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

Evidence that growth factors IGF-I, IGF-II and EGF can stimulate nuclear maturation of porcine oocytes via intracellular protein kinase A

The aim of our in vitro experiments was to study the role of growth factors and protein kinase A (PKA)-dependent intracellular mechanisms in the control of nuclear maturation of porcine oocytes. Oocytes were cultured with or without growth factors (IGF-I, IGF-II, EGF; 10 ng$\cdot$mL$^{-1}$ medium) and inhibitors of PKA (Rp-cAMPS or KT5720; 100 ng$\cdot$mL$^{-1}$). Stages of meiosis were determined from the structure of chromosomes after staining with Giemza. Intracellular levels of PKA were evaluated immunocytochemically using primary antisera against the PKA regulatory and catalytic subunits and by Western immunoblotting using primary antiserum against the PKA catalytic subunit. It was found that after 24 h culture the majority of oocytes had resumed nuclear maturation (they were at a stage of meiosis after diplotene) and that after 48 h culture the majority of cells had completed maturation (they had reached metaphase II of meiosis). Addition of IGF-I, IGF-II or EGF, or a combination of IGF-I and EGF, significantly increased the proportion of oocytes which resumed and completed meiosis. Immunocytochemistry demonstrated a significant increase in the proportion of cells containing catalytic and, in some cases, the regulatory subunits of PKA after addition of IGF-I, IGF-II and EGF. Immunoblotting showed the presence of 2 forms of the PKA catalytic subunit within the oocytes (MW approximately 52 and 40 kD). EGF, but not IGF-I or IGF-II, increased the content of both isoforms. Inhibitors of PKA, when given alone, did not substantially influence the proportion of oocytes which resumed or completed meiosis. However, Rp-cAMPS and KT5720 both prevented the stimulatory effects of IGF-I, IGF-II and EGF on the resumption and completion of oocyte maturation. The present observations suggest (1) that IGF-I, IGF-II and EGF are potent stimulators of both resumption and completion of porcine oocyte nuclear maturation, (2) that PKA is present in oocytes, and (3) that PKA-dependent intracellular mechanisms can mediate the action of growth factors on porcine oocytes.Les facteurs de croissance IGF-I, IGF-II et EGF peuvent stimuler la maturation nucléaire de l'ovocyte de porc par la voie intracellulaire de la protéine kinase A. Ces expériences ont pour but d'étudier in vitro le rôle des facteurs de croissance et des mécanismes intracellulaires dépendant de la protéine kinase A (PKA) vis-à-vis du contrôle nucléaire des ovocytes porcins. Des ovocytes ont été cultivés avec ou sans facteurs de croissance (IGF-I, IGF-II, EGF ; 10 ng$\cdot$mL$^{-1}$ de milieu) et avec ou sans des inhibiteurs de la PKA (Rp-cAMPS, KT5720 ; 100 ng$\cdot$mL$^{-1}$). Les stades de la méiose ont été déterminés par examen des chromosomes après coloration par le Giemza. Les niveaux intracellulaires de PKA ont été évalués par immunocytochimie en utilisant un anticorps primaire de la sous-unité régulatrice et de la sous-unité catalytique de PKA et par Western immunoblot avec des anticorps primaires dirigé contre la sous-unité catalytique. Après 24 h de culture, la majorité des ovocytes a repris sa maturation nucléaire (stade diplotène dépassé) et elle l'a terminée (stade métaphase II atteint) après 48 h. L'addition de IGF-I, IGF-II ou d'EGF, ou une combinaison de IGF-I et d'EGF, accroît significativement la proportion d'ovocytes qui ont repris et terminé leur méiose. L'addition de IGF-I, IGF-II ou d'EGF s'accompagne d'un accroissement significatif des cellules exprimant la sous-unité catalytique et dans certains cas la sous-unité régulatrice de la PKA (immunocytochimie). La présence de deux formes de la sous-unité catalytique à l'intérieur des ovocytes est montrée en immunoblot (PM 52 et 40 kD environ). EGF mais non IGF-I ou IGF-II augmente le contenu des deux isoformes. Les inhibiteurs de la PKA, utilisés seuls, ne modifient pas la proportion des ovocytes qui reprennent ou terminent leur méiose. Cependant ils préviennent les effets stimulants de IGF-I, IGF-II et EGF. Ces observations suggèrent que : (1) IGF-I, IGF-II et EGF sont des stimulateurs efficaces de la reprise et de l'achèvement de la maturation nucléaire chez le porc, (2) PKA est présente dans les ovocytes, et (3) les facteurs de croissance peuvent agir sur les ovocytes porcins par des mécanismes intracellulaires dépendant de la PKA

Sperm quality of Zemplin Rabbit and Liptov Bold-Spotted Rabbit breeds

Zemplin Rabbit and Liptov Bold-Spotted Rabbit are Slovak national breeds. Our aim was to characterize the sperm quality of these two breeds, as these characteristics are important for artificial insemination and cryopreservation as biodiversity conservation tools. For this evaluation, sperm samples of sexually mature Zemplin Rabbit (ZR) males (n = 6) and Liptov Bold-Spotted Rabbits (LR) (n = 4) were used. According to progressive motility (PM) data (CASA), samples were divided into two groups: A (>30% PM) and B (<30% PM). In addition to PM (ZR-A: 48.6±3.8%, ZR- B: 16±3.2%, LR-A: 35.38±2.6%, LR-B: 4±2.2%), total motility (TM) (ZR-A: 69.1±4.1%, ZR-B: 35.5±4.1%, LR-A: 59.1±3%, LR-B: 26.9±4.1%) and morphological abnormalities (ZR-A: 29.7±1.2%, ZR-B: 40±4%, LR-A: 34.3±4.3%, LR-B: 48.3±4.7%) were also assessed using the CASA system. The proportion of dead/live, apoptotic and oxidative damaged spermatozoa (Spz) was assessed by flow cytometry using fluorescent dyes: SYBR-14, Sytox Green, Yo-Pro-1 and CellROX Green, respectively. The results of flow cytometry correspond to the values of motility and morphometry. Sperm with PM less than 30% does not show proper quality values, while the sperm with PM higher than 30% is suitable for further analysis and use