Preimplantation Embryo Development Research Articles

Cd26 Knockdown Negatively Affects Porcine Parthenogenetic Preimplantation Embryo Development.

cd26 is ubiquitously distributed in the body, particularly in the endothelial and epithelial cells, with the highest expression in the kidney, liver, and small intestine. In humans, cd26 serves as a marker for the embryo implantation phase. However, little is known about the role of cd26 in porcine pre-implantation embryo development. Here, we aimed to examine siRNA-induced cd26 downregulation in the cytoplasm of MII oocytes, to determine whether cd26 is involved in the regulation of porcine pre-implantation embryonic development. The cd26 siRNA was micro-injected into the cytoplasm of MII oocytes, which were then parthenogenetically activated electrically in a medium containing 0.3M Mannitol. Inhibition of the cd26 expression did not affect cleavage but stopped development in the blastocyst stage. Additionally, the cd26 siRNA-treated blastocysts had significantly more apoptotic cells than the untreated blastocysts. Among the 579 transcripts evaluated with transcriptome resequencing, 38 genes were differentially expressed between the treatment and control blastocysts (p < 0.05). Twenty-four genes were upregulated in cd26 siRNA-injected blastocysts, whereas 14 were downregulated. These genes are involved in apoptosis, accumulation of reactive oxygen species, and aberrant expression of ribosomal protein genes. Our results indicate that cd26 is required for proper porcine parthenogenetic activation during embryonic development.

Glutamate can act as a signaling molecule in mouse preimplantation embryos†.

Free amino acids are present in the natural environment of the preimplantation embryo, and their availability can influence early embryo development. Glutamic acid is one of the amino acids with the highest concentrations in female reproductive fluids, and we investigated whether glutamic acid/glutamate can affect preimplantation embryo development by acting through cell membrane receptors. Using reverse transcription-polymerase chain reaction, we detected 15 ionotropic glutamate receptor transcripts and 8 metabotropic glutamate receptor transcripts in mouse ovulated oocytes and/or in vivo developed blastocysts. Using immunohistochemistry, we detected the expression of two α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits, three kainate receptor subunits, and member 5 metabotropic glutamate receptor protein in blastocysts. Extracellular concentrations of glutamic acid starting at 5 mM impaired mouse blastocyst development, and this fact may be of great practical importance since glutamic acid and its salts (mainly monosodium glutamate) are widely used as food additives. Experiments with glutamate receptor agonists (in combination with gene expression analysis) revealed that specific AMPA receptors (formed from glutamate receptor, ionotropic, AMPA3 [GRIA3] and/or glutamate receptor, ionotropic, AMPA4 [GRIA4] subunits), kainate receptors (formed from glutamate receptor, ionotropic, kainate 3 [GRIK3] and glutamate receptor, ionotropic, kainate 4 [GRIK4] or glutamate receptor, ionotropic, kainate 5 [GRIK5] subunits), and member 5 metabotropic glutamate receptor (GRM5) were involved in this effect. The glutamic acid-induced effects were prevented or reduced by pretreatment of blastocysts with AMPA, kainate, and GRM5 receptor antagonists, further confirming the involvement of these receptor types. Our results show that glutamic acid can act as a signaling molecule in preimplantation embryos, exerting its effects through the activation of cell membrane receptors.

TEAD4 regulates trophectoderm differentiation upstream of CDX2 in a GATA3-independent manner in the human preimplantation embryo.

TEAD4 regulates trophectoderm differentiation upstream of CDX2 in a GATA3-independent manner in the human preimplantation embryo.

Environment to embryo: intersections of contaminant exposure and preimplantation embryo development in agricultural animals†.

Environmental impacts on reproductive function are well documented in humans, yet little information is known about the effects on large animals. The interface of environment and reproduction has evolved prudently with a concerted effort to ensure global food sustainability tightly integrated with the application of technological advances in agriculture production that include nutrient and resource management. Exposure to environmental toxicants through chemical pesticide application and industry practices has coincided with a decline in cattle and human fertility. The increased adoption of agriculture animals for human biomedical models further emphasizes the importance of understanding the consequences of livestock exposure to environmentally and physiologically relevant levels of contaminants to preimplantation embryo development. In addition, increased awareness of paternal contributions to the early embryo that include both genetic and nongenetic factors supports the need to define environmental interactions from gamete to genome. Herein we summarize current knowledge of common environmental contaminants on reproductive function including direct and indirect effects on embryo development success in livestock. Information obtained from a diverse number of species including humans is presented to illustrate gaps in knowledge within livestock directly pertaining to agriculture success, sustainability, clinical practice, and biomedical research.

