Female Brain Maintained by Methylation

Development of female sexual behaviors requires DNA methylation in the preoptic area of the rodent brain. 

By Anna Azvolinsky | March 30, 2015

WIKIMEDIA, RAMADifferences in male and female rodent sexual behaviors are programmed during brain development, but how exactly this occurs is not clear. In the preoptic area (POA) of the brain—a region necessary for male sex behavior—the female phenotype results from repression of male-linked genes by DNA methylation, according to a study published today (March 30) in Nature Neuroscience.

There is very little known about how the brain is masculinized—and even less about how it is feminized—even though the question has been studied for more than 50 years, said Bridget Nugent, study author and now a postdoctoral fellow at the University of Pennsylvania.

These sex differences in the brain are programmed toward the end of fetal development, through to one week after birth in rodents. In males, testicular hormones drive masculinization of the brain; this was thought to occur by direct induction of gene expression by hormone-associated transcription factors. Because a feminized brain occurred in the absence of ovarian hormone signals, most researchers assumed that the female brain and behavior was a sort of default state, programmed during development when no male hormones are present. But the downstream mechanisms of how hormones can modify gene expression were not previously known.

“This study reveals that DNA methylation plays an important role in regulating sexual differentiation,” said Nirao Shah, who also studies the neural basis for sex-specific behaviors at the University of California, San Francisco, but was not involved with the work.

“Our understanding that the female state of the brain is the default still stands. What changes now, because of this study, is our thinking as to how the default state is preserved,” said Geert de Vries, director of the Neuroscience Institute at Georgia State University who studies how sex influences the developing brain but was not involved in the work. “The authors show that there is active repression of the masculine brain program, and that is really a novel idea.”

In other words, male hormones unleash the male program. “It’s an emancipation, you might say, of these genes that are suppressed by the female. So, evolutionarily, this evolved a lot differently than we thought,” said study author Margaret McCarthy, a professor of pharmacology at the University of Maryland School of Medicine.

Nugent, McCarthy, and their colleagues demonstrated that activity of DNA methyltransferase (Dnmt) enzymes—which control the methylation of DNA, and therefore gene repression—was lower in the POA of the brain in male versus female rats during the sensitive period. Treating newborn female rats with male hormones resulted in male-level Dnmt activity, but had no effect on older rats. Typical female rats also had twice the levels of fully methylated CpG sites throughout their genomes compared to either male or masculinized female rats.

The team was then able to drive both male morphology within the POA and male copulation behavior in female rats by administering small molecule inhibitors of Dnmts directly into the brain at birth.

While giving male hormones to females only modified female rodent sexual behavior during the critical period, inhibiting Dnmts led to a male phenotype even after the sensitive window had closed, 10 days after birth. The researchers also found that knocking out one of the Dnmt enzymes in a mouse model several days after birth—outside of the sensitive development window—resulted in female mice exhibiting male copulation behaviors.

Because disrupting DNA methylation even after female brain programming has occurred can result in masculinization, the results imply that after brain development, the female brain state still needs to be maintained by methylation patterns, said Nugent.

“Male hormones need to be there during the critical period to masculinize the brain, whereas interfering with methylation can actually override patterns of methylation that had previously been established and masculinize the female,” Nugent explained. “Females need to keep high methylation levels in the brain to maintain the female phenotype and suppress their inner male.”

“This is the first study to identify a molecular component of sexual differentiation that appears to be part of the causal change of testosterone masculinizing the male brain,” Marc Breedlove who studies sexual differentiation of the developing brain at the Michigan State University told The Scientist in an e-mail. “The idea that testosterone might be acting by decreasing methylation, thereby releasing many genes from epigenetic repression, is an important contribution, and is likely to spur a lot of new research lines,” wrote Breedlove, who was not involved in the research.

Using whole-genome RNA sequencing, the authors also showed global differences in not only RNA levels but in the expression of male and female-specific splice variants and differential promoter usage. These differences accounted for more variability between males and female mice compared to differences in gene expression, said McCarthy.

“We thought there would be spectacular differences only in those brain areas that control different sex behaviors, but its much more pervasive,” said de Vries. Taken together, these latest findings suggest that there may be more sex differences in the rodent brain than previously thought. “The brain is really a mosaic of different stories of sexual differentiation,” de Vries said.

B.M. Nugent al., “Brain feminization requires active repression of masculinization via DNA methylation,” Nature Neuroscience, doi:10.1038/nn.3988, 2015. 


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Avatar of: James V. Kohl

James V. Kohl

Posts: 516

March 30, 2015

...programmed during brain development, but how exactly this occurs is not clear. In the preoptic area (POA) of the brain...

