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Further Development 6.9: Brain Sex and Gender

Sex Determination and Gametogenesis

Neither fully Mars nor fully Venus

“Brain sex” is a controversial topic, and when it comes to behavior, there may be a great deal of difference between animal models and human subjects. Whereas rodents, fish, and birds appear to have numerous sex-specific behaviors (McCarthy 2015, 2016), humans do not appear to have “male brains” and “female brains,” and most of the behaviors overlap between the sexes. Men and women have brain areas that are structurally different, and these regions (as summarized below) generally control the hormones involved in reproductive physiology (such as the menstrual cycle) and gamete production.

Recent neurological data (Joel et al. 2015) have shown that one cannot classify brains into two statistical male/female categories, as one can classify genitalia. Even in brain areas where there are statistically significant differences between the brains of men and women, these differences can be altered by environmental agents. Moreover, each man and woman has some areas of the brain tending toward the male-prevalent and some tending to the female-prevalent; and “brains with features that are consistently at one end of the “maleness-femaleness” continuum are rare (Joel et al 2015). Rather, most brains appear to be unique ‘mosaics’ of phenotypes, some more common in females compared with males, some more common in males compared with females, and some common in both males and females. Needless to say, this idea has generated controversy (see Chekroud et al 2016; Rosenblatt 2016; Del Giudice et al 2016; Joel et al 2016).

Intrinsic sex differences

Scientists have known for a long time that the brain, like other tissues, is responsive to the steroid hormones produced by the gonads. New evidence suggests that there may even be some sex differences in the brain that become evident before the gonads mature. These regions of the brain may experience direct regulation by the X and Y chromosomes (Arnold and Burgoyne 2004).

The first indication that something besides testosterone and estrogen was important in forming sexually different structures in the brain came from studies on Parkinson disease during which embryonic rat brains were dissected before the gonads matured. These studies indicated that brains from XX embryos had more epinephrine-secreting neurons than XY embryonic brains (Beyer et al. 1991). Later studies, using microarrays and PCR, demonstrated that more than 50 genes in the mouse brain are expressed in sexually dimorphic patterns before gonad differentiation has occurred (Dewing et al. 2003). Moreover, the mouse Sry gene, in addition to being expressed in the embryonic testes, is also expressed in the fetal and adult brain (Lahr et al. 1995; Mayer et al. 1998, 2000). In humans, SRY is specifically active in the substantia nigra of the male hypothalamus, where it helps regulate the gene for tyrosine hydroxylase, an enzyme that is critical for the production of the neurotransmitter dopamine (Dewing et al. 2006). The SRY protein can have several functions, and some of them involve epigenetic regulation by histone and DNA methylation. The possibilities that SRY expression in the brain may lead to different gene expression patterns is being considered for several sex-associated normal and disease phenotypes (Sekido 2014).

Numerous regions of the brain that are not involved in sexually specific functions have been found to be sexually dimorphic, and in these instances, the sex chromosomes produce effects as important as hormones. Therefore, McCarthy and Arnold (2011) postulated that rather than there being a few hormonally dependent dimorphic structures in an otherwise “monomorphic” brain, the entire brain might be “male” or “female” as a result of sex chromosome differences. By creating XX mice with or without an SRY transgene attached to an autosome, as well as XY mice with or without Sry, Arnold and Chen (2009) were able to determine which sexually dimorphic phenotypes in mice might be caused without hormones and which by other actions of the sex chromosomes. While hormones were seen to be responsible for many of the anatomical and behavioral differences, the chromosomes themselves appeared to have some roles in sexually dimorphic differences in metabolism and in aggressive and pain-sensing behaviors (Chen et al 2013).

Hormone-associated brain sex differences

Genetic, hormonal, and environmental agents all play roles in generating sexual behaviors (McCarthy and Arnold 2011; Ngun et al. 2011). Several areas of the mammalian brain are known to be involved in sex-specific behaviors, and these brain regions are thought to be regulated by gonadal steroid hormones. The cyclic secretion of luteinizing hormone (involved in ovulation) by the pituitary in adult female rats is dependent on a lack of testosterone during the first week of the animal’s life. The luteinizing hormone secretion of female rats can be made noncyclical by giving them testosterone 4 days after birth. Conversely, the luteinizing hormone secretion of males can be made cyclical by removing their testes within a day of birth (Barraclough and Gorski 1962).

