Zinc Chloride Supplemented during Ovarian Tissue Vitrification Improves In Vitro Follicle Development and Fertilization in Pigs

Ovarian tissue vitrification is a promising method to preserve follicles and gametes, but can be improved with mineral supplementation to the vitrification medium. The objective of this study was to determine the effects of supplementing 0.5 mg/mL zinc chloride to the media during cryopreservation of pig ovarian tissue. After thawing, the following criteria were evaluated: (1) follicular development and damage, (2) in vitro fertilization (IVF) characteristics, and (3) embryonic development. The number of damaged antral follicles (72.0 ± 3.8%) was less (p < 0.05) in ovarian tissue vitrified in media supplemented with zinc chloride compared to those not supplemented with zinc chloride (86.7 ± 4.1%). Oocytes obtained from the antral follicles on ovarian tissue vitrified in media supplemented with zinc chloride had less (p < 0.05) polyspermic penetration and higher (p < 0.05) male pronuclear formation during IVF than oocytes obtained from ovarian tissue not supplemented with zinc chloride. There were no statistical differences in embryonic development rates. Based on these results, supplementing zinc chloride during the vitrification protocol improves follicular development and subsequent IVF in pigs.

Nuclear-to-cytoplasmic ratios of 1PN and 2PN zygotes after in vitro fertilization of mouse oocytes

Summary Numerous studies have reported comparisons of the nuclear-to-cytoplasmic (NC) ratio during mitosis. However, little information is known about how the pronuclear size is regulated and determined at the end of meiosis II in mammalian zygotes. The present study aims to analyze the NC ratio of female and male pronuclei, and also to compare the size of single pronuclei using photographs that were obtained during experiments to create chimeric hermaphrodites from 2-cell oocytes. The volume of both the female and the male pronucleus was found to correlate with the volume of the oocyte cytoplasm. The NC ratio of the male pronucleus was greater than that of the female pronucleus. The NC ratio of the average volume of the female and male pronuclei was greater than the NC ratio of the mononucleate oocytes. The occurrence of 1PN oocytes was significantly higher when the volume of cytoplasm was lower than the cut-off value. These results indicated that the NC ratio is retained during pronuclear formation. A higher NC ratio in male compared with the female pronucleus indicated structural and/or molecular difference between the two pronuclei. 1PN formation may occur when sperm enters close to the MII spindle.

Paternal hypoxia exposure impairs fertilization process and preimplantation embryo development

Summary Environmental hypoxia exposure causes fertility problems in human and animals. Compelling evidence suggests that chronic hypoxia impairs spermatogenesis and reduces sperm motility. However, it is unclear whether paternal hypoxic exposure affects fertilization and early embryo development. In the present study, we exposed male mice to high altitude (3200 m above sea level) for 7 or 60 days to evaluate the effects of hypoxia on sperm quality, zygotic DNA methylation and blastocyst formation. Compared with age-matched controls, hypoxia-treated males exhibited reduced fertility after mating with normoxic females as a result of defects in sperm motility and function. Results of in vitro fertilization (IVF) experiments revealed that 60 days’ exposure significantly reduced cleavage and blastocyst rates by 30% and 70%, respectively. Immunohistochemical staining of pronuclear formation indicated that the pronuclear formation process was disturbed and expression of imprinted genes was reduced in early embryos after paternal hypoxia. Overall, the findings of this study suggested that exposing male mice to hypoxia impaired sperm function and affected key events during early embryo development in mammals.

PSV-10 Effects of l-α-amino Butyrate Supplementation on Oocyte Maturation

L-α-amino butyrate is a low-molecular weight thiol compound that acts to increase the levels of glutathione in the oocyte. Glutathione acts as an antioxidant during oocyte maturation and promotes male pronuclear formation during fertilization. Supplementing the L-α-amino butyrate helps to decrease polyspermic penetration rates and improve early embryonic development in swine. However, it is unknown if L-α-amino butyrate supplementation affects the environment of the oocyte or the oocyte directly. Therefore, the objective of this study was to determine if L-α-amino butyrate supplementation to the maturation media acted on the oocyte or had alternative beneficial effects in the surrounding environment. Oocytes were randomly assigned to a maturation media containing an amino acid transport inhibitor, quisqualic acid (QA) (0 or 1 mM) and then supplemented with L-α-amino butyrate (0 or 3.3 mM). Oocytes were evaluated for stage of meiosis (n=380) and cumulus cell expansion (n=411) at the end of maturation. The remaining oocytes were fertilized and evaluated for cortical granule exocytosis (n=400) and IVF kinetics (n=456). Supplementation of L-α-amino butyrate with or without QA significantly increased (P &lt; 0.05) cumulus cell expansion, cortical granule exocytosis and male pronuclear formation compared to no supplementation or QA supplementation. There was no difference in meiotic progression, fertilization or polyspermic penetration rates between the treatment groups. Results suggest that when L-α-amino butyrate is supplemented during maturation, it improves the maturation of the oocyte by acting directly on the oocyte and not through the surrounding environment of the oocyte.

