Oviductal extracellular vesicles miRNA cargo varies in response to embryos and their quality.

BACKGROUND: Increasing evidence points to an active role of oviductal extracellular vesicles (oEVs) in the early embryo-maternal dialogue. However, it remains unclear whether oEVs contribute to the recognition of the presence of embryos and their quality in the oviduct. Hence, we examined whether the molecular cargo of oEVs secreted by bovine oviduct epithelial cells (BOEC) differs depending on the presence of good (≥ 8 cells, G) or poor (< 8 cells, P) quality embryos. In addition, differences in RNA profiles between G and P embryos were analyzed in attempt to distinguish oEVs and embryonic EVs cargos. METHODS: For this purpose, primary BOEC were co-cultured with in vitro produced embryos (IVP) 53 h post fertilization as follows: BOEC with G embryos (BGE); BOEC with P embryos (BPE); G embryos alone (GE); P embryos alone (PE); BOEC alone (B) and medium control (M). After 24 h of co-culture, conditioned media were collected from all groups and EVs were isolated and characterized. MicroRNA profiling of EVs and embryos was performed by small RNA-sequencing. RESULTS: In EVs, 84 miRNAs were identified, with 8 differentially abundant (DA) miRNAs for BGE vs. B and 4 for BPE vs. B (P-value < 0.01). In embryos, 187 miRNAs were identified, with 12 DA miRNAs for BGE vs. BPE, 3 for G vs. P, 8 for BGE vs. GE, and 11 for BPE vs. PE (P-value < 0.01). CONCLUSIONS: These results indicated that oEVs are involved in the oviductal-embryo recognition and pointed to specific miRNAs with signaling and supporting roles during early embryo development.

Nobiletin as a novel agent to enhance porcine in vitro embryo development and quality.

In vitro embryo production (IVP) is of great importance to the porcine industry, as well as for basic research and biomedical applications. Despite the large efforts made in laboratories worldwide to address suboptimal culture conditions, porcine IVP remains inefficient. Nobiletin (Nob, 5,6,7,8,3',4' hexamethoxyflavone) supplementation to in vitro culture (IVC) medium, enhances in vitro embryo development in various species. However, its impact on the quality and developmental capacity of in vitro-produced pig embryos is yet to be established. This study evaluated the effects of different concentrations (2.5 and 5 µM) of Nob during the early culture of in vitro-produced pig embryos on embryo developmental competence, mitochondrial activity, lipid content, intracellular Reactive Oxygen Species (ROS) and Glutathione (GSH) content, Total Cell Number (TCN) per blastocyst, and expression of genes related to embryo development, quality and oxidative stress. Embryos cultured in medium without Nob supplementation and in medium supplemented with 0.01 % dimethyl sulfoxide (DMSO-vehicle for Nob) constituted the Control and DMSO groups, respectively. Embryo development rates were evaluated on Days 2, 6 and 7 of IVC. Additionally, a representative group of embryos was selected to assess mitochondrial activity, lipid, ROS and GSH content (on Days 2 and 6 of IVC), TCN assessment and gene expression analyses (on Day 6 of IVC). No significant differences were observed in any of the parameters evaluated on Day 2 of IVC. In contrast, embryos cultured under the presence of Nob 2.5 showed higher developmental rates on Days 6 and 7 of IVC. In addition, Day 6 embryos showed increased mitochondrial activity, with decreased levels of ROS and GSH in the Nob 2.5 group compared to the other groups. Both Nob 2.5 and Nob 5 embryos showed higher TCN compared to the Control and DMSO groups. Furthermore, Nob 2.5 and Nob 5 upregulated the expression of Superoxide dismutase type 1 (SOD1) and Glucose-6-phosphate dehydrogenase (G6PDH) genes, which could help to counteract oxidative stress during IVC. In conclusion, the addition of Nob during the first 48 h of IVC increased porcine embryo development rates and enhanced their quality, including the upregulation of relevant genes that potentially improved the overall efficiency of the IVP system.

