Construction and evaluation of the novel DNA vaccine harboring the inhibin α (1-32) and the RF-amide related peptide-3 genes for improving fertility in mice.

To further improve fertility of animals, a novel gene RFRP-3 (RF-amide related peptide-3, RFRP-3) was used to construct DNA vaccines with INH α (1-32) (inhibin, INH) fragment for the first time. The aim of this study was to evaluate the effects of novel DNA vaccines on fertility in mice. Synthesized SINH and SRFRP (INH and RFRP genes were separately ligated to the C-terminus of the small envelope protein of the hepatitis B virus (HBV-S) gene) fragments were inserted into multiple cloning site of pIRES vector to develop p-SINH/SRFRP. The synthesized tissue plasminogen activator (TPA) signal sequence was then inserted into the p-SINH/SRFRP to construct p-TPA-SINH/TPA-SFRFP. Meanwhile, p-SINH was prepared and considered as positive control. Forty Kunming mice were equally divided into four groups and respectively immunized by electroporation with p-SINH, p-SINH/SRFRP and p-TPA-SINH/TPA-SRFRP vaccine (three times at 2 weeks interval) and saline as control. Results showed that the average antibodies (P/N value) of anti-INH and anti-RFRP in mice inoculated with p-TPA-SINH/TPA-SFRFP were significantly higher (P<0.05) than those inoculated with p-SINH/SRFRP and the positive rates were 100% (anti-INH) and 90% (anti-RFRP) respectively, at 2 weeks after the third immunization. Litter size of mice immunized with the three recombinant plasmids was higher (P<0.05) than that of the control, and litter size of mice immunized with p-TPA-SINH/TPA-SRFRP significantly increased (P<0.05) compared with p-SINH. These results suggested that the p-TPA-SINH/TPA-SRFRP harboring INH and RFRP genes was successfully constructed and had good immunogenicity, and might effectively increase litter size.

Introduction

The release of inhibin inhibits the secretions of gonadotropin, follicular development and spermatogenesis. Immunoneutralization of endogenous inhibin is considered to be an effective method for induction of superovulation [13, 16, 25, 41]. DNA immunization usually leads to in vivo expressions of the target antigen by a plasmid DNA [17]. This process is reported to be safe and easier on large-scale production and storage compared with the use of purified proteins [28]. The small envelope protein of the hepatitis B virus (HBV-S) is used as a carrier molecule for DNA vaccine, which are regarded as safe and can induce high immune responses [23, 39]. Therefore, in our previous studies, the INH-α (1–32) fragment was inserted into the HBV-S gene to construct two DNA vaccines (pcIS and pcISI), which effectively induced immune responses [10, 42]. However, the effect of these vaccines on improving fertility, particularly in large animals, was inadequate. The limitation could be due to other key factors that inhibit gonadotropin secretion and follicular development, which may result in low fertility.

A novel hypothalamic neuropeptide was found in quail and termed gonadotropin-inhibitory hormone (GnIH) for inhibiting gonadotropin release from cultured quail anterior pituitary gland [33]. The GnIH precursor is composed of 173 amino acid residues and cleaved into three mature peptides, namely, GnIH,GnIH related peptide-1 and GnIH related peptide-2, which possess an LPXRF amide sequence at their C-termini. Subsequently its analog was found in mammals, and named as RF-amide related peptide (RFRP) possessing LPXRF amide sequence at the C-terminus, and most mammalian RFRP genes encode two peptides, namely, RFRP-1 and RFRP-3 [7, 37]. GnIH and RFRP-3 are the most effective in acting in the hypothalamus-pituitary-gonad axis. In addition to the mRNA expression of GnIH (RFRP-3) in the hypothalamus, its expression was also found in other organs such as, ovary, testis, uterus, kidney, eye, and adrenal gland [19, 26, 30, 44]. GnIH (RFRP-3) plays major physiological roles by binding to its specific receptor, G-protein-coupled receptor147 (GPR147), which is also known as Npffr1 or OT7T022 [4, 12]. GnIH could inhibit the release of LH and FSH from the pituitary glands of chicken and quail [5, 38]. Intravenous administration of RFRP-3 decreased the level of peripheral blood gonadotropin in gonadectomized male rats and inhibited testicular steroidogenesis and spermatogenesis in adult mice [1, 27]. In sheep, intravenous injection of RFRP-3 reduced LH pulse amplitude and suppressed LH and FSH secretion [8]. In addition, it has also been shown that GnIH suppressed follicular development and steroidogenesis in chicken [2, 22]. These findings suggest that GnIH may direct or indirect inhibit follicular development and ovulation. We infer that if innate INH and GnIH (RFRP-3) are simultaneously neutralized through specific immunization in animals, then it might further improve litter size of animals.

