Abstract
Cancer treatments such as chemotherapy can have gonadotoxic effects. In order to preserve and restore the fertility of prepubertal patients with cancer, testicular biopsies are frozen and could theoretically be later matured in vitro to produce spermatozoa for assisted reproductive technology. A complete in vitro spermatogenesis has been obtained from prepubertal testicular tissue in the mouse model, although the sperm yield was low. Since steroid hormones play an essential role in spermatogenesis, it appears necessary to ensure that their synthesis and mechanisms of action are not altered in in vitro cultured tissues. The aim of this study was therefore to investigate steroidogenesis as well as androgen and estrogen signaling during in vitro maturation of mouse prepubertal testicular tissues.
Histological, RT-qPCR, Western blot analyses, measurements of cholesterol, steroid hormones levels and aromatase activity were performed on fresh or frozen/thawed in vitro cultured mouse testicular tissues from 6.5 days postpartum (dpp) mice as well as on age-matched in vivo controls.
A similar density of Leydig cells (LC) was found after 30 days of organotypic culture (D30) and at 36.5 dpp, the corresponding in vivo time point. However, LC were partially mature after in vitro culture, with decreased Sult1e1 and Insl3 mRNA levels (adult LC markers). Moreover, the transcript levels of Cyp11a1, Cyp17a1 and Hsd17b3 encoding steroidogenic enzymes were decreased in vitro. Increased amounts of progesterone and estradiol and reduced androstenedione intratesticular levels were observed at D30. Furthermore, androgen signaling was altered at D30, with decreased transcript levels of androgen target genes (Rhox5, Septin12). Moreover, the expression and activity of aromatase and estrogen signaling were impaired at D30. The addition of hCG to the organotypic culture medium induced an elevation in androgen production but did not improve sperm yield.
In conclusion, this study reports partial LC maturation, disturbed steroidogenic activity of LC, abnormal steroid hormone content as well as altered androgen and estrogen signaling in cultures of fresh and frozen/thawed prepubertal mouse testicular tissues. The organotypic culture system will need to be further improved to increase the efficiency of in vitro spermatogenesis and allow a clinical application.
Competing Interest Statement
The authors have declared no competing interest.
