To advance the use of embryo vitrification technology in veterinary practice, we developed a syst... more To advance the use of embryo vitrification technology in veterinary practice, we developed a system in which embryo vitrification, warming, and dilution can be performed within a straw. An in-straw embryo cryopreservation method reduces the need for equipment and technical skills and can facilitate direct embryo transfer to the uterus. This study proposes the use of a new device named “Sarah” that is designed to permit all in-straw embryo cryopreservation procedures. Ovine in vitro-produced (IVP) embryos were vitrified at either early blastocyst stage (EB, n = 65, 6 days post-IVF) or fully expanded blastocyst stage (FB, n = 168, 7 days post-IVF). The vitrification procedure using Sarah constituted a 0.25-mL straw with a capsule having 50-µm pores inserted at one end. Embryos at each stage (EB and FB) were divided into 2 subgroups and vitrified by 1 of 2 methods: (1) multi-step (MS) group-a straw containing 2 embryos was sequentially loaded vertically into 1.5-mL tubes containing 6 different vitrification solutions: 10, 20, 40, 60, 80, or 100% ES (with 100% ES being 7.5% DMSO +7.5% EG + 20% FCS in TCM-199; 90 s each step) followed by 30 s each in 75 and 100% VS (100% VS being 18% DMSO +18% EG + 0.5 M trehalose + BSA in TCM-199); and (2) two-step (TS) group-the straw (2 embryos/straw) was loaded with 100% of ES (5 min), followed by 100% VS solution for 30 s. For both methods, at the end of the preparation steps, the straws were plunged directly into liquid N2. Non-vitrified embryos were maintained in in vitro culture as a control group (n = 102). The warming procedure consisted of placing the straws directly into 5-mL tubes containing 100, 50, 25% WS (WS = 1 M sucrose in TCM-199+ 20% FCS) at 38.6°C (for first solution) and at room temperature for all the rest (5 min each), before being placed into the holding medium. Embryos were recovered from the straws, incubated at 38.6 C in 5% CO2 in air in TCM 199 + 5% FCS, and evaluated for blastocoel re-expansion, embryo survival, and hatching rate at 2, 14, 48 h post-warming. Blastocyst re-expansion (2 h) after warming increased as the developmental stage progressed and was not affected by the vitrification method. In fact, it was significantly (P < 0.05) higher for FB vitrified in the MS and TS methods (77.90% and 71.25%, respectively) compared with the EB method (62.5% and 48.50%, respectively). At 24 h, survival rate of vitrified FB was significantly higher (P < 0.05) in the MS system (95.35%) compared with those in TS (86.25%). Survival rates of FB embryos for both methods (MS and TS) were significantly higher (P < 0.001) than EB embryos vitrified in MS (56.25%) and TS (56.55) methods. After 48 h of culture, the hatching rate for FB vitrified in the MS system (87.21%) was comparable with TS (77.5%) and control (85.3%) groups but significantly higher (P < 0.001) than vitrified EB in MS (43.75%) and TS (36.36%). In conclusion, we showed that a high survival rate of IVP embryos can be achieved by this new in-straw vitrification and warming device (“Sarah”), with hatching rates in vitro comparable with that of control fresh embryos. This method has the potential for use in direct embryo transfer in field conditions.
Ultrasonography has become the method of choice for monitoring gestation in veterinary obstetric.... more Ultrasonography has become the method of choice for monitoring gestation in veterinary obstetric. We provided a chronological description of the development of the ovine conceptus between day 30 and 100 , based on 3 different ultrasonographic morphometric foetal parameters: CRL (Crown-rump length), th th BPD (Biparietal diameter) and TL (Tibia length) and compared these data with ones registered during foetal growth of vitrified/warmed IVP embryos transferred into recipients ewes. Our results showed a regular increasing of the CRL and BPD whereas TL showed a regular increasing trend from day 54 to 89 , when it th th starts a faster raise until 100 day. No significant differences were observed between the parameters of NM th (natural mating) group and V/W (Vitrified/Warmed embryos) group. On the other hand we could highlight an increase of the gestation period in the vitrified/warmed group and the absence of spontaneous lambing in 35%. Newborns derived after transfer of IVP vitrified...
