Recombinant Gonadotropin-releasing Hormone Research Articles

Intracellular production of recombinant GnRH1 in yeast, Pichia pastoris, and its potential as oral treatment to advance gonadal development in juvenile orange-spotted grouper, Epinephelus coioides

Late maturation and large size at maturity are significant challenges when administering hormonal treatments in captive broodstock. Here, we report the potential of yeast, Pichia pastoris, as a vehicle to orally deliver a recombinant gonadotropin-releasing hormone (rGnRH1) aimed at stimulating gonadal development in juvenile orange-spotted grouper, Epinephelus coioides. Two recombinant GnRH1 constructs were designed utilizing pPIC3.5, the yeast expression vector lacking a signal sequence, hence retaining the recombinant within the yeast. The first construct (rGnRH1_1xGAP) comprised of GnRH1 decapeptide and the GnRH-associated peptide (GAP) separated by a cleavage site. The second construct (rGnRH1_10x) consisted of ten GnRH1 decapeptides, each separated by a cleavage site. Expression of the two recombinant peptides was confirmed by mass spectrometry. ELISA for the GnRH analogue (GnRHa) was validated to determine the level of rGnRH1 from the yeast extracts. Parallelism between the serially diluted GnRHa and serially diluted extracts from recombinant yeast confirmed validity of the assay. A luciferase reporter assay showed stimulation of tilapia GnRH type 3 receptor by the yeast extract, suggesting biological activity in vitro. In a short term experiment, lyophilised yeast loaded in gelatine capsules and fed once to juvenile orange-spotted grouper resulted in a significantly higher plasma GnRH compared with the control. In a 4-week experiment, lyophilized yeast containing either rGnRH1_1xGAP or rGnRH1_10x was incorporated in fish pellets and fed daily at a dose of 1 μg rGnRH1/kg fish body weight/day. The mean gonadosomatic index and oocyte diameter did not vary between the treated and control fish. The mean plasma levels of FSH, LH and GnRH in the rGnRH1-fed and control fish did not also vary significantly, however there were individuals in the rGnRH1-fed groups that had increased level of the hormones. Together with histological evidence showing oocyte development in a proportion of fish fed with the recombinant, results of the present study point to a biological activity in vivo of the rGnRH1 in yeast. With further refinement, this technology has the potential to provide a non-invasive method for broodstock management of large, late-maturing species of fish and of small, delicate endangered species that require breeding in captivity.

The effects of recombinant GnRH with dopamine antagonist on reproduction performance, sex steroid levels, and stress response in female koi carp (Cyprinus carpio)

In this study, the effect of different doses of recombinant gonadotropin-releasing hormone (rGnRH) and ovaprim (OVP) was investigated on reproductive function, sex steroid levels, and stress response in female koi carp, Cyprinus carpio, females. For this purpose, female koi carps (980.66 ± 23.46 g) were divided into five experimental groups and injected with the following preparation procedure: 0.9% NaCl with 5 mg/kg body weight (BW) of domperidone (Dop) (Control, C), 0.5 ml/kg BW of ovaprim (OVP); 10 μg kg BW−1 of rGnRH with 5 mg/kg of Dop (rGn10), 25 μg/kg BW of rGnRH with 5 mg/kg of Dop (rGn25), and 50 μg/kg BW of rGnRH 5 with mg/kg of Dop (rGn50). To evaluate sex steroids and stress indicators in the females, blood samples were collected before injection and 6, 12, and 24 h post-injection. The results indicated that the females were significantly different in terms of the number of spawned females, with the highest numbers in OVP and rGn25 treatments and the lowest number in the control treatment. Significant differences were observed between hormone-treated fish at the latency period, and the highest latency was obtained in the rGn10 treatment compared to shorter latencies in the other treatments. Sex steroid levels 17β estradiol (E2), testosterone (T), and 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) were affected by the hormonal treatments. Levels of E2 decreased in rGnRH and OVP treatments at 6 and 12 h after the injection. T and DHP levels increased in rGnRH and OVP treatments at 6 and 12 h after the injection and then decreased at 24 h post-injection. Cortisol levels increased at 6 and 12 h post-injection and decreased 24 h after the injection. No changes were observed in lactate and glucose levels at 6 h after the injection, but its levels were significantly higher in hormone-injected treatments than in the control treatment at 12 h after the injection. rGnRH could induce spawning, and the highest spawning rate and efficiency were measured in the rGn25 treatment. According to the results, rGnRH may be suggested as an alternative to other hormones used for artificial propagation in koi carp.

