Sheep Preantral Follicles Research Articles

Effect of the acidified extract of Moringa oleifera leaves as a supplement in the in vitro culture medium of sheep preantral follicles

This study was conducted to evaluate the effects of the acidified extract of M. oleifera leaves as a supplement into the base medium for in vitro culture of sheep isolated secondary follicles. Follicles were isolated and cultured for 12 days in α-MEM+(supplemented with bovine serum albumin, insulin, glutamine, hypoxanthine, transferrin, selenium, and ascorbic acid) with or without 0.1; 0.2 or 0.4 mg/ml of the acidified extract of M. oleifera. Follicle morphology, antral cavity formation, follicular and oocyte diameter, glutathione (GSH) concentration, mitochondrial activity and meiotic resumption were evaluated. After 12 days of culture, there was no significant difference among treatments in relation to follicular morphology, antral cavity formation, diameter and mitochondrial activity. Nevertheless, oocytes from follicles cultured in α-MEM+ showed greater GSH concentration than media containing M. oleifera extract. Furthermore, the concentration of 0.4 mg/ml M. oleifera extract significantly increased the percentage of fully grown oocyte (≥ 110 µm) when compared to the other treatments. In conclusion, the concentration of 0.4 mg/ml M. oleifera extract as a supplement of the culture medium, maintained the survival, and increased the percentage of fully grown oocytes.

Effect of reduced water intake on ovarian reserve, leptin immunoexpression and impact of leptin on the in vitro culture of sheep secondary follicles

This study evaluated the effect of reduced water intake on survival, apoptosis and immunoexpression of leptin in sheep preantral follicles, activation of primordial follicles, serum levels of leptin, estradiol (E2) and progesterone (P4), and in vitro maturation (IVM) of oocytes antral follicles, as well evaluated the effects of leptin on in vitro culture of secondary follicles isolated these animals. Ewes (n = 32) were divided into four groups: water ad libitum (Control – 100%), 80%; 60% and 40% of ad libitum intake. Blood was collected to determine, leptin, E2 and P4, before and after experiment. After the slaughter, ovarian cortex was used to histological and immunohistochemistry analysis and oocytes IVM. Moreover, isolated secondary follicles were cultured in vitro for 12 days in control medium (α-MEM+) or α-MEM+ with 10 or 25 ng/mL leptin. The reduction of water intake caused a linear decreasing effect on the percentages of normal preantral follicles, especially of primordial (P < 0.05), increased the apoptosis (P < 0.05) and decreased leptin expression in preantral follicles. The treatment with 60% of water intake showed greater total growth rate of isolated secondary follicles cultured with 25 ng/L leptin (P < 0.05), compared to those cultured in α-MEM+ . In conclusion, reduced water intake impaired the number of normal sheep preantral follicles, especially of primordial follicles, increased apoptosis and decreased leptin expression in preantral follicles. Moreover, secondary follicles from of ewes that receive 60% water intake increased follicular growth after in vitro culture with 25 ng/mL leptin.

Immunolocalization of melatonin receptor type 1 in the sheep ovary and involvement of the PI3K/Akt/FOXO3a signaling pathway in the effects of melatonin on survival and in vitro activation of primordial follicles.

This study characterized the expression of melatonin receptor type 1 (MT1 ) protein in sheep ovaries, evaluated melatonin effects on primordial follicle survival and development after in vitro culture of ovarian tissue and verified the possible involvement of the phosphatidylinositol-3-kinase/protein kinase B/forkhead box O3a (PI3K/Akt/FOXO3a) pathway in the melatonin actions. Ovine ovarian fragments were cultured in α-modified minimum essential mediumalone (α-MEM+ ) or supplemented with 100, 500, or 1000 pg/ml melatonin for 7 days. PI3K inhibition was performed through pretreatment of ovarian fragments with LY294002. Thereafter, immunohistochemistry was performed to evaluate the expression of cleaved caspase-3, Akt, phosphorylated-Akt, and phosphorylated-FOXO3a(p-FOXO3a). The immunohistochemical localization of the MT1 receptor protein was documented in sheep preantral and antral follicles. After in vitro culture, 100 pg/ml melatonin showed higher follicular survival and activation than α-MEM+ and other melatonin concentrations. After PI3K inhibition, there was an increase in cleaved caspase-3-positive follicles, and a decrease in the primordial follicle activation, Akt phosphorylation, and nuclear exclusion of p-FOXO3a. In conclusion, MT1 receptor protein is present in the sheep ovary. Furthermore, 100 pg/ml melatonin maintains survival and stimulates activation of primordial follicles through the PI3K/Akt/FOXO3a signaling pathway after in vitro culture of sheep ovarian tissue.

