Sexual differentiation of the rodent hypothalamus: hormonal and environmental influences

Brain sexual differentiation is a complex developmental phenomenon influenced by the genetic background, sex hormone secretions and environmental inputs, including pollution. The main hormonal drive to masculinize and defeminize the rodent brain is testosterone secreted by the testis. The hormone does not influence sex brain differentiation only in its native configuration, but it mostly needs local conversion into active metabolites (estradiol and DHT) through the action of specific enzymatic systems: the aromatase and 5alpha-reductase (5alpha-R), respectively. This allows the hormone to control target cell gene expression either through the estrogen (ER) or the androgen (AR) receptors. The developmental profile of testosterone metabolizing enzymes, different in the two sexes, is therefore of the utmost importance in affecting the bioavailability of the steroids active in brain differentiation. Widely diffused pollutants, like polychlorinated biphenyls (PCBs) are able to affect the production and/or action of testosterone metabolites, exerting detrimental influences on reproduction and sex behavior. The main studies performed in our and other laboratories concerning the pattern of expression and the control of the enzymatic systems involved in brain androgen action and metabolism are shortly reviewed. Some recent data on the influence exerted by PCBs on these metabolic systems are also reported.     

Sexual differentiation and the Kiss1 system: hormonal and developmental considerations

The nervous system (both central and peripheral) is anatomically and physiologically differentiated between the sexes, ranging from gender-based differences in the cerebral cortex to motoneuron number in the spinal cord. Although genetic factors may play a role in the development of some sexually differentiated traits, most identified sex differences in the brain and behavior are produced under the influence of perinatal sex steroid signaling. In many species, the ability to display an estrogen-induced luteinizing hormone (LH) surge is sexually differentiated, yet the specific neural population(s) that allows females but not males to display such estrogen-mediated "positive feedback" has remained elusive. Recently, the Kiss1/kisspeptin system has been implicated in generating the sexually dimorphic circuitry underlying the LH surge. Specifically, Kiss1 gene expression and kisspeptin protein levels in the anteroventral periventricular (AVPV) nucleus of the hypothalamus are sexually differentiated, with females displaying higher levels than males, even under identical hormonal conditions as adults. These findings, in conjunction with accumulating evidence implicating kisspeptins as potent secretagogues of gonadotropin-releasing hormone (GnRH), suggest that the sex-specific display of the LH surge (positive feedback) reflects sexual differentiation of AVPV Kiss1 neurons. In addition, developmental kisspeptin signaling via its receptor GPR54 appears to be critical in males for the proper sexual differentiation of a variety of sexually dimorphic traits, ranging from complex social behavior to specific forebrain and spinal cord neuronal populations. This review discusses the recent data, and their implications, regarding the bi-directional relationship between the Kiss1 system and the process of sexual differentiation.     

Sexual differentiation of brain and behavior in quail and zebra finches: Studies with a new aromatase inhibitor, R76713

In many species of vertebrates, major sex differences affect reproductive behavior and endocrinology. Most of these differences do not result from a direct genomic action but develop following early exposure to a sexually differentiated endocrine milieu. In rodents, the female reproductive phenotype mostly develops in the absence of early steroid influence and male differentiation is imposed by the early action of testosterone, acting at least in part through its central conversion into estrogens or aromatization. This pattern of differentiation does not seem to be applicable to avian species. In Japanese quail (Coturnix japonica), injection of estrogens into male embryos causes a permanent loss of the capacity to display male-type copulatory behavior when exposed to testosterone in adulthood. Based on this experimental result, it was proposed that the male reproductive phenotype is “neutral” in birds (i.e. develops in the absence of endocrine influence) and that endogenous estradiol secreted by the ovary of the female embryo is responsible for the physiological demasculinization of females. This model could be recently confirmed. Females indeed display a higher level of circulating estrogens that males during the second part of their embryonic life. In addition, treatment of female embryos with the potent aromatase inhibitor, R76713 or racemic vorozole™ which suppresses the endogenous secretion of estrogens maintains in females the capacity to display the full range of male copulatory behaviors. The brain mechanisms that control this sexually differentiated behavior have not been identified so far but recent data suggest that they should primarily concern a sub-population of aromatase-immunoreactive neurons located in the lateral parts of the sexually dimorphic preoptic nucleus. The zebra finch (Taeniopygia guttata) exhibits a more complex, still partly unexplained, differentiation pattern. In this species, early treatment with exogenous estrogens produces a masculinization of singing behavior in females and a demasculinization of copulatory behavior in males. Since normal untreated males sing and copulate, while females never show these behaviors even when treated with testosterone, it is difficult to understand under which endocrine conditions these behaviors differentiate. In an attempt to resolve this paradox, we recently treated young zebra finches with R76713 in order to inhibit their endogenous estrogens secretion during ontogeny and we subsequently tested their behavior in adulthood. As expected, the aromatase inhibitor decreased the singing frequency in treated males but it did not affect the male-type copulatory behavior in females nor in males. In addition, the sexuality differentiated brain song control nuclei which are also masculinized in females by early treatment with estrogens, were not affected in either sex by the aromatase inhibitor. In conclusion, available data clearly show that sexual differentiation of reproductive behaviors in birds follows a pattern that is almost opposite to that of mammals. This difference may be related to the different mechanisms of sex determination in the two taxa. In quail, the ontogeny of behavioral differentiation is now well understood but we only have a very crude notion of the brain structures that are concerned. By contrast, in zebra finches, the brain mechanisms controlling the sexually differentiated singing behavior in adulthood have been well identified but we do not understand how these structures become sexually dimorphic during ontogeny.     

