Slc26a1 is not essential for spermatogenesis and male fertility in mice

Generation of Slc26a1^−/− mice using CRISPR/Cas9 technology

C57BL/6J healthy mice were purchased from Cyagen Biosciences Inc and raised at the Experimental Animal Center of Nanjing Medical University under conditions of 30–70% humidity and 26 °C. The mice were allowed free access to food and water throughout the experiment. The food contains 20% protein and 4% fat and was provided Xietong Pharmaceutical Bio-engineering (Nanjing, China). All the cages were exactly of the same size and material. Animal house was specific pathogen-free (Murray et al., 2021) and 12 h light-12 h darkness-cycles. At the end of the study, the mice were anesthetized with carbon dioxide.

This study was approved by the Animal Ethics and Welfare Committee of Nanjing Medical University (No. IACUC-2004020) and was conducted in accordance with the Guide for the Care and Use of Laboratory Animals. Slc26a1 KO mice were generated using CRISPR/Cas9 genome editing technology, as previously described (Shen et al., 2014; Wang et al., 2013). Single guide (sg)RNA target sequences for Slc26a1 are as follows: 5′-CATGGTGGTCCACACATGGT-3′. The combination of sgRNA and Cas9mRNA was microinjected into the fertilized eggs of C57BL/6J mice and then immediately transplanted into the oviduct of pseudopregnant females of the same strain. The mutant mice have been registered in the MGI database as Slc26a1<em1Njrml> (MGI:7529731).

Genotype identification

Slc26a1^−/− mutation was identified by Sanger sequencing. Polymerase chain reaction (PCR) and Sanger sequencing were performed using the forward primer, 5′-GGCTGGGCTTCGTGTCTACCTA-3′, and the reverse primer, 5′-GCTCTTGGTTGGCACTGACAGA-3′.

Fertility test

Slc26a1 (WT/KO) adult males were respectively mated with WT females at a ratio of 1:2 for 3 months. The mice were raised at 26 °C. Female mice, whether pregnant or not, were replaced once a week. The numbers of litters and born mice per litter were recorded. The genetic background of WT controls was C57BL/6J (from the same batch as KO mice but not littermates).

Cell culture and transfection

Mouse germ cell lines GC-1spg (GC-1) and GC-2(spd)ts (GC-2) were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) under the conditions of 37 °C and 5% CO₂. SLC26A1 and negative control small interfering RNAs (si-NC) were synthesized by GenePharma (Suzhou, China). GC-1 and GC-2 cells were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). At 6 h post-transfection, cells were transferred to culture medium; at 48 h post-transfection, cells were collected for analysis. The sequences of the small interfering RNAs (siRNAs) were:

si-NC:5′-CACUCAAGAUUGUCAGCAA-3′;

si-Slc26a1 #1:5′-GGAAUACCUAGCAGGUGAU-3′;

si-Slc26a1 #2:5′-GCCAAUACCCACAGAGUUA-3′

Cell viability and migration analysis

After transfection with siRNA, GC-1 and GC-2 cells were seeded in 96-well plates at a density of 4 × 10³ cells/well. Cell viability was evaluated using a cell counting kit-8 (CCK-8; Beyotime Institute of Biotechnology, Nantong, China), as previously described (Chen et al., 2022a; Xue et al., 2022; Yu et al., 2022). In the colony formation assay, 1,000 transfected cells were placed in 6-well plates; 2 mL DMEM containing 10% FBS was added to each well and changed after 6 d. After 2 weeks, the cells were fixed with methanol and stained with 0.1% crystal violet (Beyotime, Jiangsu, China) for 30 min, and the visible proliferating clumps were counted under a microscope (Carl Zeiss, Oberkochen, Germany).

In the migration assay, 300 μL serum-free DMEM was added to the top of a culture chamber with an 8-mm membrane (14831; Corning, Corning, NY, USA), and the cells were then added; 700 μL DMEM containing 10% FBS was added to the lower part of the chamber. After 24–48 h, the cells below the membrane were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Finally, photographs were taken under a microscope (Carl Zeiss, Oberkochen, Germany).

