Proteomic Analysis of Domestic Cat Blastocysts and Their Secretome Produced in an In Vitro Culture System without the Presence of the Zona Pellucida.

Domestic cat blastocysts cultured without the zona pellucida exhibit reduced implantation capacity. However, the protein expression profile has not been evaluated in these embryos. The objective of this study was to evaluate the protein expression profile of domestic cat blastocysts cultured without the zona pellucida. Two experimental groups were generated: (1) domestic cat embryos generated by IVF and cultured in vitro (zona intact, (ZI)) and (2) domestic cat embryos cultured in vitro without the zona pellucida (zona-free (ZF group)). The cleavage, morula, and blastocyst rates were estimated at days 2, 5 and 7, respectively. Day 7 blastocysts and their culture media were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). The UniProt Felis catus database was used to identify the standard proteome. No significant differences were found in the cleavage, morula, or blastocyst rates between the ZI and ZF groups (p > 0.05). Proteomic analysis revealed 22 upregulated and 20 downregulated proteins in the ZF blastocysts. Furthermore, 14 proteins involved in embryo development and implantation were present exclusively in the culture medium of the ZI blastocysts. In conclusion, embryo culture without the zona pellucida did not affect in vitro development, but altered the protein expression profile and release of domestic cat blastocysts.

Adenosine promoted angiogenesis mediated by the release of small extracellular vesicles from human endothelial progenitor cells.

Endothelial progenitor cells (EPCs) are stem cells mainly derived from bone marrow; from where they migrate to repair and regenerate damaged tissues. eEPCs have been classified into two sub-populations, early (eEPC) and late EPCs (lEPC), depending on maturation stages in vitro. In addition, eEPC release endocrine mediators, including small extracellular vesicles (sEVs), which in turn may enhance the eEPC-mediated wound healing properties. Nevertheless, adenosine contributes to angiogenesis by recruiting eEPC at the injury site. However, whether ARs may enhance the secretome of eEPC, including sEVs, is unknown. Therefore, we aimed to investigate whether AR activation increase the release of sEVs in eEPC, which in turn has paracrine effects on recipient endothelial cells. Results shown that 5'-N-ethylcarboxamidoadenosine (NECA), a non-selective agonist, increase both the protein levels of the vascular endothelial growth factor (VEGF), and the number of sEVs released to the conditioned medium (CM) in primary culture of eEPC. Importantly, CM and EVs harvested from NECA-stimulated eEPC promote in vitro angiogenesis, without changes in cell proliferation, in recipient ECV-304 endothelial cells. This constitutes the first evidence showing that adenosine enhances sEVs release from eEPC, which has pro-angiogenic capacity on recipient endothelial cells.

Zona pellucida removal modifies the expression and release of specific microRNAs in domestic cat blastocysts.

The in vitro culture of domestic cat embryos without the zona pellucida affects their implantation capacity. MicroRNAs (miRNAs) have an important role in embryo-maternal communication and implantation. The objective of this study was to evaluate the expression of specific miRNAs in domestic cat blastocysts cultured without the zona pellucida. Two experimental groups were done: (1) domestic cat embryos cultured with the zona pellucida (zona intact control group, ZI); and (2) cultured without the zona pellucida (zona free group, ZF). The cleavage, morula and blastocyst rates were evaluated. The blastocysts and their spent medium were used for miRNA expression analysis using RT-qPCR (miR-21, miR-24, mi25, miR-29, miR-96, miR-98, miR-103, miR-191, miR-196, miR-199, miR-130, miR-155 and miR-302). The pre-mature microRNAs (pre-miRNAs) and miRNAs were evaluated in the blastocysts and only miRNAs were evaluated in the spent medium. No differences were observed in the cleavage, morula and blastocyst rates between the ZF and ZI groups (P > 0.05). For miRNAs analysis, miR-103 and miR-191 had the most stable expression and were selected as internal controls. ZF blastocysts had a higher expression of miR-21, miR-25, miR-29 and miR-199 and a lower expression of miR-96 than their ZI counterparts (P < 0.05). Furthermore, higher levels of miR-21, miR-25 and miR-98 were detected in the spent medium of ZF blastocysts (P < 0.05). In conclusion, in vitro culture of domestic cat embryos without the zona pellucida modifies the expression of miR-21, miR-25, miR-29, miR-199 and miR-96 at the blastocyst stage and the release of miR-21, miR-25 and miR-98.