Sexual experience modulates neuronal activity in male Japanese quail

After an initial increase, repeated exposure to a particular stimulus or familiarity with an event results in lower immediate early gene expression levels in relevant brain structures. We predicted that similar effects would occur in Japanese quail after repeated sexual experience within brain areas involved in sexual behavior, namely, the medial preoptic nucleus (POM), the bed nucleus of stria terminalis (BST), and the nucleus taeniae of the amygdala (TnA), an avian homolog of medial amygdala. High experience subjects copulated with a female once on each of 16 consecutive days, whereas low experience subjects were allowed to copulate either once or twice. Control subjects were never exposed to a female. High experience subjects were faster to initiate sexual interaction, performed more cloacal contacts, and completed each cloacal contact faster than low experience subjects. Low experience subjects showed an increase in egr-1 (ZENK) expression, an immediate early gene product used as marker of neural activation in birds, in the areas of interest. In contrast, in high experience animals, egr-1 expression in the POM, BST, and the periaqueductal gray (PAG) was not different than the level of expression in unmated controls. These results show that experience modulates the level of immediate early gene expression in the case of sexual behavior. Our results also indicate that immediate early gene expression in specific brain areas is not necessarily related to behavioral output but depends on the behavioral history of the subjects.     

Sexual differentiation in higher plants

As in all organogenetic studies, sex differentiation implies three fields of research: the identification of macsomalecular markers specific for stamens or carpels }expression program); the analysis of the signals (such as phytohormones{ inducing them: and finally the knowledge of the regulator genes (sex detsrmination genes).
In dioecious plants, sex determination (male and female genes or combinations of heterochromosomes) occurs at the fertilization stage. These regulators probably act early by means of inducers; their action is perceptible mainly when reproductive organs develop, and the existence of target cells able to respond to the inductive message is obvious. Experimental control of sex expression by phytohormones leads to the repression of the normal organogenetic program (induced by the presence of the sex genes) arrd to the induction of the opposite program (normally incompatible with these genes).
Analogous mechanisms occur in monoecious plants, but here the male program is always expressed before the female one. Experimental control of sex expression suggests that the successive induction of first male and then the female program results from inverse gradients of male and female signals. Sex differentiation studies of monaeciousness are more difficult than studies on dioeciousness since, in all the cells. not only male and female programs co-exist as in dioeciousness, but also male and female sex penes and signals.
The present model has been tested in the dioecious Mercurialisannua (2n = 16) and some other species, and the results that have been established are described.     

Triiodothyronine influences quail myoblast proliferation and differentiation*

The influence of triiodothyronine (T3) on avian myoblast proliferation and differentiation was studied in secondary cultures using plating densities of 2500 and 7000 cells/cm². Culture media were depleted of T3 (control myoblasts) and increasing amounts were then added to concentrations of 0.6, 3 and 15 nM T3 (treated myoblasts). Independent of the cell density, T3 induced a dose-related decrease in myoblast proliferation measured by cell number, doubling time and ³H-thymidine incorporation. However, with the lower plating density, this influence was delayed, occurring only after the third day of culture for 0.6 nM T3-treated myoblasts and simultaneous with the onset of myosin heavy chain accumulation. Moreover, when myoblasts were exposed to BrdU for 48 h, the T3 growth inhibitory effect disappeared, thus showing that this effect was clearly linked to differentiation. In addition, we have shown that T3 induced an early fusion of myoblasts: 65% of the maximal value of the fusion index was reached on day 3 in the T3-treated cells in comparison to 25% in the control myoblasts. This hormone also enhanced accumulation of muscle-specific proteins (connectin, acetylcholine receptors, myosin heavy chain), tested by cytoimmunofluorescence, ELISA, binding experiments and Western blot. All these results show that T3 increased myoblast differentiation through a pathway including myoblast withdrawal from the cell cycle. The influence of T3 could partly explain its previously reported positive effect on the number of muscle fibers.     

