Dnajb8, a target gene of SOX30, is dispensable for male fertility in mice

Background The DNAJ family of molecular chaperones maintains protein homeostasis in mitotic and postmeiotic cells, especially germ cells. Recently, we found that the transcription factor SOX30 initiates transcription of Dnajb8 during late meiosis and spermiogenesis in mouse testes. Methods We used the CRISPR/Cas9 system to generate Dnajb8 mutant mice and analyze the phenotype of the Dnajb8 mutants. Results AlthoughDnajb8 is an evolutionarily conserved gene, it is not essential for spermatogenesis and male fertility. We provide this phenotypic information, which could prevent duplicative work by other groups.


INTRODUCTION
Mammalian spermatogenesis is a complicated cellular process during which diploid spermatogonial stem cells (SSCs) generate haploid spermatozoa. The process takes place in seminiferous epithelium of the testis and includes three successive developmental phases: spermatogonial proliferation and differentiation, two meiotic divisions to produce haploid round spermatids, and the differentiation of spermatids into sperm (Nishimura & L'Hernault, 2017). During spermatogenesis, the accurate regulation of protein folding and sorting is fundamental for the production of high-quality spermatozoa (Meccariello et al., 2014).
The DNAJ family is the largest molecular chaperone family and plays a key role in protein folding, trafficking, aggregation, homeostasis and conformation (Zarouchlioti et al., 2018). The human DNAJ proteins are divided into three subgroups based on a highly conserved domain structure containing a J domain that interacts with HSP70 (type III), a Gly/Phe-rich region (type II) and a cysteine-rich region (type I) (Cheetham & Caplan, 1998). Mutations in DNAJ proteins occur frequently in neurodegenerative diseases, such as cerebellar ataxia, distal hereditary motor neuropathy and Parkinson's disease (Koutras & Braun, 2014). Nevertheless, DNAJ proteins has also been implicated in tumorogenesis and spermatogenesis (Kusumoto et al., 2018;Meccariello et al., 2014;Nishizawa et al., 2012). As there are functional differences among the DNAJ proteins, it is unclear whether deficiencies in each DNAJ protein could lead to male infertility.
DNAJB8, a member of the DNAJB family of proteins, plays a role in suppressing protein expression by interacting with HDACs (Hageman et al., 2010). A recent study also showed that DNAJB8 is a cancer-testis (CT) antigen carrying tumor-initiating ability (Nishizawa et al., 2012). Among mouse tissues, DNAJB8 is highly expressed in the testis, especially in postmeiotic germ cells (Nishizawa et al., 2012). Notably, we previously found that Dnajb8 was directly regulated by the transcription factor SOX30, which controls the expression of a core set of postmeiotic genes (Bai et al., 2018). However, whether DNAJB8 affects spermatogenesis and male fertility is still not clear. Because in vitro spermiogenesis has still not been established, we generated a Dnajb8 knockout (KO) mouse via Cas9/RNA-mediated gene targeting to understand the function of DNAJB8 in spermatogenesis.

Animals
Animals were housed in Laboratory Animal Center at University of Science and Technology of China under specific pathogen-free conditions with free access to food and water. All mice were treated humanely and euthanized by cervical dislocation to collected testis and epididymal samples for further analyses. The use of animals and the experimental design were approved by the Institutional Animal Care and Use Committees of the University of Science and Technology of China (No. 2019-N(A)-061), Hefei, China.

Generation of an anti-DNAJB8 polyclonal antibody
DNAJB8 polyclonal antibody was prepared by ABclonal (Wuhan, China) and derived from rabbits. Recombinant fusion protein contained a sequence corresponding to a fragment (amino acids 116-132) of mouse DNAJB8. We have used this antibody for western blot.

Generation of Dnajb8 knockout mice with the CRISPR/Cas9 System
Dnajb8 knockout mice were generated using CRISPR/Cas9 technology. The Dnajb8 exon containing the J-domain was targeted by sgRNAs. The sgRNA was synthesized by Genescript (Nanjing, China) and the sequences were as follows: 5 -ACCTGTCCCTGAGAACGGGG-3 and 5 -CATAGTAGTCGCAACCTAAC-3 . The Cas9 expressed vector pX330 was obtained from Addgene, linearized with NotI (New England Biolabs, USA), transcribed using T7 ULTRA Transcription Kit (Ambion AM1345, USA) and purified using MEGAclear TM Kit (Ambion AM1908, USA). Cas9 mRNA and sgRNAs were coinjected into fertilized eggs for KO mouse production. The pups were genotyped by genomic PCR followed by Sanger sequencing. After genotyping, the F0 mice went through serial mating to generate homozygous mutants.