In vitro growth and maturation of sheep preantral follicles: Influence of luteinizing hormone at stage-specific time points

In vitro growth and maturation of sheep preantral follicles: Influence of luteinizing hormone at stage-specific time points

Effect Of Retinol In The Vitrification Medium On Viability Of Vitrified Ovine Preantral Follicles And Expression Of Key Developmental And Apoptosis Related Genes

BACKGROUND: Vitrification increases the production of reactive oxygen species (ROS) and the antioxidants in the vitrification solution may be beneficial by reducing excessive ROS production. OBJECTIVE: To evaluate the effect of retinol supplementation in vitrification solution on viability, apoptosis and development-related gene expression in vitrified sheep preantral follicles. MATERIALS AND METHODS: Preantral follicles were isolated and randomly assigned into one of five groups: Group1, control fresh preantral follicles; Group 2, vitrification treatment; Group 3, vitrification + 2 μM retinol; Group 4, vitrification + 5 μM retinol ; Group 5, vitrification + 10 μM retinol . Preantral follicles were placed in vitrification solutions and then plunged into liquid nitrogen (-196°C). After a week, the follicles were thawed and analyzed for follicular viability by trypan blue exclusion method and for gene expression. RESULTS: Vitrification with 5 μM retinol positively affected viability in comparison with vitrification without retinol (P &lt; 0.05). There was no significant difference in viability among the Group 1, Group 2, Group 3 and Group 5. Expression of apoptotic genes BAX and Casp 3 were higher in the vitrified group, and vitrification with 5 μM retinol (Group 4) is comparable to the control fresh. Expressions of other apoptosis-related genes (i. e., BCL2L1, BAD and BAK) showed significant difference between the control fresh group and the vitrification group with 5 μM retinol. Expression of Annexin5 was also significantly different among various groups. The expression of development competence genes GDF-9 and BMP-15 were higher (P &lt; 0.05) in the Group vitrified with 5 μM retinol. CONCLUSION: The supplementation of 5 μM retinol in vitrification solution was beneficial for the vitrification of ovine preantral follicles.

Role of Melatonin as a Survival Factor for In vitro Development of Sheep Preantral Follicles

Background: Melatonin, a powerful free radical scavenger and broad-spectrum antioxidant may directly affect ovarian function by regulating folliculogenesis, maintenance of follicular integrity, oocyte quality and maturation capacity. Therefore, we aimed to study effects of melatonin and its interaction with growth factors in sheep preantral follicles. Methods: The influence of different concentrations of Melatonin (5-500 pM) on in vitro culture of preantral follicles (PFs’) isolated from sheep ovaries was studied. Experiments I and II were conducted to standardize the optimum concentration of Melatonin that supports better development of preantral follicles. Experiment III was conducted with the optimum level of Melatonin derived in the Experiments I and II to evaluate the effect of melatonin at 100pM in combination with various growth factors. Result: Overall follicular development was found to be the best in the PFs’ cultured in medium supplemented with 100pM of Melatonin. Melatonin supplementation showed positive effects on the preantral follicular development in combination with different growth factors.

​Effects of Time Specific Supplementation of Insulin Like Growth Factor-I (IGF-I) on in vitro Development of Sheep Preantral Follicles

Background: In in vitro culture systems IGF-I was known to promote follicular maturation, granulosa cell proliferation and increase overall cellular function. The expression patterns of developmentally important markers for IGF-I are expressed differentially during the transition of preantral follicles to the ovulatory stage. Therefore, we aimed to investigate the influence of time specific addition of Insulin like growth factor-I (IGF-I) on in vitro development of sheep preantral follicles (PFs’). Methods: Mechanically isolated sheep preantral follicles were cultured for six days either in TCM 199B or standard medium supplemented with IGF-I at different points during the culture period and were subsequently subjected to in vitro maturation (IVM) for additional 24hrs to evaluate different follicular development parameters. Conclusion: Based on the results, it is concluded that: 1) IGF-I supplementation was needed during early rather than later stages of culture period, therefore, role of IGF-I in time/stage specific manner in cultured preantral follicles is confirmed. 2) Supplementation of TCM 199B with IGF-I simultaneously for the first two days followed by TCM 199B alone without any growth factor (s) in later days (Treatment 1) supported better development of PFs’ in vitro.

