Extracellular vesicles of follicular fluid from heat-stressed cows modify the gene expression of in vitro-matured oocytes

While follicular fluid (FF) is well known to provide an optimal environment for oogenesis, its functional roles following its release into the oviduct during ovulation are currently elusive. We hypothesized that FF and FF-derived extracellular vesicles (EVs) may be conveyors of signals capable of inducing functionally-relevant transcriptional responses in oviductal cells. The aim of this study was, therefore, to evaluate the effect of FF and FF-derived EVs on the transcriptome of primary bovine oviductal epithelial cells (BOECs). We examined the gene expression of BOECs in three conditions: BOECs cultured with FF, FF-derived EVs, and without supplementations. For each condition, cells were cultured for 6 and 24 h. RNA sequencing results revealed that FF had a stronger effect on BOECs gene expression compared to EVs. We detected 488 and 1998 differentially expressed genes (DEGs) with FF treatment in 6 and 24 h, respectively, whereas only 41 DEGs were detected at 6 h following EV treatment. Pathway analysis of the FF-induced DEGs showed that several pathways were highly enriched, notably oxidative phosphorylation, thermogenesis, arachidonic acid metabolism, and steroid hormone biosynthesis. Some of these pathways have a role in sperm survival, fertilization, and early embryo development. In conclusion, the findings of our study demonstrate for the first time that bovine FF and FF-derived EVs can induce changes in the gene expression of the bovine oviductal cells which, although observed in vitro, may be reflective of in vivo responses which may contribute to a favorable periconceptional microenvironment for sperm survival, fertilization, and early embryo development.

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135 Effect of different concentrations of follicular fluid exosome-like extracellular vesicles on in vitro oocyte maturation and embryo development in cattle

Reproduction Fertility and Development ◽

10.1071/rdv34n2ab135 ◽

2022 ◽

Vol 34 (2) ◽

pp. 305

Author(s):

G. N. Singina ◽

E. N. Shedova ◽

R. E. Uzbekov ◽

S. Uzbekova

Keyword(s):

Embryo Development ◽

Extracellular Vesicles ◽

Oocyte Maturation ◽

Follicular Fluid ◽

In Vitro Oocyte Maturation

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Expression of mRNAs for Pro-and Anti-Apoptotic Factors in Granulosa Cells and Follicular Fluid of Women Undergoing in Vitro Fertilization. A Pilot Study

10.21203/rs.3.rs-330029/v1 ◽

2021 ◽

Author(s):

Jozsef Bodis ◽

Endre Sulyok ◽

Akos Varnagy ◽

Viktória Prémusz ◽

Krisztina Godony ◽

...

Keyword(s):

Gene Expression ◽

In Vitro Fertilization ◽

Mrna Expression ◽

Granulosa Cells ◽

Follicular Fluid ◽

Vitro Fertilization ◽

Expression Levels ◽

The Impact ◽

Apoptotic Factors

Abstract BackgroundThis observational clinical study evaluated the expression levels and predictive values of some apoptosis-related genes in granulosa cells (GCs) and follicular fluid (FF) of women undergoing in vitro fertilization (IVF).Methods GCs and FF were obtained at oocyte retrieval from 31 consecutive patients with heterogeneous infertility diagnosis (age: 34.3±5.8 years, body mass index: 24.02±3.12 kg/m2, duration of infertility: 4.2±2.1 years). mRNA expression of pro-apoptotic (BAX, CASP3, CASP8) and anti-apoptotic (BCL2, AMH, AMHR, FSHR, LHR, CYP19A1) factors was determined by quantitative RT-PCR using ROCHE LightCycler 480. Results No significant difference in GC or FF mRNA expression of pro- and anti-apoptotic factors could be demonstrated between IVF patients with (9 patients) or without (22 patients) clinical pregnancy. Each transcript investigated was detected in FF, but their levels were markedly reduced and independent of those in GCs. The number of retrieved oocytes was positively associated with GC AMHR (r=0.393, p=0.029), but the day of embryo transfer was negatively associated with GC LHR (r=-0.414, p=0.020) and GC FSHR transcripts (r=-0.535, p=0.002). When pregnancy positive group was analysed separately the impact of apoptosis- related gene expressions on some selected measures of IVF success could be observed. Strong positive relationship was found between gene expression levels of pro- and anti-apoptotic factors in GCs.ConclusionOur study provides only marginal evidences for the apoptosis dependence of IVF outcome and suggests that the apoptosis process induces adaptive increases of the anti-apoptotic gene expression to attenuate apoptosis and to protect cell survival.

