Prepubertal Sheep Oocytes Research Articles

Roscovitine use for the delay of meiotic progression in prepubertal sheep oocytes

Abstract: The objective of this work was to evaluate the efficiency of roscovitine on reversibly inhibiting oocytes from prepubertal sheep at the germinal vesicle (GV) stage, and to investigate the kinetics of meiosis progression after inhibitor removal. Cumulus-oocyte complexes, recovered from Sarda breed lambs aged 30-40 days, were cultured for 6 hours in a maturation medium (control) containing 75 μmol L-1 roscovitine (Rosco) at 38.5°C and 5% CO2. Then, the complexes were subjected to in vitro maturation (IVM) for 18 or 23 hours, in an inhibitor-free medium supplemented with gonadotropins. The evaluation of nuclear configuration by Hoescht staining, under a fluorescence-inverted microscope, showed that 88.7% of the lamb oocytes treated with roscovitine remained at the GV stage, as observed for the immature ones (97.3%) stained after collection. The inhibitory action was reversible; however, the proportion of oocytes (83.3%) at the metaphase-II stage, after 23 hours of IVM, was significantly higher than that observed after 18 hours (29.5%), in which meiosis was still in progression with 34.2% oocytes at metaphase-I, 11.6% oocytes at anaphase-I, and 18.5% oocytes at telophase-I. Roscovitine is efficient to arrest the nuclear maturation in oocytes from prepubertal sheep; however, despite the reversibility, meiosis progression is delayed, requiring more time to be completed.

Kompetensi Maturasi dan Fertilisasi Oosit Domba Prapubertas Secara In Vitro (DEVELOPMENTAL COMPETENCE OF MATURATION AND FERTILIZATION PREPUBERTAL SHEEP OOCYTES IN VITRO)

The objective of this study was to evaluate the maturation and fertilization ability of oocytes frompre-pubertal sheep ovary in vitro. Prepubertal ovary were collected based on the absence of corpus luteumor absence of corpus albicans in both of the ovaries. Oocytes were collected by slicing methode. Only theoocytes which categories of homogeneous cytoplasm and compact cumulus cells were used and it wasmatured for 24 hours in CO2 incubator with temperature 39oC. Oocytes fertilized in vitro used post thawedspermatozoa with concentration 5x106 and incubated for 12-14 hours. Oocytes were evaluated on numberof oocytes reached MII and number of PN formation. Result of the experiment revealed that there was nosignificant difference in the percentage of MII oocytes after in vitro maturation (89% vs 90,7%, PA 0,05)between prepubertal sheep and pubertal sheep. Meanwhile, the fertilization rate was significantly lower(PÂ 0,05) in prepubertal sheep oocytes compared to pubertal sheep oocytes (60% vs 77,7%). The incidenceof polispermic fertilization was higher in prepubertal sheep oocytes than pubertal sheep oocytes (21,8% vs7,4%) (PA0,05). In conclusion, prepubertal sheep oocytes and pubertal sheep oocytes have similar in vitromaturation ability, even though the ability to be fertilized of the prepubertal sheep oocytes is lower thanthe pubertal sheep.

The effect of conspecific ampulla oviductal epithelial cells during in vitro maturation on oocyte developmental competence and maturation-promoting factor (MPF) activity in sheep

The acquisition of fertilization ability by oocytes is one of the prerequisites for successful in vitro embryo production. In the present study, we examined the influence of conspecific ampulla oviductal epithelial cells incubated with cumulus-oocyte complexes (COCs) throughout the IVM phase on the developmental competence and maturation-promoting factor (MPF) activity of sheep oocytes. There were six experimental groups in this study, namely four groups with and two groups without oviductal epithelial cells added to IVM media: adult COCs matured in vitro with the ampulla oviductal epithelial cells obtained from adult (AAE; G1) or prepubertal donors (prepubertal sheep ampulla oviductal epithelial cells [PAE]; G4), COCs obtained from prepubertal animals cocultured with AAE (G2) or PAE (G3), and adult (C1) and prepubertal sheep COCs (C2) matured without oviductal epithelial cells. Coincubation of oocytes retrieved from both adult and sexually immature donors with AAE (G1 and G2) resulted in significantly improved rates of metaphase-II (M-II) attainment (G1: 85.1 ± 2.0 and G2: 40.2 ± 1.3) and blastocyst formation (G1: 42.2 ± 1.1 and G2: 21.2 ± 1.0) as well as blastocyst development (total cell count; G1: 130.3 ± 7.8, G2: 70.2 ± 3.5) compared with their respective controls (C1: 94.3 ± 4.1 and C2: 49.7 ± 2.0). Prior to IVM, the activity of MPF was greater (P < 0.05) for oocytes obtained from ewes (G1, G4, and C1) compared with those from ewe lambs (G2, G3, and C2). The greatest increment in MPF activity was recorded in G2 (MPF activity before IVM/MPF activity after IVM = 3.62) followed by C2 and G3 (2.22 and 2.20, respectively), and then all remaining groups of oocytes (C1: 1.89, G1: 1.87, and G4: 1.86). In summary, coincubation with AAE during the 24-hour IVM had a positive impact on ovine oocyte competence and ensuing in vitro embryo production efficiency. A significant increase in MPF activity following IVM of G2 oocytes could be responsible, at least partly, for the improved rate of blastocyst formation after IVF of prepubertal sheep oocytes.

