The optimum oxygen tension for culturing mammalian embryos has been widely debated by the scientific community. While several laboratories have moved to using 5% as the value for oxygen tension, the majority of modern in vitro fertilization (IVF) laboratory programmes still use 20%. Several in vivo studies have shown the oxygen tension measured in the oviduct of mammals fluctuates between 2% and 8% and in cows and primates this values drops to <2% in the uterine milieu. In human IVF, a non-physiological level of 20% oxygen has been used in the past. However, several studies have shown that atmospheric oxygen introduces adverse effects to embryo development, not limited to numerous molecular and cellular physiology events. In addition, low oxygen tension plays a critical role in reducing the high level of detrimental reactive oxygen species within cells, influences embryonic gene expression, helps with embryo metabolism of glucose, and enhances embryo development to the blastocyst stage. Collectively, this improves embryo implantation potential. However, clinical studies have yielded contradictory results. In almost all reports, some level of improvement has been identified in embryo development or implantation, without any observed drawbacks. This review article will examine the recent literature and discusses ongoing efforts to understand the benefits that low oxygen tension can bring to mammal embryo development in vitro.

The effect of different dilution rates and equilibration times on the cryopreservation of Bactrian camel spermatozoa was evaluated in the current study. Semen samples from four healthy adult males were collected, processed and pooled. They were then subjected to a completely randomized 4×2 factorial design including four dilution rates (DR; 1:1, 1:2, 1:4 or 1:8; v:v with SHOTOR diluent) and two equilibration times (ET; 1 or 2 h at 5ºC). After freezing and thawing, sperm kinematic parameters as well as viability, plasma membrane integrity, abnormality and seminal malondialdehyde level were assessed. According to the results, four-fold diluted samples recorded significantly higher values (P < 0.05) for sperm total (39.58 vs 31.83 and 33.33,%) and progressive motility (19.50 vs 14.00 and 14.25,%), viability (55.37 vs 43.50 and 48.75,%) and plasma membrane integrity (46.75 vs 37.25 and 37.37,%) than those of both less (1:1) and high (1:8) concentrated samples, respectively. By contrast, the percentage of abnormal spermatozoa and the concentration of seminal malondialdehyde were comparable among all treated groups. Moreover, ET revealed that 1 h equilibration had significantly higher sperm motility (37.04 vs 33.33%), linearity (42.29 vs 32.26%), beat cross-frequency (13.15 vs 8.70 Hz), plasma membrane integrity (42.25 vs 39.75%) and viability (51.37 vs 48.12%) compared with 2 h of ET (P < 0.05). Taken together, a four-fold dilution along with 1 h equilibration can be an optimal procedure to cryopreserve Bactrian camel sperm.

Endometrial injury is an important cause of intrauterine adhesion (IUA), amenorrhea and infertility in women, with limited effective therapies. Recently, stem cells have been used in animal experiments to repair and improve injured endometrium. To date, our understanding of adipose-derived stem cells (ADSCs) in endometrial injury repair and their further therapeutic mechanisms is incomplete. Here, we examined the benefit of ADSCs in restoration of injured endometrium by applying a rat endometrial injury model. The results revealed by immunofluorescence showed that green fluorescent protein (GFP)-labelled ADSCs can differentiate into endometrial epithelial cells in vivo. At 30 days after ADSCs transplantation, injured endometrium was significantly improved, with increased microvessel density, endometrial thickness and glands when compared with the model group. Furthermore, the fertility of rats with injured endometrium in ADSCs group was improved and had a higher conception rate (60% vs 20%, P = 0.014) compared with the control phosphate-buffered saline (PBS) group. However, there was no difference in the control group compared with the sham group. In addition, expression levels of the oestrogen receptor Eα/β (ERα, ERβ) and progesterone receptor (PR) detected by western blot and enzyme-linked immunosorbent assay (ELISA) were higher in the ADSCs group than in the PBS group. Taken together, these results suggested that ADSC transplantation could improve endometrial injury as a novel therapy for IUA.

The present study evaluated the effects of low salinity on the early larval development of Oreochromis niloticus, specifically histological damage to white muscle, morphology of the yolk-sac surface and trunk area, and molecular expression of apoptosis and cell proliferation biomarkers. Newly hatched larvae were submitted to four salinity treatments for a period of 48 or 72 h, in duplicate: (S0) freshwater, (S2) 2 g l−1, (S4) 4 g l−1, and (S6) 6 g l−1NaCl. Larval development was examined using histology, electron microscopy, enzyme-linked immunosorbent assay (ELISA), and morphometry. At the yolk-sac surface, larvae of S4 and S6 displayed alterations to the apical opening of chloride cells that may be related to osmotic expenditure caused by the increased salinity. Caspase-3 expression did not differ significantly among treatments, however significantly lower proliferating cell nuclear antigen (PCNA) expression (P < 0.05) suggested minor cell proliferation in larvae of S4 and S6 compared with S0 and S2. Furthermore, there was a significant reduction in both trunk area and percentage of normal white muscle fibres (WF) in larvae of S4 and S6. Vacuolated areas and myofibrils concentrated at the cell periphery and found in the white muscle from larvae exposed to saline environments suggested disturbance to muscle development. Oedema and mononuclear infiltrate were also observed in the white muscle of S4 and S6 larvae. Together these results indicated that treatments with 4 and 6 g l−1 NaCl may cause osmoregulation expenditure, morphological alterations to the yolk-sac surface and histological damage to skeletal muscle that negatively affected the early larval development of O. niloticus.

