Overexpression of human SPATA17 protein induces germ cell apoptosis in transgenic male mice
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- Nie, D., Liu, Y., Juan, H. et al. Mol Biol Rep (2013) 40: 1905. doi:10.1007/s11033-012-2246-z
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SPATA17 is a new testis-specific-expressed gene that is involved in Spermatogenesis process. Previous studies show that SPATA17 was involved in acceleration of cell apoptosis in GC-1 cell lines. To further investigate specific roles of SPATA17 in Spermatogenesis in vivo, we generated transgenic mice in which the human SPATA17 gene was expressed specifically in spermatocytes using the human phosphoglycerate kinase 2 (PGK2) promoter. The SPATA17 transgenic mice did not show any significant defect in gross testicular anatomy as well as in fertility. However, a significant increase was observed in defective spermatogenic cells, such as apoptotic cells in the SPATA17 transgenic mice. These results revealed that elevated production of the SPATA17 protein disturbed the normal development of male germ cells.
KeywordsCell apoptosisSPATA17SpermatogenesisTransgenic Mouse
During normal spermatogenesis, apoptosis is believed to play an important role in controlling germ cell numbers and eliminating defective germ cells to produce functional spermatozoa [1–4]. The apoptosis that occurs during spermatogenesis is a highly complex process that involves genes for various factors, such as the Bcl-2 family, Fas and p53 [1–4]. Germ cell apoptosis can also be induced by various pathological conditions such as heat stress, exposure to ionizing radiation , toxic substances , hormonal depletion  and loss of stem cell factor (SCF) signaling [8, 9]. Thus, the specific molecular mechanisms that govern germ cell apoptosis under different apoptotic conditions have not yet been characterized.
SPATA17 gene, also known as MSRG-11, is predominantly expressed in testis . Immunohistochemical analysis revealed that SPATA17 protein was most abundant in the cytoplasm of round spermatids and elongating spermatids within seminiferous tubules of the adult testis. Overexpression of SPATA17 protein in the GC-1 cell line could accelerate GC-1 cell apoptosis and its effect increases with the increasing of the transfected dose of pcDNA3.1(-)/SPATA17 . These results suggest that SPATA17 may play an important role in the development of testes and is a candidate gene of testis-specific apoptosis.
At present, Transgenic mice represent a powerful tool to study genetic, molecular, biochemical, and physiological events in the whole animal, organ, tissue, or cell in vivo, as well as in real time, with resolution and specificity similar to that obtained in cell cultures. To further elucidate the function of human SPATA17 (GenBank No.: AY963797) in vivo, we generated transgenic mice in which the human SPATA17 gene was expressed specifically in spermatocytes using the human phosphoglycerate kinase 2 (PGK2) promoter. The SPATA17 transgenic mice did not show any significant defect in gross testicular anatomy as well as in fertility. However, we observed significant increases in defective spermatogenic cells, such as apoptotic cells and giant degenerating cells in the SPATA17 transgenic mice. The above results revealed that overexpression of the SPATA17 protein disturbed the normal development of male germ cells.