See the section on molecular epigenetics in our 1996 Hormones and Behavior review: From Fertilization to Adult Sexual Behavior

Excerpt: "Small intranuclear proteins also participate in generating alternative splicing techniques of pre-mRNA and, by this mechanism, contribute to sexual differentiation in at least two species, Drosophila melanogaster and Caenorhabditis elegans (Adler and Hajduk, 1994; de Bono, Zarkower, and Hodgkin, 1995; Ge, Zuo, and Manley, 1991; Green, 1991; Parkhurst and Meneely, 1994; Wilkins, 1995; Wolfner, 1988). That similar proteins perform functions in humans suggests the possibility that some human sex differences may arise from alternative splicings of otherwise identical genes."

Nutrient-dependent RNA-directed DNA methylation and RNA-mediated amino acid substitutions now extend what is currently known about the biophysically constrained chemistry of protein folding to cell type differentiatioin in all cells of all genera via conserved molecualr mechanisms.  See: RNA-mediated:  "Here you will find information that links physics, chemistry, and molecular epigenetics via RNA-mediated events such as the de novo creation of olfactory receptor genes in order to encourage a public discussion of a paradigm shift."

For a historical perspective, Elekonich and Robinson (2000) linked our model to insects and Elekonich and Roberts (2005) linked it to the life history transitions of the honeybee model organism that now extend to what is known about a single amino acid substitution and cell type differentiation in the human brain during a life history transition. See: Oppositional COMT Val158Met effects on resting state functional connectivity in adolescents and adults

This links RNA-mediated metabolic networks to genetic networks in the context of what is currently known about nutrigenomics and pharmacogenomics. Clinically Actionable Genotypes Among 10,000 Patients With Preemptive Pharmacogenomic Testing

The preoptic area of the mammalian brain appears to be the source for generation of the gonadotropin releasing hormone (GnRH) pulse, which links the nutrient-dependent pheromone-controlled cell type differentiation and reproduction of yeasts to mammals. See: Feedback loops link odor and pheromone signaling with reproduction (2005)

...programmed during brain development, but how exactly this occurs is not clear.

It is perfectly clear to anyone who is not a human pheromone-denier, because the conserved molecular mechanisms of cell type differentiation do not vary accross species. The conserved molecular mechanisms cause the differences in nutrient-dependent pheromone-controlled morphological phenotypes and behavioral phenotypes manifested in the biodiversity of life on this planet. The only way to deny the involvement of human pheromones is to define them in terms used by theorists who would typically rather discuss mutations and evolution, not biologically-based cause and effect.

Avatar of: James V. Kohl

James V. Kohl

Posts: 516

Replied to a comment from James V. Kohl made on March 30, 2015

March 30, 2015

A colleague posted a link to this article in a group where one of my 1996 co-authors was banned from participation and my posts are censored. Many of the other members are biologically uninformed.

Marc Breedlove, who is mention above is an exception. See, for example: Organizational and activational effects of hormones on insect behavior 

Excerpt: "Effects of hormones on brain and behavior occur through three mechanisms: (1) behaviors both organized and activated by hormones, (2) behaviors only organized by hormones, and (3) behaviors only activated by hormones (reviewed in Arnold and Breedlove, 1985; Diamond et al., 1996)."

Diamond et al., 1996 is our Hormones and Behavior review. I'm not sure if Marc or anyone else from the group besides Simon LeVay has cited my award-winning journal article and book chapter:  The Mind's Eyes: Human pheromones, neuroscience, and male sexual preferences.

LeVay (2011) dismissed the model with claims that there is no compelling evidence for the primacy of one sense over another.

"This model is attractive in that it solves the "binding problem" of sexual attraction. By that I mean the problem of why all the different features of men or women (visual appearance and feel of face, body, and genitals; voice quality, smell; personality and behavior, etc.) attract people as a more or less coherent package representing one sex, rather than as an arbitrary collage of male and female characteristics. If all these characteristics come to be attractive because they were experienced in association with a male- or female-specific pheromone, then they will naturally go together even in the absence of complex genetically coded instructions."

"Still, even in fruit flies, other sensory input besides pheromones -- acoustic, tactile, and visual stimuli -- play a role in sexual attraction, and sex specific responses to these stimuli appear to be innate rather than learned by association 36.. We simply don't know where the boundary between prespecified attraction and learned association lie in our own species, nor do we have compelling evidence for the primacy of one sense over another."

