Molecular Biology Reports

, Volume 40, Issue 2, pp 1905–1910

Overexpression of human SPATA17 protein induces germ cell apoptosis in transgenic male mice

Authors

    • Department of Chemistry and Chemical EngineeringHunan Institute of Science and Technology
  • Yu Liu
    • Department of Chemistry and Chemical EngineeringHunan Institute of Science and Technology
  • He Juan
    • The Second People’s Hospital of YueYang
  • Xiang Yang
    • Department of Chemistry and Chemical EngineeringHunan Institute of Science and Technology
Article

DOI: 10.1007/s11033-012-2246-z

Cite this article as:
Nie, D., Liu, Y., Juan, H. et al. Mol Biol Rep (2013) 40: 1905. doi:10.1007/s11033-012-2246-z

Abstract:

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.

Keywords

Cell apoptosisSPATA17SpermatogenesisTransgenic Mouse

Introduction

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 [14]. 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 [14]. Germ cell apoptosis can also be induced by various pathological conditions such as heat stress, exposure to ionizing radiation [5], toxic substances [6], hormonal depletion [7] 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 [10]. 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 [11]. 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

Animals

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 PGK2SPATA17 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 [12]. 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.

Sperm counts

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.

Immunohistochemical analysis

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 [14]. 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

Our previous study revealed that SPATA17 protein was most abundant in the cytoplasm of round spermatids and elongating spermatids within seminiferous tubules of the adult testis and can accelerate cell apoptosis in GC-1 cells [10], which prompted us to investigate the in vivo function of this gene during spermatogenesis. For this purpose, the fusion gene PGK2-SPATA17 transgenic construct (Fig. 1A) contains 1.4 kb of the human PGK2 promoter which has been successfully used to direct spermatocyte-specific expression of a CAT report gene [15], SV40 TAg gene [12] and Peroxisomal testis-specific 1 gene (Pxt1) [13]. Down-stream of the SPATA17 ORF, the 3′ untranslated region (UTR) of human growth hormone 1 (GH1) and SV40 poly(A) sequence were located in order to ensure the proper posttranscriptional proceeding of the transgenic mRNA. A total of 106 2-cell stage embryos that had survived the microinjection procedure were transplanted to pseudopregnant foster mothers and 12 founder mice were born, of which 9 mice survived, its survive rate was 75 %. The positive founder mice were detected by PCR using genomic DNA. As can been seen in Fig. 1B, A 1104-bp fragment was observed only in transgenic founders (TR/WT), but not in wild-type (WT/WT) animals. Southern blot analysis was further used to demonstrate the PCR positive founder mice, a 1.5-kb band in the transgenic (TR) corresponding to the PGK2-SPATA17 transcript in TR testis was observed in Fig. 1C. Totally, three animals (33.3 %) were positive by PCR and Southern blot hybridization of tail DNA.
https://static-content.springer.com/image/art%3A10.1007%2Fs11033-012-2246-z/MediaObjects/11033_2012_2246_Fig1_HTML.gif
Fig. 1

Generation and expression analysis of PGK2-SPATA17 transgenic mice. A Schematic representation of the PGK2-SPATA17 transgenic construct. The construct consists of a 1.4-kb part of the PGK2 promoter, c-myc-tag, complete ORF of the human SPATA17 gene, 3′UTR of the GH1 and poly(A) signal of SV40. Start codon (ATG) and stop codon are given. B Genotyping PCR of the transgenic founders using transgenic construct-specific primers. A 1104-bp product (lane 2, 3 and 5) could be observed in transgenic founders in upper panel, but not in wild-type animals in lane 1. lane M pUCmix8 DNA molecular weight marker, lane 2–6 stand for five transgenic founders. Lane 1 wild-type mice. C Southern blot analysis of the PGK2-SPATA17 transgenic expression in transgenic founders using the SPATA17-specific probe, a 1.5-kb band was detected in the transgenic (TG) corresponding to the PGK2-SPATA17 mRNA transcript in TG testis (lane 2, 7 and 9). Lane 1 DNA marker, lane 2–9 stand for eight PGK2-SPATA17 transgenic mice; lane 10 blank control

Expression of the PGK2-SPATA17 in the testis

Nine lines of transgenic mice with the PGK2-SPATA17 constructs were generated and there positive mice were further analyzed by Northern blot hybridization to determine expression of the SPATA17 transgenes in the testis. The results showed that the endogenous mouse SPATA17 mRNA was detected both in the transgenic mice and in their littermates, whereas the transgene mRNA products were detected only in the transgenic mice (Fig. 2 upper panels). The levels of the endogenous SPATA17 mRNA were higher than those of the transgenic mRNA, suggesting that the promoter activity of the endogenous SPATA17 gene was stronger than that of the exogenous PGK2 promoter in male germ cells. Since the transgene constructs lacked the 3′ untranslated sequence of the SPATA17 cDNA, their mRNA products appeared smaller than the endogenous SPATA17 mRNA. The protein products of the SPATA17 transgenes were detected with the immunoblot analysis using anti–myc tag antibody (Fig. 3). The expected 43.5-kDa band corresponding to the size predicted for PGK2-SPATA17 fusion protein was detected in the testes of transgenic mice but not in the analyzed wild-type tissues. These results showed that PGK2 promoter can correctly direct the expression of SPATA17 in transgenic testes.
https://static-content.springer.com/image/art%3A10.1007%2Fs11033-012-2246-z/MediaObjects/11033_2012_2246_Fig2_HTML.gif
Fig. 2