Analysis of trophectoderm markers in domestic cat blastocysts cultured without zona pellucida.

Domestic cat embryos generated by in vitro fertilization (IVF) and cultured without the zona pellucida have a reduced implantation capacity after embryo transfer at the blastocyst stage. The objective of this study was to evaluate the expression of trophectoderm markers in domestic cat blastocysts cultured without the zona pellucida. Two experimental groups were selected: (1) domestic cat embryos generated by IVF and cultured in vitro normally (zona intact group, ZI); and (2) domestic cat embryos generated by IVF and cultured in vitro without a zona pellucida (zona-free group, ZF). In the ZF group, the zona pellucida of the presumptive zygote was removed and these were cultured using the well of the well (WOW) system. In vitro culture was carried out for 7 days. The cleavage, morula and blastocyst rates were estimated. Finally, the relative expression levels of the trophectoderm markers TEAD4, YAP1, CDX2 and EOMES, the cell adhesion marker E-cadherin and the apoptosis marker CASP3 were evaluated by RT-qPCR in the blastocysts. The Wilcoxon test was used to evaluate differences (P < 0.05). No differences were observed in the cleavage, morula and blastocyst rates between the ZF and ZI groups. No differences were found in the expression of TEAD4, CDX2, E-cadherin and CASP3 between groups. The expression of YAP1 and EOMES was higher in ZF blastocysts than in ZI blastocysts. In conclusion, the in vitro culture without the zona pellucida generates an overexpression of YAP1 and EOMES in the domestic cat blastocysts. More studies are needed to confirm if this overexpression might affect in vivo development.

Evaluation of extracellular vesicles and gDNA from culture medium as a possible indicator of developmental competence in human embryos.

Human embryos generated in vitro have a high incidence of chromosomal abnormalities that negatively affect pregnancy rate. Embryos generated in vitro secrete extracellular vesicles (EVs) into the culture medium that could be used potentially as indicators of embryo competence. This research aimed to evaluate the concentration and size of EVs and their gDNA content as an indicator of developmental competence in human embryos. Human embryos generated by intracytoplasmic sperm injection (ICSI) were classified morphologically as of either TOP, FAIR or POOR quality. Culture medium and developmentally arrested embryos (which were not able to be used for embryo transfer) were collected. Microvesicles, exosomes (MV/Exo) and apoptotic bodies (ABs) were isolated from culture medium. Nanoparticle tracking analysis (NTA) and array comparative genomic hybridization (aCGH) analysis were performed to evaluate EVs and their gDNA content. From NTA, the diameter (mean) of MVs/Exo from TOP quality embryos was higher (112.17 nm) compared with that of FAIR (108.02) and POOR quality embryos (102.78 nm) (P < 0.05). aCGH analysis indicated that MVs/Exo and ABs carried gDNA with the presence of 23 chromosome pairs. However, when arrested embryos were compared with their respective MVs/Exo and ABs, the latter had an increased rate of chromosomal abnormalities (24.9%) compared with embryos (8.7%) (P < 0.05). In conclusion, the size of EVs from culture medium might be an alternative for evaluating competence of human embryos, however more studies are needed to validate the use of gDNA from EVs as an indicator of embryo competence.

MicroRNAs from Extracellular Vesicles Secreted by Bovine Embryos as Early Biomarkers of Developmental Competence.

During early development, embryos secrete extracellular vesicles (EVs) that participate in embryo-maternal communication. Among other molecules, EVs carry microRNAs (miRNAs) that interfere with gene expression in target cells; miRNAs participate in embryo-maternal communication. Embryo selection based on secreted miRNAs may have an impact on bovine breeding programs. This research aimed to evaluate the size, concentration, and miRNA content of EVs secreted by bovine embryos with different developmental potential, during the compaction period (days 3.5-5). Individual culture media from in vitro-produced embryos were collected at day 5, while embryos were further cultured and classified at day 7, as G1 (conditioned-culture media by embryos arrested in the 8-16-cells stage) and G2 (conditioned-culture media by embryos that reached blastocyst stages at day 7). Collected nanoparticles from embryo conditioned culture media were cataloged as EVs by their morphology and the presence of classical molecular markers. Size and concentration of EVs from G1 were higher than EVs secreted by G2. We identified 95 miRNAs; bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were upregulated in G1, whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were downregulated. The most significant upregulated pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The characteristics of EVs secreted by bovine embryos during the compaction period vary according to embryo competence. Embryos that reach the blastocyst stage secrete fewer and smaller vesicles. Furthermore, the loading of specific miRNAs into the EVs depends on embryo developmental competence.

