Journal of Advanced Research

Journal of Advanced Research

Volume 49, July 2023, Pages 115-126
Journal of Advanced Research

Original Article
Connexin-43 is a promising target for lycopene preventing phthalate-induced spermatogenic disorders

https://doi.org/10.1016/j.jare.2022.09.001Get rights and content
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Highlights

  • Phthalate induced spermatogenic disorders and destroyed BTB integrity.

  • Phthalate caused cell migration capacity decline and cytoskeletal disorganization.

  • LYC alleviated DEHP-induced disruption of intercellular junctions in mouse testes.

  • LYC improved DEHP-induced spermatogenic disorders by regulating BTB integrity.

  • LYC ameliorated DEHP-induced BTB disruption by modulating Cx43 expression.

Abstract

Introduction

Male infertility is a multifactorial pathological condition and may be a harbinger of future health. Phthalates are ubiquitous environmental contaminants that have been implicated in the global decline in male fertility. Among them, di-(2-ethylhexyl) phthalate (DEHP) is the most prevalently used. Lycopene (LYC) is a possible preventive and therapeutic agent for male infertility owing to its antioxidant properties. The blood-testis barrier (BTB) is formed between Sertoli cells where it creates a unique microenvironment for spermatogenesis.

Objectives

We hypothesize that phthalate caused male infertility and LYC plays an important role in phthalate-induced male fertility disorders.

Methods

Hematoxylin-eosin (H&E) staining, ultrastructure observation, and fluorescence microscopy were used to examine the morphological changes. RNA-Seq, and western blotting were conducted to detect gene and protein levels. Routine testing for sperm morphology and sperm-egg binding assay were conducted to examine the morphological structure and function of sperm. Cell scratch assay and transepithelial electrical resistance (TER) were used to detect cell migration capacity and barrier integrity.

Results

In vivo experiments, we showed that LYC prevented DEHP-induced impairment of BTB integrity, which provided a guarantee for the smooth progress of spermatogenesis. LYC improved DEHP-induced change in sperm parameters and fertilization ability. Subsequent in vitro experiments, LYC alleviated MEHP-induced disruption of intercellular junctions in mouse Spermatogonia cells (GC-1 cells) and mouse Sertoli cells (TM4 cells). In MEHP-induced BTB impairment models of Sertoli cells, treatment with LYC or overexpressing connexin-43 (Cx43) promoted cell migration capacity and normalized BTB integrity. Cx43 knockdown inhibited cell migration capacity and perturbed BTB reassembly in LYC preventing DEHP-induced BTB impairment.

Conclusion

Our study provides evidence for the role of LYC in phthalates-induced spermatogenic disorders and points to Cx43 as a potential target for male fertility.

Graphical abstract

The present study identified a critical role of Cx43 in LYC maintaining BTB integrity, which is a prerequisite for normal spermatogenesis. This study reveals the role of Cx43 in LYC preventing phthalate-induced spermatogenic disorders, and Cx43 regulation may provide the basis for future therapeutic male infertility.

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Keywords

Lycopene
Di (2-ethylhexyl) phthalate
Connexin-43
Blood-testis barrier
Spermatogenic disorders

Abbreviations

DEHP
di-2-ethylhexyl phthalate
LYC
lycopene
BTB
blood-testis barrier
Cx43
connexin-43
PVC
polyvinyl chloride
EPA
Environmental Protection Agency
EDCs
endocrine disrupting chemicals
TJ
tight junctions
AJ
adherent junction
GJ
gap junction
NEAU
Northeast Agricultural University
FITC
fluorescein isothiocyanate
PMSG
pregnant mare serum gonadotropin
hCG
human chorionic gonadotrophin
RIPA
radio-immunoprecipitation assay
SDS-PAGE
sodium dodecyl sulfate polyacrylamide gel electrophoresis
NC
nitrocellulose
IF
immunofluorescence
DMEM
ulbecco's Minimal Essential Medium
FBS
fetal bovine serum
MEHP
mono(2-ethylhexyl) phthalate
THF
Tetrahydrofuran
BHT
butylated hydroxytoluene
DMSO
dimethyl sulfoxide
CCK8
cholecystokinin 8
AO/EB
acridine orange/ethidium bromide
SCs
Sertoli Cells
WT-1
Wilms tumor protein 1
TER
transepithelial electrical resistance
SD
standard deviation
ZO-1
Zona occludens 1
TZI
teratozoospermia index
SDI
sperm deformity index
ATCC
American Type Culture Collection

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Peer review under responsibility of Cairo University.