Therefore, we constructed a novel plasmid DNA vaccine p-SINH/SRFRP carrying both RFRP-3 and INH gene by using a commercial mammalian expression vector (pIRES), which contains internal ribosomal entry sites (IRES) and allows the expression of two genes of interest from the same bicistronic mRNA transcript. Because TPA could enhance immunogenicity of antigen and elicit stronger humoral immune responses when it was introduced into DNA vaccine, we constructed another novel DNA vaccine, p-TPA-SINH/TPA-SRFRP fused with TPA, and p-SINH for positive control. All mice were grouped and immunized with p-TPA-SINH/TPA-SRFRP, p-SINH/SRFRP, p-SINH vectors and saline by electroporation. The immunogenicity of p-TPA-SINH/TPA-SRFRP, p-SINH/SRFRP and p-SINH was investigated, and the effects of immunization with these vaccines on the fertility of mice were also evaluated.

Materials and Methods

Construction of recombinant plasmids

The eukaryotic expression vector pIRES was preserved in our laboratory. The fragment of SINH (swine INH-α (1–32) NM_214189) was ligated to the C-terminus of HBV-S gene) was synthesized by Generay Biotech Co., Ltd., which separately contains NheI and EcoRI in its ends. Likewise, the fragment of SRFRP (bovine RFRP-3 (BD317666.1) was ligated to the C-terminus of the HBV-S) was synthesized, which separately contains SalI and NotI in its ends. In addition, TPA signal sequences (E02360.1) containing NheI and XmaI/SalI also were synthesized. The fragments of SINH and SRFRP were separately sub-cloned into pIRES vector using NheI/EcoRI and SalI/NotI restriction sites and were named p-SINH and p-SINH/SRFRP, respectively (Fig.1A). Next, two TPA fragments were separately inserted into p-SINH/SRFRP by using NheI and XmaI/SalI restriction enzymes and the plasmid was named p-TPA-SINH/TPA-SRFRP (Fig. 1B).

Fig. 1.

Construction maps of the recombinant plasmid p-SINH/SRFRP and p-TPA-SINH/TPA-SRFRP. (A) p-SINH/SRFRP: the SINH gene encoding HBV-S and INH-α (1–32) fragment, and SRFRP gene encoding HBV-S and RFRP-3 gene were separately inserted into the pIRES vector by using different restriction sites. (B) p-TPA-SINH/TPA-SRFRP: TPA signal sequences were inserted into the N-terminal of SINH and SRFRP genes.

Enzyme digestion and sequencing of recombinant plasmids

A single clone of p-TPA-SINH/TPA-SRFRP, p-SINH/SRFRP and p-SINH plasmids was selected and inoculated into the liquid broth medium with ampicillin. After 12 h, the plasmids were extracted through TIANprep Rapid Mini Plasmid Kit, identified by restriction analysis and sequenced by Takara Biotechnology Co., Ltd.

