Deletion of lncRNAs lead to various defects in spermatogenesis. (A) LncRNA knockout mutants cause malformation and obstruction of the testis: (top rows) whole testis; (middle rows) seminal vesicle stained with DAPI; (bottom rows) sperm in seminal vesicle. Scale bars are noted. Testes of CR44455/6−/−, lncRNA:TS23−/−, and CR43282−/− contained an accumulation of abnormal white flocculus, although these lncRNA mutants had a normal spherical testis shape. Seminal vesicles from CR44455/6−/− males contained smaller sperm relative to wild type. lncRNA:TS23−/− contained significantly reduced numbers of mature sperm in seminal vesicles, whereas the numbers in CR43282−/− were comparable to those in the wild type. (B) LncRNA knockouts affect male germ cell development. Testes squash preparations were stained with DAPI to visualize DNA of the wild-type and lncRNA mutants. In the wild type, the initially round spermatids nuclei elongated and condensed to form long, straight, and needle-shaped mature sperm. In lncRNA:TS1−/−, some mature-stage sperm adopted a tadpole shape in which the nucleus was concentrated at one end of the spermatid head. Deletion of CR42858 led to scattered or curled sperm in which some nuclei did not fully condense. CR45542−/− exhibited some round uncondensed sperm at a very mature stage. (C) Chromatin condensation defects of lncRNA mutants appear in late spermatogenesis. HIS2AV-RFP and Protamine B-GFP were used to distinguish early spermatid nuclei and mature sperm, respectively. The round uncondensed nuclei of CR44420−/− and the bent sperm of CR43416−/− were labeled by Protamine B-GFP but not HIS2AV-RFP, indicating that these abnormal germline cell phenotypes appeared in late elongate to mature sperm stage. (D) Spermatogenesis in wild type and CR44455/6−/− (male infertility). Spermatids in CR44455/6−/− were smaller than those in the wild type from the meiotic stages onward. The nuclei of mature sperm in CR44455/6−/− were half the size of those in wild-type sperm. (E) LncRNA mutants exhibit individualization defects. Phalloidin was used to stain investment cones (ICs) in wild-type and lncRNA mutants. Wild-type testis contained ordered and associated ICs. In CR43484−/− and CR43282−/−, ICs were severely disorganized or lagged, and individual actin cone structures were scattered. Scale bars are noted.
