Search

Pregnancy maintenance in dromedary camels poses significant challenges, including early embryonic loss in the left uterine horn (LH) and unsuccessful pregnancy in the right uterine horn (RH), suggesting a potential asynchrony between conceptus signaling and uterine receptivity. The transition of the uterine epithelium from a pre-receptive to a receptive state requires a delicate balance of adhesion-promoting and anti-adhesion molecules. Mucin-1 (MUC1) acts as an anti-adhesive molecule on the uterine luminal (LE) and glandular (GE) epithelium. Downregulation of MUC1 is believed to be crucial for successful embryo attachment in various mammals. This study aimed to investigate the temporospatial expression of MUC1 in the LH and RH on Days 8, 10, and 12 pregnant dromedaries and their conceptuses. Quantitative real-time polymerase chain reaction (qrt-PCR), Western blot analysis, immunohistochemistry, and immunofluorescence techniques were employed to assess MUC1 expression at the mRNA and protein levels. The results demonstrated a reduction in MUC1 mRNA expression on Day 8, then increased on Day 10, followed by a decrease on Day 12 in LH. While the RH exhibited progressive increases, peaking on Day 12. However, MUC1 expression constantly exhibited higher levels in RH than in LH in all days. Two bands were detected at 150-kDa and 180-kDa, with the highest intensity observed on Day 10. Spatially, MUC1 was localized in the apical, cytoplasmic, and lumen of uterine glands only. MUC1 was barely detectable on Day 8 but gradually increased on Days 10 and 12 in both horns. Likewise, the RH exhibited higher MUC1 signals than the LH on Days 10 and 12. In the conceptuses, MUC1 mRNA increased on Day 8, peaked on Day 10, and declined on Day 12. Notably, MUC1 protein was detected in both the trophectoderm and endoderm, with high expression observed on Day 10 and reduced by Day 12. In conclusion, the decrease in MUC1 expression on Day 8 in the LH may be associated with maternal recognition of pregnancy (MRP), and the increase on Day 10 may related to embryo protection and movement, while the subsequent decrease on Day 12 could be linked to the embryo attachment and preparation for the implantation. Conversely, the increase of MUC1 in the RH implies a role in the anti-adhesion mechanism. These findings contribute to understanding MUC1's involvement in reproductive processes and provide insights into the complex mechanisms underlying successful pregnancy establishment and maintenance in dromedary camels. • Differential expression patterns of MUC1 in LH and RH of dromedary camels suggest potential functional specialization. • MUC1 reduction in LH and conceptus on Day 8 may be associated with MRP. • MUC1 elevation in LH and conceptus on Day 10 may play role in embryo movement. • MUC1 reduction in LH and conceptus on Day 12 may contribute to embryo attachment. • The increase of MUC1 in RH implies a crucial role as anti-adhesion mechanism. [ABSTRACT FROM AUTHOR]

Early embryonic mortality caused by maternal-fetal recognition failure in the three weeks after fertilization represents a major cause of reproductive inefficiency in the cattle industry. Modifying the amounts and ratios of prostaglandin (PG) F 2α and PGE 2 can benefit the establishment of pregnancy in cattle. Adding conjugated linoleic acid (CLA) to endometrial and fetal cells culture affects PG synthesis, but its effect on bovine trophoblast cells (CT-1) is unknown. The aim of this study was to determine the effects of CLA (a mixture of cis - and trans -9, 11- and −10,12-octadecadienoic acids) on PGE 2 and PGF 2α synthesis and the expression of transcripts involved with maternal-fetal recognition of bovine trophectoderm. Cultures of CT-1 were exposed to CLA for 24, 48 and 72 h. Transcript abundance was determined by qRT-PCR and hormone profiles were quantified by ELISA. The PGE 2 and PGF 2α concentrations were reduced in the culture medium of CLA-exposed CT-1 compared to that of unexposed cells. Furthermore, CLA supplementation increased the PGE 2 :PGF 2α ratio in CT-1 and had a quadratic effect (P < 0.05) on the relative expression of MMP9, PTGES2, and PTGER4. The relative expression levels of PTGER4 were reduced (P < 0.05) in CT-1 cultured with 100 μM CLA than in the unsupplemented and 10 μM-CLA groups. Treatment of CT-1 with CLA decreased PGE 2 and PGF 2α synthesis but a biphasic effect of CLA was observed on the PGE 2 :PGF 2α ratio and relative abundance of transcripts with 10 μM CLA providing maximal improvements in each endpoint. Our data suggest that CLA may influence eicosanoid metabolic process and extracellular matrix remodeling. • Fatty acid supplementation affects prostaglandin production. • CLA increases PGE 2 :PGF 2α ratio in bovine trophoblast cells. • CLA regulates eicosanoids and extracellular matrix remodeling pathways. [ABSTRACT FROM AUTHOR]

