Background
Spermatogenesis is characterized by successive periods of regulated cell proliferation, meiosis, and haploid differentiation. Abnormalies during any step of spermatogenesis could cause male infertility. It is estimated that approximately 2,000 genes regulate the process of spermatogenesis, and most of these genes are present on the autosomes, while approximately 30 genes are found on the Y chromosome [
1]. Recent studies have shown that
Septin12[
2],
Fank1[
3],
CKT2[
4],
RGS22[
5] and
NANOS2[
6] are specifically expressed in the testis and are functionally involved in spermatogenesis. Identification of these genes and studies on their spatial and chronological expression patterns are essential for understanding the mechanisms of spermatogenesis and male infertility [
7‐
9].
Recently, using Affymetrix Genechips, we identified 2,058 up-regulated transcripts during the developmental period from postnatal day 4 to 6 months in mice [
10] Among these transcripts were 292 testis-specific genes [
11], including
TSG23[
12],
TSC21[
13],
TSC24[
14] and
TSC77[
15]. In the present study, another gene, Dickkopf-Like1 (
DKKL1/Dkkl1) was identified using Affymetrix mouse and human Genechips.
DKKL1/Dkkl1 was identified independently as a distant homologue to the Dickkopf (Dkk) family of proteins that modulate WNT/β-catenin signalling [
16]. In contrast to conventional Dkks, Dkkl1 does not modulate WNT/β-catenin canonical signalling [
17]. Several reports have concluded that
Dkkl1 mRNA is expressed at high levels in adult mice testis in the spermatogenic epithelium of the seminiferous tubules [
18] and in developing spermatocytes where
Dkkl1 accumulates first in developing acrosomes and then in the acrosome of mature sperm [
19]. This suggests that
Dkkl1 may play a role in spermatocyte development and maturation in mice. However, little is known about the character and function of
DKKL1 in human testes. Therefore, the present study was set out to explore the spatial and chronological expression of
DKKL1/Dkkl1 in human and mouse testes and to compare the mRNA and protein expression levels of
DKKL1/Dkkl1 in fertile and infertile human testes. A clearer understanding of the role of
DKKL1/Dkkl1 in testes may help elucidate the biological principles underlying the increasing rate of male infertility and may provide targets for the development of a male contraceptive.
Methods
Sources of samples
Male and female Balb/c mice were obtained from the Animal Laboratory Centre of South Medical University (Guangzhou, China) and maintained in a temperature and humidity-controlled room. All animals had free access to standard mouse chow and water. Male and female mice (1:3) were mated naturally, and the day of birth was designated as day 1. Testes were individually collected from Balb/c mice on days 4, 9, 18, 35, and 54, as well as at 6 months (m 6). Testis samples at postnatal days 4 (n = 30), 9 (n = 20), 18 (n = 15), 35 (n = 8), and 54 (n = 4), as well as at m 6 were collected. Other organs including the brain, heart, liver, spleen, lung, kidney, muscle, stomach, intestine, bladder and epididymis were also collected from adult mice (n = 4).
Testis biopsy material from male infertility patients aged 20–40 years with Sertoli cell only syndrome, cryptorchidism or spermatogenic arrest were obtained from Peking University Shenzhen Hospital, Shenzhen, China. A sample of fertile human testis was obtained from an adult male patient (aged 27 yr) undergoing bilateral orchiectomy for the treatment of prostate carcinoma, and a sample of foetal testis was obtained from a naturally aborted embryo (aged 6 m). In addition, human tissues, including ovary, kidney, uterus, prostate, thyroidea, stomach and oesophagus, were also collected. All samples were frozen in liquid nitrogen and then immediately stored at −80°C. All patients signed consent forms approved by the Committee on Human Rights in Research of the Ethics Committee at Peking University Shenzhen Hospital, Shenzhen, China. Animal experiments were approved by the Animal Test Centre of China.