308 VERBASCOSIDE TREATMENT DURING IN VITRO MATURATION IMPROVES THE EMBRYO DEVELOPMENT OF PREPUBERTAL OVINE OOCYTES

Verbascoside (VB), a bioactive polyphenol from olive mill wastewater with known antioxidant activity, was shown to act as a pro-oxidant molecule, by impairing energy/redox status and embryo developmental competence of prepubertal ovine oocytes when added at micromolar concentrations in a continuative 24-h in vitro maturation (IVM) exposure protocol (1). The aim of the present study was to determine whether a lower (nanomolar) VB concentration and a shorter exposure time (2 v. 24 h) during IVM may improve the maturation rates of prepubertal ovine oocytes and their subsequent embryonic development in vitro. Cumulus-oocyte complexes derived from the ovaries of slaughtered 1-mo-old prepubertal sheep oocytes underwent IVM in TCM 199 with 10% oestrus sheep serum, 0.1 IU mL–1 of FSH/LH, and 100 µM cysteamine, in 5% CO2 in air at 38.5°C for 24 h. Based on our previous results (Dell'Aquila et al. 2014 Biomed. Res. Int. 2014, 878062), VB was added in the IVM medium at 1.03 nM, and 2 incubation times (24 and 2 h) were tested. In the 2-h exposure group, after 2 h of exposure to VB, oocytes were washed and cultured up to 24 h without VB. A group of oocytes were cultured in absence of VB, as controls. Matured oocytes were fertilized with frozen-thawed ram semen in SOF medium for 22 h and zygotes were cultured in vitro for 8 days. Metaphase II (MII) cleavage and blastocyst rates were analysed by Chi-squared test. Embryo quality was evaluated by staining and total cell count of the blastocyst and analysis of variance (ANOVA) was applied. Differences were considered to be significant when P &lt; 0.05. Compared to controls, VB treatment at the concentration of 1.03 nM and 24 h of exposure had no effect on MII rates (196/268, 73% v. 226/323, 70% MII/cultured oocytes; P &gt; 0.05). However, this treatment allowed to obtain significantly higher rates of cleaved embryos/MII oocytes (156/196, 80% v. 165/226, 73%; respectively; P &lt; 0.05), blastocyst yield/cleaved embryos (59/156, 38% v. 45/165, 27%, respectively; P &lt; 0.05), and total blastocyst cell numbers (108.62 ± 19.87 v. 89.61 ± 26.32, respectively; P &lt; 0.05) compared to control oocytes. The VB treatment at the same concentration but for 2 h induced only significantly higher cleavage rate (196/210, 93% v. 165/226, 73%; P &lt; 0.05). In conclusion, our results showed that VB treatment at 1.03 nM during 24 h of IVM exerted a positive effect on in vitro embryo development of prepubertal ovine oocytes by increasing the blastocyst yield and their quality. The hypothesis that VB at nanomolar concentrations may improve cumulus-oocyte energy/redox status is under investigation.The authors acknowledge support by the Regione Autonoma della Sardegna (LR 7, Agosto 2007, no. 7, CRP-17602). The authors thank Dr D. Bebbere and L. Falchi, Dept. Veterinary Medicine, Sassari, for statistical analysis.

