Forkhead-associated phosphopeptide binding domain 1 (FHAD1) deficiency impaired murine sperm motility

Animal studies

All mice were obtained from and maintained under specific pathogen-free (SPF) conditions in the Laboratory Animal Center of Nanjing Medical University. The mice were maintained in a pathogen-free animal housing (20–22 °C, 50–70% humidity, 12 h light/dark cycle) with free food and water access. All mice were treated humanely and all efforts were made to minimize suffering. When appropriate, mice were euthanized with CO₂ and cervical dislocation prior to tissue sample collection. Each ventilated cage housed 4–5 mice, and cages were routinely cleaned, with bedding being added at a consistent density. On study completion, all mice were euthanized with CO₂. There were no surviving animals at the end of the study.

Animal ethics

The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):

The use of the mice and their treatment were checked and authorized under the Animal Ethical and Welfare Committee (Approval No. IACUC-1601117). Protocols for mouse feeding and use are under the guiding principles of the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University.

Fhad1 ^−/− mouse production

Fhad1^−/− mice were generated using CRISPR/Cas9 as in a prior report (Shen et al., 2013). To knock out the Fhad1 gene, a pair of guide RNAs (gRNA 1: 5′-gtgcagagaagttggtcacaggg-3′ and gRNA 2: 5′- gtgaggggcaggtgggaccatgg-3′) were designed onto the exon3 of the Fhad1 gene (NC_000070). Cas9 and sgRNA plasmids were linearized with AgeI and DraI, followed by purification with the MinElute PCR Purification Kit (Qiagen, Hilden, Germany). These sequences were then respectively prepared using the MESSAGE mMACHINE T7 Ultra Kit (Ambion, Austin, TX, USA) and the MEGA Shortscript and Clear Kit (Ambion, Austin, TX, USA). Male C57BL/6N mice and superovulated wildtype females were mated to generate zygotes into which the Cas9 mRNA and sgRNA constructs were injected. The injected embryos were then transferred into the uteri of recipient pseudo-pregnant females. Sanger sequencing was used to identify the genotype of the founder mouse and its offspring.

Genotyping

To mitigate the potential for off-target phenotypes, Fhad1^+/+ mice were mated with genome-edited founder mice lacking Fhad1 expression for a minimum of three generations. PCR amplification and Sanger sequencing was employed for the genotyping of all offspring with the primers detailed in Table S1.

qPCR

TRIzol (Vazyme, Xuanwu Qu, China) was used according to the provided instructions to extract tissue RNA, after which cDNA was generated with the PrimeScript Reverse Transcription Mix (Vazyme, Xuanwu Qu, China). A 7,500 real-time PCR instrument (Applied Biosystems, Waltham, MA, USA) and SYBR Green (Q131; Vazyme, Xuanwu Qu, China) were utilized for qPCR analyses, with individual 20 µL reactions containing 1 μL of cDNA, 0.8 μL of each primer, 7.4 μL of sterile water and 10 μL of SYBR Green Master Mix. The thermocycler settings were 95 °C for 5 min, followed by 40 cycles of alternating 95 °C and 60 °C steps. Melt curves were additionally generated to confirm the presence of only a single peak for each primer pair. Relative expression was assessed with the 2^−ΔΔCt method, using 18S rRNA as a comparison. Primers used for these analyses are documented in Table S1.

Cell lines and reagents

This study utilized the TM4 and 293T cell lines. Both TM4 and 293T cells were obtained from the Chinese Academy of Science Cell Bank (Shanghai, China). TM4 cells were grown in high-glucose DMEM (Invitrogen, Waltham, MA, USA) with 5% FBS (HyClone, Logan, UT, USA), while 293T cells were grown in RPMI-1640 (Invitrogen, Waltham, MA, USA) with 10% FBS (HyClone, Logan, UT, USA). Both media contained 1% antibiotic-antimycotic, and 10 mg/mL gentamicin (Life Technologies, Carlsbad, CA, USA). Cells were grown at 37 °C and 5% CO₂. The spermatogonial stem cell (SSC) cDNA used for Fhad1 detect was a gift from Prof. Zheng’s lab (Shen et al., 2019).