We performed the exposure of bovine oocytes to anethole during in vitro maturation (0 or 300 µg/ml), during in vitro embryo production (0, 30, 300 or 2000 µg/ml), or during both periods to determine the rates of 2−4 cells embryos, blastocysts rates and cells numbers, as well as the production of reactive oxygen species (ROS). Bovine ovaries (n = 240) were collected from a local abattoir after slaughter and cumulus–oocyte complexes (COCs) with homogeneous and non-dark cytoplasm, surrounded by two or more compact layers of cumulus cells, and an intact zona pellucida were selected for in vitro maturatuion (IVM). Mature oocytes were then submitted to in vitro fertilization (IVF) and in vitro embryo production (IVP) in culture medium supplemented or not with different concentrations of anethole, as described above. Although IVM medium supplementation with 300 µg/ml anethole improved the rates of bovine blastocysts formation, we demonstrated that IVP medium supplementation with 30 µg/ml anethole, regardless of IVM medium enrichment, considerably enhanced blastocysts rates. Furthermore, ROS levels were decreased only when anethole was added to the IVP medium without previous IVM medium supplementation.

The complexity of predicting embryo development potential at the cleavage stages and the emergence of epigenetic risks during prolonged in vitro culture of pre-implantation embryos made it more advantageous to transfer embryos at the morula stage to the uterine cavity. The criteria for estimating embryos at this stage that allow prediction of cryopreservation outcomes have been poorly described. All day 4 embryos (n = 224) were graded 1, 2, 3, 4 or 5 according to blastomere compaction degree (BCD = 100, 75, 50, 25 or 0%, respectively) and the survival and blastocyst formation rate of these morulae were studied after cryopreservation. An inverse dependence was found between survival rate and BCD. Excluded fragments were characterized by low osmotic reaction during exposure to cryoprotective medium and, after freeze-thawing, they were destroyed. As damaged necrotic areas of the embryo can affect their further development rate we proposed blastomeres and biopsy fragments of incomplete compacted morula be removed before embryo cryopreservation. This step led to significant increase in the post-thawing survival rate up to 93.1 ± 4.1%, 75 ± 8.8% and blastocyst formation rate up to 85.2 ± 10.4%, 59.4 ± 5.2% in grade 2 and grade 3 embryos, respectively. There was no significant difference in grade 4 embryos. Therefore the removal of blastomeres and biopsy fragments in incomplete compacted morulae can improve cryopreservation outcomes of grade 2 and grade 3 embryos with BCD.

Reference gene selection in mouse oocytes is an important task required to perform further adequate analysis of target gene expression levels. In the current work we have analyzed expression stability of the seven most commonly used reference genes (Actb, Eef1e1, Gapdh, H2afz, Ppia, Rpl4 and Ubc) in mouse oocytes at the germinal vesicle (GV) stage. We have performed analysis of expression stability of the above-mentioned reference genes with the three most commonly used software tools: geNorm, BestKeeper and NormFinder. Taking into account the results obtained from all of these programmes Gapdh, Rpl4 and H2afz seem to be suitable candidate reference genes in GV oocytes of mouse.

Quality of in vitro-produced embryos is influenced by changes in gene expression in response to adverse conditions. Gene markers for predicting ‘good embryos’ do not exist at present. We propose that the expression of pluripotency markers OCT4–SOX2–NANOG in D9 (day 9) bovine demi-embryos correlated with development at D13 (day 13). Day 8 in vitro-produced blastocysts were split in two cloned halves, one half (D9) was subjected to analysis of pluripotency markers and the other was kept in culture until D13 of development. Embryo development was scored and correlated with its own status at D9 and assigned to one of two categories: G1, arrested/dead; or G2, development up to D13. SOX2 and NANOG expression levels were significantly higher in embryos from G1 and there was also negative correlation between SOX2 and embryo survival to D13 (G3; r = −0.37; P = 0.03). We observed a significant reduction in the expression of the three studied genes from D9 to D13. Furthermore, there was a correlation between the expression of pluripotency markers at D9 and embryo diameter and the expression of trophoblastic markers at D13 (TP1–EOMES–FGF4–CDX2–TKDP1). Finally, the quotient between the relative expression of SOX2 and OCT4 in the D9 blastocysts from G1 and G2 showed that embryos that were considered as competent (G2) had a quotient close to one, while the other group had a quotient of 2.3 due to a higher expression of SOX2. These results might indicate that overexpression of SOX2 at the blastocyst stage had a negative effect on the control of embryonic developmental potential.