Development and cell differentiation are driven by complex epigenetic mechanisms that regulate ch... more Development and cell differentiation are driven by complex epigenetic mechanisms that regulate chromatin structure and specific gene transcription programs. We recently demonstrated that it is possible to modify the epigenetic signature of terminally differentiated cells, switching their phenotype into one of higher plasticity, through the use of molecules that remove epigenetic marks from DNA and histones (Pennarossa et al. 2013 Proc. Natl. Acad. Sci. 110, 8948–8953; Brevini et al. 2014 Stem Cell Rev. 10, 633–642). Here we drive mammalian fibroblasts into a high plasticity state using the epigenetic eraser, 5-aza-cytidine (5-aza-CR), and investigate whether the simultaneous use of a micro-bioreactor culture system is able to promote three-dimensional (3D) cell rearrangement, boost the induction of high plasticity, and stably maintain it. To this purpose, fibroblasts were either plated on plastic dishes (Group A) or encapsulated in a liquid marble micro-bioreactor (polytetrafluoroet...
The objective of this work was to evaluate the efficiency of roscovitine on reversibly inhibiting... more 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% CO 2 . 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.
Journal of Assisted Reproduction and Genetics, Jul 1, 2020
PurposeThe age-associated decline in female fertility is largely ascribable to the decrease in oo... more PurposeThe age-associated decline in female fertility is largely ascribable to the decrease in oocyte quality. The subcortical maternal complex (SCMC) is a multiprotein complex essential for early embryogenesis and female fertility and functionally conserved across mammals. The present work evaluated expression dynamics of its components during folliculogenesis in relation to maternal age in sheep.MethodsThe expression of the SCMC components (KHDC3/FILIA, NLRP2, NLRP5/MATER, OOEP/FLOPED, PADI6, TLE6 and ZBED3) was analyzed by real-time PCR in pools of growing oocytes (GO) of different diameters (70–90 μm (S), 90–110 μm (M), or 110–130 μm (L)) derived from non-hormonally treated adult (Ad; age < 4 years), prepubertal (Pr; age 40 days), or aged ewes (age > 6 years).ResultsSpecific expression patterns associated with donor age were observed during folliculogenesis for all genes, except ZBED3. In oocytes of adult donors, the synthesis of NLRP2, NLRP5, PADI6, and ZBED3 mRNAs was complete in S GO, while FILIA, TLE6, and OOEP were actively transcribed at this stage. Conversely, Pr GO showed active transcription of all mRNAs, except for ZBED3, during the entire window of oocyte growth. Notably, aged GO showed a completely inverse pattern, with a decrease of NLRP2, TLE6, FILIA, and PADI6 mRNA abundance during the latest stage of oocyte growth (L GO). Interestingly, MATER showed high expression variability, suggesting large inter-oocyte differences.ConclusionOur study describes the SCMC expression dynamics during sheep oogenesis and reports age-specific patterns that are likely involved in the age-related decline of oocyte quality.
The efficiency of in vitro sheep embryo production is still low compared to that observed in vivo... more The efficiency of in vitro sheep embryo production is still low compared to that observed in vivo and in other species. In this context, meiotic inhibition strategies emerged as a promising alternative to improve this biotechnology. So, this study aimed to evaluate, for the first time, the effects of roscovitine on in vitro maturation of sheep oocytes and their subsequent embryo development. For this, cumulus-oocyte complexes (COCs) were cultured for 6 h in the presence (Rosco) or absence (Control) of 75 μm roscovitine and, subsequently, in vitro matured (IVM) for 18 h with gonadotropins. At 0 (Immature), 6 and 24 h of culture, the nuclear status of oocytes was evaluated by Hoechst staining. Embryo cleavage and blastocyst formation were recorded 30 h after in vitro fertilization and on day 7 of culture, respectively. Blastocyst quality was evaluated by differential staining. At 6 h, the GV rate in the Rosco treatment (93.8%) was similar to that observed in the Immature oocytes (94.9%) and significantly higher compared to Control (41.3%). After IVM for 18 h, a high and similar proportion of oocytes from Rosco (93.6%) and Control (88.4%) reached the MII stage. In both treatments, approximately 70% of oocytes cleaved and 50% of them developed up to blastocyst. The mean percentage of blastocyst cells, embryoblast, trophoblast and pyknosis did also not differ between Control and Rosco. In conclusion, roscovitine, at the studied experimental conditions, was efficient to reversibly inhibit the meiosis of adult sheep oocytes without detrimental effect on development and quality of the in vitro produced embryos.
