Ram Sperm Motility Parameters under The Influence of Epidermal Growth Factor

Epidermal growth factor (EGF) is one of the important cytokines that play a role in fertility. It is known that EGF affects both male and female reproduction, but its effect on sperm parameters is not fully understood. Up to the present, the effect of EGF on ram sperm motility parameters has not been published. We analyzed motility parameters of ejaculates after 24, 48, and 72 hours from the EGF addition. EGF was added to chilled ram sperm at concentrations of 0, 100, 200, and 400 ng·ml−1. Analyses were realized using computer, assisted semen analyzer (CASA)—Hamilton Thorn motility analyzer (version 7). The effect of EGF was already visible after 30 min of incubation. Significant effect on ram sperm total motility and progressive movement was observed at higher EGF concentrations after 48 h of incubation. Our results show that EGF affects sperm motility parameters depending on concentration and time of exposure

State of actin cytoskeleton and development of slow-frozen and vitrified rabbit pronuclear zygotes

[EN] This study was focused on the effect of cryopreservation on the state of actin cytoskeleton and development of rabbit pronuclear zygotes. Zygotes were collected from superovulated females and immediately used for 1) slow-freezing in a solution containing 1.5 M 1,2-propanediol and 0.2 M sucrose, or 2) vitrification in a solution containing 42.0% (v/v) of ethylene glycol, 18.0% (w/v) of dextran and 0.3 M sucrose as cryoprotectants. After thawing or warming, respectively, zygotes were evaluated for 1) actin distribution, 2) in vitro or 3) in vivo development to blastocyst. Comparing actin filaments distribution, a significantly higher number of vitrified zygotes with actin distributed in cell border was observed (55 ± 7.7 vs. 74 ± 6.1% for slow-frozen vs. vitrified, respectively). After 24 and 72 h of in vitro development, significant differences in the cleavage and morula rate among the groups were observed (9 ± 2.4 and 3 ± 1.3 vs. 44 ± 3.0 and 28 ± 2.7% for slow-frozen vs. vitrified, respectively). None of the slow-frozen zygotes reached the blastocyst stage, in contrast to the vitrified counterparts (11 ± 1.9%). Under in vivo culture conditions, a significant difference in blastocyst rate was observed between vitrified and fresh embryos (6 ± 1.5 vs. 35 ± 4.4% respectively). Our results showed that alterations in actin cytoskeleton and deteriorated development are more evident in slow-frozen than vitrified pronuclear zygotes. Vitrification method seems to be a more effective option for rabbit zygotes cryopreservation, although pronuclear zygotes manipulation per se resulted in a notable decrease in embryo development.This research was supported by the projects: UGAVIII/16/2015, VEGA 1/0611/15, by the Spanish Research project AGL2014-53405-C2-1-PComision Interministerial de Ciencia y Tecnologia (CICYT), Generalitat Valenciana research program (Prometeo II 2014/036), grant of Slovak Research and Development Agency: APVV-14-0043 and by the European Community under project no 26220220180: Building Research Centre "AgroBioTech". B. Kulikova received fellowship from a Collaborative European Network on Rabbit Genome Biology (RGB-Net) (COST-STSM-TD1101)Kulikova, B.; Jiménez-Trigos, ME.; Makarevich, AV.; Chrenek, P.; Vicente Antón, JS.; Marco-Jiménez, F. (2016). State of actin cytoskeleton and development of slow-frozen and vitrified rabbit pronuclear zygotes. Cryobiology. 72(1):14-20. https://doi.org/10.1016/j.cryobiol.2015.11.009142072

Presumptive mediators of growth hormone action on insulin-like growth factor I release by porcine ovarian granulosa cells

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Effect of growth factors on proliferation, apoptosis and protein kinase A expression in cultured porcine cumulus oophorus cells

