Cryopreservation of sturgeon egg mitochondria and their replacement in germline: A novel strategy for maternal genetic preservation in sturgeons

Abstract

Nearly all sturgeon species are critically endangered, necessitating the development of innovative approaches to preserve their genetic diversity and support population recovery. Unlike sperm, the cryopreservation of fish eggs or embryos remains technically unscalable because of the inherent structural and compositional barriers. Mitochondria are abundant in germ plasm and indispensable in the formation of primordial germ cells (PGCs). Transplanting isolated mitochondria into the vegetal pole of sturgeon embryos, where germ plasm is located, represents a promising conservation strategy. The transplanted mitochondria integrate into the germ plasm and are subsequently incorporated into the germline. Considering seasonality and long generation intervals of sturgeon reproduction, a reliable method for long-term storage of sturgeon egg mitochondria would be advantageous. This study optimized the cryopreservation of sturgeon egg-derived mitochondria, with subsequent validation of mitochondria structural and functional integrity. Various concentrations of dimethyl sulfoxide (DMSO), glycerol, and bovine serum albumin (BSA), combined with different freezing protocols were tested. Mitochondria functionality was assessed through four keys indicators: adenosine triphosphate synthesis, reactive oxygen species production, mitochondrial membrane potential, and integrity. For the first time, we demonstrated that sturgeon egg mitochondria can be successfully cryopreserved, recovered using transplantation and incorporated into the germline. Therefore, we restored maternal germplasm in sturgeon embryos. This work establishes a vital technological advance for conservation biology, offering an alternative approach to preserving and restoring maternal genetic information, which is currently unachievable through other methods in fish. Future research can leverage mitochondrial applications to advance germline preservation, surrogate reproduction, fertility enhancement, and reproductive biotechnology in aquaculture species.