Electromagnetic radiation (EMR) has deleterious effects on sperm motility and viability, as well as oocyte membrane and organelle structure. The aim was to assess the effects of cell phone radiation on preimplantation embryo morphokinetics and blastocyst viability in mice. For superovulation, 20 female mice were treated with intraperitoneal (IP) injections of 10 IU pregnant mare’s serum gonadotropin (Folligon® PMSG), followed by 10 IU of human chorionic gonadotropin (hCG) after 48 h. The zygotes (n = 150) from the control group were incubated for 4 days. The experimental zygotes (n = 150) were exposed to a cell phone emitting EMR with a frequency range 900–1800 MHz for 30 min on day 1. Then, all embryos were cultured in the time-lapse system and annotated based on time points from the 2-cell stage (t2) to hatched blastocyst (tHDyz), as well as abnormal cleavage patterns. Blastocyst viability was assessed using Hoechst and propidium iodide staining. Significant increases (P < 0.05) were observed in the cleavage division time points of t2, t8, t10, and t12 of the experimental group compared with the controls. In terms of blastocyst formation parameters, a delay in embryo development was observed in the experimental group compared with the controls. Data analysis of the time intervals between the two groups showed a significant difference in the s3 time interval (P < 0.05). Also, the rates of fragmentation, reverse cleavage, vacuole formation, and embryo arrest were significantly higher in the experimental group (P < 0.05). Furthermore, the cell survival rate in the experimental group was lower than the control group (P < 0.05). Exposure to EMR has detrimental consequences for preimplantation embryo development in mice. These effects can manifest as defects in the cleavage stage and impaired blastocyst formation, leading to lower cell viability.

Abnormalities in the zona pellucida (ZP) adversely affect oocyte maturation, embryo development and pregnancy outcomes. However, the assessment of severity is challenging. To evaluate the effects of different degrees of ZP abnormalities on embryo development and clinical outcomes, in total, 590 retrieval cycles were scored and divided into four categories (control, mild, moderate and severe) based on three parameters: perivitelline space, percentage of immature oocytes and percentage of oocytes with abnormal morphology. As the severity of abnormal ZP increased, both the number of retrieved oocytes and mature oocytes decreased. The fertilization rate did not differ significantly among groups. The rates of embryo cleavage and day-3 high-quality embryos in the mild group and the moderate group did not vary significantly between the two groups but were significantly higher than those in the severe group. The blastulation rates of the abnormal ZP groups were similar; however, they were lower than those of the control group. Moreover, the cycle cancellation rate of the severe abnormal ZP group was as high as 66.20%, which was significantly higher than that of the other three groups. Although the rates of cumulative clinical pregnancy and live births were lower than those in the control group, they were comparable among the abnormal ZP groups. There were no differences in the neonatal outcomes of the different groups. Together, ZP abnormalities show various degrees of severity, and in all patients regardless of the degree of ZP abnormalities who achieve available embryos, there will be an opportunity to eventually give birth.

The low maturation rate of oocytes is an important reason for female infertility and failure of assisted pregnancy. The germinal vesicle breakdown (GVBD) is a landmark event of oocyte maturation. In our previous studies, we found that zona pellucida 3 (ZP3) was strongly concentrated in the nuclear region of germinal vesicle (GV) oocytes and interacted with aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) and lamin A to promote GVBD. In the current study, we found that lamin A is mainly concentrated in the nuclear membrane. When ZP3 is knocked down, lamin A will be partially transferred to the nucleus of oocytes. The prelamin A is increased in both the nuclear membrane and nucleus, while phosphorylated lamin A (p-lamin A) is significantly reduced. AIPL1 was also proved to accumulate in the GV region of oocytes, and ZP3 deletion can significantly inhibit the aggregation of AIPL1 in the nuclear region. Similar to ZP3 knockdown, the absence of AIPL1 resulted in a decrease in the occurrence of GVBD, an increase in the amount of prelamin A, and a significant decrease in p-lamin A in oocytes developed in vitro. Finally, we propose the hypothesis that ZP3 can stabilize farnesylated prelamin A on the nuclear membrane of AIPL1, and promote its further processing into mature lamin A, therefore promoting the occurrence of GVBD. This study may be an important supplement for the mechanism of oocyte meiotic resumption and provide new diagnostic targets and treatment clues for infertility patients with oocyte maturation disorder.

