In vitro maturation of guinea pig oocytes supplemented with Epidermal Growth Factor and Insulin-Like Growth Factor I

Insights in oocyte maturation process in guinea pigs are essential for the development of in vitro culture systems in this species, since it represents an interesting animal model in reproduction field (Suzuki et al. Mol Reprod Dev 2003; 64, 219–25). The goal of this study was to elucidate the influence of both Epidermal Growth Factor (EGF) and Insulin-like Growth Factor I (IGF-I) on in vitro oocyte maturation (IVM) medium of guinea pig. We assessed meiotic and cytoplasmic oocyte maturation, in terms of cortical granules (CG) and mitochondrial distribution, apoptotic rate and steroidogenic response of cumulus-oocyte-complexes (COCs) after IVM. A pool of 500 COCs from adult guinea pigs were cultured in groups of 40 COCs in four replicates in TCM-199 with 2 mM/mL glutamine, 0.1 mg/mL sodium pyruvate and 0.003% BSA for 17h (38ºC, 5%CO2) (Sigma Chemical Company)

Effect of epidermal growth factor on nuclear and cytoplasmic in vitro maruration of guinea pig oocytes

The guinea pig may represent an animal model for research on ovarian infertility and improvement of the in vitro maturation (IVM) conditions is needed in this species. The aim of the present work was to immunolocalize the Epidermal Growth Factor (EGF)-Receptor in the guinea pig ovaries and to study the effect of EGF on meiotic and cytoplasmic maturation, and apoptotic rate in cumulus-oocyte-co mplexes (COCs). Immunohistochemistry was performed in paraffined ovaries using a rabbit polyclonal antibody EGF-R (1:100; Santa Cruz Biotechnology) and the ABC Vector Elite kit (Vector Laboratories). For the IVM, COCs were collected by aspiration of follicles >700μm under a stereoscopic microscope

Nobiletin enhances the development and quality of bovine embryos in vitro during two key periods of embryonic genome activation

In vitro culture can alter the development and quality of bovine embryos. Therefore, we aimed to evaluate whether nobiletin supplementation during EGA improves embryonic development and blastocyst quality and if it affects PI3K/AKT signaling pathway. In vitro zygotes were cultured in SOF + 5% FCS (Control) or supplemented with 5, 10 or 25 µM nobiletin (Nob5, Nob10, Nob25) or with 0.03% dimethyl-sulfoxide (CDMSO) during minor (2 to 8-cell stage; MNEGA) or major (8 to 16-cell stage; MJEGA) EGA phase. Blastocyst yield on Day 8 was higher in Nob5 (42.7 ± 1.0%) and Nob10 (44.4 ± 1.3%) for MNEGA phase and in Nob10 (61.0 ± 0.8%) for MJEGA phase compared to other groups. Mitochondrial activity was higher and lipid content was reduced in blastocysts produced with nobiletin, irrespective of EGA phase. The mRNA abundance of CDK2, H3-3B, H3-3A, GPX1, NFE2L2 and PPARα transcripts was increased in 8-cells, 16-cells and blastocysts from nobiletin groups. Immunofluorescence analysis revealed immunoreactive proteins for p-AKT forms (Thr308 and Ser473) in bovine blastocysts produced with nobiletin. In conclusion, nobiletin supplementation during EGA has a positive effect on preimplantation bovine embryonic development in vitro and corroborates on the quality improvement of the produced blastocysts which could be modulated by the activation of AKT signaling pathway.This work was funded by the Spanish Ministry of Science and Innovation (PID2019-111641RB-I00 to D.R. and RTI2018-093548-B-I00 to A.G.-A). Y.N.C. was supported by a predoctoral fellowship from the Secretaría Nacional de Educación Superior, Ciencia, Tecnología e Innovación (Convocatoria abierta 2017, SENESCYT-Ecuador). C.L.V.L. was supported by a BPE grant from Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil (FAPESP #2017/20339-3).Peer reviewe

Guinea pig sperm is capable of fertilising bovine zona-intact oocytes in vitro

Peer reviewe

Fertilising capacity of guinea pig spermatozoa by heterologous fertilisation with zona-intact murine oocytes