The proliferation, apoptosis and protein kinase A (PKA) in porcine cumulus oophorus (CO) before and after 40 h of culture together with oocytes in the presence of IGF-I, IGF-II and EGF (all at 10 ng.mL$^{-1}$ medium) were compared. Cellular proliferation, apoptosis and PKA contents were evaluated by immunocytochemistry using specific antibodies against PCNA, TUNEL and catalytic (C-alpha) and regulatory (RI) subunits of PKA. The in-vitro culture of oocyte-CO complexes in a basal medium was accompanied by a decrease in the proportion of PCNA-positive CO cells (from 51 to 36%, $p < 0.05$). The addition of either IGF-I or EGF to the culture medium prevented this process and increased the proliferation rate (64 and 67% respectively, $p < 0.001$). During culture, the percentage of apoptotic (TUNEL-positive) CO cells increased from 42 to 57% ($p < 0.01$). The addition of IGF-I or EGF resulted in the inhibition of apoptosis to 36 and 12% respectively ($p < 0.001$). IGF-II and EGF reduced the amount of PKA catalytic subunits in the CO (percentage of cells with immunoreactive PKA catalytic subunits (28%, $p < 0.05$ and 27%, $p < 0.05$ respectively; versus control -41%), whilst the effect of IGF-I on this index was insignificant (31%). The expression of the PKA regulatory subunit was increased by EGF (51% compared with 29% in the control, $p < 0.05$), but not by IGF-I or IGF-II (30 and 29%).Our observations demonstrate that 40 h of culture of porcine CO resulted in a decrease in the proliferation and development of apoptosis in CO cells. IGF-I or EGF can stimulate proliferation and inhibit apoptosis. The influence of growth factors on the PKA content of the CO suggests that cAMP/PKA may be a mediator of the action of growth factors on these cells. The differential effects of IGFs and EGF on the regulatory subunit of PKA may indicate differences between their mechanisms of action.Effets des facteurs de croissance sur la prolifération, l'apoptose et l'expression de la protéine kinase A des complexes “ cumulus-ovocyte " de porc en culture. Des complexes “ cumulus-ovocyte " de porc (COC) ont été mis en culture en présence d'IGF-I, IGF-II, EGF (10 ng.mL$^{-1}$ de milieu). Leurs effets sur la prolifération cellulaire, l'apoptose et la protéine kinase A (PKA) ont été évalués par immuno-cytochimie à l'aide d'anticorps spécifiques dirigés contre PCNA, TUNEL et les sous-unités catalytique (C-alpha) et régulatrice (RI) de PKA. En milieu témoin, les cellules des COC montrent une diminution de prolifération (de 51 à 31 % de cellules PCNA positives, $p < 0,05$). Avec IGF-I ou EGF dans le milieu, on constate au contraire une augmentation de la prolifération par comparaison aux niveaux de départ (64 et 67 % respectivement, $p < 0,001$). Pendant la culture, les cellules apoptotiques (TUNEL-positives) ont augmenté de 42 à 57 % ($p < 0,01$) ce qui est inhibé par l'addition d'IGF-I ou d'EGF (36 et 12 % des témoins respectivement, $p < 0,001$). IGF-II et EGF ont réduit de façon significative les quantités de la sous-unité catalytique de PKA (cellules immuno-détectables pour cette sous-unité : 28 et 27 % respectivement par comparaison aux témoins : 41 %, $p < 0,05$) alors que l'effet d'IGF-I n'a pas été significatif (31 %). EGF a provoqué une augmentation de la sous-unité régulatrice de PKA (51 % versus 29 % chez les témoins, $p < 0,05$) alors que IGF-I et IGF-II ont été sans effet sur cette dernière (30 et 29 % respectivement). Nos observations montrent que les cellules de COC de porc ont une activité de prolifération en diminution et une apoptose en augmentation lorsqu'elles sont cultivées pendant 40 h. IGF-I et EGF peuvent stimuler la prolifération et inhiber l'apoptose. L'influence de ces facteurs de croissance sur PKA suggère que cAMP/PKA peut être un médiateur de ces facteurs sur ces types cellulaires. Les effets différenciés des IGFs et de l'EGF sur les sous-unités de PKA peuvent indiquer des différences de leurs modes d'action

Glutathione during Post-Thaw Recovery Culture Can Mitigate Deleterious Impact of Vitrification on Bovine Oocytes

Vitrification of bovine oocytes can impair subsequent embryo development mostly due to elevated oxidative stress. This study was aimed at examining whether glutathione, a known antioxidant, can improve further embryo development when added to devitrified oocytes for a short recovery period. Bovine in vitro matured oocytes were vitrified using an ultra-rapid cooling technique on electron microscopy grids. Following warming, the oocytes were incubated in the recovery medium containing glutathione (0, 1.5, or 5 mmol L−1) for 3 h (post-warm recovery). Afterwards, the oocytes were lysed for measuring the total antioxidant capacity (TAC), activity of peroxidase, catalase and glutathione reductase, and ROS formation. The impact of vitrification on mitochondrial and lysosomal activities was also examined. Since glutathione, added at 5 mmol L−1, significantly increased the TAC of warmed oocytes, in the next set of experiments this dose was applied for post–warm recovery of oocytes used for IVF. Glutathione in the recovery culture did not change the total blastocyst rate, while increased the proportion of faster developing blastocysts (Day 6–7), reduced the apoptotic cell ratio and reversed the harmful impact of vitrification on the actin cytoskeleton. These results suggest that even a short recovery culture with antioxidant(s) can improve the development of bovine devitrified oocytes

Enrichment of Rabbit Primitive Hematopoietic Cells via MACS Depletion of CD45+ Bone Marrow Cells

Hematopoietic stem and progenitor cells (HSC/HPCs) of human or few animal species have been studied for over 30 years. However, there is no information about rabbit HSC/HPCs, although they might be a valuable animal model for studying human hematopoietic disorders or could serve as genetic resource for the preservation of animal biodiversity. CD34 marker is commonly used to isolate HSC/HPCs. Due to unavailability of specific anti-rabbit CD34 antibodies, a novel strategy for the isolation and enrichment of rabbit HSC/HPCs was used in this study. Briefly, rabbit bone marrow mononuclear cells (BMMCs) were sorted immunomagnetically in order to remove all mature (CD45+) cells. The cells were depleted with overall purity about 60&ndash;70% and then cultured in a special medium designed for the expansion of CD34+ cells. Quantitative Polymerase Chain Reaction (qPCR) analysis confirmed the enrichment of primitive hematopoietic cells, as the expression of CD34 and CD49f increased (p &lt; 0.05) and CD45 decreased (p &lt; 0.001) at the end of culture in comparison to fresh BMMCs. However, cell culture still exhibited the presence of CD45+ cells, as identified by flow cytometry. After gating on CD45&minus; cells the MHCI+MHCII&minus;CD38+CD49f+CD90&minus;CD117&minus; phenotype was observed. In conclusion, rabbit HSC/HPCs might be isolated and enriched by the presented method. However, further optimization is still required