Extracellular vesicles secreted during blastulation show viability of bovine embryos.

Extracellular vesicles (EVs) secreted by blastocysts may be clinically relevant, as indicator of embryo viability on in vitro fertilization. We tested if the characteristics of EVs secreted during blastulation are related to embryo viability. Morulae were individually cultured in SOF media depleted of EVs until day 7.5 post IVF. Viable embryos were determined by a system of extended in vitro culture of bovine embryos until day 11 (post-hatching development). Afterward, a retrospective classification of blastocyst and culture media was performed based on blastulation time (early blastulation (EB) or late blastulation (LB)) and post-hatching development at day 11 (viable (V) or non-viable embryo (NV)). A total of 254 blastocysts and their culture media were classified in four groups (V-EB, NV-EB, V-LB, NV-LB). Group V-EB had a larger blastocyst diameter (170.8 µm), higher proportion of good-quality blastocysts (77%) and larger mean size of population of EVs (122.9 nm), although the highest concentration of EVs (5.75 × 109 particles/mL) were in group NV-EB. Furthermore, small RNA sequencing detected two biotypes, miRNA (86-91%) and snoRNA (9-14%), with a total of 182 and 32 respectively. In differential expression analysis of miRNAs between V versus NV blastocysts, there were 12 miRNAs upregulated and 15 miRNAs downregulated. Binary logistic regression was used to construct a non-invasive novel model to select viable embryos, based on a combination of variables of blastocyst morphokinetics and EVs characteristics, the ROC-AUC was 0.853. We concluded that characteristics of EVs secreted during blastulation vary depending on embryo quality.

Effect of BMP15 and/or AMH during in vitro maturation of oocytes from involuntarily culled dairy cows.

The high metabolic activity to which the dairy cattle are exposed to maintain milk production altered steroid metabolism that affects reproductive physiology and reduce oocyte competence. Our aims were (a) to characterize the competence of immature oocytes collected from dairy cattle based on the expression of genes in cumulus cells (CCs) and (b) to improve oocyte competence to support preimplantation embryo development by the supplementation of maturation medium with bone morphogenetic protein 15 (BMP15) and/or anti-mullerian hormone (AMH). Oocyte donors were identified at the moment of ovary collection and grouped by involuntarily culled dairy cows (Holstein breed) or beef cattle. The embryo development speed to blastocyst of the cull dairy cattle versus beef cattle (control group) was lower. Besides, <10% of oocytes (with CC biopsies) derived from dairy cattle were able to develop to the blastocyst stage. In addition, a higher level of expression and a positive correlation were observed in the expression of most of the genes evaluated (LUM, KRT18, KRT8, CLIC3, BMPR1B, and SLC38A3) in the cumulus-oocyte complexes that produced blastocysts versus those which did not develop correctly (arrested development). Further, use of BMP15 in the maturation of oocytes from dairy cattle seems to increase competence, modulating the expression of OCT4, SOX2, CDX2, GATA6, and TP1 in resulting blastocysts.

eCG stimulation in domestic cats increases the expression of gonadotrophin-induced genes improving oocyte competence during the non-breeding season.

The objective of this study was to evaluate the eCG stimulation on domestic cat oocyte competence during the non-breeding season. Four experimental groups were made: (a) untreated cycling cats (Breeding season group; BS), (b) untreated anestrous cats (Non-breeding group; NB), (c) anestrous cats treated with 200 IU of eCG (eCG group) and (d) anestrous cats treated with 200 IU of eCG and 100 IU of hCG four days later (hCG group). In the BS, NB and eCG groups, grade I and II immature cumulus-oocyte complexes (COCs) were subjected to in vitro maturation or used for the gene expression analysis of FSHR, LHCGR, EGFR, EGR1, ESR2, PTGS2, GDF9, BMP15 and GATM. The in vitro matured oocytes from the BS, NB and eCG groups and the in vivo matured oocytes from the hCG group were subjected to parthenogenetic activation. The grade I and II COCs from the eCG group had an increased expression of FSHR, LHCGR, EGFR, EGR1 and ESR2 and a higher maturation rate than the BS and NB groups (p < 0.05). After parthenogenetic activation, the blastocyst rate from the hCG, eCG and BS groups was higher than the NB group (p < 0.05). However, no significant improvement was observed in the blastocyst rate in the hCG group compared to the eCG group (p > 0.05). In conclusion, the eCG treatment increases the expression of specific genes improving the oocyte competence during the cat non-breeding season, which is reflected in an enhanced in vitro maturation and in vitro embryo development after parthenogenetic activation.