Influence of TCM 199B, α-MEM, Waymouth MB 752/1 culture media, VEGF, Estradiol-17β, GDF-9 and FGF on in vitro development of preantral follicles in sheep.

A total of 2792 preantral follicles (PFs’) isolated from 750 ovaries of sheep were cultured in four different experiments. The efficacy of three commercially available culture media viz., TCM 199B, α-MEM and Waymouth MB 752/1 on the growth of sheep PFs’ was tested in experiment I. Among the three media TCM 199B supported better development of PFs’ in culture. The remaining experiments established the best concentrations of vascular endothelial growth factor (VEGF), Estradiol-17β (E2), GDF-9, Fibroblast growth factor (FGF) and their best combinations for the in-vitro development of PFs’. Inclusion of VEGF at 10 ng/mL, Estradiol-17β at 5 ng/mL, GDF-9 at 10 ng/mL or FGF at 10 ng/mL individually in a standard medium (SM) (containing FSH, IGF-I, GH and T4) supported better nuclear maturation of the oocytes to MII stage. Different combinations of VEGF, Estradiol-17β, GDF-9 and FGF supplemented in the SM promoted similar overall follicular growth. However, (a) SM + VEGF(10 ng/mL) + E2(5 ng/mL) supported higher increase in the diameter, (b) SM without any supplements induced antrum formation in greater proportion of follicles, and (c) SM + VEGF(10 ng/mL) + GDF 9(10 ng/mL) or SM + E2 (5 ng/mL) + FGF(10 ng/mL) supported high proportion of oocytes to reach MII stage. To conclude, TCM 199B appeared to be a better medium for development of sheep PFs’. VEGF, Estradiol-17 β, GDF-9 and FGF have beneficial influence on the development of sheep PFs’ when supplemented in TCM 199B

Effect of LH and Estradiol-17β Supplementation at Different Time Points on In vitro Development of Preantral Follicles in Sheep

Background: Ovarian follicular development and growth are controlled by many hormones and growth factors. Despite the fact that LH and estradiol-17β have been utilized for the in vitro culture of preantral follicles yet, the suitable time points of supplementation of LH and estradiol-17β is not known. Therefore this study aimed to investigate the influence of addition of LH and estradiol-17β at different time points on in vitro development of preantral follicles (PFs’) in sheep. Method: Preantral follicles isolated from the ovarian cortical slices using micro dissection method were cultured for six days in Bicarbonate buffered Tissue culture medium 199B (TCM 199B) or in a standard culture medium supplemented with LH (2 μg/ml) and estradiol-17β (5 ng/ml) at different points during the culture period. COCs isolated from the follicles at the end of six day culture in different treatments were subjected to in vitro maturation for additional 24h. Result: Supplementation of LH and estradiol-17β during last two days of the culture supported better proportion of PFs’ exhibiting growth whereas supplementation of LH and estradiol-17β during first two days of the culture supported better average increase in diameter and proportion of PFs’ exhibiting antrum formation at the end of six day culture. Further the oocytes in COCs isolated at the end of culture in these treatments and subsequently subjected to in vitro maturation (IVM) for 24hr developed at a higher frequency to MII (metaphase II) stage. Supplementation of LH and estradiol-17β to TCM 199B culture medium in early stages followed by standard medium alone in later stages supports better development of PFs’ in vitro. Following supplementation with LH and estradiol-17β for the first two days culture of PFs’ in standard medium appears to be advantageous for the development of preantral follicles in vitro.