Cell sorting

A modified version of a previously published approach was used to purify germ cells (Bastos et al., 2005). Briefly, after collecting testis tissue from 8-week-old mice, the tissue samples were digested for 15 min using 1 mg/mL type IV collagenase (17104-019; Invitrogen, Waltham, MA, USA), digested for 10 min using 0.25% trypsin/1 mM EDTA (25200-072; Gibco, Waltham, MA USA), and suspended and stained for 30 min with PBS containing 0.1% Hoechst 33342 at 37 °C to enable the identification of haploid, diploid, and tetraploid cells. After three washes with PBS, the cells were sorted using a FACSAria Fusion SORP instrument (Becton Dickinson, Franklin Lakes, NJ, USA).

Analyses of fertility

Adult male Fhad1^+/+ and Fhad1^−/− mice were caged separately for 16 weeks with 2 Fhad1^+/+ C57BL/6N females. Data on vaginal plugs, number of pups, and birth dates of litters were logged to assess fertility-related outcomes. The knockout male mice were subjected to the same feeding conditions as controls. All mice used for the fertility test were 8–12 weeks old.

Histological staining

Epididymal and testicular tissue samples were collected from a minimum of three mice per genotype, fixed for 24 h in modified Davidson’s fluid, and stored in 70% EtOH. An EtOH gradient was used for sample dehydration, after which they were paraffin-embedded, cut to produce 5 μm sections, and mounted on glass slides. Before histological examination, samples were deparaffinized and underwent hematoxylin and eosin (H&E) staining. Sperm were spread on glass slides and air-dried, after which they were fixed in 4% paraformaldehyde (PFA) at room temperature for 30 min, followed by H&E staining and morphological observation.

Computer-assisted sperm analysis

Analyses of sperm characteristics were conducted as described (Castaneda et al., 2017). For each mouse, the distal cauda region of the right epididymis was clamped, excised, and rinsed with warmed PBS, before placing in an Eppendorf tube with 200 μl of fresh human tubal fluid (HTF) media (Millipore, Burlington, MA, USA) containing 10% fetal bovine serum (FBS) that had been pre-warmed to 37 °C. After removing the clamps, the cauda was pierced with a scalpel, and sperm were able to diffuse into the surrounding media for 5 min during which they were incubated at 37 °C, after which 10 μl of the sperm suspension was used to assess sperm motility using computer-assisted sperm analysis (CASA) (Hamilton Thorne Research Inc., Beverly, MA, USA).

Immunofluorescence

Tissue sections were deparaffinized, rehydrated, washed three times using PBS (10 min per wash), and boiled in a microwave in 10 mM citrate buffer solution (pH 6.0) for antigen retrieval. After three 10-min washes with PBS, samples were blocked with blocking buffer (1% BSA in PBS with 0.1% Triton X-100) for 2 h, followed by overnight incubation with the primary anti-γH₂AX antibody (ab81299, 1:1,000; Abcam, Cambridge, UK) at 4 °C. To test sperm samples, the sperm were spread directly on slides and allowed to air-dry. The sperm samples were then fixed in 4% PFA for 30 min, washed three times with PBS, blocked with 1% BSA in PBS containing 0.1% Triton X-100, and then incubated with anti-AC-TUBULIN (FNab00082, 1:1,000; FineTest, Palm Coast, FL, USA) at 4 °C overnight. Cell lines were grown on chamber slides precoated with fibronectin (FC010; Sigma, Burlington, MA, USA). The cells were washed three times with PBS and then fixed with 4% PFA for 15 min; after three times washes with PBS, the samples were blocked with 1% BSA in PBS containing 0.1% Triton X-100 and then incubated with anti-γH₂AX (ab81299, 1:1,000; Abcam, Cambridge, United Kingdom) at 4 °C overnight. Secondary antibody staining was performed for 2 h at room temperature. Nuclear counterstaining was performed for 5 min using Hoechst 33342. Sections were then washed with PBS, mounted with VectaShield or Immu-Mount, and imaged with an LSM800 confocal microscope (Carl Zeiss AG, Oberkochen, Germany).