cDNA microarray hybridization
The screen for
Dkkl1 was undertaken by hybridizing cDNA from mouse testes at six developmental stages with commercially available Affymetrix mouse Genechips, which contain 45,000 pairs of probes including 39,000 transcripts, as previously described [
10]. The homologous human gene,
DKKL1, was also screened for by comparing the expression profiles of human adult and foetal testis using Affymetrix human Genechips containing 47,000 transcripts derived from approximately 38,500 well-substantiated human genes. All of these procedures were carried out as described by Affymetrix. After hybridization, the array was washed, stained with streptavidin phycoerythrin using the Affymetrix Genechip Fluidics Workstation 400, and scanned on a Hewlett-Packard gene array scanner (Hewlett-Packard Co., Palo Alto, CA, USA). After the arrays were scanned, the signals generated were quantified and analyzed using MAS 5.0 software. Absolute and comparison analyses were also performed using MAS 5.0. After normalization of these data, experimental arrays were compared with baseline arrays to detect changes in the expression of transcripts across samples targeted to different arrays (see
http://www.Affymetrix.com for details on the statistics of these analyzis).
Semi-quantitative RT-PCR
Semi-quantitative RT-PCR was performed to analyse and confirm the expression of the
DKKL1/Dkkl1 genes. Total RNA (2 μg) was reverse-transcribed into cDNA in a reaction primed by an oligodeoxynucleotide (dT)
18 primer using RevertAid
TMM-Mulv Reverse Transcriptase (Fermentas, Glen Burnie, MD, USA) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) primers for
DKKL1/Dkkl1,
β-actin and
GAPDH were synthesized by Shanghai Bioengineering Inc. (Shanghai, China; Table
1). The PCR reaction was initiated by hot start at 94°C for 4 min, followed by 33 cycles of 94°C for 30 s, 64°C for 30 s and 72°C for 40 s, followed by extension at 72°C for 5 min. PCR products were run out on a 1% agarose gel in 0.5x TBE buffer (30 min at 100 V) and analyzed using a Rapid Agarose Gel Electrophoresis System (Wealtec Corp.,Sparks, NE, USA).
Table 1
Oligonucleotide sequences used in RT-PCR analysis
DKKL1
| 58 | 299 | Sense: TGCTGCTCCTCTCTACCCT |
Antisense: CTCTCCTGTCTTGTTGTCGG |
Dkkl1
| 55 | 217 | Sense: TCGTGTCCTCCTCTGCTCTCT |
Antisense: TTGCCCATTCTGTGCTCCT |
β-actin
| 55 | 281 | Sense: AACAGTCCGCCTAGAAGCAC |
Antisense: CGTTGACATCCGTAAAGACC |
GAPDH
| 58 | 100 | Sense: GCTCTCTGCTCCTCCTGTTC |
Antisense: GACTCCGACCTTCACCTTCC |
DKKL1 transcription analysis in the testes of patients with male infertility
Testicular tissues were obtained via biopsy from 15 patients with male infertility at the Peking University Shenzhen Hospital (Shenzhen, China). The clinical diagnoses based on testicular biopsy were Sertoli-cell-only syndrome (SCOS), cryptorchidism, and spermatogenic arrest at different stages. Total RNA (about 2 μg) was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA). Reverse transcription and PCR were performed as described above.
Protein extraction and Western blot analysis
Human and mouse tissues were lysed with lysis buffer in the presence of a protease inhibitor cocktail (Merck, USA) and kept on ice for 1 h. After centrifugation at 12,000 g for 20 min at 4°C, the resulting supernatant was collected for Western blot analysis. After the protein concentration was determined by BCA protein assay (Thermo Fisher Scientific Inc., USA), supernatant fractions from the lysate were mixed with 6x SDS sample buffer and boiled for 10 min. Samples were reduced with 5% β-mercaptoethanol and stored at −20°C until used.
Extract samples containing approximately 30 μg protein were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the extracts were then transferred onto polyvinylidene difluoride (PVDF) membranes (MilliPore, Bedford, MA, USA). The membranes were blocked in TBST (5 mmol Tris–HCl, pH 7.4; 136 mmol NaCl; and 0.05% Tween20) containing 2% BSA overnight at 4°C. The next day, the membranes were hybridized at room temperature for 4 h with rabbit anti- DKKL1/Dkkl1 antibody (ABGENT, USA) at a dilution of 1 μg/ml and rabbit anti-GAPDH (Abcam) as an internal control, followed by three washes for 10 min with TBST. Next, the blots were incubated for 1 h at room temperature with HRP-conjugated goat anti-IgG rabbit antibody (1:5000; Abcam), and washed three times with TBST. Bound antibodies were detected by electro-chemiluminescence using SuperSignal West Dura substrates (Pierce Biotechnology Inc.) according to the manufacturer's recommendations and visualized by fluorescence detection equipment (ChemiDoc XRS, BIO-RAD, Hercules, CA, USA).