Effects of a Recombinant Gonadotropin-Releasing Hormone Vaccine on Reproductive Function in Adult Male ICR Mice.

Gonadotropin-releasing hormone (GnRH) regulates the reproductive endocrine system in mammals. The GnRH immunocontraception vaccine can aid animal population control and management. We evaluated a recombinant GnRH fusion protein with the adjuvant MONTANIDE ISA 206 VG as a GnRH vaccine in adult male ICR mice by evaluating anti-GnRH antibodies; concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone; testis size and histomorphology; and semen quality. Response was assessed after intramuscular administration of the vaccine to mice in weeks 0, 4, and 8. The vaccine induced specific antibody response by week 5, with peak of antibody levels observed by week 13 and a declining level thereafter until the end of the study at week 24. Furthermore, it reduced serum FSH, LH, and testosterone concentrations. The vaccinated mice exhibited testicular atrophy and reduced sperm quality, concentration, morphology, and viability compared to control males. The outcomes of pairings of treated males with untreated females revealed reduced mating, pregnancy rates and number of litters compared to control pairings. Assessment of this GnRH vaccine in different species could assist its development for future applications.

Induction of immunocontraceptive effects in both male and female mice immunized with GnRH vaccine.

BackgroundGonadotropin‐releasing hormone (GnRH) plays a pivotal role in regulating the reproductive endocrine system.ObjectiveAn immunocontraception vaccine aimed at inhibiting the functions of GnRH is tested as a potential tool for controlling animal populations.MethodsWe developed a recombinant immunocontraceptive vaccine composed of GnRH‐I and GnRH‐II (GnRH I+II), which was conjugated with Salmonella typhimurium flagellin. Forty‐eight BALB/c mice aged 4 weeks were divided into four groups (each group had n = 12): non‐vaccinated male (NVM), non‐vaccinated female (NVF), vaccinated male (VM), and vaccinated female (VF). Mice in the vaccinated groups were vaccinated twice by intramuscular injection at 0 and 2 weeks with 300 μg of the recombinant GnRH protein complex per mouse. Mice in the non‐vaccinated groups were injected with saline and served as the unimmunized controls. Twenty‐four pairs of male and female mice were mated for 10–12 weeks after initial immunization in four groups: 6 NVF × 6 NVM, 6 VF × 6 NVM, 6 NVF × 6 VM, and 6 VF × 6 VM. Results: An increase (p < 0.001) in antibody titers in VM and VF mice was observed. The testosterone levels and the number of spermatocytes were lower (p < 0.001) in VM mice than those in the control mice. The progesterone levels and the number of corpora lutea were lower (p < 0.001) than those in the control mice. Mating results in both VM and VF mice confirmed a 60% reduction in pregnancy rates and offspring numbers.ConclusionsThe recombinant GnRH vaccine can be used for birth control in both male and female animals.

Is the use of recombinant cGnRH may be a future alternative to control the fish spawning? Let us go with the goldfish example.