Splitting of IVP bovine blastocyst affects morphology and gene expression of resulting demi-embryos during in vitro culture and in vivo elongation.

Embryo splitting might be used to increase offspring yield and for molecular analysis of embryo competence. How splitting affects developmental potential of embryos is unknown. This research aimed to study the effect of bovine blastocyst splitting on morphological and gene expression homogeneity of demi-embryos and on embryo competence during elongation. Grade I bovine blastocyst produced in vitro were split into halves and distributed in nine groups (3 × 3 setting according to age and stage before splitting; age: days 7-9; stage: early, expanded and hatched blastocysts). Homogeneity and survival rate in vitro after splitting (12 h, days 10 and 13) and the effect of splitting on embryo development at elongation after embryo transfer (day 17) were assessed morphologically and by RT-qPCR. The genes analysed were OCT4, SOX2, NANOG, CDX2, TP1, TKDP1, EOMES, and BAX. Approximately 90% of split embryos had a well conserved defined inner cell mass (ICM), 70% of the halves had similar size with no differences in gene expression 12 h after splitting. Split embryos cultured further conserved normal and comparable morphology at day 10 of development; this situation changes at day 13 when embryo morphology and gene expression differed markedly among demi-embryos. Split and non-split blastocysts were transferred to recipient cows and were recovered at day 17. Fifty per cent of non-split embryos were larger than 100 mm (33% for split embryos). OCT4, SOX2, TP1 and EOMES levels were down-regulated in elongated embryos derived from split blastocysts. In conclusion, splitting day-8 blastocysts yields homogenous demi-embryos in terms of developmental capability and gene expression, but the initiation of the filamentous stage seems to be affected by the splitting.

Correction: Blastulation time measured with time-lapse system can predict in vitro viability of bovine blastocysts.

[This corrects the article DOI: 10.1371/journal.pone.0289751.].

Short preconditioning with TGFß of equine adipose tissue-derived mesenchymal stem cells predisposes towards an anti-fibrotic secretory phenotype: A possible tool for treatment of endometrosis in mares.

Endometrosis in mares is a disease resulting from chronic inflammation characterized by peri glandular fibrosis. There is no effective treatment so far, which opens the door for exploring the use of stem cells as a candidate. Transforming growth factor beta (TGFß) is crucial for the establishment and progression of fibrosis in mare's endometrosis. We aimed to develop regenerative approaches to treat endometrosis by using mesenchymal stem cells (MSC), for which understanding the effect of TGFß on exogenous MSC is crucial. We isolated and characterized equine adipose MSC from six donors. Cells were pooled and exposed to 10 ng/ml of TGFß for 0, 4, and 24 h, after which cells were analyzed for proliferation, migration, mesodermal differentiation, expression of fibrosis-related mRNAs, and prostaglandin E2 secretion. At 24 h of exposition to TGFß, there was a progressive increase in the contraction of the monolayer, leading to nodular structures, while cell viability did not change. Exposure to TGFß impaired adipogenic and osteogenic differentiation after 4 h of treatment, which was more marked at 24 h, represented by a decrease in Oil red and Alizarin red staining, as well as a significant drop (p < 0.05) in the expression of key gene regulators of differentiation processes (PPARG for adipose and RUNX2 for osteogenic differentiation). TGFß increased chondrogenic differentiation as shown by the upsurge in size of the resulting 3D cell pellet and intensity of Alcian Blue staining, as well as the significant up-regulation of SOX9 expression (p < 0.05) at 4 h, which reached a maximum peak at 24 h (p < 0.01), indicative of up-regulation of glycosaminoglycan synthesis. Preconditioning MSC with TGFß led to a significant increase (p < 0.05) in the expression of myofibroblast gene markers aSMA, COL1A1, and TGFß at 24 h exposition time. In contrast, the expression of COL3A1 did not change with respect to the control but registered a significant downregulation compared to 4 h (p < 0.05). TGFß also affected the expression of genes involved in PGE2 synthesis and function; COX2, PTGES, and the PGE2 receptor EP4 were all significantly upregulated early at 4 h (p < 0.05). Cells exposed to TGFß showed a significant upregulation of PGE2 secretion at 4 h compared to untreated cells (p < 0.05); conversely, at 24 h, the PGE2 values decreased significantly compared to control cells (p < 0.05). Preconditioning MSC for 4 h led to an anti-fibrotic secretory phenotype, while a longer period (24 h) led to a pro-fibrotic one. It is tempting to propose a 4-h preconditioning of exogenous MSC with TGFß to drive them towards an anti-fibrotic phenotype for cellular and cell-free therapies in fibrotic diseases such as endometrosis of mares.