Androgenetic embryonic stem (AgES) cells offer a possible tool for patient-specific pluripotent stem cells that will benefit genomic imprinting studies and clinic applications. However, the difficulty in producing androgenetic embryos and the unbalanced expression of imprinted genes make the therapeutic applicability of AgES cells uncertain. In this study, we produced androgenetic embryos by injecting two sperm into an enucleated metaphase II (MII) oocyte. By this method, 88.48% of oocytes survived after injection, and 20.24% of these developed to the blastocyst stage. We successfully generated AgES cell lines from the androgenetic embryos and assayed the expression of imprinted genes in the cell lines. We found that the morphological characteristics of AgES cells were similar to that of fertilized embryonic stem cells (fES), such as expression of key pluripotent markers, and generation of cell derivatives representing all three germ layers following in vivo and in vitro differentiation. Furthermore, activation of paternal imprinted genes was detected, H19, ASC12 and Tss3 in AgES cell activation levels were lower while other examined genes showed no significant difference to that of fES cells. Interestingly, among examined maternal imprinted genes, only Mest and Igf2 were significantly increased, while levels of other detected genes were no different to that of fES cells. These results demonstrated that activation of some paternal imprinted genes, as well as recovery of maternal imprinted genes, was present in AgES cells. We differentiated AgES cells into a beating embryoid body in vitro, and discovered that the AgES cells did not show significant higher efficiency in myocardial differentiation potential.

Human embryo studies have proposed the use of additional morphological evaluations related to the moment of the first cell divisions as relevant to embryo viability. Nevertheless, there are still not enough data available related to morphokinetic analysis and its relationship with lipid composition in embryos. Therefore, the aim of this study was to address the lipid profile of bovine embryos with different developmental kinetics: fast (four or more cells) and slow (two or three cells) at 40 h post-insemination (hpi), at three time points of in vitro culture (40, 112 and 186 hpi) and compare these to profiles of in vivo embryos. The lipid profiles of embryos were analyzed by matrix-assisted laser desorption ionization mass spectrometry, which mainly detected pools of membrane lipids such as phosphatidylcholine and sphingomyelin. In addition to their structural function, these lipid classes have an important role in cell signalling, particularly regarding events such as stress and pregnancy. Different patterns of lipids in the fast and slow groups were revealed in all the analyzed stages. Also, differences between in vitro embryos were more pronounced at 112 hpi, a critical moment due to embryonic genome activation. At the blastocyst stage, in vitro-produced embryos, despite the kinetics, had a closer lipid profile when compared with in vivo blastocysts. In conclusion, the kinetics of development had a greater effect on the membrane lipid profiles throughout the embryo culture, especially at the 8–16-cell stage. The in vitro environment affects lipid composition and may compromise cell signalling and function in blastocysts.

In response to gonadotropins and androgens, testicular cells produce various molecules that control proper proliferation and differentiation of spermatogenic cells through their paracrine and autocrine actions. However, molecules functioning downstream of the hormonal stimulation are poorly understood. Leukaemia inhibitory factor (Lif) is known to maintain the pluripotency of stem cells including embryonic stem cells and primordial germ cells at least in vitro, but its actual roles in vivo remain to be elucidated. To clarify the function of Lif in teleost (medaka) testes, we examined the effects of Lif on spermatogenesis in a newly established cell culture system using a cell line (named Mtp1) derived from medaka testicular somatic cells as feeder cells. We found that addition of baculovirus-produced recombinant medaka Lif to the culture medium or co-culture with Lif-overexpressing Mtp1 cells increased the number of spermatogonia. In situ hybridization and immunohistochemical analyses of the medaka testes showed that mRNAs and proteins of Lif are expressed in spermatogonia and the surrounding Sertoli cells, with higher expression levels in type A (undifferentiated) spermatogonia than in type B (differentiated) spermatogonia. Our findings suggest that Lif regulates spermatogonial cell proliferation in the medaka.

Long-term heat stress (HS) induced by testicular insulation generates oxidative stress (OS) on the testicular environment; consequently activating antioxidant enzymes such as superoxide dismutase (SOD), glutathione reductase (GR) and glutathione peroxidase (GPx). The aim of this work was to immunolocalize antioxidant enzymes present in different cells within the seminiferous tubule when rams were submitted to HS. Rams were divided into control (n = 6) and treated group (n = 6), comprising rams subjected to testicular insulation for 240 h. After the testicular insulation period, rams were subjected to orchiectomy. Testicular fragments were submitted to immunohistochemistry for staining against SOD, GR and GPx enzymes. We observed immunolocalization of GPx in more cell types of the testis after HS and when compared with other enzymes. In conclusion, GPx is the main antioxidant enzyme identified in testicular cells in an attempt to maintain oxidative balance when HS occurs.