Hormonal differentiation of sexual behavior in Japanese quail

The effect of hormones on the development of Japanese quail during the postembryonic period was examined. First, subcutaneous implants of estradiol monobenzoate (EB) and testosterone propionate (TP) were implanted 6–12 hr after hatching. EB and TP had no effect on the differentiation of sexual behavior in genetic males or females. However, EB had marked feminizing effects on plumage in genetic males. Second, the role of gonadal hormones during development was examined by gonadectomizing males and females 6–12 hr after hatching and treating them intramuscularly with EB or TP as adults. EB-treated adult females displayed sexual behavior typical of the genetic female and developed female plumage. A significant proportion of TP-treated females (57%) displayed male sexual behavior patterns. Cloacal gland development and male-type vocalizations were induced. EB-treated males displayed either male or female sexual patterns depending on the stimulus conditions. Third, to test whether bisexuality in gonadectomized males and females is maintained despite steroid treatment and expression of sexual behavior in adulthood, gonadectomized quail which were originally treated with EB received TP and vice versa. The results indicate that in the absence of gonadal hormones after hatching female quail remain bisexual until exposed to estrogen, whereas gonadectomized male quail retain behavioral bisexuality irrespective of prior estrogen or androgen exposure.     

Sexual determination and differentiation in teleost fish

The present work reviews the latest information on the cellular, molecular and physiological aspects of sexual determination and differentiation in teleost fish. The group exhibits a large variety of mechanisms of sexual determination. These may be genetic, or depend on environmental conditions such as temperature, pH, and social factors, all of which can influence the proportion of the sexes. Additionally, sex steroids play an important role in the regulation of sexual differentiation. The patterns of gonadal sexual differentiation are diverse, and species may be hermaphroditic or gonochoristic, some of the latter displaying juvenile hermaphroditism. In recent years, several genes involved in the sexual determination and differentiation pathways in vertebrates, particularly in mammals, have also been characterized in teleosts. Conserved as well as diversified functions have been proposed.     

Effects of hormonal pretreatment of cardiac necrosis in the Japanese quail.

Male Japanese quail castrated and treated with beta-estradiol-3-benzoate for 3 wk were more resistant than sham-operated males to the necrogenic effects of massive doses of isoproterenol. The estrogen-treated castrate also weighed significantly more and had significantly lower hematocrit ratios than either the sham-operated male or the intact male. These results indicate that there is a definite sex-related protective effect this type of cardiac necrosis which cannot be explained on the basis of differences in body weight.     

Gene controlling a differentiation step in the quail melanocyte

Albino mutation in animals blocks pigmentation owing to a deficiency in tyrosinase, although it does not affect the differentiation of colorless melanocytes from the neural crest. In the albino Japanese quail (al, sex-linked), it was demonstrated that morphologically normal melanocytes differentiated from neural crest cells in culture and that these cells contained unmelanized melanosomes as expected for the mutant cells. The mutant melanocytes, however, were shown to exhibit tyrosinase activity in the Golgi-endoplasmic reticulum-lysosome region and in the Golgi vesicles. Our results seem to indicate that the mutation at the al locus affects the transport of tyrosinase from the Golgi area to melanosomes.     

Sexual differences in hormonal control of release calls in bullfrogs

Release calls in anuran amphibians are given when animals are inappropriately clasped by others. Since other call types, such as mate calls, are sexually dimorphic in frogs and toads, sonogram analysis was used to determine whether release call characteristics might also be sexually dimorphic in bullfrogs (Rana catesbeiana). Only intercall intervals differed significantly between males and females. Call duration, dominant frequency characteristics, and the display of secondary or tertiary harmonic frequencies were similar in both sexes. In the spring, but not the fall, calling rates were significantly lower in female bullfrogs, compared to males. Females also had significantly lower plasma androgen concentrations and higher plasma estrogen in the spring, compared to males. In both sexes, plasma androgen and estrogen were significantly higher in the spring, compared to the fall. The neuropeptide arginine vasotocin significantly decreased release call rates in females in the spring while it significantly increased rates in males. Vasotocin had no significant effect in the fall. Prostaglandin E2 significantly inhibited release calling in both males and females. On the other hand, prolactin significantly inhibited calling in female bullfrogs, but had no affect in males. Thus, although acoustic characteristics of release calls were similar in male and female bullfrogs, hormonal control of call rates was sexually dimorphic and seasonally variable.     