Fertility test
Every 8-week-old Dnajb8 +/+ and Dnajb8 −/− male mouse was caged with two 8-week-old Dnajb8 +/+ female mice for at least 8 weeks. During the breeding test, the number of pups was counted at birth, and the average litter size for each mouse line was recorded.

Histology analysis
Testes and epididymides from 10-week-old Dnajb8 +/+ and Dnajb8 −/− male were fixed in Bouin's solution overnight, dehydrated with increasing concentrations of ethanol (70%-100%) and embedded in paraffin. The tissue was cut into 5-µm-thick sections and mounted onto glass slides and followed by Hematoxylin and Eosin (H&E; Sigma-Aldrich, USA) staining. For sperm staining, epididymal sperm from the of 8-week-old male mice were extruded, fixed in 4% PFA at 4 • C overnight, and immobilized onto glass slides. The sections were stained with H&E. All sections were analyzed microscopically (LEICA DM2500, Germany).

Sperm parameters analysis
Sperm parameters assays were performed using 10-week-old Dnajb8 +/+ and Dnajb8 −/− male. Cauda epididymal sperm were extracted, incubated in PBS, fixed in 4% of PFA, followed by sperm counting using a hemocytometer. For sperm motility assays, cauda epididymal sperm were released into HTF medium at 37 • C and measured using computeraided sperm analysis (CASA) system (Hamilton Thorne Biosciences, USA).

Western blotting
Tissues and spermatozoa that separated from seminal plasma were rinsed with PBS and lysed in cold RIPA buffer supplemented with phosphatase inhibitor and protease inhibitor cocktail tablets. Lysates were incubated on ice and centrifuged at 13000 rpm for 15 min at 4 • C. The protein concentration was determined by the Bicinchoninic Acid (BCA) Assay (E11201, Vazyme, China) according to the manufacturer's instructions. In total, 20 µg of proteins was separated on 10% SDS-PAGE gels. The primary antibodies used were as follows: anti-DNAJB8 (diluted 1:1000 in TBST, ABclonal, China) and anti-α-TUBULIN (diluted 1:5000 in TBST, 11224-1-AP, Proteintech, China).

Phylogenetic analyses
Multiple amino acid sequence alignments and phylogenetic trees were constructed by the MEGA program. The amino acid sequences were downloaded from the NCBI database.

Statistical analysis
All data are reported as mean ± SD. Significance was tested using a two-tailed unpaired Student's t test using Prism 7.0 software. A p value < 0.05 was considered statistically significant. NS means not significant.

SOX30 regulates Dnajb8 transcription
In a previous study, we combined bioinformatics analyses of the transcription factor SOX30 RNA-seq and ChIP-seq datasets to reveal that SOX30 directly regulates expression of a core set of postmeiotic genes (Bai et al., 2018). Among these direct targets, most have been reported to be involved in haploid germ cell development, such as Tnp1, Hils1, Ccdc54 and Tsks, while the function of some haploid cell-enriched genes during spermatogenesis, including Dnajb8, remained unclear. We reanalyzed our published SOX30 RNA-seq datasets and found that Dnajb8 was downregulated in both Sox30 null pachytene spermatocytes and round spermatids compared with wild-type cells (Figs. 1A,  1B). In addition, our published SOX30 ChIP-seq datasets also showed that strong binding peaks were observed at the Dnajb8 promoter, indicating that Dnajb8 is a direct downstream target of SOX30 (Fig. 1C).

Dnajb8 is a conserved and testis-enriched gene
Phylogenetic analyses demonstrated that DNAJB8 protein is conserved between a variety of mammalian species ( Fig. 2A). We further found that Dnajb8 transcripts were highly and exclusively in the testis using published RNA-seq data generated from different mouse tissues (Fig. 2B) (Li et al., 2017). Moreover, Dnajb8 transcripts displayed dynamic expression patterns during spermatogenesis (Soumillon et al., 2013). As shown in Fig. 2C, Dnajb8 transcripts began to increase in pachytene spermatocytes and plateaued in round spermatids, but they were reduced in spermatozoa.

Generation of Dnajb8 knockout mice
To explore the function of DNAJB8, Dnajb8 mutant mice with a Danjb8 allele containing an 888-bp deletion were generated by CRISPR-Cas9 system (Figs. 3A, 3B). Homozygous mutant Dnajb8 alleles were obtained by selective breeding and determined by genotyping PCR (Fig. 3C).