The use of recombinant gonadotropin-releasing hormone (rGnRH) has very rarely been tested in fish to promote spawning. This study evaluated the impact of recombinant chicken gonadotropin-releasing hormone (rcGnRH) with metoclopramide on the release of sex steroids and final maturation induction in goldfish (Carassius auratus) broodstock. For this purpose, goldfish broodstock was divided into four groups and treated with 0.9% NaCl with 20mg/kg metoclopramide (Met) (C); 10μg/kg body weight (BW) rcGnRH with 20mg/kg metoclopramide (rcGn10); 15μg/kg BW rcGnRH with 20mg/kg metoclopramide (rcGn15); and 20μg/kg BW rcGnRH with 20mg/kg metoclopramide (rcGn20). The capability of the rcGnRH for eliciting biological response was tested in vivo by evaluating the changes of 17β estradiol (E2), testosterone (T), and 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) and the induced spawning. Blood samples were obtained at 0h, 12h, and 24h after injection. The rcGn10, rcGn15, and rcGn20 treatments induced lower E2 concentration, especially 24h post-injection. T levels were significantly higher in rcGn10, rcGn15, and rcGn20 treatments 12h post-injection than at 0h and then decreased at 24h post-injection. Furthermore, the rcGnRH tested significantly enhanced DHP secretion in rcGn10, rcGn15, and rcGn20 treatments 12h post-injection before a decline at 24h post-injection. No significant difference between the sampling times was found in the C treatment for the 3 sex steroids tested. The results also displayed that rcGnRH at 10-20µg/kg of body weight can trigger spawning with the highest speed and efficiency of spawning at 20µg/kg. The obtained results represent a possible strategy for enhancing the artificial reproduction and ovulation of broodstock fish by rGnRH and further support the use of recombinant hormones to promote reproduction in aquaculture.

Immunogenicity of a Recombinant Gonadotropin-releasing Hormone Associated to the B Subunit of Escherichia coli Heat-labile Enterotoxin Expressed in Pichia pastoris and Escherichia coli Platforms

HIGHLIGHTS P. pastoris and E. coli systems were able to express GnRH/LTB; Fusion of singles GnRH and LTB molecules are immunogenic in mice; GnRH vaccine induces histological changes on mice gonads; GnRH/LTB molecule is a promising antigen for an immunocontraceptive vaccine.

Spawning induction in Sterlet sturgeon (Acipenser ruthenus) with recombinant GnRH: Analysis of hormone profiles and spawning indices

In the present study, the biological function of recombinant gonadotropin-releasing hormone associate peptide (rGnRH) was tested for the induction of ovulation and spawning in Sterlet sturgeon (Acipenser ruthenus) broodstock. For this purpose, Sterlet sturgeon broodstock were divided into four treatments and treated with following preparing procedure: 0.9% NaCl (C); 2.5 μg kg body weight−1 (BW) LHRH-A2 (luteinizing hormone-releasing hormone-A2, L2.5); 2.5 μg kg BW−1 rGnRH (rGn2.5); 10 μg kg BW−1 rGnRH (rGn10). The capability of the rGnRH for eliciting biological response was studied in vivo by evaluating the changes of 17β estradiol (E2), progesterone (P) and testosterone (T) and by its ability for inducing final maturation. Blood samples were collected at the time of first injection, 10 h after the first injection and at ovulation time. The L2.5 or rGn10 treatments led to significantly lower E2 concentration 10 h after the first injection and at ovulation time compared to the baseline measures. E2 levels increased in the rGn2.5 treatment 10 h after the first injection and by ovulation time compared to the time of first injection. T levels were significantly increased in the L2.5 or rGn10 treatments 10 h after the first injection compared to the time of first injection and it further decreased at ovulation time. No significant differences were found in C and rGn2.5 treatments between the different sampling times. P secretion was significantly higher in the rGn10 or L2.5 treatments compared to C and rGn2.5 treatments 10 h after the first injection and at ovulation time. No significant difference was found in P secretion in the C and rGn2.5 treatments at different sampling times. The results showed that the rGnRH at the highest dose (10 μg kg BW−1) was more efficient than the other treatments in the induction of spawning and this dose caused P secretion and spawning in vivo. Our results present the possibility of using rGnRH in artificial reproduction of sturgeons and it can be introduced as a specific homologue to treat reproductive disorders in fish.

