The Genetics Behind Sex Determination and Differentiation – Part 2 of Where Do Ovaries (and Testes) Come From?

Pomegranates as a metaphor for ovaries

I began this series of posts as a background with the goal of discussing Polycystic Ovary Syndrome in trans men.  I thought that in order to understand what happens when things go wrong in ovarian function, we first need to talk about what happens when things go right. 

So first, we had a discussion about where ovaries come from, and we learned that the gonads arise from the genital ridges, indifferent embryonic structures that are bi-potential.  The genital ridges can become ovaries or testes depending on the genetic signals that push their development.

In addition, we discussed how the nascent gonads are populated by primordial germ cells (PGCs) that migrate from the yolk sac.  PGCs can become either oogonia or spermatogonia, again, depending on the genes that drive their differentiation.

In support of the young germ cells are undifferentiated somatic (i.e. non-germ) cells that eventually become supportive Sertoli cells in the testis and granulosa cells in the ovary.  In the embryonic testis, the early Sertoli cells encase the germ cells in sex cords which eventually develop into seminiferous tubules, which is where the sperm cells develop in the adult.  In the ovary, the pre-granulosa cells form follicles by joining in a single layer to encapsulate the germ cells, which then enter meiosis, subsequently becoming arrested part way through the process.

All in all, gonadal development is a relatively intriguing phenomenon when you think about it.  The primordial gonads are bi-potential and can develop into either ovaries or testes.  And the germ cells don’t even arise from within the nascent gonads!  They migrate in from outside the embryo, and are also bi-potential — they can become immature eggs or sperm.  These steps require genetic orchestration and direction, where some signaling pathways are turned on while others are inhibited — a genetic balancing act, so to speak.

So how does this all work?  And what happens when things don’t go according to plan?

In this post, we will learn about the genes and signaling pathways responsible for sex determination of the gonads.  In a subsequent post, we will talk about what happens when the genes and signals that drive gonadal development and differentiation deviate from the “master plan.”  In those cases, the results are intersex conditions, also known as disorders of sexual development (DSDs).

This series of posts is about the ovary, with the goal of talking about Polycystic Ovary Syndrome in trans men.  But in order to understand ovarian development, it’s easier to first talk about development of the testes, because it’s the absence or inhibition of signals that occur in testicular differentiation that contribute, in part, to ovarian development.

Differentiation of the Embryonic Testis

Male sex determination, as we currently know it, begins when the somatic cells differentiate into the supportive Sertoli cells by expressing the gene SRY, which, in humans, begins at 40-50 days of gestation.    The young Sertoli cells proliferate and form the sex cords around the primordial germ cells (PGCs).  The germ cells differentiate into spermatogonia through contact with the Sertoli cells. 

The gene SRY, called sex-determining region Y, was discovered in humans and mice in 1990 (Gubbay et al., 1990; Sinclair et al., 1990).  Located on the Y-chromosome, the SRY gene encodes a protein that contains sequences that both target it to the nucleus and allow it to bind to a particular DNA sequence.  This type of protein is a transcription factor in that it can turn genes on by binding to the DNA of the promoter sequences of other genes and initiate transcription of the genes’ DNA into RNA.

In essence, SRY is a master switch — it’s expression sets off a series of events that includes turning on a host of other genes and induces differentiation of cells in the genital ridge to go down the male path, ultimately resulting in the production of hormones that direct male sexual differentiation and development, including regression of the female Müllerian ducts, production of testosterone, development of the male Wolfian ducts and development of secondary sex glands and sex characteristics (reviewed in Ostrer, 2003 and Wainright & Wilhelm, 2010).  I have discussed some of these steps in another post.

[We know that there are genes and signaling factors upstream of the SRY gene that regulate its expression and turn it on when it's time for the bipotential gonad to commit to differentiation, but I will not go into those details here.  Readers are invited to read the brief review in Wainwright & Wilhelm (2010) for that information.]

So there is SRY, the master switch for male sex determination — its expression in the early Sertoli cells of the human embryonic testis reaches a peak at the 44th day of gestation and then declines to low levels up until at least 18 weeks of gestation (Hanley et al., 2000).  Its expression turns on the gene SOX9 (SRY-related HMG BOX gene 9; Sekido & Lovell-Badge, 2008) in Sertoli cells.

The expression timing and pattern of SOX9 in the developing testis is similar to that of SRY, beginning between Days 41 to 44 of gestation and continuing until at least 18 weeks (Hanley et al., 2000).  Also similar to SRY, the protein encoded by the gene SOX9 is a transcription factor that binds to DNA and turns on other genes.  Two important products of SOX9 up-regulation during male sex determination are FGF9 (fibroblast growth factor 9) and PGD2 (prostaglandin D2).

