Maintenance of the spinal nucleus of bulbocavernosus and perineal muscles in female Albino Swiss rats treated with perinatal dihydrotestosterone

Female Albino Swiss rats were exposed to 5 alpha-dihydrotestosterone benzoate (DHTB) for the last 4 days of gestation, or for 4 days after birth, or both. Post-natal DHTB exposure (as well as pre-plus post-natal exposure) resulted in a 2-3-fold increase in the number of motor neurones forming the spinal nucleus of bulbocavernosus (SNB); numbers were intermediate between those found in normal females (approximately 40) and males (approximately 200). These DHTB-treated groups also possessed perineal muscles which were approximately 25% of the weight of those in normal males. Transverse sections of one of the muscles (levator ani) showed that it had approximately half the muscle fibres of normal males. Females exposed prenatally to DHTB showed a small (but significant) rise in SNB numbers, but had no recognizable perineal muscles.     

Actions of esters of testosterone, dihydrotestosterone, or estradiol on sexual behavior in castrated male guinea pigs

Prepuberally castrated male guinea pigs were treated in adulthood with estradiol benzoate, testosterone propionate, dihydrotestosterone propionate or corn oil (vehicle control). Both corn oil and estradiol benzoate were ineffective in augmenting or inducing any aspect of adult male sexual behavior. Dihydrotestosterone propionate and testosterone propionate were both effective in establishing the complete male sexual behavior pattern, although they differed in the manner in which they affected specific components. For example, males treated with testosterone propionate showed more non-intromissive but not more intromissive mounts than males treated with dihydrotestosterone propionate. In addition, the average frequency of thrusts per intromission was greater for males treated with dihydrotestosterone propionate than for males treated with testosterone propionate.     

Testosterone metabolites differentially maintain adult morphology in a sexually dimorphic neuromuscular system

The lumbar spinal cord of rats contains the sexually dimorphic, steroid‐sensitive spinal nucleus of the bulbocavernosus (SNB). Androgens are necessary for the development of the SNB neuromuscular system, and in adulthood, continue to influence the morphology and function of the motoneurons and their target musculature. However, estrogens are also involved in the development of the SNB system, and are capable of maintaining function in adulthood. In this experiment, we assessed the ability of testosterone metabolites, estrogens and nonaromatizable androgens, to maintain neuromuscular morphology in adulthood. Motoneuron and muscle morphology was assessed in adult normal males, sham‐castrated males, castrated males treated with testosterone, dihydrotestosterone, estradiol, or left untreated, and gonadally intact males treated with the 5α‐reductase inhibitor finasteride or the aromatase inhibitor fadrozole. After 6 weeks of treatment, SNB motoneurons were retrogradely labeled with cholera toxin‐HRP and reconstructed in three dimensions. Castration resulted in reductions in SNB target muscle size, soma size, and dendritic morphology. Testosterone treatment after castration maintained SNB soma size, dendritic morphology, and elevated target muscle size; dihydrotestosterone treatment also maintained SNB dendritic length, but was less effective than testosterone in maintaining both SNB soma size and target muscle weight. Treatment of intact males with finasteride or fadrozole did not alter the morphology of SNB motoneurons or their target muscles. In contrast, estradiol treatment was completely ineffective in preventing castration‐induced atrophy of the SNB neuromuscular system. Together, these results suggest that the maintenance of adult motoneuron or muscle morphology is strictly mediated by androgens. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 70: 206–221, 2010.     

