Deletion of Ck2β gene causes germ cell development arrest and azoospermia in male mice

Abstract Objectives In humans, non‐obstructive azoospermia (NOA) is a major cause of male infertility. However, the aetiology of NOA is largely unknown. Previous studies reported that protein CK2β was abundantly and broadly expressed in spermatogenic cells. Here, we investigate whether protein CK2β participates in spermatogenesis. Materials and Methods In this study, we separated spermatogenic cells using STA‐PUT velocity sedimentation, analysed the expression pattern of protein CK2β by immunoblotting, specifically deleted Ck2β gene in early‐stage spermatogenic cells by crossing Ck2βfl mice with Stra8‐Cre+ mice and validated the knockout efficiency by quantitative RT‐PCR and immunoblotting. The phenotypes of Ck2βfl/Δ;SCre+ mice were studied by immunohistochemistry and immunofluorescence. The molecular mechanisms of male germ cell development arrest were elucidated by immunoblotting and TUNEL assay. Results Ablation of Ck2β gene triggered excessive germ cell apoptosis, germ cell development arrest, azoospermia and male infertility. Inactivation of Ck2β gene caused distinctly reduced expression of Ck2α′ gene and CK2α′ protein. Conclusions Ck2β is a vital gene for germ cell survival and male fertility in mice.


| INTRODUC TI ON
Infertility affects about 15% of couples, 1 and male factor contributes to approximately 50% of all infertility cases in humans. 2 Azoospermia is a major cause of male infertility, including obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). NOA, caused by testicular failure, is the most severe form of male infertility. NOA accounts for about 60% of azoospermia and affects about 10% of infertile men. 3,4 Clinically, NOA can be categorized into hypospermatogenesis, germ cell arrest and Sertoli cell-only syndrome. 5 Complex genetic factors contribute to testicular failure, including autosomal chromosome abnormalities, 6 microdeletions of the Y chromosome [7][8][9] and single gene mutations. Mutations on dozens of genes, including SOHLH1, 10 NR5A1, 11 TAF4B, 12 ZMYND15, 12 MCM8, 13 SYCE1, 14 TEX11, 15,16 TDRD7, 17 TDRD9, 18 MEIOB, 19 TEX14, 19 DNAH6, 19 MAGEB4, 20 TEX15, 21,22 FANCM, 23,24 DMC1 25 and XRCC2, 26 have been identified to cause NOA by pedigree analysis. However, these genes can explain only a part of male infertility, and more related genes need to be explored. Protein kinase CK2, previously known as casein kinase Ⅱ, is a serine/threonine kinase in the form of tetramer with 2 catalytic subunits (α and α') and 2 regulatory β subunits, 27 functioning in many biological processes like cell proliferation, 28 apoptosis 29-31 and DNA damage repair. 32 We have reported that CK2 is vital for oogenesis by participating in apoptosis and DNA damage repair process 33 ; CK2β is essential for follicle survival, depletion of which causes massive follicle atresia and eventually premature ovarian failure at young adulthood. 33 In mice, immunoblotting shows that CK2α, CK2α´ and CK2β are expressed in testis, 34 and the expression of CK2β is significantly lower than CK2α and CK2α´ in spermatozoa. 35 Immunohistochemistry reveals that CK2α is localized at the acrosome area of spermatids; CK2α´ appears in the acrosomal and cytoplasmic region of spermatids, whereas CK2β is expressed in spermatogonia and spermatocytes. 35 Immunofluorescence in spermatozoa reveals that CK2α is localized at the acrosome and mid-piece; CK2α´ is localized at acrosome, while CK2β displays a weak staining at the acrosome and a strong staining at the mid-piece. 35 Ablation of the CK2α' coding gene Ck2α′ leads to infertility of male mice, with decreased sperm count and increased spermatozoa with head abnormality. 34,36 Considering the distinct localization of CK2β and CK2α', it is quite necessary to clarify whether CK2β functions in spermatogenesis and how it functions.
Here, we specifically depleted Ck2β gene in spermatogonia by crossing Ck2β fl mice with Stra8-Cre + mice. We found that loss of Ck2β gene resulted in germ cell differentiation arrest and male infertility.
CK2β is likely to be an essential anti-apoptotic factor participating in regulating spermatocyte survival.

| Mice
To obtain Ck2β fl/Δ ;SCre + males, we crossed Stra8-Cre 37 with previously reported Ck2β fl/fl mice, 38   Chinese Academy of Sciences. All mice were housed in a temperature-controlled room with a 12D:12L cycle.

