Metabolic cooperation between Tfm and wild-type cells in mosaic mice after induction of DNA synthesis

Mosaic mice composed of androgen-insensitive Tfm and androgen-sensitive wild-type cells are constructed by virtue of the natural X inactivation: XX mice heterozygous for X-linked testicular feminization (Tfm) are reverted to males by the sex reversal (Sxr) mutation. After stimulation with testosterone, in the epididymis of the mosaic mice, the incorporation of ³H-thymidine is compared in both cell fractions. The labeling index of Tfm and wild-type cells is in the same order of magnitude. The result indicates that the stimulus for DNA synthesis exerted by testosterone is conveyed from the androgen-sensitive wild-type to the androgen-insensitive Tfm cells by metabolic cooperation on tissue level.On the other hand, the proportion of Tfm cells in the epididymal mosaic is far less than expected from X inactivation. The reason is that in the mosaic, in spite of normal proliferation, the undifferentiated Tfm cells die off steadily. Typical dense bodies are observed as signs of physiological cell death.     

Genetically directed preferential X-activation seen in mice

The nature of the O^hv mutation of the X chromosome of the mouse is defined. This locus exerts a cis position effect upon the expression of genes Tfm and Blo mapping in the same region; genes on the same chromosome as the O^hv mutation are preferentially activated and genes on the other X chromosome are usually inactive. Following the proportion of Tfm cells in the kidneys of heterozygotes confirms that the variegation seen of the locus Blo in the coat is matched in inner tissues. By introducing the sex reversal gene Sxr into these stocks, a situation can be created in which wildtype kidney cells have a selective advantage over Tfm cells in the embryonic Wolfflan duct and urogenital sinus. In spite of this difference in cell advantages, Blo coat variegation and Tfm Wolffian duct cell preponderance continue to exhibit a good correlation. This excludes the possibility that the variegation depends upon a selective advantage of cells carrying Tfm alleles after random X-inactivation and therefore reinforces the conclusion that the O^hv mutation is directly concerned in the X-activation process. A model is presented in which this locus acts as a receptor site recognized by molecules which activate one X chromosome.     

Steroid metabolism in testicular tissue of genetic mutant mice

The conversion of testosterone and progesterone to other products was studied in testes of normal BALB/C mice, testieular, feminized (Tfm o⁺) and testicular feminized mice carrying the protective (o^hv) gene. The formation of testosterone from progesterone was 11 ± 1.3% in normal mice, 2.20 ± 0.8% in (Tfm o^hv) and 0.8 ± 0.2% in (Tfm o⁺ mice. With androstenedione as substrate, both (Tfm o⁺) and (Tfm o^hv) synthesized testosterone equally well. Testicular progesterone metabolism was also compared between normal and sex reversed (sxr) mice. The data suggested that the 17β hydroxysteroid dehydrogenase was more active in normal mice whereas the testes from the sex reversed (Tfm o^hv) gave higher conversions to androstenedione. Some conversion to dihydrotestosterone and androstanediol was found in all the testicular incubations.     

Cytosol androgen receptor from kidney of normal and testicular feminized (Tfm) mice

Steroid binding has been studied in cytoplasmic extracts of normal mouse kidney, an androgen sensitive organ, and of kidney from mice affected with testicular feminization (Tfm mutant) that have inherited androgen resistance. Macromolecules that bind ³H-5α-dihydrotestosterone (DHT, the presumed active androgen in most testosterone target organs) and sediment in glycerol gradient at 8–9S can be observed in cytosol from kidney of mice of different sex, age, and hormonal history. The 8–9S component from normal females is heat labile, pronase sensitive, and dissociated by high salt to a lower molecular weight entity. The apparent equilibrium dissociation constant (K_d) for the DHT-receptor complex is 1.4 × 10^?9M, and there are about 1500 binding sites per testosterone-sensitive kidney proximal tubule cell. Cytosol from Tfm/Y ^ animals also shows a sharp peak of ³H-DHT-binding activity at 8–9S. The Tfm protein, however, has reduced affinity for DHT and binds only 10–25% as much ³H-DHT as wild-type receptor at ³H-DHT concentrations from 5 × 10^?11M to 1.2 × 10^?8M. Scatchard analysis, and studies involving competition with unlabeled steroids, relative binding of various androgens, and dissociation of the ³H-DHT-binding protein complex after extensive dialysis have led to the conclusion that Tfm kidney contains very little, if any, androgen receptor with properties similar to that found in normal kidney.     