FGF9 and PGD2 feedback to the early Sertoli cells and reinforce their development, and also signal to adjacent somatic cells in the nascent testis and induce their proliferation and development to Sertoli cells (reviewed in Nef & Vasselli, 2009 and DiNapoli & Capel, 2008). These signals result in expansion of the supporting cells and growth of the developing testis.  In addition, these two factors inhibit “female” signals that would induce ovarian differentiation and development.

[FGF9 belongs to a 22-member family of proteins that have quite a list of effects on processes in both the developing embryo/fetus and adult.  PGD2 is a member of a family of lipid signaling molecules that are formed through the action of cyclooxygenase enzymes.  Both factors play important roles in normal physiology and disease.  Interested readers can learn about them in free reviews by Krejci et al., 2009 and Smyth et al., 2009.]

Differentiation of the Embryonic Ovary

When I was a baby scientist, I was taught that there were two sexes and ‘female’ was that default state.  Now we scientists know better.  We know that there are signals that drive ovarian development just as there are signals that drive testicular development.  There is no true “default” when it comes to biological sex.

In the XX embryonic gonads, SRY is absent and, unlike the testis where one pathway determines male development (i.e. SRY), differentiation of the ovary involves multiple signaling pathways (reviewed in Schlessinger et al., 2010).  One of the first steps occurs when retinoic acid from the mesonephros turns on the STRA8 gene (Stimulated by Retinoic Acid Gene 8) in the primordial germ cells, inducing them to become oogonia and enter meiosis.  This is a step that is obligatory for formation of ovarian structure (reviewed in Nef & Vassalli, 2009; Kocer et al., 2009) — without the oogonia, follicles will not form. 

Retinoic acid cannot induce differentiation of germ cells in the XY embryonic gonad because the up-regulation of SOX9 in nascent Sertoli cells induces expression of CYP26B1, a gene that encodes an enzyme that breaks down retinoic acid, thereby stopping the germ cells from entering meiosis.  In the XX embryonic ovary that does not have SOX9, there is also no CYP26B1 and so the retinoic acid is not degraded and can push the germ cells into meiosis, down the female pathway (reviewed in Piprek, 2010).

One of the main differentiation genes for the ovary is R-spondin1 (RSPO1; Roof plate-specific Spondin 1).  The gene is turned on in the somatic (pre-granulosa) cells of the nascent ovary, and the protein encoded by this gene is secreted by the cells to modulate other genes, such as WNT4 and the β-catenin pathways.  The RSPO1 pathway acts to both promote ovarian differentiation and also to suppress testis formation by silencing the SOX9 and FGF9 genes (reviewed in Nef & Vasselli, 2009).

[The Wnt family of signaling proteins were initially characterized by developmental biologists due to their importance during embryogenesis, but they also play roles in adult processes and during cancer. β-catenin is one of a group of proteins that is important in cell growth and adhesion. Wikipedia, the most accurate source for information in the world, has information about Wnt and β-catenin family members and signaling pathways.  RSPO1 is a member of the R-spondin family of genes which are important during development, particularly in the Wnt/β-catenin signaling pathway.  In addition to sex determination, RSPO1 is involved in skin differentiation and the regulation of growth of epithelial cells of intestinal crypts.]

Another ovary-promoting differentiation gene in the embryonic gonad is FOXL2 (Forkhead box L2), which encodes a winged-helix/forkhead transcription factor that is expressed in the nascent granulosa cells (reviewed in Schlessinger et al., 2010).  FOXL2 is believed to act in concert to RSPO1 to push the XX bipotential gonad in the diretion of the ovary and to block genes that promote differentiation in the direction of the testis (reviewed in Nef & Vasselli, 2009).

[FOXL2 belongs to the family of Forkhead Box (“FOX”) transcription factors that are involved in the regulation of genes important during development and other processes where there is cellular growth, proliferation and differentiation.]

 

From Nef & Vassalli, 2009

So in essence, with these competing pathways, there is a push and pull between male and female, testis and ovary.  Regarding differentiation of the gonad, Nef and Vassalli (2009) refer to these signaling processes as “complementary pathways” but DiNapoli and Capel (2008) categorize them as more of a balancing act:

“This precarious balance of the gonad between these two developmental pathways is likely what confers its dual potential.  Normally, the system employs complex cell signaling loops that reinforce a single fate decision in the supporting cell lineage and recruit all gonadal cells behind the testicular or ovarian pathways.  Defects in these reinforcing signaling loops may explain many disorders of incomplete sexual development that manifest as gonadal dysgenesis, ovotestis formation, ambiguous ductal or genitalia development, or a combination of these features.” 