Oxidative stress is an undesirable effect of in vitro culture, which requires antioxidant supplementation. This study investigated the analogue of resveratrol (RA33) as an alternative to resveratrol, an antioxidant molecule, for the in vitro culture of in vitro-fertilized bovine embryos. The effect of different concentrations of RA33 on embryo development was evaluated and a comparison between RA33 and resveratrol was performed. The cleavage rate was higher (P < 0.05) with 2.5 μM (69.0 ± 4.4%) than at 0, 0.1 or 0.5 μM RA33 (62.1 ± 2.0%, 60.7 ± 5.9% and 56.7 ± 5.8%, respectively). The blastocyst rates on days 7 and 8 post-fertilization with 2.5 μM RA33 (19.4 ± 3.3% and 24.6 ± 3.3%, respectively) were higher (P < 0.05) than for 0 μM (12.4 ± 2.5% and 15.2±2.5%, respectively). When 2.5 μM RA33 was compared with 0.5 μM resveratrol, similar (P > 0.05) cleavage and blastocyst rates were found between them, but the cleavage rate was higher (P < 0.05) in the control (80.8 ± 3.4%) than for the resveratrol treatment (76.4 ± 3.6%). The numbers of apoptotic cells and the apoptotic index were lower (P < 0.05) with RA33 (6.5 ± 0.6 cells and 6.4 ± 0.7%, respectively) and resveratrol (5 ± 0.8 cells and 5.5 ± 1.0%, respectively) than in the control group (9.8 ± 1.2 cells and 8.9 ± 1.1%, respectively). In conclusion, RA33 can enhance the preimplantation development of in vitro-fertilized bovine embryos and be an alternative to resveratrol in embryo culture medium.

The cause of hybrid sterility and inviability has not been analyzed in the fin-fish hybrid, although large numbers of hybridizations have been carried out. In this study, we produced allo-diploid hybrids by cross-fertilization between female goldfish (Carassius auratus) and male golden venus chub (Hemigrammocypris rasborella). Inviability of these hybrids was due to breakage of the enveloping layer during epiboly or due to malformation with serious cardiac oedema around the hatching stage. Spontaneous allo-triploid hybrids with two sets of the goldfish genome and one set of the golden venus chub genome developed normally and survived beyond the feeding stage. This improved survival was confirmed by generating heat-shock-induced allo-triploid hybrids that possessed an extra goldfish genome. When inviable allo-diploid hybrid cells were transplanted into goldfish host embryos at the blastula stage, these embryos hatched normally, incorporating the allo-diploid cells. These allo-diploid hybrid cells persisted, and were genetically detected in a 6-month-old fish. In contrast, primordial germ cells taken from allo-diploid hybrids and transplanted into goldfish hosts at the blastula stage had disappeared by 10 days post-fertilization, even under chimeric conditions. In allo-triploid hybrid embryos, germ cells proliferated in the gonad, but had disappeared by 10 weeks post-fertilization. These results showed that while hybrid germ cells are inviable even in chimeric conditions, hybrid somatic cells remain viable.

