The mutation c.346-1G > A in SOHLH1 impairs sperm production in the homozygous but not in the heterozygous condition

Abstract

Non-obstructive azoospermia (NOA) is an important cause of male infertility, and the genetic pathogenesis is still incompletely understood. The previous study reported that heterozygous mutation of c.346-1G > A in spermatogenesis and oogenesis specific basic helix-loop-helix 1 (SOHLH1) was identified in two NOA patients and suggested it is the pathogenic factor for NOA. However, in our research, this heterozygous mutation was confirmed in three Chinese infertile patients who suffered from teratozoospermia, but they had normal sperm number. Intriguingly, a homozygous mutation of c.346-1G > A in SOHLH1 was detected in a severe oligozoospermia (SOZ) patient, characterized with severely decreased sperm count. Notably, we unprecedently revealed that this homozygous mutation of c.346-1G > A in SOHLH1 leads to the sharp decrease in various germ cells and spermatogenesis dysfunction, which is similar to the phenotype of SOHLH1 knockout male mice. Moreover, western blotting confirmed that the homozygous mutation declined SOHLH1 protein expression. Additionally, we correlated the good prognosis of intracytoplasmic sperm injection (ICSI) in the patients carrying the mutation of c.346-1G > A in SOHLH1. Thus, we suggested that the heterozygous mutation of c.346-1G > A in SOHLH1 is responsible for teratozoospermia, and this homozygous mutation in SOHLH1 impairs spermatogenesis and further leads to the reduced sperm count, eventually causing male infertility, which unveils a new recessive-inheritance pattern of SOHLH1-associated male infertility initially.

Figure 1

A recurrent heterozygous mutation in SOHLH1 identified in three teratozoospermia patients. Pedigrees of three families with the c.346-1G > A variant in SOHLH1. Sanger sequencing confirmed the SOHLH1 mutation in these families. Squares indicate males, and circles represent females; the mutation site is designated by a red arrow, and the wild-type site is designated by a black arrow.

Figure 2

The morphology of spermatozoa from the teratozoospermia patients by electron microscopy. (A) The abnormal sperm phenotypes were observed using SEM (scale bars, 5 μm). (B) TEM shows abnormal ultrastructure of the head from the patients’ spermatozoa compared to normal control (scale bars, 100 nm).

Figure 3

Ultrastructure of the spermatozoa flagella of teratozoospermiac patients by TEM. In cross sections, the ultrastructural abnormalities were observed in the patients compared with the normal control by TEM (scale bars, 100 nm).

Figure 4

The role of the homozygous c.346-1G > A mutation in SOHLH1 in male infertility. (A) The homozygous variant in SOZ patient was confirmed by Sanger sequencing. (B) Western immunoblotting indicated the decrease of SOHLH1 expression in the testicular tissues of the SOZ patient compared to the normal control. (C) H&E staining of testicular sections from a normal control and SOZ patient. Compared with the normal control, we observed sparse cellular arrangements in patient’s sperm at different spermatogenic stages. i, ii, iii, iv and v show testicular sections from SOZ patient at higher magnification. Red arrowheads denote various germ cells. SPG, spermatogonia; PS, primary spermatocytes; RS, round spermatids; ES, elongated spermatids (scale bar, 20 μm). (D) Staining of SOX9, KI67, SYCP1 and PNA in the testes from a normal control and SOZ patient. The results showed an obvious signal decline in KI67, SYCP1 and PNA in the testis of patient A, while the SOX9 signal was indistinguishable from that of the normal control. SOX9 marks Sertoli cells; KI67 marks spermatogonia; SYCP1 marks spermatocytes and PNA marks round spermatids (red, SOX9; yellow, KI67; green, SYCP1; pink, PNA; blue, DAPI; scale bar, 50 μm). SOZ, Severe oligozoospermia.