Porcine spermatozoa are stored in the oviductal isthmus after natural mating, and the number of spermatozoa is increased in the oviductal ampulla when the mature cumulus-oocyte complexes (COCs) are transferred into the ampulla. However, the mechanism is unclear. Herein, natriuretic peptide type C (NPPC) was mainly expressed in porcine ampullary epithelial cells, whereas its cognate receptor natriuretic peptide receptor 2 (NPR2) was located on the neck and the midpiece of porcine spermatozoa. NPPC increased sperm motility and intracellular Ca2+ levels, and induced sperm release from oviduct isthmic cell aggregates. These actions of NPPC were blocked by the cyclic guanosine monophosphate (cGMP)-sensitive cyclic nucleotide-gated (CNG) channel inhibitor l-cis-Diltiazem. Moreover, porcine COCs acquired the ability to promote NPPC expression in the ampullary epithelial cells when the immature COCs were induced to maturation by epidermal growth factor (EGF). Simultaneously, transforming growth factor-β ligand 1 (TGFB1) levels were dramatically increased in the cumulus cells of the mature COCs. The addition of TGFB1 promoted NPPC expression in the ampullary epithelial cells, and the mature COC-induced NPPC was blocked by the transforming growth factor-β type 1 receptor (TGFBR1) inhibitor SD208. Taken together, the mature COCs promote NPPC expression in the ampullae via TGF-β signaling, and NPPC is required for the release of porcine spermatozoa from the oviduct isthmic cells.

Premature ovarian insufficiency (POI) is a clinical disease that is diagnosed by the loss of ovarian function before the age of 40. Despite recent progress in molecular diagnosis, the genetic etiology of POI is not well established. The aim of this study is to reveal pathogenic genetic variants involved in POI.
To reveal pathogenic genetic variants involved in POI, whole exome sequencing was performed in nonconsanguineous family members with POI. Constitutional variants were filtered against population databases and a missense mutation of natriuretic peptide C (NPPC) (c.131A>G, p.Q44R) was selected as a convincing candidate mutation among 14 heterozygous mutant alleles in 13 genes.
The wild-type NPPC and mutant NPPC (NPPC131A>G) were expressed in HeLa cells, and cells expressing NPPC131A>G secreted unique peptides. The ProP 1.0 Server, a neural network prediction tool, predicted the presence of a cleavage site at the substituted arginine residue (p.Q44R) of NPPC. The molecular weight of predicted cleaved peptides processed from mutant NPPC precursor corresponded to that of the actual mutant peptide. The cGMP synthetic activity of NPR2-expressing cells was significantly decreased by interaction with the mutant NPPC peptide compared with wild-type NPPC.
The peptide generated by a rare mutation of NPPC might influence paracrine C-type natriuretic peptide (CNP)-mediated preantral follicle development and/or sustain meiotic arrest in oocytes. We therefore suggest that a mutation of the NPPC gene is involved in the pathogenesis of POI.

Acute Kidney Injury (AKI) is an often neglected but crucial element of clinical nephrology. The aim of the Nephrology Public Policy Committee (NPPC) of the European Renal Association - European Dialysis and Transplant Association (ERA-EDTA) is to promote several key aspects of European nephrology. One of the targets proposed by NPPC was to advance European nephrology involvement in AKI. We undertook literature analyses to define the current position of European nephrology in the field of AKI compared to other regions, and about how different European countries compare to each other. It appeared that vis-à-vis countries with a comparable socio-economic status (the US, Australia, New Zealand, Canada), the European contribution was almost 50% lower. Within Europe, Central/Eastern Europe and countries with a lower gross domestic product (GDP) showed lower scientific output. Nephrologists contributed to less than half of the output. There was no trend for a change over the last decade. It is concluded that there is room to improve the contribution of European nephrology in the field of AKI. We propose a model on how to promote clinical collaboration on AKI across Europe, the creation of a pan-European nephrology network of interested units is proposed, to improve clinical outcomes, increase nephrologist involvement and awareness outside nephrology, and stimulate research on AKI in Europe. Accordingly, we also propose a list of research priorities and stress the need for more European funding of AKI research.

Differences in reproductive physiology between cattle breeds may help to explain distinct responses to assisted reproductive techniques and to define breed-specific protocols with improved efficiency. Germinal vesicle (GV) stage oocytes are characterized by increasing levels of chromatin compaction enclosed within the nucleus (graded from GV0 to GV3), associated with different developmental competence. The first objective of this study was to characterize chromatin configuration of GV stage oocytes recovered by OPU at random days of the estrous cycle from Nelore (Bos indicus) and Holstein (Bos taurus) cows. In Nelore 90% of the oocytes presented advanced stages of chromatin compaction associated with higher developmental competence (GV2 and GV3), while in Holstein, only 65% of the oocytes were at these stages. Then, aiming to obtain a more homogeneous population of oocytes in Holstein, we tested two synchronization protocols combining aspiration of all visible follicles at a random day (day 0), two IM injections of FSH 12 h apart on day 2, and OPU on day 4 (OPU/D4) or 5 (OPU/D5). The protocol OPU/D4 provided around 45% of the oocytes with low chromatin compaction (GV1), while the protocol OPU/D5 provided 70% of the oocytes at GV2 and 20% at GV3. Finally, we assessed the effects of a culture system known to prevent meiotic resumption on chromatin configuration of the GV2 enriched oocyte population obtained with the protocol OPU/D5. After 9 h of culture most oocytes transited from GV2 to GV3, with 90% of the oocytes at GV3 stage. This study demonstrates differences between Nelore and Holstein cows regarding patterns of chromatin configuration that may account for their different performance in IVM/IVF. In addition, it provides novel references for the design of protocols aiming to regulate oocyte quality before IVM for the optimization of IVF outcomes.

