In Mexico, the northwest region concentrates the largest fishing and aquaculture production in the country. However, several of the species used in fishery production are overexploited. Most fish species in this region exhibit late sexual reproduction, approximately four to six years to mature in a natural environment. Therefore, maintenance and management in captivity makes it difficult to obtain reproduction protocols. In view of this problem, biotechnological strategies such as in vitro culture and transplantation of stem germ cells or “surrogacy reproduction” are an alternative for the conservation and reconstitution of species. The stem germ cells (spermatogonia or oogonia) in addition to having the capacity to reconstitute species, they also present characteristics such as sexual plasticity and the ability to transfer genetic information to the following generations. For this reason, a protocol for cryopreservation of spermatogonia stem cell was initially established in the yellowtail amberjack (Seriola lalandi). Therefore, the objective of this work was to establish the conditions for the in vitro culture of spermatogonia stem cell that promote cell proliferation, from previously cryopreserved tissue.
Previously cryopreserved testicular tissue was subjected to the enzymatic disaggregation process at 0.3% trypsin. For in vitro culture, the protocol established in rainbow trout (Oncorhynchus mykiss) was used. A cell concentration of 0.6 × 104 cells/cm2 was seeded in 6-well plates coated with 0.1% gelatin with 2 mL of medium. Half the volume of the medium will be changed every 3 days. The cells will be incubated in an oven at a temperature of 28°C with a 5% CO2 atmosphere. The growth factors that will be added to the basal medium are Leibovitz’s L-15 medium Hepes (25 mM), Penicillin (50 U/mL), Ampicillin (50 µg/mL), Streptomycin (50 µg/mL), FBS (1%), Bovine serum albumin (0.5%), L-aspartic acid (20 µg/mL), L-cystine (20 µg/mL), L-proline (20 µg/mL), L-glutamic acid (20 µg/mL), Adenosine (200 µM), fish serum (0.25%), 2-mercaptoetanol (50 µM), Ascorbic acid (50 µM), bovine insulin (25 µg/mL), bFGF human (1 ng/mL), fish embryo extract (1 µg/mL). The percentage of stem spermatogonia will be validated after 6, 12, 21 and 27 days of culture. In each collection, cell concentration, mitotic activity and cell viability will be validated through flow cytometry.
The results of the present work are being analyzed, but from them it is expected to optimize the in vitro culture conditions for stem spermatogonia in the yellowtail amberjack (S. lalandi). This study would represent the first step towards the establishment of stem spermatogonia cell lines, aiming in the future at germ cell transplantation for sterile recipients with intermittent reproduction