Fig 2: Defective elongation of round spermatids in the Spindoc-null mice. (A) Immunofluorescence staining of testicular sections from WT and KO mice at 5 months of age demonstrating no significant loss of germ cells in the KO mice. Scale bar, 20 μm. Quantification of germ cells was performed in each seminiferous tubule of WT(n = 20) and KO(n = 23) at 5 months of age (right panel). Data were presented as the mean ± SD, p > 0.1. (B) HE staining of seminiferous tubules from the WT and KO mice at the age of 2 months, indicating interrupted transition from round spermatids to elongated spermatids. Between stages IX- XII, round spermatid-like (RSL) cells were present in the KO testes; Pl, pre-leptotene; L, leptotene; Z, zygotene; P, pachytene; RS, round spermatids; RSL, round spermatid-like; CS, condensed spermatids; ES, elongated spermatids; M, metaphase. Scale bar, 20 μm. (C) HE staining of seminiferous tubules from the WT and KO mice at the age of 5 months, indicating defective elongation of round spermatids (stages IX-XII) in the KO mice. Scale bar, 20 μm

Fig 3: Spindoc is predominantly expressed in testis. (A) Violin plots showing the mRNA expression pattern of Spindoc in multiple human organs in the GTEx database (TPM, Transcripts Per Million). (B) RT-qPCR analyses of Spindoc mRNA levels across ten organs from adult wild-type (WT) mice. Data are presented as mean ± SEM, n = 3. (C) Western blot showing the expression levels of Spindoc protein among ten organs of WT adult mice. GAPDH served as a loading control. (D) Densitometric quantification of Spindoc protein levels as in (C). Data are presented as mean ± SEM, n = 3. (E) RT-qPCR analyses of Spindoc mRNA levels in developing testes. Testes at postnatal Day 3 (P3), P5, P7, P10, P12, P14, P17, P21, P35, P42 and P56 were analyzed. Data are presented as mean ± SEM, n = 3. (F) Western blot illustrating the Spindoc protein levels in developing testes. Testes at postnatal Day 3 (P3), P5, P7, P10, P12, P14, P17, P21, P35, P42 and P56 were analyzed. GAPDH served as a loading control. (G) Dynamic expression pattern of Spindoc mRNA from single cell RNA-seq analyses in adult human testes [18]. SSC, Spermatogonial Stem cells. (H) Dynamic expression levels of Spindoc mRNA from single cell RNA-seq analyses in RA-synchronized testicular cells [19]. A1, type A1 spermatogonia; In, intermediate spermatogonia; BS, S phase type B spermatogonia; ePL, early preleptotene; mPL, middle preleptotene; lPL, Late preleptotene; L, leptotene; Z, zygotene; eP, early pachytene; mP, middle pachytene; lP, late pachytene; D, diplotene; MI, metaphase I; MII, metaphase II; RS1–2, steps 1–2 spermatids; RS3–4, steps 3–4 spermatids; RS5–6, steps 5–6 spermatids; RS7–8, steps 7–8 spermatids

Fig 4: Spindoc KO caused impaired sperm production leading to male subfertility. (A) Number of pups per litter from male mice (> 8 weeks) naturally crossed with WT female mice (> 6 weeks) for 4 months. Data are presented as the mean ± SD, n = 5, p < 0.0001 by student t-test. (B) Histological analysis of the cauda epididymis from the WT and KO mice. The average number of sperm released from the cauda in KO mice was less than that in WT mice. Scale bar = 50 μm. The histogram showed the number of sperm retrieved from one cauda epididymis of WT and KO mice. Data were presented as mean ± SEM, n = 3. P < 0.01 by student t-test. (C) Histological analysis showing the morphology of sperm from cauda epididymis in WT and KO mice. Scale bar, 10 μm. (D) The histogram showing the percentage of sperm with abnormal morphology in WT and KO mice at the age of 6 months. Data are presented as mean ± SEM, n = 3. P < 0.001 by student t-test. (E) HE staining of sperm with normal morphology in the WT mice. Sperm with head defects, including irregular shape (F, G, H), coiled mid-piece tail (I, J, K), and tail defect (L), were more frequently observed in the KO mice. Scale bar, 5 μm

Fig 5: Generation of Spindoc knockout mouse models. (A) Schematic diagram shows the targeting strategy for generating Spindoc knockout (KO) mice using the CRISPR/Cas9 system. Blue boxes represent exons of the Spindoc gene on mouse chromosome 19. The genomic sequence targeted by a pair of sgRNAs are underlined with PAM sequences being highlighted in purple color. The red arrowheads point to the cut sites around exon 2 of Spindoc gene. Two F0 founder lines were generated - Line 1 carried 1 nt (T) insertion, while Line 2 carried a combination of 22 bp deletions as indicated; (B, C) Sanger sequencing of genomic DNA from mouse tail clips showing the frameshift variant (+ 1 nt) of KO mice (Line 1) (B), and the other frameshift variant (− 22 nt) (Line 2) (C); (D) Representative western blot analyses validated the Spindoc protein levels in WT and KO adult testes. GAPDH was used as a loading control. (E) Quantitative analysis of Spindoc protein levels in WT and KO adult testes. Data are presented as mean ± SEM, n = 3. P < 0.001 by student t-test. (F) Representative morphology of testes and epididymis from WT, Heterozygote (HET) and KO mice. The testis of KO was smaller than that of the WT; the epididymis of KO was more transparent than that of the WT. (G) Histogram showing the testis weights in WT and KO adult mice. Data are presented as mean ± SEM, n = 3. p < 0.001 by student t-test