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Platelet Derived Extracellular Vesicles Promote in Vitro Gingival Wound Healing.

10.21203/rs.3.rs-889480/v1 ◽

2021 ◽

Author(s):

Miquel Antich-Rosselló ◽

Marta Munar-Bestard ◽

Maria Antònia Forteza-Genestra ◽

Javier Calvo ◽

Antoni Gayà ◽

...

Keyword(s):

Gene Expression ◽

Wound Healing ◽

Extracellular Vesicles ◽

Size Exclusion ◽

Matrix Remodeling ◽

Platelet Concentrates ◽

Ldh Activity ◽

Exclusion Chromatography ◽

Good Biocompatibility

Abstract Purpose: Gingival regeneration aims at restoring the architecture and functionality of oral damaged tissue. Different biomaterials or biological materials have been tested for tissue repair, such as platelet concentrates like platelet lysate (PL). In this article, the use of extracellular vesicles (EVs) derived from PL and their combination with hyaluronic acid biomaterials (HA) in wound healing was investigated.Methods: EVs were isolated by size exclusion chromatography from PL. In addition, HA gels were formulated with PL or EVs. EVs or HA combined with EVs (HA-EVs) were tested in vitro for biocompatibility (LDH activity and metabolic activity) and by a wound healing assay and gene expression analysis.Results: EVs and EVs-HA treatments were biocompatible and showed an increase in wound healing compared to control. Moreover, changes in gene expression related to extracellular matrix remodeling were observed in gingival keratinocytes and gingival fibroblasts after the treatment with EVs.Conclusion: EVs can be combined with HA biomaterials, showing good biocompatibility and preserving their activity and functionality. Therefore, platelet derived EVs emerge as promising candidates for oral regeneration with the possibility to combine them with biomaterials in order to enhance their application in clinical use.

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The Oviductal Extracellular Vesicles’ RNA Cargo Regulates the Bovine Embryonic Transcriptome

International Journal of Molecular Sciences ◽

10.3390/ijms21041303 ◽

2020 ◽

Vol 21 (4) ◽

pp. 1303 ◽

Cited By ~ 7

Author(s):

Stefan Bauersachs ◽

Pascal Mermillod ◽

Carmen Almiñana

Keyword(s):

Gene Expression ◽

Extracellular Vesicles ◽

Sequencing Analysis ◽

Rna Seq ◽

High Throughput Analysis ◽

Positive Effects ◽

Early Embryos ◽

And Control ◽

The Impact

Oviductal extracellular vesicles (oEVs) are emerging as key players in the gamete/embryo–oviduct interactions that contribute to successful pregnancy. Various positive effects of oEVs on gametes and early embryos have been found in vitro. To determine whether these effects are associated with changes of embryonic gene expression, the transcriptomes of embryos supplemented with bovine fresh (FeEVs) or frozen (FoEVs) oEVs during in vitro culture compared to controls without oEVs were analyzed by low-input RNA sequencing. Analysis of RNA-seq data revealed 221 differentially expressed genes (DEGs) between FoEV treatment and control, 67 DEGs for FeEV and FoEV treatments, and minor differences between FeEV treatment and control (28 DEGs). An integrative analysis of mRNAs and miRNAs contained in oEVs obtained in a previous study with embryonic mRNA alterations pointed to direct effects of oEV cargo on embryos (1) by increasing the concentration of delivered transcripts; (2) by translating delivered mRNAs to proteins that regulate embryonic gene expression; and (3) by oEV-derived miRNAs which downregulate embryonic mRNAs or modify gene expression in other ways. Our study provided the first high-throughput analysis of the embryonic transcriptome regulated by oEVs, increasing our knowledge on the impact of oEVs on the embryo and revealing the oEV RNA components that potentially regulate embryonic development.