Leptin induced in vitro development of ovarian follicles in sheep is related to the expression of P450 aromatase and steroidogenesis

The objective of this study was to test the hypothesis that Leptin induced in vitro growth in preantral follicles in sheep involves modulation of P450 aromatase expression and steroidogenesis. Accordingly, the expression of P450 aromatase gene was studied in the cumulus cells and oocytes isolated from different stages of preantral follicles (PFs') grown in vivo, cultured in TCM 199B, TCM 199B + Leptin (10 ng/ml) (TCM199BL) or a standard PF culture medium supplemented with Leptin (10 ng/ml) (SML). Ovarian follicles grown in vivo or in SML expressed P450 aromatase both in cumulus cells and oocytes at all the development stages. In the oocytes from PFs' grown in vitro, P450 expression was consistently lower than in those from in vivo grown follicles at all except the preantral stage. The patterns of expression of aromatase gene in the cumulus cells from in vivo grown and the PFs' cultured in TCM 199BL were similar. Significantly higher levels of progesterone production were supported by SML at all the development stages than the other two media. Oestradiol concentration in the spent TCM 199B and SML showed a significant increase as the development progressed from preantral to large antral stage. However, such increase was not sustained beyond early antral stage in the PFs’ cultured in TCM199BL. It is concluded that Leptin modulates the expression P450 aromatase while supporting the in vitro development of the ovarian follicles in sheep.

Kaempferol promotes primordial follicle activation through the phosphatidylinositol 3-kinase/protein kinase B signaling pathway and reduces DNA fragmentation of sheep preantral follicles cultured in vitro.

The aim of this study was to evaluate the effects of kaempferol on the morphology, follicular activation, growth, and DNA fragmentation of ovine preantral follicles cultured in situ, and the effects of a phosphatidylinositol 3 kinase (PI3K) inhibitor and the expression of phosphorylated protein kinase B (pAKT) after culture. Ovine ovarian fragments were fixed for histological and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analyses (fresh control) or cultured in α-MEM+ alone (control) or with different concentrations of kaempferol (0.1, 1, 10, or 100 μM) for 7 days. Follicles were classified as normal or atretic, primordial or growing, and the oocyte and follicle diameters were measured. Proliferating cells were analyzed and DNA fragmentation was evaluated by the TUNEL assay. Inhibition of PI3K activity was performed through pretreatment in media added with 50 µM LY294002 for 1 hr and pAKT immunohistochemistry was performed after culture in the absence or presence of LY294002. After culture, the percentage of normal follicles was similar among the treatments (p > 0.05), except for 100 µM kaempferol, which had less normal follicles (p < 0.05). Moreover, kaempferol at 10 μM showed a higher percentage of follicular activation and cell proliferation than the other treatments (p < 0.05) and a percentage of TUNEL-positive cells similar to that in the fresh control and lower than other treatments (p < 0.05). LY294002 significantly inhibited primordial follicle activation stimulated by α-MEM+ and 10 μM kaempferol and reduced pAKT expression in those follicles. In conclusion, 10 μM kaempferol promotes primordial follicle activation and cell proliferation through the PI3K/AKT pathway and reduces DNA fragmentation of ovine preantral follicles cultured in vitro.

Morphological-metric, ultrastructural and immunohistochemical effects of gossypol on cultured granulosa cells and oocytes of ewes using MOEPF

Manipulation of oocytes enclosed in preantral follicles (MOEPF) allows for analyzing follicular development and use of this biotechnology in the pre-analysis of the beneficial or toxic effects of bio-products on granulosa cells and oocytes at different developmental stages. In this study, there was evaluation of the effects of gossypol by culturing granulosa cells and oocytes in ewe ovarian tissues. Ovarian tissues were cultured with gossypol at 37 °C, in humidified air and 5% CO2. Variables that were evaluated were morphology, morphometry, ultrastructure and abundance of estradiol receptor α (α-ER). There were no differences in developmental characteristics when there was treatment with any of the gossypol doses that were evaluated. Immunostaining indicated that when the gossypol dose increases, the abundance of α-ER also increases in the cytoplasm, nucleus, and granulosa cells. Findings with the ultrastructural analysis indicated that for granulosa cells there was fewer cells and greater disorganization and a lack of structural integrity of follicular cell layers as a result of all gossypol treatments. The culture of oocytes in preantral ovarian follicles in presence of gossypol did not affect the morphological-metric structure at the doses evaluated. The findings with evaluation of ultrastructural and immunohistochemical structures indicated granulosa cells and α-ER were affected by the treatments with gossypol indicating there were effects of this compound on ovarian function in sheep. This study indicated there is a toxic action of gossypol when using the biotechnology, MOEPF. Thus, gossypol negatively affects granulosa cell development and structural integrity of preantral follicles in sheep.