| Isolation of mouse spermatogenic cells
Considering the sequential and synchronized occurrence of Sertoli cells, spermatogonia, spermatocytes and round spermatids in the postnatal mouse testis, isolation was performed from samples at 6, 8 and 17 days postpartum (dpp) and adult mice, respectively. 39 The isolation procedures were described previously. 39,40 Briefly, testes from Ck2β fl/Δ and Ck2β fl/Δ ;SCre + mice were dissected and decapsulated. The seminiferous tubules were minced into pieces and incubated in 8 mL phosphate-buffered saline (PBS) containing 100 μL 1 mg/mL collagenase (Sigma, C5138) and 100 μL 1 mg/mL hyaluronidase (Sigma, H3506) at 37℃ for 15 minutes with gentle shaking.
After centrifugation at 4℃, 200 g for 5 minutes, the cells were collected, washed with PBS, incubated in 15 mL PBS with 0.25% trypsin (Gibco, 25200-072) and 1 mg/mL DNase I (AppliChem; A3778,0050) at 37℃ for 15 minutes with gentle shaking. After filtration using a 40-μm nylon cell strainer and sedimentation for 3 hours, the cells were separated by 2%-4% bovine serum albumin (BSA) gradient in PBS. The cell pools (10 mL/pool) were collected separately in numbered tubes and centrifuged at 4℃, 600 g for 5 minutes, and then the supernatant was removed. Adding 1 mL PBS to each odd-numbered tube, the cells were resuspended, and then, 60 µL suspension of each tube was added to 96-cell plate for further observation. The cellular purity and cell types were identified under phase contrast microscope based on morphological evaluation and the diameter of cells. 39 Expected cell types with cellular purity (≥90%) were collected for immunoblotting.

| Immunoblotting
The tunica albuginea of testes was removed, the seminiferous tubules were homogenized using a homogenizer in RIPA buffer (25 mM Tris-HCl, pH 7.6, 350 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate and 0.1% sodium dodecyl sulphate) (Solarbio Life Sciences; R0010) supplemented with protease and phosphatase inhibitor cocktail (Roche Diagnostics, 04693116001). Spermatogenic cells isolated from testes were resuspended in the above-described RIPA buffer. After transient sonication, the lysates were incubated on ice for 30 minutes and then centrifuged at 4℃, 14 000 g for 20 minutes. The supernatant was transferred to a new tube, and equal volume loading buffer was added. After being boiled at 95℃ for 10 minutes, the protein lysates were used for immunoblotting analysis. Immunoblotting was performed as described previously. 41 Briefly, the separated proteins in SDS-PAGE were electrically transferred to a polyvinylidene fluoride membrane. After incubation with primary and secondary antibodies, the membranes were scanned with Bio-Rad ChemiDoc XRS+.

| Quantitative RT-PCR
Total RNA of testes from Ck2β fl/Δ and Ck2β fl/Δ ;SCre + male mice was extracted using the RNeasy Micro Kit (Qiagen, 74004), and the firststrand cDNA was generated with cDNA synthesis kit (Invitrogen; 11754050). Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) or β-actin was used as internal control to normalize the cDNA level of the samples. The experiment was conducted by using UltraSYBR Mixture (CoWin Biosciences; CW0957) in Roche LightCycler480 II detection system. The relative gene expression was calculated by the 2 −ΔΔCt method. The primers used were as follows.
Caudal epididymides were fixed in 4% paraformaldehyde (pH 7.4) overnight at 4℃. The tissues were then dehydrated in a graded ethanol series, cleaned in xylene and embedded in paraffin. The paraffinembedded tissues were sectioned into 5 μm and mounted on glass slides. After adequately drying at 48℃, the sections were deparaffinized in xylene, hydrated in a graded ethanol series and stained with haematoxylin and eosin for histological analysis.
Testes for immunofluorescent staining were fixed in 4% paraformaldehyde (pH 7.4) overnight at 4℃, dehydrated and embedded in paraffin. The paraffin-embedded testes were sectioned into 5 μm and mounted on glass slides. The sections were then deparaffinized in xylene, hydrated in a graded ethanol series, immersed in sodium citrate buffer (pH 6.0) and heated for 15 minutes in a microwave oven for antigen retrieval. After blocking with 5% donkey serum albumin, the sections were incubated with primary antibody at 4℃ overnight and appropriate TRITC-conjugated secondary antibody. The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Images were captured using a laser scanning confocal microscope (Zeiss 780 META).