Departamento de Reproducción Animal​.The guinea pig is an important meat production species in South America and a valuable animal model for the study of reproduction in humans and mammals. In vitro fertilisation (IVF) in this species is poorly developed mainly because of the limited accessibility to homologous (Ho) oocytes outside of South America. Thus, heterologous (He) IVF represents an alternative to improve the procedure. We aimed to evaluate the fertilising capacity of guinea pig sperm using two capacitation protocols in He IVF with murine oocytes. Spermatozoa were collected from the vas deferens of three guinea pigs and processed separately using two protocols: (A) spermatozoa were isolated by flushing the lumen of the vas deferens with 2 mL of 0.15 m NaCl and the sperm suspension was washed twice by centrifugation at 600 × g for 3 min and then incubated in minimal culture medium with 0.25 mM Na-pyruvate, 20.0 mM Na-lactate, and 5.56 mM glucose (MCM-PLG), during 2 h at 5% CO2 and 37°C; (B) the vas deferens was gently minced with fine scissors and spermatozoa suspended in 500 μL of HTF medium supplemented with 1% bopvine serum albumin (BSA), and incubated for 1 h at 5% CO2 and 37°C. Zona-intact murine oocytes collected from superovulated female mice were used for He IVF with spermatozoa capacitated through protocol A (HeA, n = 285) or B (HeB, n = 296) into drops of 500 µL of MCM-PLG with 2.7 mM KCl and 0.3% BSA or HTF medium, respectively. In parallel, Ho IVF (n = 243) and parthenogenesis (non-fertilised oocytes, n = 75) was performed in HTF medium. In vitro-matured oocytes were co-incubated for 5 h with 1 × 106 spermatozoa mL−1 and then presumptive zygotes or (parthenogenetic) oocytes were cultured in KSOM medium at 37°C and 5% CO2 to complete a total of 48 h of incubation. Sperm-oocyte interaction was assessed at 2.5 h post-insemination (hpi) by evaluating the number of bound spermatozoa. Presumptive zygotes were fixed and stained with Hoechst at 6, 18, and 22 hpi to assess polyspermy and pronuclear formation (PrF) by widefield fluorescence microscope with structured illumination. Cleavage rate was evaluated at 24 and 48 hpi. Data obtained from three replicates were analysed using one-way ANOVA. The number of bound sperm was similar between groups (Ho = 0.9 ± 0.1; HeA = 0.8 ± 0.1; HeB = 0.8 ± 0.1). No differences were seen in PrF at any time point (ranged from 24.4 ± 0.6 to 25.1 ± 0.9%) or cleavage rate between HeA or HeB IVF at 24 hpi (25.6 ± 0.9 and 25.7 ± 2.9%, respectively) or 48 hpi (24.2 ± 1.7 and 25.0 ± 1.3%). Homologous IVF was associated with higher percentages of PrF at 6 hpi (79.8 ± 2.1%) compared to HeA (24.4 ± 0.6%) and HeB (25.1 ± 0.9) IVF, and no polyspermy was detected. As expected, the cleavage rate at 24 or 48 hpi was higher (P < 0.05) in Ho (80.8 ± 0.9 and 81.4 ± 1.0, respectively) than He IVF. In addition, spontaneous parthenogenetic activation in mature unfertilised oocytes from mice at 24 and 48 hpi was observed (1.9 ± 1.9 and 2.6 ± 2.6%, respectively). In conclusion, He IVF is a promising method to assess the fertilising ability of guinea pig spermatozoa obtained from epididymis, revealing their ability to penetrate zona-intact murine oocytes, leading to hybrid embryo formation.Peer reviewe

Twelve novel HGD gene variants identified in 99 alkaptonuria patients: focus on ‘black bone disease’ in Italy

Alkaptonuria (AKU) is an autosomal recessive disorder caused by mutations in homogentisate-1,2-dioxygenase (HGD) gene leading to the deficiency of HGD enzyme activity. The DevelopAKUre project is underway to test nitisinone as a specific treatment to counteract this derangement of the phenylalanine-tyrosine catabolic pathway. We analysed DNA of 40 AKU patients enrolled for SONIA1, the first study in DevelopAKUre, and of 59 other AKU patients sent to our laboratory for molecular diagnostics. We identified 12 novel DNA variants: one was identified in patients from Brazil (c.557T>A), Slovakia (c.500C>T) and France (c.440T>C), three in patients from India (c.469+6T>C, c.650–85A>G, c.158G>A), and six in patients from Italy (c.742A>G, c.614G>A, c.1057A>C, c.752G>A, c.119A>C, c.926G>T). Thus, the total number of potential AKU-causing variants found in 380 patients reported in the HGD mutation database is now 129. Using mCSM and DUET, computational approaches based on the protein 3D structure, the novel missense variants are predicted to affect the activity of the enzyme by three mechanisms: decrease of stability of individual protomers, disruption of protomer-protomer interactions or modification of residues in the region of the active site. We also present an overview of AKU in Italy, where so far about 60 AKU cases are known and DNA analysis has been reported for 34 of them. In this rather small group, 26 different HGD variants affecting function were described, indicating rather high heterogeneity. Twelve of these variants seem to be specific for Italy