Fig 2: Cct6b-/- mice exhibit normal spermatogenesis.(A) The average number of pups per litter for Cct6+/+ and Cct6b-/- mice, n = 3. (B) Images of testis and epididymis samples from Cct6b+/+ and Cct6b-/- adult mice. (C) Average testis weight normalized to body weight, n = 3. (D) Testis sections from Cct6+/+ and Cct6b-/- mice following Periodic-acid Schiff staining. (E) The relative composition ratios of different cell types in testis sections at spermatogenic stages VIII and XI. P-L, pre-leptotene; Z, zygotene; P, pachytene; D, diplotene; RS, round spermatids; ES, elongated spermatids.

Fig 3: Cct6b-/- mice exhibit normal spermatogenic marker expression.(A) DNA double-strand break formation during meiosis, as measured via ?H2AX staining, appeared normal. Spermatids from these knockout mice also exhibited normal acrosomal structures (PNA); Z, zygotene; P, pachytene; D, diplotene; n = 3. (B)–(C) Spermatogonia proliferation (LIN28A) did not differ significantly when comparing CCT6b-/- and CCT6b+/+ mice; Spg, spermatogonia, n = 3. (D)–(E) Sertoli cells (SOX9) numbers in testis sections were comparable in Cct6+/+ and Cct6b-/- mice. Ser, Sertoli cell, n = 3.

Fig 4: Cct6b distribution patterns in mice.(A) The expression of Cct6b in different murine tissues (12 weeks) was assessed via qPCR. n = 3. (B) The expression of Cct6b in murine testes at different developmental stages, including 0, 1, 2, 3, 4, 5, 6, and 7 weeks, was assessed via qPCR. 18s served as a normalization control for all qPCR analyses.

Fig 5: Cct6b-/- mouse generation.(A) An overview of the CRISPR/Cas9 knockout targeting strategy. (B) Sanger sequencing indicated the presence of a 5-bp deletion in Cct6b-/- mice. (C) Cct6b expression was confirmed to be reduced via qPCR in Cct6b-/- mice. (D) Western blotting confirmed that testis samples from Cct6b-/- mice did not exhibit a band of the expected size (58.1 kD). ß-TUBULIN served as a normalization control.