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 report significant declines in sperm parameters over recent decades⁵. Recent global analyses further confirm widespread declines in sperm quality across multiple regions⁶. While factors such as smoking, alcohol use, radiation, and chemical exposure are established risks⁷, PM2.5 has emerged as an additional reproductive toxicant targeting the testes⁸.

The testes are highly sensitive to environmental toxicants due to their unique physiological structure and continuous spermatogenic activity. Previous research has indicated that PM2.5 exposure can induce testicular damage through multiple pathways, such as oxidative stress, inflammatory responses, and disruption of the blood-testis barrier⁹. These acute effects are characterized by reduced sperm quality, abnormal testicular histology, and hormonal imbalances¹⁰. However, most studies focus on immediate injury, leaving the long-term or delayed effects poorly characterized.

Current PM2.5 exposure models have inherent limitations: whole-body inhalation mimics natural exposure but requires specialized equipment and lacks dose precision¹¹, while intratracheal instillation ensures dose control but is invasive and fails to replicate physiological particle deposition¹². Our time-resolved intranasal model avoids these drawbacks-its non-invasive delivery aligns with upper respiratory deposition patterns, enables precise dose control, and supports long-term multi-time-point sampling to capture delayed injury, which exceeds the capacity of acute-focused traditional models¹³.

Our 5 mg/kg/day PM2.5 dose aligns with rodent subacute exposure paradigms proven to elicit reproductive responses, and via body surface area conversion, approximates a human equivalent dose ~6-fold higher than ambient levels-standard for accelerating long-term toxicity manifestations¹⁴. Species considerations apply: while rats share conserved testicular physiology with humans, their shorter spermatogenic cycle may accelerate injury progression observed in our timepoints¹⁵, and intranasal delivery differs from human lower respiratory deposition, though systemic toxin dissemination to testes is confirmed.

The delayed effects of environmental toxicant exposure are critical for assessing long-term reproductive risks, as they may reveal irreversible damage that is not evident in short-term observations¹⁶. For instance, some toxicants can cause latent injuries to the testes, which gradually manifest or worsen over time, even after exposure stops, potentially leading to chronic reproductive dysfunction¹⁷. Given the ubiquity and persistence of PM2.5, determining whether its reproductive toxicity continues or worsens post-exposure is critical¹⁸.

Sustained inflammation, oxidative stress, and impaired repair mechanisms are plausible drivers of delayed effects. PM2.5-induced upregulation of IL-1β, IL-6, and TNF-α can maintain chronic inflammation, disrupt spermatogenesis, and promote germ cell apoptosis¹⁹. Damage to key junction proteins, such as Connexin-43 and Occludin, compromises the blood-testis barrier and may prevent functional recovery²⁰. Activation of the c-Jun N-terminal kinase (JNK) pathway, a mediator of stress and apoptotic signaling, has also been implicated²¹.

Despite the growing recognition of PM2.5's reproductive toxicity, there is a significant knowledge gap regarding the temporal dynamics of testicular damage after exposure cessation. Specifically, it remains unclear how long the testicular injuries persist, whether they worsen over time, and what molecular pathways drive these delayed effects²². Addressing these questions is crucial for developing targeted preventive and therapeutic strategies to mitigate PM2.5-induced male reproductive harm.

Significant gaps remain regarding the duration, progression, and mechanisms of PM2.5-induced testicular injury after exposure cessation. This study addresses these gaps by assessing reproductive endpoints; histopathology; hormone profiles-specifically follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2); inflammation; apoptosis; and barrier-protein expression at multiple time points after exposure. The findings provide insight into the long-term reproductive risks of PM2.5 and inform public health strategies for protecting male fertility.

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 throughout the study.

PM2.5 material and handling
A PM2.5 standard reference material was used for dosing. Following established particulate-preparation procedures, dry powder was suspended in sterile PBS at the required concentration and sonicated in a water bath for 10-15 min immediately before use, without surfactant. Working suspensions were prepared fresh each day. Endotoxin levels were evaluated by limulus amoebocyte lysate assay and were undetectable. When necessary, polymyxin-B controls were included to exclude LPS-driven effects.