Effect of leptin on in vitro development of ovine preantral ovarian follicles

The influence of human or ovine leptin on in vitro culture of preantral follicles (PFs) isolated from sheep ovaries was investigated. Among the 12 different concentrations (0–1000 ng/mL) of human leptin tested, proportion of PFs exhibiting growth, mean increase in diameter, antrum formation, and maturation of the oocytes to MII stage were the best in 10 ng/mL. Culture of sheep ovarian PFs in TCM 199 supplemented with 10 ng/mL of human or ovine leptin FSH (2.5 μg/mL), thyroxine (1 μg/mL), insulinlike growth factor I (10 ng/mL), and GH (1 mIU/mL) resulted in significantly (P ≤ 0.05) greater average increase in diameter (11 and 9 vs. 6 μm), better proportions of PFs exhibiting growth (66% and 58% vs. 48%), antrum formation (51% and 51% vs. 34%), and maturation of oocytes to MII stage (24% and 22% vs. 7%) than the control medium. It is concluded that (1) the optimum dose of leptin for the growth of sheep PFs in vitro was 10 ng/mL, (2) human or ovine leptin supported similar development in vitro of PFs in sheep, (3) inclusion of leptin along with FSH, thyroxine, insulinlike growth factor I, and GH resulted in only a marginal further improvements in in vitro development of sheep PFs'.

P.1.k.007 From genes to temperament to culture: differential distribution of affective temperaments parallels cultural index scores

A consistent observation during in vitro culture of preantral follicles (PFs) of mammals, was that the rates of in vitro maturation and embryogenesis of the oocytes obtained from the cultured PFs was low relative to the oocytes obtained from antral follicles. It was hypothesized that the suboptimal development potential of the oocytes from cultured PFs may be related to the altered expression patterns of developmentally important genes. Expression of P450 aromatase gene is needed for the synthesis of steroid hormones in the ovarian follicles as they develop from preantral to Graafian follicle stage. The present study was undertaken to compare the quantitative expression of P450 aromatase gene in the sheep oocytes and granulose cells obtained from in vivo grown and cultured ovarian follicles from preantral to Graafian follicle stages.P450 aromatase expression was found in granulose cells obtained from all the stages of in vivo grown follicles. In the in vitro cultured follicles aromatase expression was found in the granulose cells from 2 day cultured follicles only. Similarly the P450 aromatase expression was observed in the oocytes from all the in vivo grown stages of follicles but from none of the in vitro grown stages.It is concluded that the culture system for sheep PFs decreased P450 aromatase expression leading to compromised development potential of the oocytes. Present results provide support to the hypothesis that the compromised development potential of the oocytes from cultured ovarian follicles is related to aberrant expression of developmentally important genes.

Quantification of P450 aromatase gene expression in cultured and in vivo grown ovarian follicles in sheep

A consistent observation during in vitro culture of preantral follicles (PFs) of mammals, was that the rates of in vitro maturation and embryogenesis of the oocytes obtained from the cultured PFs was low relative to the oocytes obtained from antral follicles. It was hypothesized that the suboptimal development potential of the oocytes from cultured PFs may be related to the altered expression patterns of developmentally important genes. Expression of P450 aromatase gene is needed for the synthesis of steroid hormones in the ovarian follicles as they develop from preantral to Graafian follicle stage. The present study was undertaken to compare the quantitative expression of P450 aromatase gene in the sheep oocytes and granulose cells obtained from in vivo grown and cultured ovarian follicles from preantral to Graafian follicle stages.P450 aromatase expression was found in granulose cells obtained from all the stages of in vivo grown follicles. In the in vitro cultured follicles aromatase expression was found in the granulose cells from 2 day cultured follicles only. Similarly the P450 aromatase expression was observed in the oocytes from all the in vivo grown stages of follicles but from none of the in vitro grown stages.It is concluded that the culture system for sheep PFs decreased P450 aromatase expression leading to compromised development potential of the oocytes. Present results provide support to the hypothesis that the compromised development potential of the oocytes from cultured ovarian follicles is related to aberrant expression of developmentally important genes.