In these regions of the brain, sex hormones probably act during the fetal or neonatal stage of a mammal’s life to organize the nervous system in a sex-specific manner. Then, during adult life (especially puberty), the same hormones may have transitory motivational (or “activational”) effects. This model of the hormonal basis of sex-specific brain development and behavior is called the organization/activation hypothesis. Perinatal exposure of mice to testosterone alters the DNA methylation pattern. Remarkably, these changes in DNA methylation and subsequent gene transcription are not seen in immediately after the administration of the hormone. Rather, they are seen in the adult mice.

Ironically, the hormone chiefly responsible for determining the male neural pattern is estradiol, a form of estrogen.* Testosterone from fetal or neonatal blood can be converted into estradiol by the enzyme aromatase. This conversion occurs in the hypothalamus and limbic system—two areas of the brain known to regulate hormone secretion and reproductive behavior (Reddy et al. 1974; McEwen et al. 1977). When the estrogen receptors have been knocked out in mice, male sexual behavior has been lost completely (Ogawa et al. 2000; Kudwa et al. 2005). Thus, in rodents, testosterone exerts its effects on the nervous system by being converted into estradiol in the brain.

But the fetal environment is rich in estrogens from the gonads and placenta. What stops these estrogens from masculinizing the nervous system of a female fetus? In both male and female rats, fetal estrogen is bound by α-fetoprotein, which binds and inactivates estrogen, but not testosterone. Relationships among estradiol, aromatase, and α-fetoprotein have been analyzed by observing sexual behaviors in mice that have loss-of-function mutations for aromatase and α-fetoprotein. The brain and the behaviors of mice lacking α-fetoprotein have been defeminized, showing that α-fetoprotein prevents the female brain from receiving circulating estrogens.

Indeed, female mice whose α-fetoprotein genes have been knocked out are sterile because the brain genes controlling ovulation (such as those for gonadotropin-releasing hormone) are downregulated. However, this lack of ovulation can be reversed (and the normal female pattern of gene expression established) if such mice are also given drugs that block aromatase. Similarly, the amount of lordosis (a swayback posture taken by female rodents that permits males to mate with them) is almost completely abrogated in female mice lacking functional α-fetoprotein genes. This behavior, too, can be restored by treating the mice prenatally with aromatase inhibitors (Figure 1B; Bakker et al. 2006; De Mees et al. 2006; Bakker and Baum 2008).

While the prenatal lack of estrogen and testosterone may be critical for the formation of female brains, the feminization of the rodent brain may require estrogens after birth. This is suggested by the behavioral phenotypes of mice whose aromatase genes have been knocked out. Their female-specific behaviors (e.g., lordosis; the ability to discriminate male pheromones) are also impaired (Bakker and Baum 2008).

Stunning demonstrations that sexual dimorphism in the brain can be caused before gonadal hormone synthesis come from natural and experimental conditions in birds. One big difference between male and female finches is that large regions of the male brain are devoted to producing songs. Male finches sing; the females do not. While testosterone is important in the formation of the song centers in finches (and, when added experimentally, can cause female birds to sing), blocking those hormones in males does not prevent normal development of the song centers or singing. Genetically male birds form these brain regions even without male hormones (Mathews and Arnold 1990).

A natural experiment presented itself in the form of a bird that was half male and half female, divided down the middle. Such animals, where some body parts are male and others female, are called gynandromorphs (Greek gynos, “female”; andros, “male”; morphos, “form”). Agate and colleagues (2002) showed that the gynandromorph finch had ZZ (male) sex chromosomes on its right side and ZW (female) sex chromosomes on its left. Its testes produced testosterone, and the bird sang like a male and copulated with females. However, although many brain structures were similar on both sides, some brain regions differed between the male and female halves. The song circuits on the right side had a more masculine phenotype than similar structures on the left, showing that both intrinsic and hormonal influences were important.

Unlike mammals, sex in birds is cell-autonomous, with each cell making its own sexual decision. Circulating hormones do not usually integrate the sexual phenotype (Zhao et al. 2010).

Pheromones and the hormonal pathway

Pheromones—sex-specific chemicals secreted into the atmosphere—play major roles in sexual behaviors in rodents. If the vomeronasal organ (which is responsible for sensing pheromones in rodents and some other mammals, although such an organ is not present in humans) or the genes involved in pheromone recognition are removed from male mice, they fail to discriminate between males and females and attempt to mate with both. If this pheromone recognition system is removed from female mice, they lack certain female behaviors and acquire the full set of male courtship behaviors (including mounting, pelvic thrusting, and solicitation of females).