DNA Content in Embryonic Extracellular Vesicles Is Independent of the Apoptotic Rate in Bovine Embryos Produced In Vitro.

Pre-implantation embryos release extracellular vesicles containing different molecules, including DNA. The presence of embryonic DNA in E-EVs released into the culture medium during in vitro embryo production could be useful for genetic diagnosis. However, the vesicles containing DNA might be derived from embryos suffering from apoptosis, i.e., embryos of bad quality. This work intended to confirm that embryos release DNA that is useful for genotyping by evaluating the effect of embryonic apoptosis on DNA content in E-EVs. Bovine embryos were produced by parthenogenesis and in vitro fertilization (IVF). On Day 5, morulae were transferred to individual cultures in an EV-depleted SOF medium. On Day 7, embryos were used to evaluate cellular apoptosis, and each culture medium was collected to evaluate E-EV concentration, characterization, and DNA quantification. While no effect of the origin of the embryo on the apoptotic rate was found, arrested morulae had a higher apoptotic rate. E-EVs containing DNA were identified in all samples, and the concentration of those vesicles was not affected by the origin or quality of the embryos. However, the concentration of DNA was higher in EVs released by the arrested parthenogenetic embryos. There was a correlation between the concentration of E-EVs, the concentration of DNA-positive E-EVs, and the concentration of DNA. There was no negative effect of apoptotic rate on DNA-positive E-EVs and DNA concentration; however, embryos of the best quality with a low apoptotic rate still released EVs containing DNA. This study confirms that the presence of DNA in E-EVs is independent of embryo quality. Therefore, E-EVs could be used in liquid biopsy for noninvasive genetic diagnosis.

Development and characterization of a novel variant of long-acting bovine follicle-stimulating hormone (brscFSH).

Assisted reproduction is a key aspect of modern animal breeding, providing valuable assistance in improving breeding programs. In this field, the administration of exogenous hormones, such as follicle-stimulating hormone (FSH), plays a crucial role in the induction of multiple ovulations. However, commercial FSH used in veterinary practice has been derived primarily from pituitary glands, obtained mostly from pigs for nearly four decades. Although these hormones have contributed significantly to the advancement of assisted reproductive techniques, they have certain limitations that warrant further improvements. These limitations include contamination with luteinizing hormone (LH), the potential risk of pathogen contamination, the potential to trigger an immune response in non-pig species, and the short half-life in circulation, requiring the implementation of complex 8-dose superovulation schedules. Our research team has developed and characterized a new variant of bovine follicle-stimulating hormone (bscrFSH) to address these limitations. The new hormone is produced recombinantly in CHO cell cultures, with a specific productivity of about 30 pg/cell/day. The bscrFSH can be purified to a high purity of 97 % using a single step of immobilized metal affinity chromatography (IMAC). N-glycan analysis of bscrFSH showed that approximately 74 % of the glycans corresponded to charged structures, including mono-, di-, tri-, and tetra-sialylated glycans. Superovulation trials conducted in cattle revealed that bscrFSH, administered at a total dose of about 0.5 µg per kg of body weight, using a decrescent schedule of 4 doses with 24-h intervals, resulted in an average yield of 8-12 transferable embryos per animal. Further research is required; however, the preliminary findings indicate that bscrFSH, currently packaged under the provisional brand name of Cebitropin B, holds potential as a commercial product for assisted reproduction in ruminants.