Construction of animal models in vivo
Seventy-two SPF male Sprague-Dawley rats (2 months old, 190 ± 10 g) were housed under standard conditions and randomized into nine groups (n = 8 per group), consisting of a control group, vehicle groups sampled at 24 h, 1, 2, and 4 months, and PM2.5-exposed groups at the same time points. PM2.5 was administered intranasally at 5 mg∙kg^-1∙day^-1 in 10-20 µL per naris under light isoflurane anesthesia once daily for 7 days. After administration, rats were placed in a supine position for 1-2 min to facilitate inhalation. Animals were maintained until euthanasia at the designated post-exposure time points, and body mass was recorded for organ coefficient calculations. Endpoints included organ coefficients, sperm quality, histology and ultrastructure, serum reproductive hormones, and molecular assays.

Reproductive organ morphology and sperm quality assessment
Testes and epididymides were dissected under deep pentobarbital anesthesia, blotted, and weighed to calculate organ coefficients. Sperm were collected from the cauda epididymis by mincing in pre-warmed buffer at 37 °C. Concentration was determined using a hemocytometer. Motility was assessed by examining at least 200 sperm across five or more non-overlapping fields on pre-warmed slides. Morphology was evaluated after fixation, smearing, and staining with eosin-nigrosin or Diff-Quik, with at least 200 sperm scored per rat. All assessments were blinded, and technical replicates were averaged to obtain one value per animal.

Histological and ultrastructural analysis of testicular tissue
Testes were fixed in 10% neutral-buffered formalin, processed through graded ethanol and xylene, embedded in paraffin, and sectioned at 5 µm for HE staining. Stained sections were evaluated using systematic random sampling, with exclusions applied for oblique cuts, artifacts, or poor fixation. Tubule diameters were calculated from orthogonal axes, and epithelial thickness was measured at evenly spaced points. Analyses were blinded, and a minimum of 10 tubules per rat were evaluated.

For TEM, small tissue blocks were fixed in glutaraldehyde, post-fixed in osmium tetroxide, dehydrated, infiltrated with resin, and polymerized. Ultrathin sections were stained with uranyl acetate and lead citrate and imaged at 60-120 kV under consistent settings. QC included assessment of membrane and mitochondrial integrity and avoidance of extraction artifacts.

Enzyme-linked immunosorbent assay (ELISA)
Blood was collected from the abdominal aorta under anesthesia within a fixed morning window to control for circadian variation. After clotting and centrifugation, serum was aliquoted and stored at −80 °C. Testosterone, FSH, LH, and E2 were quantified using a validated immunoassay platform. Samples were run in duplicate with appropriate controls, and assay precision adhered to the platform's specifications. Hemolyzed or lipemic samples were excluded.

Real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from testicular tissue using phenol-chloroform and purified through isopropanol precipitation and ethanol washing. RNA quality was verified spectrophotometrically. cDNA was synthesized, and RT-qPCR was performed with dye-based chemistry under standard cycling conditions, followed by melt curve analysis. Expression levels were normalized to GAPDH using the 2^−ΔΔCq method.

Western blot analysis
Protein lysates were prepared in RIPA buffer with protease inhibitors, quantified by BCA assay, and denatured in loading buffer. SDS-PAGE and PVDF transfer were performed under controlled conditions. Membranes were blocked, incubated with primary antibodies for Connexin-43, Occludin, JNK, and GAPDH, followed by HRP-conjugated secondary antibodies. Bands were detected by chemiluminescence, and densitometry involved background subtraction, normalization to GAPDH, and scaling to the control group.

Statistical analysis
Data were reported as mean ± SD. One-way ANOVA with Tukey's post hoc test or Student's t-test was used where appropriate, with non-parametric data analyzed by Mann-Whitney U test. A p-value < 0.05 was considered significant.

Completion and Cleanup
All hazardous chemical waste was logged and disposed of appropriately. Raw data, including instrument outputs and analysis files, were archived with corresponding notes to ensure traceability.

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