Effect of α-linolenic acid on oocyte maturation and embryo development of prepubertal sheep oocytes

The purpose of this study was to evaluate the effect of omega-3 α-linolenic acid (ALA) added to the IVM medium on embryo development of prepubertal sheep oocytes. Experiment 1 investigated the effect of ALA at different concentrations (0 [control], 50, 100, and 200 μM) and DMSO (100 μM) in IVM media on cumulus cell expansion and oocyte nuclear maturation and on synthesis of prostaglandins (PGE2 and PGF2α). Experiment 2 investigated the effects of ALA at different concentrations in the IVM medium on oocyte fertilization, cleavage, and developmental potential to blastocyst stage and changes in estradiol and progesterone concentrations in the spent IVM media. IVM oocytes were fertilized with frozen-thawed spermatozoa capacitated in a serum-free sperm medium. Presumptive zygotes were cultured 8 days in synthetic oviductal fluid (SOF) medium without serum. Blastocyst quality was assessed by counting total cell number and the number of apoptotic cells using Hoechst and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Nuclear maturation of oocytes and the number of fully expanded cumulus cells were reduced after treatment with 200 μM of ALA compared with other groups (P ≤ 0.05). Supplementation with ALA increased both PGE2 and PGF2α concentrations in the spent media (P ≤ 0.05). No differences were observed in blastocyst development among control (12.2%) and 50, 100, and 200 μM ALA groups (6.9%, 11.5% and 14.0%, respectively). However, the total cell number (46.50 ± 5.85, 67.94 ± 6.71, 45.20 ± 6.37, and 59.80 ± 5.51, respectively; P ≤ 0.05) and apoptotic cell number (6.45 ± 0.89, 2.48 ± 0.81, 4.02 ± 1.15, and 3.67 ± 1.15, respectively; P ≤ 0.05) were significantly improved. After IVM, estradiol concentration was lower and progesterone concentration was higher in ALA groups compared with the control group (P ≤ 0.05). In conclusion, these results revealed that ALA affects prepubertal sheep embryo quality associated with alteration of releasing reproductive hormones.

Mitochondria and vesicles differ between adult and prepubertal sheep oocytes during IVM

Oocytes from prepubertal animals have a reduced ability to undergo normal embryo development and produce viable offspring. The correct quantity, activity and cytoplasmic distribution of oocyte organelles are essential for oocyte maturation, fertilisation and subsequent embryo development. The aim of this study was to quantify the ultrastructural differences between oocytes from prepubertal lamb and adult ewes using electron microscopy and stereology. We also determined whether quantitative polymerase chain reaction (qPCR) methods give comparable estimates of mitochondrial number to stereology. Mean storage vesicle volume was greater in adult compared with lamb oocytes before IVM and decreased during maturation in both adult and lamb oocytes. Mitochondrial volume and number increased in adult oocytes during maturation; however, no increase was observed in lamb oocytes. Mitochondrial DNA copy number measured by qPCR showed no differences between adult and lamb oocytes. A different distribution of mitochondria was observed in lamb oocytes before maturation, while the percentage of hooded mitochondria increased during maturation in adult oocytes and decreased in the lamb. In conclusion, the present study has identified differences in the vesicles and mitochondria between adult and lamb oocytes from ewes that may contribute to reduced developmental competence in prepubertal oocytes.

AY9944 A-7 promotes meiotic resumption and preimplantation development of prepubertal sheep oocytes maturing in vitro

Follicular fluid meiosis-activating sterol (FF-MAS), an intermediate in the cholesterol biosynthetic pathway, has been identified as a compound that induces the resumption of meiosis in mammalian oocyte. FF-MAS is converted to testis meiosis-activating sterol by a sterol Δ14-reductase. An inhibitor of Δ14-reductase and Δ7-reductase, AY9944 A-7, causes accumulation of FF-MAS by inhibiting its metabolism. The objective of this study was to determine the effects of AY9944 A-7 supplementation to oocyte maturation media on prepubertal sheep oocyte meiotic resumption and subsequent preimplantation development of embryos. Prepubertal sheep oocytes isolated at the germinal vesicle stage from their follicles were cultured with 0, 10, 20, 30, and 40 μM AY9944 A-7 for 24 hours in media with or without a meiotic inhibitor hypoxanthine (Hx, 4 mM). The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown and the first polar body (PBI) extrusion. After maturation for 24 hours, oocytes with PBI were inseminated in vitro, and the percentages developing to the two-cell stage and blastocyst stage were measured as indicators of early embryonic developmental competence. AY9944 A-7 induced maturation of sheep cumulus-oocyte complexes with optimal concentrations of 10 and 20 μM both in Hx-inhibited meiotic maturation and spontaneous maturation, whereas AY9944 A-7 with any concentrations had no significant effect on that of denuded oocytes and split cumulus-oocyte complexes. Furthermore, maturing oocytes treated with either 10 or 20 μM AY9944 A-7 dramatically increased the percentages of ovine embryos developing to the two-cell stage and blastocyst stage. Higher concentrations of AY9944 A-7, 30 and 40 μM, were detrimental to oocytes and led to their degeneration. The present findings indicated for the first time that AY9944 A-7 was not only able to promote meiotic maturation, both Hx-inhibited and spontaneous, but also enhanced preimplantation developmental competence of prepubertal sheep oocytes maturing in vitro.