Blastulation time measured with time-lapse system can predict in vitro viability of bovine blastocysts.

The objective of this study was to evaluate the time of blastulation monitored by time-lapse technology to predict in vitro viability of bovine blastocysts. This technology can be a powerful tool for bovine embryos selection with higher implantation capacity and competence. Also, in humans an early blastulation is associated with higher quality and pregnancy rate. Cumulus oocyte complexes (COCs) were matured for 20 to 22 h and then fertilized by co-incubation of COCs and spermatozoa (10,000 sperm per oocyte) for 18 h. Presumptive zygotes were placed individually in microwells, in droplets of commercial culture medium. The Primo Vision TL system (EVO+; Vitrolife) captured digital images of developing embryos every 15 minutes. The time frame from IVF to the start of blastulation (tSB) and to blastocyst development (tB) was recorded. After day 7.5, the blastocysts were in vitro culture for 48 h until day 9.5 after IVF to evaluate post hatching development. In vitro viability was evaluated at day 9.5: those with a diameter greater than 200 µm and a total cell count greater than 180 were classified as viable (value 1), while the rest were classified as non in vitro viable (value 0). The area under the ROC curve (AUC) was estimated to determine the predictive power of in vitro viability through blastulation time. In addition, binary logistic regression analysis was used to generate a mathematical model with morphokinetic variables that allow the best prediction of in vitro viability. In 13 sessions, the blastocyst production rate was 46.2% (96/208). The cut-off time to discriminate early or late blastulation was 149.8 h. The post-hatching development of the embryos with early blastulation was 63.3% (31/49), being statistically superior (p = 0.001) than the late blastulation group 14.9% (7/47). Likewise, the time of blastulation showed an accuracy of 90.8% (p < 0.001) in predicting in vitro viability of bovine blastocysts. In conclusion, the selection of blastocysts based on blastulation time (< 155 h) and blastocyst diameter measured on day 7.5 after IVF (> 180 µm) maximizes the in vitro viability.

Bovine embryos release extracellular vesicles with differential miRNA signature during the compaction and blastulation stages.

Pre-implantation embryos release extracellular vesicles (EVs) to extracellular environment. In this work it is hypothesized that the EVs miRNA cargo will vary during pre-implantation development due to the constant changes in gene expression that take place through this period. The concentration, size and miRNA cargo of EVs secreted by competent bovine embryos during the period from compaction to blastulation (Day 3-7) were analyzed. For this analysis tow developmental windows were defined: W2 from 8-cells (D3) to morula (D5) and W3 from morula (D5) to blastocyst (D7). For W2, in vitro produced embryos were individually cultured in EVs-depleted medium from D3 to D5; culture media were collected and assigned to Group W2. Morulae were kept in culture up to blastocyst stage to determine the developmental competence. For W3, D5 morulae were collected and cultured individually in EVs-depleted medium up to blastocyst stage; culture media were assigned to Group W3, and blastocysts were kept in culture up to day 11 to define their competence. The mean size of EVs was similar between groups, however, EVs concentration was lower in W2. A total of 140 miRNAs were identified. From them, 79 were differentially expressed between the groups, 28 upregulated and 51 downregulated. miRNAs differentially detected between both developmental windows participate in the regulation of signaling pathways which crucial for embryonic development. It was concluded that the secretion of EVs is regulated by the developmental progress of the embryo during the pre-implantation period.

Mare stromal endometrial cells differentially modulate inflammation depending on oestrus cycle status: an in vitro study.