In vitro fertilization (IVF)
Female mice were superovulated, and the stage MII oocytes were collected from mice oviducts, as described [
20]. Mouse spermatozoa from cauda epididymis were capacitated in Human Tubal Fluid medium (HTF; In-Vitro Fertilization. Inc., USA) within 30 min, and the spermatozoa (in drops of 50 μl with a concentration of 5 × 10
4/ml) were incubated for 30 min with either HTF or HTF containing anti-
Dkkl1 antibody at a dilution of 1 μg/ml. The treated spermatozoa (50 μl) were deposited into Cleavage medium (In-Vitro Fertilization. Inc., USA) containing 30–35 mouse oocytes and incubated for 2 h. The unbound spermatozoa were washed away. To analyze the IVF rate, two pronuclear cells were examined 6 h after fertilization. The zygote cleavages were counted at 42 h [
21].
Immunohistochemistry
The specimens were fixed for 4 hr in 10% formalin and then embedded in paraffin, sectioned at 5 μm, and mounted on silane-coated slides. For immunohistochemistry, sections were dewaxed and rehydrated through descending grades of alcohol to distilled water, followed by incubation in 2% hydrogen peroxide to quench the endogenous peroxidase activity and then washed in PBS. Subsequently, nonspecific binding was blocked with goat serum (Fuzhou Maixin Biotechnology, China) for 2 h, followed by incubation with polyclonal rabbit anti- DKKL1/Dkkl1 antibody (ABGENT, USA) overnight at 4°C. Following three washes in PBS, the sections were incubated with horseradish peroxidase (HRP) conjugated goat anti-rabbit secondary antibody (Fuzhou Maixin Biotechnology, China) for 1 h at room temperature. Immunoreactive sites were visualized with diaminobenzidine (DAB) and mounted for bright field microscopy (DMLB; Leica Microsystems, Germany). Negative control sections were incubated with the buffer 1% BSA in place of the primary antibody.
Discussion
It has been previously shown that spermatogenesis is mainly regulated by testis-specific gene activation. Investigation of testis-specific genes is expected to lead to a broader and more thorough understanding of spermatogenesis. Many genes related to human and mouse spermatogenesis have been identified by our previous research [
12‐
15]. The present study focuses on the characterization of a newly recognized gene,
DKKL1/Dkkl1.
As spermatogenesis is divided into three major phases, namely proliferation and differentiation of spermatogonia, meiosis and spermiogenesis [
23], the expression patterns of
Dkkl1 in mouse testes at different developmental stages were first investigated using a gene chip approach. The six selected developmental stages represent the major stages of germ cell development during the first wave of spermatogenesis: day 4, cells with stem cell properties; day 9, spermatogonia mitosis; day 18, spermatocyte meiosis; day 35, round spermatid production and elongated spermatid formation, also called spermiogenesis; day 54, normal postpubertal spermatogenesis; 6 months, elongated spermatids and immature sperm [
24]. As demonstrated in the mouse gene chip results, the expression of
Dkkl1 was detected on days 18, 35, and 54, as well as at 6 months, but it was not detected on days 4 and 9. This was further verified by RT-PCR and Western blot analysis. Analysis revealed that
Dkkl1 was weakly expressed in mouse testis on day 18, with expression increasing after day 18 and remaining stable after day 54. Based on the expression characteristics of
Dkkl1 in mice, we suggest that the expression of
Dkkl1 mRNA and protein are associated with the postmeiotic phase of spermatogenesis and with the generation of late pachytene spermatocytes and round spermatids. In addition, the results of multi-tissue RT-PCR showed that this gene was highly expressed in testis and weakly expressed in the epididymis. A possible reason for the weak expression of
Dkkl1 in the epididymis of mice is that some immature and mature sperm are stagnated in the epididymis.
