Dynamic cues guiding postnatal germline development in marmoset

• Applicants: Dr. Sandra Laurentino; Professorin Dr. Nina Neuhaus
• Subject Area: Reproductive Medicine, Urology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 560245430

Survival rates among childhood cancer patients have continuously increased over the past decades. This leads to an increasing number of patients facing the long-term effects of pediatric oncological treatments, including permanent infertility. For adult male patients, cryopreservation of sperm prior to treatment is routinely offered, as these cells can later be used for in vitro fertilization. This approach, however, is not applicable to patients prior to puberty, as spermatogonial stem cells have not yet begun their differentiation into sperm. To create options for the reproductive future of these patients, cryopreservation of immature testicular tissues, containing spermatogonia as the precursors of sperm, is offered. This is done in spite of the fact that protocols to derive sperm from spermatogonia remain under development, as the regulatory pathways governing germ cell differentiation are largely unknown. Since access to healthy immature human testicular tissues is limited, the marmoset monkey is the best available non-human primate model. It shares key features with the human such as the downregulation of pluripotency markers in gonocytes soon after birth and a pubertal phase, during which germ cell differentiation is initiated. Capitalizing on our unique research environment, we have generated unprecedented single cell RNA-seq (scRNA-seq) datasets of marmoset testes representing over 70.000 cells from distinct developmental time points (n=3 neonatal, n=3 prepubertal, n=3 pubertal, and n=4 adult). This unparalleled dataset forms the basis of this application. We hypothesize that male germ cell fate decisions during postnatal life, from silencing of pluripotency programs after birth to initiation and maintenance of spermatogenesis after puberty, are guided by cell-cell communication cues from the somatic environment. Using the already generated datasets and applying sophisticated bioinformatics analysis combined with quantitative analysis of multispectral immunofluorescence stainings of testicular tissues and functional in vitro studies, we will identify the regulatory networks from the testicular microenvironment that regulate the switch from pluri- to unipotency in neonatal testes. To unveil the mechanisms controlling the commitment to differentiation of spermatogonia and initiation of spermatogenesis, we will identify regulatory networks arising from these cell-cell communication cues by integrated bioinformatic analyses of scRNA-seq datasets from prepubertal, pubertal, and adult animals, as well as quantitative in situ analyses. Crucially, methylome analyses of germ cells from prepubertal, pubertal, and adult animals will uncover the epigenetic changes associated with this germ cell maturation process. The identification of those pathways relevant for male germ cell differentiation in primates is the foreseen key achievement of this project, which will have direct translational impact in the field of male fertility preservation.

• DFG Programme: Research Grants