Much effort has been devoted to improving the efficiency of animal cloning. The aim of this study was to investigate the effect of BRG1 contained in Xenopus egg extracts on the development of cloned mouse embryos. The results showed that mouse NIH/3T3 cells were able to express pluripotent genes after treatment with egg extracts, indicating that the egg extracts contained reprogramming factors. After co-injection of Xenopus egg extracts and single mouse cumulus cells into enucleated mouse oocytes, statistically higher pronucleus formation and development rates were observed in the egg Extract− co-injected group compared with those in the no egg extract-injected (NT) group (38–66% vs 18–34%, P<0.001). Removal of BRG1 protein from Xenopus egg extracts was conducted, and the BRG1-depleted extracts were co-injected with single donor cells into recipient oocytes. The results showed that the percentages of pronucleus formation were significantly higher in both BRG1-depleted and BRG1-intact groups than that in the nuclear transfer (NT) group (94, 64% vs 50%, P<0.05). Furthermore, percentages in the BRG1-depleted group were even higher than in the BRG1-intact group (94% vs 64%). More confined expression of Oct4 in the inner cell mass (ICM) was observed in the blastocyst derived from the egg extract-injected groups. However, Nanog expression was more contracted in the ICM of cloned blastocysts in the BRG1-depleted group than in the BGR1-intact group. Based on the present study, BRG1 might not play an essential role in reprogramming, but the factors enhancing pronucleus formation and development of cloned mouse embryos are contained in Xenopus egg extracts.

The aim of our study was to compare the viability of sperm cells from transgenic (mWAP-hFVIII gene) or non-transgenic (normal) rabbit males as assessed by viability (SYBR-14/PI) and apoptosis (annexin V) tests. These results were evaluated using female conception rates following insemination with the respective sperm samples. No significant differences were found in concentration and motility between transgenic and non-transgenic spermatozoa. Spermatozoa from both transgenic (63.05 ± 20.05%) or non-transgenic (65.75 ± 22.15%) males, stained with SYBR-14 (green), were found to be morphologically normal. In both groups, the highest proportion of annexin V-positive sperm staining was found in the post-acrosomal part of the sperm head (8.66 and 27.53%). The percentage of sperm that stained with SYBR-14/PI or with annexin V/DAPI was correlated with liveborn in transgenic rabbits (R2 = 0.6118 and R2 = 0.2187, respectively) or non-transgenic rabbits (R2 = 0.671 and R2 = 0.3579, respectively). These data indicate that there was no difference in the viability of rabbit transgenic and non-transgenic spermatozoa when determined by both fluorescence assays.

The present study aimed to use an in vitro follicle culture (IVFC) biotechnique as a tool to evaluate the influence of whole flaxseed as a feed supplementation in the diet on the in vitro development of caprine early antral follicles (EAFs) and further embryo production. In total, 18 adult goats were homogeneously allocated into two diet groups: Control and Flaxseed. EAFs from both experimental groups (300–400 µm) were isolated and cultured in vitro for 18 days. After IVFC, recovered cumulus–oocyte complexes were submitted to in vitro maturation, and subsequently to IVF and in vitro embryo culture. The endpoints evaluated were follicular growth and morphology, oocyte recovery rate and diameter, sperm penetration, pronuclei formation, embryo development, and estradiol production. The addition of the whole flaxseed in the diet did not affect (P > 0.05) follicular growth and diameter. A higher (P < 0.05) percentage of oocytes ≥ 110 µm was recovered from the flaxseed treatment. However, the sperm penetration rate was higher (P < 0.05) in the control treatment when compared with the flaxseed treatment, but no differences were found regarding the rate of fertilization nor cleaved embryos. In conclusion, dietary flaxseed increased the recovery rate of fully grown oocytes, but it did negatively affect the sperm penetration rate, even though there was no further effect on the cleavage rate.

The sea urchin embryo is a National Institutes of Health model system that has provided major developments, and is important in human health and disease. To obtain initial insights to identify glycans that mediate cellular interactions, Lytechinus pictus sea urchin embryos were incubated at 24 or 30 h post-fertilization with 0.0009–0.03 M alpha-cyclodextrin, melibiose, l(–)-rhamnose, trehalose, d(+)-xylose or l(–)-xylose in lower-calcium artificial sea water (pH 8.0, 15°C), which speeds the entry of molecules into the interior of the embryos. While α-cyclodextrin killed the embryos, and l(–)-xylose had small effects at one concentration tested, l(–)-rhamnose caused substantially increased numbers of unattached archenterons and exogastrulated embryos at low glycan concentrations after 18–24 h incubation with the sugar. The results were statistically significant compared with the control embryos in the absence of sugar (P < 0.05). The other sugars (melibiose, trehalose, d(+)-xylose) had no statistically significant effects whatsoever at any of the concentrations tested. In total, in the current study, 39,369 embryos were examined. This study is the first demonstration that uses a live embryo assay for a likely role for l(–)-rhamnose in sea urchin gastrula cellular interactions, which have interested investigators for over a century.