The global use of herbicides accounts for more than 48% of total pesticide usage. Picolinafen is a pyridine carboxylic acid herbicide that is predominantly used to control broadleaf weeds in wheat, barley, corn, and soybeans. Despite its widespread use in agriculture, its toxicity in mammals has rarely been studied. In this study, we first identified the cytotoxic effects of picolinafen on porcine trophectoderm (pTr) and luminal epithelial (pLE) cells, which are involved in the implantation process during early pregnancy. Picolinafen treatment significantly decreased the viability of pTr and pLE cells. Our results demonstrate that picolinafen increased the number of sub-G1 phase cells and early/late apoptosis. In addition, picolinafen disrupted mitochondrial function and resulted in the accumulation of intracellular ROS, leading to a reduction in calcium levels in both the mitochondria and cytoplasm of pTr and pLE cells. Moreover, picolinafen was found to significantly inhibit the migration of pTr. These responses were accompanied by the activation of the MAPK and PI3K signal transduction pathways by picolinafen. Our data suggest that the deleterious effects of picolinafen on the viability and migration of pTr and pLE cells might impair their implantation potential. [Display omitted] • Picolinafen decreases cell viability and alters cell cycle progression in trophectoderm. • Picolinafen causes ROS accumulation and disrupts intracellular calcium regulation. • Picolinafen interferes with mitochondrial integrity in trophectoderm and uterine cells. • Picolinafen alters the transcriptional regulation of genes involved in implantation. [ABSTRACT FROM AUTHOR]

Cellular differentiation induces various morphological changes, including elongation, in mitochondria. Preimplantation embryos have round-shaped mitochondria, characteristic of undifferentiated cells. However, there is controversy regarding the precise mitochondrial morphology in blastocyst embryos, which are generated from two cell lineages: undifferentiated inner cell mass (ICM) and differentiated trophectoderm (TE). This study attempted to precisely determine mitochondrial morphology in these two blastocyst regions. Transmission electron microscopy analyses were conducted using more than 1000 mitochondria from blastocyst embryos. No significant differences were observed in the configuration of mitochondrial cristae and frequencies of hooded mitochondria, which are specific to embryos of livestock animals, between the ICM and TE. To accurately compare mitochondrial roundness between the ICM and TE, oblateness was calculated based on both the major and minor axes. Average oblateness was significantly greater in the TE than in the ICM (P < 0.01). These results indicate tissue-specific mitochondrial maturation with complete elongation in the TE at the blastocyst stage. Since mitochondrial elongation is closely associated with cellular metabolism and differentiation, the present study provides new insights for better understanding of early embryonic development in cattle. • Mitochondrial ultrastructure of bovine blastocysts was determined by TEM analysis. • Evaluation of mitochondrial morphology by TEM was based on the oblateness value. • Mitochondria in the trophectoderm were more elongated than those in the inner cell mass. [ABSTRACT FROM AUTHOR]

Trophectoderm (TE) barrier function is an essential prerequisite for blastocyst development. CLAUDIN7 (CLDN7), a member of CLAUDINS family, is involved in regulating intercellular exchange and cell polarity in epithelium cells. However, the role of CLDN7 in porcine early embryo development is yet to be explored. Here, we found that CLDN7 was highly conserved in different species and was widely expressed in different tissues. Remarkably, CLDN7 expression maintained a low level from GV oocyte to 4-cell stage whereas its expression exhibited a higher level from 8-cell stage onwards. Microinjection of siRNA into cytoplasm effectively knocked down expression of CLDN7 mRNA and protein in porcine embryos. CLDN7 knockdown not only significantly reduced blastocyst rates of embryos derived from parthenogenetic activation and in vitro fertilization, but also reduced number of total cells and TE cells in the resulting blastocysts. Furthermore, CLDN7 knockdown led to a significant reduction in expression of multiple genes associated with tight junction assembly and fluid accumulation. A permeability assay revealed that CLDN7 knockdown disrupted tight junction assembly and paracellular sealing in the TE epithelium. Taken together, these results demonstrate that CLDN7 regulates porcine blastocyst development via modulating trophectoderm barrier function. • CLDN7 modulates expression of multiple genes associated with tight junction assembly and fluid accumulation. • CLDN7 is required for paracellular sealing in the trophectoderm epithelium. • CLDN7 is involved in regulating porcine blastocyst development. [ABSTRACT FROM AUTHOR]

