Assessment of in vitro maturation of fresh and frozen prepubertal testicular tissue at the single cell level – sc-SpermInVitro

Testicular tissue freezing (TTF) is proposed to preserve the fertility of prepubertal boys before highly gonadotoxic cancer treatment. In vitro spermatogenesis is one of the potential approaches that could be used to restore male fertility after recovery. Because of the scarcity of human prepubertal testicular tissue available for research, upstream research has to be performed in animal models. A complete in vitro spermatogenesis has been successfully achieved in the mouse model. However, the spermatogenic yield is reduced in in vitro matured prepubertal mouse testicular tissue and the freezing-thawing process induces additional detrimental effects on the ability of spermatogonial stem cells (SSCs) to differentiate in vitro.
According to the preliminary data obtained in our prepubertal mouse model of in vitro spermatogenesis and in order to improve the in vitro spermatogenic yield, the objectives of the project will be: (i) To promote SSCs self-renewal before differentiation and investigate their ability to differentiate in vitro, (ii) To explore the androgen/estrogen synthesis and signaling pathways and identify potential deregulations observed during in vitro spermatogenesis compared to the physiological conditions, (iii) To analyze the nuclear quality of spermatozoa obtained in vitro, using fresh and frozen prepubertal mouse testicular tissue, the epigenetics of early embryos and the health of the progeny, (iv) To assess the best culture condition for in vitro maturation (IVM) of human prepubertal testicular tissue and to explore the identity and functionality of the human SSC pool.
This is the first study that will examine at the single cell level paracrine and endocrine pathways in the developing testis in order to promote in vitro spermatogenesis. The originality of our project is also to reproduce the complete sequence that could be proposed to prepubertal boys who benefit from fertility preservation before gonadotoxic cancer treatment. Our sequence will include: (i) prepubertal TTF, (ii) in vitro maturation of fresh and frozen testicular tissue, (iii) analysis of the genome and epigenome of in vitro generated spermatozoa, (iv) analysis of the development and DNA methylation of intracytoplasmic sperm injection (ICSI) embryos and (v) evaluation of the offspring’s health. Indeed, we believe that such optimization of the culture conditions with improvement of the culture medium as well as oxygen and nutrient delivery will greatly contribute to the promotion of in vitro sperm production not only in the mouse model but also within the context of a human application. Indeed, we will assess in the second part of the project the possibility to achieve in vitro spermatogenesis up to the haploid stage in cultures of frozen-thawed human prepubertal testicular tissues. Even in case the strategy will not lead to the expected results in terms of improvement of the in vitro spermatogenic yield, this study could lead to the identification of other deregulated factors during in vitro spermatogenesis in mice and human and to other potential modifications of the culture conditions. Additionally, our project will lead to novel data not only on the DNA integrity of in vitro generated mouse spermatozoa, known to be of crucial importance for the balanced transmission of genetic information to future generations, but also on the quality of the progeny obtained after ICSI using these spermatozoa.