Molecular basis of sperm reservoir formation in the oviduct and the effects of scrotal hyperthermia

Fertility declines during the summer months have been widely observed in farm animals, posing significant challenges to production. This issue is particularly crucial with the use of artificial insemination, where infertility in one male can affect hundreds or thousands of females. Although the impact of high testicular temperature on sperm production has been explored extensively, the precise molecular mechanisms disrupted by heat stress remain unclear. This study aims to identify the molecular pathways affected by testicular hyperthermia and develop sperm biomarkers to enhance fertility assessments. For this study, heat stress was induced in the experimental group by covering the scrotum with insulated sacs for 48 hours, while non-insulated sacs were used in the control group. Temperature monitoring showed the insulated sacs increased scrotal temperature by 3–4°C. Semen was collected three times per week for two months to avoid sperm mixing in the epididymis. Sperm motility was analyzed with computer-assisted semen analysis (CASA), and morphology was assessed using Coomassie Blue G-250 staining. Proteomic analysis was performed using protein profiles of sperm from before insulation (baseline) and on days 6, 17, 24, and 60 after removing the insulation. Boars subjected to elevated scrotal temperatures exhibited reduced sperm motility and progressive motility at days 24 and 37 (P < 0.05). Morphologically normal sperm percentages also decreased on day 24 (P < 0.05). Among 881 proteins detected in at least three samples, the majority were categorized as catalytic proteins (45%), binding proteins (38%), and molecular function regulators (5.5%). Protein profile changes were observed throughout the study, with four proteins (VDAC1, TOMM34, SPATA18, and GPX4) consistently upregulated compared to baseline. Overall, our findings suggest that elevated testicular temperature negatively affects sperm motility, morphology, and protein expression, with implications for metabolism, protein degradation, translation, and extracellular matrix functions. Another important factor for male fertility is how sperm are transported and stored in the female reproductive tract. Sperm are retained and stored in the oviduct by binding to specific glycans. PKDREJ has been identified in porcine sperm as a potential receptor for oviduct glycans, specifically those containing Lewis X (LeX) trisaccharide and multi-antennary 6-sialylated motifs. PKDREJ is a homolog of sea urchin receptor for egg jelly (suREJ) and a member of the polycystin-1 gene family. Previous studies in the mouse demonstrated that sperm lacking PKDREJ showed normal morphology and motility but had reduced fertility, observable only when competing with wild-type sperm due to the delay in zona pellucida-induced acrosome reaction. In this study, we explored the role of PKDREJ in the porcine sperm reservoir formation and fertility by generating PKDREJ-deficient boars using CRISPR/Cas9. The genotypes of male founders were verified using DNA from ear notches and sperm. Three mosaic males with multiple mutant alleles and no detected wild-type alleles were used. All pigs had mutations in PKDREJ that encoded truncated versions of protein lacking the transmembrane domain. Western blotting confirmed a lack of PKDREJ protein in their sperm, while immunofluorescence revealed reduced PKDREJ on sperm heads. Sperm from PKDREJ-deficient boars showed no differences in motility or morphology compared to control sperm. Binding assays showed that PKDREJ-deficient sperm adhered equally well to oviduct spheroids and cumulus-free zona pellucida-encased oocytes. However, a fertility trial using artificial insemination (AI) revealed that PKDREJ-deficient sperm failed to achieve any pregnancies, in contrast to a 65% pregnancy rate with control sperm. In a competitive insemination trial, where equal amounts of control and PKDREJ-deficient sperm were used to inseminate seven females, only offspring from control sperm were produced, confirming severely reduced fertility in PKDREJ-deficient boars. These findings establish that PKDREJ is essential for porcine male fertility following artificial insemination. This study is the first to generate PKDREJ-deficient pigs and study PKDREJ function in porcine sperm. The results indicate the importance of PKDREJ in male fertility although its specific role is still uncertain.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Animal Sciences

Grantor

University of Illinois Urbana-Champaign

Year dc:date

2025

Author dc:creator

• Doungkamchan, Kankanit

Contributors dc:contributor

• Miller, David J
• Knox, Robert V
• Ko, Chemyong
• Bahr, Janice M

• SPERM
• SPERM RESERVOIR
• OVIDUCT
• OVIDUCT GLYCAN
• SCROTAL HYPERTHERMIA
• PKDREJ

Statement dc:rights

• Copyright 2025 Kankanit Doungkamchan

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/129484

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F129484