A prenatal source for defeminization of female rats is the maternal ovary

Sexual behavior in laboratory rats is influenced by a variety of factors in the perinatal environment. Male rats are masculinized and defeminized in response to circulating testosterone perinatally. Females undergo a process of feminization but in some cases are exposed to testosterone. Previous work has shown that during prenatal development female rats normally undergo a partial masculinization and defeminization of sexual behavior as reflected by altered responsiveness to gonadal hormones in adulthood. In the present study we investigated whether the maternal ovary influences adult females' responsiveness to gonadal hormones. Pregnant rats were ovariectomized on Day 10 of pregnancy and their offspring tested for sexual behavior in adulthood. Following ovariectomy pregnancies were maintained by administration of systemic progesterone. In addition the ovariectomized pregnant rats were given one of three daily treatments (Days 10-21): 0.2 microgram estradiol benzoate in sesame oil and 0.1 cc propylene glycol, 5 mg of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) in 0.1 cc propylene glycol, or 0.1 cc propylene glycol. A control group was generated from SHAM operated mothers given daily control injections of propylene glycol and sesame oil. Offspring were ovariectomized in adulthood and tested for display of feminine sexual behavior in response to estradiol benzoate and progesterone or estradiol benzoate alone. Masculine sexual behavior was measured in response to testosterone propionate (TP). Feminine sexual behavior was enhanced in offspring from ovariectomized mothers given only progesterone replacement during pregnancy. Offspring from mothers treated with ATD displayed the greatest elevations in feminine sexual behavior. Estradiol treatments of ovariectomized mothers prevented the increase in feminine potential seen in offspring in the other groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neonatal sex steroids affect ventilatory responses to aspartic acid and NMDA receptor subunit 1 in rats.

We hypothesized that administration of estradiol benzoate to males and testosterone propionate to female neonatal rat pups alters sex-specific ventilatory responses to aspartic acid with correspondent changes in N-methyl-D-aspartate receptor subunit 1 (NR1) expression determined by Western blot in specific brain regions. One-day-old rat pups received estradiol benzoate, testosterone propionate, or vehicle and were studied at weanling and adulthood. Different groups had distinct patterns of changes in tidal volume and frequency of breathing after aspartic acid administration. NR1 expression in hypothalamus was altered by age, sex, and treatment. Medullary and pontine NR1 expression correlated with baseline ventilation and magnitude of the ventilatory response to aspartic acid in some groups. Thus 1) tidal volume and breathing frequency patterns in response to aspartic acid are gender, age, and treatment dependent; 2) sex, age, and exogenous steroid hormones affect NR1 expression primarily in the hypothalamus; and 3) there is correlation between NR1 expression in pons and medulla with ventilatory parameters.     

Induction of male mating behavior in androgen-insensitive (tfm) and Normal (King-Holtzman) male rats: effect of testosterone propionate, estradiol benzoate, and dihydrotestosterone

Castrated androgen-insensitive rats exhibited mounting and intromission patterns in response to testosterone propionate (TP), estradiol benzoate (EB), or EB combined with dihydrotestosterone (DHT) treatment in adulthood. Treatment with DHT alone was ineffective in stimulating male mating behavior in the mutant rats. Since androgen-insensitive rats, like normal males, have the potential to show mounting behavior following hormone treatment in adulthood, the neural substrate underlying this behavior must be masculinized during development. The effectiveness of gonadal hormones in activating the entire copulatory sequence in castrated littermate males (King-Holtzman) was also examined. TP treatment induced mating behavior in the control rats. DHT also stimulated the complete copulatory pattern, although it was not as effective as TP. The administration of EB, however, did not induce ejaculation in control rats. These results do not support the hypothesis that the activation of male mating behavior by testosterone requires its metabolite estrogen (aromatization hypothesis).     

Effect of ovarectomy of females and oestrogen administration to males during the neonatal critical period on salt intake in adulthood in rats.

The effect of interference in the neonatal critical period on water and salt solution (3% NaCl) intake by adult rats given a free choice of these fluids was studied. Consumption was expressed per animal, per 100 g body weight and as the NaCl concentration in the total daily fluid intake volume [NaCl]I. Newborn female rats were ovarectomized or sham-ovarectomized. Ovarectomy markedly reduces consumption and [NaCl]I in adulthood and brought these values close to the values in normal males. Newborn male rats were injected with 1 mg oestradiol propionate dissolved in oil or just with oil. Oestradiol propionate severely inhibited growth, but produced no changes in fluid intake per animal. The [NaCl]I values were likewise unaltered, but owing to the lower body weight comsumption values per 100 g b.w. were higher in rats given oestradiol propionate than in those given oil. The relationship of the given growth changes to the regulation of salt intake and to hypothalamic function and its sexual correlates is discussed.     

Differential effects of testosterone metabolites upon the size of sexually dimorphic motoneurons in adulthood.