81 Pronuclear formation and SMARCA4 incorporation after intracytoplasmic sperm injection (ICSI) or assisted ICSI in pig zygotes

Pigs are considered an important experimental model for their biological similarities to humans, including their potential as organ donors in xenotransplantation. Unfortunately, in this species conventional invitro fertilization results in high polyspermic rates. ICSI avoids polyspermy and ICSI-mediated gene edition could be a powerful technique to produce genetically modified pigs. However, ICSI is not yet efficient in pigs. Moreover, the ATP-dependent chromatin remodeller, SMARCA4, translocates to the pronuclei soon after fertilization and its mislocalization or reduction leads to poor embryo development. The aim of this study was to assess whether assisted activation or the use of the piezo drill (PD) during ICSI improves pronuclear (PN) formation rates and to analyse SMARCA4 intensity levels in pronuclei. First, cumulus–oocyte complexes were collected from slaughterhouse ovaries and matured invitro for 44h. Matured and denuded oocytes were subjected to (1) ICSI (n=47), (2) ICSI assisted by PD (ICSIp, n=21), (3) ICSI assisted by electrical activation (ICSIe, n=39), and (4) electrical activation as an haploid parthenogenetic control (HAP, n=21). Presumptive zygotes were fixed for 20min in 4% formaldehyde solution 18h after injection or activation and incubated with SMARCA4 antibody (1:100) and Alexa Fluor (1:1000) as a secondary antibody. Then, the zygotes were classified according to the presence of PN in 2 PN (2-PN), 1 PN with the presence of a semi-condensed or condensed sperm (1-PN), and semi-condensed or condensed sperm with no evidence of PN (no activation). Zygotes that exhibited a different pattern were included in the “other” category. A region of interest was drawn around each PN and the average pixel intensity of SMARCA4 was determined with ImageJ image processing software. Data were analysed by Fisher’s exact test and Kruskal–Wallis test using GraphPad software (GraphPad Inc.). Differences were considered significant at P&lt;0.05. We found no significant differences in 2-PN formation rates among groups after ICSI (ICSI n=16, 34.04%; ICSIe n=10, 25.64%; ICSIp n=6, 28.57%). As expected, the majority of the HAP zygotes exhibited 1 PN (n=14, 66.67%). In contrast, in most of the zygotes of all experimental groups, SMARCA4 was found to be localised in both PN, being absent in polar bodies, metaphase plate, or condensed sperm. Interestingly, out of the total 2-PN porcine ICSI zygotes of all experimental groups (n=25), 7 zygotes (28%) showed clear asymmetric intensity levels between PN. The rest of the ICSI zygotes (n=18, 72%) showed a similar SMARCA4 intensity level between PN. In conclusion, our results suggest that neither the use of piezo drill or electrical activation improves PN formation or SMARCA4 pattern. It remains to be determined whether the asymmetric levels of SMARCA4 between PN observed in some zygotes could be associated with a lower embryo developmental competence.

PSVII-17 Effects of zinc chloride supplementation during ovarian cortex vitrification prior to in vitro fertilization on embryo development in pigs

Ovarian tissue has an increased risk of damage during vitrification due to the presence of different cell types and water permeability levels within the tissue. Supplementation of antioxidant-like compounds such as zinc chloride improves follicular integrity and increases antral follicle development post-thawing. However, the effects of zinc chloride supplementation on the oocytes within the follicles are unknown. Therefore, the objective of this study is was to determine the effects of 5 μg/mL zinc chloride supplementation during the vitrification process on post-thawing fertilization characteristics and embryonic development. Ovarian cortex samples (5 x 5 mm; n=27) were isolated from ovaries extracted from cycling gilts. Cortical pieces were loaded onto a 25-gauge needle and incubated for five min in equilibrium solution followed by five min in vitrification solution, both supplemented with 5 μg/mL zinc chloride. Following incubation, the cortexes were place in liquid nitrogen for 7 d. Cortexes were then thawed and oocytes were aspirated from antral follicles. Oocytes (n=162) were incubated in maturation media for 40-44 h and then subjected to IVF and embryo culture. Frozen-thawed semen from three boars was used (30 oocytes/well, 200 sperm/oocyte). Post-IVF, a portion of the potential embryos (n=25) were evaluated for penetration, polyspermy, and male pronuclear formation rates. The remaining embryos (n=137) were evaluated 48 h after IVF for cleavage and 144 h for blastocyst formation. Data were analyzed using ANOVA and Tukey’s test. There were no differences between treatment groups when comparing penetration, 2-cell and blastocyst formation rates. However, polyspermic penetration rates were significantly lower (P &lt; 0.05) and male pronuclear formation were significantly higher (P &lt; 0.05) in the zinc chloride supplemented treatment group compared to no supplementation. These results indicate that supplementing zinc chloride during ovarian cortex freezing improves fertilization characteristics of their oocytes post-thawing and maturation.

