Delayed and Progressive Post Exposure Testicular Injury in Rats Due to Fine Particulate Matter (PM2.5)

Summary

PM2.5 exposure induces delayed testicular injury in rat models through sustained inflammatory responses, enhanced germ cell apoptosis, downregulation of Connexin-43/Occludin, and signaling consistent with activation of the JNK pathway, highlighting its long-term threat to male reproductive health.

Abstract

Fine particulate matter (PM2.5) is increasingly implicated in male reproductive impairment. However, because most research remains acute-focused, delayed, persistent, or progressive post-exposure testicular damage-critical for long-term risk assessment-remains poorly understood. We established a time-resolved rat model to determine whether injury persists or worsens after exposure. Seventy-two male Sprague-Dawley rats were randomized into nine groups (control; vehicle at 24 h, 1, 2, and 4 months; PM2.5 at the same time points) and received intranasal PM2.5 at 5 mg/kg/day for 7 days. We quantified sperm quality and reproductive organ indices, examined testicular histology and ultrastructure, measured serum testosterone, FSH, LH, and E2, profiled inflammatory cytokine mRNAs (IL-1β, IL-6, TNF-α, IFN-γ), assessed germ-cell apoptosis by TUNEL, and evaluated junctional and stress-related proteins (Connexin-43, Occludin, JNK). PM2.5 exposure resulted in sustained declines in sperm concentration and motility, along with increased malformations and testicular and epididymal atrophy that did not recover over the 4-month post-exposure period. Histology showed progressive thinning of the seminiferous epithelium and ultrastructural degeneration. Endocrine disruption was also evident, with reduced testosterone and altered gonadotropins. Mechanistically, PM2.5 exposure maintained elevated IL-1β and IL-6 expression, promoted germ cell apoptosis, and downregulated Connexin-43 and Occludin while exhibiting patterns consistent with activation of the JNK pathway. Together, these findings demonstrate delayed and progressive testicular toxicity after PM2.5 exposure ends and suggest that preserving junctional integrity and targeting JNK may mitigate long-term reproductive harm.

Introduction

PM2.5, a complex mixture of fine particulate matter with an aerodynamic diameter of≤2.5µm, originates from industrial emissions, vehicle exhaust, and biomass combustion, and has emerged as a major global environmental pollutant¹. Epidemiological studies link PM2.5 exposure to respiratory and cardiovascular disease², as well as reproductive system impairments³. The impact on male fertility is of particular concern due to evidence that air pollution can reduce sperm quality⁴. Male factors contribute to over half of infertility cases globally, and multiple studies r....

Protocol

All animal procedures were conducted in accordance with institutional regulations and were approved by the Animal Ethics Committee of Fujian Health College (Project Approval No.: DW2024-01).

Safety and hazardous waste
Toxic reagents including formalin, osmium tetroxide, and chloroform were handled in a certified fume hood while wearing appropriate personal protective equipment. Halogenated organic waste and osmium-containing solutions were collected in designated containers and disposed of according to institutional requirements. Pentobarbital sodium was logged as a controlled substance, and sharps were handled safely....

Results

Persistent deterioration of male reproductive function in rats, characterized by reduced sperm quality and impaired reproductive organ development due to PM2.5 exposure

To investigate the impact of PM2.5 on male reproductive function, sperm quality, reproductive organ coefficients, and related indicators were examined across nine groups of male rats. Analyses were performed relative to the 24 h postexposure time point. Although a Day-7 sampling timepoint was not included, infl.......

Discussion

Short-course PM2.5 exposure was followed by progressive, post-exposure deterioration of male reproductive function over 1-4 months. These findings support time-dependent reproductive toxicity that persists after dosing ceases and extend prior observations of PM2.5-associated reproductive impairment²³.

