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Bovine C-Type Natriuretic Peptide Elisa Kit (CNP

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[#E11C0339] Bovine C-Type Natriuretic Peptide Elisa Kit (CNP


E11C0339 | Bovine C-Type Natriuretic Peptide Elisa Kit (CNP, 96 Tests/kit
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(1) Luteinizing hormone upregulates NPPC and downregulates NPR3 mRNA abundance in bovine granulosa cells through activation of the EGF receptor.[TOP]

Pubmed ID :29960164
Publication Date : //
During folliculogenesis, the luteinizing hormone (LH) surge triggers dynamic events in granulosa cells that culminate with ovulation. The aim of this study was to evaluate if the epidermal growth factor receptor (EGFR) is required for ovulation in cattle, and if it regulates the expression of the natriuretic peptide (NP) system in granulosa cells after gonadotropin-releasing hormone (GnRH)/LH stimulation. It was observed that GnRH induces amphiregulin (AREG) and epiregulin (EREG) mRNA at 3 and 6 h after in vivo treatment, but the expression of these genes was not regulated by atrial (ANP) and C-type (CNP) NPs in granulosa cells cultured in vitro. The abundance of mRNA encoding the NP receptors (NPR1, 2 and 3) was not altered by LH supplementation and/or EGFR inhibition (AG1478; AG) in granulosa cells after 6 h of in vitro culture. However, in the same conditions, mRNA encoding the natriuretic peptide precursor C (NPPC) was upregulated by LH, whereas AG (0.5 and 5 μM) inhibited the LH effect. In order to confirm those results, 5 μM AG or saline were intrafollicularly injected in preovulatory follicles and cows were simultaneously treated with GnRH intramuscularly. Granulosa cells harvested at 6 h after GnRH injection revealed higher NPR3 and lower NPPC mRNA levels in AG-treated, compared to control cows. However, intrafollicular injection of AG did not inhibit GnRH-induced ovulation. In granulosa cells cultured in vitro, ANP associated with LH increased prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA abundance. In conclusion, we inferred that LH modulated NPPC and NPR3 mRNA abundance through EGFR in bovine granulosa cells, but ovulation in cattle did not seem to depend on EGFR activation.

Authors : Dos Santos Joabel T, De Cesaro Matheus P, Ferst Juliana G, Pereira Dau Andressa M, da Rosa Paulo R A, Pasqual Bruno M, Antoniazzi Alfredo Q, Gasperin Bernardo G, Bordignon Vilceu, Gonçalves Paulo B D,

(2) Supplementing in vitro embryo production media by NPPC and sildenafil affect the cytoplasmic lipid content and gene expression of bovine cumulus-oocyte complexes and embryos.[TOP]

Pubmed ID :29361408
Publication Date : //
In our study, we added natriuretic peptide type C (NPPC) and/or sildenafil during in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COCs) followed by in vitro culture (IVC) of embryos with or without sildenafil. We evaluated the effects on the lipid content (LC) of oocytes and embryos and also verified the expression of 96 transcripts related to competence in matured COCs and 96 transcripts related to embryo quality in blastocysts. After IVM, LC was decreased in oocytes by NPPC while sildenafil did not affect LC in oocytes. The genes involved in lipid metabolism and lipid accumulation (DGAT1, PLIN2and PLIN3) were not affected in COCs after treatment during IVM, although the expression of PTX3 (a cumulus cells expansion biomarker) was increased and the hatched blastocyst rate was increased by NPPC during IVM. During IVM, sildenafil increased the mRNA relative abundance of HSF1 and PAF1 and decreased REST in blastocysts. The use of sildenafil in IVC increased the LC of blastocysts. The mRNA abundance in blastocysts produced during IVC with sildenafil was changed for ATF4, XBP1, DNMT3A, DNMT3B, COX2, and SOX2. Although NPPC reduced the LC of oocytes after IVM and upregulated markers for cumulus expansion, embryo production was not affected and the produced blastocysts were able to regain their LC after IVC. Finally, the use of sildenafil during IVC increased the cytoplasmic LC of embryos but did not affect embryo quality, as measured by analysis of 96 transcripts related to embryo quality.