Nucleoporin37 may play a role in early embryo development in human and mice.

Maternal-effect genes (MEGs) play an important role in maintaining the survival and development of mammalian embryos at the cleavage stage after fertilization. Despite long-term efforts, the MEGs that regulate preimplantation embryo development remain largely unknown. Here, using whole-exome sequencing and homozygosity mapping, we identified a potential candidate gene associated with early embryo development: nucleoporin37 (NUP37), a nucleoporin gene that encodes a member of the nuclear pore complexes and regulates nuclear pore permeability and nucleocytoplasmic transport. Moreover, we determined the temporal and spatial expression patterns of Nup37 in mouse oocytes and early embryos, and explored the role of NUP37 in oocyte maturation and preimplantation embryo development. Immunoprecipitation assays confirmed that yes-associated protein-1 (YAP1) binds to TEA domain transcription factor 4 (TEAD4) and NUP37. Furthermore, Nup37 gene knockdown reduced the nuclear import of YAP1 and down-regulated the expression of YAP1-TEAD pathway downstream genes Rrm2 and Rpl13 in early embryos. Our study provides evidence that maternal NUP37 contributes to the nuclear import of YAP1 and then activates the YAP1-TEAD pathway, a signalling pathway essential for zygotic genome activation. Nup37 may be a key gene involved in preimplantation embryo development in mammals.

Linoleic acid reduces apoptosis via NF-κB during the in vitro development of induced parthenogenic porcine embryos

Fatty acid has a various role in preimplantation embryo development. Especially, Linoleic acid, polyunsaturated fatty acid, has been reported to affect the apoptosis pathway via nuclear transcription factor-kappa B. But to date, the function of NF-κB has not been demonstrated in porcine preimplantation embryos. We demonstrated that linoleic acid had a positive effect on embryo development at a certain concentration(25 μM), but developmental failure was observed at higher concentration. Furthermore, the expression level of NF-κB increased, unlike that of IL-6, as the concentration of linoleic acid increased. Interestingly, the concentration of NF-κB was found to increase even at the concentration of linoleic acid at which embryo development decreased. We found that pro-apoptotic gene expression was downregulated in the linoleic acid-treated group. It was also found that MCL-1, an anti-apoptotic gene known to be unaffected by IL-6, was found to be increased at the mRNA level in the linoleic acid-treated group. As the concentration of NF-kB increased, the nuclear translocation of C-JUN gradually increased dependent on the linoleic acid concentration. It was confirmed that NF-κB is an important factor in porcine embryos by treated ammonium pyrrolidinedithiocarbamate (APDC 0.1 μM, an inhibitor of NF-κB) affected NF-κB protein expression, IL-6 expression, and blastocyst production. These data supported porcine embryos can use exogenous linoleic acid as a metabolic energy source via NF-κB.

Ppan is essential for preimplantation development in mice†.

PETER PAN (PPAN), located to nucleoli and mitochondria, is a member of the Brix domain protein family, involved in rRNA processing through its rRNA binding motif and mitochondrial apoptosis by protecting mitochondria structure and suppressing basal autophagic flux. Ppan is important for cell proliferation and viability, and mutation of Ppan in Drosophila caused larval lethality and oogenesis failure. Yet, its role in mammalian reproduction remains unclear. In this study, we explored the function of Ppan in oocyte maturation and early embryogenesis using conditional knockout mouse model. Deficiency of maternal Ppan significantly downregulated the expression level of 5.8S rRNA, 18S rRNA, and 28S rRNA, though it had no effect on oocyte maturation or preimplantation embryo development. However, depletion of both maternal and zygotic Ppan blocked embryonic development at morula stage. Similar phenotype was obtained when only zygotic Ppan was depleted. We further identified no DNA binding activity of PPAN in mouse embryonic stem cells, and depletion of Ppan had minimum impact on transcriptome but decreased expression of 5.8S rRNA, 18S rRNA, and 28S rRNA nevertheless. Our findings demonstrate that Ppan is indispensable for early embryogenesis in mice.