Age-associated decline in female fertility is largely attributable to decrease in oocyte quality.... more Age-associated decline in female fertility is largely attributable to decrease in oocyte quality. However, the molecular mechanisms that shape oocyte developmental competence, and that may be involved in reproductive aging, are yet to be elucidated. The subcortical maternal complex (SCMC) is a multiprotein complex located in the subcortex of oocytes that is essential for early embryogenesis and female fertility. It appears to be functionally conserved across mammals; aberrant expression of its members was observed in several animal models of differential competence, and mutations in human SCMC genes were associated with certain human reproductive disorders. At least seven proteins contribute to the complex: KH domain-containing 3 like (KHDC3/FILIA), NLR family pyrin domain-containing 2 (NLRP2), NLRP5 (MATER), oocyte expressed protein (OOEP), peptidyl arginine deiminase 6 (PADI6), transducin-like enhancer of split 6 (TLE6), and zinc finger BED-type-containing 3 (ZBED3), all encoded by maternal effect genes (MEGs). The aim of the present work was to evaluate expression dynamics of the SCMC components during folliculogenesis in relation to maternal age in sheep. Total RNA was isolated and reverse-transcribed from pools of denuded growing oocytes (GO) of different diameters (70-90μm (small, S), 90-110μm (medium, M), or 110-130μm (large, L)) derived from nonhormonally treated prepubertal (Pr, age 40 days), adult (Ad, age &amp;amp;amp;lt;4 years), or aged (Aged, age &amp;amp;amp;gt;6 years) animals (5 pools of 30 oocytes per experimental group). The SCMC expression was assessed by real-time PCR (PCR efficiency of 90-110% and correlation coefficient r2&amp;amp;amp;gt;0.99). Data were normalized against oocyte number and an exogenous spike-in mRNA, Luciferase, as reference gene. Expression dynamics were analyzed within each age group (general linear model ANOVA). Strikingly, patterns specifically associated with donor age were observed during folliculogenesis for six of the seven SCMC components. The Pr group showed active transcription of all mRNA, except ZBED3, during the entire window of oocyte growth (P&amp;amp;amp;lt;0.05). On the contrary, the similar abundance of NLRP2, NLRP5, PADI6, and ZBED3 in Ad S, M, and L GO suggests earlier storage during folliculogenesis; FILIA, OOEP, and TLE6 showed an increase between Ad S and M GO (P&amp;amp;amp;lt;0.05), indicating that the synthesis of these transcripts is complete at this stage (M GOs). Notably, oocytes derived from Aged donors showed a completely inverse expression pattern, with a decrease in abundance of NLRP2, TLE6, FILIA, and PADI6 mRNAs during the last stage of oocyte growth (L GO; P&amp;amp;amp;lt;0.05). Interestingly, MATER showed very high variability in expression (standard error (SE) ranging from 0.79 to 1.13 quantitation cycles (Cq)) in Aged GO, compared to Ad GO (SE 0.16-0.24 Cq) or Pr GO (SE 0.16-0.26 Cq), suggesting large inter-oocyte differences. In conclusion, age affects the storage of the MEGs encoding the SCMC during folliculogenesis. The observed depletion in SCMC transcripts in GO of aged donors is likely to be involved in the age-related decline in oocyte quality.
Abstract A complete assessment of morphological and functional characteristics of ram semen durin... more Abstract A complete assessment of morphological and functional characteristics of ram semen during refrigeration is necessary to optimize the process of semen manipulation and storage. The aim of this study was to describe changes in main predictive parameters of ram semen diluted in a commercial soy lecithin-based extender (OVIXcell), during long term liquid storage at 4 °C. Ejaculates of 5 Sarda rams were collected, pooled and diluted in OVIXcell. Samples were cooled at 4 °C and stored at this temperature until 96 h. At 0-24-48-72-96 h semen samples were analysed for the following parameters: motility [computer assisted sperm analysis (CASA)]; integrity of cytoplasm membrane and acrosome (PI/PSA staining); DNA fragmentation index [DFI(%); sperm chromatin structure assay (SCSA)]; levels of reactive oxygen species (ROS; H2DCFDA staining). Effect of time of storage on main parameters and correlations among them were assessed respectively by ANOVA and Pearson's correlation coefficient. Primary and secondary motility parameters were significantly affected by time of storage (P 0.05). Long term storage did not affect the levels of DFI(%) (P > 0.05) while ROS production significantly increased from 48 to 96 h (P
Journal of Assisted Reproduction and Genetics, Aug 14, 2019
PurposeTesticular tissue cryopreservation prior to gonadotoxic therapies is a method to preserve ... more PurposeTesticular tissue cryopreservation prior to gonadotoxic therapies is a method to preserve fertility in children. However, the technique still requires development, especially when the tissue is immature and rather susceptible to stress derived from in vitro manipulation. This study aimed to investigate the effects of vitrification with a new cryodevice (E.Vit) on cell membrane integrity and gene expression of prepubertal testicular tissue in the ovine model.MethodsPieces of immature testicular tissue (1 mm3) were inserted into “E.Vit” devices and vitrified with a two-step protocol. After warming, tissues were cultured in vitro and cell membrane integrity was assessed after 0, 2, and 24 h by trypan blue exclusion test. Controls consisted of non-vitrified tissue analyzed after 0, 2, and 24 h in vitro culture (IVC). Expression of genes involved in transcriptional stress response (BAX, SOD1, CIRBP, HSP90AB1), cell proliferation (KIF11), and germ- (ZBDB16, TERT, POU5F1, KIT) and somatic- (AR, FSHR, STAR) cell specific markers was evaluated 2 and 24 h after warming.ResultsPost-warming trypan blue staining showed the survival of most cells, although membrane integrity immediately after warming (66.00% ± 4.73) or after 2 h IVC (59.67% ± 4.18) was significantly lower than controls (C0h 89.67% ± 1.45). Extended post-warming IVC (24 h) caused an additional decrease to 31% ± 3.46 (P < 0.05). Germ- and somatic-cell specific markers showed the survival of both cell types after cryopreservation and IVC. All genes were affected by cryopreservation and/or IVC, and moderate stress conditions were indicated by transcriptional stress response.ConclusionsVitrification with the cryodevice E.Vit is a promising strategy to cryopreserve prepubertal testicular tissue.