Effect of the mouse mutants testicular feminization and sex reversal on hormone-mediated induction and repression of enzymes

The mouse mutants testicular feminization and sex reversal have been used to investigate hormone-mediated induction and repression of enzymes. Tfm/Y animals were already known to be androgen insensitive, rendering the androgeninducible enzymes ADH and -glucuronidase noninducible becuase of an inherited deficiency of a cytosol androgen-receptor complex. The animals display female secondary sexual characteristics. Sxr/+,XX animals display male primary and secondary sexual characteristics with small testes. We demonstrate (1) that the Tfm mutation is pleiotropic, preventing repression of an androgenrepressible enzyme (ornithine aminotransferase) as well as induction of androgen-inducible enzymes, (2) that an estrogen-inducible enzyme (histidine decarboxylase) is not affected by the Tfm mutation, and (3) that Sxr/+,XX animals produce enough androgen for malelike activities of androgen-sensitive enzymes. It was also discovered that histidine decarboxylase repressed by androgen in normal animals, rather than being unaffected by it in Tfm/Y animals, is in fact induced. This unexpected phenomenon is discussed and an explanation is suggested for it.This work was supported by a grant from the MRC.     

Neonatal neural organizing effects of exogenous corticosteroids on sexual differentiation of the brain in the female rat

Testosterone, deoxycorticosterone, or vehicle was administered neonatally to female Long-Evans rats. Parameters expressing the reproductive physiology and behavior of the adult animals were studied. It was found that neonatal administration of testosterone produced the expected "defeminization" and "masculinization" of the brain, affecting both the reproductive behavior and cyclicity of these females. In contrast, neonatal administration of the adrenal steroid did not affect cyclicity although it "defeminized" and "masculinized" sexual behavior, albeit to a lesser degree than testosterone. The results suggest a dichotomy in the neuroregulation of reproductive physiology and sexual behavior.     

Androgen receptors are required for full masculinization of nitric oxide synthase system in rat limbic-hypothalamic region

The neuronal nitric oxide synthase (nNOS) is involved in the control of male and female sexual behavior and its distribution in several regions of the limbic–hypothalamic system, as well as its coexistence with gonadal hormones' receptors, suggests that these hormones may play a significant role in controlling its expression. However, data illustrating the role of gonadal hormones in controlling the nNOS expression are, at present, contradictory, even if they strongly suggest an involvement of testosterone (T) in the regulation of nNOS. The action of T may be mediated through androgen (AR) or, after aromatization to estradiol (E₂), through estrogen receptors.To elucidate the role of AR on nNOS expression, we compared male and female rats with a non-functional mutation of AR (Tfm, testicular feminization mutation) to their control littermates. We investigated some hypothalamic and limbic nuclei involved in the control of sexual behavior [medial preoptic area (MPA), paraventricular (PVN), arcuate (ARC), ventromedial (VMH) and stria terminalis (BST) nuclei]. In BST (posterior subdivision), VMH (ventral subdivision), and MPA we detected a significant sexual dimorphism in control animals and a decrease of nNOS positive elements in Tfm males compared to their littermate. In addition, we observed a significant increase of nNOS positive elements in BST (posterior) of Tfm females. No significant changes were observed in the other nuclei. These data indicate that, contrary to current opinions, androgens, through the action of AR may have a relevant role in the organization and modulation of the nNOS hypothalamic system.     

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)     

Identification of prostaglandin E2 receptors mediating perinatal masculinization of adult sex behavior and neuroanatomical correlates

