MP01-11 IN VITRO PROPAGATION OF UNDIFFERENTIATED SPERMATOGONIA ISOLATED FROM A 46, XX MALE TESTIS
Abstract
INTRODUCTION AND OBJECTIVE:
The SRY-positive 46, XX genotype is a testicular disorder of sex development found in 1/20,000 males. Patients are phenotypically male with small testes, variable hypogonadism, and azoospermia. Current literature has not yet documented spermatogonia in 46 XX male patients. This study aimed to identify and propagate spermatogonia in vitro in a 46, XX male.
METHODS:
A 28-year-old Caucasian male was initially suspected of having Klinefelter Syndrome (XXY), but karyotype and FISH analysis found a 46, XX genotype with a Y chromosome translocation containing the SRY gene onto the short distal arm of the X chromosome. He underwent experimental bilateral microscopic testicular biopsy and tissue preservation under IRB protocol. Fresh tissue was sent for histopathologic analysis using H&E staining and immunostaining of undifferentiated spermatogenic markers, including PGP 9.5/UCHL1 and ZBTB16. Biopsy portions from bilateral testes were used for characterization. Digital PCR and Real-time Quantitative (RT-Q) PCR were performed with ZBTB16 (undifferentiated spermatogonia), STAR (Leydig cell), CD34 (Peritubular cell), and SOX9 (Sertoli cell) target primers on testis samples from both sides. Isolated cells from the right testicular biopsy were cultured in the StemPro medium for four weeks. RT-QPCR was performed with target primers for undifferentiated spermatogonia (ZBTB16, UCHL1, THY1, CD9, ITGA6, ITGB1), Sertoli (SOX9, GATA4, CYP19a1), Leydig (STAR, CALB2), and peritubular cells (CD34). Digital PCR was performed using ZBTB16 target primer to quantify undifferentiated spermatogonia. Flow cytometry on cultured cells identified the spermatogonia stem cell (SSC) population as negative for HLA-ABC and positive for CD9 and CD49f.
RESULTS:
Testes exhibited Leydig cell hyperplasia, seminiferous tubule hyalinization like in Klinefelter Syndrome, and tubules filled mainly with Sertoli cells. Rare cells in seminiferous tubules were weakly positive for PGP9.5 and ZBTB16, representing undifferentiated spermatogonia. Digital and RT-QPCRs of testis tissue and cultured cells found evidence of all major testicular somatic cells and undifferentiated spermatogonia. Digital PCR for ZBTB16 identified 7.61% of cultured cells as undifferentiated spermatogonia and 0.71% as SSCs. Flow cytometry identified 2.52% of cultured cells as SSCs.
CONCLUSIONS:
This is the first study suggesting the presence of SSCs in 46 XX male testes. Future work will focus on further characterization of cultured testicular cells and In Vitro or In Vivo differentiation of SSCs from XX male testis.
Source of Funding:
WFIRM