Transmission electron microscopy

Transmission electron microscopy (TEM) analysis was performed in accordance with previous reports (Wu et al., 2023). The samples were first fixed in 1% osmium tetroxide, dehydrated in a series of ethanol solutions (50%, 70%, 90%, and 100%), followed by dehydration in 100% acetone. After infiltration with acetone and SPI-Chem resin and embedding via Epon812, the samples were sectioned using an ultra-microtome and stained with uranyl acetate and lead citrate. Visualization and image capture were conducted using a Talos L120C G2 electron microscope (Thermo Fisher, Waltham, MA, USA).

TUNEL staining

An In Situ Cell Death Detection Kit (Roche Diagnostics,11684795910, Indianapolis, IN, USA) and a TUNEL Bright-Green Apoptosis Detection Kit (A112-03; Vazyme, Jiangsu, China) were used to perform TUNEL staining, as reported previously (Hua et al., 2019).

Statistical analyses

Data were analyzed with GraphPad Prism 9.0. Results are presented as the mean ± standard deviation, and were compared with two-tailed Student’s t-tests. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Analyses of time- and tissue-dependent patterns of murine Fhad1 expression

To begin exploring the possible functional roles that FHAD1 plays in male fertility, Fhad1 expression patterns in murine tissues were assessed using the NCBI database (Yue et al., 2014). Although Fhad1 was also expressed in small amounts in the lungs, it was highly enriched in the testes compared to other tissues. Subsequent qPCR-based analyses of mice confirmed that Fhad1 was expressed at high levels in testis samples, whereas it was largely undetectable in other tissue types except the lungs (Fig. 1A). Longitudinal analyses of the Fhad1 mRNA levels in wild-type male mice revealed that it underwent progressive upregulation from a postnatal age of 2 weeks, when the most advanced germ cells are beyond the zygotene stage (Fig. 1B). Next, fluorescence-activated cell sorting was used to purify germ cells with different ploidy from wild-type mouse testes. In addition, the qPCR analysis showed high levels of Fhad1 mRNA in both haploid and tetraploid cells, indicating the expression of Fhad1 in primary spermatocytes and spermatids (Fig. 1C). The specific expression of Fhad1 in the testis may suggest that it plays key roles in the spermatogenic process after meiotic initiation.

Fhad1 ^−/− mouse development

To test the functional role that FHAD1 plays in male fertility, a CRISPR/Cas9 strategy was utilized to knock out the Fhad1 gene in C57BL/6 mice, yielding a stable mutant mouse line in which 28 nucleotides were deleted (Fig. 1D). Sanger sequencing was used to confirm the deletion of this DNA fragment (Fig. 1E), and qPCR analyses revealed the reduced expression of Fhad1 in these Fhad1^−/− mice (Fig. 1F). However, no changes in the viability, growth, or behavior of Fhad1^−/− mice were noted, thus confirming the successful establishment of a mouse line in which this gene of interest had been deleted.

Fhad1 ^−/− mice exhibit normal fertility phenotypes

To assess the fertility of male Fhad1^−/− mice, they were housed individually with Fhad1^+/+ females for at least 16 weeks, and the average litter size and number of pups were assessed for each of the males (Fig. 2A). No differences in these fertility-related parameters were observed when comparing Fhad1^+/+ and Fhad1^−/− males, nor did the testis sizes differ between these groups (Figs. 2B and 2C). The loss of Fhad1 expression was thus not associated with reduced male fertility.

Spermatogenesis occurs in a unidirectional manner, with germ cells proceeding through developmental stages that are subject to strict temporal regulation. In the testes, the level of spermatid development can be used to classify the seminiferous epithelial cycle into 12 stages (stages I to XII) (Ahmed & de Rooij, 2009). These stages were thus used to evaluate spermatogenic cell distributions and arrangements in Fhad1^−/− mice, revealing no abnormal changes in the seminiferous tubule architecture of these mice and no evidence of dysregulated spermatogenesis, with visible germ cells from spermatogonia to elongated spermatids. The phenotypes of these mice were comparable to those in the testes of Fhad1^+/+ controls (Figs. 2D and 2E).

