Effects of blueberry anthocyanins on growth of mouse testicular organoids and spermatogonial stem cell fate

<bold>Objective</bold> This study aimed to investigate the effects of blueberry anthocyanins on the growth and development of mouse testicular organoids and the fate of spermatogonial stem cells（SSCs），thereby providing scientific evidence for the application of anthocyanin supplements in improving the reproductive capacity of breeding male livestock. <bold>Method</bold> Mouse testicular organoids with seminiferous tubular-like structures，constructed in vitro，were used as experimental materials.Blueberry anthocyanins were added at five concentration gradients：0，50，100，200，and 500 μg/mL.Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling（TUNEL）assay was performed to detect cell apoptosis in the organoids，immunofluorescence staining was used to evaluate the structure changes of organoid and the quantities of <italic>Sertoli</italic> cells and germ cells，and the reverse transcription-polymerase chain reaction（RT-PCR）was applied to analyze the fate of SSCs within the organoids. <bold>Result</bold> （1）Results of the organoid construction experiment showed that the constructed mouse testicular organoids had the structural characteristics of testicular tissue，including seminiferous tubule-like structures and important cell types such as Sertoli cells，germ cells，peritubular myoid cells，and Leydig cells.（2）Results of the tissue structure experiment showed that when the concentration of blueberry anthocyanins was 200 μg/mL，the early growth rate of the organoid diameter was significantly lower than that of the control group（<italic>P</italic>&lt;0.01）.However，the growth accelerated in the later stage，and the diameter became comparable to that of the control group by day 16.Additionally，at 200 μg/mL，the quantities of germ cells and <italic>Sertoli</italic> cells were significantly higher than those in the control group（<italic>P</italic>&lt;0.01）.In contrast，at 500 μg/mL，the number of <italic>Sertoli</italic> cells decreased significantly（<italic>P</italic>&lt;0.01）.（3）Results of the cell apoptosis experiment showed that at the concentration of 500 μg/mL，the intensity of apoptosis signals in testicular organoids was significantly higher than that in the other groups（<italic>P</italic>&lt;0.01）.（4）Results of the spermatogonial stem cell fate determination experiment showed that， at 200 μg/mL，the expression level of <italic>Plzf</italic>（a marker for undifferentiated spermatogonia）was significantly increased（<italic>P</italic>&lt;0.05），while the expression level of <italic>Stra8</italic>（a marker for differentiation spermatogonia）significantly decreased（<italic>P</italic>&lt;0.05）. <bold>Conclusion</bold> Adding an appropriate concentration of blueberry anthocyanins during the culture of testicular organoids can enhance the self-renewal ability of SSCs and promote the growth and development of organoids.However，an excessive dose of blueberry anthocyanins may cause irreversible cell damage and reduce the efficiency of organoid formation.