Similar fertilization rates and preimplantation embryo development among testosterone-treated transgender men and cisgender women

What are the effects of testosterone treatment on oocyte fertilization and preimplantation embryo development among transgender men who have undergone fertility preservation? A retrospective study was undertaken in a university-affiliated tertiary hospital between April 2016 and November 2021. Embryos were divided into three groups by source: 210 embryos from 7 testosterone-exposed transgender men, 135 from 10 cisgender women who cryopreserved embryos, and 276 from 24 cisgender women who underwent fertility treatment. Statistical analyses compared assisted reproductive technology outcomes between the group of transgender men and both groups of cisgender women. Morphokinetic and morphological parameters were compared between the embryos derived from these three groups. The transgender men (30.2±3.5 years of age) were significantly younger than the cisgender women who cryopreserved embryos (35.1±1.8 years; P=0.005) and the cisgender women who underwent fertility treatment (33.8±3.2 years; P=0.017). After adjusting for participant age, the fertilization rate was comparable between the transgender men and both groups of cisgender women (P=0.391 and 0.659). There were no significant differences between the transgender men and the cisgender women who preserved fertility in terms of number of cryopreserved embryos (7.2±5.1 and 3.5±2.6; P=0.473) or the distribution of embryo age at cryopreservation (P=0.576). All morphokinetic parameters evaluated by time-lapse imaging, as well as the morphological characteristics, were comparable for the embryos in all three groups. Testosterone exposure among transgender men has no adverse impact upon fertilization rates or preimplantation embryo development and quality.

Fetal resorption coincides with dysregulated LH secretion in AMH-overexpressing mice

Female anti-Müllerian hormone (AMH) overexpressing (Thy1.2-AMHTg/0) mice experience fetal resorption (miscarriage) by mid-gestation. This study examined whether the ovary, uterine implantation sites and hypothalamus are potential sites of AMH action, as AMH type-2 receptor (AMHR2) expression is reported in each tissue. Pregnancy in Thy1.2-AMHTg/0 mice was compared to wild-type (WT) mice via histological examination of implantation sites, hormone assays, embryo culture and embryo transfer. Uterine AMH and AMHR2 expression was examined by RT-qPCR and immunohistochemistry. The first signs of fetal resorption in the Thy1.2-AMHTg/0 dams occurred at embryonic day 9.5 (E9.5) with 100% of fetuses resorbing by E13.5. Cultured embryos from Thy1.2-AMHTg/0 dams had largely normal developmental rates but a small proportion experienced a minor developmental delay relative to embryos from WT dams. However, embryos transferred from WT donor females always failed to survive to term when transferred into Thy1.2-AMHTg/0 dams. Amh and Amhr2 mRNA was detected in the gravid uterus but at very low levels relative to expression in the ovaries. Progesterone and estradiol levels were not significantly different between WT and Thy1.2-AMHTg/0 dams during pregnancy but luteinizing hormone (LH) levels were significantly elevated in Thy1.2-AMHTg/0 dams at E9.5 and E13.5 relative to WT dams. Collectively, these experiments suggest that AMH overexpression does not cause fetal resorption through an effect on oocytes or preimplantation embryo development. The Thy1.2-AMHTg/0 fetal resorption phenotype is nearly identical to that of transgenic LH overexpression models, suggesting that neuroendocrine mechanisms may be involved in the cause of the miscarriage.

