O-257 IGF2BP3 induces apoptosis of spermatogenic cells by regulating the stability of Tcf4 mRNA in an m6A-dependent manner, thereby affecting the occurrence of azoospermia
Authors: Dong M , Xue Z , Weihao S , Jichun T 
Published: June 2023

Abstract

Study question
Does IGF2BP3 regulate Tcf4 mRNA stability in an N6-methyladenosine (m6A)-dependent manner, affect spermatogenesis, and lead to idiopathic non-obstructive azoospermia (iNOA)?

Summary answer
IGF2BP3 induces apoptosis of spermatogenic cells by regulating the stability of Tcf4 mRNA in an m6A-dependent manner, which in turn affects spermatogenesis leading to iNOA.

What is known already
Azoospermia is the most serious phenotype of male infertility, affecting approximately 10-15% of male infertility patients. iNOA refers to a class of non-obstructive azoospermia of unknown etiology for which there is no effective treatment. The etiology of iNOA is poorly understood, and its genetic and molecular mechanisms are poorly studied. IGF2BP3 was found to be highly expressed in tumor tissues and to be a post-transcriptional regulator in cancers. The study found that IGF2BP3 is an m6A reading protein that recognizes mRNA m6A modification and enhances mRNA stability and translation.

Study design, size, duration
We collected testicular tissue from 120 iNOA and 120 patients with obstructive azoospermia through prospective studies to analyze differentially expressed genes by high-throughput RNA sequencing.

Participants/materials, setting, methods
IGF2BP3 knockout and overexpression mouse models were constructed. Spermatogenesis was assessed by immunostaining, HE staining, spermatogenic specific gene expression, and computer-assisted sperm analysis (CASA). The stability of target mRNA was conducted by ActD. Merip-qRCR was used to detect IGF2BP3 binds to the target gene in an m6A-dependent manner; the m6A modification site of Tcf4 was predicted by SRAMP and verified by double luciferase reporting experiments. Overexpression Tcf4 to analyze its effect on spermatogenesis.

Main results and the role of chance
RNA-seq showed that IGF2BP3 was lowly expressed in iNOA testicular tissue which was verified by qRT-PCR and immunohistochemistry. After knocking down IGF2BP3, the spermatogenesis process is impaired, HE showed that the arrangement of spermatogenic cells in the seminiferous tubule was disordered and reduced, the spermatogonia and spermatoblasts were significantly reduced, and there were very few sperm in the seminiferous tubule. CASA showed that sperm density was significantly lower than that of the control group (p &lt; 0.05). Western blot and qRT-PCR showed that spermatogenic cells-specific genes Nanos3, Sycp3, Tnp1, proliferation indexes PLZF and PCNA were all reduced compared with the control group. Overexpression of IGF2BP3 can reverse spermatogenesis in azoospermia mice; after knocking down IGF2BP3, the fertility of mice in vivo and in vitro was impaired; apoptosis of spermatogenic cells increased in testicular tissues of mice after knocking down IGF2BP3 and decreased in mouse testicular tissue after knocking down IGF2BP3. IGF2BP3 induces apoptosis of spermatogenic cells by regulating the stability of Tcf4 mRNA in an m6A-dependent manner, resulting in impaired spermatogenesis; overexpression of Tcf4 in GC1 cells knocked down IGF2BP3 reduces apoptosis and increases cell proliferation.

Limitations, reasons for caution
In this study, we constructed knockdown and overexpression mice models by in situ injection of IGF2BP3 lentivirus in the testes, and the results were more accurate if conditional knockout mice model was used.

Wider implications of the findings
We found direct clinical and in vivo animal model evidence for the causes of male infertility in patients with idiopathic non-obstructive azoospermia, and for the first time found that IGF2BP3 affects spermatogenesis by regulating the stability of target genes in an m6A-dependent manner.

Trial registration number
not applicable