SIRT5 is associated with asthenozoospermia and regulates GC‑2 spd cell proliferation and apoptosis

Abstract

Infertility is a multifactorial condition that affects ~7% of the male population. The mechanism of male infertility primarily involves intrinsic and environmental factors, of which genetic defects are key for its occurrence. Sirtuin 5 (SIRT5) is primarily localized in mitochondria and associated with male reproduction. SIRT5 is downregulated in asthenozoospermic sperm compared with normal sperm. To explore the role of SIRT5 in male reproduction, Cell Counting Kit‑8, EdU, wound healing, and apoptosis assayswere performed in GC‑2 spd cells (mouse spermatocytes) which revealed that SIRT5 inhibited apoptosis and promoted cell proliferation and migration. Western blotting was performed and the results showed that SIRT5 was involved in the proliferation of GC‑2 spd cells by regulating the PI3K/AKT signaling pathway. The present study demonstrated a mechanism of male infertility, which may aid in its treatment.

Keywords: GC‑2 cell; PI3K/AKT signaling pathway; asthenozoospermia; sirtuin 5.

Figure 1.

Expression of SIRT5 in human and mouse sperm. (A) Previous proteomics analysis revealed that SIRT5 expression was significantly decreased in asthenozoospermia compared with normal sperm (26). (B) Analysis of the GSE160749 data set revealed that the mRNA expression of SIRT5 in asthenozoospermia and asthenoteratozoospermia sperm was decreased compared with normal sperm. (C) Expression of SIRT5 in asthenozoospermia sperm samples was lower compared with that in 21 normal samples. (D) SIRT5 protein expression between normal and asthenozoospermia sperm samples. Immunofluorescent co-localization of SIRT5 and (E) marker of spermatocytes in meiosis (γH2AX) in murine testes and (F) mitochondria. Scale bar, 100 µm. (G) SIRT5 expression in testicular cell species. *P<0.05 vs. N; ^##P<0.01 vs. NZS. SIRT5, sirtuin 5; N, normal; A, asthenozoospermia; NZS, normozoospermic; AZS, asthenozoospermic; TPM, transcripts per million.

Figure 2.

Knockdown of SIRT5 inhibits proliferation of GC-2 spd cells. Following transfection with siRNAs, SIRT5 expression was suppressed in GC-2 spd cells at both (A) transcriptional and (B) translational levels. After infection with OE-SIRT5 lentivirus, SIRT5 expression was markedly increased in GC-2 spd cells at both (C) transcriptional and (D) translational levels. (E) Viability of GC-2 spd cells treated with siRNAs or lentivirus. (F) EdU assays revealed the proliferation capacity of GC-2 spd cells treated with siRNAs or lentivirus. **P<0.01, ***P<0.001 vs. NC; ^#P<0.05, ^###P<0.001 vs. Ctrl. Ctrl, control; si, small interfering; NC, negative control; OD, optical density; SIRT5, sirtuin 5; OE, overexpression.

Figure 3.

SIRT5 facilitates migration in GC-2 spd cells. (A) Wound healing assay of GC-2 spd cells transfected with SIRT5 (A) siRNA and (B) OE lentivirus. ***P<0.001 vs. NC; ^##P<0.01 vs. Ctrl. SIRT5, sirtuin 5; Ctrl, control; NC, negative control; siRNA, small interfering RNA; OE, overexpression.

Figure 4.

SIRT5 inhibits apoptosis in GC-2 spd cells. (A) Proportion of apoptotic SIRT5 (A) knockdown and (B) OE cells. **P<0.01, ***P<0.001 vs. NC; ^##P<0.01 vs. Ctrl. SIRT5, sirtuin 5; Ctrl, control; NC, negative control; siRNA, small interfering RNA; OE, overexpression.

Figure 5.

SIRT5 regulates the PI3K/AKT signaling pathway in GC-2 spd cells. (A) Western blot analysis of PI3K, p-AKT, AKT, Bcl-2, Bax and SIRT5 in GC-2 spd cells treated with siRNA or lentivirus. (B) Semi-quantitative analysis of western blot results. (C) Co-IP using a PI3K and FLAG antibody on GC-2 spd cells. *P<0.05, **P<0.01, ***P<0.001 vs. NC; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. Ctrl. p-, phosphorylated; SIRT5, sirtuin 5; Ctrl, control; NC, negative control; ns, not significant; siRNA, small interfering RNA; OE, overexpression; IB, immunoblot; IP, immunoprecipitation.