To enhance reproducibility, we standardized four elements. First, exposure consistency was maintained by normalizing dose to body mass, controlling intranasal volume and p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1% osmium tetroxide	TCI, China	O0308	For post - fixation in TEM analysis of testicular tissue
10% neutral buffered formalin	Solarbio, China	PC0020	For fixing testicular tissue samples in HE staining
2.5% glutaraldehyde	Solarbio, China	G1102	For fixing testicular tissue samples in TEM analysis
75% ethanol	Acros Organics, USA	61509-0040	For purifying precipitated RNA in RT - qPCR
Automatic Chemiluminescence Imager	Bio-Rad, USA	ChemiDoc	Used for automatic imaging of chemiluminescent signals in Western blotting.
BCA protein assay kit	Solarbio, China	PC0020	For determining total protein concentration in Western blot analysis
Blocking buffer	Not specified	T7131A	Used in Western blot analysis to eliminate non - specific binding
Chloroform	Acros Organics, USA	327270010	Used in RNA separation during RT - qPCR
Connexin - 43	Abcam, USA	ab11370	Detects the expression and distribution of a key blood - testis barrier protein to help assess the integrity of the barrier
ECL kit	Biosharp, China	BL520B	For visualizing protein bands in Western blot analysis
ELISA kits (for testosterone, FSH, LH, E2)	Abcam, UK	ab108666; ab108702; ab108651; ab108667	For detecting serum reproductive hormones
Eosin	Solarbio, China	G1002	Used in HE staining of testicular tissue
Epoxy resin	Solarbio, China	GP2001	For embedding testicular tissue samples in TEM analysis
GAPDH	Abcam, USA	ab128915	Evaluates the apoptosis of testicular cells induced by PM2.5 and provides a morphological basis for studying its impact on testicular cell apoptosis
Hematoxylin	Solarbio, China	G1077	Used in HE staining of testicular tissue
Imaging System	Nikon, Japan	Nikon DS-U3	Used for imaging samples under a microscope.
Isopropanol	Acros Organics, USA	AC447080025	Used in RNA precipitation during RT - qPCR
JNK	Abcam, USA	ab124956	Explores the molecular mechanism of testicular injury caused by PM2.5 and identifies the cell types in which it is activated
Microplate Reader	MOLECULAR DEVICES, USA	SpectraMax iD3	Used for detecting signals in microplates (e.g., absorbance, fluorescence).
Occludin	Abcam, USA	ab31721	Understands the damage degree of the blood - testis barrier and explores the interactions between relevant proteins
Optical Microscope	Chongqing Opto - Electronic Instrument , China	BDS200	Used for general optical microscopic observation.
Pathological Microtome	Shanghai Kaiyu Instrument, China	RM2016	Used for cutting sections of pathological tissues.
Pentobarbital sodium	Solarbio, China	G1102	Used for intraperitoneal injection in rats before blood collection
PM2.5 standard reference material	National Institute of Metrology, China	GBW13643	For PM2.5 exposure in experimental group
Polyvinylidene difluoride (PVDF) membrane	Thermo Fisher, USA	88585	Used in Western blot analysis for protein transfer
PrimeScript RT reagent Kit	TAKARA, Japan	RR037Q	For cDNA synthesis in RT - qPCR
Real-Time PCR System	Roche, Switzerland	LightCycler 480	Used for real - time polymerase chain reaction for gene expression analysis.
RIPA lysis buffer	TAKARA, Japan	RR9161	For extracting proteins in Western blot analysis
Shaker	Thermo, USA	MAXQ-4000	Used for shaking samples in experiments.
SPSS	IBM, USA	28	For data analysis
Sterile saline	Solarbio, China	S8160	Used in vehicle group treatment
TB Green Premix Ex Taq II kit	TAKARA, Japan	RR820S	Used in RT - qPCR
Transmission Electron Microscope (TEM)	Hitachi, Japan	HT7800/HT7700	Used for ultrastructural observation at the nanoscale.
Trizol reagent	Ambion, China	15596018	For extracting total RNA in RT - qPCR
Ultramicrotome	Leica, Germany	Leica UC7	Used for cutting ultra - thin sections for TEM.
Universal Protein Transfer System / Protein Transfer Blot Machine	Bio-Rad, USA	Trans-Blot Turbo	Used for protein transfer in Western blotting.
Upright Optical Microscope	Nikon, Japan	Nikon Eclipse E100	Used for upright optical microscopic observation.
Uranyl acetate	Merck, Germany	201030	For staining ultrathin sections in TEM analysis of testicular tissue