Kit ligand and insulin-like growth factor I affect the in vitro development of ovine preantral follicles

We investigated the effect of KL and IGF-I, alone or in combination, on the in vitro survival, growth and antrum formation of sheep preantral follicles, and subsequent oocyte developmental competence. Preantral follicles ∼200μm were isolated and cultured for 18 days in basic medium (control) added or not by KL, IGF-I or both. Every six days follicular survival, growth and antrum formation was evaluated. At the end of the culture period, the oocytes underwent in vitro maturation (IVM) and their viability and chromatin configuration were assessed. To evaluate oocyte quality and its developmental competence, parthenogenetic activation and in vitro fertilization (IVF) of the oocytes recovered from in vitro grown preantral follicles were performed. Medium supplementation with KL improved follicular integrity and antrum formation. IGF-I promoted the increase in follicular diameter at the end of the in vitro culture when compared to control, but not differently from KL. Follicles cultured in the presence of KL presented the highest percentages of meiosis resumption (92.3%). Therefore, these oocytes were submitted to parthenogenetic activation and IVF resulting in a percentage of 35.6% of parthenotes and 23.1% of 2- or 4-cells embryos. Medium supplementation with KL results in increased rates of antrum formation, as well as in meiosis resumption, resulting in the production of embryos using parthenogenetic activation or IVF.

In vitro development of ovine preantral follicles and oocyte cleavage rate are not affected by long-term ingestion of detoxified castor meal

In the present study, we evaluated the effect of long-term ingestion of detoxified castor meal on in vitro development of sheep preantral follicles as well as on the developmental competence of oocytes from antral follicles. Ovarian fragments were cultured for one or seven days and further analyzed by histology and fluorescence microscopy (experiment 1). The cumulus oocyte complexes (COCs) obtained from antral follicles were matured in vitro and activated by parthenogenesis or fertilization in vitro (experiment 2). Even after 1 day of culture, in both tested groups, a significant reduction in the percentage of primordial follicles and a concomitant increase in the percentage of intermediate follicles were observed when compared to non cultured tissue. After 7 days of culture, the percentage of primary follicles in both tested groups was significantly higher when compared to the non cultured tissue or the tissue cultured for 1 day (P<0.05). The number of in vitro embryos produced was similar between the tested groups. However, for those animals fed with detoxified castor meal, the parthenogenesis method resulted in a higher number of embryos (morulae) when compared to in vitro fertilization (IVF) activation. In conclusion, detoxified castor meal can be used as an alternative protein source without affecting the ovine preantral and antral folliculogenesis.

Eight-Cell Parthenotes Originated From In Vitro Grown Sheep Preantral Follicles

We investigated the effect of the leukemia inhibitory factor (LIF) alone or in association with follicle-stimulating hormone (FSH) on the in vitro growth and antrum formation of sheep preantral follicles. To evaluate oocyte quality, parthenogenetic activation of the oocytes recovered from in vitro grown preantral follicles was performed. Preantral follicles >110 μm in diameter were isolated and cultured for 18 days in basic medium either alone (control) or supplemented with LIF (10 or 50 ng/mL) in the absence or presence of FSH. Every 6 days the follicular survival, growth, and antrum formation were evaluated. When compared to control (P < .05), antrum formation was increased in follicles cultured in the presence of LIF10 and FSH. At the end of the culture, the oocytes underwent in vitro maturation (IVM); their viability and chromatin configuration were assessed. Although IVM was not affect by the treatments (P > .05), the numerically highest maturation rates (29.63%) were obtained when follicles were cultured in 50 ng/mL LIF (LIF50). Therefore, their oocytes were submitted to parthenogenetic activation; from which 58.3% of the mature oocytes resulted in 8-cell stage parthenotes. In conclusion, although LIF10 + FSH increases antrum formation when compared to a nonsupplemented medium (minimum essential medium), oocytes from sheep preantral follicles are capable of growing and maturing in vitro independent of LIF addition to the medium, which resulted in the formation of 8-cell parthenotes.