Prostaglandin E2 (PGE2) mediates the organization of male rat sexual behavior and medial preoptic area (MPOA) neuroanatomy during a sensitive perinatal window. PGE2 is up-regulated in response to estradiol, and initiates a two-fold increase in dendritic spines densities on neurons. All the four receptors for PGE2 and EP1-4 are present in developing POA, a critical region controlling male sexual behavior. Previous studies explored that EP receptors are involved in PGE2-induction of neonatal levels of spinophilin protein, a surrogate marker for dendritic spine formation, but did not assess behavioral masculinization. Here, we used two approaches, suppression of EP receptor expression with antisense oligonucleotides and activation of EP receptors with selective agonists, to test which receptors are necessary and sufficient, respectively, for the effects of PGE2 on behavior and neuronal morphology. In female rats, neonatal treatment with antisense oligonucleotides against EP2 or EP4 but not EP1 or EP3 completely prevented the expression of adult behavior organized by PGE2 exposure. The effects of ONO-DI-004, ONO-AE-259-01, ONO-AE-248, and ONO-AE1-329 (EP1-4 agonists respectively) were equivalent to PGE2 treatment, which suggests activating any EP receptor neonatally suffices in masculinizing sex behavior. When given alone, not all EP agonists increased neonatal POA spinophilin levels; yet giving each agonist neonatally increased adult levels. Moreover, adult spinophilin levels significantly correlated with two measures of male sexual behavior. The body of evidence suggests that EP2 and EP4 are both necessary and sufficient for PGE2-induced masculinization of sex behavior, whereas EP1 and EP3 provide redundant roles.     

Exogenous androgen during development alters adult partner preference and mating behavior in gonadally intact male rats

In the rat, neonatal administration of testosterone propionate to a castrated male causes masculinization of behavior. However, if an intact male is treated neonatally with testosterone (hyper-androgen condition), male sexual behavior in adulthood is disrupted. There is a possibility that the hyper-androgen treatment is suppressing male sexual behavior by altering the male's partner preference and thereby reducing his motivation to approach the female. If so, this would suggest that exposure to supra-physiological levels of androgen during development may result in the development of male-oriented partner preference in the male. To test this idea, male rats were treated either postnatally or prenatally with testosterone, and partner preference and sexual behavior were examined in adulthood. The principal finding of this study was that increased levels of testosterone during early postnatal life, but not prenatal, decreased male sexual behavior and increased the amount of time a male spent with a stimulus male, without affecting the amount of time spent with a stimulus female during partner preference tests. Thus, the reduction in male sexual behavior produced by early exposure to high levels of testosterone is not likely due to a reduction in the male's motivation to approach a receptive female.     

The role of neonatal NMDA receptor activation in defeminization and masculinization of sex behavior in the rat

Normal development of the male rat brain involves two distinct processes, masculinization and defeminization, that occur during a critical period of brain sexual differentiation. Masculinization allows for the capacity to express male sex behavior in adulthood, and defeminization eliminates or suppresses the capacity to express female sex behavior in adulthood. Despite being separate processes, both masculinization and defeminization are induced by neonatal estradiol exposure. Though the mechanisms underlying estradiol-mediated masculinization of behavior during development have been identified, the mechanisms underlying defeminization are still unknown. We sought to determine whether neonatal activation of glutamate NMDA receptors is a necessary component of estradiol-induced defeminization of behavior. We report here that antagonizing glutamate receptors during the critical period of sexual differentiation blocks estradiol-induced defeminization but not masculinization of behavior in adulthood. However, enhancing NMDA receptor activation during the same critical period mimics estradiol to permanently induce both defeminization and masculinization of sexual behavior.     

The role of neonatal NMDA receptor activation in defeminization and masculinization of sex behavior in the rat

Normal development of the male rat brain involves two distinct processes, masculinization and defeminization, that occur during a critical period of brain sexual differentiation. Masculinization allows for the capacity to express male sex behavior in adulthood, and defeminization eliminates or suppresses the capacity to express female sex behavior in adulthood. Despite being separate processes, both masculinization and defeminization are induced by neonatal estradiol exposure. Though the mechanisms underlying estradiol-mediated masculinization of behavior during development have been identified, the mechanisms underlying defeminization are still unknown. We sought to determine whether neonatal activation of glutamate NMDA receptors is a necessary component of estradiol-induced defeminization of behavior. We report here that antagonizing glutamate receptors during the critical period of sexual differentiation blocks estradiol-induced defeminization but not masculinization of behavior in adulthood. However, enhancing NMDA receptor activation during the same critical period mimics estradiol to permanently induce both defeminization and masculinization of sexual behavior.     