Sexual differentiation and development in the malaria parasite

The protozoan parasites belonging to the genus Plasmodium have a complex life cycle in which the asexual multiplication of parasites in the vertebrate host alternates with an obligate sexual reproduction in the mosquito. Gametocytes (male and female) produced in the vertebrate host are responsible for transmitting parasites to mosquitoes. Although our understanding of the biology and genetics of sexual differentiation in Plasmodium is expanding, the most basic questions concerning molecular mechanisms of sexual differentiation and sex determination still remain unanswered. Recently, insight into the control of this complex process in P. falciparum and P. berghei has come from studying parasite mutants with aberrant capacities for gametocyte production. Here, Cheryl-Ann Lobo and Nirbhay Kumar review these analyses in P. falciparum.     

Smelling wrong: hormonal contraception in lemurs alters critical female odour cues

Animals, including humans, use olfaction to assess potential social and sexual partners. Although hormones modulate olfactory cues, we know little about whether contraception affects semiochemical signals and, ultimately, mate choice. We examined the effects of a common contraceptive, medroxyprogesterone acetate (MPA), on the olfactory cues of female ring-tailed lemurs (Lemur catta), and the behavioural response these cues generated in male conspecifics. The genital odorants of contracepted females were dramatically altered, falling well outside the range of normal female variation: MPA decreased the richness and modified the relative abundances of volatile chemicals expressed in labial secretions. Comparisons between treatment groups revealed several indicator compounds that could reliably signal female reproductive status to conspecifics. MPA also changed a female''s individual chemical ‘signature’, while minimizing her chemical distinctiveness relative to other contracepted females. Most remarkably, MPA degraded the chemical patterns that encode honest information about genetic constitution, including individual diversity (heterozygosity) and pairwise relatedness to conspecifics. Lastly, males preferentially investigated the odorants of intact over contracepted females, clearly distinguishing those with immediate reproductive potential. By altering the olfactory cues that signal fertility, individuality, genetic quality and relatedness, contraceptives may disrupt intraspecific interactions in primates, including those relevant to kin recognition and mate choice.     

Bismuth autometallography: protocol, specificity, and differentiation.

We provide a detailed protocol of the autometallographic bismuth technique and evaluate the specificity of the technique. We show by the multi-element technique "proton-induced X-ray microanalysis" (PIXE) that the autometallographic grains contain silver, bismuth, and sulfur, proving that autometallography can be used for specific tracing of bismuth bound as bismuth sulfide clusters in tissue sections from Bi-exposed animals or humans. In sections from animals exposed concurrently to selenium and bismuth, the autometallographic grains also contain selenium. This demonstrates that, if present in excess in the organisms, selenium will bind to exogenous bismuth, creating bismuth selenide clusters. As a further possible control for specificity and as a tool for differentiating among autometallographically detectable metals in sections containing more than one, we describe how bismuth sulfide clusters can be removed from Epon-embedded tissue sections by potassium cyanide.     

Sexual differentiation in Asparagus officinalis L.

Summary Using an immunological method we assayed the levels of auxin, abscisic acid and three cytokinins (transzeatin riboside, dihydrozeatin riboside, isopentenyladenosine) in flowers of female and male plants of Asparagus officinalis L. at different stages of development. The largest differences between the sexes were found for auxin: auxin content was found to be about three times higher in young male flowers than in female flowers at a corresponding developmental stage. In order to identify some of the biochemical markers linked to sex differentiation, we also examined peroxidase isoenzyme patterns during flower development. We found five flower-specific peroxidase bands, three of which appear to be localized in the anthers. In young flowers still sexually undifferentiated in their morphology these bands are present in both sexes. They subsequently rapidly disappear in the female flower (approximately at the same time as when anther development is blocked), while they persist for a much longer time in the male. The temporary presence of these peroxidase isoenzymes in female young flowers together with the large difference in auxin content indicate that the stage of the young flower is a crucial moment in the process of sex determination.     

Sexual differentiation in Asparagus officinalis L.

Summary Sexual dimorphism in the dioecious plant Asparagus officinalis L. was examined by two-dimensional (2-D) electrophoresis of both total proteins and newly synthesized proteins from cladophylls (leaves), whole mature flowers and homologous sex organs (i.e. true female ovaries and small sterile ovaries from male flowers). Polypeptides isolated from cladophylls of male and female plants were practically indistinguishable; the flowers, however, showed a distinct set of specific proteins, some of which differed between the two sexes. While the total protein profiles of isolated ovaries from male and female plants were very similar, the patterns were strikingly different after the tissues were pulsed with ³⁵S-methionine: mature male ovaries showed a number of newly synthesized proteins, while in female ovaries only a few molecular species were actively synthesized.