Western blot

The proteins of transfected GC-1 and GC-2 cells were extracted using RIPA buffer (RIPA, Beyotime, Jiangsu, China) and denatured at 100 °C. The denatured proteins were then separated on 10% sodium dodecyl sulfate-polyacrylamide gels, transferred to a polyvinylidene difluoride membrane, and sealed with 5% skim milk at room temperature for 1 h. The primary antibodies were anti-SLC26A1 (1:1,000, NBP1-84897; Novus, St. Charles, MO, USA) and anti-tubulin antibody (1:1,000, MA1-91878, Thermo Fisher Science, Waltham, MA, USA). The membranes were then incubated with secondary antibody (1:3,000, A0208, Beyotime, Jiangsu, China) for 1 h at room temperature. Finally, a quantitative analysis was performed using a SuperSignal West Femto chemiluminescence substrate detection system (Thermo Fisher Scientific, Waltham, MA, USA).

RNA extraction and reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted from testicular tissue and transfected GC-1 and GC-2 cells using TRIZOL^™ reagent (Vazyme, Nanjing, China) according to the manufacturer’s instructions. The RNA was then reverse transcribed into complementary cDNA by using a PrimeScript RT reagent Kit (Vazyme, Nanjing, China) and quantified using a real-time PCR system (Applied Biosystems, Foster City, CA, USA). The reaction system was set up as follows: SYBR Green Mix (10 μL), forward primer (0.4 μL), reverse primer (0.4 μL), cDNA template (1.0 μL), ddH2O (8.2 μL), resulting in a total volume of 20 μL. 18S rRNA was used as an internal control. Target gene expression was calculated using the 2^−ΔΔCT method: ΔΔCT = (CT_Target − CT_18srRNA)_Sample − (CT_Target − CT_18srRNA)_Control. The primers used are summarized in Table S1.

Histology and sperm phenotyping

Testes and epididymides from Slc26a1-WT and Slc26a1-KO mice aged 8–12 weeks were immediately fixed in modified Davidson’s solution for 48 h, dehydrated in a series of graded ethanol solutions, embedded in paraffin, and cut into sections (thickness, 4 μm). The sections were rehydrated and stained for histological analysis using hematoxylin-eosin (H&E) or periodic acid Schiff (PAS) stain, as previously described (Shen et al., 2021; Wu et al., 2022; Yu et al., 2021). For sperm malformation analysis, we selected and quantified the head and tail deformities of sperm, respectively. Then, we performed computer-assisted sperm analysis (CASA). Cauda epididymides were suspended in human tubal fluid culture medium (InVitroCare, Inc., Frederick, MD, USA), incubated at atmospheric pressure and 37 °C for 10 min, and analyzed for semen quality using a Ceros^™II sperm analysis system (Hamilton Thorne, Beverly, MA, USA).

Immunofluorescence

Immunofluorescence staining of mouse testis was performed as previously described (Gao et al., 2020; Zhao et al., 2019). The paraffin sections were boiled in 10 mM citrate buffer (pH 6.0), soaked in 3% hydrogen peroxide, blocked with bovine serum albumin, and incubated with primary antibody (Table S2) at 4 °C. Subsequently, the slides were rinsed three times with phosphate-buffered saline, then incubated with Alexa-Fluor secondary antibody (Thermo Fisher Scientific, Waltham, USA) at 37 °C and stained with 4′,6-diaminodiphenylindole. Fluorescent staining images of all sections were obtained under a confocal microscope (Zeiss, Oberkochen, Germany). Images of 50 tubules per male were used for quantification.

Terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) assay

Apoptosis was detected using the TUNEL assay kit (Vazyme, Nanjing, Jiangsu, China), according to the manufacturer’s instructions. Paraffin sections were rehydrated and treated with proteinase K, then reacted with TUNEL labeling mix buffer at 37 °C. Images were obtained under a confocal microscope (Zeiss, Oberkochen, Germany). Fifty tubules per male were analyzed.

Statistical analysis

All relevant statistical analyses were performed in triplicate. Unpaired student’s t-tests or one-way ANOVA were used to verify differences between Slc26a1-WT and Slc26a1-KO mice. Data are the mean ± Standard Deviation (SD). A P-value < 0.05 was considered to indicate a significant difference.

Generation of Slc26a1-KO mice

To investigate the effect of SLC26A1 on male fertility, CRISPR/Cas9 gene editing technology was used to introduce a mutation consisting of a 10 bp deletion in exon 3 of Slc26a1 (Fig. 1A), which is predicted to lead to frame-shift mutations. This mutation causes a complete deletion of the Sulfate Transport Anti-Sigma antagonist (STAS) domain (Fig. 1B). PCR and Sanger sequencing were used to confirm the changes (Fig. 1C). Immunofluorescence staining of sperm showed that SLC26A1 was specific localized in the acrosome of spermatids of WT mice, but not in the Slc26a1-KO spermatids (Fig. 1D). Localization of Slc26a1 in mice suggested that Slc26a1 may affect acrosome during spermatogenesis.

