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Effects of α-Linolenic Acid on Seminiferous Tubule Morphology and UCHL1-Positive Cells in Cultured Testicular Tissue Fragments from a 3-Month-Old Hu Ram: A Pilot Study
State Key Laboratory of Herbage Improvement and Grassland Agro-Ecosystems, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, China
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Animals 2026, 16(18), 2873; https://doi.org/10.3390/ani16182873
Submission received: 11 August 2026
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Revised: 2 September 2026
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Accepted: 9 September 2026
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Published: 12 September 2026
(This article belongs to the Special Issue Male Germ Cell Development in Animals)
Simple Summary
Alpha-linolenic acid is an essential fatty acid that may benefit testicular function. In sheep, most dietary alpha-linolenic acid is metabolized during digestion before it reaches the testis. Studies of isolated cells cannot show how intact testicular tissue responds. This pilot study compared the structure of cultured sheep testicular tissue, the number of UCHL1-positive cells, and gene expression between control and alpha-linolenic acid-supplemented cultures. Testicular tissue fragments from one 3-month-old Hu ram were cultured for 50 days in control medium or medium supplemented with alpha-linolenic acid. Culture with alpha-linolenic acid was associated with greater seminiferous tubule diameter and a lower proportion of empty space within the tubules on days 30 and 50. The number of UCHL1-positive cells per seminiferous tubule was also higher on day 50. Gene expression analysis showed that gene sets related to immune and inflammatory responses were enriched in the control fragments, whereas gene sets related to DNA replication and germ cell processes were enriched in the alpha-linolenic acid-treated fragments. However, all tissue fragments were obtained from the same ram. Therefore, these findings are preliminary and require confirmation using tissues from multiple rams.
Abstract
α-linolenic acid (ALA) has been reported to benefit testicular function. However, dietary ALA is extensively metabolized in the rumen, whereas isolated cell models do not retain seminiferous tubule structure or interactions among testicular cells. This pilot study examined the effects of ALA on seminiferous tubule morphology, UCHL1-positive cells, and gene expression in cultured sheep testicular tissue fragments. Tissue from one 3-month-old Hu ram (n = 1) was cultured for 50 d in control medium (CK) or medium containing 5 μM ALA. Three independently cultured fragments per condition were analyzed. Histology, UCHL1 immunofluorescence, RNA sequencing, and qRT-PCR were performed. ALA-treated fragments showed greater seminiferous tubule diameter, a lower proportion of empty space, and more UCHL1-positive cells per seminiferous tubule. Gene sets related to ATP-dependent chromatin remodeling, DNA replication, and male meiotic nuclear division were enriched in ALA-treated fragments, whereas immune gene sets were enriched in CK fragments. The relative expression levels of SYCP3 and MKI67 were higher in ALA-treated fragments than in CK fragments. Overall, these preliminary findings suggest that ALA treatment was associated with morphological and transcriptional changes in cultured sheep testicular tissue. Further studies using tissues from multiple rams are needed to confirm these findings.