The effectiveness of the use of natriuretic peptide C (NPPC) in the blocking of meiosis has already been proven in several species. However, there are no reports on the use of NPPC in the activation of metabolic processes in embryos. Whereas modulations of cAMP concentrations alter the lipid metabolism of bovine oocytes, the present study aims to evaluate the effect of NPPC on the development, lipid content and transcript levels of genes related to lipid metabolism of IVP bovine embryos. For this purpose, ovaries were obtained from a slaughterhouse, and oocytes were fertilized in vitro (D0). From D5 of in vitro culture, embryos were treated with 100 nM NPPC (NPPC group) or with no NPPC (Control group) and evaluated in terms of Blastocyst (D7) and hatching rates (D10). For the assessment of the cytoplasmatic lipid amounts, blastocysts were stained with Sudan Black B dye. The embryonic lipid profile was investigated by electrospray ionization desorption-mass spectrometry (DESI-MS). The abundance of nine transcripts related to lipid metabolism were assessed using the Biomark HD system. For statistical analysis, blastocyst and hatching rates, lipid content by the Sudan Black B and variation of gene expression between groups were compared by Student t-test. For lipid profile analysis, principal component analysis (PCA) and fold-change were performed. The embryo lipid content was similar between NPPC (881 ± 3.7) and Control (883 ± 5.2) groups (p > 0.05). However, cholesteryl esters and TAGs were downregulated by NPPC at multiple levels according to the DESI-MS profiles. Of the analyzed genes, ELOVL6 and SREBF1 showed an up-regulation in the control group (p < 0.05), while CPT2 was observed to be up-regulated in the NPPC-treated embryos. There was no significant difference in the blastocyst production rate between NPPC (44.4%) and Control (42.4%), however the hatching rate at D10 was higher (p < 0.05) in the NPPC group (69.77%) when compared to the Control group (48.33%). These findings demonstrate that NPPC alters the mRNA expression of genes related to lipid metabolism and that it exerts a positive effect on the hatching rates of IVP Bos taurus indicus embryos.

In mammalian follicles, oocytes are arrested at the diplotene stage of prophase I until meiotic resumption following the LH surge. C-type natriuretic peptide (CNP), encoded by natriuretic peptide precursor type C (NPPC), was found to be reduced by the LH surge in the follicle, and then lead to meiotic resumption by decreasing the level of cAMP in the oocyte. As a wide-spread cytokine, macrophage colony-stimulating factor (M-CSF) takes part in the oocyte development to maturation and ovulation. Our study describes the expression curve of M-CSF and its receptor and investigates the impact on the levels of CNP/NPPC to explore the possible mechanism for meiotic resumption in both vivo and vitro. The result shows after the LH/HCG surge, the expressions of M-CSF and its receptors decline significantly inside ovarian follicles, thus leading to transduction of a range of signals. Consequently, the expression of CNP reaches the peak at 2 h and immediately declines to a relatively low level.

The heart is an endocrine organ, as cardiomyocytes (CMs) secrete natriuretic peptide (NP) hormones. Since the discovery of NPs, no other peptide hormones that affect remote organs have been identified from the heart. We identified osteocrin (Ostn) as an osteogenesis/chondrogenesis regulatory hormone secreted from CMs in zebrafish. ostn mutant larvae exhibit impaired membranous and chondral bone formation. The impaired bones were recovered by CM-specific overexpression of OSTN. We analyzed the parasphenoid (ps) as a representative of membranous bones. In the shortened ps of ostn morphants, nuclear Yap1/Wwtr1-dependent transcription was increased, suggesting that Ostn might induce the nuclear export of Yap1/Wwtr1 in osteoblasts. Although OSTN is proposed to bind to NPR3 (clearance receptor for NPs) to enhance the binding of NPs to NPR1 or NPR2, OSTN enhanced C-type NP (CNP)-dependent nuclear export of YAP1/WWTR1 of cultured mouse osteoblasts stimulated with saturable CNP. OSTN might therefore activate unidentified receptors that augment protein kinase G signaling mediated by a CNP-NPR2 signaling axis. These data demonstrate that Ostn secreted from the heart contributes to bone formation as an endocrine hormone.

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.

Chromosomal translocation of 2q37.1 just distal to the NPPC gene coding for C-type natriuretic peptide (CNP) and subsequent overproduction of CNP have been reported to cause a skeletal overgrowth syndrome. Loeys-Dietz syndrome (LDS) is one of marfanoid overgrowth syndromes, of which subtype IV is caused by haploinsufficiency of transforming growth factor beta 2 (TGFB2). We report on a girl with clinical phenotypes of overgrowth syndrome, including long and slim body habitus, macrodactyly of the big toe, scoliosis, ankle valgus deformity, coxa valga, slipped capital femoral epiphysis, and aortic root dilatation. Karyotyping revealed a balanced chromosomal translocation between 1q41 and 2q37.1, and the breakpoints could be mapped by targeted resequencing analysis. On chromosome 2q37.1, the translocation took place 200,365 bp downstream of NPPC, and serum level of the amino terminal of CNP was elevated. The contralateral site of translocation on chromosome 1q41 disrupted TGFB2 gene, presumed to cause its haploinsufficiency. This case supports the concept that NPPC is overexpressed because of the loss of a specific negative regulatory control in the normal chromosomal location, and demonstrates the effectiveness of targeted resequencing in the mapping of breakpoints.