Recombinant HBsAg-S and RFRP-3 DNA vaccine promotes reproduction hormone secretion in sheep.

RF-amide related peptides (RFRP) have been proposed as critical regulators of gonadotropin secretion in mammals. This study was designed to construct a DNA vaccine and investigate the effect of vaccine encoding RFRP-3 on reproduction physiology in ewe. A recombinant vaccine was constructed using two copies of the RFRP-3 gene and HBsAg-S that generate a fusion protein to induce an immunology response. Results showed this recombinant vaccine could produce a significant antibody titer in the treated animals (P < 0.05). The specific RFRP-3 antibody response induced by the vaccine was detected at week 2 with a peak at week 6 after the initial immunization. Furthermore, we found that ewes inoculated with pVAX-tPA-HBsAg-S-2RFRP-asd vaccine significantly raised the concentration of GnRH, LH and E2 in serum compared to the control group. LH and E2 concentration in the treated ewes (Group T) was significantly higher than that in control ewes (Group C) at weeks 10, 12 and 14 after the initial immunization, respectively (P < 0.05). Therefore, RFRP-3 can be used as a target for DNA immunization to promote reproductive hormone secretion in ewes and RFRP-3 gene immunization might be a candidate tool to regulate mammal reproduction.

Multiple-Pathway Synergy Alters Steroidogenesis and Spermatogenesis in Response to an Immunocastration Vaccine in Goat.

BACKGROUND: Animal reproduction performance is crucial in husbandry. Immunocastrated animals serve as an ideal animal model for studying testicular function. During androgen suppression, the testis undergoes dramatic developmental and structural changes, including the inhibition of hormone secretion and spermatogenesis. METHODS: To characterize this process, we investigated the effects of castration using a recombinant B2L and KISS1 DNA vaccine, and then identified functional genes in the testes of Yiling goats using RNA-seq and WGS. The experimental animals were divided into three groups: the PVAX-asd group (control), PBK-asd-immunized group, and surgically castrated group. RESULTS: The results demonstrated that the administration of the recombinant PBK-asd vaccine in goats elicited a significant antibody response, and reduced serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH), resulting in smaller scrotal circumferences and decreased sexual desire compared to the control group. In addition, RNA transcriptome sequencing (RNA-seq) analysis of the testes revealed that the biological processes after immunocastration mainly focused on the regulation of cell matrix adhesion, histone acetylation, negative regulation of developmental processes, apoptosis, and activation of the complement system and the thrombin cascade reaction system. Then, we integrated the whole-genome sequencing and testis transcriptome, and identified several candidate genes (FGF9, FST, KIT, TH, TCP1, PLEKHA1, TMEM119, ESR1, TIPARP, LEP) that influence steroidogenesis secretion and spermatogenesis. CONCLUSIONS: Multiple pathways and polygenic co-expression participate in the response to castration vaccines, altering hormone secretion and spermatogenesis. Taken together, our atlas of the immunocastration goat testis provides multiple insights into the developmental changes and key factors accompanying androgen suppression, and thus may contribute to understanding the genetic mechanism of testis function. Joint analysis of whole genome sequencing and RNA-seq enables reliable screening of candidate genes, benefiting future genome-assisted breeding of goats.

New trends in immunocastration and its potential to improve animal welfare: a mini review.

Castration is frequently used to reduce aggressive behavior and improve the meat quality of animals. Traditionally, surgical and mechanical castration are used to sterilize the animals, but these approaches are associated with a high level of pain, stress, long recovery periods, and post-operative infections. Immunocastration is a new animal-friendly, painless alternative castration technique that is used to prevent undesired sexual behavior, reduce aggressive behavior, prevent unwanted pregnancy, control wildlife populations and wandering species, enhance growth performance, improve meat quality, and treat various sex hormone-dependent disorders. The mechanism of immunocastration includes the immunological block of the hypothalamic-pituitary-gonadal axis (HPG axis) which inhibits gonadotropin secretions, causes atrophy of gonadal tissues, and inhibits gametogenesis, resulting in infertility in both female and male mammals. By the mid-1990s, various immunocastration vaccines have been tested in different animal models to achieve successful castration effects. Recently, genetic immunocastration especially DNA vaccine has gained increasing attention due to its safety, being animal-friendly, and being easy to use. This review aims to evaluate the potential of traditional castration methods, as well as the current status of immunocastration vaccines, their effects, and future prospective.

