Palmitoyltransferase ZDHHC19 regulates histone-to-protamine exchange during spermiogenesis in mice

Abstract

Spermiogenesis, the final phrase of spermatogenesis, involves replacing histones by protamines, a process known as histone-to-protamine exchange, which is crucial for chromatin condensation. While this exchange is essential, the precise mechanisms remain incompletely understood. In this study, we discovered that the palmitoyltransferase ZDHHC19 functions as a key regulator of mouse spermiogenesis. Loss of Zdhhc19 leads to male infertility and abnormal sperm morphology. Zdhhc19-deficient sperm exhibit impaired histone-to-protamine exchange, leading to retention of histones and misdistribution of protamines. Similarly, the palmitoyltransferase catalytic site mutant Zdhhc19 C142S knock-in mice show reduced fertility, sperm abnormalities, and histone retention. Mechanistically, ZDHHC19 mediates histone H3 palmitoylation at cysteine-110, weakening H3-H4 interactions and increasing chromatin accessibility. Palmitoylation of H3 facilitates histone-to-protamine exchange. This study highlights the essential role of ZDHHC19's palmitoyl-transferase activity in histone-to-protamine exchange during spermiogenesis and underscores the broader role of histone palmitoylation in chromatin remodeling.

Fig. 1.. Zdhhc19 knockout in germ cell impairs spermatogenesis.

(A) Expression of Zdhhc19 mRNA levels in several mouse organs. Error bars represent means ± SEM. ***P < 0.001, by Student’s t test. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) Immunoblot of ZDHHC19 in testes from adult mice as indicated genotypes (top). Quantitative of grayscale statistical graph of ZDHHC19 (bottom) (n = 3). Error bars represent means ± SEM. n.s., not significant. *P < 0.05; ***P < 0.001, by Student’s t test. (C) Fertility analysis of Zdhhc19 knockout and conditional knockout mice compared to that of control mice. FCP, frequency of copulatory plug, ratio of count of plugs to total count of females; FC, frequency of conception, ratio of count of litters to count of plugs. (D) The Diff-Quick staining of cauda epididymal sperm from adult mice. (E) Percentage of bent head sperm from Zdhhc19 knockout and conditional knockout males by Diff-Quick staining. Error bars represent means ± SEM. ***P < 0.001, by Student’s t test.

Fig. 2.. Loss of Zdhhc19 impairs spermiogenesis by disrupting chromatin condensation.

(A) Fluorescent staining of sperm and proportion of bent head sperm analysis from Zdhhc19^+/+ and Zdhhc19^−/− epididymis cauda (n = 4). Red, mito tracker for mitochondrion; green, peanut agglutinin (PNA) for acrosome; blue, Hoechst 33342 [4′,6-diamidino-2-phenylindole (DAPI)] for nuclei. Error bars represent means ± SEM. ***P < 0.001, by Student’s t test. (B) Scanning electron microscopy of sperm and proportion of bent head sperm analysis from Zdhhc19^+/+ and Zdhhc19^−/− epididymis cauda (n = 3). Error bars represent means ± SEM. ***P < 0.001, by Student’s t test. (C) TEM of sperm from Zdhhc19^+/+ and Zdhhc19^−/− epididymis cauda. (D) SCSA revealed the chromatin integrity of Zdhhc19^+/+ and Zdhhc19^−/− sperm (n = 3). Error bars represent means ± SEM. **P < 0.01, by Student’s t test.

Fig. 3.. Zdhhc19 deletion disrupts histone-to-PRM exchange in sperm.

(A) Representative confocal images of immunofluorescence using anti-H2A, H2B, H3, and TNP2 antibodies in mature sperm from Zdhhc19^+/+ and Zdhhc19^−/− mice. (B) Representative confocal histological section images of Zdhhc19^+/+ and Zdhhc19^−/− epididymis cauda using anti-PRM1 and anti-PRM2 antibodies (red). (C) Immunoblot of core histones, TNPs, and PRMs from Zdhhc19^+/+ and Zdhhc19^−/− mature sperm.

Fig. 4.. The PAT activity of ZDHHC19 is essential for spermiogenesis.

(A) Schematic representation of Zdhhc19 C142S knock-in mouse model. (B) The transcription and protein level of Zdhhc19 in Zdhhc19^+/+ and Zdhhc19^C142S/C142S testes (n = 3). Error bars represent means ± SEM. ***P < 0.001, by Student’s t test. (C) Verified palmitoylation level of ZDHHC19 in Zdhhc19^C142S/C142S mice testis. (D) Pregnancy rate and number of pups per litter (the average number of offspring that pregnant females give birth to) were compared between Zdhhc19^+/+ and Zdhhc19^C142S/C142S male mice. n is the number of tested males. Error bars represent means ± SEM. ***P < 0.001, by Student’s t test. (E) Morphology of sperm and the percentage of bent head sperm from Zdhhc19^+/+ (n = 12) and Zdhhc19^C142S/C142S (n = 17) mice. The red arrows indicate the bent head sperm. Error bars represent means ± SEM. ***P < 0.001, by Student’s t test. (F) Motility and several velocities of sperm from Zdhhc19^+/+ (n = 8), Zdhhc19^C142S/+ (n = 8), and Zdhhc19^C142S/C142S (n = 6) males were analyzed by CASA. VAP, average path velocity; VSL, straight line velocity; VCL, curve line velocity. Error bars represent means ± SEM. ***P < 0.001, by Student’s t test. (G) Immunoblot of core histones, TNPs, and PRMs from Zdhhc19^+/+ and Zdhhc19^C142S/C142S sperm. (H) Representative confocal images of immunofluorescence using anti-H2B and PRM1 antibodies in sperm from Zdhhc19^+/+ and Zdhhc19^C142S/C142S mice.

