AZFc deletions do not affect the function of human spermatogonia in vitro

Abstract

Azoospermic factor c (AZFc) deletions are the underlying cause in 10% of azoo- or severe oligozoospermia. Through extensive molecular analysis the precise genetic content of the AZFc region and the origin of its deletion have been determined. However, little is known about the effect of AZFc deletions on the functionality of germ cells at various developmental steps. The presence of normal, fertilization-competent sperm in the ejaculate and/or testis of the majority of men with AZFc deletions suggests that the process of differentiation from spermatogonial stem cells (SSCs) to mature spermatozoa can take place in the absence of the AZFc region. To determine the functionality of AZFc-deleted spermatogonia, we compared in vitro propagated spermatogonia from six men with complete AZFc deletions with spermatogonia from three normozoospermic controls. We found that spermatogonia of AZFc-deleted men behave similar to controls during culture. Short-term (18 days) and long-term (48 days) culture of AZFc-deleted spermatogonia showed the same characteristics as non-deleted spermatogonia. This similarity was revealed by the same number of passages, the same germ cell clusters formation and similar level of genes expression of spermatogonial markers including ubiquitin carboxyl-terminal esterase L1 (UCHL1), zinc finger and BTB domain containing 16 (ZBTB16) and glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRA1), as well as germ cell differentiation markers including signal transducer and activator of transcription 3 (STAT3), spermatogenesis and oogenesis specific basic helix-loophelix 2 (SOHLH2), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) and synaptonemal complex protein 3 (SYCP3). The only exception was melanoma antigen family A4 (MAGEA4) which showed significantly lower expression in AZFc-deleted samples than controls in short-term culture while in long-term culture it was hardly detected in both AZFc-deleted and control spermatogonia. These data suggest that, at least in vitro, spermatogonia of AZFc-deleted men are functionally similar to spermatogonia from non-deleted men. Potentially, this enables treatment of men with AZFc deletions by propagating their SSCs in vitro and autotransplanting these SSCs back to the testes to increase sperm counts and restore fertility.

Keywords: AZFc; SSC; infertility; spermatogonia.

Figure 1

Confirmation of the absence of the complete AZFc region in testis tissues of all AZFc-deleted patients. The complete absence or presence of the AZFc region in testicular genomic DNA of all patients and controls was performed in a multiplex PCR by testing six STS markers. Primer set 1 contains sY142, sY1191 and sY1197. Primer set 2 contains sY1206, sY1291 and sY1201. Absence of sY1191, sY1206 and sY1291 in the presence of sY142, sY1197 and sY1201 (LANES 4–9) confirmed the AZFc deletion in our azoospermic patients. All controls had no deletion (LANES 1–3).

Figure 2

Expression of Basic charge Y-linked, 2 (BPY2), Chromo domain on Y (CDY1), Deleted in azoospermia (DAZ), Golgi autoantigen Golgin subfamily A2 like Y (GOLGA2LY) and Chondroitin sulfate proteoglycan 4 like Y (CSPG4LY) in testicular tissue; non-cultured; short-term (18 days) cultured and long-term (48 days) cultured spermatogonia from three men with no deletion of AZFc (URO0077, URO0034 and URO0059). Except for total testicular tissue, MACS sorted ITGA6+ cells were used.

Figure 3

Relative gene expression of germ cell markers in non-deleted (controls) and AZFc-deleted short-term and long-term cultured and MACS sorted HLA⁻ germ cells. (A) Spermatogonial marker expression: ubiquitin carboxyl-terminal esterase L1 (UCHL1), zinc finger and BTB domain containing 16 (ZBTB16), GDNF family receptor alpha 1 (GFRA1) and melanoma antigen family A4 (MAGEA4). (B) Germ cell differentiation marker expression: signal transducer and activator of transcription 3 (STAT3), spermatogenesis and oogenesis specific basic helix-loop-helix 2 (SOHLH2), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) and synaptonemal complex protein 3 (SYCP3). Polymerase (RNA) II polypeptide A (POLR2A) was used as the reference gene. Data expressed as mean ± SEM compared with the short-term or long-term cultured control cells. Asterisks highlight significant differences with P< 0.05.