This study examined the effect of testosterone and two of its metabolites on the size of motoneurons in the sexually dimorphic spinal nucleus of the bulbocavernosus (SNB) in adult male rats. Treatment of castrates with either testosterone or dihydrotestosterone maintained SNB cell size, although testosterone was more effective in this regard. However, estradiol, either alone or in conjunction with dihydrotestosterone treatment, had no effect on the size of the somata or nuclei of SNB motoneurons. These results indicate that testosterone affects SNB cell size by interacting with androgen receptors and that aromatized metabolites of testosterone are not involved in this aspect of motoneuronal plasticity in adulthood. Because the penile reflexes mediated by the SNB neuromuscular system are also sensitive to androgen but not estrogen treatment, morphological changes in SNB cells may contribute to the androgenic modulation of these reflexes.     

Differential effects of dihydrotestosterone and estrogen on the development of motoneuron morphology in a sexually dimorphic rat spinal nucleus

The rat lumbar spinal cord contains a sexually dimorphic motor nucleus, the spinal nucleus of the bulbocavernosus (SNB), whose motoneurons innnervate perineal muscles involved in copulatory reflexes. Dendritic development of SNB motoneurons is biphasic and androgen dependent. During the first 4 postnatal weeks, SNB dendrites grow exuberantly, and subsequently retract to mature lengths by 7 weeks of age. After early postnatal castration, SNB dendrites fail to grow, and testosterone replacement restores this growth. In other systems, testosterone and its metabolites, dihydrotestosterone and estrogen, are important for somatic and neural sexual differentiation. The purpose of the present study was to examine the effects of castration and dihydrotestosterone or estrogen replacement on the growth of SNB motoneuron somata and dendritic arbors. Male rat pups were castrated on postnatal (P) day 7 and treated daily with either dihydrotestosterone propionate (DHTP; 2 mg) or estradiol benzoate (EB; 100 μg) until P28 or P49. By using cholera toxin horseradish peroxidase (BHRP) histochemistry, the soma size, dendritic length, dendritic extent, and arbor area of BHRP-labeled SNB motoneurons were measured and analyzed. Both DHTP and EB treatment supported the initial exuberant growth of SNB dendrites through P28, but EB treatment was ineffective in maintaining mature, adult lengths at P49. The possible sites of hormone action and functional implications of these hormonal treatments are discussed. 1994 John Wiley & Sons, Inc.     

Neonatal dihydrotestosterone and estrogen stimulation: Effects on sexual behavior of male rats

Male rats castrated on the second day after birth (Day 2) were, for the next 10 days, given daily injections of one of five steroids or steroid combinations: 200 μg of testosterone propionate (TP); 200 μg of dihydrotestosterone propionate (DHTP); 5 μg of estradiol benzoate (EB); 5 μg of estradiol benzoate plus 200 μg of dihydrotestosterone propionate; oil vehicle (VH). Control male rats castrated on Day 90 received a sham castration and oil vehicle in the neonatal period. All animals were given TP in adulthood and tested for male sexual behavior. There was no difference in mounting activity among the subjects. Day 2 DHTP subjects displayed intromissions but were incapable of ejaculating. The more frequent display of intromissions by Day 2 DHTP animals in comparison to Day 2 VH animals could be solely due to their larger and more highly developed penes. On the other hand, the ejaculatory failure of the Day 2 DHTP subjects was attributed to some deficiency in central neural processes controlling ejaculatory mechanisms rather than inadequate penile development. Equivocal results were obtained with the Day 2 EB and Day 2 EB-DHTP animals in that only a few animals in both groups showed an ejaculatory pattern.     

Induction of vaginal mucification in rats with testosterone and 17beta-hydroxy-5alpha-androstan-3-one.

Based on histological criteria, Kingsley and Bogdanove (3) reported that the benzoate ester of 17beta-hydroxy-5alpha-androstan-3-one (5alpha-DHT), unlike testosterone propionate, is unable to induce vaginal mucification when given subcutaneously to rats. In contrats, Kennedy (4) found in estrogen-pretreated rats that both 5alpha-DHT and testosterone induced vaginal mucification as indicated by increased vaginal sialic acid concentration.To determine if esterification of these androgens altered their ability to induce vaginal mucification, ovariectomized rats, pretreated for 3 days with 0.25 mug estradiol-17beta, were treated for 8 days with either sesame oil or 7 mumoles of testosterone, 5alpha-DHT and their respective propionate and benzoate esters. All treatments except 5-alpha-DHT benzoate increased vaginal weight and vaginal mucification, as assessed histochemically and biochemically. 5alpha-DHT propionate was less effective than 5alpha-DHT while testosterone benzoate, but not propionate was less effective than testosterone. To determine if estrogens are necessary for the vaginal effects of androgens, ovariectomized and ovariectomized-adrenalectomized rats were treated with testosterone or 5alpha-DHT. Adrenalectomy did not significantly affect the vaginal response to either androgen. It is therefore concluded that androgens are capable of inducing vaginal mucification in the absence of estrogens.     