Thus, it appears that the neural circuitry for both male and female behaviors exists in every mouse brain, but the interpretation of pheromone signals is what distinguishes male from female brains. In females, the “feminine” pattern of behavior is activated (sexual receptivity to males, lactating behavior with pups), while the “masculine” pattern (if it’s male, fight it; if it’s female, mount it) is repressed. In males, the pheromones activate this “masculine” pattern, while the “feminine” pathway is suppressed (Kimchi et al. 2007). The interpretation of pheromone signals is thought to take place in the medial preoptic area/anterior hypothalamus region of the brain, and we know this region to be sexually dimorphic as a result of prenatal estrogen exposure. Thus, the organizational abilities of testosterone may act largely to effect changes in this small area of the brain, and once this region is organized, it will interpret the pheromone signals to activate either the male or the female sets of neurons (Baum 2009).

The roles of experience

Usually we think of DNA as controlling neural anatomy, and neural anatomy as controlling behaviors. This is the lesson that genetic mental retardation syndromes have taught us. But new research is claiming that the pathway is not one-way and that behaviors can control both gene expression and nervous system anatomy. One of the most sexually dimorphic regions of the rat central nervous system is the spinal nucleus of the bulbocavernosus (SNB). This controls the pelvic thrusting muscles during mating, and it is larger in the male. The SNB is also testosterone-sensitive, and it shrinks when rats are castrated (unless the rats are given replacement testosterone). Interestingly, the size of SNB neurons changes with sexual behavior, becoming smaller as male rats mate more frequently. “It is possible,” noted Breedlove (1997), “that differences in sexual behavior cause, rather than are caused by, differences in brain structure.”

The roles of experience in causing changes in brain gene expression and behavior were highlighted in a series of studies involving the effects of maternal care on the behaviors of rats. Maternal care during the first week of life involves grooming and licking the young pups. Those female rat pups that experience such maternal care when young will provide such maternal care to their own offspring, whereas female pups that do not receive such maternal attention will not. The licking and grooming responses are largely regulated through the estrogen-responsive neurons of the medial preoptic area (MPOA), a sexually dimorphic region of the brain. When estrogen binds to its receptors in MPOA neurons, these neurons activate the genes that encode receptors for oxytocin, the hormone involved with nursing and grooming.

So how is this trait inherited? It turns out that the key player is the experience of being licked and groomed. Licking and grooming by the mother alters the DNA methylation pattern of brain-specific enhancers in the major estrogen receptor gene (ERa) in the pups (Meaney and Szyf 2005; Champagne et al. 2006). In the MPOA neurons, licking and grooming decreases the amount of DNA methylation. This enables the Stat5 transcription factor to bind and permit the estrogen receptor gene to be transcribed at high levels. This ensures the high levels of estrogen receptors needed to stimulate licking and grooming behaviors. Thus, mothers that lick and groom their offspring tend to have daughters that will lick and groom their offspring. Cross-fostering (giving the newborn pups of “high licking and grooming” mothers to “low licking and grooming mothers” and vice versa) has demonstrated that this neonatal experience does indeed cause the gene expression differences (Cameron et al. 2008).

Interestingly, in another area of the brain, the anterior paraventricular nucleus (PVN), rat pups that experience high levels of licking and grooming have a highly methylated promoter on this gene, thereby downregulating the estrogen receptor in this region. The PVN helps regulate gonadotropins. Rats experiencing low levels of maternal licking and grooming have high levels of estrogen receptors in the PVN and correspondingly high levels of gonadotropins. As they mature, these rats are predisposed to a suite of sexual behaviors that include precocious puberty, heightened sexual activity, and lack of attention to their pups (Cameron et al. 2008). Thus, experience can create changes in gene expression and neuroanatomy. Moreover, inherited variation can come about by experience-induced changes of DNA methylation. The distinction between nature and nurture disappears in this environmental regulation of gene expression.

The human element

The mammalian examples above are exclusively from rodents. It is a very risky business extrapolating from such rodent studies to humans. Human fetuses, for instance, do not make a strong estrogen-binding protein and have a much higher level of free estrogen than do rodent embryos (see Nagel and vom Saal 2003). So although the organization/activation hypothesis explains many of the hormonal effects on rodent development, one of its fundamental assumptions—that α-fetoprotein strongly binds estrogens during prenatal development—does not in fact hold true for humans.