Northern blot analysis of the PGK2-SPATA17 transgene expression Using the SPATA17-specific probe, a 2.0-kb band was detected, representing the endogenous SPATA17 transcript in the testis of wild type (WT) and transgenic (TG) and a 1.6-kb band corresponding to the PGK2-SPATA17 transcript only in the transgenic testis (lanes 1, 4, 5). Lane 1, lane 4 and lane 5 stand for the testes of three PGK2-SPATA17 transgenic mice. Lane 2, lane 3, lane 6, lane 7, lane 8 and lane 9 stand for the lung, liver, heart, spleen, kidney and brain of PGK2-SPATA17 transgenic mice, respectively. RNA quality and integrity was checked using the G3PDH as control (bottom)

https://static-content.springer.com/image/art%3A10.1007%2Fs11033-012-2246-z/MediaObjects/11033_2012_2246_Fig3_HTML.gif
Fig. 3

Western blot analysis of SPATA17 protein in wild-type (WT) and transgenic (TG) testicular lysates. Results are representative of three separate experiments with 3 WT (lanes 1–3) and three transgenic (TG) (lanes 4–6) mice. A 43.5-kD band corresponding to human SPATA17 protein is detected in SPATA17 transgenic TG, but not in WT animals. β -actin was used as control

Body and testis weights

The PGK2-SPATA17 mice did not show any obvious phenotype in the preliminary analysis; the animals were viable and grew normally. Since SPATA17 is normally expressed in developing spermatogonia and spermatocytes, overproduction of SPATA17 can only accelerate or increase its function in testis development. Although the body weights were similar in the wild type and PGK2-SPATA17 transgenic mice, there was a clear difference in the weight of their testes (Fig. 4A), the absolute and relative testicular weights were decreased by 25 % (P < 0.001) and 23 % (P < 0.001) respectively in PGK2-SPATA17 transgenic mice compared with the wild-type littermates (Table 1). PGK2-SPATA17 transgenic mice also exhibited a tendency for diminished production of spermatozoa as their epididymal sperm counts were 10 % lower when compared with wild-type mice (Table 1). However, spermatozoa of PGK2-SPATA17 transgenic mice were motile, and Acridine Orange staining did not reveal any gross disturbances in the spermatozoa DNA structure (results not shown).
https://static-content.springer.com/image/art%3A10.1007%2Fs11033-012-2246-z/MediaObjects/11033_2012_2246_Fig4_HTML.gif
Fig. 4

Cell apoptosis assay in testes of control and SPATA17 transgenic mice. Analysis of tubule diameter and number of TUNEL-positive cells. A Comparison of testicular sizes in wild type (WT) and PGK2-SPATA17 transgenic mice (TG). Scale bars = 1 mm. B Diameter of stage VII–VIII tubules was measured with the help of a scale bar. Values in wild type and transgenic mice are presented as mean ± SEM. (C and D) Polyclonal rabbit anti-SPATA17 and biotin peroxidase-conjugated goat anti-rabbit antibodies were used and the presence of SPATA17 was revealed by brown staining. No significant differences were detected in spermatogonia and spermatozoa in the seminiferous tubules of the wild type mouse D and transgenic mouse C. Scale Bars = 100 μm. E Apoptotic cells were visualized with TUNEL staining. Values are the number of TUNEL-positive cells per 100 tubules in WT and TG mice. F a TUNEL assay was performed to investigate whether the degenerated cells undergo apoptosis,. No significant increase in the number of apoptotic cells was observed (Fa); in contrast, strong apoptosis induction was observed in the transgenic animals (Fb). Arrows indicate positive cells. Scalebars = 100 μm

Table 1

Body weight, testis weights, and epididymal sperm count of wild-type and transgenic male mice. Values are mean ± SEM

Genotype

Body weight (g)

Testis weight (mg)

Testis weight (% of body weight)

Sperm count (106 per epididymis)

Wild type

20.7 ± 1.5

102.7 ± 2.2

0.49 ± 0.01

8.76 ± 0.55

Transgenic

20.2 ± 1.0

77.8 ± 3.0*

0.38 ± 0.03*

7.87 ± 0.76

*P<0.001

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 [16] and could accelerate GC-1 cell apoptosis in vitro [10], 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 [19]; (3) apoptosis inhibitor Bcl-2 and apoptosis inducer Bax at apoptosis effector stage [20]; (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 [21]. 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 [24]. 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.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (Grant No.: 30971570 and No.: 31171196).

Copyright information

© Springer Science+Business Media Dordrecht 2012