The effects of the paternal breed on early embryo and later pre- and postnatal development are well documented. Several recent studies have suggested that such paternal effects may be mediated by the paternally induced epigenetic modifications during early embryogenesis. The objective of this study was to investigate the effects of the paternal breed on the early embryonic development and relative expression of the maternally imprinted gene, IGF-II, and the apoptosis-related genes BAK1 and BCL2-L1 in in vitro produced (IVP) bovine embryos derived from two unrelated paternal breeds (Holstein and Brown Swiss). The degree of correlation of IGF-II expression pattern with embryo developmental competence and apoptosis-related genes was also investigated. The relative abundance of IGF-II, BCL2-L1 and BAK1 transcripts in day 8 embryos was measured by quantitative reverse-transcription polymerase chain reaction using the comparative Cp method. Our data revealed that the paternal breed did not influence cleavage rate, blastocyst rate and relative abundance of IGF-II, BAK1 and BCL2-L1 in day 8 blastocysts (P > 0.05). Nevertheless, IGF-II expression levels were highly correlated with embryonic developmental competence (r = 0.66, P < 0.1), relative expression of BCL2-L1 (r = 0.72, P < 0.05) and ratio of BCL2-L1/BAK1 (r = 0.78, P < 0.05). In conclusion, our data show that IGF-II, BCL2-L1 and BAK1 expression is not related to the chosen combination of paternal breed, but that IGF-II expression is correlated with embryonic viability and apoptosis-related gene expression.

Pronuclear morphology seems to be an important predictive value of zygote development and integrity. In this study we want to evaluate the effect of insemination technique, male factor and oocyte cryopreservation on pronuclear morphology of zygotes derived from sibling oocytes in our Centre of Reproductive Medicine, Department of Obstetrics and Gynecology, Arcispedale S. Maria Nuova, Reggio Emilia, Italy. Subjects (n = 190) were submitted to IVF cycles with non-frozen and frozen sibling oocytes. Morphological evaluations were assessed using zygote pronuclear morphology (pronuclei, nucleoli and axis) in four groups: Group 1: 144 zygotes from 85 conventional IVF cycles with non-frozen oocytes; Group 2: 164 zygotes from 85 intracytoplasmic sperm injection (ICSI) cycles with Group 1 patients' sibling frozen oocytes; Group 3: 221 zygotes from 123 ICSI cycles with non-frozen oocytes; Group 4: 197 zygotes from 123 ICSI cycles with Group 3 patients' sibling frozen oocytes. No differences between Group 1 and Group 2 were seen. Group 3 was statistically different from Group 4 in relation to the nucleolar morphology. Oocyte cryopreservation procedure modified the nucleolar morphology of zygotes only in the presence of poor semen quality.

The announcement in 2019 of a new coronavirus disease that quickly became a major pandemic, is an exceptional challenge to healthcare systems never seen before. Such a public health emergency can largely influence various aspects of people’s health as well as reproductive outcome. IVF specialists should be vigilant, monitoring the situation whilst contributing by sharing novel evidence to counsel patients, both pregnant women and would-be mothers. Coronavirus infection might adversely affect pregnant women and their offspring. Consequently, this review paper aims to analyse its potential risks for reproductive health, as well as potential effects of the virus on gamete function and embryo development. In addition, reopening fertility clinics poses several concerns that need immediate addressing, such as the effect of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) on reproductive cells and also the potential risk of cross-contamination and viral transmission. Therefore, this manuscript summarizes what is currently known about the effect of the SARS-CoV-2 infection on medically assisted reproductive treatments and its effect on reproductive health and pregnancy.

