, two typically androgen-dependent responses. This suggests that sexual behavior is not controlled by exactly the same mechanism as crowing or cloacal gland growth. In another experiment, estradiol (E2) alone activated sexual behavior but it is only at very high doses which had clear toxic effects that a significant behavioral activation could be observed. This questions the role of E2 as the physiological agent stimulating copulation in intact birds unless it is assumed that centrally administered E2 would be much more active compared to peripheral E2 which is exposed to a very intense peripheral catabolism. In the last two experiments, a clear synergism could be detected between 5 alpha-dihydrotestosterone (5 alpha-DHT) and E2 in the activation of sexual activity and doses of hormones could be defined which had almost no activity by themselves but significantly stimulated sexual behavior when given simultaneously. It was however impossible to define a hormonal treatment with T metabolites which restored behavior to its precastration level, a result very easily achieved with T treatments. Taken together, these data suggest that activation of sexual behavior in quail does not depend only on E2, nor 5 alpha-DHT nor even on their combined action. Considering that specific T receptors which probably do not bind 5 alpha-DHT are present in the brain, it would seem justified to reconsider the possible role played by T itself in the activation of behavior.     

Maternal behavior in the mouse: effect of estrogen and progesterone

The administration of 100 μg and 50 μg of estradiol benzoate for 5 consecutive days to adult, nulliparous. Rockland-Swiss mice that previously exhibited maternal behavior resulted in a loss of such behavior. The effect of estradiol was temporary since normal maternal activities were exhibited when the animals were retested two weeks after the termination of hormone treatment. Treatment with 5 μg and 0.5 μg estradiol benzoate did not significantly affect maternal behavior. Progesterone treatment at dosages of 1000 μg and 500 μg was without affect.     

Interferon as a modifier of estrogen receptors

Interferon (IFN-alpha) increased estrogen receptor (ER) activity in human breast cancer tissue, human uterus (endometrium), and rabbit uterus. The optimum response was obtained with three different doses of IFN-alpha 10, 100, and 1000 U per ml. Escalation of the dose up to 5000 U per ml did not increase further ER activity. Cytosol fractions with zero or low binding activity (below 10 fm per mg) did not respond to IFN-alpha treatment. Addition of antibodies against IFN-alpha resulted in abrogation of IFN-alpha effect. Use of recombinant IFN-alpha or highly purified lymphoblastoid IFN-alpha showed similar effects on ER. The mechanism of the augmentation effect of ER activity by IFN-alpha is not clear. The increase of ER activity may have important clinical applications if the same is obtained in vivo.