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Can extracellular vesicles from bovine ovarian follicular fluid modulate the in-vitro oocyte meiosis progression similarly to the CNP-NPR2 system?

Theriogenology ◽

10.1016/j.theriogenology.2020.06.031 ◽

2020 ◽

Vol 157 ◽

pp. 210-217 ◽

Cited By ~ 2

Author(s):

Elisa M. Pioltine ◽

Mariana F. Machado ◽

Juliano C. da Silveira ◽

Patrícia K. Fontes ◽

Ramon C. Botigelli ◽

...

Keyword(s):

Extracellular Vesicles ◽

Follicular Fluid ◽

Oocyte Meiosis ◽

Ovarian Follicular Fluid

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99 EXTRACELLULAR VESICLES OF BOVINE OVIDUCTAL FLUID MODIFY THE GENE EXPRESSION ON BOVINE IN VITRO-DERIVED EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab99 ◽

2016 ◽

Vol 28 (2) ◽

pp. 179 ◽

Cited By ~ 3

Author(s):

R. Lopera-Vasquez ◽

M. Hamdi ◽

V. Maillo ◽

C. Nunez ◽

M. Yanez-Mo ◽

...

Keyword(s):

Gene Expression ◽

Embryo Development ◽

Extracellular Vesicles ◽

Membrane Trafficking ◽

Dna Methyltransferase ◽

De Novo ◽

Expression Patterns ◽

Water Channel ◽

Developmental Competence

Extracellular vesicles (EVs) act as intercellular communicators through their protein, lipid, and mRNA content. The interaction of EVs from oviducal environment and the first stages of embryo development is currently an enigma. The aim of the present study was to evaluate the developmental competence and the expression profile of bovine blastocysts cultured with previously purified EVs recovered from ampullary and isthmic oviducal fluid (OF) under different centrifugal forces. OF-EVs recovered from oviducts of slaughtered heifers in early luteal phase were quantified with a nanoparticle tracking analysis system, and their integrity and size were assessed by electron microscopy. In vitro-produced zygotes were cultured in SOF+3 mg mL–1 BSA (C–), C– with 3 × 105 OF-EVs/mL from the ampulla (A) and isthmus (I) isolated at 1 × 103 (A10k and I10k, respectively) and 1 × 105 (A100k and I100k, respectively) × g. A control culture group of SOF+5% FCS (C+) was included. Blastocyst development was recorded on Day 7, 8, and 9 (D0: day of fertilization). Blastocysts on Days 7/8 cultured in C–, C+, I10k, and I100k were used to measure the relative mRNA expression of genes related with membrane trafficking (AQP3, AQP11, and ATP1A1), metabolism (LDLR and LDHA), and epigenetics (DNMT3A, IGF2R, GRB10, and SNRPN) by RT-qPCR. One-way ANOVA was used for statistical analysis. The size of ampullary and isthmic OF-EVs was similar with a mean of 220 nm. The concentration of I10k was significantly lower compared with A100k (3.6 × 108 v. 10.5 × 108 EVs/mL, respectively; P < 0.05); however, no differences were found in the rest of the groups with a mean concentration of 7.6 × 108 EVs/mL. EVs and C– groups showed a delayed embryo development at Day 7 compared with C+ (range: 12.0–13.8 v. 20.6%, respectively, P < 0.05); however, it was compensated at Days 8 and 9 (Day 9 range: 28.5–30.8%). The water channel related protein AQP3, associated with blastocoel formation, water, and cryoprotectant movement during cryopreservation, was up-regulated in I10k and I100k blastocysts compared with C+. The lipid receptor LDLR, proposed as a regulator of lipid uptake in blastocysts, was significantly down-regulated in C+ compared with the other groups, a possible consequence of a higher concentration of lipids in the C+ group. The de novo DNA methyltransferase DNMT3A and the imprinting gene SNRPN were down-regulated in the C+ compared with I100k, suggesting alterations in imprinting. In conclusion, bovine isthmic OF-EVs supplementation in in vitro embryo culture has a positive effect on gene expression patterns of developmental related genes compared with serum supplementation, suggesting an association between the oviducal environment and the developing embryo. Funded by the Spanish Ministry of Science and Innovation (AGL2012–37510 and AGL2012–39652-C02–01).