Immunocastration with gene vaccine (KISS1) induces a cell-mediated immune response in ram testis: A transcriptome evaluation.

Immunocastration vaccines achieve their effects through neutralization of the endogenous hormone by the humoral antibody produced against the immunized genes, but there is little information regarding cell-mediated immune response on the gonadal function of the immunized model is available. In this study, we used ram as a model animal to identify the cellular immune response in testicular tissues of rams immunized with intranasal KISS1 gene vaccine. The immune castration model was evaluated by sexual behaviours, spermatogenesis and serum hormone profiles after the KISS1 gene immunization. Transcriptome analysis of testicular tissues was carried out to identify the expressions of protein-coding genes involved in cellular immunity. The results showed that we successfully constructed the KISS1 immune castration ram model, in which testicular growth and development, testosterone and kisspeptin-54 levels, and sexual function were suppressed in immunized rams (p < .05). Using Hiseq™ 2000 high sequencing for ram testicular, we identified 21 differentially expressed genes (DEGs) related to cellular immunity, of which, 14 genes were upregulated and seven genes were downregulated in the testis of the immunized group (p < .05). The Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment showed that these differentially expressed genes were enriched in the antigen presentation process mediated by MHC class I and the cytotoxic pathway mediated by natural killer cells. It is concluded that KISS1 gene vaccine induced the cell-mediated immune response in testicular tissue to suppress reproductive activities in rams.

Expression of cell proliferation regulatory factors bricd5, tnfrsf21, cdk1 correlates with expression of clock gene cry1 in testes of Hu rams during puberty.

BACKGROUND: Cryptochrome 1 (cry1), the core regulator of the circadian clock, is essential for ontogeny and mammalian reproduction. Unlike in other tissues, the cry1 gene have noncircadian functions in spermatogenesis, which implies the unique role of cry1 gene in the development of testis. The role of cry1 during the puberty has not been described yet. This study aimed to explore the relationship between cry1 expression and spermatogenic cell numbers. METHODS AND RESULTS: We analyzed testicular tissues from Hu sheep aged 0-180 days by hematoxylin and eosin staining, measured cry1 and cell proliferation regulatory factors (bricd5, tnfrsf21, cdk1) expression by quantitative real-time PCR and characterized the transcription factor in the 5' flanking region of cry1 gene. The data revealed that the number of spermatocytes and early spermatocytes increased rapidly from 90 to 120 dpp (day postpartum). Correspondingly, there was a marked variation in the cry1 and cell proliferation related genes (bricd5, tnfrsf21, cdk1) mRNA expression in the testes from the age of 90 days to 180 days (p < 0.05). We also identified some transcription factors (tcfl5) related to cell proliferation. CONCLUSIONS: There is a significant causal relationship between the transcription level of cry1 gene in Hu sheep testes and the number of spermatogenic cells. It is speculated that cry1 gene may regulate the proliferation of spermatogenic cells by regulating the expression of cell proliferation related genes such as bricd5, tnfrsf21 and cdk1.

Effect of Kisspeptin-54 immunization on performance, carcass characteristics, meat quality and safety of Yiling goats.

This study aimed to evaluate the effects of kisspeptin-54 immunocastration vaccine on performance, carcass characteristics, meat quality, and safety of Yiling goats. Thirty buck goats were randomly assigned into three groups: PVAX-B2L-Kisspeptin-54-asd immunized (PBK-asd), control, and surgically castrated. PBK-asd immunization significantly stimulated serum anti-kisspeptin antibody production and reduced testosterone hormone compared with the control group (p < .05). Interestingly, PBK-asd plasmid did not integrate into the host genome and had no significant effect on growth hormone, body weight, and average daily gain (ADG). Conversely, surgical castration significantly reduced ADG and carcass weight compared to the control group. Furthermore, PBK-asd immunization did not affect carcass characteristics (dressing percentage, loin area, and fat thickness) and meat quality traits (pH, color, cooking loss, drip loss, and shearing force). These results indicate that the Kisspeptin-54 DNA vaccine is safe and has potential to be used as an alternative to surgical castration for goats without negatively affecting carcass and meat quality.

Local expressions and function of Kiss1/GPR54 in goats' testes.