Organizational effects of testosterone,estradiol, and dihydrotestosterone on vasopressin mRNA expression in the bed nucleus of the stria terminalis

In adulthood, male rats express higher levels of arginine vasopressin (AVP) mRNA in the bed nucleus of the stria terminalis (BST) than do female rats. We tested whether this sex difference is primarily due to differences in neonatal levels of testosterone. Male and female rats were gonadectomized on the day of birth and treated with testosterone propionate (TP) or vehicle on postnatal days 1, 3, and 5 (P1, P3, and P5). Three months later, all rats were implanted with testosterone-filled capsules. Two weeks later, brains were processed for in situ hybridization to detect AVP mRNA. We found that neonatal TP treatment significantly increased the number of vasopressinergic cells in the BST over control injections. We then sought to determine the effects of testosterone metabolites, estradiol and dihydrotestosterone, given alone or in combination, on AVP expression in the BST. Rat pups were treated as described above, except that instead of testosterone, estradiol benzoate (EB), dihydrotestosterone propionate (DHTP), a combination of EB and DHTP (EB+DHTP), or vehicle was injected neonatally. Neonatal treatment with either EB or EB+DHTP increased the number of vasopressinergic cells in the BST over that of DHTP or oil treatment. However, treatment with DHTP also significantly increased the number of vasopressinergic cells over that of oil treatment. Hence, in addition to bolstering evidence that estradiol is the more potent metabolite of testosterone in causing sexual differentiation of the brain, these data provide the first example of a masculinizing effect of a nonaromatizable androgen on a sexually dimorphic neuropeptide system.     

Adult plasticity in hormone-sensitive motoneuron morphology: methodological/behavioral confounds

Changes in androgen levels can alter the structure of motoneurons in the spinal nucleus of the bulbocavernosus (SNB), a motor nucleus that innervates perineal muscles involved in copulatory behavior. While sexual activity can alter androgen levels in normal males, it has no effect on SNB motoneuron soma size or dendritic morphology (Beversdorf, Kurz, and Sengelaub, 1990). However, Breedlove (1997) reported reductions in the size of SNB somata, nuclei, and target muscles of copulating versus noncopulating castrated rats maintained on subphysiological testosterone. To reconcile the results obtained using intact versus implant paradigms, we tested the hypothesis that the implant/behavior paradigm could produce differences in hormone levels, potentially confounding sexual behavior effects on the morphology of this androgen-sensitive neuromuscular system. Young adult male rats were castrated and immediately given 5-mm Silastic implants containing crystalline testosterone. One week later, blood samples were drawn and the males were housed with receptive females (copulators) or nonreceptive females (noncopulators) or housed alone (singles). After 27 days, blood samples were drawn again, and SNB target muscles and spinal cords removed. No differences in target muscle weight or SNB somata and nuclei size were observed between copulators, noncopulators, or singles; as expected, all measures were significantly reduced relative to intact males. Radioimmunoassay showed that testosterone declined differentially over the course of the behavioral manipulation across groups, being greatest in copulators and least pronounced in single males. These data indicate that differences in sexual or housing experience can alter testosterone titers under these implant conditions, potentially confounding hormone-sensitive measures of morphology.     