Authors : Botigelli Ramon Cesar, Razza Eduardo Montanari, Pioltine Elisa Mariano, Fontes Patricia Kubo, Schwarz Kátia Regina Lancellotti, Leal Cláudia Lima Verde, Nogueira Marcelo Fábio Gouveia,

(3) Natriuretic peptide receptor 2 (NPR2) localized in bovine oocyte underlies a unique mechanism for C-type natriuretic peptide (CNP)-induced meiotic arrest.[TOP]

Pubmed ID :29080478
Publication Date : //
Meiosis is of prime importance for successful gametogenesis, and insufficient maintenance of oocyte meiotic arrest compromises oocyte developmental competence. Recent studies have demonstrated that the C-type natriuretic peptide (CNP)-Natriuretic peptide receptor 2 (NPR2) pathway can inhibit mammalian oocyte meiotic resumption. In mouse and porcine, the inhibitory effect of mural granulosa cell (MGC)-derived CNP on oocyte meiotic resumption is mediated by NPR2 localized in cumulus cells (CCs) surrounding the oocytes. However, in the present study, we identified a novel mechanism for CNP-induced meiotic arrest that appears to be unique to bovine oocytes. Unlike mouse and porcine, bovine NPR2 not only localizes in CCs, but also in oocyte membranes. We also showed that CNP can directly activate intra-oocyte cGMP production via NPR2 localized in oocyte membranes, in parallel with the CC-mediated pathway. Furthermore, we demonstrated that Npr2 expression in bovine CCs and oocytes were synergistically regulated by estradiol and oocyte-derived growth factors. Finally, based on the profound inhibitory effect of CNP on meiotic resumption, we established a natural factor synchronized in vitro oocyte maturation (NFSOM) system, which can significantly improve the developmental competence of matured oocytes, thereby resulting in higher in vitro embryo production efficiency. Taken together, our study not only provides new insight into understanding the crosstalk between oocytes and follicular somatic cells in mammals, but also presents a promising strategy for improving the in vitro oocyte maturation systems of assisted reproductive technology (ART).

Authors : Xi Guangyin, An Lei, Jia Zhenwei, Tan Kun, Zhang Jiaxin, Wang Zhuqing, Zhang Chao, Miao Kai, Wu Zhonghong, Tian Jianhui,

(4) Steroid hormones interact with natriuretic peptide C to delay nuclear maturation, to maintain oocyte-cumulus communication and to improve the quality of in vitro-produced embryos in cattle.[TOP]

Pubmed ID :28356185
Publication Date : //
In vivo, oocyte maturation is triggered by the ovulatory LH surge, whereas in vitro it is precociously induced when the cumulus-oocyte complex is removed from the follicle. Natriuretic peptide C (NPPC) delays germinal vesicle breakdown (GVBD) while increasing oocyte-cumulus communication during in vitro maturation (IVM) in cattle. In the present study we first tested the hypothesis that steroids secreted by the follicle (17β-oestradiol, progesterone and androstenedione) interact with NPPC to delay GVBD and to maintain oocyte-cumulus communication as assessed by transfer of a dye (Lucifer Yellow) from the oocyte to cumulus cells. Then, we assessed the effects of steroid hormones and NPPC, alone and in combination in a pre-IVM culture, on embryo production. The combination of NPPC with steroids delayed GVDB, increased natriuretic peptide receptor 2 (NPR2) mRNA abundance in cumulus cells during culture, and maintained oocyte-cumulus communication at levels not different from non-cultured controls. The addition of steroids and/or NPPC to a pre-IVM culture did not alter blastocyst rates after IVF, but supplementation with steroids increased blastocyst total cell number. The present study provides evidence, for the first time in cattle, that steroids interact with NPPC to regulate oocyte nuclear maturation and oocyte-cumulus communication, and improve oocyte developmental competence.

Authors : Soares Ana Caroline S, Lodde Valentina, Barros Rodrigo G, Price Christopher A, Luciano Alberto M, Buratini José,

(5) Meiotic arrest as an alternative to increase the production of bovine embryos by somatic cell nuclear transfer.[TOP]

Pubmed ID :27780485
Publication Date : //
This study aimed to evaluate the effect of meiotic arrest using phosphodiesterase type 3A (PDE 3A) inhibitors, cilostamide and C-type natriuretic peptide (NPPC), on pre-maturation (PM) of oocytes to be used in the production of cloned embryos. Nuclear maturation, in vitro embryo production (IVP), somatic cell nuclear transfer (SCNT) and parthenogenetic activation (PA), and total cells number of cloned embryos were evaluated. The results were analysed by chi-squared and Kruskal-Wallis test with a P-value 0.05) between control and PM, both for cleavage (78.2% and 76.9%) and blastocyst (35.5% and 29.3%) rates. After SCNT, cleavage rate was also similar (P > 0.05) between control and PM (66% and 51.9%) however, blastocyst rate was lower (P < 0.05) in the PM group than in the control group (7.4% and 30.2%). After 6 h of PM with 100 nM of NPPC, approximately 84.9% of the oocytes remained at GV. No difference was found between control and PM in cleavage (69.2% and 76.1%) and blastocyst rates (37,4% and 35%) after IVP. Similarly, no differences between PM and control groups were observed for cleavage (69.2% and 68.4%) and blastocyst (24.4% and 21.5%) rates. SCNT and PA embryos from control or PM oocytes had similar total cell number. It can be concluded that PM for 6 h with 100 nM NPPC is feasible for cloned embryo production without affecting embryo outcome.

