Phylogenetic tree of the clasp gene family.

(a) Schematic experimental design outline for organs dissected from adult zebrafish (Danio rerio). (b) Schematic experimental design outline for organs dissected from an adult guppy (Poecilia reticulata). (c) qPCR analysis of clasp2 in the brain, eye, kidney, testis, and ovary of adult zebrafish. Clasp2 is most transcribed in the testis. Statistical analysis was completed using a one-way ANOVA with Tukey–Kramer post hoc tests, adjusted for multiple comparisons (n = 3 animals used). * p < 0.01; ** p < 0.001; *** p < 0.0001. ns—not significant. (d) qPCR analysis of clasp2 in the brain, eye, kidney, testis, and ovary of an adult guppy. Clasp2 is most transcribed in the testis. Statistical analysis was completed using a one-way ANOVA with Tukey–Kramer post hoc tests, adjusted for multiple comparisons (n = 3 animals used). * p < 0.01; ** p < 0.001; *** p < 0.0001.

Clasp2 mRNA transcription pattern in adult zebrafish testis at widefield fluorescence microscopy. (a) DAPI staining marks cell nuclei. (b) Fluorescence in situ hybridization of clasp2 in adult zebrafish testis. (c) Fluorescence in situ hybridization of clasp2 and cell nuclei (DAPI). (d) High magnification of spermatozoa cell nuclei (DAPI). (e) High magnification of clasp2 expressing cells. (f) High magnification of fluorescence in situ hybridization of clasp2 and cell nuclei (DAPI). Abbreviations: Spz: spermatozoa; Spc: spermatocytes; Spg: spermatogonia (inside the dashed line) in adult zebrafish testis. Scale bars: 100 µ (a–c) and 25 µ (d–f).

Clasp2 transcription pattern in adult zebrafish testis at confocal microscopy. (a) DAPI staining marks cell nuclei. (b) Fluorescence in situ hybridization of clasp2 in adult zebrafish testis. (c) Fluorescence in situ hybridization of clasp2 and cell nuclei (DAPI) in adult zebrafish testis. (d) DAPI staining marks cell nuclei at high magnification. (e) Fluorescence in situ hybridization of clasp2 in adult zebrafish testis at high magnification. (f) Fluorescence in situ hybridization of clasp2 and cell nuclei (DAPI) in adult zebrafish testis at high magnification. Abbreviations: Spz; spermatozoa. Spt; spermatids. Scale bars: 100 µ (a–c) and 25 µ (d–f).

Clasp2 transcript distribution in sections of adult guppy testis. (a) In this section, we identified the organization of the testis. Cysts containing germ cells in the early stages of spermatogenesis (spermatogonia and spermatocytes) are located near the periphery of the testis, while cysts with numerous spermatids at different stages of spermiogenesis are located deeper, near the efferent ducts. In the same region, there are present spermatozeugmata cysts, in which spermatozoa are tightly packed with the sperm heads oriented towards the Sertoli cells and flagella oriented towards the center of the cyst. Spermatozeugmata is also found inside the efferent ducts (located in the central region of the testis), where mature spermatozoa are released. Chromogenic in situ hybridization of clasp2 in this section shows a clear marking at the periphery of the spermatozeugmata. The peripheral region of spermatozeugmata cysts located inside the efferent ducts shows weak staining (Sg: spermatogonia; Sc: spermatocytes; St: spermatids; Sz: spermatozeugmata; Ed: efferent ducts). (b) The strong marking of spermatozeugmata. (Sz: spermatozeugmata). Scale bars: 100 µ (a); 50 µ (b).

Clasp2 mRNA distribution in adult guppy testis, confocal microscopy. (a) Fluorescence in situ hybridization of clasp2 shows the labeling concentrated at the periphery of numerous spermatozeugmata. (b) High magnification of a portion of spermatozeugmata in a white rectangle is shown in panel a. (c) Fluorescence in situ hybridization of clasp2 is seen at the periphery of spermatozeugmata cysts. (d) DAPI staining marks all the cell nuclei of the testis. (e) The merge of fluorescence in situ hybridization of clasp2 and DAPI in adult guppy testis. Abbreviation: Sz; spermatozeugmata. Scale bars are 50 µ (a); 10 µ (b); and 30 µ (c–e).