Sexual behavior and penile reflexes of neonatally castrated male rats treated in infancy with estrogen and dihydrotestosterone

Male rats castrated neonatally and treated with a combination of 0.5 μg estradiol benzoate (EB) plus 50μg dihydrotestosterone propionate (DHTP) for the next 14 days displayed normal sexual behavior when injected with testosterone propionate (TP) in adulthood. Neither EB nor DHTP alone had this developmental effect inasmuch as only 20–25% of the neonatal castrates treated with just 0.1, 0.5, or 10 μg EB, or 50 μg DHTP, displayed ejaculatory responses. The periodic application of mildly painful electric shock, which has been previously shown to markedly facilitate ejaculatory responding in normal male rats, failed to improve sexual performance in these latter subjects. This was true even of the castrates treated neonatally with DHTP which frequently intromitted. Castrates treated with EB or DHTP alone neonatally were subjected to spinal transection (after testing of sexual behavior) for examination of penile reflexes. Those treated with DHTP showed normal reflexes, characterized by numerous erections and flips, indicating the presumably nonaromatizable DHTP has developmental effects on penile reflexes similar to those of testosterone. Subjects treated with EB, including four animals that had ejaculated at least once, displayed very few, if any, erections on reflex tests and no flips. These results show that sometimes intromissive and ejaculatory patterns can occur even though the animal appears to have little or no capacity for penile reflexes.     

Gonadal hormones in experimental Ancylostoma caninum infections in male Swiss albino mice

Ismail Bhai and Pandey A. K. 1982. Gonadal hormones in experimental Ancylostoma caninum infections in male Swiss albino mice. International Journal for Parasitology12: 589–591. Orchiectomy in mice increased their resistance to Ancylostoma caninum infection. Testosterone propionate increased the susceptibility of castrated animals to infection and also increased the survival of larvae in muscles, compared with sham-operated placebo-treated controls with intact testes. Estradiol benzoate increased the resistance of castrated mice as demonstrated by differential larval recoveries. The possible involvement of the small intestine as a barrier to infection and the influence of testosterone on host susceptibility to infection is discussed.     

Estrogen alters excitability but not morphology of a sexually dimorphic neuromuscular system in adult rats

In rats, motoneurons of the spinal nucleus of the bulbocavernosus (SNB) innervate the bulbocavernosus (BC) muscle, which surrounds the base of the penis. The SNB/BC is a sexually dimorphic, steroid-sensitive neuromuscular system, which is critically important in male reproductive behavior. Androgens are necessary for the development, morphology, and function of the SNB/BC system. However, estradiol (E) is also necessary for the development of the SNB/BC system, and E is capable of maintaining BC EMG activity in adulthood. In this study, we used electrophysiological and anatomical methods to examine estrogenic effects on BC EMG activity. We used a modified H-reflex testing method to investigate polysynaptic reflex characteristics in intact males, castrates, and castrates treated short term with estradiol benzoate (EB). Measures of EMG activity, response latency, and spike count were altered in castrates, but maintained in EB-treated castrates to the levels of intact males. Furthermore, estrogenic effects were found in EMG activity that could be isolated to the periphery of the SNB/BC system. BC NMJ size and muscle fiber area have been demonstrated to be hormone sensitive, and we examined these for possible correlates of E's effects on BC EMG activity. BC muscles of intact males, castrates, and short-term EB-treated castrates were fixed and stained with zinc iodide and osmium tetroxide. NMJ size and muscle fiber area did not differ between groups. Together, these data suggest that E treatment results in changes in the neuromuscular periphery that maintain BC EMG activity, but this effect cannot be accounted for by changes in NMJ size or muscle fiber area.     

Effect of neonatal testosterone and oestradiol treatment on the development of the hypothalamo-hypophysial axis in the female rat.

The hypothalamic LH-RH content and the concentrations of pituitary and plasma LH were measured at various ages in female rats treated daily with 10 micrograms testosterone propionate or 10 micrograms oestradiol-17beta from birth to Day 15. Persistent vaginal oestrus was induced in all the treated rats. Both hormones significantly reduced the hypothalamic LH-RH content and pituitary and plasma LH concentrations. Hypothalamic LH-RH increased after cessation of treatment but pituitary LH did not return to normal levels. Plasma LH levels were significantly lower than those in control rats. It is concluded that testosterone propionate and oestradiol-17beta (1) have a direct negative feed-back influence on the hypothalamus in the neonatal female rat; (2) alter the normal pattern of plasma and pituitary LH in developing female rats; (3) prevent the cyclic secretion of plasma LH after maturity; and (4) probably cause a chronic impairment in the release of LH-RH.     