In the next post of this series, we will look at what happens when this balance is lost and things don’t go according to the genetic plan of sex determination.

–ATM

PS – I apologize for the delay in making this post.  I came down with a cold, got buried by work and am now traveling again.  You should see the next couple of posts come more quickly in this series.  Thank you for your patience and for your interest in this blog!

References 

DiNapoli L, Capel B, 2008. SRY and the standoff in sex determination.  Mol Endocrinol 22:1-9

Gubbay J, Collignon J, Koopman P et al., 1990.  A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes.  Nature 346:245-250

Hanley NA, Hagan DM, Clement-Jones M, et al., 2000.  SRY, SOX9, and DAX1 expression patterns during human sex determination and gonadal development.  Mech Devel 91:403-407

Kocer A, Reichmann J, Best D, Adams IR, 2009.  Germ cell sex determination in mammals.  Mol Hum Reprod 15:205-213

Nef S & Vassalli J-D, 2009.  Complementary pathways in mammalian female sex determination.  J Biol 8:74

Ostrer H, 2003.  Genetics of Sexual Differentiation.  In:  Gynecology and Obstetrics, Sciarra JJ, ed, Vol 5, Chapt 78

Piprek R, 2010.  Molecular and cellular machinery of gonadal differentiation in mammals.  Int J Dev Biol 54:779-786

Schlessinger D, Garcia-Ortiz J-E, Forabosco A et al., 2010.  Determination and stability of gonadal sex.  J Androl 31:16-25

Sekido R & Lovel-Badge R, 2008.  Sex determination involves synergistic action of SRY and SF1 on a specific SOX9 enhancer.  Nature 453:930-934

Sinclair AH, Berta P, Palmer MS et al., 1990.  A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif.  Nature 346:240-244

Smyth EM, Grosser T, Wang M, Yu Y, FitzGerald GA, 2009.  Prostanoids in health and disease.  J Lipid Res 50 Suppl:S423-428

Wainright EN & Wilhelm D, 2010.  The game plan:  cellular and molecular mechanisms of mammalian testis development.  Curr Topics Devel Biol 90:231-262

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23 Responses to The Genetics Behind Sex Determination and Differentiation – Part 2 of Where Do Ovaries (and Testes) Come From?

  1. Lloyd says:

    This is really a fantastic series! I look forward to the rest of this series!

  2. j says:

    Do you think my ex-doctor knows all this? I really wonder! She used to give me holy pamphlets and tell me to “take it to the lord in prayer”. In other words she was trying to tell me there was no worldly cure. Charlatan!

    Sir, Thank You for doing this. You’ve taken a lot of effort. No one has done this.
    With your permission I’m putting down this link to a website for holistic health management for PCOS patients. The site helped me drop about 20 kilos and just be a healthier happier person. Weight on the waist is especially bad and it’s the first thing that has to go, BTW for those readers who dont know.

    http://www.ovarian-cysts-pcos.com/weight-loss.html

    (read the other chapters also; and get the newsletter.. its great)
    PCOS I hear from my FTM friends, can actually come in the way of transitioning. If you are overweight and 40-60% of PCOS patients move on to becoming obese, then it comes in the way of transitioning. The meds to reign in PCOS can cause blood clots in some individuals and thyroid issues. And the whole scenario is just bad news after bad news. Which is why I feel a holistic approach is most effective. If you can stop PCOS from going to the second stage which is thyroid problems (hyper/hypo), then that is what you should be aiming for. What bugs me no end is that PCOS is treated so casually by the med fraternity. Patients are left alone to deal with it. Doctors only ‘solution’ is “swallow meds”. For women PCOS is life altering enough. I’m guessing for men it’s just as bad. Especially since they are told they cannot transition.
    BTW, hello lloyd.

    Sir, after this series you have to do one for FTMs with hypo / hyper thyroid problems. How does it come in the way of their transitioning. Anyway there is hair loss with hyperthyroid and with T that worsens right?
    Take care Sir; come back soon. Look after yourself.

  3. Chuck says:

    I enjoyed your blog… right up until that one line when the science left the building and gender studies took over. That line is: “When I was a baby scientist, I was taught that there were two sexes and ‘female’ was that default state. Now we scientists know better. ” and you go on to state that sex is a spectrum.

    I have no quibble with the female default state bit being wrong and outdated and I have no trouble understanding that birth defects happen in the complicated process of sex differentiation. I do have trouble with the way you state something that is in deep dispute as a given as if the vast majority of scientists agree that sex in humans is a spectrum and we layman shouldn’t even question it. It is bad form to represent your opinion as a widely accepted scientific fact and to use the scientific community to validate your opinion when that isn’t the sceintific consensus by a long shot.