Although
Dkkl1 has been previously suggested to be important for male fertility i
n vitro[
25], surprisingly,
Dkkl1−/−mice are not only viable and fertile, but both male and female Dkkl1−/− mice produce offspring at efficiencies comparable to wild-type animals [
17,
26]. These studies made it impossible to evaluate the contribution of
Dkkl1. So what role might
Dkkl1 play in fertilization? In the present study, we used an
in vitro fertilization assay to investigate the role of
Dkkl1. Following the use of a
Dkkl1 neutralizing antibody, the fertilization rate was significantly reduced. This result is consistent with a previous study [
25] that found that
Dkkl1 is required for efficient fertilization
in vitro. It is likely that
Dkkl1 plays a role in the penetration of spermatozoa into oocytes, and during this time window, the neutralizing antibody has a chance to block its action. However, IVF provides an assay for detecting fertilization-related problems that are not apparent in vivo. More likely is the possibility that a delay in fertilization caused by the absence of
Dkkl1 is compensated by other factors during spermatogenesis or preimplantation development.
Given that
Dkkl1 was closely linked to mouse spermatogenesis, what is its relationship to human spermatogenesis? To address this question, we next conducted hybridization of adult and foetal testes samples to a human gene chip. The results indicated that
DKKL1 was expressed at a higher level in adult testis than in foetal testis. The relative hybridization signal intensity of
DKKL1 in adult testis was 405.56 times that in foetal testis. There are only Sertoli cells and undifferentiated spermatogonia cells in the seminiferous tubules of the foetal testis, whereas the seminiferous tubules of the adult testis contain not only Sertoli cells and spermatogenous cells, but also various spermatogenic cells. In other words, there are many developmental stages of germ cells represented in adult testis that are not found in foetal testis. Many genes expressed in the testis are developmental stage specific or cell type specific, which reflects the demands of tissue development [
27]. It has been demonstrated that other genes with differential expression between adult and foetal testis, namely
Spef1[
28],
Akap4[
29], and
Rosbin[
30], are spermatogenesis-specific. Thus, the results of our gene chips analysis provided an important clue that
DKKL1 might be associated with testis development and spermatogenesis in humans.
Whether the expression of testis development and spermatogenesis genes were altered in male infertility? Previous study have shown that patients with SCOS or spermatogenesis arrest at spermatocyte do not express a novel human testis-specific and spermatogenesis protein NYD-SP12 [
31]. Recently, it has been reported that the signal of the human testis developmental gene
SPATA12 was not detected in patients with cryptorchidism or SCOS [
32]. To further validate the function and role of
DKKL1 in male infertility, we examined the
DKKL1 mRNA transcript levels as well as protein levels among the patients with SCOS, cryptorchidism or spermatogenic arrest. Most of them had no or insufficient expression of
DKKL1 protein in the testes. Only the control fertile male testis sample, in which every stage of spermatogenic cell was represented, fully expressed
DKKL1. The trend of increasing expression with the presence of the more mature spermatogenic cells in testis is consistent with the development-dependent characteristics of
DKKL1 mRNA. It was revealed that decreased expression of
DKKL1 was associated with spermatogenic failure in infertile men.
Acknowledgement
We are grateful to Elsevier Language Editing Services for editorial assistance with the manuscript. We would like to thank Miss Xiaoyan Zhang, Mr. Zhenmin Zhang, Mr. Yong Wang and Mr. Wenjie Li for their technical assistance. This work was supported by grants from the National Natural Science Foundation of China (No.30972992, No.81170613, and No.81101922), the Shenzhen Important Foundation of Science & Technology (201001015), the Shenzhen Basic Research Funds for Distinguished Young Scientists, and the Qingyuan Foundation of Science & Technology (2010B001, 2010B006).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
QY, XW and CC participated in the design of the study, collected the materials, and carried out all experiments. QY drafted the manuscript. RD, YL, JH, JC and ZY collected the materials and helped to carry out the cDNA microarray hybridization, RT-PCR, immunohistochemistry, and Western blot analyses. AT, YG and ZC conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.