MicroRNA-148b secreted by bovine oviductal extracellular vesicles enhance embryo quality through BPM/TGF-beta pathway

Background Extracellular vesicles (EVs) and their cargoes, including MicroRNAs (miRNAs) play a crucial role in cell-to-cell communication. We previously demonstrated the upregulation of bta-mir-148b in EVs from oviductal fluid of cyclic cows. This miRNA is linked to the TGF-β pathway in the cell proliferation. Our aim was to verify whether miR-148b is taken up by embryos through gymnosis, validate its target genes, and investigate the effect of miR-148b supplementation on early embryo development and quality. Methods Zygotes were cultured in SOF + 0.3% BSA (Control) or supplemented with: 1 μM miR-148b mimics during: D1-D7 (miR148b) or D1-D4 (miR148b-OV: representing miRNA effect in the oviduct) or D4-D7 (miR148b-UT: representing miRNA effect in the uterus) or 1 μM control mimics was used during: D1-D7 (CMimic). Embryos at ≥ 16-cells and D7 blastocysts (BD7) were collected to examine the mRNA abundance of transcripts linked to the TGF-β pathway (TGFBR2, SMAD1, SMAD2, SMAD3, SMAD5, BMPR2, RPS6KB1, POU5F1, NANOG), total cell number (TC), trophectoderm (TE), and inner cell mass (ICM) were also evaluated. One-way ANOVA was used for all analyses. Results We demonstrated that miR-148b can be taken up in both 16-cell embryos and BD7 by gymnosis, and we observed a decrease in SMAD5 mRNA, suggesting it's a potential target of miR-148b. Cleavage and blastocysts rates were not affected in any groups; however, supplementation of miR-148b mimics had a positive effect on TC, TE and ICM, with values of 136.4 ± 1.6, 92.5 ± 0.9, 43.9 ± 1.3 for miR148b and 135.3 ± 1.5, 92.6 ± 1.2, 42.7 ± 0.8, for miR148b-OV group. Furthermore, mRNA transcripts of SMAD1 and SMAD5 were decreased (P ≤ 0.001) in 16-cell embryos and BD7 from miR148b and miR148b-OV groups, while POU5F1 and NANOG were upregulated (P ≤ 0.001) in BD7 and TGFBR2 was only downregulated in 16-cell embryos. pSMAD1/5 levels were higher in the miR148b and miR148b-OV groups. Conclusions Our findings suggest that supplementation of bta-miR-148b mimics during the entire culture period (D1 - D7) or from D1 - D4 improves embryo quality and influences the TGF-β signaling pathway by altering the transcription of genes associated with cellular differentiation and proliferation. This highlights the importance of miR-148b on embryo quality and development.

Oviductal epithelial cells transcriptome and extracellular vesicles characterization during thermoneutral and heat stress conditions in dairy cows.

In this study, the transcriptome of oviductal epithelial cells and certain characteristics of their extracellular vesicles of dairy cows were described under thermoneutral and heat stress conditions. Twenty cows were compared in springtime at THI = 65.6 ± 0.90 and in summertime at THI = 78.36 ± 2.73. During each season, the estrous cycles of the cows were synchronized, and on day 3 of the ensuing cycle, a blood sample was collected for progesterone determination, while their oviducts were collected after slaughter. Epithelial cells and oviductal fluid were collected from the oviduct ipsilateral and contralateral to the corpus, respectively. For the gene expression study, a comparative transcriptomic approach, using RNASeq, was performed on cells collected from the ipsilateral and the contralateral oviducts. The size and the concentration of extracellular vesicles (EVs) at both seasons were analyzed using Transmission Electron Microscopy and Nanoparticle tracking analysis and specific proteins were detected by Western blotting. Progesterone concentration was higher during the thermoneutral period. Between seasons, divergent expression of genes related to immune system, contractility, gamete protection and lncRNAs was found. The size and the concentration of the EVs did not differ between seasons, however, the concentration in the ipsilateral oviduct tended to be lower (p = 0.09) from the contralateral one in the summer, but not in the spring. Our results show for the first time that HS could be involved with alterations in the oviductal cells' gene expression and in the changes in concentration of EVs in the oviductal lumen. Our results imply that the altered oviductal environment during HS could be associated with the suppressed summer fertility in dairy cows.

Nobiletin-induced partial abrogation of deleterious effects of AKT inhibition on preimplantation bovine embryo development in vitro†.

