Evaluating in vitro spermatogenesis in Atlantic salmon using single-cell transcriptomics

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Abstract
Understanding how to maintain and direct spermatogenesis in vitro is central to advancing reproductive biotechnologies in aquaculture species where the ability to generate gametes outside the organism could facilitate selective breeding, genetic modification, and germline preservation. However, current culture systems remain poorly defined in farmed fish species. In Atlantic salmon ( Salmo salar ), progress has been further limited by the absence of a comprehensive reference atlas of testicular cell types, making it difficult to determine how cells maintained in culture relate to their native counterparts. To address this, we first established a single-cell RNA atlas of the Atlantic salmon testis from freshly isolated tissue, resolving somatic and germ cell populations across all major stages of spermatogenesis. Primary testicular cells were then cultured under distinct conditions designed to promote either proliferation or differentiation for 14 days and subsequently subjected to single-cell RNA sequencing. To assign cell identities in cultured samples, the transcriptional profiles of cultured cells were computationally mapped onto the atlas, allowing direct comparison of cultured and native cell states. This approach revealed pronounced, condition-specific shifts in cellular composition. Proliferation medium supplemented with epidermal growth factors (EGF) and insulin-like growth factor (IGF) enriched spermatogonial populations, indicating preferential support of undifferentiated and actively dividing germ cells. In contrast, basal medium favoured the preferential survival of Sertoli cells in the absence of defined growth cues. A differentiation medium containing hormones that stimulate male gonad development (gonadotropins and androgens) failed to robustly promote meiotic progression. Further, comparative analysis of Sertoli cells across different conditions ( in vivo and in vitro ) revealed a loss of canonical identity markers and induction of stress-associated transcriptional programs in vitro compared to in vivo , indicating a shift away from specialised somatic function. Together, these findings establish the first single-cell reference atlas of Atlantic salmon testis and provide a framework for evaluating and optimising testis culture systems in salmonids. While early germ cell populations could be maintained and enriched in vitro, progression through later stages of spermatogenesis remained limited, indicating that important biological requirements of the native testicular environment are not yet fully recapitulated under current culture conditions.

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