Ram sperm and seminal plasma alter immune transcriptome and cytokine secretion in ovine cervical explants ex vivo

Jessica P Rickard ¹, Sophie Warr ¹, Simon P de Graaf ¹

⁰School of Life and Environmental Sciences, Faculty of Science, The University of Sydney, Sydney, New South Wales, Australia.

Reproduction (cambridge, England). Supplement +

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October 6, 2025

View abstract on PubMed

Summary

Seminal plasma significantly alters gene expression in the ovine cervix, impacting immune response and sperm communication. This research provides a model to study sperm-immune tolerance mechanisms in the cervix ex vivo.

Area Of Science

Reproductive immunology
Animal reproduction
Genomics

Background

The ovine cervix poses a barrier to frozen-thawed ram spermatozoa, potentially reducing pregnancy success after artificial insemination (AI).
This barrier is hypothesized to stem from altered sperm molecular profiles after seminal plasma (SP) exposure and cryopreservation, affecting female reproductive tract interactions.
The direct impact of different sperm types on the cervix itself remains understudied due to challenges in in vivo interaction profiling.

Purpose Of The Study

To investigate the impact of spermatozoa and seminal plasma (SP) on the ovine cervix transcriptome using an ex vivo cell culture model.
To explore changes in gene expression and cytokine secretion within cervical explants upon exposure to various sperm types and SP.
To establish a model for studying sperm-immune tolerance mechanisms within the ovine cervix.

Main Methods

Ovine cervical explants from Merino ewes (n=6) were cultured ex vivo with epididymal spermatozoa, epididymal spermatozoa + SP, frozen spermatozoa, frozen spermatozoa + SP, SP alone, or media control for 6 hours.
Gene expression was analyzed using RNA sequencing.
Cytokine levels in culture supernatants were quantified via multiplex ELISAs; cervical epithelial cell presence was confirmed using Cytokeratin and H&E staining.

Main Results

Epididymal sperm alone induced differential expression of 18 genes compared to the control.
The addition of SP to epididymal sperm significantly altered 781 genes, implicating pathways in inflammation, signaling, and ATP synthesis.
SP exposure to frozen sperm resulted in significantly increased IL1A and reduced TGFB1 cytokine production compared to other treatment groups.

Conclusions

The presence of ram seminal plasma and spermatozoa significantly alters the cervical immune response and gene expression.
These findings highlight the crucial role of seminal plasma in modulating the cervical environment for sperm interaction.
The established ex vivo model is validated for studying the mechanisms underlying sperm-immune tolerance in the ovine cervix, paving the way for identifying key molecular candidates.

Keywords:

cryopreservation mRNA sheep sperm survival

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