Simply put, the fact that RNA-mediated cell type differentiation is linked from sexual orientation in yeasts to humans in our model may not mean that Feedback loops link odor and pheromone signaling with reproduction in humans. Humans could be the only species on the planet in which biologically-based cause and effect does not link the nutrient-dependent physiology of pheromone-controlled behavior to our personal preferences. Indeed, others have suggested that snake-centric evolution of primate brains may have somehow occurred to explain our responses to visual input. 

For comparison, see the link from olfaction and GnRH to behavior in octopuses. Role of olfaction in Octopus vulgaris reproduction

Excerpt: "... olfactory organ could exert regulatory action on the OL via epigenetic effects of nutrients and pheromones on gene expression (Kohl, 2013; Elekonich and Robinson, 2000).

Elekonich and Robinson (2000) takes us back to Arnold and Breedlove (1985) and to Diamond, Binstock, and Kohl (1996), and Kohl (2013) brings us forward to what is currently known about nutrient-dependent RNA-directed DNA methylation;  RNA-mediated amino acid substitutions; and cell type differentiation in species from microbes to humans. It would be great if the forward momentum would continue.

From The Scientist (1995) The book, which Kohl wrote with Robert T. Francoeur, a professor of human sexuality at Farleigh Dickinson and New York universities, explains that odors can accelerate puberty, control women's menstrual cycles, and influence sexual orientation.

Avatar of: James V. Kohl

James V. Kohl

Posts: 516

March 31, 2015

Reported at ScienceDaily as: Brain's 'gender' may be quite flexible: Mechanism that plays key role in sexual differentiation of brain described

Excerpt: Prof. McCarthy is now doing additional research on the links between the immune system and brain sex differences.

See also: An immune hypothesis of sexual orientation Excerpt: "...this ICS-hypothesis is consistent with the fact that many mfTSs can have children and does not preclude effects of odors and pheromones, but elucidates why those substances are less effective for individuals with sexual orientation altered by immune-mediated mechanisms. Importantly, Peeters et al. elegantly demonstrated that familial does not necessarily imply genetic (148). Their findings are relevant to immune-mediated alterations of SoGo because ± in the context of fetal and postnatal critical periods of immune development ± infections during pregnancy and/or early infancy might lastingly shape an individual's sexually significant ICS."

From the 1996 review she co-authored: "Parenthetically it is interesting to note even the yeast Saccharomyces cerevisiae has a gene-based equivalent of sexual orientation (i.e., a-factor and alpha-factor physiologies). These differences arise from different epigenetic modifications of an otherwise identical MAT locus (Runge and Zakian, 1996; Wu and Haber, 1995)."

RNA-directed DNA methylation links nutrient-dependent RNA-mediated cell type differentiation via amino acid substitutions in all cell types of all individuals of all genera via their physiology of reproduction. In species from microbes to man, cell type differentiation is nutrient-dependent and pheromone-controlled via fixation of the amino acid substitutions in the organized genome.

The human pheromone-deniers delayed the scientific progress on RNA-mediated cell type differention by at least two decades. During the same time they have delayed explanations of biologically-based cause and effect  that link metabolic networks and genetic networks via the conserved molecular mechanisms of the biophysically constrained chemistry of protein folding.

What's known has been detailed by others in the context of nutrigenomics and pharmacogenomics. It is great to see those who study sexual differentiation of the brain begin to catch up.

If someone asks why they did not know this, perhaps they will read Dobzhansky (1973) "...the so-called alpha chains of hemoglobin have identical sequences of amino acids in man and the chimpanzee, but they differ in a single amino acid (out of 141) in the gorilla" (p. 127).

Avatar of: James V. Kohl

James V. Kohl

Posts: 516

April 2, 2015

DNA can't explain all inherited biological traits, research shows

Excerpt: "The finding demonstrates for the first time that DNA is not solely responsible for how characteristics are inherited."

Unraveling a mystery in the 'histone code' shows how gene activity is inherited

Excerpt: "...a single amino acid difference in the structure of histone H3.3 enables it to serve as a kind of memory device for the cell, marking genes that need to remain active."

In my model of ecological adaptation, single amino acid substitutions arise in the context of nutrient-dependent RNA-directed DNA methylation, which links metabolic networks to genetic networks in all genera via the biophysically constrained chemistry of RNA-mediated protein folding.

Fixation of the RNA-mediated amino acid substitutions in the cell types of all cells of all individuals of all genera occurs via the physiology of their nutrient-dependent reproduction. In animals, the nutrient-dependent physiology of reproduction is controlled by the metabolism of nutrients to species-specific pheromones.

Nutrient-dependent pheromone-controlled ecological adaptations: from atoms to ecosystems

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