Dulbecco’s modified Eagle’s medium (DMEM)/F-12 and fetal bovine serum (FBS) were purchased from HyClone Laboratories, Inc., (Logan, UT, USA). The SYBR^® PrimeScript™ RT-PCR kit (Perfect Real Time) was purchased from Takara Biotechnology (Dalian, China). The Bicinchoninic Acid (BCA) Protein Assay kit, Protein Extraction kit, BeyoECL Plus Western Blotting Detection kit, and Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection kit were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Testosterone Immunoassay kits were obtained from USCNK (Wuhan, China). Antibody for 3β-HSD1 (sc-30821) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The In Situ Hybridization Detection kit was purchased from Haoyang (Tianjing, China). All other chemicals were purchased from Sangon Biotech (Shanghai, China).

A total of 24 male Balb/c mice at 18 days, 4 months, and 16 months of age (n=8 in each age group) and a New Zealand white rabbit were obtained from the Chongqing Medical University Animal Center (Chongqing, China). Mice were sacrificed by intraperitoneal injection of 10% chloral hydrate solution at the dose of 8 ml/kg. Immediately after euthanasia, the testes were removed and washed with saline (0.9% NaCl). The left testis was fixed in a 4% paraformaldehyde solution for in situ hybridization and immunohistochemistry, while the right testis was used for protein and RNA extraction. The animal protocols conformed to those approved by the Chongqing Medical University Animal Care and Use Committee.

TM3 Leydig cells were obtained from The Cell Bank of the Chinese Academy of Sciences (Shanghai, China)) and 293FT cells were from Invitrogen (Carlsbad, CA, USA). TM3 Leydig cells were cultured in F12/DMEM supplemented with 10% FBS. 293FT cells were cultured in DMEM containing 10% FBS. All cells were placed in an incubator containing 95% air and 5% CO₂ at 37°C, and the culture media were replaced every 2 days.

A polyclonal anti-PLP-J antibody was prepared to detect PLP-J protein expression. Based on the amino acid sequence of PLP-J (NP_038794.1; http://www.ncbi.nlm.nih.gov/protein/7305407), the following peptide fragments were designed: RYDRKSNEEI, IQPGIEENNE, KTNEDLLK, KMYKILD, VLTHLGSYDGMM, RELRSSKKSK and FFYCLRKDTK. These fragments were joined, along with connecting residues, to yield the final sequence: RYDRKSNEEIGSIQPGIEENNEAKTNEDLLKKMYKILDGSVLTHLGSYDGMMGSRELRSSKKSKFFYCLRKDTK. The amino acid sequence was converted to a nucleotide sequence, which was generated via gene synthesis following incorporation of KpnI and HindIII sites, and cloned into pET-30a (Novagen, Madison, WI, USA) via these two sites. The resulting plasmid was used to transform BL21-competent cells prior to the selection of positive clones. Protein production was facilitated by isopropyl β-D-1-thiogalactopyranoside induction.

To immunize a single New Zealand white rabbit, 200 μg of the recombinant protein was subcutaneously injected followed by three boosters of 100 μg recombinant protein at 21 days, 42 days and 63 days. Subsequent to the last booster, a blood sample was obtained from an ear vein for ELISA analysis to determine the titer. If the desired level (titer of antibody >12,800) was reached, blood was drawn from the carotid artery and centrifuged at 5,000 × g for 10 min to collect antiserum, which was subjected to protein G affinity purification. The purity of the antiserum was determined by western blot analysis. The antiserum was divided into aliquots, and the pre-immune serum was used as a control.

The expression of PLP-J mRNA in testis tissues was assessed by RT-PCR. The primer designs were based on the sequences of mouse PLP-J (NM_013766; http://www.ncbi.nlm.nih.gov/nuccore/7305406). The primer sequences were as follows: Forward, 5′-ACCAAGATGTGCCAAACCATTTCTA-3′; and reverse, 5′-CAGCTCTTGTCATCATCCCATCA-3′. The predicted size of the RT-PCR product was 163 bp. For mouse β-actin amplification, the primer sequences were as follows: Forward, 5′-TCGTGCGTGACATCAAAGAG-3′; and reverse, 5′-CAAGAAGGAAGGCTGGAAAA-3′. The length of the RT-PCR product was 177 bp.

