Ether-lipid deficiency remodels the testicular interstitium beyond spermatogenic arrest in Gnpat-knockout mice

Ether lipids, including plasmalogens and the germ-cell glycolipid seminolipid, require the peroxisomal enzyme GNPAT. Gnpat-knockout mice are male-infertile, but their testis has been described only qualitatively. We immunostained testis sections from 12-week-old wild-type (WT) and Gnpat-knockout (KO) mice (n = 3 per genotype) for SOX9 and {gamma}H2AX with DAPI and analysed 24 fields (22,560 nuclei) with an automated pipeline and a conservative animal-level analysis. Seminiferous tubules fell from 90% to 43% of the section, and the interstitium expanded five-fold (from 11% to 54%). Interstitial cells were 1.6-fold larger but not more densely packed, and half of them, against a quarter in WT, showed lipofuscin-like autofluorescence. Spermatid-sized nuclei fell from 38% to 10%, with the loss confined to the adluminal compartment. The architectural, interstitial and germ-cell changes held at the animal level (P [≤] 0.02). Anucleate SOX9-immunoreactive bodies, likely residual bodies, were 2.2-fold rarer, and {gamma}H2AX was not increased, which argues against a DNA-damage checkpoint arrest. Ether-lipid deficiency thus arrests germ cells, most likely through loss of seminolipid, and remodels the interstitium into an expanded compartment of enlarged, autofluorescent cells, a second target relevant to rhizomelic chondrodysplasia punctata.