Epigenetic modifiers during in vitro maturation as a strategy to increase oocyte competence in bovine

Oocyte in vitro maturation (IVM) is a key procedure for the in vitro production (IVP) of bovine embryos; however, the efficiency of this step is limited by the intrinsic developmental competence of oocytes. This study aimed to investigate possible epigenetic changes resulting from using of histone deacetylase (HDAC) inhibitors in the maturation of bovine oocytes and subsequent embryo development. Initially, we investigated the meiotic progression of bovine oocytes matured in vitro in the presence of trichostatin A (TSA). We then evaluated histone H3k9 acetylation levels in oocytes exposed to different TSA concentrations, and the relative expression of genes linked to oocyte competence. Finally, we studied pre-implantation embryonic development by analyzing the cleavage, blastocysts, and hatching rates. Acetylation levels of H3k9 increased (p < 0.05) when oocytes were exposed to 50 nM or 100 nM TSA during IVM, but there were no significant changes in the relative expression of the evaluated genes p34cdc2, cyclin B1, MAPK, GDF9, G6PDH, and HSP70. We found that 5 nM TSA promoted the attenuation of meiotic progression and positively affected pre-implantation embryo development in bovine species, allowing a 10% increase in the blastocyst rate. We concluded that TSA treatment during IVM was efficient in promoting changes in H3k9 acetylation levels from 50 nM and promoted attenuated meiotic progression in bovine oocytes at all concentrations evaluated, with a positive impact on pre-implantation development when used at low concentrations.

Maternal underweight does not adversely affect the outcomes of IVF/ICSI and frozen embryo transfer cycles or early embryo development

Objective To compare assisted reproductive technology (ART) outcomes and preimplantation embryo development between underweight and normal-weight women. Methods This retrospective cohort study included 26 underweight women (body mass index [BMI] < 18.50 kg/m2) and 104 normal-weight women (BMI >20 and <24.9 kg/m2) who underwent a total of 204 in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles and 358 fresh/frozen embryo transfers (ET) in our institution between January 2016 and December 2018. Statistical analyses compared selected ART outcomes (ovarian stimulation, fertilization, and pregnancy) between both weight groups. Morphokinetic and morphological parameters were also compared between 346 and 1467 embryos of underweight and normal-weight women, respectively. Results The mean ± standard deviation age of the underweight and normal-weight women was similar (31.6 ± 4.17 vs 32.4 ± 3.59 years; p = .323). There were no differences in the peak estradiol levels, the number of retrieved oocytes, the number of metaphase II oocytes, and the oocyte maturity rates between the two groups. The IVF/ICSI fertilization rates and the number of embryos suitable for transfer or cryopreservation were similar for both groups. All morphokinetic parameters that were evaluated by means of time-lapse imaging as well as the morphological characteristics were comparable between low and normal BMI categories. There were no significant differences in pregnancy achievement, clinical pregnancy, live births, and miscarriage rates between the suboptimal and optimal weight women. Conclusion Underweight status has no adverse impacts on the outcomes of IVF/ICSI with either fresh or frozen ET or on preimplantation embryo development and quality.

Placental Abnormalities are Associated With Specific Windows of Embryo Culture in a Mouse Model.

Assisted Reproductive Technologies (ART) employ gamete/embryo handling and culture in vitro to produce offspring. ART pregnancies have an increased risk of low birth weight, abnormal placentation, pregnancy complications, and imprinting disorders. Embryo culture induces low birth weight, abnormal placental morphology, and lower levels of DNA methylation in placentas in a mouse model of ART. Whether preimplantation embryos at specific stages of development are more susceptible to these perturbations remains unresolved. Accordingly, we performed embryo culture for several discrete periods of preimplantation development and following embryo transfer, assessed fetal and placental outcomes at term. We observed a reduction in fetal:placental ratio associated with two distinct windows of preimplantation embryo development, one prior to the morula stage and the other from the morula to blastocyst stage, whereas placental morphological abnormalities and reduced imprinting control region methylation were only associated with culture prior to the morula stage. Extended culture to the blastocyst stage also induces additional placental DNA methylation changes compared to embryos transferred at the morula stage, and female concepti exhibited a higher loss of DNA methylation than males. By identifying specific developmental windows of susceptibility, this study provides a framework to optimize further culture conditions to minimize risks associated with ART pregnancies.

Novel Homozygous PADI6 Variants in Infertile Females with Early Embryonic Arrest.