Briefly, total RNA from the mouse testis was extracted using TRIzol reagent according to the manufacturer’s instructions. First-strand cDNA was synthesized using the SYBR^® PrimeScript™ RT-PCR kit (Perfect Real Time) in the thermal cycler (CFX96; Bio-Rad, Hercules, CA, USA). Each RT-PCR reaction was performed in triplicate in a total volume of 20 μl reaction mixture [2 μl cDNA, 6.8 μl H₂O, 10 μl SYBR^® Premix Ex Taq™, 0.6 μl of each of the forward and reverse primers (10 μM)]. The PCR program consisted of 1 min at 95°C, followed by 40 cycles of 10 sec at 95°C and 30 sec at 60°C. Data were analyzed according to the 2^−ΔΔCt method (14). The expression level of PLP-J mRNA was normalized to the concurrent measurement of β-actin mRNA.

In concordance with the gene sequence of murine PLP-J, a probe with the sequence 5′-GGCAGTCACATATACCCACAGGAACAT-3′ was designed in the current study, which was then synthesized at Sangon Biotech. To identify the cell type expressing PLP-J mRNA, in situ hybridization was performed on 5-μm tissue sections. Paraffin-embedded tissues were dewaxed and blocked in H₂O₂ solution for 20 min at room temperature to prevent endogenous catalase activity. After rinsing with 0.01 M phosphate-buffered saline (PBS) 3 times, acetylation solution was added dropwise until it covered the surface of the tissues, which were then incubated at room temperature for 10–30 min. The specimens were washed with PBS 3 times and rinsed once with 0.2X saline sodium citrate (SSC) buffer. Prehybridization buffer was added dropwise until it covered the tissues, which were then incubated at 37°C for 2 h. The samples were rinsed 3 times with 0.2X SSC for 5 min. Hybridization buffer was added dropwise until it covered the tissues, which were incubated at 37°C for 8 h and then washed with SSC. Mouse Anti-Digoxin-Biotin Antibody solution was added and the samples incubated at 37°C for 45 min. The specimens were rinsed 3 times with 0.01 M PBS for 5 min. A working solution of high-performance streptavidin peroxidase complex (3 ml) was added to the specimens, incubated at 37°C for 45 min, and then washed with PBS. The slides were stained with 3,3′-diaminobenzidine and counter-stained with hematoxylin, which stained the nuclei blue. The slides were then observed with a BX51 microscope (Olympus, Tokyo, Japan).

To examine whether PLP-J is expressed in Leydig cells, the cellular colocalization of PLP-J with 3β-hydroxysteroid dehydrogenase (HSD) 1 was examined in the testes of adult mice using a double immunofluorescence staining method. Briefly, the sections were incubated for 48 h at 4°C with a mixture of rabbit anti-PLP-J IgG and goat anti-3β-HSD1 IgG. Subsequently, the sections were rinsed in 0.01 M PBS (pH 7.4) and then incubated for 24 h at 4°C with a mixture of FITC-conjugated anti-rabbit IgG and tetramethyl rhodamine isothiocyanate-conjugated anti-goat IgG (ZSGB-Bio, Beijing, China). The tissues were then examined under the BX51 microscope.

For immunofluorescent staining of PLP-J in TM3 cells, the TM3 cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, followed by permeabilization in PBS containing 0.5% Triton X-100 at room temperature for 10 min. The cells were then immersed in blocking solution for 1 h. The cells were immunolabeled with rabbit anti-PLP-J primary antibody overnight at 4°C. Following 3 washes with PBS, the cells were incubated with FITC-conjugated anti-rabbit IgG at room temperature for 1 h. The slides were then observed and analyzed using a TE-2000U fluorescence microscope (Nikon, Tokyo, Japan).

In order to isolate protein, tissues or cells were homogenized and lysed with radioimmunoprecipitation assay buffer. Insoluble material was removed by centrifugation at 12,000 × g for 10 min at 4°C, and the supernatant was collected. The protein concentration was determined using the BCA assay. For western blotting, samples were subjected to SDS-PAGE and then transferred to a polyvinylidine fluoride membrane, followed by incubation with anti-PLP-J or anti-β-actin polyclonal antibody. Membranes were washed briefly in Tris-buffered saline with Tween 20 and incubated with secondary antibodies. The antigen-antibody complexes were then detected using the BeyoECL kit according to the manufacturer’s instructions.

To investigate the biological activities of PLP-J in vitro, a PLP-J-expressing lentivirus was generated, which was transfected into TM3 cells to induce upregulation of PLP-J expression. The PLP-J sequence was obtained from Genbank (NM_013766.2; http://www.ncbi.nlm.nih.gov/nuccore/NM_013766.2), synthesized, and then cloned into the LV5 vector (GenePharma Co., Ltd., Shanghai, China). Lentiviruses were then produced in 293T cells, titrated, and used to transfect the TM3 cell line.