Proteins expression of recombinant plasmids in HeLa cells

Three recombinant plasmids (p-SINH, p-SINH/SRFRP and p-TPA-SINH/TPA-SRFRP) were transfected into HeLa cells for 48 h. The cells were then washed with cold PBS twice and lysed in RIPA buffer (Beyotime, Nantong, China) containing protease inhibitor cocktail and PMSF (Sigma, ST. Louis, USA). After incubation for 5 min on ice, cell suspension was collected in EP tubes, placed on ice for 20 min, and centrifuged at 13,000 × g for 15 min. The supernatant was carefully transferred to a new EP tube. Total protein concentration was measured using a BCA kit (Beyotime, Nantong, China). The protein samples were separated on 12% polyacrylamide gel and transferred to PVDF membranes (Millipore, MA, USA). The membranes were blocked in TBST (10 mM Tris (pH7.5), 150 mM NaCl; and 0.05% Tween-20) supplemented with 5% skimmed milk (Sigma-Aldrich, USA) and incubated overnight at 4°C with primary antibodies (anti-inhibin-α 1:200, abDSerotec, USA , MCA951S; anti-RFRP 1:500, Santa Cruz, USA , sc-32377, and anti-actin, 1:1,000, Santa Cruz, USA , sc-1616). After incubation, the membranes were washed three times with TBST and then incubated with second antibody (1:3,000, HRP labeled goat anti-mouse, Abbkine, USA, A21010) for 1 h at room temperature. After washing with TBST, the membranes were developed on an ECL Western blot detection system (Beyotime, Nantong, China), and then exposed to X-ray film for visualization of the protein bands.

DNA vaccination

Forty Female Kunming mice (6 weeks old) were purchased from the Hubei Center for Disease Control and Prevention (Wuhan, China). All experiments involving mice were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mice were randomly divided into four groups (10 mice per group). Each group was respectively injected with 20 µg of p-TPA-SINH/TPA-SRFRP (dissolved in 100 µl of 0.9% saline; group P), 20 µg of p-SINH/SRFRP (group S), 20 µg of p-SINH (group T, and positive control), and 100 µl of 0.9% saline (group C, and control) in the quadricep muscles. After that, two needle electrodes (each spaced by approximately 5 mm) were inserted immediately into the muscle at the site of injection, and electroporation treatment was performed under specific parameters (36 V, with 50 ms duration of electrical pulse duration for six consecutive times at1 s interval). Mice were immunized thrice (similar to the primary immunization), with an interval of 2 weeks. Blood samples were collected from the tail vein on days 0, 14, 28 and 42. Subsequently, serum was separated after clotting and stored at −80°C until further analysis.

Detection of antibodies against INH and RFRP

Specific IgG antibodies were detected using indirect ELISA with synthesized INH-α (1–32) and RFRP-3 antigens (Apeptide Co., Shanghai, China) as standard antigen. Briefly, 96-well ELISA plates (Costar-3590, USA) were coated with 100 ng/100 µl antigen and stored at 4°C overnight. The following day, the wells were washed three times with PBS supplemented with 0.1% Tween-20 (PBST) and blocked with 5% (w/v) skimmed milk in PBST for 1 h at 37°C (250 µl/ well). Subsequently, 100 µl serum samples (diluted in 1:100 dilutions with PBST) were added into the wells and incubated for 1 h at 37°C. After washing five times with PBST, antibodies were assayed by adding a second antibody (HRP-labeled goat anti-mouse IgG antibody, Abbkine, USA), which was diluted in PBST (1/5,000), and then incubated for 1 h at 37°C. After that, each well was saturated with tetramethylbenzidine [18] and placed in an incubator at 37°C for 20 min. After the reaction was terminated with 2 M H2SO4 (50 µl/well), optical density values was determined at 450 nm by using a microplate reader (Bio-Rad, Imark680, USA). Each sample was tested in triplicate. ELISA results were analyzed with P/N ratios, where P and N denote the OD values of the tested and negative control samples, respectively. P/N ratios higher than 2.0 and OD value over 0.2 were considered positive [10, 43].