During preimplantational embryo development, PI3K/AKT regulates cell proliferation and differentiation and nobiletin modulates this pathway to promote cell survival. Therefore, we aimed to establish whether, when the AKT cascade is inhibited using inhibitors III and IV, nobiletin supplementation to in vitro culture media during the minor (2- to 8-cell stage, MNEGA) or major (8- to 16-cell stage, MJEGA) phases of EGA is able to modulate the development and quality of bovine embryos. In vitro zygotes were cultured during MNEGA or MJEGA phase in SOF + 5% FCS or supplemented with: 15 µM AKT-InhIII; 10 µM AKT-InhIV; 10 µM nobiletin; nobiletin + AKT-InhIII; nobiletin + AKT-InhIV; 0.03% DMSO. Embryo development was lower in treatments with AKT inhibitors, while combination of nobiletin with AKT inhibitors was able to recover their adverse developmental effect and also increase blastocyst cell number. The mRNA abundance of GPX1, NFE2L2, and POU5F1 was partially increased in 8- and 16-cell embryos from nobiletin with AKT inhibitors. Besides, nobiletin increased the p-rpS6 level whether or not AKT inhibitors were present. In conclusion, nobiletin promotes bovine embryo development and quality and partially recovers the adverse developmental effect of AKT inhibitors, which infers that nobiletin probably uses another signaling cascade that PI3K/AKT during early embryo development in bovine.

Anti-Müllerian Hormone Concentrations Are Inversely Associated With Subclinical Atherosclerosis in Premenopausal Women.

Anti-Müllerian hormone (AMH), which is secreted by granulosa cells of late preantral and small antral follicles, is a marker of ovarian reserve. The association of ovarian reserve with subclinical atherosclerosis in women of reproductive age is currently unknown. We primary investigated whether AMH levels are associated with markers of subclinical atherosclerosis in healthy, normally menstruating women. In this cross-sectional study, vascular structure and function were assessed by measurement of carotid and femoral intima-media thickness (IMT), flow-mediated dilation, carotid-femoral pulse wave velocity and augmentation index. Lipid profile and serum AMH concentrations were also measured. Seventy premenopausal women, aged 32.7 ± 6.5 years, were included. Mean AMH levels were lower in smokers than in non-smokers and negatively associated with total cholesterol (TC) levels. An inverse association between mean AMH concentrations and femoral and carotid IMT in all segments was observed. No correlation with other markers of subclinical atherosclerosis or established cardiovascular (CV) risk factors was found. After multivariable adjustment, the association between AMH concentrations and combined carotid IMT or carotid bulb IMT remained significant. In conclusion, in healthy, normally ovulating women, AMH concentrations are negatively associated with subclinical atherosclerosis indices and TC levels, independently of established CV risk factors.

An approach to study the local embryo effect on gene expression in the bovine oviduct epithelium in vivo.

This study aimed to examine the local embryo effect on the transcriptomic response of the epithelial cells of the oviduct in vivo. Fifteen heifers were synchronized and artificially inseminated to a standing heat. All heifers were slaughtered on Day 2.5 after oestrus. The oviducts from 13 animals were isolated, trimmed free of tissue and divided between ampulla/isthmus. The ipsilateral isthmus was divided into smaller sections (2 cm). Each section was sequentially flushed until the embryo was located (4/13) and then opened and scraped longitudinally to obtain the epithelial cells. Cells were snap-frozen in LN2 for gene expression analysis. All recovered embryos were found at the beginning of the isthmus. The 2 cm sections selected for the transcriptomic analysis were as follows: embryo section (in which the embryo was found); proximal section (through which the embryo had passed); distal section (on the uterine side of the embryo); and contralateral section (section from the contralateral isthmus). The expression pattern of eight genes (STK32A, KERA, QRFPR, MCTP1, PRELP, VAT1L, SOCS3 and CCL20) differentially expressed between the isthmus of pregnant (multiple embryo model) and cyclic heifers were assessed by RT-qPCR. One-way ANOVA and t test was used for statistical analysis. Comparisons between ipsilateral and contralateral oviduct or along the ipsilateral oviduct resulted in no differences for all genes. Despite the failure to detect a site-specific response of a single embryo on the abundance of distinct transcripts in the bovine oviduct in vivo on Day 2.5, the current methodology with proposed modifications would be useful for future studies to examine the local embryo effect.

Gene expression and metabolic response of bovine oviduct epithelial cells to the early embryo.