Materials and methods
C57BL/6 mice were purchased from Experimental animal Center of Hunan. All animals were housed under controlled environmental conditions (21 °C, 12-h light/12-h dark cycle) with free access to standard mouse chow and tap water. All of the experimental procedures were carried out in accordance with the principles and procedures of the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
Generation of the PGK2-SPATA17 transgenic mice
The PGK2–SPATA17 transgenic construct contains the promoter region of human phosphoglycerate kinase 2 (PGK2), c-myc tags, the complete open reading frame (ORF) of human SPATA17, 3′ untranslated region (UTR) of human growth hormone 1 (hGH1), and SV40 poly(A) sequence. The transgenic cassette was generated according to Tascou et al. and Karina et al. [12, 13] with pBluescript II SK (±) vector (Stratagene, CA) containing a 1.4-kb region of PGK2 promoter flanked by XhoI/HindIII restriction sites. The 351-bp fragment, including leader sequence and five copies of c-myc tag, was amplifed by PCR using pCS2–3′ plasmid as a template with primers C-myc-F (5′-CCGAATTCCGTCGGAGCAAGCTTGATTTA-3′) and C-myc-R (5′-TTGCGGCCGCCTTTTGCTCCATGGTGAGGTC-3′). The 1083-bp SPATA17 coding sequence was amplifed by RT-PCR with human testis cDNA using primer SPATA17-F (5′-GGGCGGCCGCATGGCCACGTTAGCCCGG-3′) and SPATA17-R (5′-GGGTCGACTTATACAATCTGTCCAGC-3′) and was cloned downstream of the c-myc tag. The 161-bp 3′ UTR of human GH1 was obtained after PCR on human genomic DNA using hGH-3UTR-F (5′-TTCCGCGGCTGCCCGGGT GGCATCCC-3′) and hGH-3UTR-R (5′-TACCGCGGCATACCACCCCCCTCCAC-3′) and afterward was cloned downstream of the SPATA17 ORF. The 128-bp SacII/SacI fragment containing SV40 poly(A) was amplifed by PCR using pEGFP-N1 vector as a template and primer polA-SV40-F (5′-TTCCGCGGGGTTACAAATAAAGCAATAGCATCAC-3′) and polA-SV40-R primer (5′-TCCGAGCTCCGCTTACAATTTACGCCTTAAGAT-3′). Finally, the SV40 polyA signal was included at the 3′ end of the transgenic construct. After sequencing, the obtained 3.12-kb transgenic cassette was excised from pBluescript II SK (±) vector by XhoI/SacI digestion and then purified from agarose gel (Qiagen). Subsequently the construct was diluted to a concentration of 20 μg/ml in TE buffer (5 mM Tris, pH 7.4, and 0.1 mM EDTA, pH 8.0) and microinjected into the pronucleus of one-cell stage mouse embryos. The embryos were transferred to pseudopregnant foster mothers. Genomic DNA was extracted (Promega) from tail biopsies at 4–5 weeks of age and the resulting founder mice were analyzed for the presence of the transgene by PCR of tail DNA using primers SPATA17-F and hGH-3UTR-R2(5′-GATGCCACCCGGGCAGCCGCG-3′). Amplification was performed for 33 cycles using an annealing temperature of 57 °C. Detection of transgenes in founder mice was also done by Southern blot analysis using the 1086-bp SPATA17 cDNA fragment as the probe. The extracted DNA was then digested with EcoRI/SalI. Hybridization with [a-32P] dCTP Probes was performed in hybridization mix (5 × SSC, 2.5 × Denhardt’s reagent, 5 mM EDTA, 0.1 % SDS, 10 % dextran sulfate, 100 mg/ml salmon sperm DNA) for overnight at 42 °C, and after washing with 0.2 × SSC/0.1 % SDS, the filters were exposed to X-ray film.
Northern blot analysis
For total RNA preparation, tissues were frozen in liquid nitrogen and the total RNA was isolated using the acid guanidinium thiocyanate–phenol–chloroform method. 10 μg total RNA was electrophoresed in a denaturing 0.85 % agarose gel containing 2.2 M formaldehyde, transferred to a nitrocellulose membrane, and hybridized with the SPATA17 probe. The G3PDH cDNA probe was used as an internal control for equal loading. The probes were labeled with [a-32P] dCTP by random priming. Prehybridizations and hybridizations were performed at 42 °C in a solution containing 50 % formamide, 5x Denhardt solution, 5xSSPE, 0.5 % SDS, and 200 μg/ml salmon sperm DNA. The filters were washed once for 30 min at room temperature and twice for 10 min at 65 °C with 1xSSC, 0.1 % SDS, and then exposed to Fuji Medical X-ray film after quantitation by phosphorimager analysis.