Abstract This work examined the downstream fetal and placental outcomes of introducing a cocktail of uterine-derived growth factors during bovine embryo culture. Abattoir-derived bovine oocytes were matured and fertilized in vitro. On day 4 post-fertilization, ≥ 8-cell embryos were harvested, pooled and exposed to an embryokine mix, termed EFI, which contained recombinant human epidermal growth factor (10 ng/ml), bovine fibroblast growth factor-2 (10 ng/ml) and human insulin-like growth factor 1 (50 ng/ml) or to a carrier-only control treatment (CON). On day 7, individual, transfer-quality embryos were transferred to recipients. Timed ovulation was completed in mature, non-suckled commercial beef cows. Cows either were artificial inseminated (AI) or received an embryo (ET) on day 7 post-estrus (n = 23–31 cows/treatment over 4 replicate studies). The percentage of grade 1 and 2 morulae and blastocysts was greater (P < 0.05) for EFI-treated embryos than CON. The percentage of pregnant cows diagnosed by transrectal ultrasonography did not differ among the AI and ET groups on days 28, 42 and 56 post-estrus. There also were no differences in the ratio of male to female fetuses determined on day 60 post-estrus by transrectal ultrasonography. On day 21 post-estrus, the relative abundance of three interferon-stimulated gene (ISG) transcripts in peripheral leukocytes were not different based on AI/ET group or the sex of the conceptus. Circulating pregnancy-associated glycoprotein (PAG) concentrations differed (P < 0.05) among days. Also, a difference in PAG concentrations (P < 0.05) were detected between male and female pregnancies in the CON-ET group but not in the AI or EFI-ET groups. Crown-rump length was not affected by AI/ET group on day 42 but were less (P < 0.05) in the CON and EFI-ET groups than the AI group on day 56. These findings implicate EFI supplementation as a means for improving transferable embryo production in a bovine IVP system, but it is not clear if this treatment improves embryo competency after ET. Highlights • Supplementing a mix of uterine-produced growth factors improves in vitro bovine embryo production. • Growth factor treatment altered circulating pregnancy-associated glycoprotein concentration in a fetal sex-dependent manner. • Supplementing these embryokines did not correct the early fetal growth restriction observed with in vitro embryo culture. [ABSTRACT FROM AUTHOR]

In the uterine environment, the pre-implantation embryo adapts to low oxygen concentrations through intracellular responses including modification of gene expression, progression via the cell cycle and metabolism. In this study, we determined mechanisms underlying the adaptation of pig embryos to oxygen deficiency in the maternal-conceptus microenvironment in in vitro experiments using our established porcine trophectoderm (pTr) cells in culture. The transition from G 1 to S phase in pTr cells was reduced in response to 2% oxygen during a short period (<24 h), and the hypoxia-induced G 1 arrest was reversible during prolonged hypoxia exposure. Acute hypoxia up-regulated expression of transcription factors p21 and p27 and down-regulated cell cycle regulators associated with the G 1 /S phase transition including cyclin D1, cyclin E1 and E2F1 mRNAs and proteins. Furthermore, hypoxia exposure for 24 h markedly increased the abundance of HIF-1α protein. Even under acute hypoxia, by HIF-1α silencing reduced the hypoxia-induced transient G 1 arrest and expression of p21 and p27 genes was restored. Contrary to acute hypoxia, the accumulation of HIF-1α protein decreased as the length of the hypoxic period increased. Overall results of the present study suggest that increases in HIF-1α are responsible for initial response to hypoxia that results in a transient cell cycle arrest in pTr cells and cell cycle progression is restored by increasing degradation of HIF-1α during prolonged hypoxia. These findings advance understating of cellular adaptation of developing pre-implantation porcine conceptuses to hypoxic stress. [ABSTRACT FROM AUTHOR]