During its journey through the oviduct, the bovine embryo may induce transcriptomic and metabolic responses, via direct or indirect contact, from bovine oviduct epithelial cells (BOECs). An in vitro model using polyester mesh was established, allowing the study of the local contact during 48 h between a BOEC monolayer and early embryos (2- or 8-cell stage) or their respective conditioned media (CM). The transcriptomic response of BOEC to early embryos was assessed by analyzing the transcript abundance of SMAD6, TDGF1, ROCK1, ROCK2, SOCS3, PRELP and AGR3 selected from previous in vivo studies and GPX4, NFE2L2, SCN9A, EPSTI1 and IGFBP3 selected from in vitro studies. Moreover, metabolic analyses were performed on the media obtained from the co-culture. Results revealed that presence of early embryos or their CM altered the BOEC expression of NFE2L2, GPX4, SMAD6, IGFBP3, ROCK2 and SCN9A. However, the response of BOEC to two-cell embryos or their CM was different from that observed to eight-cell embryos or their CM. Analysis of energy substrates and amino acids revealed that BOEC metabolism was not affected by the presence of early embryos or by their CM. Interestingly, embryo metabolism before embryo genome activation (EGA) seems to be independent of exogenous sources of energy. In conclusion, this study confirms that early embryos affect BOEC transcriptome and BOEC response was embryo stage specific. Moreover, embryo affects BOEC via a direct contact or via its secretions. However transcriptomic response of BOEC to the embryo did not manifest as an observable metabolic response.

Cultured bovine embryo biopsy conserves methylation marks from original embryo.

A major limitation of embryo epigenotyping by chromatin immunoprecipitation analysis is the reduced amount of sample available from an embryo biopsy. We developed an in vitro system to expand trophectoderm cells from an embryo biopsy to overcome this limitation. This work analyzes whether expanded trophectoderm (EX) is representative of the trophectoderm (TE) methylation or adaptation to culture has altered its epigenome. We took a small biopsy from the trophectoderm (30-40 cells) of in vitro produced bovine-hatched blastocysts and cultured it on fibronectin-treated plates until we obtained ∼4 × 104 cells. The rest of the embryo was allowed to recover its spherical shape and, subsequently, TE and inner cell mass were separated. We examined whether there were DNA methylation differences between TE and EX of three bovine embryos using whole-genome bisulfite sequencing. As a consequence of adaptation to culture, global methylation, including transposable elements, was higher in EX, with 5.3% of quantified regions showing significant methylation differences between TE and EX. Analysis of individual embryos indicated that TE methylation is more similar to its EX counterpart than to TE from other embryos. Interestingly, these similarly methylated regions are enriched in CpG islands, promoters and transcription units near genes involved in biological processes important for embryo development. Our results indicate that EX is representative of the embryo in terms of DNA methylation, thus providing an informative proxy for embryo epigenotyping.

Bovine embryo-oviduct interaction in vitro reveals an early cross talk mediated by BMP signaling.

Signaling components of bone morphogenetic proteins (BMPs) are expressed in an anatomically and temporally regulated fashion in bovine oviduct. However, a local response of this signaling to the presence of the embryo has yet to be elucidated. The aim of the present study was to evaluate if early embryo-oviduct interaction induces changes in the gene expression of BMP signaling components. For this purpose, we used an in vitro co-culture system to investigate the local interaction between bovine oviductal epithelial cells (BOEC) from the isthmus region with early embryos during two developmental periods: before (from the 2-cell to 8-cell stage) or during (from the 8-cell to 16-cell stage) the main phase of embryonic genome activation (EGA). Exposure to embryos, irrespective of the period, significantly reduced the relative abundance of BMPR1B, BMPR2, SMAD1, SMAD6 and ID2 mRNAs in BOEC. In contrast, embryos that interacted with BOEC before EGA showed a significant increase in the relative abundance of SMAD1 mRNA at the 8-cell stage compared to embryos cultured without BOEC. Moreover, embryos at the 16-cell stage that interacted with BOEC during EGA showed a significant increase in BMPR1B, BMPR2 and ID2 mRNA. These results demonstrate that embryo-oviduct interaction in vitro induces specific changes in the transcriptional levels of BMP signaling, causing a bidirectional response that reduces the expression levels of this signaling in the oviductal cells while increases them in the early embryo. This suggests that BMP signaling pathway could be involved in an early cross talk between the bovine embryo and the oviduct during the first stages of development.

Sexually Dimorphic Gene Expression in Bovine Conceptuses at the Initiation of Implantation.