Early embryonic arrest denotes premature termination of development in preimplantation embryos, which is one of the major phenotypes of recurrent assisted reproduction failure. Padi6 is proven to be a member of the subcortical maternal complex (SCMC) in mice, which is essential in oocyte maturation and embryogenesis. We and other groups previously found that biallelic mutations in PADI6 caused female infertility manifesting as early embryonic arrest. In this study, we identified two novel homozygous variants (p.Cys163Arg, and p. Trp475*) of PADI6 in two infertile patients from a cohort of 75 females with the phenotype of early embryonic arrest. An in vitro expression study indicated severe decrease of PADI6, which might destruct the stability of SCMC. Our study expands the mutational spectrum of PADI6 and further supports the causality between PADI6 mutations and female infertility.

DNA methylation profiles after ART during human lifespan: a systematic review and meta-analysis.

The many manipulations and processes used in ART coincide with the timing of epigenetic reprogramming and imprinting during female gametogenesis and pre-implantation embryo development, leading to concerns that the actual ART could negatively affect epigenetic reprogramming and imprinting in gametes and early embryos. A growing body of literature suggests that ART may affect epigenetic marks, such as DNA methylation, in the fetus and placenta. Potentially, this may be responsible later in life for the increased risk of adverse outcomes associated with ART. Unfortunately, the conclusions are inconsistent and, despite the increasing usage of ART, its safety at the epigenetic level is still not established. To examine whether ART is associated with DNA methylation modifications and if these modifications persist throughout life, we provide an update on the current understanding of epigenetic reprogramming in human gametes and embryos, and then focus on the assessment of fetal and postnatal DNA methylation modifications that may remain until adulthood following the use of ART in humans. We reviewed studies using targeted or epigenome-wide techniques to assess the DNA methylation patterns of the conceptus after ART compared with natural conceptions. A search for relevant studies was performed in the PubMed and EMBASE databases on 15 July 2021 with an extensive search equation. Studies on animals, gametes and embryos were subsequently excluded. After an in-depth review of full-text articles, studies on specific populations with imprinting disorders were removed and not further discussed. Before comprehensive analysis, the risk of bias of each included study was assessed with the Newcastle-Ottawa scale and quality of evidence was graded using the Grading of Recommendations, Assessment, Development and Evaluations criteria. In total, 928 records were initially identified, and 51 were finally included in the systematic review. Given the variability in the genomic scale at which DNA methylation was measured in the different studies, they were separated into two categories: targeted DNA methylation or genome-wide DNA methylation study. The present systematic review has made it possible to assess a substantial number of children since more than 4000 DNA methylation profiles of ART concepti were compared to more than 7000 controls. There is evidence that ART conception is associated with aberrant DNA methylation in imprinted loci and other genes in various tissues. One isolated modification notably occur in the paternally expressed gene 1/mesoderm-specific transcript homologue (PEG1/MEST) region, and we cannot rule out other studied sequences owing to the heterogeneity of the evidence base. Differences in DNA methylation after ART conceptions are modest, and the functional relevance in adult tissues is unknown. Functional effects in terms of gene expression as well as the roles of other epigenetic marks need to be further explored. Moreover, there is little overlap of findings obtained in targeted and genome-scale analyses owing to the lack of comparability of CpGs analyzed between both techniques. This issue also stems from small sample sizes and marked differences in methodology and cohort characteristics. Standardization of methodologies and large collaborative efforts are required to reduce the inconsistency of results and increase the robustness of findings. Finally, further studies are required to determine the contribution of parental infertility per se from the ART treatment.

EP267: De novo COL11A1 and COL9A2 variants corresponding to Stickler syndrome phenotype