Stimulation of growth by testosterone via the mesenchyme

Summary To establish testosterone-dependent growth in organ-culture, anlagen of preputial glands from normal wild-type and from androgeninsensitive mouse embryos carrying the testicular feminization mutation (Tfm) were explanted and cultured in the presence of testosterone. Within six days a size difference developed between Tfm and wild-type explants involving length of hair follicle, amount of preputial gland tissue, and overall size.Anlagen from Tfm and wild-type preputial glands were then separated into epithelial bud and mesenchyme. Reciprocal recombinants were prepared and cultured with testosterone. In the recombinants development of hair follicles and gland tissue was inconsistent. Nevertheless, the effect of testosterone was expressed in the overall size of the explants. The size correlated with the type of mesenchyme used, but not with the type of epithelium: Androgen-insensitive Tfm epithelium combined with wild-type mesenchyme reached the same size as whole wild-type glands and wild-type/wild-type recombinants. Wild-type epithelium with Tfm mesenchyme resulted in small explants, which were in the range of the whole androgen-insensitive Tfm glands. Tfm/Tfm recombinants showed very poor growth, probably related to the fact that in this group no hair or gland structures developed.     

Testosterone-“Regulon” in the Mouse Kidney

Proximal tubule cells of the mouse kidney (metanephros) are normally extremely responsive to testosterone and its intracellular metabolites. The X-linked Tfm mutation recovered by Lyon and Hawkes seems to represent an i^s (repressor noninducible) mutation of the regulatory locus.     

The analysis of Lyon's hypothesis through preferential X-activation

Lyon's hypothesis of random X-inactivation or activation can be represented as a problem of compound binomials: (p + q)ⁿ, where p = q = 0.5 and n is the number of early cells representing a future embryo, expresses initial clonal compositions of XX-embryos immediately after the random but irreversible decision on the fate of individual X-chromosomes. Clonal compositions of a given somatic cell type in the adult body, on the other hand, can be expressed as (p′ + q′)^n′ where p′ and q′ are now variables determined by the initial clonal composition, while n′ is the number of embryonic cells which serve as the immediate progenitors of that somatic cell type. Since n is considerably larger than 10 even in the mouse, a paradox is created because so long as the initial decision process remains random, experimental tests of the hypothesis also remain impractical.We have determined the frequency with which the entire somatic cell type becomes Tfm + (0^hv) Blo monoclonal on a number of divergent organs of Tfm + (0^hv) Blo/ + + (0⁺) + heterozygotes. The results are compatible with parameters of (0.8 + 0.2)⁶⁰, determining the initial clonal compositions of these heterozygotes. Alleles, such as (0^hv) and (0⁺) of Cattanach's “controlling element” site on the X-chromosome, apparently determine the values which p and q take in the initial decision process.     

Nuclear DHT-Receptor in <Emphasis Type="Italic">Tfm</Emphasis>/<Emphasis Type="Italic">Y</Emphasis> Kidney Cell

OUR previous studies on the X-linked testicular feminization (Tfm) mutation¹ of the mouse^2–4 showed that the so-called cytosol and nuclear 5αx-dihydrotestosterone (DHT) receptor protein^5–7 might be a regulatory protein specified by the Tfm locus. The dual role of being a translational repressor in the cytoplasma and a mediator of hypertrophy in the nucleus was envisaged⁸. We found, however, another class of androgen-receptor in the polysome fraction of kidney proximal tubule cells which seems better qualified to be a translational regulator. Since a single gene locus specifies only one kind of polypeptide chain, we re-examined whether the cytosol and nuclear DHT-receptor protein underwent a true mutational change in Tfm/Y individuals.     

The role of aromatization in the development of sexual behavior of the female hamster (Mesocricetus auratus)

An aromatization inhibitor, ATD (1,4,6-andostatrien-3,17-dione) was used to test the hypothesis that aromatization of testosterone to estradiol is necessary for behavioral masculinization and defeminization of female hamsters. Pups received either 0.5 or 1.0 mg ATD or propylene glycol along with either 50 or 100 μg testosterone, 2μg estradiol, or sesame oil. Both hormones and aromatization inhibitor were given on Days 2 through 4 after birth. ATD blocked masculinization of sexual behavior produced by testosterone but did not block the masculinizing effects of estradiol. ATD also blocked the defeminizing effect of testosterone but not estradiol. These data support the aromatization hypothesis.     