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183 GENE EXPRESSION OF IN VITRO-MATURATED OOCYTES CAN BE MODULATED BY FOLLICLE EXOSOMES FROM COWS KEPT UNDER THERMONEUTRAL OR HEAT STRESS CONDITIONS

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab183 ◽

2017 ◽

Vol 29 (1) ◽

pp. 200 ◽

Cited By ~ 1

Author(s):

F. M. Dalanezi ◽

F. C. Destro ◽

R. A. Ferrazza ◽

H. D. Mogollon García ◽

F. F. Franchi ◽

...

Keyword(s):

Gene Expression ◽

Heat Stress ◽

Cell Viability ◽

Oocyte Maturation ◽

Follicular Fluid ◽

Stress Conditions ◽

Standard Medium ◽

Humidity Index ◽

Lower Expression

There are several intrafollicular agents that have the ability to interfere with the metabolism and development of the oocyte, among these we highlight the exosomes (EXO). Thus, the aim of this study was to evaluate the capacity of EXO extracted from the follicular fluid of cows kept under thermoneutral or heat stress conditions to modulate oocyte maturation in vitro. Twenty-four Holstein cows were subjected to the following treatments for 14 days: heat stress (HS; n = 12), 38°C, 60% RH, temperature-humidity index = 88; and thermo-neutral (TN; n = 12), 24°C, 60% RH, temperature-humidity index = 71. Cows had their follicles aspirated when their diameter reached 9 to 12 mm; all follicles with this diameter were aspirated. All follicular fluid aspirated from cows subjected to HS or TN was pooled forming the groups (HS and TN). The EXO were obtained by ultracentrifugation of follicular fluid (120,000 × g for 70 min at 4°C, twice) and had their presence confirmed by transmission electron microscopy. Bos indicus cumulus-oocyte complexes (COC) collected from ovaries obtained in commercial slaughterhouse, were pooled in groups of 20 COC and randomly subjected to 1 of the following treatments: Control, matured in standard medium (TCM 199, supplemented with Earle’s salts, glutamine, NaHCO3, pyruvate, FSH, and amikacin); HS-EXO, matured in standard medium added with 10 µL of a solution of follicular EXO from HS cows; and TN-EXO, matured in standard medium added with 10 µL of a solution of follicular EXO from TN cows. The procedures were repeated 4 times, always with 20 COC per treatment in each replica. After 22 h of maturation, COC were recovered and the expression of genes related to apoptosis protection (BCL2), cell viability (STAT3), cell maintenance (RPL15), oocyte competence (BMP15), oxidative stress (CPT1B), cumulus cell expansion (HAS2), cell cycle (CDCA8), and heat stress protection (HSF1) were assessed. Oocyte genes were differentially expressed according to the source of EXO. Groups were statistically analysed using ANOVA and Tukey tests. All genes, except CPT1B, showed lower expression in TN-EXO oocytes when compared with control and HS-EXO (P < 0.05). CPT1B showed a higher expression in HS-EXO oocytes (P < 0.05). The results showed that the addition of EXO from exogenous follicles can modulate the expression of oocytes genes related to cell viability and survival. The lower expression of these genes in TN-EXO suggested that the EXO obtained in TN conditions attenuate several genes related to the oocytes maturation and viability. Surprisingly, the control oocytes showed a similar gene expression pattern of the HS-EXO. In conclusion, EXO derived from follicular fluid of cows submitted to TN or HS conditions can modulate the gene expression of oocytes matured in vitro. These results open new perspectives for the use of theses EXO as a tool to increase the efficiency of in vitro oocyte maturation. Financial support from FAPESP #12/18297–7.

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