Kisspeptin, encoded by the Kiss1 gene, and its receptor GPR54 have a central regulatory role in the male reproduction. However, their functions in peripheral tissues, such as testes, remain unclear. This study investigated the local expressions and function of Kiss1/GPR54 in goats' testes. The mRNA expression of Kiss1/GPR54 in pubertal goat Leydig cells was detected through reverse transcriptase PCR (RT-PCR), and its protein expression in Leydig cells or the testis was examined by immunohistochemistry and Western blot analysis. Isolated and cultured Leydig cells were treated with different concentration of kisspeptin (0, 1, 10 and 100 µM) and kisspeptin antagonist for 4, 24 or 72 h. The direct effect of kisspeptin on testosterone secretion and Kiss1/GPR54 mRNA expression was evaluated by ELISA and RT-PCR. Kiss1/GPR54 mRNA and protein were expressed in Leydig cells and spermatids, and GPR54 were expressed in Sertoli cells. Kisspeptin treatment significantly stimulated testosterone secretion in Leydig cells, with the highest levels found under 24 h of treatment with 10 µM kisspeptin. Treatment with kisspeptin + kisspeptin antagonist significantly reduced the kisspeptin-stimulated testosterone secretion in Leydig cells. Kisspeptin treatment significantly enhanced the expression of Kiss1/GPR54 mRNA in Leydig cells. These data suggest the local expressions of Kiss1/GPR54 in goats' testes and its autocrine role in Leydig cells, which is helpful in understanding the regulation role of kisspeptin/GPR54 system in other peripheral tissues.

Recombinant B2L and Kisspeptin-54 DNA Vaccine Induces Immunity Against Orf Virus and Inhibits Spermatogenesis In Rats.

Orf is a highly contagious zoonotic disease of small ruminants caused by Parapoxvirus. Kisspeptin, encoded by the KISS1 gene with its cognate receptor GPR-54 is recognized as an upstream orchestrator in the hypothalamic-pituitary-gonadal axis. This study was designed to construct a DNA vaccine that produces a fused peptide composed of a major immunodominant protein of the orf virus (B2L) and kisspeptin-54, a neuropeptide with recognized roles in mammalian reproductive biology. The administration of this recombinant vaccine is shown to produce a significant antibody and cell-mediated immune response directed against B2L compared to the control group (p < 0.05). Furthermore, we found that rats inoculated with PBK-asd vaccine up-regulated antigen-mediated splenocyte proliferation and significantly raised antigen-specific tumor necrosis factor-alpha (TNFα-), interferon-gamma (IFN-ϒ) and interleukin (IL-2) compared to the control group (p < 0.05). This recombinant vaccine also stimulated antibody responses to kisspeptin and decreased serum luteinizing hormone and testosterone levels. Moreover, the current recombinant vaccine caused testicular atrophy and arrested spermatogenesis. It is concluded that this recombinant B2L and Kisspeptin-54 vaccine could be a promising approach for construction of bivalent orf virus and immunocastration vaccine. Furthermore, we concluded that the orf virus envelope protein (B2L) could be used as an immunomodulator for kisspeptin-54 to produce a strong antibody response.

Kisspeptin recombinant oral vaccine: A master gene vaccine inhibiting the reproductive physiology and behavior of ram lambs.

The KISS1 gene product, kisspeptin, stimulates gonadotrophic steroid hormone (GNRH) neuronal signaling through the G-protein coupled receptor, kiss1r. Disturbance of this signaling pathway causes hypogonadotropic hypogonadism in mammals. As part of this cutting-edge research project, we analyzed the efficacy of an oral kisspeptin recombinant vaccine on the reproductive physiology and behavior of ram lambs. Ten 56-day old ram lambs were randomly divided into treatment and control groups to receive the experimental recombinant vaccines, C500/pKS-asd or C500/pVAX-asd (aspartate-ß semialdehyde dehydrogenase), respectively. The vaccines were orally administered at day 0, 28 and 56 and blood samples were taken and scrotal circumference data recorded at 14-day intervals (days 0, 14, 28, 42, 56, 70, and 84). At the end of the experimental period, day 98, sexual behaviors were assessed, scrotal circumferences were measured, and blood samples were collected. Testicular samples were also collected after the animals were sacrificed. Anti-kisspeptin antibody and testosterone serum levels were measured by indirect ELISA. Results demonstrated that the levels of anti-kisspeptin antibodies were significantly higher in the treatment group compared to controls (P<0.05, P<0.01 and P<0.001). However, serum testosterone levels were lower in the treatment group (P<0.01). Interestingly, vaccine administration contributed to a significant reduction (P<0.01) in sexual behavior propensity. These results suggest that the kisspeptin recombinant oral vaccine regulates and inhibits the reproductive physiology and behavior of ram lambs.

