Gametogenesis commencement in recipient gonads using germ cells retrieved from dead fish

Abstract

When valuable fish kept in captivity dies due to facility accidents or disease infection, maintaining their genetic resources becomes impossible. However, if the gonads from the dead fish can be used for germ cell transplantation, it would be possible to manufacture gametes obtained from postmortem fish. To examine the feasibility of this scenario, in this study, the viability and transplantability of germ cells obtained from postmortem rainbow trout (Oncorhynchus mykiss) were investigated. Nine-month-old vasa-green fluorescent protein (gfp) transgenic male rainbow trout were killed through a high-dose of anesthesia and left at 10.5 °C of flowing water for zero, six, 12, and 24 h. Histological analyses were conducted to evaluate the deterioration of the testes. As a result, pyknosis occurred in type-A spermatogonia (ASG), especially when the postmortem time was extended to 24 h. Furthermore, the number of germ cells recovered from the postmortem fish was quantified after their enzymatic dissociation. However, there was no significant difference in retrieved germ cell numbers in each time group. Next, to evaluate their ability to be incorporated into genital ridges and start gametogenesis, 104 testicular cells were transplanted into the body cavity of 30 d post-fertilization larvae of rainbow trout. The transplantation efficiencies were analyzed using dissecting recipients, and their gonads were observed at 25, 90, and 150 d post-transplantation (dpt). The transplantation efficiency of zero, six, 12, and 24 h post-death (hpd) testes were 90.61 ± 5.26%, 82.22% ± 11.76%, 73.33% ± 3.33%, and 6.68 ± 6.66%, respectively. Furthermore, fluorescence observation and immunohistochemistry showed that ASG incorporated into recipient gonads, were proliferated in male gonads and differentiated into oocytes possessing a diameter of >100-μm in female gonads of 90 and 150-dpt recipients. Thus, if germ cells can be retrieved from fish within 12 h post-death, there is still the possibility of creating the next generation from the dead fish. This research suggests that germ cell transplantation can be a potential emergency tool for preserving the genetic resources of postmortem fish.