Design, production and purification of a novel recombinant gonadotropin-releasing hormone associated peptide as a spawning inducing agent for fish

GnRH is a neuropeptide known to regulate reproduction in vertebrates. The purpose of this study was to design and produce recombinant gonadotropin-releasing hormone associated peptide (rGnRH/GAP) as an alternative of the previous GnRHs and native extracted hormone from tissue, to induce final maturation in fish. Decapeptide as well as GAP area sequences were compared between GnRH1, GnRH2, and mGnRH from Acipenser sp and Huso huso, respectively. Considering the conserved amino acids and the replacement of un-stable amino acids with those that were more stable against proteolytic digestion as well as had a longer half-life, the sequence was designed. The sequences of decapeptide and GAP region were synthesized and then cloned on pET28a expression vector and transformed into expression host Escherichia coli BL21(DE3). The supernatant of cultured recombinant bacteria was used for purification using TALON Metal affinity resin. The purity of the GnRH/GAP was confirmed by single 8 kDa band on SDS-PAGE and Western blot. Bioinformatics studies were performed for evaluation of homology between GnRH protein sequences and prediction of 3D protein structure using Swiss Model. The result showed that the structure prediction of the recombinant GnRH decapeptide was relatively similar to decapeptide of GnRH2 from Beluga (Huso huso). The GAP structure was similar to GAP1 of Nile tilapia (Oreochromis niloticus) and sturgeon and GnRH2 of Chinese sturgeon (Acipenser sinensis). The mass analysis showed that the sequence was exactly the same as designated sequence. Biology activity of rGnRH/GAP was tested in mature goldfish (Carassius auratus) and results showed that rGnRH/GAP had a positive effect in final maturation. Indeed 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) was increased 17 h and 24 h after injection with rGnRH/GAP and spawning stemmed from that injection. These novel findings introduce the potential of utilizing rGnRH/GAP in aquaculture.

Immunization against recombinant GnRH-I alters ultrastructure of gonadotropin cell in an experimental boar model

BackgroundGonadotropin cell is the main responsible for the secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH), and immunocastration reduces the concentrations of serum FSH and LH. A few studies have reported the histological structure of gonadotropin cells obtained from immunocastration animals at the light microscopy level. However, the ultrastructure of gonadotropin cells remains largely unexplored. The aim of this study was to evaluate and to compare ultrastructure of gonadotropin cell in gonadally intact boars and immunologically castrated male animals.FindingsIn this study, serum and adenohypophysis tissue were collected from nine gonadally intact boars and nine male pigs treated with recombinant gonadotropin releasing hormone I (GnRH-I). Anti-GnRH-I antibodies in serum and the ultrastructure of gonadotropin cell in adenohypophysis were determined by enzymelinked immunosorbent assay and electron microscopy, respectively. The results demonstrated that active immunization against recombinant GnRH-I increased serum GnRH-I antibody levels (P<0.05). Ultramicroscopic analysis of gonadotropin cell revealed a decrease (P<0.05) in the number and size of the large granules and small granules in the recombinant GnRH-I immunized animals.ConclusionsWe conclude that immunization against recombinant GnRH-I induces severe atrophy of granules in gonadotropin cell of boars, possibly reflecting GnRH-I regulation of gonadotropin cell.

Androgênios em más respondedoras

Treatment of poor-responder patients to controlled ovarian stimulation is a challenge in reproductive technology. ObjectiveTo evaluate some synergistic effects in folliculogenesis by improving the ovarian sensitivity to FSH (Follicle Stimulating Hormone) and follicular response to gonadotrophins. MethodA literature review that evaluated the use of androgens in ovarian response of pretreatment of ovarian stimulation on low-responder IVF (In Vitro Fertilization) patients. ResultsThe most articles showed an increase on ovarian response in patients who used transdermic testosterone before ovarian stimulation. Those women achieved greater number of recovered oocites and more mature ones in women who used transdermal testosterone. In addition, it increased clinical pregnancy and live birth rates. Women who received DHEA (dehydroepiandrosterone), aromatase inhibitors, rLH (recombinat luteinizing hormone) or rhCG (recombinant gonadotropin releasing hormone) showed no significant differences in studied parameters. ConclusionPretreatment with transdermal testosterone seems to be beneficial to ovarian response on poor-responder patients. It has increased clinical pregnancy and live birth rates. There were no benefits with use of rLH, hCG, DHEA or aromatase inhibitors proved in those studies.