Activation and differentiation of sexual behavior and translocation of hypothalamic estrogen receptors in rats by 6α-fluorotestosterone

Androgens classified as nonaromatizable in placental assay systems typically do not mimic testosterone's effects on sexual behavior in rats. 6α-Fluorotestosterone is an exception. To pursue this challenge to the aromatization hypothesis, we compared several behavioral and neuroendocrine effects of 6α-fluorotestosterone propionate (6α-fluoro-TP) with those of testosterone propionate (TP). Even at a very low dose (6.25 μg/100 g/day), 6α-fluoro-TP maintained most aspects of male sexual behavior as well as TP. It was slightly less potent than TP for inhibiting gonadotropin secretion (testicular development) in prepubertal males. Given neonatally, these androgens were equally likely to induce anovulatory sterility. 6α-Fluoro-TP defeminized sexual development in females and neonatally castrated males half as effectively as TP based on lordosis:mount ratios following estrogen and progesterone therapy in adulthood. Neither androgen masculinized sexual behavior. The behavioral effects of 6α-fluoro-TP correspond to its ability to inhibit cell nuclear accumulation of 17β-[³H]estradiol in the hypothalamuspreoptic area. When injected on a schedule like that used to activate male sexual behavior, the two androgens reduced estrogen uptake equally. When injected into adult castrates on a schedule like that used to defeminize sexual development, 6α-fluoro-TP blocked estrogen uptake half as well as TP. 6α-Fluorotestosterone did not alter estrogen uptake when injected simultaneously with 17β-[³H]estradiol. These data suggest that 6α-fluorotestosterone activates male behavior and defeminizes development because it translocates estrogen receptors in the brain, probably via an aromatized metabolite. Hence androgen aromatizability in the placenta may not reflect neural metabolism and cannot predict the behavioral or neuroendocrine effects of androgens.     

Copulatory behavior of adult tamarins (Saguinus fuscicollis) castrated as neonates or juveniles: Effect of testosterone treatment

The sexual interactions of Saguinus fuscicollis males castrated as neonates, at 37 days of age, or prepubertally with adult intact females were studied. Prepubertally castrated males were observed while receiving testosterone, and while being treated with saline. Males castrated neonatally or at 37 days of age were observed while receiving testosterone. Neonatal castrates had previously been studied without hormone treatment and therefore no control condition was included for these animals. Prepubertally castrated males showed Mounts, Mounts with Thrusts, and Sexual Tongue Flicking when treated with saline only. In three of the four males, all measures of sexual behavior increased with testosterone treatment. Neonatally castrated males had failed to display any mounting or thrusting without testosterone treatment during a previous study. During the present study, three of the four males did not respond to testosterone treatment with sexual behavior. The fourth male and one male castrated at 37 days of age displayed some sexual behavior. These results suggest that most neonatally castrated males are not able to respond to testosterone with the activation of copulatory behavior. The findings are consistent with the hypothesis that in callitrichids the sensitive period for behavioral differentiation is shifted into neonatal life. However, some neonatally castrated males show a weak response to testosterone. This may reflect an extended and perhaps partially prenatal period of sensitivity.     

Putative androgen receptors distinguished in wild-type and testicular-feminized (Tfm) mice.

The reduced level of putative androgen receptor in the mouse mutant, testicular feminization (Tfm), chromatographs on DNA-cellulose differently from the bulk of wild-type receptors. While the elution maximum for extracts of Tfm/Y kidney is in the 180–190 mM NaCl range, wild-type kidney extracts exhibit two maxima of elution at 140–150 mM NaCl and 180–190 mM NaCl, respectively. For hypothalamus-preoptic area, Tfm/Y has one elution maximum at approximately 180 mM NaCl, while the wild-type exhibits a major elution maximum at 140–150 mM NaCl, with a minor peak at approximately 180 mM NaCl. Mixing experiments between wild-type and Tfm/Y cytosols reveal that the different characteristic elution patterns are intrinsic to the binding complexes and are not conveyed simply by other soluble factors. The distinctive pattern for Tfm indicates that the mutation does not cause merely a reduced level of wild-type receptor. Rather the residual receptor of the mutant may be either an abnormal protein or a minor form of wild-type receptor, not readily seen in wild-type tissue due to the presence of more preponderant species. Differences in the elution profiles of androgen receptor species of wild-type kidney with the two bound androgens, testosterone and dihydrotestosterone, are also presented. A model of the androgen receptor system is proposed which includes several binding classes for androgen ligands and metabolites. In light of aromatization of androgens to estrogens and its probable role in some androgenic responses, we include the “estrogen receptor” in this mechanism.