Organizational effects of testosterone,estradiol, and dihydrotestosterone on vasopressin mRNA expression in the bed nucleus of the stria terminalis

In adulthood, male rats express higher levels of arginine vasopressin (AVP) mRNA in the bed nucleus of the stria terminalis (BST) than do female rats. We tested whether this sex difference is primarily due to differences in neonatal levels of testosterone. Male and female rats were gonadectomized on the day of birth and treated with testosterone propionate (TP) or vehicle on postnatal days 1, 3, and 5 (P1, P3, and P5). Three months later, all rats were implanted with testosterone‐filled capsules. Two weeks later, brains were processed for in situ hybridization to detect AVP mRNA. We found that neonatal TP treatment significantly increased the number of vasopressinergic cells in the BST over control injections. We then sought to determine the effects of testosterone metabolites, estradiol and dihydrotestosterone, given alone or in combination, on AVP expression in the BST. Rat pups were treated as described above, except that instead of testosterone, estradiol benzoate (EB), dihydrotestosterone propionate (DHTP), a combination of EB and DHTP (EB+DHTP), or vehicle was injected neonatally. Neonatal treatment with either EB or EB+DHTP increased the number of vasopressinergic cells in the BST over that of DHTP or oil treatment. However, treatment with DHTP also significantly increased the number of vasopressinergic cells over that of oil treatment. Hence, in addition to bolstering evidence that estradiol is the more potent metabolite of testosterone in causing sexual differentiation of the brain, these data provide the first example of a masculinizing effect of a nonaromatizable androgen on a sexually dimorphic neuropeptide system. © 2003 Wiley Periodicals, Inc. J Neurobiol 54: 502–510, 2003     

Effects of steroid hormones and xenobiotics on the pubertal development of UDP-glucuronosyltransferase activities towards androsterone and 4-nitrophenol in Wistar rats.

Oestradiol benzoate, testosterone propionate, progesterone, corticosterone, 3-methylcholanthrene and phenobarbital were administered to Wistar rats at the pubertal period, and their effects on hepatic UDP-glucuronosyltransferase activities were determined. Pretreatment with oestradiol benzoate had a temporary suppressive effect on androsterone UDP-glucuronosyltransferase activity in rats with the high-activity phenotype of androsterone glucuronidation. The effect was marked in 40-day-old rats, but was not found in older rats. Androsterone UDP-glucuronosyltransferase activity was induced by phenobarbital in rats with the high-activity phenotype, but not in rats with the low-activity phenotype. Foster-feeding experiments showed that breast milk did not alter the genetically determined expression of androsterone UDP-glucuronosyltransferase activity in Wistar rats. In contrast, 4-nitrophenol UDP-glucuronosyltransferase activity was not affected by steroid hormones, but was highly induced by 3-methylcholanthrene.     

Hormonally mediated plasticity of motoneuron morphology in the adult rat spinal cord: a cholera toxin-HRP study.

The dorsolateral nucleus (DLN) and the spinal nucleus of the bulbocavernosus (SNB) of the rat lumbar spinal cord are sexually dimorphic groups of motoneurons that innervate striated perineal muscles involved in male copulatory behavior. Androgens control the development of these motoneurons and their target muscles, and continue to influence the system in adulthood. Given that several features of SNB motoneuron morphology have been shown to be androgen sensitive in adult male rats, we examined the effects of androgen manipulations on the morphology of motoneurons in the DLN in adult rats. Adult male rats were castrated and implanted with testosterone-filled or blank implants, or were subjected to a sham-castration procedure. Six weeks after treatment, motoneurons in the DLN were retrogradely labeled with cholera toxin-horseradish peroxidase (HRP) after injection into the ischiocavernosus (IC) muscle and their morphology assessed. Measures of the radial extent and coverage of the dendritic arbor of DLN motoneurons projecting to the IC (DLN-IC motoneurons) were similar across the groups, indicating comparable degrees of HRP transport. However, DLN-IC motoneurons in castrates with blank implants possessed both shorter dendritic lengths and smaller somas than those of castrates treated with testosterone. Castrates with testosterone implants had DLN-IC motoneurons that were significantly larger than those of sham castrates in dendritic length and soma area. These results suggest that motoneurons in the DLN, like those in the SNB, possess a significant degree of structural plasticity in adulthood which is influenced by androgens.