Evaluation of effects on fertility of mice after immunization

After final blood collection, all mice were housed in different cages (two mice per cage). Male mice were introduced in each cage until female mice became pregnant. Litter size of three successive generations were respectively recorded.

Statistical analysis

Statistical analysis was performed using SPSS (Version 19.0) software. All data were expressed as mean ± SD and differences among groups were determined with ANOVA and least significant difference test. P values <0.05 were considered significant.

Results

Construction and identification of recombinant plasmids

All recombinant plasmids including p-SINH, p-SINH/SRFRP and p-TPA-SINH/TPA-SRFRP were separately identified by double digestion with NheI and EcoRI endonucleases. The target fragment SINH (789 bp) was confirmed on agarose gel (Fig. 2A). The p-SINH/SRFRP and p-TPA-SINH/TPA-SRFRP plasmids were identified separately by double digestion with SalI/NotI and XmaI/NotI endonucleases. The target fragments (778 bp for SRFRP and 847 bp for TPA-SRFRP) were separately confirmed on agarose gel (Figs. 2B and C). Sequence analysis further showed the successful construction of the three recombinant plasmids.

Fig. 2.

Identification of plasmids by restriction endonuclease digestion. (A) The p-SINH (Lane 1), p-SINH/SRFRP (Lane 2) and p-TPA-SINH/TPA-SRFRP (Lane 3) were separately double digested with NheI and EcoRI. M1:DL7,000 bp DNA marker. M2:DL1,000 bp DNA marker. The target band was of 789 bp. (B) Lane1& 2: p-SINH/SRFRP was double digested with SalI and NotI, and the target band was of 778 bp. M1:DL7,000 bp DNA marker. M2:DL1,000 bp DNA marker. (C) Lane1&2: p-TPA-SINH/TPA-SRFRP was double digested with XmaI and NotI, and the target band was of 847 bp. M3: DNA marker IV. M2: DL 1,000 bp DNA marker.

Assay of fusion protein

The protein expression of SINH and SRFRP was analyzed through western blot. The bands of SINH (about 29 kDa) and SRFRP (about 29 kDa) proteins were separately observed using their specific antibodies (Fig.3). The results showed that these proteins were expressed in HeLa cells.

Fig. 3.

The fusion proteins of SINH and SRFRP were expressed in HeLa cells. Lanes 1: pIRES; Lanes2: p-SINH; Lanes 3: p-TPA-SINH/TPA-SRFRP; and Lanes 4: p-SINH/SRFRP.

Effect of INH immunization on anti-INH level in mouse sera

After immunization with respective vaccines, the level of anti-INH antibodies increased with each immunization in all groups except in the control. Anti-INH antibody levels peaked at 2 weeks after the third immunization. After the primary and second immunizations, mice immunized with the three vaccines (p-SINH, p-SINH/SRFRP and p-TPA-SINH/TPA-SRFRP) developed high levels of antibody. No significant difference was found among groups T, S, and P, but group P developed significantly higher antibody level than the two other groups 2 weeks after of the third immunization (P<0.05) (Table 1). In addition, the percentage of positive-immunized mice for INH was higher in group P (60%) than that in groups T and S (both 40%), 2 weeks after immunization and then peaked after the third immunization (Table 1).

Table 1.

The P/N value of antibody against INH and ratio of mice with positive antibody after immunization of p-SINH (T), p-SINH/SRFRP (S), p-TPA-SINH/TPA-SRFRP (P), Saline (C)

Group	2 weeks			4 weeks			6 weeks
	P/N value	(%) Ratio of positive antibody		P/N value	(%) Ratio of positive antibody		P/N value	(%) Ratio of positive antibody
T	2.114 ± 0.598a	40 (4/10)		2.301 ± 0.591a	50 (5/10)		2.764 ± 0.869b	70 (7/10)
S	1.943 ± 0.487a	40 (4/10)		2.333 ± 0.667a	50 (5/10)		2.679 ± 0.949b	80 (8/10)
P	2.294 ± 0.431a	60 (6/10)		2.91 ± 1.132a	70 (7/10)		3.578 ± 0.798a	100 (10/10)
C	0.987 ± 0.169c	0 (0/10)		1.1 ± 0.301c	0 (0/10)		1.174 ± 0.379c	0 (0/10)