Cysteamine in Maturation Medium Enhances Nuclear Maturation and Fertilization Rate of Sheep Oocytes In Vitro

Low nuclear maturation and fertilization rate is one obstacle in the in vitro embryo production which decrease embryo yield. This problem is presumable related with high production of reactive oxygen species (ROS) during maturation process. Glutathione (GSH) as an antioxidant is well known to overcome effect of ROS production. GSH synthesis in the cytosol part of the oocyte cytoplasm is influenced by cysteine availability. It is therefore, this research was conducted to evaluate the ability of cysteamine to provide cysteine availability as GSH precursor on the nuclear maturation and fertilization rate of sheep oocytes. Results of this experiment revealed additional cysteamine at 150 µm and 200 µm could significantly improve nuclear maturation rate. On the other side, although additional of cysteamine at 50 µm could not improve nuclear maturation rate, however 50 µm cysteamine in the maturation medium could significantly improve the fertilization rate. Based on those experiment results, it seems that the additional cysteamine might be improve not only GSH availability but also the oocyte quality which characterized the ability of pronuclear formation. This finding strongly suggested that additional cysteamine in the maturation medium could improve nuclear maturation and fertilization rate of sheep oocytes.

Coculture of porcine cumulus–oocyte complexes with porcine luteal cells during IVM: effect on oocyte maturation and embryo development

Coculture with somatic cells is an alternative to improve suboptimal invitro culture conditions. In pigs, IVF is related to poor male pronuclear formation and high rates of polyspermy. The aim of this study was to assess the effect of a coculture system with porcine luteal cells (PLCs) on the IVM of porcine cumulus–oocyte complexes (COCs). Abattoir-derived ovaries were used to obtain PLCs and COCs. COCs were matured invitro in TCM-199 with or without the addition of human menopausal gonadotrophin (hMG; C+hMG and C-hMG respectively), in coculture with PLCs from passage 1 (PLC-1) and in PLC-1 conditioned medium (CM). In the coculture system, nuclear maturation rates were significantly higher than in the C-hMG and CM groups, but similar to rates in the C+hMG group. In cumulus cells, PLC-1 coculture decreased viability, early apoptosis and necrosis, and increased late apoptosis compared with C+hMG. PLC-1 coculture also decreased reactive oxygen species levels in cumulus cells. After IVF, monospermic penetration and IVF efficiency increased in the PLC-1 group compared with the C+hMG group. After invitro culture, higher blastocysts rates were observed in the PLC-1 group. This is the first report of a coculture system of COCs with PLCs. Our model could be an alternative for the conventional maturation medium plus gonadotrophins because of its lower rates of polyspermic penetration and higher blastocysts rates, key issues in porcine invitro embryo production.

Homozygous mutations in REC114 cause female infertility characterised by multiple pronuclei formation and early embryonic arrest

BackgroundAbnormal pronuclear formation during fertilisation and subsequent early embryonic arrest results in female infertility. In recent years, with the prevalence of assisted reproductive technology, a few genes have been identified that are involved in female infertility caused by abnormalities in oocyte development, fertilisation and embryonic development. However, the genetic factors responsible for multiple pronuclei formation during fertilisation and early embryonic arrest remain largely unknown.ObjectiveWe aim to identify genetic factors responsible for multiple pronuclei formation during fertilisation or early embryonic arrest.MethodsWhole-exome sequencing was performed in a cohort of 580 patients with abnormal fertilisation and early embryonic arrest. Effects of mutations were investigated in HEK293T cells by western blotting and immunoprecipitation, as well as minigene assay.ResultsWe identified a novel homozygous missense mutation (c.397T>G, p.C133G) and a novel homozygous donor splice-site mutation (c.546+5G>A) in the meiotic gene REC114. REC114 is involved in the formation of double strand breaks (DSBs), which initiate homologous chromosome recombination. We demonstrated that the splice-site mutation affected the normal alternative splicing of REC114, while the missense mutation reduced the protein level of REC114 in vitro and resulted in the loss of its function to protect its partner protein MEI4 from degradation.ConclusionsOur study has identified mutations in REC114 responsible for human multiple pronuclei formation and early embryonic arrest, and these findings expand our knowledge of genetic factors that are responsible for normal human female meiosis and fertility.