Authors : Caixeta F M C, Sousa R V, Guimarães A L, Leme L O, Sprícigo J F W, Netto S B Senna, Pivato I, Dode M A N,

(6) Effect of C-type natriuretic peptide pretreatment on in vitro bovine oocyte maturation.[TOP]

Pubmed ID :27761784
Publication Date : //
C-type natriuretic peptide (CNP) has been considered as a physiological meiotic inhibitor that stimulates the cGMP production by cumulus cell natriuretic peptide receptor 2 (NPR2), which inhibits oocyte phosphodiesterase type 3 activity and increases cAMP. In this study, we explored the effect of CNP pretreatment on the in vitro maturation (IVM) of bovine oocytes by examining changes in cleavage rate, blastocyst formation, mitochondrial DNA (mtDNA) copy number, reactive oxygen species (ROS) level, glutathione (GSH) content, and redox state. Our results showed that 200 nM CNP could effectively maintain meiotic arrest of bovine oocytes in vitro within 6 h. The two-step IVM system in which oocytes were pretreated with 200 nM CNP for 6 h and then cultured IVM for 28 h yielded a significantly (P < 0.05) increased blastocyst rate and cell number after in vitro fertilization (IVF) while compared to the conventional one-step IVM method. In addition, in comparison with the conventional 24-h matured oocyte, oocytes pretreated with 200 nM CNP for 6 h followed by 28 h IVM resulted in significantly (P < 0.05) higher mtDNA copy number and ROS levels in oocytes, while GSH level significantly (P < 0.05) decreased. Remarkably, regardless of treatment, no changes were observed in FAD++, NAD(P)H autofluorescence intensity, and redox ratio (FAD++/NAD(P)H) within the oocytes, maintaining a healthy metabolic equilibrium of redox throughout the two-step IVM. In conclusion, these results indicate that CNP pretreatment could dramatically improve the quality of bovine oocytes during in vitro maturation.

Authors : Zhang Tong, Zhang Chunqiang, Fan Xiaomei, Li Ruilan, Zhang Jiaxin,

(7) Effect of kit ligand on natriuretic peptide precursor C and oocyte maturation in cattle.[TOP]

Pubmed ID :27492081
Publication Date : //
In vitro maturation (IVM) of oocytes in cattle is inefficient, and there is great interest in the development of approaches to improve maturation and fertilization rates. Intraovarian signalling molecules are being explored as potential additives to IVM media. One such factor is kit ligand (KITL), which stimulates the growth of oocytes. We determined if KITL enhances oocyte maturation in cattle. The two main isoforms of KITL (KITL1 and KITL2) were expressed in bovine cumulus-oocyte complexes (COC), and levels of mRNA increased during FSH-stimulated IVM. The addition of KITL to the culture medium increased the percentage of oocytes that reached meiosis II but did not affect cumulus expansion after 22 h of IVM. Addition of KITL reduced the levels of mRNA encoding natriuretic peptide precursor C (NPPC), a protein that holds oocytes in meiotic arrest, and increased the levels of mRNA encoding YBX2, an oocyte-specific factor involved in meiosis. Removal of the oocyte from the COC resulted in increased KITL mRNA levels and decreased NPPC mRNA levels in cumulus cells, and addition of denuded oocytes reversed these effects. Taken together, our results suggest that KITL enhances bovine oocyte nuclear maturation through a mechanism that involves NPPC, and that the oocyte regulates cumulus expression of KITL mRNA.