In cattle, maternal recognition of pregnancy occurs on Day 16 via secretion of interferon tau (IFNT) by the conceptus. The endometrium can distinguish between embryos with different developmental competencies. In eutherian mammals, X-chromosome inactivation (XCI) is required to ensure an equal transcriptional level of most X-linked genes for both male and female embryos in adult tissues, but this process is markedly different in cattle than mice. We examined how sexual dimorphism affected conceptus transcript abundance and amino acid composition as well as the endometrial transcriptome during the peri-implantation period of pregnancy. Of the 5132 genes that were differentially expressed on Day 19 in male compared to female conceptuses, 2.7% were located on the X chromosome. Concentrations of specific amino acids were higher in the uterine luminal fluid of male compared to female conceptuses, while female conceptuses had higher transcript abundance of specific amino acid transporters (SLC6A19 and SLC1A35). Of note, the endometrial transcriptome was not different in cattle gestating a male or a female conceptus. These data support the hypothesis that, far from being a blastocyst-specific phenomenon, XCI is incomplete before and during implantation in cattle. Despite differences in transcript abundance and amino acid utilization in male versus female conceptuses, the sex of the conceptus itself does not elicit a different transcriptomic response in the endometrium.

Extracellular Vesicles from BOEC in In Vitro Embryo Development and Quality.

To evaluate the effect of conditioned media (CM) and Extracellular Vesicles (EVs) derived from bovine oviduct epithelial cell (BOEC) lines on the developmental capacity of bovine zygotes and the quality of embryos produced in vitro, presumptive zygotes were cultured under specific conditions. In experiment 1, zygotes were cultured either on monolayers from BOEC extended culture (E), together with fresh BOEC suspension cells, or with BOEC-CM from fresh or E-monolayers. In experiment 2, EVs were isolated from BOEC-CM and characterized (150-200 nm) by Nanosight® and electron microscopy. Zygotes were cultured in the presence of 3x10(5) EVs/mL, 1.5x10(5) EVs/mL or 7.5x10(4) EVs/mL of fresh or frozen BOEC-EVs. In experiment 3, zygotes were cultured in absence of FCS but with EVs from BOEC-E that had been cultured in different culture media. In experiment 4, zygotes were cultured in SOF+5% normal-FCS, or EV-depleted-FCS. In all cases, cleavage rate (Day 2) and blastocyst development (Day 7-9) was assessed. Blastocysts on Days 7/8 were used for quality evaluation through differential cell count, cryotolerance and gene expression patterns. No differences were found among all FCS-containing groups in cleavage rate or blastocyst yield. However, embryos derived from BOEC-CM had more trophectoderm cells, while embryos derived from BOEC-EVs, both fresh and frozen, has more trophectoderm and total cells. More embryos survived vitrification in the BOEC-CM and BOEC-EV groups. In contrast, more embryos survived in the EV-depleted-FCS than in normal-FCS group. Gene expression patterns were modified for PAG1 for embryos cultured with EVs in the presence of FCS and for IFN-T, PLAC8, PAG1, CX43, and GAPDH in the absence of FCS. In conclusion, EVs from FCS have a deleterious effect on embryo quality. BOEC-CM and EVs during in vitro culture had a positive effect on the quality of in vitro produced bovine embryos, suggesting that EVs have functional communication between the oviduct and the embryo in the early stages of development.

An efficient system to establish biopsy-derived trophoblastic cell lines from bovine embryos.

Trophoblastic cells play a crucial role in implantation and placentogenesis and can be used as a model to provide substantial information on the peri-implantation period. Unfortunately, there are few cell lines for this purpose in cattle because of the difficulty of raising successive cell stocks in the long-term. Our results show that the combination of a monolayer culture system in microdrops on a surface treated with gelatin and the employment of conditioned media from mouse embryonic fibroblasts support the growth of bovine trophoblastic cells lines from an embryo biopsy. Expression profiles of mononucleate- and binucleate-specific genes in established trophoblastic cells lines represented various stages of gestation. Moreover, the ability to expand trophoblastic cell lines for more than 2 yr together with pluripotency-related gene expression patterns revealed certain self-renewal capacity. In summary, we have developed a system to expand in vitro trophoblastic cells from an embryo biopsy that solves the limitations of using amplified DNA from a small number of cells for bovine embryo genotyping and epigenotyping and, on the other hand, facilitates the establishment of trophoblastic cell lines that can be useful as peri-implantation in vitro models.

Radiofrequency ablation assisted resection for hepatocellular carcinoma: morbidity, mortality and long term survival.