The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) links the folate cycle that produces one-carbon units with the methionine cycle that converts these into S-adenosylmethionine (SAM), the universal methyl donor for almost all methyltransferases. Previously, MTHFR has been shown to be regulated by phosphorylation, which suppresses its activity. SAM levels have been shown to increase substantially soon after initiation of meiotic maturation of the mouse germinal vesicle (GV) stage oocyte and then decrease back to their original low level in mature second meiotic metaphase (MII) eggs. As MTHFR controls the entry of one-carbon units into the methionine cycle, it is a candidate regulator of the SAM levels in oocytes and eggs. Mthfr transcripts are expressed in mouse oocytes and preimplantation embryos and MTHFR protein is present at each stage. In mature MII eggs, the apparent molecular weight of MTHFR was increased compared with GV oocytes, which we hypothesized was due to increased phosphorylation. The increase in apparent molecular weight was reversed by treatment with lambda protein phosphatase (LPP), indicating that MTHFR is phosphorylated in MII eggs. In contrast, LPP had no effect on MTHFR from GV oocytes, 2-cell embryos, or blastocysts. MTHFR was progressively phosphorylated after initiation of meiotic maturation, reaching maximal levels in MII eggs before decreasing again after egg activation. As phosphorylation suppresses MTHFR activity, it is predicted that MTHFR becomes inactive during meiotic maturation and is minimally active in MII eggs, which is consistent with the reported changes in SAM levels during mouse oocyte maturation.

This study aimed to optimize the derivation of trophectoderm from in vitro-produced camel embryos under feeder-free culture conditions using the basement membrane matrix Matrigel. Trophoblastic vesicles were obtained through mechanical microdissection of in vitro-produced camel (Camelus dromedarius) embryos. Supplementing the culture medium with 10 ng/ml of epidermal growth factor and 10 ng/ml fibroblast growth factor improved the attachment and subsequent outgrowths of cultured trophoblastic vesicles when compared with the control group and the groups supplemented individually with each growth factor. The expression levels of pluripotency genes octamer-binding transcription factor 4 (Oct4), sex determining region Y-box 2 (Sox2), myelocytomatosis proto-oncogene (c-Myc) and anti-apoptotic gene B-cell lymphoma 2 (Bcl2) were increased in trophoblastic vesicles supplemented with both growth factors when compared with the control group. Conversely, both growth factors decreased the expression of apoptotic genes tumour protein p53 (p53) and Bcl-2-associated X protein (Bax). To the best of our knowledge, this may be the first report describing the derivation of trophoblast stem cells from in vitro-produced camel embryos.

In this study, we built on our previous research that discovered that autophagy activated the metaphase I stage during porcine oocytes in vitro maturation. We investigated the relationship between autophagy and oocyte maturation. First, we confirmed whether autophagy was activated differently by different media (TCM199 and NCSU-23) during maturation. Then, we investigated whether oocyte maturation affected autophagic activation. In addition, we examined whether the inhibition of autophagy affected the nuclear maturation rate of porcine oocytes. As for the main experiment, we measured LC3-II levels using western blotting after inhibition of nuclear maturation via cAMP treatment in an in vitro culture to clarify whether nuclear maturation affected autophagy. After autophagy inhibition, we also counted matured oocytes by treating them with wortmannin or a E64d and pepstatin A mixture. Both groups, which had different treatment times of cAMP, showed the same levels of LC3-II, while the maturation rates were about four times higher after cAMP 22 h treatment than that of the 42 h treatment group. This indicated that neither cAMP nor nuclear status affected autophagy. Autophagy inhibition during in vitro oocyte maturation with wortmannin treatment reduced oocyte maturation rates by about half, while autophagy inhibition by the E64d and pepstatin A mixture treatment did not significantly affect the oocyte maturation. Therefore, wortmannin itself, or the autophagy induction step, but not the degradation step, is involved in the oocyte maturation of porcine oocytes. Overall, we propose that oocyte maturation does not stand upstream of autophagy activation, but autophagy may exist upstream of oocyte maturation.