Authors : Lima Paula F, Ormond Cinthia M, Caixeta Ester S, Barros Rodrigo G, Price Christopher A, Buratini José,

(8) MAPK3/1 is conducive to luteinizing hormone-mediated C-type natriuretic peptide decrease in bovine granulosa cells.[TOP]

Pubmed ID :26655567
Publication Date : //
C-type natriuretic peptide (CNP) plays a role as an oocyte maturation inhibitor (OMI) in many species, including the bovine. However, the effects of luteinizing hormone (LH) on CNP expression and its potential mechanisms have not reported in the bovine. In the present study, we aimed to study the effects of LH on CNP expression and to illuminate the potential molecular mechanism in this process. Our results showed that LH induced epidermal growth factor receptor (EGFR) phosphorylation, mitogen-activated protein kinase3/1 (MAPK3/1) activation and CNP mRNA decrease in cultured bovine granulosa cells. Further study revealed that LH suppressed CNP expression via the MAPK3/1 signaling pathway, which was activated by the EGFR pathway. In conclusion, our research suggested that MAPK3/1 is involved in LH-mediated decrease of CNP and that this process is related to the EGFR and MAPK3/1 signal pathways.

Authors : Yang Lei, Wei Qiang, Ge Junbang, Zhao Xiaoe, Ma Baohua,

(9) Natriuretic peptides stimulate oocyte meiotic resumption in bovine.[TOP]

Pubmed ID :26051611
Publication Date : //
The aim of the present study was to evaluate the expression of mRNA encoding natriuretic peptides (NPs) and their receptors in the cumulus-oocyte complex in cattle, a monovular mammalian species, and also to investigate the role of NPs in oocyte meiotic resumption in vitro. mRNA was observed for the NP precursor type-A (NPPA), type-C (NPPC), NP receptor-1 (NPR-1), receptor-2 (NPR-2) and receptor-3 (NPR-3) in bovine cumulus cells, and NPR-2 mRNA was observed in oocytes. These results are different from those obtained in mouse and pig models. The effects of NPPA, NP precursor type-B (NPPB) and NPPC on the resumption of arrested meiosis maintained by forskolin were studied at three different doses (10, 100 and 1000nM) with a 12h culture system. The germinal vesicle breakdown rates were greater (P≤0.05) in oocytes that were cultured in the presence of one or a combination of NPs (from 44% to 73%) than the negative control (from 24% to 27%). Additionally, it was demonstrated that the concentration of cyclic guanosine 3',5'-monophosphate (cGMP) is increased by NPPA and NPPC in oocytes and cumulus cells after 3h of in vitro maturation. However, in both groups, the concentration of cyclic adenosine 3',5'-monophosphate (cAMP) in the oocyte did not increase between 3 and 6h of culture, even when forskolin was used. In summary, we observed the presence of mRNA for NPs and their receptors in the bovine cumulus-oocyte complex and demonstrated that, in vitro, NPPA, NPPB and NPPC stimulate oocyte meiotic resumption in a monovular species.

Authors : De Cesaro Matheus P, Macedo Mariana P, Santos Joabel T, Rosa Paulo R A, Ludke Charles A, Rissi Vitor B, Gasperin Bernardo G, Gonçalves Paulo B D,

(10) Controlled release of C-type natriuretic peptide by microencapsulation dampens proinflammatory effects induced by IL-1β in cartilage explants.[TOP]

Pubmed ID :25556565
Publication Date : //
C-type natriuretic peptide (CNP) exhibits potent anti-inflammatory effects in chondrocytes that have the potential to repair cartilage damage observed in osteoarthritis (OA). However, treatments for OA have been challenging due to poor targeting and delivery of therapeutics. The present study fabricated polyelectrolyte microcapsules loaded with CNP and examined whether the layer-by-layer (LbL) approach could have protective effects in cartilage explants treated with the pro-inflammatory cytokine, interleukin-1β (IL-1β). SEM showed uniform, 2 to 3 μm spherical microcapsules with morphological characteristic similar to templates loaded with or without CNP. The protein was localized around the external surface of the microcapsules with encapsulation efficiencies >82.9%. CNP release profiles were broadly similar following 9 days of culture. The presence of CNP microcapsules did not significantly affect cell viability (80%) with DNA values that remained stable throughout the culture conditions. Confocal imaging showed clustering of microcapsules in chondrocytes to natriuretic peptide receptor (Npr) 2 and 3. Treatment of cartilage explants with CNP microcapsules led to concentration-dependent inhibition of NO release in response to IL-1β and restoration of matrix synthesis. In summary, we demonstrate controlled delivery of CNP to dampen pro-inflammatory effects induced by IL-1β in cartilage explants. The LbL approach has the potential to promote cartilage repair in vivo.

Authors : Peake Nick J, Pavlov Anton M, D'Souza Alveena, Pingguan-Murphy Belinda, Sukhorukov Gleb B, Hobbs Adrian J, Chowdhury Tina T,