Western blot analysis
Protein extraction and Western blotting were performed as previously described . Briefly, 80 μg of protein was resolved on a 12 % SDS-PAGE gel at 120 V. Equal loading was examined by running a duplicate gel and staining with Coomassie blue. Proteins were transferred to 0.45 μm nitrocellulose membranes in cold transfer buffer (25 mM Tris-base, 190 mM glycine, 20 % methanol) at 100 V for 1 h. Membranes were blocked in 5 % nonfat dried milk in TTBS (0.9 % NaCl, 0.1 % Tween 20, 100 mM Tris–HCl, pH 7.5) and then incubated with rabbit polyclonal anti–c-myc antibodies. Following 5–10 min washes in TTBS, membranes were incubated with horseradish peroxidase-conjugated donkey anti-rabbit (Amersham Life Science Inc) secondary antibody at a 1:1000 dilution. For immunodetection, membranes were incubated with SuperSignal Chemiluminescent Substrate (Pierce, Rockford, Ill) and exposed to Fuji X-ray film.
Epididymis was excised from 3-month-old mice and pierced with a 25-gauge needle in a cell-culture dish containing 0.5 ml Brinster’s BMOC-3 medium (Invitrogen, USA). Sperm suspensions were incubated at 37 °C in a humidified 5 % CO2/95 % air environment for 30 min. Sperm suspensions were diluted 1:10 in the medium and sperm counts were determined using a hemocytometer. Sperm count is expressed as the number of sperm cells per epididymis. Diameter of seminiferoustubules was measured from tubules at stage VII–VIII of the seminiferous epithelial cycle with the help of a scale bar.
A wild type mouse and a transgenic mouse were euthanized by CO2 inhalation, and dissected testes were fixed in Bouin’s fluid overnight, extensively washed in 70 % ethanol, dehydrated in ethanol, embedded in paraffin and sectioned into sections of 5 μm thickness, then rehydrated. Immunohistochemistry was performed according to the procedure described in the manufacturer’s instructions (SABC kit; Boster). The peroxidase activity was detected using a DAB kit (Boster).Testicular sections were counterstained with methyl green (Sigma), then observed and photographed under a fluorescence microscope.
Histological analysis and TUNEL assay
Histological and TUNEL analyses were performed as previously described . In brief, testes were dissected, fixed in 4 % paraformaldehyde and embedded in paraffin. For analysis of apoptosis, deparaffinized and rehydrated sections were subjected to TUNEL assay after pretreatment with proteinase K (Roche, USA) using an ApoTag Plus peroxidase kit (Oncor, USA) according to the manufacturer’s instruction. Within testis cross-sections, apoptosis was quantified by counting the number of TUNEL-positive and TUNEL-negative tubules per section and TUNEL-positive cells in each tubule. Data are presented as average number of TUNEL-positive cells per tubuli ± SD. For each animal 10–15 fields were counted, at least 3 animals were used for each group. Slides were analyzed under a light microscope (BX-60, Olympus).
Results and discussion
Construction of the PGK2-SPATA17 transgenic mice
Expression of the PGK2-SPATA17 in the testis
Body and testis weights
Body weight, testis weights, and epididymal sperm count of wild-type and transgenic male mice. Values are mean ± SEM
Body weight (g)
Testis weight (mg)
Testis weight (% of body weight)
Sperm count (106 per epididymis)
20.7 ± 1.5
102.7 ± 2.2
0.49 ± 0.01
8.76 ± 0.55
20.2 ± 1.0
77.8 ± 3.0*
0.38 ± 0.03*
7.87 ± 0.76
Overexpression of SPATA17 leads to increased germ cell apoptosis
Histological examination of testes of 3-month-old transgenic mice revealed no apparent abnormalities in their spermatogenesis (Fig. 4C, D). However, in 3-month-old transgenic mice, the average diameter of stage VII–VIII seminiferous tubule was decreased by 7 % (Fig. 4B), with both seminiferous tubule lumen size and wall thickness being reduced. We next performed TUNEL staining to examine whether overexpression of SPATA17 gene influenced testicular cell apoptosis. We measured the number of TUNEL-positive cells in cross-sectioned testes and observed that only a few TUNEL-positive cells were observed in control animals (Fig. 4F–a). However, the number and signal density of positive cells significantly increased in SPATA17 transgenic mice (Fig. 4F–b). The index of apoptotic cells was 32.8 % in wild type mice, while 56.4 % in transgenic mice (Fig. 4E). The number of apoptotic cells was increased by 42 % (P<0.05) in SPATA17 transgenic mice compared with wild-type mice. Most of the TUNEL positive cells were primary spermatocytes both in wild-type and SPATA17 transgenic mice. This result showed that overexpression of SPATA17 can accelerate testicular cell apoptosis in vivo.
