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Culturing ovarian follicles to foster in vitro gametogenesis

A recent study has demonstrated that the generation of functional ovarian follicle structures from mouse pluripotent stem cells in culture is able to support oocyte production, representing a milestone for in vitro gametogenesis.

In vitro gametogenesis

In vitro gametogenesis is the recapitulation of germ cell development in culture from pluripotent stem cells. Achieving this process would accelerate our understanding of reproductive biology. In the last decade, scientists have made headway in generating functionally mature germ cells. They have successfully converted pluripotent stem cells into functional primordial germ cell-like cells (PGCLCs) in mice and humans. However, there has been little success in reconstituting the later stages of gametogenesis, when the primordial germ cells differentiate into mature gametes.

This stage depends on the surrounding somatic cell environment. In female mammalian embryos, primordial germ cells develop into mature oocytes in the genital ridges, where the ovaries develop. Crucially, ovarian somatic cells in follicle structures create a niche to support oocyte maturation. The ovarian follicular cells provide numerous signals and components necessary for oocyte development.

The lack of in vitro-derived ovarian follicular cells has hindered the generation of functional oocytes in the laboratory. Attempts to overcome this have included transplanting PGCLCs back into the ovaries in vivo or co-culturing them with dissociated mouse gonadal somatic cells. However, these methods are highly complicated and not scalable, rendering them incompatible with human cell-based systems.  

Producing ovarian follicular cells in vitro

A study, recently published in Science, described a breakthrough method that can generate the ovarian follicular cell environment using pluripotent mouse embryonic stem cells (mESCs). To achieve this, researchers led by Kyushu University’s Graduate School of Medical Sciences generated culture conditions that mimicked the in vivo signalling events necessary for the differentiation of ESCs into embryonic ovaries.

The development of embryonic ovaries occurs in steps in the genital ridge, each directed by distinct signalling events. The researchers first introduced reporter constructs to monitor gene expression throughout ovarian development. They then determined a series of culture conditions that induced the expression of key genes at each step.

To recreate the in vivo differentiation trajectory of mESCs, the researchers applied several morphogens in culture in a stepwise manner. Morphogens are key signalling molecules that regulate cell fate. Specifically, the researchers used WNT (wingless-related integration site), BMP (bone morphogenetic protein), SHH (sonic hedgehog), and RA (retinoic acid).

Under these conditions, the mESCs differentiated into foetal ovarian somatic cell-like cells (FOSLCs). The FOSLCs adopted cell identities and diversities, akin to those in foetal ovarian follicles.

When the researchers cultured FOSLCs with murine PGCLCs, the FOSLCs developed into follicles and PGCLCs developed into mature oocytes. Remarkably, the PGCLC-derived oocytes were capable of fertilisation and developed into healthy, fertile offspring following transplantation of the embryo into female mice.

Prospects

Despite its success in producing functional oocytes, this method still needs further refinement. FOSLCs are much less efficient at generating healthy oocytes compared to mouse gonadal somatic cells. Furthermore, the study did not investigate whether there were differences between the cytoplasmic contents or the genetic and epigenetic profiles of in vitro-derived and in vivo-produced oocytes.

Nonetheless, this technical breakthrough has great potential for accelerating germ cell research. The study has demonstrated that FOSLCs may act as a viable replacement for embryonic ovarian tissue during in vitro gametogenesis. As such, this culture system can be used to study the molecular mechanisms underlying the later events of oogenesis.

When applied in humans, this method may even enhance assisted reproductive technologies. The system would first have to be modified since human primordial germ cell development differs from that of mice. Human gametogenesis also occurs over a longer time scale and will likely require a different supporting niche. More importantly, the ethical conflicts that will inevitably arise from this prospect will need to be considered before clinical application. 

Image credit: freepik – Freepik


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