The modulation of inflammation is pivotal for uterine homeostasis. Here we evaluated the effect of the oestrus cycle on the expression of pro-inflammatory and anti-inflammatory markers in a cellular model of induced fibrosis. Mare endometrial stromal cells isolated from follicular or mid-luteal phase were primed with 10 ng/mL of TGFß alone or in combination with either IL1ß, IL6, or TNFα (10 ng/mL each) or all together for 24 h. Control cells were not primed. Messenger and miRNA expression were analyzed using real-time quantitative PCR (RT-qPCR). Cells in the follicular phase primed with pro-inflammatory cytokines showed higher expression of collagen-related genes (CTGF, COL1A1, COL3A1, and TIMP1) and mesenchymal marker (SLUG, VIM, CDH2, and CDH11) genes; p < 0.05. Cells primed during the mid-luteal overexpressed genes associated with extracellular matrix, processing, and prostaglandin E synthase (MMP2, MMP9, PGR, TIMP2, and PTGES; p < 0.05). There was a notable upregulation of pro-fibrotic miRNAs (miR17, miR21, and miR433) in the follicular phase when the cells were exposed to TGFß + IL1ß, TGFß + IL6 or TGFß + IL1ß + IL6 + TNFα. Conversely, in cells from the mid-luteal phase, the treatments either did not or diminished the expression of the same miRNAs. On the contrary, the anti-fibrotic miRNAs (miR26a, miR29b, miR29c, miR145, miR378, and mir488) were not upregulated with treatments in the follicular phase. Rather, they were overexpressed in cells from the mid-luteal phase, with the highest regulation observed in TGFß + IL1ß + IL6 + TNFα treatment groups. These miRNAs were also analyzed in the extracellular vesicles secreted by the cells. A similar trend as seen with cellular miRNAs was noted, where anti-fibrotic miRNAs were downregulated in the follicular phase, while notably elevated pro-fibrotic miRNAs were observed in extracellular vesicles originating from the follicular phase. Pro-inflammatory cytokines may amplify the TGFß signal in the follicular phase resulting in significant upregulation of extracellular matrix-related genes, an imbalance in the metalloproteinases, downregulation of estrogen receptors, and upregulation of pro-fibrotic factors. Conversely, in the luteal phase, there is a protective role mediated primarily through an increase in anti-fibrotic miRNAs, a decrease in SMAD2 phosphorylation, and reduced expression of fibrosis-related genes.

Effects of Extra-Long-Acting Recombinant Bovine FSH (bscrFSH) on Cattle Superovulation.

Over the last few years, several commercial FSH products have been developed for cattle superovulation (SOV) purposes in Multiple Ovulation and Embryo Transfer (MOET) programs. The SOV response is highly variable among individuals and remains one of the main limiting factors in obtaining a profitable number of transferable embryos. In this study, follicle stimulating hormone (FSH) from different origins was included in two SOV protocols, (a) FSH from purified pig pituitary extract (NIH-FSH-p; two doses/day, 12 h apart, four consecutive days); and (b) extra-long-acting bovine recombinant FSH (bscrFSH; a single dose/day, four consecutive days), to test the effects of bscrFSH on the ovarian response, hormone profile levels, in vivo embryo production and the pluripotency gene expression of the obtained embryos. A total of 68 healthy primiparous red Angus cows (Bos taurus) were randomly distributed into two experimental groups (n = 34 each). Blood sample collection for progesterone (P4) and cortisol (C) level determination was performed together with ultrasonographic assessment for ovarian size, follicles (FL) and corpora lutea (CL) quantification in each SOV protocol (Day 0, 4, 8, and 15). Moreover, FSH profiles were monitorised throughout both protocols (Day 0, 4, 5, 6, 7, 8, 9, 10, and 15). In vivo embryo quantity and quality (total structures, morulae, blastocysts, viable, degenerated and blocked embryos) were recorded in each SOV protocol. Finally, embryo quality in both protocols was assessed by the analysis of the expression level of crucial genes for early embryo development (OCT4, IFNt, CDX2, BCL2, and BAX). P4 and cortisol concentration peaks in both SOV protocols were obtained on Day 15 and Day 8, respectively, which were statistically different compared to the other time-points (p < 0.05). Ovarian dimensions increased from Day 0 to Day 15 irrespective of the SOV protocol considered (p < 0.05). Significant changes in CL number were observed over time till Day 15 irrespective of the SOV protocol applied (p < 0.05), being non- significantly different between SOV protocols within each time-point (p > 0.05). The number of CL was higher on Day 15 in the bscrFSH group compared to the NIH-FSH-p group (p < 0.05). The number of embryonic structures recovered was higher in the bscrFSH group (p = 0.025), probably as a result of a tendency towards a greater number of follicles developed compared to the NIH-FSH-p group. IFNt and BAX were overexpressed in embryos from the bscrFSH group (p < 0.05), with a fold change of 16 and 1.3, respectively. However, no statistical differences were detected regarding the OCT4, CDX2, BCL2, and BCL2/BAX expression ratio (p > 0.05). In conclusion, including bscrFSH in SOV protocols could be an important alternative by reducing the number of applications and offering an improved ovarian response together with better embryo quality and superior performance in embryo production compared to NIH-FSH-p SOV protocols.