Morphologic, viability and ultrastructural analysis of vitrified sheep preantral follicles enclosed in ovarian tissue

The main objective was to compare the efficiency of vitrification techniques and solutions on the preservation of morphology, ultrastructure and viability of sheep preantral follicles enclosed in ovarian tissue fragments. The fragments were cryopreserved by using macrotube vitrification (MTV), solid-surface vitrification (SSV) or conventional vitrification (CV). These techniques were combined with one of the six solutions containing 6M ethylene glycol (EG) and with or without sucrose (SUC) (0.25 or 0.50M) and with or without fetal calf serum (FCS) (10%). After one week, samples were warmed and histological analysis was performed, showing that the percentage of normal follicles after CV (66.20±8.87%) using a solution containing 6M EG, 0.25M SUC and 10% FCS (vitrification solution 4 – VS4) was similar to fresh control (79.40±7.83%), MTV (53.40±10.60%) and SSV (56.75±15.33%), all of them with the same vitrification solution (P<0.05). For follicular viability evaluation, ovarian fragments were vitrified as described above. After warming, follicles were assessed by trypan blue dye. Controversially, the highest percentage of viable follicles was observed in MTV (97.06%) and was similar to fresh control (92.62%) (P<0.05), but was significantly different from SSV (81.08%) and CV (83.81%) (P<0.05). These results were validated by transmission electron microscopy that showed normal follicles observed in MTV and in fresh control. In addition, to verify the MTV with VS4 (a combination of the best technique plus the best solution), follicle viability was evaluated after 48h in vitro culture. The viability assay was performed by fluorescence microscopy (calcein-AM and ethidium homodimer-1) analysis as follows: follicles isolated from fresh tissue were forthwith analyzed or underwent 48h in vitro culture before analysis, whereas others fragments were vitrified/warmed and immediately analyzed or underwent 48h in vitro culture before analysis. These results showed that, although follicular viability after MTV/VS4 (65%) was reduced when compared to the non-vitrified follicles at day 0 (100%), follicular viability after MTV/VS4 at day 2 (36.5%) was similar to follicles vitrified at day 0 (65%) and similar to non-vitrified follicles at day 2 (62.5%) (P>0.05). As the decrease of viability in non-vitrified follicles at day 2 was similar to the decrease of MTV/VS4 in the same time, follicle viability at day 2 is not affected by MTV/VS4. In conclusion, using the experimental conditions of the present study, an efficient solution (VS4: 6M EG, 0.25M SUC and 10% FCS) and technique (MTV) were successfully used to vitrify ovine ovarian tissue.

In Vitro Grown Sheep Preantral Follicles Yield Oocytes with Normal Nuclear-Epigenetic Maturation

BackgroundAssisted reproductive technologies allow to utilize a limited number of fully grown oocytes despite the presence in the ovary of a large pool of meiotically incompetent gametes potentially able to produce live births. In vitro folliculogenesis could be useful to recruit these oocytes by promoting their growth and differentiation.Methodology/Principal Findings In vitro folliculogenesis was performed starting from sheep preantral (PA) follicles to evaluate oocyte nuclear/epigenetic maturation. Chromatin configuration, quantification of global DNA methylation, and epigenetic remodelling enzymes were evaluated with immunocytochemistry, telomere elongation was assessed with the Q-FISH technique, while the DNA methylation status at the DMRs of maternally IGF2R and BEGAIN, and paternally H19 methylated imprinted genes was determined by bisulfite sequencing and COBRA. Specifically, 70% of PA underwent early antrum (EA) differentiation and supported in culture oocyte global DNA methylation, telomere elongation, TERT and Dnmt3a redistribution thus mimicking the physiological events that involve the oocyte during the transition from secondary to tertiary follicle. Dnmt1 anticipated cytoplasmic translocation in in vitro grown oocytes did not impair global and single gene DNA methylation. Indeed, the in vitro grown oocytes acquired a methylation profile of IGF2R and BEGAIN compatible with the follicle/oocyte stage reached, and maintained an unmethylated status of H19. In addition, the percentage of oocytes displaying a condensed chromatin configuration resulted lower in in vitro grown oocytes, however, their ability to undergo meiosis and early embryo development after IVF and parthenogenetic activation was similar to that recorded in EA follicle in vivo grown oocytes.Conclusions/SignificanceIn conclusion, the in vitro folliculogenesis was able to support the intracellular/nuclear mechanisms leading the oocytes to acquire a meiotic and developmental competence. Thus, the in vitro culture may increase the availability of fertilizable oocytes in sheep, and become an in vitro translational model to investigate the mechanisms governing nuclear/epigenetic oocyte maturation.