Stickler Syndrome is a rare clinically and genetically heterozygous connective tissue disorder with variable clinical presentation including serious vision (eg, myopia, cataract, and retinal detachment), hearing loss (eg, sensorineural, mixed and/or conductive), craniofacial abnormalities (midface underdevelopment and cleft palate) and joint problems (eg, precocious arthritis, spondyloepiphyseal dysplasia). Stickler syndrome is associated with pathogenic variants in the COL2A1, COL11A1, COL11A2, COL9A1, COL9A2 and COL9A3 genes are present. This syndrome is mainly inherited as an autosomal dominant pathology. We are presenting the case of a 15-year-old female who was referred to genetics by cardiology due to hypoglycemia, tachycardia and to rule out Marfan syndrome due to Marfanoid habitus. Upon physical examination there was evidence of joint hyperlaxity and decreased muscle tone. She also presented mild thoracolumbar dextroscoliosis of 25 degrees in curvature documented by spine X-rays. A connective tissue disorder gene panel was requested. The results indicate that the patient is heterozygous for variants in three genes: COL11A1, COL9A2 and NEPRO. The COL11A1 gene is responsible for providing the blueprint to synthesize a component of type XI collagen, which is commonly located in cartilage. Type XI collagen can also be found in the inner ear, the vitreous and in the nucleus pulposus. A sequence variant c.4718C>T in the COL11A1 gene was identified in this patient. This change is expected to cause an amino acid substitution p.Ser1573Leu, which has not been reported in the literature. Pathogenic variants in COL11A1 have been associated with autosomal dominant Stickler syndrome type II, autosomal dominant Marshall syndrome, autosomal dominant deafness 37 as well as autosomal recessive fibrochondrogenesis 1. COL9A2 gene provides the blueprint for synthesizing a part of a large molecule called type IX collagen, which is more flexible than other types of collagen. A sequence variant designated as c.1553G>T was documented in this gene and is expected to result in an amino acid substitution designated as p.Arg518Leu. Pathogenic variants in COL9A2 have been linked with autosomal recessive Stickler syndrome type V and autosomal multiple epiphyseal dysplasia 2. This variant has not been reported in the literature. The NERPRO (Nucleolus and neural progenitor protein) gene could be involved in cortex development as part of the Notch signaling pathway and it may also be involved in preimplantation embryo development. A NEPRO gene sequence variant designated as c.871G>A was documented as well. This sequence variant is expected to result in an amino acid substitution p.Val291Met. This variant has not been reported in the literature. Fifteen years old female with genetic variants on COL11A1, COL9A2 and NEPRO genes. The patient presents scoliosis, myopia, and amblyopia, micrognathia, hyperlaxed joints and hypotonia. The COL11A1 and the COL9A2 genes has been associated with autosomal dominant and autosomal recessive Stickler syndrome phenotypes respectively. Stickler syndrome is a connective tissue disorder that can include ocular findings of myopia, cataract, and retinal detachment; hearing loss that is both conductive and sensorineural; midfacial underdevelopment and cleft palate (either alone or as part of the Robin sequence). The COL11A1 variant has not been reported in the medical literature associate to the disease however predictive algorithms suggest the possibility of a deleterious effect on the protein. The patient also has a genetic variant on the COL9A2 gene that has not been associate to the disease but haploinsufficiency mechanisms may also be playing a role in this patient phenotype.

Changes of the zona pellucida patterns during oocyte maturation, fertilization and embryo development in mammals: mini-review

Abstract The mammalian zona pellucida (ZP) is an extracellular matrix that surrounds immature and mature oocytes and early embryos until the stage of a blastocyst and its implantation. This mini-review summarizes basic information on the ZP and its morphologic and functional changes during in vitro oocyte maturation and fertilization and in vivo pre-implantation embryo development.

Effects of embryonic stem cell-conditioned medium on the preimplantation development of mouse embryos.

The production of high-quality embryos in the laboratory and a successful pregnancy are closely related to the condition and contents of oocyte and embryo culture media. In this study, we investigated the effects of embryonic stem cell-conditioned medium (ESCCM) and embryonic stem cells growth medium (ESCGM) compared with potassium-enriched simplex optimized medium (KSOM) on preimplantation embryo development stages during natural or in vitro fertilization (IVF). Birth rate of pups was measured. To obtain mature oocytes, and 2-cell and 8-cell embryos, human chorionic gonadotropin (HCG) was injected 48 h after i.p. injection of 5 units of pregnant mare serum gonadotropin. Mature oocytes were obtained from non-mated female mice 14 h after HCG injection. To obtain 2-cell and 8-cell embryos, mated female mice, 1 day and 3 days, respectively, after HCG injection, were used. Mature oocytes were fertilized in HTF medium. Embryos obtained from natural or in vitro fertilization were cultured in experimental media, ESCCM and ESCGM, or KSOM as the control culture medium. Embryos that developed to the blastocyst stage were transferred to the uteri of pseudopregnant mice and effects of the experimental media on embryo viability were determined. ESCCM and ESCGM could not pass the embryo after the 2-cell stage, but they were suitable for the development of the embryo from the 8-cell stage to the blastocyst. It can be concluded that the embryo has various requirements at different stages of development.

