Flagella-Forming Potency of Cultured Round Spermatids (R-ST)

Abstract

Objective: For R-ST injection (ROSI) in primary azoospermia where spermatogenic differentiation is arrested at the R-ST stage, immature male reproductive cells, including primary and secondary spermatocytes can be selected. If R-ST can be successfully cultured to form flagella, we will be able to avoid mis-selection. The results from our experiments, focusing on the flagella-forming potency of cultured R-ST, offer practical applications. Design: Retrospective review of flagella forming potency of cultured human, mouse or rat R-ST. Materials and Methods: (1) Incubation in cultured medium: Normal mouse, rat or human testis tissue was gently disentangled in RBC lysing buffer for hemolysis. Each sample was transferred into HTF containing BSA (5 μg/ml) and gently disentangled again. The seminiferous tubules were cut into fine pieces and then rinsed out through pipetting. R-ST were picked up with a micromanipulator and separately incubated in culture medium (Ham-F 12: Leibovitz = 1:1) supplemented with epinephrine (1 μg/ml) and norepinephrine (1 μg/ml) at 32.5°C in 5% CO2 in air. (2) Co-culture with Sertoli’s cells: After the removal of tunica albuginea, 14 day-old mouse tissue was gently disentangled with tweezers in PBS containing collagenase (1 mg/ml) and deoxyribonuclease (1 mg/ml) at 4°C. Through pipetting, the seminiferous tubules were further disentangled and filtered out using a nylon mesh filter. Precipitated cells were dispensed into collagen-coated dishes at 106 cells/well and cultured. Within the first week of culture, Sertoli’s cells adhered to the bottom of the wells to form a monolayer on which the reproductive cells clustered. After eliminating these reproductive cells, mouse R-ST were cultured on the monolayer of Sertoli’s cells. (3) Incubation in rat rete testicular fluids: In order to collect the efficient volume of rete testicular fluids, the tip (100–150 μm) of a micropipette was inserted into the rete testis several times. Rat R-ST, isolated by the same procedures (Method I), were cultured in the drop of filtered rete testicular fluids at 32.5°C in 5% CO2 in air. Results: (1) After 48 hrs of in vitro cultivation, 22.2% (38/171) of human R-ST had formed flagella. Flagella developed as early as 16 hrs after culture and continued to elongate over the next 30∼40 hrs. The length of flagella was 15∼25 μm and its width was the same along the length. No movement of flagella was observed. (2) R-ST from mouse cultured in medium also showed similar morphological changes to those in human R-ST. Flagella formation, however, was found in only 6.7% (10/146). (3) In 3.9% (6/153) of R-ST from mouse, co-cultured with Sertoli’s cells, flagella was formed. (4) No rat R-ST incubated in rete testicular fluid could produce flagella. Conclusion: (1) The generation of flagella by isolated populations of purified R-ST indicates that this process is independent of other cellular influences of the seminiferous epithelium. (2) For in-vitro cultured R-ST derived from cases of maturation arrest at the R-ST stage, the occurrence of flagella formation and the advance of differentiation may be useful labels for their identification.