    I’m looking forward to reading more about science on your blog.

    • Thanks Chuck for your comment. It helps me identify a place in my writing that is not clear. I will try to cover more of the bases in my response.

      First, addressing your reference to ‘gender studies,’ I am not talking in any way with these posts about gender, gender expression or gender-based behavior. I am only talking about development of the gonads in this series of posts, although I think that might not have been your point anyway.

      Also, when I talk about ‘male sex’ and ‘female sex’ in this particular post series, I am only talking about the gonads, only testis and ovary. I am not talking about other reproductive organs such as the uterus or the prostate. The signals that drive differentiation of those organs, although intertwined with gonadal development in some ways, are a separate issue, at least for the purposes of this blog series. So in these posts, I am focusing on the differentiation of the testes and the ovaries, independent of any other organs. But again, I’m not sure that was your concern.

      And when I speak about “default” development of the gonads, I mean that we now know that differentiation in one direction or the other (testis or ovary) happens with genetic drivers that push differentiation in either direction (to testis or ovary). But you did not appear to have an issue with that concept either.

      So, when I talk about a ‘spectrum of sex,’ I am still only talking about the gonads. I am not talking about the external secondary sex characteristics, nor am I talking about any reproductive organs other than the gonads. And clearly, there is a spectrum of human gonadal development, with ovotestes (organs that have characteristics of both ovaries and testes) being in the middle of the spectrum. You might wish to refer to development of ovotestes as a “birth defect,” or perhaps you prefer to refer to ovotestes as a third sex? Whatever the terminology, that does not take away the diversity, the spectrum of gonadal development that occurs in humans. The majority of the cases of gonadal differentiation in humans follow the ‘master plan’ of development into either testes or ovaries, but there are intersex cases that are not just one or the other, not simply testes or ovaries, but have physiological, histological and morphological characteristics of both. Perhaps you are referring to Anne Fausto-Sterling’s paper “The Five Sexes: Why Male and Female Are Not Enough”? If so, I would not argue with that and would suggest that perhaps we are saying the same thing, just with different words.

      I’m not sure what you mean when you say that there is no scientific consensus, as intersex cases are clearly documented, and I’m not sure exactly what is in “deep dispute” that you refer to. Perhaps it’s just a difference in terminology that we choose to use? You will have to let me know.

      Have I addressed your point? If I haven’t, please tell me more about your point of view and I’ll respond. (It could be an interesting discussion.)

      • Chuck says:

        No, you haven’t addressed my point at all. I am speaking about sex, not gender, nor am I advocating 5 sexes or multigender or any of that stuff. Sticking with biology, I’m addressing your characterization of gonadal sex as a spectrum. I disagree that it is a spectrum but I do acknowledge that there are birth defects and anomolys just as in most other aspects of biology.

        You assert that it makes no difference scientifically to refer to something that goes wrong in sex differentiation as proof of a spectrum. I think that’s misleading to what has really happened. When something has gone wrong, a whole alternate viable sex hasn’t been created, nor has a true middle sex been created. This is no more a third type of sex as a person born with a third leg is a new type of tri-ped human within the human spectrum from non-ped to quad-ped. If you want to call our difference on this subject simple a difference in terminology, that’s not quite it and besides, terminology is important.

        • Thanks for the clarification. That helps.

          Regarding your example of a human with a third leg, I wouldn’t consider that a good comparison for gonadal sex. That would be like saying a person has a third ovary or a third testis. On the other hand, if a bi-potential limb could develop into either an arm or a leg, and sometimes the limb develops as something with both arm and leg characteristics, then I’d say you would have a fair comparison with gonadal development.

          Also, I am not saying that “a whole alternate viable sex” has been created. I’m only talking about the architecture of the gonads.

          And so when some of the somatic cells in the bi-potential gonad develop as Sertoli cells (testis) and some develop as granulosa cells (ovary), and there are seminiferous tubules in part of the gonad and follicles in another, all within the same organ, an ovotestis, then I’d say that’s something in between 100% testis and 100% ovary. Or perhaps calling an ovotestis a “mixed gonad” would be better?

          As you state that terminology is important, what terminology would you use?

  4. Chuck says:

    I think your change of my example of the limbs doesn’t work for the same reason that your previous statement doesn’t work. You’re emphasizing the potential of a thing over the mechanism that affects that potential and it’s normal functioning. Basically, you’re emphasizing the exceptions and biological mishaps over the function. The rare exception in sex that is a result of a malfunction does not a spectrum make.