PURPOSE: Hepatic resection is the mainstay of the curative treatment of primary hepatic tumors, with constantly improving short and long term results. Radiofrequency ablation (RFA)-assisted liver resection is a relatively new method of transection of the liver parenchyma with favorable intra- and perioperative results. The aim of this study was to investigate the oncological efficacy (long term overall survival/OS and disease free survival/DFS) and to confirm the favorable short term morbidity and mortality. METHODS: Between May 2004 and January 2007, 28 patients underwent 32 resections with removal of 50 hepatocellular carcinoma (HCC) lesions. The technique of parenchymal transection has been described previously as RFA-assisted liver resection. RESULTS: Thirty-day morbidity and mortality were 42.8 and 0%, respectively. Blood transfusion was necessary for 28.5% of the patients. The median hospital stay was 16.5 days (range 5-34). The 1- and 3-year OS were 92.9 and 65.7%, respectively. The 1- and 3-year DFS were 62.3 and 54.6- respectively. No patient developed metastatic disease or local recurrence at the margin site. Twelve patients (42.9%) developed in-the-liver recurrence away from the resection area. CONCLUSION: RFA-assisted liver resection is a safe and oncologically efficacious method for the surgical treatment of HCC with results comparable to other surgical techniques.

Transcriptomic changes in the bovine conceptus between the blastocyst stage and initiation of implantation.

Conceptus-maternal communication is vital for the successful establishment and maintenance of pregnancy, yet relatively little information exists for many of the mechanisms and the nature of the conceptus signals responsible for this cross-talk. Sub-optimal communication, resulting from impairment of conceptus development and/or from abnormal uterine receptivity, contributes to a high incidence of embryonic mortality. Therefore, detailed examination of the mechanisms regulating both pre- and peri-implantation conceptus development are necessary to fully understand the factors regulating successful post-hatching development, pregnancy recognition and implantation signaling. Despite significant progress in understanding of the temporal changes in the transcriptome of the uterine endometrium, there is only a rudimentary knowledge of the genes and pathways governing growth and development of the cattle conceptus. Furthermore, although there are a large number of studies describing gene expression profiles in bovine embryos focused mainly during the earlier preimplantation stages (up to and including Day 7), very little information exists for the post-hatching embryo and elongating conceptus. This period of development is arguably more important as a significant proportion of all embryonic loss occurs between Days 8 and 16 of pregnancy in cattle, corresponding to the time of hatching of the blastocyst from the zona pellucida and its subsequent elongation coincident with the time of maternal recognition of pregnancy. Given that this is a critical period in development leading up to maternal recognition and establishment of pregnancy, the identification of key genes and pathways regulating these crucial developmental events is essential.

Effect of the post-fertilization culture environment on the incidence of chromosome aberrations in bovine blastocysts.

We have previously shown that the postfertilization embryo culture environment has a significant influence on the quality of the resulting bovine blastocyst measured in terms of its cryotolerance and relative abundance for several developmentally important gene transcripts. Using three different culture conditions known to produce blastocysts of differing quality, the objective of this study was to examine whether the postfertilization culture environment had an effect on the incidence of mixoploidy in bovine blastocysts. Presumptive zygotes, produced by in vitro maturation and fertilization, were cultured in vitro in synthetic oviduct fluid (SOF) medium in the absence or presence of fetal calf serum (FCS), or in vivo in the ewe oviduct. Blastocysts were recovered from the three systems at Day 7 and the incidence of mixoploidy was assessed using fluorescence in situ hybridization with chromosome 6- and chromosome 7-specific probes. A total of 10 025 nuclei were scored in 122 blastocysts. The frequency of normal, diploid, blastocysts was 8.8%, 21.4%, and 34.8% in embryos derived from culture in SOF+FCS, SOF, and the ewe oviduct, respectively, the remainder showing some degree of mixoploidy. The incidence of mixoploidy was apparently not related to the presence of serum; omission of serum from SOF resulted in a reduction in the incidence of mixoploidy (91.2% vs. 78.6%), although this difference was not significant. Culture in vivo, however, resulted in a significant (P < 0.01) reduction in the incidence of mixoploidy compared with culture in vitro in the presence of serum (65.2% vs. 91.2%, respectively). Among the mixoploid blastocysts, the majority contained less than 10% polyploid cells, irrespective of culture group (SOF, 69.7%; SOF+FCS, 64.5%; ewe oviduct, 60.0%). More than one type of polyploidy was frequently observed in mixoploid blastocysts. Overall, diploidy-triploidy was the most frequent abnormality, but diploid-tetraploid and diploid-triploid-tetraploid mosaics were also observed. A significantly higher proportion (P < 0.05) of blastocysts derived from SOF+FCS had more than one type of abnormality (80.6%, 25/ 31) compared with those derived from SOF (45.4%, 15/33) or in vivo culture (53.3%, 16/30). In conclusion, the postfertilization culture environment of the developing embryo can affect the incidence and severity of mixoploidy in the resulting blastocyst.