Folate receptor-1 is vital for developmental competence of goat embryos.

Folate is essential for DNA synthesis and methylation via one-carbon (C1) metabolism during embryonic development. It is transported into the developing oocytes via folate receptors (FOLR1 and FOLR2) and transporters (RFC1) for utilization during embryo development. However, the role of folate receptors during pre-implantation stages of embryos is not well known. Thus, the present study aimed to investigate the expression of folate transport genes and proteins in mature oocytes and pre-implantation embryos and the effect of FOLR1 knockdown in zygotes on blastocyst outcome. For this, immature goat oocytes were matured in maturation medium followed by in vitro fertilization and culture at standard conditions. A group of zygotes was transfected with esiRNA against FOLR1 and in vitro cultured for blastocyst outcome assessment. The transcripts and proteins for FOLR1, FOLR2 and RFC1 were present in oocytes as well as all the stages of pre-implantation embryos. Immunofluorescence revealed the presence of FOLR1 in the nuclei of embryos but not in the metaphase (matured) oocytes. The knockdown of FOLR1 in embryos was effective and significantly reduced the blastocyst production rate. The present study demonstrates the existence of active folate transport in oocytes and pre-implantation goat embryos. FOLR1 is vital for pre-implantation embryo development and may aid in the progression by functioning as a transcription factor.

Protein Expression Landscape Defines the Formation Potential of Mouse Blastoids From EPSCs.

Preimplantation embryo development is a precisely regulated process organized by maternally inherited and newly synthesized proteins. Recently, some studies have reported that blastocyst-like structures, named blastoids, can be generated from mouse ESCs (embryonic stem cells) or EPSCs (extended pluripotent stem cells). In this study, to explore the dynamic expression characteristics of proteins and their PTMs in mouse EPS blastoids, we revealed the protein expression profile of EPS blastoids and metabolite characteristics by TMT-based quantitative mass spectrometry (MS) strategy. Furthermore, the protein phosphorylation sites were identified to show the phosphoproteomic analysis in blastoids compared with mouse early embryos. Above all, our study revealed the protein expression profile of EPS blastoids compared with mouse embryos during preimplantation development and indicated that glucose metabolism is key to blastoid formation.

Repro-protective role of royal jelly in phenylhydrazine-induced hemolytic anemia in male mice: Histopathological, embryological, and biochemical evidence.

To estimate the repro-protective effect of royal jelly (RJ) on phenylhydrazine (PHZ)-induced anemia's detrimental effects, 24 mature mice were divided into control group (0.10mL normal saline; intra-peritoneally), RJ group (100 mg/kg/day; orally), experimental anemia (EA) group that received only PHZ (6mg/100 g/48 h; intra-peritoneally), and RJ + EA (according to the previous prescription) group. After 35 days, testicular histoarchitecture, RNA damage in germinal cells, sperm characteristics, testicular total anti-oxidant capacity and malondialdehyde as well as serum testosterone levels, pre-implantation embryo development and cyclin D1 and c-myc mRNA levels at two-cell, morula and blastocyst stages were analyzed. Spermatogenesis indices were ameliorated following RJ co-administration. Moreover, RJ co-treatment reduced germinal cells RNA damage, improved sperm characteristics, boosted pre-implantation embryo development and restored androgenesis, and oxidant/anti-oxidant status. Co-administration of RJ also decreased mRNA levels of cyclin D1 and up-regulated those of c-myc in two-cell embryos, morulas and blastocysts. The findings suggest that RJ can play a repro-protective role in PHZ-induced anemia in mice through anti-oxidant defense system reinforcement and androgenesis restoration as well as cyclin D1 and c-myc expressions regulation.