Immunization against Kisspeptin-54 perturb hypothalamic-pituitary-testicular signaling pathway in ram lambs.

Kisspeptin, a peptide product of KISS1 gene, recently identified as essential upstream gatekeeper in hypothalamic-pituitary-gonadal (HPG) axis. This study was designed to investigate the effect of immunization against kisspeptin-54 on hypothalamic-pituitary-testicular signaling pathway. A total of ten intact 56-days-old ram lambs were used and randomized into the treatment and control groups, which were, respectively immunized by kisspeptin-54 based vaccine and the empty plasmid via intramuscular route. We employed indirect enzyme-linked immunosorbent assay and quantitative real-time PCR to characterize the difference in serum kisspeptin, luteinizing hormone, testosterone hormone concentration and mRNA expression of reproductive-related genes in HPG axis across kisspeptin-54 immunized and control ram lambs. Serum kisspeptin, luteinizing hormone and testosterone concentration in the treatment group was lower (p < 0.05) than that of the control group. Compared with the control group, the mRNA expression of the hypothalamic androgen receptor (AR), KISS1, G protein-coupled receptor (GPR54) and gonadotropin-releasing hormone (GnRH) was altered in the immunized group (p < 0.05). Moreover, mRNA expression of pituitary luteinizing hormone beta (LHß), follicle stimulating hormone beta (FSHß), and GnRH receptor as well as, testicular LH receptor and FSH receptor, were remarkably lower (P < 0.05) in the treatment group. We concluded that immunization against kisspeptin-54 reduced serum kisspeptin levels thereby, the normal hypothalamic-pituitary-testicular signaling pathway disrupted. This data provides a great insight for the use of kisspeptin to regulate reproduction.

Potent effect of KISS1-54 DNA vaccine compared with KISS1-10 DNA vaccine in inhibiting the fertility of female rats.

BACKGROUND: Most studies on immunocastration currently focused on male animals. However, immunization of male animals does not completely inhibit sexual behavior and fertility. This study aimed to compare the immunocastration effect of KISS1 DNA vaccines encoding different lengths of kisspeptins in female rats for effective castration effects on both male and female rats. METHODS: Fifteen female rats were randomly divided into three groups. The rats in T1 group or T2 group was orally given respectively KISS1-54 or KISS1-10 DNA vaccines with fused tPA signal peptide, and the control group (Group C) was orally administered with empty vector vaccine, at a dose of 5 × 109 CFU/rat at weeks 0, 3 and 6 of the study. Blood samples were collected by retroorbital bleeding before primary immunization and at weeks 3 and 9 after primary immunization. RESULTS: Both KISS1-54 and KISS1-10 DNA vaccines induced the body's humoral immune response, and the anti-kisspeptin antibody titres in the T1 group were significantly higher than that in T2 and C groups (p < 0.05). The rats in T1 group has lower serum kisspeptin and estradiol levels than those in T2 and C groups and smaller litter size of rats than those in the control group after mating (p < 0.05). No significant difference was observed between T2 and C groups. The levels of KISS1 and GPR54 mRNA in the hypothalamus and ovaries of the T1 group were significantly lower than that in control group. However, the levels of KISS1 mRNA in the T2 group were significantly lower than that in the control group only in ovaries (p < 0.05). CONCLUSION: The oral KISS1-54 DNA vaccine with fused tPA signal peptide was more effective than that KISS1-10 DNA vaccine in suppressing fertility of female rats.

Reversibility and safety of KISS1 metastasis suppressor gene vaccine in immunocastration of ram lambs.

OBJECTIVE: The aim of this study was to investigate the reversibility and safety of KISS1 metastasis suppressor (KISS1) gene vaccine in immunocastration. METHODS: Six eight-week old ram lambs were randomly divided into vaccinated and control groups. The vaccine (1 mg/ram lamb) was injected at weeks 0, 3, and 6 of the study. Blood samples were collected from the jugular vein before primary immunization and at weeks 2, 4, 6, 10, 14, 22, and 30 after primary immunization. All ram lambs were slaughtered at 38 weeks of age, and samples were collected. RESULTS: The specific anti-KISS1 antibody titers in vaccinated animals were significantly higher and the serum testosterone level was significantly lower than those in the control groups from week 4 to 14 after primary immunization (p<0.05). No significant difference was observed at weeks 22 and 30 after the primary immunization. Similar results were also found for scrotal circumference, testicular weight, length, breadth, and spermatogenesis in seminiferous tubules in week 30 after primary immunization. KS (KISS1-hepatitis B surface antigen S) fusion fragment of KISS1 gene vaccine was not detected in host cell genomic DNA of 9 tissues of the vaccinated ram lambs by polymerase chain reaction. CONCLUSION: The effects of KISS1 gene vaccine in immunocastration were reversible and no integration events were recorded.