Fig. 5.. ZDHHC19 specifically catalyzes palmitoylation of Histone H3 at cysteine-110.

(A) Immunoblot analysis of palmitoylation level of H3 in Zdhhc19^+/+, Zdhhc19^−/−, and Zdhhc19^C142S/C142S testes. (B) Quantitative of grayscale statistical graph of palmitoylated H3 in (A) (n = 3). Relative palm-H3 is calculated by palm/input ratios and normalized by the ratio of Zdhhc19^+/+. Error bars represent means ± SEM. n.s., not significant. *P < 0.05, by Student’s t test. (C) Palmitoylation level analysis (left) and quantitative of grayscale statistical graph (right) of corresponding proteins in Zdhhc19^+/+ and Zdhhc19^−/− testes (n = 3). Error bars represent means ± SEM. n.s., not significant, by Student’s t test. (D and E) Palmitoylation level of endogenous H3 after overexpression MIG (vector), and ZDHHC19^WT and ZDHHC19^C142S protein in GC1 cells (D) and 3T3 (E) cells. (F) Palmitoylation level (left) and quantitative of grayscale statistical graph (right) of ectopic WT and mutant Flag-tagged H3 after overexpression ZDHHC19^WT protein in 3T3 cells (n = 3). Relative palm-H3 is calculated by palm/input ratios and normalized by the ratio of WT group. Error bars represent means ± SEM. n.s., not significant. ***P < 0.001, by Student’s t test.

Fig. 6.. ZDHHC19-mediated H3 palmitoylation facilitates histone removal during spermiogenesis.

(A) Histological sections of elongating spermatid stage seminiferous epithelium from Zdhhc19^+/+, Zdhhc19^−/−, and Zdhhc19^C142S/C142S testes were stained with H3 (red), PNA (green), and DAPI (blue). (B) CoIP assay with anti-H3 antibody in haploid spermatids from Zdhhc19^+/+, Zdhhc19^−/−, and Zdhhc19^C142S/C142S testes. (C) Quantitative of grayscale statistical graph of IP-H4 in (B) (n = 4). Relative IP-H4 is calculated by IP/input ratios and normalized by the ratio of Zdhhc19^+/+. Error bars represent means ± SEM. *P < 0.05; **P < 0.01, by Student’s t test. (D) Quantitative analysis of IP-H4 in (B) using mass spectrometry (n = 2). The ratio is calculated by IP-H4/IP-H3 abundance. *P < 0.05, by Student’s t test. (E) CoIP assay with anti-Flag antibody in 3T3 cells overexpression WT and mutant Flag-tagged H3 with ZDHHC19^WT protein. (F) Quantitative of grayscale statistical graph of IP-H4 in (E) (n = 3). Relative IP-H4 is calculated by IP/input ratios and normalized by the ratio of WT group. Error bars represent means ± SEM. n.s., not significant. *P < 0.05; **P < 0.01, by Student’s t test. (G) Mass spectrometry quantitative analysis of IP-H4 in 3T3 cells overexpression WT (n = 3) and C110S (n = 2) Flag-tagged H3 with ZDHHC19^WT protein. The ratio is calculated by IP-H4/IP-H3 abundance. Error bars represent means ± SEM. *P < 0.05, by Student’s t test.

Fig. 7.. ZDHHC19-mediated H3 palmitoylation facilitates chromatin remodeling during spermiogenesis.

(A) Chromatin immunoprecipitation (ChIP) and quantitative polymerase chain reaction (qPCR) assay of haploid spermatids from Zdhhc19^+/+, Zdhhc19^−/−, and Zdhhc19^C142S/C142S mice (n = 3). Relative DNA concentration is normalized by the DNA concentration of Zdhhc19^+/+. Primers of genomic mouse GAPDH (gmGAPDH) were used in qPCR. Error bars represent means ± SEM. ***P < 0.001, by Student’s t test. (B) ChIP and qPCR assay of WT and mutant Flag-tagged H3 after overexpression ZDHHC19^WT protein in 3T3 cells. DNA concentration is normalized by the DNA concentration of WT group. Error bars represent means ± SEM. *P < 0.05; ***P < 0.001, by Student’s t test. (C) Average signal profiles and heatmap display normalized ATAC-seq signal intensity across 3-kb regions around transcription start site (TSS) in Zdhhc19^+/+ and Zdhhc19^−/− haploid spermatids (n = 2). (D) Average signal profiles and heatmap display normalized ATAC-seq signal intensity across 3-kb regions around TSS in GC1 cells after overexpression Migr (vector) or ZDHHC19^WT protein (n = 2).

Fig. 8.. Illustration of the function and mechanistic role of ZDHHC19 in spermiogenesis.

During spermiogenesis in Zdhhc19^−/− and Zdhhc19^C142S/C142S mice, H3 displayed lower palmitoylation, and histone showed more retention compared to Zdhhc19^+/+, which resulted to bent head sperm and male infertility. Created in BioRender. J. Li (2025) https://BioRender.com/7dt6uan.