The same vertical column with different letters indicate significant difference (P<0.05) between groups. 2 weeks=2 weeks after primary immunization ; 4 weeks=4 weeks after primary immunization or 2 weeks of the second immunization; 6 weeks=6 weeks after the primary immunization or 2 weeks after the third immunization. P/N: mean ± standard deviation.

Effect of RFRP immunization on the anti-RFRP level in mouse sera

The average level of anti-RFRP antibodies in group P and S reached at peak (Table 2), and the average antibodies level of mice in Group P was significantly higher than that in group S (P<0.05) at 6 weeks after the primary immunization (or 2 weeks after the third immunization). The percentages of positive immunized mice in groups P and S were 40% and 30%, respectively, 2 weeks after the primary immunization. The percentage of positive anti-RFRP mice gradually increased with each immunization in groups P and S (Table 2).

Table 2.

The P/N value of antibody against RFRP and ratio of mice with positive antibody after immunization of p-SINH/SRFRP (S), p-TPA-SINH/TPA-SRFRP (P), Saline (C)

Group	2 weeks			4 weeks			6 weeks
	P/N value	(%) Ratio of positive antibody		P/N value	(%) Ratio of positive antibody		P/N value	(%) Ratio of positive antibody
S	1.914 ± 0.345a	30 (3/10)		1.908 ± 0.423a	40 (4/10)		2.16 ± 0.372b	60 (6/10)
P	2.12 ± 0.676a	40 (4/10)		2.167 ± 0.447a	60 (6/10)		2.628 ± 0.68a	90 (9/10)
C	0.987 ± 0.169c	0 (0/10)		1.1 ± 0.301c	0 (0/10)		1.17 ± 0.379c	0 (0/10)

The same vertical column with different letters indicate significant difference (P<0.05) between groups. 2 weeks=2 weeks after primary immunization ; 4 weeks=4 weeks after primary immunization or 2 weeks of the second immunization; 6 weeks=6 weeks after the primary immunization or 2 weeks after the third immunization. P/N: mean ± standard deviation.

Effect of INH and RFRP immunization on litter size of mice

The mean litter size of three successive generations in all immunized groups was significantly higher (P<0.05) than that of control group (Table 3). Among the vaccinated groups, the p-SINH/SRFRP vaccine caused a slight increase in the mean litter size of mice compared with that of the p-SINH in three successive generations. In addition, the mean litter size in group P evidently increased (P<0.05) compared with that in group T during the entire breeding period. No significant difference was found in litter size of mice between groups P and S (Table 3).

Table 3.

Comparison of litter size of three successive generations after mice immunized with p-SINH (Group T), p-SINH/SRFRP (Group S), p-TPA-SINH/TPA-SRFRP (Group P) and saline (Group C)

Groups	The LS of the first generation	The LS of the second generation	The LS of the third generation
T	12.86 ± 1.86b	13.14 ± 1.67b	13.71 ± 1.31b
S	14.00 ± 2.00ab	14.29 ± 1.70ab	15.14 ± 1.57ab
P	14.71 ± 1.58a	15.57 ± 1.99a	16.00 ± 1.83a
C	10.86 ± 1.68c	11.00 ± 2.00c	11.80 ± 1.57c

The same vertical column with completely different letters indicate significant difference (P<0.05) between groups, and P>0.05 with same little letters. Data are presented as mean ± standard deviation.