Recent research showed that human SPATA17, also called MSRG-11, is downregulated in the testes of patients with azoospermia due to meiotic arrest  and could accelerate GC-1 cell apoptosis in vitro , which suggested that it might play a critical role in spermatogenetic cell apoptosis. To elucidate the role of SPATA17 in vivo, we have generated a transgenic line in which the PGK2-SPATA17 fusion protein is expressed under the control of the 1.4-kb region of the human PGK2 promoter. Southern blot and western blot analysis showed that PGK2 promoter could drive the expression of SPATA17 in spermatocytes in transgenic mice. The SPATA17 transgenic mice did not reveal any significant defect in gross testicular anatomy as well as in fertility and showed normal morphology compared with the wild type mice. However, we observed significant increases in apoptotic cells in the SPATA17 transgenic mice. These results demonstrated that overproduction of the SPATA17 protein disturbed the normal development of male germ cells. But the underlying mechanisms of apoptosis in spermatogenesis in SPATA17 transgenic mice are not known. Previous research data have shown that spermatocyte apoptosis is related to many factors, such as: (1) the p53 gene, which is highly expressed in spermatocytes from the leptotene to pachytene stage and is related to apoptosis of spermatogenic cells induced by heat pressure [17, 18]; (2) the FAS pathway, which is the key factor to activate the apoptosis of spermatogenic cells at initiation stage of apoptosis ; (3) apoptosis inhibitor Bcl-2 and apoptosis inducer Bax at apoptosis effector stage ; (4) protease caspase at apoptosis execution stage. SPATA17 protein is recognized as a new member of CaM binding protein family because the sequence contains three highly conservative IQ motifs (IQXXXRGXXXR). IQ motif–containing proteins are known to interact with calmodulin in a Ca2+-dependent or Ca2+-independent manner and different IQ motif proteins mediate different functions in response to intracellular Ca2+ signals . A change in calmodulin conformation induced by Ca2+ binding is known to regulate the activity of IQ motif proteins, such as myosin and L-type Ca2+ channels that modulate the ATPase activity of myosin or opening of the L-type channels. PEP-19 and RC3 are IQ motif proteins that interact with both Ca2+-bound and Ca2+-free calmodulin and, thus, modulate the availability of calmodulin by affecting its Ca2+ association and dissociation [22, 23]. Recently, a novel IQ motif protein AtBAG6 in yeast and plants can induce programmed cell death by interaction with CaM and CaM-binding IQ domain is required for AtBAG6-mediated cell death . To explore the possible mechanism of apoptosis induced by overexpression of SPATA17, we examined whether SPATA17 protein can direct bind CaM by various methods including CaM protein pulldown, immunoprecipitation and in vitro binding assays using recombinant proteins, the results demonstrated that SPATA17 protein can direct bind CaM in a Ca2+-free form (results not shown). Further identification of components associated with the CaM-SPATA17-mediated cellular response should facilitate elucidation of the underlying mechanism by which SPATA17 regulates cell apoptosis.
This work was supported by grants from the National Natural Science Foundation of China (Grant No.: 30971570 and No.: 31171196).