Brilliant Cresyl Blue stain selects largest oocytes with highest mitochondrial activity, maturation-promoting factor activity and embryo developmental competence in prepubertal sheep

The aim of this study was to test the Brilliant Cresyl Blue (BCB) stain to select prepubertal sheep oocytes for in vitro blastocyst production. Oocyte diameter, mitochondrial activity, maturation-promoting factor (MPF) activity and mRNA relative expression (RE) of genes related to metabolism (ATPase Na(+)/K(+) transporting α 1 (ATP1A1) and cytochrome c oxidase subunit 1 (COX1)) and constitutive function of the cell (cytoplasmic polyadenylation-element-binding protein (CPEB) and S100A10) were assessed. Immature oocytes were exposed to different BCB concentrations (13, 26, 39 and 52 μM) and classified according to their cytoplasm colouration as grown BCB+ (blue cytoplasm) and growing BCB- (colourless cytoplasm). Staining oocytes with 13 μM BCB during 60 min allows selection of (BCB+) the largest (123.66 μm) and most competent oocytes to develop to the blastocyst stage (21%) with a higher number of cells (69.71 ± 6.19 s.e.m.) compared with non-stained BCB- oocytes (106.82 μm, 9% and 45.91 ± 3.35 s.e.m. respectively). Mitochondrial activity, assessed by MitoTracker Orange CMTMRos probe, was significantly higher in BCB+ than in BCB- oocytes after in vitro maturation (3369 and 1565 AU respectively). MPF activity was assessed by CDC2 kinase activity assay showing significantly higher activity at metaphase II stage in BCB+ than in BCB- oocytes (1.479 ± 0.09 and 1.184 ± 0.05 optical density respectively). The genes analysed in this work, ATP1A1, COX1, CPEB and S100A 10, did not show significant effect in mRNA RE between BCB selected oocytes. In conclusion, BCB stains larger and more competent oocytes to develop to the blastocyst stage with more active mitochondria and MPF activity and higher blastocyst cell number.

251 SELECTION OF PREPUBERTAL SHEEP OOCYTES USING BRILLIANT CRESYL BLUE TEST

The aim of this study was to evaluate the utility of the brilliant cresyl blue (BCB) test as an indirect measure of oocyte growth to select competent prepubertal sheep oocytes for in vitro embryo production. The BCB stain allows the determination of glucose–6-phosphate dehydrogenase (G6PD) activity, an enzyme with decreased activity in oocytes that have finished their growth phase. Oocytes were obtained after slicing the surface of lamb ovaries (2–5 months old) obtained from a local abattoir. Oocytes with more than 3 compact cumulus layers and homogeny cytoplasm were selected and stained with different concentrations of BCB diluted in PBS (13-, 26-, 39-, and 52-μM BCB) during 60 min at 38.5°C in a humidified air atmosphere. Oocytes were classified into groups depending on their cytoplasm coloration: oocytes with blue cytoplasm or grown oocytes (BCB+) and oocytes without blue coloration or growing oocytes (BCB–). Oocytes were matured in an enriched TCM-199 medium for 24 h at 38.5°C and 5% CO2 in a humidified atmosphere. Oocyte diameter was also measured. Matured oocytes were partially denuded and transferred to fertilization medium (SOF) supplemented with 10% of oestrous sheep serum. Fresh semen was kept at room temperature (25°C) for 1 h. Highly motile spermatozoa were selected by using Ovipure density gradient (Nidacon EVB S.L.), and oocytes were fertilized with 1 × 106 sp mL–1. After 20 h postinsemination, presumptive zygotes were cultured for 8 days in SOF with 10% of fetal bovine serum at 38.5°C, 5% CO2, and 90% N2. Data was analysed by performing Fisher’s exact test for blastocyst production and ANOVA with Tukey’s post-test for oocyte diameter. Table 1 shows the percentage of BCB-stained oocytes and their embryo development. In this study oocytes exposed during 60 min to 13-μM BCB showed a higher percentage of embryos reaching blastocyst stage than did those in the control group (≤0.01). In other species such as goats (Rodriguez-Gonzalez et al. 2002 Theriogenology 57(5), 1397–1409) and cattle (Alm et al. 2005 Theriogenology 63(8), 2194–2205), the best protocol for the oocyte selection was the use of 26-μM BCB during 90 min. Oocyte diameter showed significant differences between BCB– with BCB+ and control group (110 μm, 134 μm, and 121 μm, respectively, ≤0.001). In conclusion, using 13 μM of BCB during 60 min is a suitable technique for increasing embryo blastocyst rates using lamb oocytes. Table 1.Effect of BCB1 concentration on embryo development of lamb oocytes The grant sponsor was the Spanish Ministry of Science and Innovation, Code: AGL2007-60227-CO2-01.