Human sexual behaviors differ from those of rodents in many ways, and so does brain development (see Jordan-Young 2010). Outside of physiological events such as ovulation, no sex-specific behavior has yet been identified in humans. Moreover, humans do not use pheromones as a primary sexual attractant (sight and touch being far more critical). The evidence that there are differences in brain anatomy between male homosexuals and heterosexuals has been disputed, and even so, brain anatomy can be altered by experience. No “gay gene” has been discovered, and the concordance of gender identity between identical twins is only 30%—far from the 100% expected if sexual orientation were strictly genetic (Bailey et al. 2000; CRC 2006). Moreover, behaviors that are seen as “masculine” in one culture may be considered “feminine” in another, and vice versa (see Jacklin 1981; Bleier 1984; Fausto-Sterling 1992; Kandel et al. 1995). How humans acquire gendered behaviors appears to involve a remarkably complex set of interactions between genes, hormones, nerves, and environment. As will be discussed in Chapter 25, we inherit a genome that can produce a genetically constrained range of different phenotypes. Indeed, behaviors are the “final phenotype.” They have resisted explanation because the link between genotype and behavior is relatively weak, and because behavioral phenotypes are so heavily influenced by environment.

Gender remains a very poorly understood phenotype. Moreover, it can be defined in several ways, depending on which aspect one highlights. Gender has many components, including a core gender identity (the sense of self, be it male, female, or intersex), erotic sexuality (whether one prefers or fantasizes about male, female or intersex partners), and performative gender (how one behaves in larger or smaller social groups.). This lack of clarity is especially true when it comes to sexual orientation. Not only is there no known “gay gene,” there no linear correlation between hormones and sexual orientation. What is known is that “a substantial minority of both sexes have some erotic interest in individuals of their own sex” (Hines 2011). About 5% of men in the United States report having homosexual experiences and about 18% of both men and women report experiencing sexual attraction to individuals of their same sex (Sell et al 1995). Indeed, the terms “homosexual” and heterosexual not only differ in different countries, but they depend on the individual context as well. If a male-to-female transsexual person is erotically interested in women, is she homosexual or heterosexual? (See Hines 2011).

What causes individuals to have erotic feelings towards members of their own another sex? We don’t know. This is one of the great open questions of developmental biology. We know, for instance, that prenatal hormones play a role; but not so large a role that leads to prediction or explanation. There are no measurable hormonal differences between homosexual and heterosexual men or between homosexual and heterosexual women (Mayer-Bahlberg 1977, 1979). However, hormones obviously do play some role. Those XY women who have complete androgen insensitivity are almost exclusively heterosexual, in that they prefer male sexual partners. Moreover, those women who have congenital adrenal hyperplasia and who were thus exposed in to high levels of testosterone in fetal and neonatal life have a higher percentage of homosexuality than their sisters who lack the disease. Indeed, the severity of the disease appears to correlate with the probability of homosexuality (Hines 2011.) Yet, the effects are not predictive. The most severe form of CAH is associated with homosexuality only about 50% of the time (Frisén et al 2009). Indeed, the lack of heterosexuality (and sexuality in general) may also be due to the pain and bleeding that can accompany intercourse as well as cosmetic and psychological concerns of the women.

A similar situation appears to exist with those people who have mutations in the genes regulating hormone biosynthesis. Even though raised as girls, about half those individuals whose testosterone levels rise to new levels and masculinize the body during puberty make the transition to maleness and form fertile sexual partnerships with women (Wilson et al 1993; Zucker 2002.)

Thus, there are several factors that act to determine sexual orientation. Prenatal gonadal steroids appear to be one of them, and other factors, that are not well characterized, also appear to play roles. Epigenetic alterations of histones and DNA by factors on the X and Y chromosomes may also be important players. Thus, Hines (2011) concludes, “Although a role for hormones during early development has been established, it also appears that there may be multiple pathways to a given social orientation outcome and some of these pathways my not involve hormones.” Biochemical or anatomical causes for dissatisfaction with one’s gender identity (either in homosexuality or transgendered manifestations) have still not been found (Mayer-Bahlburg 2013; Ngun and Vilain 2014.)

 

* The terms estrogen and estradiol are often used interchangeably. However, estrogen refers to a class of steroid hormones responsible (among other functions) for establishing and maintaining specific female characteristics. Estradiol is one of these hormones, and in most mammals (including humans) it is the most potent of the estrogens. The enzyme’s name, aromatase, has nothing to do with aroma (although aromas are certainly crucial to rodent sex), but refers to the destabilization of hydrogen bonds in the steroid ring structure.

 

References

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