    I would call the two sexes male and female while also acknowledging that birth defects or anomolies or the various specific names that accompany the specific types of intersexed conditions exist. We don’t call male or female a condition because it isn’t an anomoly. We call intersexed a condition because it results from something going wrong. I would stay away from the term ‘spectrum’ to describe the rare occurance of biological mishaps regardless of a potential that no longer exists.

    • Thanks Chuck.

      I can’t say that I agree with your comment about the limb example. Also, I’m not emphasizing the potential over the mechanism. The potential is part of the mechanism. For me, they have equal weight.

      Also, when I said that development of biological sex was a spectrum, I did not say that normal sex development was a spectrum. “Normal” development would be male or female, ovary or testis, but there are ‘anomalies,’ as you state. And why are there anomalies? Because of the bi-potentiality and the mechanisms of gonad sex differentiation.

      Having said all that, I’ve decided to remove that statement (about the spectrum of development) because it doesn’t belong in this particular post. This post was about sex differentiation when everything goes according to plan. The comment about a ‘spectrum of development’ is meant to describe sex differentiation and development inclusive of when things both do and don’t go according to plan and would be better written into the next post, not this one.

      Thanks again for bringing this up. It helps to clarify things.

  5. Mac says:

    I call having a body that looks female and has all the capabilities of functioning as a female, though I am male, a result of something that went terribly wrong.
    Mac

  6. Mac says:

    Yeah, I know it’s another topic, just needed to vent my frustration. Also, great work on this post. Worth the wait!

    Mac

  7. shanesdomain says:

    Hi ATM,

    Another great article! Learn something everytime I visit. Priceless!

    I have a question off topic…wondering if this avenue is one you’re willing to explore.

    I’m confused to all the research out there and the conflicting views on how sex hormones play into the building of connective tissue. Wondering if you have any views on estrogens role in building and maintaining connective tissue in both males and females? Or is estrogen not the determining factor?

    • Hey there.
      That’s an interesting question, one that I know pretty much nothing about. I have never really studied or read about connective tissue biology. Wish I had a better answer for you.

      By the way, I really like the new look of your blog.

      • shanesdomain says:

        Thank you…the black background was messing with the eyes! LOL

        Well…I’m on a mission. Hope I can interpret and convey medical studies as concise and well written as you! You have such talent my friend!

        • Well thanks. It’s a matter of practice, so the more you write about it, the better you’ll get. I’ve been doing it for over 20 years, but I do still have to work at it. That’s why it takes me so long to make a post. I have a couple colleagues who can just sit down and write this stuff out in almost-finished form on the first try. That’s an efficiency I haven’t been able to achieve yet. Maybe you will!!

  8. sirgarreth says:

    I will admit that some of the more scientific concepts are a little over my head, so I apologize if the answer to my question already lies within this post, but I believe that the question is slightly off topic anyway. I had an endo tell me that all of the theories being thrown around about hormone levels, etc. are absolutely wrong, and that being transgender is caused by fetal development in which, for whatever reason, the brain does not finish developing and lags behind sex determination. Do you know anything about this? It sounds a little…problematic to me, but I really don’t have enough of a background to even begin to judge it.

    • Hey Gareth —
      It’s nice that your endo offered you his opinion, but there is currently no biological proof of what causes transgenderism or transsexualism. We only have ‘hints’ right now.

      If you would like a little more insight into sex development, hormones and ‘brain sex,’ you can check out my posts on this topic. If you go to the side bar “ATM Blog Categories” and click on “Science/Biology,” you can find Parts 1-4 of the series “Male Gender Identity in an Individual With Complete Androgen Insensitivity Syndrome” and perhaps some informationn that will address your question.

      By the way, congrats on going on T and the on-going voice drop.

      • sirgarreth says:

        It did seem like an opinion, but she was so adamant, I wondered if I’d missed some memo announcing the “source” had been found. I will check out the series, definitely. I never know where I fall on the, “We should figure this out” vs “It’s a really bad idea to try to figure this out” spectrum in terms of finding some sort of medical cause. On the one hand, I am rather curious, but on the other hand, so many issues could arise if a definitive answer was found to that question.

        Anyway, that seems slightly off-topic from this post, actually. I don’t want to derail anything. And thank you! I am very excited, and continuously surprised, by the changes in my voice. Can’t wait to see what happens next! Hope you’re well, and enjoying your travels.

        • Yes, there’s always the concern that if The Reason was found, then someone would try to ‘cure’ us, or try to test fetuses for transsexualism in utero, or other unpleasant thoughts that override just treating us with respect and dignity for who we are.

          No worries about derailing. It’s all good.

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