Meiotic Maturation of Incompetent Prepubertal Sheep Oocytes Is Induced by Paracrine Factor(s) Released by Gonadotropin-Stimulated Oocyte-Cumulus Cell Complexes and Involves Mitogen-Activated Protein Kinase Activation

In this study, sheep oocyte-cumulus cell complexes (OCC) derived from medium (M) antral follicles (M-OCC) were in vitro matured alone or in coculture with OCC derived from small (S) antral follicles (S-OCC) to investigate the contribution of cumulus cells (CC) and oocytes to the process of oocyte meiotic maturation and cumulus expansion (CE). Experiments were conducted with or without gonadotropins (FSH/LH). Regardless of culture conditions, about 12% of S-oocytes reached the metaphase II stage, and S-CC showed a low degree of CE. In contrast, both maturational processes were significantly stimulated by gonadotropins in M-OCC. However, about 48% of S-oocytes progressed to metaphase II, and S-CC expanded after coculture with gonadotropin-stimulated M-OCC and M-CC but not with mural granulosa cells. Both maturational processes were inhibited when S-OCC were cocultured with M-denuded oocytes, or when S-denuded oocytes were cocultured with M-CC. The capacity of these paracrine factor(s) to activate the MAPK pathway in somatic and germ cells of S-complexes was investigated. It was found that MAPK kinase/MAPK phosphorylation levels in M-OCC but not in S-OCC were significantly increased by gonadotropins, first in CC and later in the oocytes. Kinase phosphorylations were activated only in S-oocytes cocultured with M-OCC or M-CC. These results demonstrate that soluble factors specifically produced by M-CC are capable to induce meiotic maturation and CE in S-complexes by acting via CC. These factors can induce MAPK activation only in S-oocytes, whose meiotic arrest could be due to the inability of surrounding CC to respond to gonadotropin stimulation.

Analysis of proteins and their synthesis in bovine oocytes and associated cumulus cells during maturation in vitro

The acquisition of fertilization ability by oocytes is one of the prerequisites for successful in vitro embryo production. In the present study, we examined the influence of conspecific ampulla oviductal epithelial cells incubated with cumulus-oocyte complexes (COCs) throughout the IVM phase on the developmental competence and maturation-promoting factor (MPF) activity of sheep oocytes. There were six experimental groups in this study, namely four groups with and two groups without oviductal epithelial cells added to IVM media: adult COCs matured in vitro with the ampulla oviductal epithelial cells obtained from adult (AAE; G1) or prepubertal donors (prepubertal sheep ampulla oviductal epithelial cells [PAE]; G4), COCs obtained from prepubertal animals cocultured with AAE (G2) or PAE (G3), and adult (C1) and prepubertal sheep COCs (C2) matured without oviductal epithelial cells. Coincubation of oocytes retrieved from both adult and sexually immature donors with AAE (G1 and G2) resulted in significantly improved rates of metaphase-II (M-II) attainment (G1: 85.1 ± 2.0 and G2: 40.2 ± 1.3) and blastocyst formation (G1: 42.2 ± 1.1 and G2: 21.2 ± 1.0) as well as blastocyst development (total cell count; G1: 130.3 ± 7.8, G2: 70.2 ± 3.5) compared with their respective controls (C1: 94.3 ± 4.1 and C2: 49.7 ± 2.0). Prior to IVM, the activity of MPF was greater (P < 0.05) for oocytes obtained from ewes (G1, G4, and C1) compared with those from ewe lambs (G2, G3, and C2). The greatest increment in MPF activity was recorded in G2 (MPF activity before IVM/MPF activity after IVM = 3.62) followed by C2 and G3 (2.22 and 2.20, respectively), and then all remaining groups of oocytes (C1: 1.89, G1: 1.87, and G4: 1.86). In summary, coincubation with AAE during the 24-hour IVM had a positive impact on ovine oocyte competence and ensuing in vitro embryo production efficiency. A significant increase in MPF activity following IVM of G2 oocytes could be responsible, at least partly, for the improved rate of blastocyst formation after IVF of prepubertal sheep oocytes.