I was telling a friend about how sex determination could be an interesting topic for my next blog and he responded asking, “Don’t we already know everything about sex determination?” So I asked an expert: Dr. Robin Lovell-Badge, the Principal Investigator of a stem cell biology and developmental genetics laboratory at the Francis Crick Institute in London, England.
Normally, in humans, sex is determined when the male passes on the X chromosome – making the child female – or the Y chromosome – making the child male. However, it was discovered in 1959 that there were some females that had only one X chromosome (XO; Turner syndrome) and some males that are XXY (Klinefelter syndrome). While both show infertility (and have other issues), this indicated that the presence or absence of a Y chromosome was important for developing into a male or female, respectively.
Turner and Klinefelter syndromes are both examples of Disorders of Sex Development (DSD). The process not going right can also lead to XY females and XX males. These individuals can look externally and internally female or male, but they are inevitably infertile. The process going awry can also lead to partial sex reversal where the individual has some male and some female anatomical characteristics, which may be internal and/or external.
Knowledge about the critical genes required for sex determination and development of the various somatic cell types, as well as the germ cells that make eggs or sperm, could be extremely important to regenerative medicine. At its simplest, these genes are good markers/indicators to know whether a specific cell type of the reproductive system can be derived and maintained.
Historically, in what was a long process of experimentation, there were multiple attempts by many laboratory teams to find the gene on the Y chromosome that triggers male development. Ultimately, all but one of the candidate genes found were wrong.
Dr. Lovell-Badge, like many others, persisted in the search for the gene. He and his collaborator, the human geneticist Dr. Peter Goodfellow ultimately were the ones to find the correct gene located in the Y-unique sequences of the Y-chromosome. This gene was named Sry, for “gene in the sex determining region of the Y chromosome.”
In a critical experiment Dr. Lovell-Badge’s lab introduced Sry into fertilized eggs of XX female mice and some of the chromosomally female embryos developed testes, rather than ovaries. This meant that Sry was not only important for testis and male development, but that it was the only gene on the Y chromosome required for this.
Subsequent research in this area has discovered much more about how SRY triggers male development. The current view is that sex determination is a “bistable” system, poised in early gonad development to go in either direction, with SRY biasing the decision to make a testis. The decision is then reinforced by other regulatory gene networks and proteins. However, there is evidence that the system remains flexible into adulthood, where loss of certain key proteins can lead to gonadal sex reversal (see below).
While many of the genes underlying the causes of DSDs in humans have been discovered, how the phenotype comes about in many DSD cases has yet to be understood. In humans, gonads start developing around six weeks into embryonic development, and experiments to investigate remaining questions cannot be done in embryos for ethical reasons.
Very recently, Dr. Lovell-Badge’s lab, in collaboration with Dr. Anu Bashamboo in Paris, has developed a way of deriving gonadal cell types in culture from pluripotent stem cells, where pluripotent stem cells from normal XY males result in Sertoli cells (cells that support sperm development), and pluripotent stem cells from normal XX females produce granulosa cells (cells that support oocyte development).
However, if they use pluripotent stem cells from a DSD individual who is chromosomally male (XY), but phenotypically female, “We actually get cells that are in a state between Sertoli and granulosa cells,” Dr. Lovell-Badge shares as he describes his yet-to-be published findings. He continues: “This is the first case where DSD stem cells have been studied in this way in vitro. We think we can use this system to study DSDs in a way that we haven’t been able to before.”
Dr. Lovell-Badge’s lab has also made some incredible insights into the role of a gene called FOXL2. A few years ago, Dr. Lovell-Badge’s lab used some genetic tricks to completely delete FOXL2 in adult female mice resulting in a gonad that looked a lot more like a testes than an ovary. Although FOXL2 is not necessary during embryonic development for the induction of ovarian fate in mice, FOXL2 becomes the only gene that is required to maintain an ovary phenotype after birth, by suppressing the male pathway. “You need FOXL2 throughout life to keep the male pathway off,” Dr. Lovell-Badge said.
In terms of humans, females who are missing one copy of the gene (heterozygous for FOXL2) run out of eggs early, and they become infertile typically in their 20s (premature ovarian insufficiency). However, they don’t show signs of testes development. There hasn’t been a case where someone is completely missing the FOXL2 gene; therefore, it’s difficult to know what the outcome of that would be.
Dr. Lovell-Badge explains that, “by using our cell culture system we can now delete both copies of FOXL2 in the pluripotent stem cells and ask if FOXL2 is simply like in the mouse – a gene involved in maintenance of ovaries – or if it’s involved in ovary determination and early ovarian differentiation.”
Discovering critical genes like FOXL2 is important for regenerative medicine as it may help direct or force the specification of a cell type, tissue or an organoid from progenitor cells or pluripotent stem cells. Manipulating key gene activity in vivo might even allow direct reprogramming of one cell type into another, which may then induce other relevant changes in adjacent cell types.
While it is far too early to know how safe and efficient any such uses might be for regenerative medicine, there are circumstances in which this may be a potential treatment for restoring or producing a gonad that has at least some function to produce sex hormones (in appropriate ways and amounts), or to allow fertility. These circumstances include the unfortunate consequence of a physical accident; clinical treatments (radiotherapy or chemotherapy); infectious disease; genetic and epigenetic disorders of sex development; or perhaps to accompany gender reassignment treatments.
There are still many things we don’t know about sex determination – especially in humans. And although the genetic data of humans and mice match very well, the relative importance of a particular gene in the sex determination pathway might be different between the two species.
And although the high levels of androgen hormones in males and estrogen hormones in females – depending on the type of gonad that develops – are commonly associated with the anatomical, physiological and behavioural differences between males and females, it turns out that the X and Y chromosomes themselves can directly impact these differences independently of hormones. More work still needs to be done in this area.
This exemplifies why sex determination is, as Lovell-Badge puts it, “A beautiful system to study cell fate – how you become one cell type over another.” Although there are only two outcomes in fate (male or female), it is nuanced and multifactorial in both its early mechanisms as well as what exactly makes the male and female different on anatomical, physiological and behavioural levels.
Krystal Jacques
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