In each oestrous cycle only a limited number of follicles are selected for ovulation whereas the remaining majority undergo atresia. The earliest and most prominent feature of atresia is the death of granulosa cells. Recent biochemical evidence has demonstrated that granulosa cell death during follicular atresia in swine (Tilly et al., 1992), bovine (Jolly et al., 1994) and rodent (Tilly et al., 1991) ovaries occurs by apoptosis, a process whereby cells die in a controlled manner. A biochemical event considered to be characteristic of apoptotic cell death is the intranucleosomal cleavage of genomic DNA into fragments 180–200 bp in size, which separate into a distinctive ladder-like pattern on agarose gel electrophoresis. Detection of this pattern of oligonucleosomes in DNA provides a marker of apoptotic cell death.

Chicken primordial germ cells (PGCs) are the primary pluripotent stem cell types that will differentiate towards germ cells. High aldehyde dehydrogenase (ALDH) activity is considered as a functional marker for the detection of cell ‘stemness’. In our study the ALDEFLUOR™ kit was used for determination of ALDH activity in PGCs. PGCs were co-stained with diethylaminobenzaldehyde (DEAB) and ALDH and analyzed by flow cytometry. Our results showed a small cell population (8.0 ± 3.3%) upon preincubation of the cells with the specific inhibitor DEAB, however cells without inhibitor staining showed a fluorescence shift as an ALDH-positive population (70.5 ± 1.6%). These findings indicate higher expression of ALDH in PGCs and ALDH activity can therefore be used as a new functional marker for the detection of cell ‘stemness’ in chicken PGCs. These results may have importance for characterization of PGCs as a potential genetic resource in poultry. Further research is necessary to elucidate the role of this functional marker in these cells.

Growth hormone (GH) plays an important role in early embryo development. It has been shown to activate multiple pathways, the most comprehensively studied being the STAT/JAK (Signal transducers and activators of transcription/Janus kinase) pathway. The objective of the present study was to investigate STAT5A gene expression during early bovine embryogenesis. Real-time polymerase chain reaction (RT-PCR) was used to measure the abundance of STAT5A transcripts. The mRNA was present at all stages of preimplantation bovine embryos investigated. The most abundant STAT5A expression occurred at the 2-cell stage. Expression was markedly reduced between the 4-cell and 8-cell stages, coinciding with the known time of embryo genome activation and loss of maternal mRNAs. This finding suggests that the embryonic STAT5A gene is primarily activated by maternal gene products.

Traditionally, in vitro oocyte and embryo culture progresses through a series of varying culture medium. To investigate simplifying the in vitro production of bovine cumulus–oocyte complexes (COCs), this study used synthetic oviductal fluid (SOF) supplemented with conjugated linoleic acid (CLA). Special interest was placed on gene expression linked to lipid metabolism and oocyte maturation. COCs were matured in different media: Medium 199 (M199 group), M199 with 100 μM CLA (M199 + CLA group), SOF (SOF group), and SOF with 100 μM CLA (SOF + CLA group). COCs matured with SOF showed a higher relative abundance of mRNA of quality indicators gremlin 1 (GREM1) and prostaglandin-endoperoxide synthase 2 (PTGS2) in oocytes, and GREM1 in cumulus cells compared with in the M199 group. SOF medium COCs had a higher relative abundance of fatty acid desaturase 2 (FADS2) compared with the M199 group, which is essential for lipid metabolism in oocytes. Furthermore, the abundance of stearoyl-coenzyme A desaturase 1 (SCD1) in oocytes matured with SOF was not influenced by the addition of CLA, whereas the relative abundance of SCD1 was reduced in M199 medium with CLA. We concluded that maturation in SOF medium results in a greater abundance of genes linked to quality and lipidic metabolism in oocytes, regardless of the addition of CLA.