Stability of plasmid and expression of a recombinant gonadotropin-releasing hormone (GnRH) vaccine in Escherichia coli

In our previous studies, the recombinant gonadotropin-releasing hormone (GnRH) peptide was constructed into a T7 RNA polymerase-based expression system. The recombinant gene encoding GnRH3-hinge-MVP, which contained three repeated GnRH units, a fragment of hinge region (225-232/225'-232'), and a T cell epitope of measles virus protein, was cloned into Escherichia coli BL21 harboring pED-GnRH3. The high activity of T7 RNA polymerase could make the expression system very powerful for high-level expression of the recombinant protein. However, during the large-scale production of recombinant protein, the productivity of the fermentation process was directly affected by many factors, such as plasmid stability, protein production, and culture conditions. In this study, we studied the effects of various culture conditions on the plasmid stability and the target protein yield including selective pressure, the time of induction by lactose, and the number of successive cultures. The results indicate that the plasmid instability may be caused by a loss of plasmid rather than structural change. However, to go down to future generations, engineered bacteria have the stability of plasmid and protein yield to a large extent. The amount of the fusion protein was also up to 40% of the total cell protein after the 50th generation. These data would be useful for the industrial production of the recombinant GnRH vaccine.

Cloning, expression, and purification of a highly immunogenic recombinant gonadotropin-releasing hormone (GnRH) chimeric peptide

To design an anti-gonadotropin-releasing hormone (GnRH) vaccine capable of eliciting strong immunogenicity, a gene fragment encoding a chimeric peptide was constructed using polymerase chain reaction and ligated into a novel expression vector for recombinant expression in a T7 RNA polymerase-based expression system. The chimeric peptide called GnRH3–hinge–MVP contained three linear repeats of GnRH (GnRH3), a fragment of the human IgG1 hinge region, and a T-cell epitope of measles virus protein (MVP). The expression plasmid contained the GnRH3–hinge–MVP construct ligated to its fusion partner (AnsB-C) via an unique acid labile Asp–Pro linker. The recombinant fusion protein was expressed in an inclusion body in Escherichia coli under IPTG or lactose induction and the target peptide was easily purified using washing of urea and ethanol precipitation. The target chimeric peptide was isolated from the fusion partner following acid hydrolysis and purified using DEAE–Sephacel chromatography. The purified GnRH3–hinge–MVP was determined to be highly homogeneous by IEF analysis and the N-terminal sequencing. Further, immunization of female mice with the recombinant chimeric peptide resulted in generation of high-titer antibodies specific for GnRH. The results showed that GnRH3–hinge–MVP could be considered as a candidate anti-GnRH vaccine.

Use of recombinant gonadotropin-releasing hormone antigens for immunosterilization of beef heifers1

The objectives of this study were to evaluate the effects of immunization against recombinant GnRH fusion proteins and growth promotants on onset of puberty, feedlot performance, and carcass characteristics of beef heifers. Heifers were immunized against an ovalbumin fusion protein containing 7 GnRH peptides (oGnRH, n = 12), a thioredoxin fusion protein containing 7 GnRH peptides (tGnRH, n = 12), a combination of oGnRH plus tGnRH (otGnRH, n = 12), or a combination of ovalbumin and thioredoxin (control, n = 11). Each heifer received a primary immunization containing 1 mg of protein in 1 mL of adjuvant injected into the mammary gland at wk 0 (mean age = 38 wk) and booster immunizations at wk 6 and 12. Six heifers within each treatment received Synovex H implants at wk -2. Weekly blood samples were collected from wk -2 to 26 for determination of serum progesterone concentrations and GnRH antibody titers. In GnRH-immunized heifers, GnRH antibody titers increased after the first booster injection, peaked after the second booster injection, and remained elevated through the end of the study (P < 0.01). Heifers immunized against oGnRH achieved greater (P < 0.05) GnRH antibody titers than tGnRH heifers but did not differ (P = 0.20) from otGnRH heifers. During the 26-wk study, ovulation was prevented (P < 0.05) in 10 out of 12, 12 out of 12, 11 out of 12, and 0 out of 11 tGnRH, oGnRH, otGnRH, and control heifers, respectively. At slaughter, uterine weights were lighter (P < 0.01) for GnRH-immunized heifers than control heifers. Synovex H-implanted heifers had greater (P < 0.05) ADG from wk -2 to 26, greater LM area, and lesser percentages of KPH, yield grade, and quality grade than nonimplanted heifers, regardless of the immunization treatment. Immunization against GnRH fusion proteins resulted in production of antibodies against GnRH that prevented ovulation in 92% of the heifers without affecting feedlot or carcass performance. Implanting heifers with Synovex H improved ADG, LM area, and yield grade. Improvements in delivery of the oGnRH vaccine may provide a feasible alternative to surgical spaying of heifers.