Slc26a1 KO mice are fertile

We subsequently performed fertility tests to study the effects of Slc26a1 on mouse fertility. The pups and litters of the experimental and control groups were counted. The results suggested that the difference in fertility between the Slc26a1-WT and Slc26a1-KO mice was not statistically significant (Fig. 2A). We also examined testicular morphology and testicular/body weight ratios of Slc26a1-KO male mice; compared with the control group, the difference was not statistically significant (Figs. 2B and 2C). The CASA results suggested that compared with WT mice, Slc26a1-KO mice had normal sperm concentrations, and percentage of sperm with motility and with progressive motility (Figs. 2D–2F). H&E staining of sperm found that the abnormal sperm of Slc26a1-KO mice was not significantly different from that of Slc26a1-W mice (Figs. 2G–2I). Therefore, Slc26a1 was not essential for mouse fertility.

Spermatogenesis is normal in Slc26a1-KO mice

H&E staining results for testes suggested that Slc26a1-KO mice had intact seminiferous tubules and spermatogenic cells at all stages (Fig. 3A). H&E staining of the epididymis found that there were no significant histological differences between the epididymides of Slc26a1-WT and Slc26a1-KO mice and that they were filled with sperm in both groups of animals (Figs. 3B and 3C).

Different spermatogenic cells are arranged in the seminiferous tubules according to special cell connections. Spermatogenesis can be divided into the three processes of spermatogonial stem cell (SSC) proliferation and differentiation, spermatocyte meiosis, and spermiogenesis. To examine the differences in spermatogenesis between Slc26a1-WT and Slc26a1-KO mice, we first analyzed the nuclei and acrosomes of the spermatogenic cells in seminiferous tubules using PAS staining. The results indicated there were no obvious morphological differences (Fig. 4A). We quantitatively analyzed spermatids in spermatogenic tubules by H&E-stained mouse testicular sections (Fig. 4B), the results suggest that there is no significant difference in the numbers of spermatids between WT and KO mice.

We also used lin-28 homolog (LIN28), SRY-box 9 (SOX9), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1(HSD-3β), TUNEL, and H2AX variant histone (γ-H2AX) immunostaining. These five marker genes are respectively located in SSCs (Figs. 4C and 4D), Sertoli cells (Figs. 4E and 4F), Leydig cells (Figs. 4G and 4H), apoptotic cells (Figs. 4I–4K), and spermatocytes (Figs. 4L and 4M). Then we quantified the number of positive cells in the testicular sections; there were no significant between-group differences. Taken together, these results suggested that Slc26a1 was not essential for spermatogenesis in mice.

Possible functional compensation from paralogs in Slc26a1-KO mice

We extracted total RNA from the testes of Slc26a1-WT and Slc26a1-KO mice. Relative transcript levels of eleven Slc26a family members were detected using RT-qPCR. The mean expression of Slc26a5 and Slc26a11 in the testes of Slc26a1-KO mice was higher than that of Slc26a1-WT mice (Fig. 5A). These results may suggest that paralog-associated functional compensation was present in the testes of Slc26a1-KO mice.

To test whether transient knockdown of the Slc26a1 gene also had a similar effect, we transiently transfected GC-1 and GC-2 cells with si-Slc26a1 #1 and #2; western blot analysis revealed reduced expression of SLC26A1 (Figs. 5B and 5C). We also extracted total RNA from GC-1 and GC-2 cells and performed RT-qPCR. The PCR results indicated there was no significant difference in relative transcription levels of Slc26a family members between Slc26a1-WT and Slc26a1-KO mice (Figs. 5D and 5E).

Since there was no compensation in these two cell lines, we assessed whether the knockdown of Slc26a1 affects their phenotype. The results of the CCK-8 and colony formation assays revealed that proliferation of GC-1 and GC-2 cells decreased with downregulation of Slc26a1 expression (Figs. 5F–5J). Transwell assays further found that downregulation of Slc26a1 inhibited cell migration (Figs. 5K–5M).

Taken together, these results indicated that the deletion of Slc26a1 could be compensated for by other Slc26a family members in some testicular cells, but transient gene knockdown in GC-1 and GC-2 cells had no such effect.