G3BP2, a stress granule assembly factor, is dispensable for spermatogenesis in mice

Animals

The C57BL/6J background mice were purchased from the animal center of Nantong University. The animals are raised in the Laboratory Animal Center of Nantong University, where they are provided with free food and water under specific pathogen-free conditions with a 12 h/12 h light/dark cycle. All mice were treated humanely and killed by carbon dioxide asphyxiation to collect testicular and epididymis samples for further analysis. The Animal Ethical and Welfare Committee (AEWC) of Nantong University approved this research (Approval No. S20200630-502).

Genotyping

Total DNA was extracted from the mouse tail by using the EasyPure Genomic DNA Kit (Transgen Biotech, Beijing, China), and the genotype was screened using PCR (EasyTaq DNA Polymerase, Transgen Biotech, Beijing, China). The primers are listed in Table 1.

Generation of the G3bp2 cKO mouse

Floxed G3bp2 mice (G3bp2^loxp/loxp) were generated from embryonic stem cells (ESCs) at Shanghai Research Center for Model Organisms. In brief, ESCs were injected with a mixture of donor (50 ng/µl), sgRNA1 (50 µg/ul), sgRNA2 (50 µg/ul), and Cas9 (100 µg/ul). The donor carried the sequence of G3bp2 Exon3 flanked by loxP sites. The gene editor mice and are C57BL/6J background. G3bp2^loxp/loxp mice were confirmed via PCR. Mice with Stra8-Cre or Ddx4-Cre in the C57BL/6J background were purchased from the Jackson Laboratory. Stra8-Cre males were first crossed with G3bp2^loxp/loxp females to generate the Stra8-iCre; G3bp2^loxp/+ male mice, then the Stra8-iCre; G3bp2^loxp/+ male mice were crossed with G3bp2^loxp/loxp female mice to obtain the Stra8-iCre; G3bp2^loxp/Δ male mice (SKO). Ddx4-Cre; G3bp2^loxp/Δ male mice (DKO) were obtained likewise.

Western blotting

Testicular extracts were prepared using RIPA lysis buffer (P0013C; Beyotime Biotechnology, Beijing, China) with the protease inhibitor PMSF (ST506; Beyotime Biotechnology, Beijing, China), and their protein content was resolved via 10% SDS-PAGE. Afterward, the protein bands were transferred from the gel onto a nitrocellulose membrane, which was then blocked with 5% nonfat milk in TBST for 1 h followed by overnight incubation with primary antibodies (Table 2) at 4 °C. Afterward, the membrane was washed three times with TBST and then incubated with secondary antibodies. The target protein bands were detected using an Amersham Typhoon 5 Biomolecular Imager (GE Healthcare Lifescience, Little Chalfont, UK).

RNA isolation and RT-qPCR

All the organs (brain, heart, kidney, liver, lung, spleen, ovary, and testis) were collected from adult C57BL/6J background mice. Total RNA was extracted from these samples by using TRIzol reagent (Ambion, Foster City, CA, USA) and then reverse-transcribed to cDNA by using the PrimeScrip 1st Strand cDNA Synthesis Kit (TaKaRa, Beijing, China). Quantitative PCR was performed on a LightCycler 96 system (Roche, Mannheim, Baden-Württemberg, Germany) by using a TB Green Premix Ex Taq (Tli RNaseH Plus) Kit (TaKaRa) according to manufacturer protocol. The relative gene expression was quantified using the comparative cycle threshold method, with the β actin expression used for normalization. The primers are listed in Table 1.

Hematoxylin-eosin (HE) staining

Mouse testis and epididymis were fixed in 4% paraformaldehyde at 4 °C for 24–48 h, embedded in paraffin wax, and sectioned at 5 μm. The sections were then deparaffinized and rehydrated as follows: Incubation in xylene for 20 min (performed twice), followed by successive incubations in 100% ethanol, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, and ddH₂O for 2 min each. HE staining was carried out using the Hematoxylin-Eosin (HE) Staining Kit (E607318; Sangon Biotech, Shanghai, China).

Immunofluorescence analysis

The testicular sections were prepared as described above for HE staining. Then antigen retrieval using the Improved Citrate Antigen Retrieval Solution (P0083; Beyotime Biotechnology, Beijing, China) was performed at 100 °C for 10 min. Subsequently, the samples were cooled and washed three times with 0.3% Triton X-100 in PBS for 10 min, followed by three PBS washes. Afterward, the samples were blocked with 1% BSA for 30 min and then incubated with primary antibodies (Table 2) in 1% BSA overnight at 4 °C. Subsequently, the samples were washed three times with PBS and then incubated with the secondary antibody for 1 h at room temperature. Next, the samples were washed three times with PBS, and DAPI (D9542) was used to stain the cell nuclei for 10 min at room temperature. Finally, the slides were mounted in glycerol (ZEISS, Oberkochen, Germany) and imaged using a ZEISS Axiocam 503 mono fluorescence microscope.

Evaluation of sperm quality

Cauda epididymis was dissected from WT, SKO, and DKO male mice (10 weeks old, n = 5) and transferred to Tyrode’s solution (T1421-500 ml: Solarbio, Beijing, China). After incubating the samples at 37 °C for 10 min, sperm quality was measured using the CASA system of the Hamilton Thorne CEROS II (Medealab™, Erlangen, Germany).

TUNEL assay

Testes were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (5 µm). The sections were then deparaffinized and rehydrated as described above, and then incubated with protease K (20 µg/ml) at room temperature for 30 min to the TUNEL assay was performed using the One-Step TUNEL Apoptosis Assay Kit (C1089; Beyotime, Beijing, China) by following the instructions of the manufacturer, and then the nuclei were stained with DAPI. Images were obtained using a ZEISS Axiocam 503 mono fluorescence microscope. We repeated the experiment five times.