Soluble Fas antigen and soluble Fas ligand in early neonatal life.

BACKGROUND: After birth, apoptosis rates might slow down, compared to those in utero. Thus, factors, attenuating the apoptotic process, like the soluble forms of Fas/FasL system, may increase. AIM-STUDY DESIGN: Soluble Fas (sFas) and soluble Fas ligand (sFasL) concentrations were measured in maternal serum (MS), umbilical cord (UC) and neonatal serum in the first (1N) and fifth (5N) days after birth in order to evaluate the alterations of these molecules during the early neonatal period. SUBJECTS AND METHODS: Soluble molecules were estimated in 35 healthy, appropriate for gestational age, full-term neonates, their mothers and in 25 healthy, nonpregnant women, age-matched to the mothers (controls), using enzyme immunoassays. RESULTS: sFas concentrations in MS (p < 0.01), UC (p < 0.0001), 1N (p < 0.0003) and 5N (p < 0.02) were lower than those in controls. Neonatal sFas concentrations showed a significant increase from UC to 5N (p < 0.001). In contrast, sFasL concentrations were significantly elevated in all neonatal samples (UC, 1N and 5N) compared to those in MS and controls (p < 0.0001), showing also a significant elevation from UC to 5N (p < 0.0001). CONCLUSION: Our results demonstrate increasing serum concentrations of the soluble molecules sFas and sFasL during the first days after birth, indicating possibly a gradual decrease of apoptosis in early neonatal life.

Relative messenger RNA abundance in bovine oocytes collected in vitro or in vivo before and 20 hr after the preovulatory luteinizing hormone surge.

In the cyclic cow, final maturation of the ovulatory follicle is initiated by the preovulatory luteinizing hormone (LH) surge. During the subsequent 24 hr period, the oocyte nucleus undergoes meiotic progression to metaphase II and several changes in cytoplasmic organization take place. We have previously shown that oocytes recovered at the time of the LH peak and matured in vitro are less competent to reach the blastocyst stage than their counterparts recovered 20 hr later following in vivo maturation, despite both groups undergoing IVF and culture in parallel. The objective of this study was to compare, using real-time quantitative RT-PCR, the relative abundance of various developmentally important gene transcripts in these oocytes. The groups used were mature bovine oocytes originating from: (1) 2-6 mm follicles from slaughterhouse ovaries; (2) preovulatory follicles punctured by ovum pick-up just before the LH surge (i.e., immature) and matured in vitro; or (3) preovulatory follicles punctured 20 hr later, just prior to ovulation (i.e., in vivo matured). In addition, immature oocytes from 2-6 mm follicles were examined. We examined the relative mRNA expression of five enzymes involved in protection against free oxygen radicals (mitochondrial Mn-superoxide dismutase, MnSOD, cytosolic Cu/Zn superoxide dismutase, Cu/ZnSOD, gamma-glutamyl-cysteine transferase, GCS, glutathione peroxidase, GPX, sarcosine oxidase, SOX), a transcript involved in follicular development (growth differentiation factor-9, GDF-9), transcripts involved in glucose metabolism (glucose-6-phosphate dehydrogenase, G6PDH, glucose transporter type-1 and -8, Glut-1, Glut-8) and genes involved in cell cycle events, Cyclin A and B, and poly(A) polymerase (PAP). Transcripts for all genes were detected, irrespective of oocyte origin. While differences were not significant in all cases, variations in levels of transcript abundance between the groups were related to developmental competence. In particular, transcripts for GDF-9 were expressed at significantly higher levels in oocytes recovered at the LH peak and matured in vitro than in those matured in vivo. The observations with GDF-9 are interesting as this gene is believed to be essential for normal folliculogenesis and may be important in the regulation of early follicle and oocyte growth. In conclusion, the results of this study demonstrate differences in the relative mRNA abundance of several developmentally important gene transcripts in bovine oocytes which may be related to developmental competence.