| TUNEL assay
TUNEL assay was performed in accordance with the DeadEnd TM Fluorometric TUNEL System (Promega Biosciences, G3250). Images were captured using a laser scanning confocal microscope (Zeiss 780 META).

| Breeding assay
C57BL/6J wild-type female mice with known fertility were mated with 8-week-old Ck2β fl/Δ and Ck2β fl/Δ ;SCre + male mice. For 6 months, the cages were monitored daily to record the number of pups and litter sizes.

| Statistical analysis
Paired two-tailed Student's t test was used for statistical analysis.

| Deletion of Ck2β by Stra8-Cre results in male infertility
To study the potential role of protein CK2β during spermatogenesis, we first isolated the spermatogonia, spermatocytes, round spermatids and Sertoli cells from mouse testes to detect the expression of CK2β. Immunoblotting analysis showed that CK2β was expressed from spermatogonia to round spermatids and the highest expression level was detected in spermatocytes, while no expression was detected in Sertoli cells ( Figure 1A and 1B). These results suggest that CK2β may participate in spermatogenesis.
To explore the potential function of protein CK2β during spermatogenesis, we mated Ck2β fl mice, in which exons I-II were targeted, with Stra8-Cre + mice to generate Ck2β fl/Δ ;Stra8-Cre + mice (referred to as Ck2β fl/Δ ;SCre + )( Figure S1). In Stra8-Cre + mice, Cre recombinase was specifically expressed in early-stage spermatogonia from 3 days after birth onward and peaked in preleptotene spermatocytes at 7 dpp. 37 Quantitative RT-PCR and immunoblotting analysis confirmed that the expression of CK2β in testes from Ck2β fl/Δ ;SCre + mice was efficiently deleted at both mRNA and protein levels (Figure 2A, 2B and Figure S2).

| Ck2β depletion causes testicular atrophy and azoospermia
To determine the causes of infertility in Ck2β fl/Δ ;SCre + males, we first examined whether the infertility was due to testicular dysfunction and the consequential functional azoospermia.

| Ck2β knockout results in spermatogenesis arrest in male mice
To clarify the cause of testicular atrophy and azoospermia in Ck2β fl/ Δ ;SCre + males, we next examined the first wave of spermatogenesis at 12 dpp, 15 dpp and 17 dpp corresponding to leptotene, zygotene and pachytene spermatocytes, respectively.
As shown in Figure 5A 42 The results demonstrated that the numbers of zygotene, pachytene and diplotene spermatocytes were significantly reduced, but that of spermatogonia and leptotene spermatocytes were unchanged in Ck2β fl/Δ ;SCre + males, compared with Ck2β fl/Δ males. These findings suggest that Ck2β knockout may not affect spermatogonial proliferation, differentiation and entrance into meiosis in mice, but severely impairs spermatocyte survival and causes spermatogenesis arrest ( Figure 6D).

| Deletion of Ck2β triggers germ cell apoptosis
To further examine the causes of decreased numbers of spermatocytes, detection of apoptosis in testes was carried out by the TUNEL assay. The results showed that more than half of the tubules had F I G U R E 6 Massive germ cell loss in the testes from Ck2β fl/Δ ;SCre + mice at 2 mo of age. A, Histological analysis of the seminiferous tubules of Ck2β fl/Δ and Ck2β fl/Δ ;SCre + mice at 2 mo of age. Scale bars, 50 μm. B, Immunofluorescence detection of MVH-positive germ cells in the seminiferous tubules from Ck2β fl/ Δ ;SCre + mice at 2 mo of age. Germ cells were labelled with anti-MVH antibody (red) and DAPI (blue). Scale bars, 10 μm. C, The diameter of the seminiferous tubules in Ck2β fl/Δ and Ck2β fl/Δ ;SCre + mice. At least 60 tubules from 3 mice of each genotype were used for the analysis. Data are presented as the mean ± SEM P < .001 (***). D, Quantification of the germ cells in the seminiferous tubules of Ck2β fl/Δ and Ck2β fl/Δ ;SCre + mice at 2 mo of age. S, spermatogonia; L, leptotene spermatocytes; Z, zygotene spermatocytes; P, pachytene spermatocytes; D, diplotene spermatocytes. At least 60 tubules from 5 mice of each genotype were used for the analysis. Data are presented as the mean ± SEM P < .01 (**), P < .001 (***) TUNEL-positive germ cells in Ck2β fl/Δ ;SCre + males compared with less than 20% of that in Ck2β fl/Δ males ( Figure 7A and 7B). Besides, the number of TUNEL-positive germ cells was also obviously increased in Ck2β fl/Δ ;SCre + males ( Figure 7A and 7C). The above data indicated that deletion of Ck2β triggered germ cell apoptosis, and consequently caused reduction in the number of spermatocytes.