To analyze the effect of PLP-J overexpression on testosterone production in TM3 cells, TM3-N cells (transfected with control lentivirus) and TM3-PLP-J cells (transfected with PLP-J lentivirus) were cultured for 48 h. The culture supernatants and cells were collected for testosterone analysis according to the manufacturer’s instructions.

Harvested TM3 cells were centrifuged at 300 × g for 10 min and the supernatant was removed. They were then washed once with 1X binding buffer. The cells were resuspended in 200 μl binding buffer prior to incubation with 5 μl Annexin V and 10 μl propidium iodide (PI) at room temperature for 15 min. Flow cytometric analysis was conducted using a FACSVantage SE flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

TM3 cells were trypsinized and centrifuged at 300 × g for 10 min. Following rinsing with PBS, the cells were fixed in 70% ethanol. TM3 cells were then centrifuged for 5 min and washed with 1X PBS. Next, the cells were incubated for 30 min in Hank’s Balanced Salt Solution containing PI (10 μg/ml) and RNase (100 μg/ml) at room temperature, followed by analysis using the FACSVantage SE flow cytometer.

Data are expressed as the mean ± standard deviation. Significant differences were evaluated using one-way analysis of variance with the Newman-Keuls method. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the production of PLP-J, polyclonal anti-PLP-J antibodies were prepared. New Zealand white rabbits were immunized 4 times with a recombinant protein containing multiple PLP-J epitopes, and the resulting antiserum was shown by ELISA to have a titer of 1:25,600. Subsequent protein G affinity purification yielded an antibody concentration of 1.66 mg/ml, as revealed by UV spectroscopy. To verify the specificity of the anti-PLP-J antibody, 293FT cells were transfected with PLP-J-expressing lentivirus or control lentivirus and analyzed via western blotting using the polyclonal anti-PLP-J antibody. The results demonstrated that 293FT cells transfected with the PLP-J lentivirus produced a specific band with the expected size, which was absent in 293FT cells transfected with control virus, indicating that the prepared anti-PLP-J antibody specifically recognized its antigen (Fig. 1) and was suitable for subsequent experiments.

The expression of PLP-J was first analyzed by RT-PCR, using RNA extracted from the testes of the mice. A single amplification product of 163 bp was sequenced, which matched perfectly with the PLP-J nucleotide sequence (data not shown), indicating the presence of PLP-J mRNA in the testes.

In situ hybridization was used to elucidate the spatial distribution of PLP-J in the mouse testis. Positive signals (brownish-yellow) were detected in the interstitial tissues of the testis, but not in Sertoli and sperm cells (Fig. 2A). No signal was observed using the sense cRNA PLP-J probe (Fig. 2B).

The localization of PLP-J in the mouse testis was further studied by immunofluorescence using sections prepared from testes of adult mice. PLP-J and 3β-HSD1 were colocalized in the cytoplasm of Leydig cells (Fig. 2C–E). As 3β-HSD1 is known to be expressed in Leydig cells, their colocalization indicated that PLP-J was also present in the Leydig cells of testis interstitial areas. PLP-J immunofluorescence of TM3 Leydig cells further revealed that this protein was localized within the cytoplasm of Leydig cells (Fig. 2F).

Using RT-PCR and western blotting, PLP-J expression levels in the testes of male mice aged 18 days, 4 months, and 16 months were evaluated (Fig. 3). The RT-PCR results indicated that PLP-J was expressed at all the time points that were examined. PLP-J expression levels were significantly higher in testes of 4-month-old mice compared with levels in 18-day-old mice. In 16-month-old mice, PLP-J expression levels were lower than those observed in 4-month-old mice, but still significantly higher than levels observed in 18-day-old mice (Fig. 3A). Western blotting was then employed to further clarify the developmental changes in PLP-J protein expression using the polyclonal anti-PLP-J antibody. Similar to the observed mRNA expression pattern, expression levels of PLP-J protein were age-dependent (Fig. 3B and C).

To investigate the function of PLP-J in TM3 cells, its expression in these cells was examined and it was discovered that the PLP-J expression levels were relatively low low (data not shown).. Thus, to accommodate the functional study, its expression was upregulated. A PLP-J-expressing lentivirus was concentrated via centrifugation to reach a titer of 1×10⁹ TU/ml, which was able to significantly boost PLP-J expression in TM3 cells (P<0.05; Fig. 4), further corroborating the specificity of the antibody.

The basal testosterone secretion level in cultured TM3 Leydig cells was not significantly different between TM3, TM3-N, and TM3-P cells (P>0.05; Fig. 5). This result implies that PLP-J is not directly involved in testosterone production in vitro.

The flow cytometry results demonstrated that PLP-J overexpression did not significantly affect cell cycle progression (Fig. 6A) or apoptosis (Fig. 6B).