KISS1 can be used as a novel target for developing a DNA immunocastration vaccine in ram lambs.

KISS1 gene-encoding kisspeptins are critical for the onset of puberty and control of adult fertility. This study investigated whether KISS1 can be used as a novel target for immunocastration. Human KISS1 was fused with the HBsAg-S gene for constructing an antibiotic-free recombinant plasmid pKS-asd that coded for 31.168 kDa target fusion protein. Six male Hu sheep lambs were divided into two equal groups, treatment and control. The vaccine (1mg/ram lamb) prepared in saline solution was injected into lambs at weeks 0, 3 and 6 of the experiment, respectively. Vaccine efficacy was evaluated in terms of KISS1-specific IgG antibody response, serum testosterone levels, scrotal circumference, testicular weight, length and breadth, extent of testicular tissue damage, and sexual behaviour changes. The specific anti-KISS1 antibody titre in vaccinated animals was significantly higher than that in controls (p<0.05). In addition, vaccinated animals showed lower serum testosterone level, testicular weight and length and smaller scrotal circumference than those in controls (p<0.05). Spermatogenesis of seminiferous tubules in vaccinated animals was suppressed; sexual behaviours in vaccinated animals were significantly lower (p<0.05) than those in controls. In conclusion, the immunization against KISS1 in this DNA vaccine induced a strong antibody response and resulted in the suppression of gonadal function and sexual behaviour in animals, demonstrating that KISS1 can be used as a novel target for developing a DNA immunocastration vaccine.

Vasoactive intestinal peptide suppresses ovarian granulosa cell apoptosis in vitro by up-regulating Bcl-2 gene expression.

Vasoactive intestinal peptide (VIP) is an endogenous peptide showing a rich profile of biological activities. Within ovaries, VIP directly regulates the ovarian functions, including granulosa cells (GCs) development. In the present study, the effects of VIP on proliferation and apoptosis in goose granulosa cells were demonstrated and its underlying mechanism investigated. A strategy of RNAi-mediated "gene silencing" of Bcl-2 (RV-Bcl-2), over-expression of Bcl-2 (JLV-Bcl-2) synthesis, and exogenous VIP was used to treat goose GCs. The results showed the amounts of Bcl-2 protein were negatively correlated with apoptosis of goose GCs in all experimental groups. Compared with other control groups, apoptosis was decreased in goose GCs following treatment of 100 nM VIP, and the amount of Bcl-2 protein was increased (P < 0.05) increased. However, VIP failed to exert an effect on cell proliferation (P > 0.05). In conclusion, the exogenous VIP plays an important role in inhibiting apoptosis of goose GCs via inducing Bcl-2 gene expression.

Effects of downregulation of inhibin alpha gene expression on apoptosis and proliferation of goose granulosa cells.

Inhibin alpha is one of the candidate genes that control the ovulation in poultry. To study the genetic effects of inhibin alpha on apoptosis and proliferation of goose granulosa cells cultured in vitro, two RNA interference (RNAi) expression vectors, psiRNA-INH alpha 1 and psiRNA-INH alpha 2, were constructed to knock down inhibin alpha gene expression. After 48 h of transfection, the efficiency of these two RNAi expression vectors was examined by fluorescence microscopy. Meanwhile, inhibin protein expression levels, apoptosis indexes (AI) and proliferation indexes (PI) of granulosa cells were analyzed by flow cytometry. In addition, the supernatants were collected to assay the concentrations of estrogen (E2) and progesterone (P) by radioimmunoassay. The results showed that the expression level of inhibin alpha in the RNAi group were decreased 30%-40% than those in the control groups (P < 0.05) and the apoptosis indexes and proliferation indexes in the RNAi groups were significantly higher than those in the control groups (P < 0.05). However, the E2 concentrations in the RNAi groups were lower than those in the control groups (P < 0.05). These results indicate that inhibin alpha has antagonistic effect on granulosa cell apoptosis.