Obesity-Induced Alterations in Sperm Quality, Testicular Morphology, and Extracellular Vesicle Localisation in Male Mice

Background: Infertility is a recognised reproductive health issue affecting individuals worldwide. An increase in male infertility has been reported, with concurrent evidence of declining semen quality. Obesity is a major contributor to infertility, with obese men presenting with decreased testosterone levels, impaired spermatogenesis and reduced sperm function. Extracellular vesicles (EVs), which facilitate cell-to-cell communication, have been discovered in various biological fluids. Specifically, in the male reproductive system, testicular-derived EVs have been reported to regulate testosterone production and spermatogenesis. There is a significant gap in knowledge regarding the effects of obesity on testicular EVs and their potential role in altering the testicular microenvironment. Therefore, examining testicular-derived EVs may provide an understanding of the association between obesity and male infertility. Methods: This study utilised a genetic mouse model of obesity (ob/ob) to investigate obesity-induced male infertility. Control (C57BL/6) and obese (ob/ob) male mice were euthanized at 16 weeks of age. Epididymides were harvested to extract sperm to measure sperm concentration, motility (total, progressive and non-progressive), kinematics (straight line velocity (VSL), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) and linearity (LIN)), and morphology via Computer-Aided Sperm Analysis (CASA). Additionally, the testes were harvested and fixed in formalin or frozen for subsequent analyses. Formalin-fixed testes underwent haematoxylin and eosin (H&E), immunohistochemistry (IHC) and immunofluorescent (IF) staining to investigate testicular morphology and assessment of EV localisation, respectively. EV-specific biomarkers such as CD63, CD9 and ANXA1 were targeted. Frozen testes were utilised for EV extraction and further western blotting (WB) analyses. Spermatogenesis was investigated via WB for spermatogenesis-related proteins (IGF1, INSL6, and PRDX2). For EV extraction, frozen testes were dissociated and underwent differential centrifugation after which WB was performed to confirm EV isolation by targeting EV-specific biomarkers. Results: At 16-weeks old, obese mice had significantly increased body weights (P<0.0001) and fasting blood glucose levels (P=0.0004). Sperm concentration remained unchanged between groups. Obese spermatozoa had significantly reduced total (P<0.0001), progressive (P=0.009) and non-progressive (P<0.0001) motility when compared to controls. Kinematic measurements indicated reduced VSL (P=0.0064), VCL (P<0.0001) and ALH (P<0.0001) parameters for obese sperm, with LIN remaining unchanged. Obese mice presented with significantly increased abnormal spermatozoa (P<0.0001) and showed significant increased head and midpiece defects (P<0.0001 and P=0.0089, respectively). Obese testicular morphology displayed decreased seminiferous tubular area (P=0.0287) and diameter (P=0.0252), decreased lumen diameter (P=0.0011), as well as a high presence of vacuolation and disorganised cellular arrangements. IHC staining showed visible differences in EV localisation between control and obese testes for CD63, CD9 and ANXA1, with IF staining showing a decreased mean fluorescent intensity for CD63 (P=0.0345) in obese testes compared to controls. WB for IGF1, INSL6 and PRDX2 remained unchanged. WB for extracted EVs (CD63, CD9 and ANXA1) could not be quantified. Conclusion: Results from this study showed obesity-induced alterations in sperm parameters. Additionally, the testes and EV localisation results suggest a significant difference in CD63 EV marker compared to controls, suggesting less EVs in the ob/ob mice. Further investigation is required to delve deeper into the association between obesity, EV dynamics and testicular functions.

Grantor dc:publisher

Stellenbosch : Stellenbosch University

Year dc:date.issued

2026

Author dc:creator

• Uys, Sharnah

Advisors dc:contributor.advisor

• Skosana, Bongekile Trisha
• Marais, Erna

Language dc:language.iso

en

Repository record dc:identifier.uri

https://scholar.sun.ac.za/handle/10019.1/135707

OAI identifier oai:identifier

oai:scholar.sun.ac.za:10019.1/135707