DNAJB8 is not essential for germ cell development
To characterize the spermatogenesis in Dnajb8 −/− mice, we next performed immunostaining for γ -H2AX-positive spermatocytes and PNA-positive acrosomes in spermatids. As shown in Fig. 6A, different stages of spermatocytes and spermatids were both observed in seminiferous tubules from adult wild-type and Dnajb8 −/− mice. We then immunostained testis sections for the Sertoli cell marker SOX9 and found that seminiferous tubules from wild-type and Dnajb8 −/− males (n = 3 for each group) contained similar numbers of Sertoli cells (Figs. 6B, 6C). The area of seminiferous tubules from Dnajb8 +/+ and Dnajb8 −/− testes (n = 3 for each group) were similar (Fig. 6D). Furthermore, TUNEL staining revealed that the numbers of apoptotic cells and tubules did not differ between Dnajb8 −/− and Dnajb8 +/+ testes (n = 3 for each group) (Figs. 6E-6G). These results show that Dnajb8 deficiency does not affect spermatogenesis in Dnajb8 −/− mice.

DISCUSSION
Here, we reanalyzed RNA-seq and ChIP-seq datasets from the transcription factor SOX30 and demonstrated that SOX30 directly regulates expression of the postmeiotic gene Dnajb8. As Dnajb8 is a highly conserved and testis-enriched gene, we further generated Dnajb8 KO mice by CRISPR/Cas9 and found that Dnajb8 is not required for male fertility, with no difference in testicular or epididymal histology and average litters sizes compared to wild-type males.
To date, several DNAJB proteins have been identified as being involved in spermatogenesis and male fertility (Meccariello et al., 2014). DNAJB1 is mainly expressed in mouse testis and is localized in the acrosomal region of the sperm head, the middle and end pieces of the sperm tail (Doiguchi et al., 2007). DNAJB3 protein is highly expressed in haploid germ cells and may be involved in vesicle fusion (Berruti & Martegani, 2001). Importantly, it has been reported that homozygous mutations in DNAJB13, a radial spoke protein of the mouse '9+2' axoneme that localized to the sperm flagella, cause male infertility, with severe oligo-astheno-teratozoospermia (El Khouri et al., 2016;Li & Liu, 2014). Moreover, heterozygous variants in DNAJB13 were correlated with male fertility in asthenozoospermia (Li et al., 2020). DNAJB13 interactions with SUN5 play a crucial role in sperm head-tail integration (Shang et al., 2018). Similar to the expression pattern of the DNAJB family, Dnajb8 is a testis-specific gene and is predominantly in spermatids. Notably, Dnajb8 was downregulated in spermatozoa of infertile men by 12-fold compared to that of normospermic individuals (Montjean et al., 2012). Hence, DNAJB8 was thought to play a role in germ cell development. However, our study showed that all stages of spermatogenic cells were detected in Dnajb8 −/− seminiferous tubules. Meanwhile, immunofluorescence staining of γ H2AX-positive spermatocytes and PNA-positive spermatids, as well as SOX9positive Sertoli cells, were not different between Dnajb8 −/− and Dnajb8 +/+ mice. These data indicate that DNAJB8 protein is not essential for spermatogenesis.
Previous studies have found that more than 54 conserved testis-enriched proteins were not essential for fertility (Miyata et al., 2016). This may be because these genes are highly covered by the redundancy of other genes. Functional redundancy of DNAJ family proteins has been shown to exist in yeast (Sahi & Craig, 2007). Interestingly, most Dnajb transcripts were mainly detected in the testis, especially the haploid germ cell-enriched genes Dnajb3 and Dnajb7 (Li et al., 2017;Soumillon et al., 2013), indicating that redundancy may be found in human DNAJs. Thus, DNAJ proteins work in parallel, and this redundancy could explain the normal male fertility of Dnajb8 KO mice.
Although we investigated litter size using co-housing assay, which provides unrestricted access of males to females, the protocol may be not detect subtle changes in Dnajb8 KO males. Nevertheless, we have shown that Dnajb8 is not essential for male fertility under normal laboratory mating conditions in this study.
Taken together, our findings demonstrate that mouse DNAJB8 is dispensable for spermatogenesis and male fertility. Even though Dnajb8 is a postmeiotic gene directly regulated by SOX30, we did not observe any defects in germ cell development in Dnajb8 −/− males. One possible explanation for this is the redundant mechanisms of DNAJ proteins that control male fertility.