Abstract: Leukemia inhibitory factor (LIF) is a cytokine that shows conflicting effects on in vitro produced (IVP) bovine embryos. Bovine LIF (bLIF) has been cloned and used in culture, but there is no commercially available bLIF. Thus, researchers use human LIF (hLIF) to supplement the culture medium for bovine embryos because of its greater sequence homology compared to murine LIF (mLIF). We compared the effects of mLIF and hLIF on the development of bovine embryos in culture with the effects described for bLIF. Oocytes were matured and fertilized in vitro and cultured in modified synthetic oviduct fluid with BSA. On Day 6 post-insemination, morulae were cultured for 48h in the presence of: (1) mLIF, 100ngml−1; (2) hLIF, 100ngml−1; or (3) no LIF. Reduced blastocyst rates were observed on Day 8 for hLIF at the middle and expanded stages, while mLIF had no effect. In contrast, Day 8 blastocysts showed decreased cell counts both in terms of inner cell mass (ICM) and ICM/total cell proportions in the presence of mLIF, while hLIF had no effect. No changes were seen in trophectoderm (TE) and total cell counts. The increased hatching rates and TE cell counts previously described for bLIF, together with the disparate effects exhibited by hLIF and mLIF during blastocyst formation indicate these compounds are inappropriate to replace bLIF. We recommend that heterospecific LIF should not be used to supplement the culture medium for bovine embryo or embryonic stem cells. [Copyright &y& Elsevier]

Abstract: Evidence indicates that oocyte/embryo quality in the sheep is affected by nutrient status during the cycle of conception. This study aimed to determine, in the superovulated ewe, if there are stages during the peri-conception period (−18 days to +6 days relative to the day of ovulation [Day 0]) when quality is more likely to be influenced by nutrition. In Experiment 1, ewes were provided with either a 0.5×maintenance (L), 1.0×maintenance (M) or 1.5×maintenance (H) diet (in terms of daily energy requirements) during the peri-conception period. Diet did not affect the mean ovulation rate (range: 15.4±1.47 to 16.1±1.55) nor the mean number of embryos collected per ewe (range: 10.9±2.05 to 12.4±1.82) but there was an increase (P &#60;0.05) in the mean number of cells per blastocyst in the L diet (74.7±1.45) compared with either the M (66.4±1.29) or H (62.0±0.84) diets. This increase was due to an increase in the number of trophectoderm (Tr) cells, resulting in a shift (P &#60;0.05) in the Tr:inner cell mass (ICM) cell ratio (range 0.69±0.03 to 0.73±0.04). In Experiment 2, six diets (HHH, MHH, MHL, MLH, MLL and LLL) were imposed during three 6-day periods commencing 12 days before and continuing until 6 days after ovulation. Although diet had minimal effect on the superovulatory response, both the mean number of cells per blastocyst and the Tr:ICM ratio were increased (P &#60;0.05) when the L diet was provided after Day 0 (diets MHL, MLL and LLL). It is concluded that the ewe is able to respond to acute changes in nutrition imposed immediately after ovulation, resulting in changes in embryo development including cell lineage differentiation. The significance of these findings, in terms of fetal development, embryo-maternal signalling and the nutritional management of the ewe is discussed. [Copyright &y& Elsevier]

Abstract: The Wnt/β-catenin signaling pathway plays essential roles in the regulation of cell fate and polarity during embryonic development of many animal species. This study investigated the possible involvement of Wnt/β-catenin signaling pathway during hatching and trophectoderm (TE) development in pig blastocysts. Results showed that β-catenin and DVL3, the key mediators of Wnt/β-catenin signaling, disappeared from the nucleus after blastocyst hatching. Specific inhibition of Wnt/β-catenin signaling pathway, by Dickkopf-1, increased the rate of blastocyst hatching, total nuclear number per blastocyst, and reduced the ratio of inner cell mass (ICM):TE (P < 0.05). In contrast, specific activation of the Wnt/β-catenin signaling pathway, by lithium chloride, reduced the rate of blastocyst hatching, total nuclear number per blastocyst, and increased the ratio of ICM:TE (P < 0.05). The change in the ICM:TE ratio was associated with the change in the number of TE cells but not the ICM cells. Activation or inhibition of Wnt/β-catenin signaling and β-catenin nuclear accumulation, by lithium chloride or Dickkopf-1, also altered the expression of CDX2. These data therefore, suggest the possible involvement of Wnt/β-catenin signaling in regulating hatching and TE fate during the development of pig blastocyst. [Copyright &y& Elsevier]