| Inactivation of Ck2β causes distinctly reduced expression of Ck2α′ but not Ck2α
To clarify the forms of CK2 functions in spermatogenesis, immunoblotting was carried out to assess protein levels of CK2α, CK2α' and CK2β in testes of Ck2β fl/Δ and Ck2β fl/Δ ;SCre + mice. Compared with Ck2β fl/Δ mice, the level of CK2β protein, as expected, was significantly reduced in testis extracts from Ck2β fl/Δ ;SCre + mice ( Figure 8A and Figure S3). Meanwhile, the level of CK2α' protein was also significantly down-regulated in testes of Ck2β fl/Δ ;SCre + mice ( Figure 8A and Figure S3). By comparison, the levels of CK2α protein showed no variation in testes of Ck2β fl/Δ and Ck2β fl/Δ ;SCre + mice ( Figure 8A and Figure S3). The results suggest that CK2 presumably functions in the forms of α' 2 β 2 in spermatogenesis and the reduced expression of CK2α' protein in testes of Ck2β fl/Δ ;SCre + is assumably due to degradation resulting from decreased stability of CK2α' protein without CK2β.
To test the hypothesis, the expression of Ck2α, Ck2α′ and Ck2β in the mRNA level was detected in testes by RT-PCR. Results found that was significantly higher than that in Ck2β fl/Δ ;SCre + ( Figure 8B and Figure S4), while the mRNA expression of Ck2α in testes of Ck2β fl/Δ was remarkably lower than it in testes of Ck2β fl/Δ ;SCre + ( Figure 8B and Figure S4).

| D ISCUSS I ON
NOA is problematic for males as it results in infertility. Accumulating studies have shown that gene mutations contribute to NOA; however, the molecular mechanisms underlying the disorder are still poorly understood. Immunoblotting revealed that protein CK2β was expressed in spermatogonia, spermatocytes and round spermatids, and no expression was seen in Sertoli cells. These findings are consistent with previous reports that CK2β protein is abundantly and broadly expressed in early spermatogenesis as shown by in situ hybridization and immunohistochemistry in mice. 34 A study reports that Ck2α -/mice die in mid-embryogenesis with severe developmental defects in the neural tube and heart, 44 but there is no specific study of its role in gametogenesis. The fertility of Ck2α′ -/females is not affected, while Ck2α′ -/males are infertile. 34 Male mice lacking Ck2α′ displayed oligozoospermia and globozoospermia; germ cells display extensive degenerative changes characterized by nuclear abnormalities at the stages from spermatogonia to early spermatids, including the first spermatogenesis wave. 34,36 Zygote-specific knockout of Ck2β results in embryonic lethality after implantation with decreased cell proliferation but no signs of apoptosis. 38 We have reported that oocyte-specific knockout of Ck2β given rise to ovarian follicle atresia and POF, which are related to down-regulated PI3K/AKT signalling and failed DNA damage response signalling. 33 In this study, using Stra8 promoter-driven Cre recombinase, Ck2β gene is effectively deleted in male mouse germ cells from the early stage of spermatogonia, which facilitated to explore the roles of Ck2β oocytes, 33 it is likely that CK2β protein functions via apoptosis or DNA damage signalling pathways in mouse spermatogenesis.
In this study, we find that deletion of protein CK2β in testes results in significant reduction in protein CK2α′. In contrast, the level of protein CK2α shows no obvious difference in Ck2β mutant testes and control testes. Considering previous reports that CK2α′ protein is indispensable to spermatogenesis, 34 CK2 presumably functions in spermatogenesis in the form of α′ 2 β 2 , and the degradation of CK2α′ protein is caused by decreased stability of CK2α' protein without CK2β. However, down-regulation of Ck2α′ at the mRNA level is observed in Ck2β mutant, indicating that Ck2β knockout disrupts Ck2α′ transcription and consequently disturbs the function of CK2α′ protein per se or as a catalytic subunit of CK2 holoenzyme. In addition, it is confusing that CK2α has no changes in the protein level while the mRNA expression is obviously elevated. From above results, the mutual regulation of the three subunits is complicated.
In summary, we identify Ck2β as a vital gene for germ cell survival and male fertility in mice, but whether it is a candidate gene for NOA in humans needs further clarification.