Contraceptive Effect of a Recombinant GnRH Vaccine in Adult Female Pigs

GonaCon ™ is a gonadotropin releasing hormone (GnRH) contraceptive vaccine developed at the USDA National Wildlife Research Center (NWRC) that contains keyhole limpet hemocyanin (KLH) coupled to GnRH. The vaccine is administered intramuscularly in an emulsion with the NWRC adjuvant, AdjuVac ™ . This first-generation vaccine, developed in 1998, has been very effective in contracepting every species tested. However, because of the high cost of KLH, NWRC has developed a second generation GnRH vaccine, replacing KLH with a less expensive mollusk protein. This second generation GnRH vaccine, called GonaCon-Blue ™ , is currently being tested in several species and has proven to be as, or more effective than the original GonaCon ™ . Scientists with the Reproductive Control Methods Project at the NWRC are also testing a recombinant GnRH antigen developed by Dr. Talwar of the Talwar Research Institute, India. The recombinant antigen was produced from plasmid gene expression grown in and extracted from E. coli . The antigen is combined with the NWRC AdjuVac ™ to form an emulsion similar to the GonaCon ™ preparation. The recombinant vaccine has been shown to be effective as a single or duel injection for contraception of adult female pigs in a short-term study at the Swine Research Facility at Penn State University. Although the long term effectiveness of the recombinant vaccine remains to be established, this small molecule of approximately 14,000 MW is of considerable interest because it may be effective in oral or transcutaneous applications. It could also be scaled-up for large field applications.

Induction of castration by immunization of male dogs with recombinant gonadotropin-releasing hormone (GnRH)-canine distemper virus (CDV) T helper cell epitope p35

Immunocastration is a considerable alternative to a surgical castration method especially in male animal species for alleviating unwanted male behaviors and characteristics. Induction of high titer of antibody specific for gonadotropin-releasing hormone (GnRH) correlates with the regression of testes. Fusion proteins composed of canine GnRH and T helper (Th) cell epitope p35 originated from canine distemper virus (CDV) F protein and goat rotavirus VP6 protein were produced in E. coli. When these fusion proteins were injected to male dogs which were previously immunized with CDV vaccine, the fusion protein of GnRH-CDV Th cell epitope p35 induced much higher antibody than that of GnRH-rotavirus VP6 protein or GnRH alone. The degeneration of spermatogenesis was also verified in the male dogs immunized with the fusion protein of GnRH-CDV Th cell epitope p35. These results indicate that canine GnRH conjugated to CDV Th cell epitope p35 acted as a strong immunogen and the antibody to GnRH specifically neutralized GnRH in the testes. This study also implies a potential application of GnRH-based vaccines for immunocastration of male pets.

Development and use of recombinant gonadotropin-releasing hormone vaccines to sterilize cattle: a review (1997)

A possible alternative to conventional castration methods is the use of vaccines that can be injected in order to sterilize animals. One promising approach involves the use of a vaccine that causes cattle to produce an immune response against one of their own reproductive hormones, gonadotropin releasing hormone (GnRH). Immunization against GnRH results in a decrease in the amount of GnRH circulating within the animal's blood. Therefore, follicle stimulating hormone (FSH) and luteinizing hormone (LH) are not stimulated to be released, and, subsequently their levels required for reproductive function. Experimentation has shown that immunization against GnRH can effectively block reproductive function in an age-independent manner but has little effect on carcass and growth parameters. However, about 10% of cattle tested do not respond when immunized against GnRH, regardless of dosing regimen. Further research is needed to improve the efficiency of potential GnRH immunocastration vaccines.; Dairy Day, 1997, Kansas State University, Manhattan, KS, 1997