P–125 Evaluation of N-acetylcysteine (NAC) effect on in vitro culture of immature mouse testis following vitrification
Authors: Nikoosokhan P , Ebrahimi B , Alizadeh A , Hajiaghalou S 
Published: August 2021

Abstract

Study question
Can the Culture of cryopreserved immature mouse testicular tissue in the presence of NAC improves the developmental process and prevent apoptosis induction during the culture?

Summary answer
An appropriate dosage of NAC in the culture medium of immature mouse testicular tissue was associated with increased cell survival and spermatogonia stem cell regeneration.

What is known already
Spermatogonial stem cells (SSCs) are the most advanced type of stem cells in the testes of prepubertal boys which are the main targets of oncological treatments. Therefore, the only possible alternative to maintain fertility in prepubertal boys is to preserve SSCs before their depletion by cryopreserving the testicular tissue. Despite the possibility of obtaining viable spermatozoa using cryopreserved testicular tissue cultivated in vitro,cryopreservation methods and damages caused by the culture procedure would be obstacles for maintaining the testicular tissueand it seems that optimizing the culture condition is vital.

Study design, size, duration
Testis tissues were harvested from 6-days-old immature NMRI male mice (n = 100) after cervical dislocation and vitrified. After 3 days testicular biopsies were warmed and distributed into control, culture Ӏ (not supplemented with NAC) and culture ӀӀ (supplemented with NAC) groups. To determine the appropriate NAC concentration 8 different dosages of NAC were evaluated in terms of cell survival and the best dose, a culture medium containing 125mmol/L NAC was selected to continue the study.

Participants/materials, setting, methods
Vitrified-warmed fragments (2mm3) obtaining from immature NMRI mice were cultured in vitro for 7 days on agar gel. The effects of culture conditions were assessed by Morphological evaluation of seminiferous tubules (using Hematoxylin-eosin staining). Cell viability, protein expression (caspase–3), and gene expression (Bax, Bcl2, Caspase–3, plzf) were evaluated by flow cytometry, immunofluorescence staining, and real time polymerase chain reaction respectively. Additionally, Malondialdehyde (MDA) concentration in the culture medium was measured by MAD Assay Kit.

Main results and the role of chance
Significant (p &lt; 0.01) increase in cell viability was observed in the culture ӀӀ group after 7 days of culture compared to the culture Ӏ. Bax/Bcl2 ratio was significantly (p &lt; 0.01) lower in the culture ӀӀ group compared to the control and culture Ӏ group. The expression of caspase–3 showed a significant (p &lt; 0.001) increase in the culture ӀӀ group while immunofluorescence analysis showed low expression of it in all groups. These results were consistent with the high level of Bcl2expression that inhibited Caspase–3 expression and consequently the inhibition of apoptosis, and on the other hand, the presence of NAC showed that plzf expressions significantly (p &lt; 0.001) increased in culture ӀӀ group compared to the control and culture Ӏ group. Although the presence of NAC did not inhibit all the deleterious effects of culture medium on tissue morphology, NAC was able to maintain better integrity of tissue and seminiferous tubules within central regions compared to the group without NAC. The decrease in MDA level in the presence of NAC (culture ӀӀ) was also a good indicator to confirm the desired results obtained from the presence of NAC in the culture medium.

Limitations, reasons for caution
Although the findings of the study were satisfactory in mice tissue after 1 week of culture, it is essential to replicate the experiments using human tissue and evaluate the quality and reproductive potential of surviving spermatogonia after long-term storage to become clinically applicable.
Wider implications of the findings: This study highlights the necessity for further experiments to improve the testicular tissue culture conditions for better spermatogonial survival and differentiation to sperm, as the prepubertal fertility restoration methods are promising to be implemented in the clinic in the near future.

Trial registration number
Not applicable