Discussion

Previous studies showed that INH could inhibit follicular development and immunization by DNA vaccine encoding inhibin α (1–32) can increase the quantity of large follicles and enhance litter size of animal slightly [10, 42]. In recent years, numerous studies have investigated GnIH (RFRP-3), which exhibits physiological function similar to that of INH and inhibit gonadotropin secretion, thereby affect the fertility of animals [8, 27]. But so far it has not been reported that the GnIH (RFRP) gene was used to construct a vaccine. In this study, we first constructed double expression DNA vaccines, namely, p-SINH/SRFRP, and p-TPA-SINH/TPA-SRFRP, which could produce better immunogenicity after immunization. The levels of INH and RFRP antibodies in sera were gradually raised with the increase in the number of immunization. The highest positive response rates of immunized mice were obtained with the p-TPA-SINH/TPA-SRFRP vaccine (100% as anti-INH and 90% as anti-RFRP, respectively) 2 weeks after the third immunization. These results have shown that fused protein (SINH, SRFRP) can elicit antibodies more efficiently, which could neutralize endogenous INH and GnIH (RFRP-3), respectively, and may promote follicular development. Interestingly, the positive response rate of mice (whether against INH or RFRP) was relatively higher compared with that reported in a study, in which pcISI vaccine harboring two copies of INH α (1–32) induced positive immunization in 22.2% of rat population, 2 weeks after the primary immunization [42]. This discrepancy may be related to the different inoculation methods employed; the previous study used intramuscular injection, whereas the present study utilized electroporation. Electroporation opens transient “pores” in the cell membrane, which allow macromolecules, such as DNA, to enter into the cytoplasm; the process is followed by the closure of the pores and retention of DNA inside the nucleus [40]. Electroporation can effectively increase the efficiency of gene expression and immunogenicity of DNA vaccines, resulting in strong antibody responses in rhesus macaques and human [3, 45], as confirmed by the current results. Furthermore, in this study, the antibodies level of anti-INH and anti-RFRP in mice immunized with p-TPA-SINH/TPA-SRFRP were significantly higher than mice immunized with p-SINH/SRFRP at 2 weeks after the third immunization. The phenomenon is probably due to TPA addition into the recombinant plasmid. Previous studies found that DNA vaccine fused TPA can induce significant high levels of IgG antibodies [9, 14, 20, 21].

INH immunization can improve follicular development and litter size of animals [11, 24, 29]. Consistent with these results, in current study, litter size of mice immunized with p-SINH was significantly higher than that of control group. In addition, some studies showed that intravenous injection of GnIH (RFRP-3) reduced the release of FSH and LH [15]. In another experiment, mice injected subcutaneously with GnIH (2 µg/day) daily for 8 days exhibited a decrease in number of follicles and corpora lutea [31]. In the present study, the mean litter size of mice inoculated with p-TPA-SINH/TPA-SRFRP expressing INH and RFRP-3 was significantly higher than that of mice with inoculated p-SINH expressing solely INH in the whole breeding. It may be due to the addition of the RFRP-3, then resulted in increase of little size after immunization with the p-TPA-SINH/TPA-SRFRP. Concurrently, it may be also due to the result of the addition of TPA, because the addition of TPA increased antibodies against both INH and RFRP-3 (as mentioned above it). In addition, the DNA vaccine encoding INH and RFRP-3 genes also exhibited certain potency in increasing litter size of mice, because the p-SINH/SRFRP induced slight increase in the litter size of mice compared with that of p-SINH during entire breeding period. Therefore, we infer that the neutralization of endogenous RFRP may promote follicular development and ovulation by either increasing the release of gonadotropins through direct action on the pituitary gland or on the gonads in a paracrine manner [6, 32, 34,35,36].

In conclusion, novel DNA vaccine p-TPA-SINH/TPA-SRFRP expressing INH-α (1–32) and RFRP-3 gene were successfully constructed, exhibited better immunogenicity, and increased litter size of mice. Further studies will be required to verify whether addition of RFRP-3 exert a significant effect in improving fertility of animals immunized with the novel p-TPA-SINH/TPA-SRFRP vaccine.

Conflict of interest

The authors have no conflict of interest regarding any matter.