Fecundity test

Two female mice (at age of 8 weeks) were caged with each test male mouse (WT, SKO, and DKO, at age of 8 to10 weeks), and copulation was evaluated by checking each morning for a vaginal plug. The number of pups was counted on the day of birth.

Heat stress assay

Adult (10 weeks) male mice (WT, SKO, and DKO) were used for the heat-treatment experiment (Kim, Cooke & Rhee, 2012). Mice were anesthetized and the lower third of the body was placed in the water bath at 42 °C for 20 min. The mice were sacrificed after heat treatment and the testis were collected for further analysis.

Statistical analysis

All data were reported as mean ± SD from at least three replicates for each experiment. Significance testing of student T-tests using Prism 7.0 software using two-tailed failure. P < 0.05 was considered statistically significant. NS means not significant.

G3bp2 is highly expressed in the mouse testis

G3bp family containing G3bp1 and G3bp2, G3bp2 is high expression than G3bp1 in adult testis by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) (Fig. 1A). To explore the spatial expression pattern of G3bp2, we used RT-qPCR and observed high expression in the spleen, brain, and testis (Fig. 1B). Next, we assessed the temporal expression pattern of G3bp2. Toward this end, the testes from adult mice and those aged 1–5 weeks were collected. RT-qPCR analysis showed G3BP2 expression in mouse testis at all stages (Fig. 1C). To evaluate the cellular expression and subcellular localization patterns, we performed immunofluorescence analysis of testis sections for G3BP2. G3BP2 was mainly detected in the cytoplasm of spermatogonia, spermatocytes, and round spermatids, and not detected signals in SKO and DKO (Fig. 1D).

G3bp2 conditional knockout (cKO) mice displayed normal spermatogenesis and fertility

Because whole-body G3bp2 knockout in the mouse leads to preweaning lethality (Bult et al., 2019), we generated germline-specific G3bp2 knockout mice. Toward this end, we inserted a loxP site at each end of exon 3 of the G3bp2 gene (G3bp2^loxp/loxp) (Fig. 2A). To explore the effect of germ-cell–specific G3bp2 knockout on spermatogenesis during different stages of post-natal development, we used stra8-iCre mice, which begin expressing Cre recombinase in germ cells at 3 days post-partum (dpp) (Sadate-Ngatchou et al., 2008), and Ddx4-Cre mice, which begin expressing Cre recombinase in germ cells at embryonic stage 15 days (Gallardo et al., 2007). G3bp2^loxp/loxp mice were crossed with these mice to generate the following two types of germline-specific G3bp2 knockout mice: Stra8-iCre; G3bp2^loxp/Δ (SKO), Ddx4-Cre; G3bp2^loxp/Δ (DKO). PCR was used to confirm testis-specific G3bp2 knockout, immunostaining, and western blotting were used to confirm the KO efficiency (Figs. 2B, 2C and 1C). There is still a very faint G3bp2 signal in KO mouse testis, which is presumably Sertoli cell protein. Furthermore, we detect a weak bands by isolate Sertoli cells protein (Fig. 2D). However, sertoli-cell–specific deletion of G3bp2 by using Amh-Cre tool mice did not impact male fertility (data not shown).

We observed that there was no significant difference in testicular morphology among wild-type (WT), SKO, and DKO mice of the same week of age (Fig. 2E). The testis body weight ratio and the fertility test showed no difference in litter size among WT, SKO, and DKO mice (Figs. 2F and 2G). Moreover, full spermatogenesis and normal epididymal structure were observed in SKO and DKO mice in comparison with the WT mice (Figs. 2H and 2I). Additionally, computer-aided sperm analysis (CASA) of the semen from WT, SKO, and DKO mice showed that they all had a similar sperm concentration and motility at 10 weeks old (Figs. 2J and 2K). In addition, sperm from these mice exhibited a similar morphology (Fig. 2L). These results indicate that G3BP2 is not essential for spermatogenesis and male fertility in the mouse.

G3BP2 deficiency-induced germ-cell apoptosis

The TUNEL assay is used to detect the DNA breaks during apoptosis. To have a deeper understanding of the influence of G3bp2 deficiency on reproduction, we performed the TUNEL assay on testicular sections from WT, SKO, and DKO mice at 10 weeks old. Surprisingly, SKO and DKO displayed more apoptotic germ cells than in WT testis (Figs. 3A and 3B), indicating that G3BP2 deficiency promotes germ-cell apoptosis. There are high levels of germ cell death in the G3BP2 KO testis, but no significant difference in sperm concentration. So we speculate there is higher spermatogonia proliferation to balance the loss of germ cells. The WT, SKO, and DKO mouse testis sections were staining PLZF, a spermatogonia marker (Song et al., 2020). The seminiferous tubules from WT, SKO, and DKO contained similar numbers of spermatogonia cells (Fig. 3C). Stress granules condense into being through liquid-liquid phase separation (LLPS), G3bp1 and G3bp2 are the core regulatory components of stress granules, so we speculated whether elimination of G3bp2 has any effect on the formation of stress granules in testis (Guillén-Boixet et al., 2020; Yang et al., 2020). WT, SKO, and DKO mice were killed immediately after being heated in a 42 °C water bath for 20 min and the heat stress testis section was staining TIA1, a stress granules marker (Kim, Cooke & Rhee, 2012; Mackenzie et al., 2017). Statistical analysis of TIA1 positive cells after heat stress, there was no significant difference in WT, SKO, and DKO, suggesting that G3bp2 deletion in not affect stress granules assembly (Fig. 3D).