Effect of Embryo Aggregation on In Vitro Development of Adipose-Derived Mesenchymal Stem Cell-Derived Bovine Clones.

Somatic cell nuclear transfer (SCNT) is a method with unique ability to reprogram the epigenome of a fully differentiated cell. However, its efficiency remains extremely low. In this work, we assessed and combined two simple strategies to improve the SCNT efficiency in the bovine. These are the use of less-differentiated donor cells to facilitate nuclear reprogramming and the embryo aggregation (EA) strategy that is thought to compensate for aberrant epigenome reprogramming. We carefully assessed the optimal time of EA by using in vitro-fertilized (IVF) embryos and evaluated whether the use of adipose-derived mesenchymal stem cells (ASCs) as donor for SCNT together with EA improves the blastocyst rates and quality. Based on our results, we determined that the EA improves the preimplantation embryo development per well of IVF and SCNT embryos. We also demonstrated that day 0 (D0) is the optimal aggregation time that leads to a single blastocyst with uniform distribution of the original blastomeres. This was confirmed in bovine IVF embryos and then, the optimal condition was translated to SCNT embryos. Notably, the relative expression of the trophectoderm (TE) marker KRT18 was significantly different between aggregated and nonaggregated ASC-derived embryos. In the bovine, no effect of the donor cell is observed on the developmental rate, or the embryo quality. Therefore, no synergistic effect of the use of both strategies is observed. Our results suggest that EA at D0 is a simple and accessible strategy that improves the blastocyst rate per well in bovine SCNT and IVF embryos and influence the expression of a TE-related marker. The aggregation of two ASC-derived embryos seems to positively affect the embryo quality, which may improve the postimplantation development.

Domestic cat embryos generated without zona pellucida are capable of developing in vitro but exhibit abnormal gene expression and a decreased implantation rate.

The removal of the zona pellucida has been used to improve the in vitro development of domestic cat embryos generated by IVF and SCNT. However, the in vivo development of domestic cat embryos generated without the zona pellucida has not been evaluated. The objective of this study was to evaluate the effects of zona pellucida removal on the in vitro and in vivo development of domestic cat embryos generated by IVF. For this purpose, two experimental groups were created: 1) domestic cat embryos cultured in vitro (Zona-intact group, ZI) and 2) domestic cat embryos cultured in vitro without the zona pellucida (Zona-free group, ZF). Domestic cat embryos were generated by IVF and cultured in vitro for 8 days. In the ZF group, the zona pellucida was removed after IVF, and embryos were cultured using the well of the well system (WOW). Cleavage, morula and blastocyst rates were evaluated in both groups. The diameter and total cell number of blastocysts were assessed. Relative expression of pluripotency (OCT4, SOX2 and NANOG), differentiation (CDX2 and GATA6) and apoptotic markers (BAX and BCL2) was evaluated in blastocysts. Finally, to evaluate in vivo development, embryos at days 5, 6 and 7 of development were transferred into recipient domestic cats, and ultrasonography was performed to evaluate implantation. No differences were observed in the cleavage, morula or blastocyst rates between embryos from the ZI and ZF groups. The diameter (mean ± SD) of blastocysts from the ZF group was greater (253.4 ± 83.3 µm) than that from the ZI group (210.5 ± 78.5 µm). No differences were observed in the relative expression of OCT4, CDX2 or GATA6. However, the relative expression of SOX2 and NANOG was significantly reduced in ZF blastocysts compared to ZI blastocysts. Furthermore, the relative expression of BAX was higher in ZF blastocysts than in ZI blastocysts. Finally, four pregnancies were confirmed after the transfer of ZI embryos (n = 110). However, no pregnancies were observed after the transfer of ZF embryos at the morula or blastocyst stage (n = 56). In conclusion, domestic cat embryos cultured without the zona pellucida were able to develop in vitro until the blastocyst stage. However, the removal of the zona pellucida negatively affected the gene expression of pluripotency and apoptosis markers, and ZF embryos were unable to implant. This might indicate that the removal of the zona pellucida is detrimental for the implantation and in vivo development of domestic cat embryos.