Given the observed expression of Fhad1 in spermatocytes, γH₂AX staining of sections from Fhad1^−/− mice was performed, revealing typical meiotic progression consistent with general meiosis remaining intact following the deletion of Fhad1 (Fig. 2F). Further, the ultrastructure of elongating spermatids was examined, with no difference observed between the groups (Fig. 2G). These data thus indicate that the absence of the testis-enriched Fhad1 gene did not adversely influence fertility or spermatogenesis in male mice.

Fhad1^-/- mice exhibit reduced sperm motility

The impact of Fhad1 knockout on spermiogenesis was next assessed by comparing epididymal spermatozoa from male Fhad1^+/+ and Fhad1^-/- mice, with no differences observed when assessing H&E-stained epididymal sections from these two groups (Fig. 3A). H&E staining also revealed that the morphology of cauda epididymal spermatozoa was not markedly affected by the absence of FHAD1 (Figs. 3B and 3C). AC-TUBULIN signals showed no significant differences between Fhad1^+/+ and Fhad1^−/− spermatozoa, suggesting that the flagellar structure of Fhad1^−/− spermatozoa was normal (Fig. 3D). Taken together, these results suggested that FHAD1 may be dispensable for the gross morphology of epididymal spermatozoa. TEM was next used to assess the ultrastructural characteristics of the flagella (Fig. 3E). Midpiece sections from both Fhad1^+/+ and Fhad1^−/− mice showed that the mitochondria formed an outer layer with a central axoneme surrounded by outer dense fibers (ODFs). Moreover, the axoneme, ODF, and fibrous sheath (FS) were clearly visible in samples from both Fhad1^+/+ and Fhad1^−/− mice, with wrapping of the membrane around the filaments in the principal piece. These intact structures suggested that Fhad1^−/− sperm exhibit no abnormal morphology.

To determine the sperm quality in Fhad1^−/− males, we examined sperm function using a CASA analysis system (Castaneda et al., 2017). Statistically, there was no significant difference in sperm counts in the cauda epididymis between Fhad1^+/+ and Fhad1^−/− males (Fig. 3F). However, the total motility of Fhad1^−/− sperm (69.4 ± 4.7), namely, the proportion of motile sperm to total sperm count, was significantly decreased compared to that in Fhad1^+/+ mice (77.3 ± 5.6) (Fig. 3G). Similarly, the progressive motility of Fhad1^−/− sperm (32.1 ± 4.8), namely, the proportion of sperm that move forward to the total sperm count, was also significantly lower than that of Fhad1^+/+ sperm (40.48 ± 3.8) (Fig. 3H). This difference was slight and did not reduce the fertility of the mice under normal conditions. Nevertheless, it does suggest that FHAD1 may play a role in sperm motility.

Together, these data show that the loss of FHAD1 expression in mice may impair sperm motility, albeit not to a degree sufficient to have a detectable adverse impact on fertility.

FHAD1 deletion triggers the apoptosis of spermatocytes during the first wave of spermatogenesis

As spermatocytes express FHAD1, to further assess its role in these cells, spermatocytes were evaluated during the first wave of spermatogenesis. TUNEL staining of the testis tissues collected from Fhad1^+/+ and Fhad1^−/− mice at P16, when the first wave of spermatogenesis proceeds to the pachytene stage, was conducted. Relative to the testes of Fhad1^+/+ mice, Fhad1^−/− mice presented with a slight increase in apoptotic cell death (Figs. 4A–4C). These results suggested that FHAD1 plays a role in meiosis such that its deletion can induce apoptotic spermatocyte death during this process.

Lastly, FHAD1 was overexpressed in 293T cells which were then treated using cisplatin to promote DNA damage. Subsequent immunostaining for the DNA damage marker γH₂AX revealed that overexpression of FHAD1 significantly inhibited DNA damage in these cells (Figs. 4D–4G), potentially supporting a role for FHAD1 as a modulator of DNA double-strand break (DSB) formation and repair during spermatocyte meiosis.