The age‐associated decline in female fertility is largely ascribable to a decrease in oocyte qual... more The age‐associated decline in female fertility is largely ascribable to a decrease in oocyte quality. This phenomenon is multifaceted and influenced by numerous interconnected maternal and environmental factors. An increase in the rate of meiotic errors is the major cause of the decline in oocyte developmental competence. However, abnormalities in the ooplasm accumulating with age — including altered metabolism, organelle dysfunction, and aberrant gene regulation — progressively undermine oocyte quality. Stockpiling of maternal macromolecules during folliculogenesis is crucial, as oocyte competence to achieve maturation, fertilization, and the earliest phases of embryo development occur in absence of transcription. At the same time, crucial remodeling of oocyte epigenetics during oogenesis is potentially exposed to interfering factors, such as assisted reproduction technologies (ARTs) or environmental changes, whose impact may be enhanced by reproductive aging. As the effects of mat...
Somatic cell nuclear transfer (SCNT) is a key technology with broad applications that range from ... more Somatic cell nuclear transfer (SCNT) is a key technology with broad applications that range from production of cloned farm animals to derivation of patient-matched stem cells or production of humanized animal organs for xenotransplantation. However, effects of aberrant epigenetic reprogramming on gene expression compromise cell and organ phenotype, resulting in low success rate of SCNT. Standard SCNT procedures include enucleation of recipient oocytes before the nuclear donor cell is introduced. Enucleation removes not only the spindle apparatus and chromosomes of the oocyte but also the perinuclear, mitochondria rich, ooplasm. Here, we use a Bos taurus SCNT model with in vitro fertilized (IVF) and in vivo conceived controls to demonstrate a ∼50% reduction in mitochondrial DNA (mtDNA) in the liver and skeletal muscle, but not the brain, of SCNT fetuses at day 80 of gestation. In the muscle, we also observed significantly reduced transcript abundances of mtDNA-encoded subunits of the...
Cryopreservation is routinely used to preserve cells and tissues; however, long time storage brin... more Cryopreservation is routinely used to preserve cells and tissues; however, long time storage brings many inconveniences including the use of liquid nitrogen. Freeze-drying could enable higher shelf-life stability at ambient temperatures and facilitate transport and storage. Currently, the possibility to freeze-dry reproductive tissues maintaining vitality and functions is still under optimization. Here, we lyophilized sheep ovarian tissue with a novel device named Darya and a new vitrification and drying protocol and assessed effects on tissue integrity and gene expression. The evaluation was performed immediately after lyophilization (Lio), after rehydration (LR0h) or after two hours of in vitro culture (IVC; LR2h). The tissue survived lyophilization procedures and maintained its general structure, including intact follicles at different stages of development, however morphological and cytoplasmic modifications were noticed. Lyophilization, rehydration and further IVC increasingly ...
Novel approach to cell sampling from preimplantation ovine embryos and its potential use in embry... more Novel approach to cell sampling from preimplantation ovine embryos and its potential use in embryonic genome analysis
To advance the use of embryo vitrification technology in veterinary practice, we developed a syst... more To advance the use of embryo vitrification technology in veterinary practice, we developed a system in which embryo vitrification, warming, and dilution can be performed within a straw. An in-straw embryo cryopreservation method reduces the need for equipment and technical skills and can facilitate direct embryo transfer to the uterus. This study proposes the use of a new device named “Sarah” that is designed to permit all in-straw embryo cryopreservation procedures. Ovine in vitro-produced (IVP) embryos were vitrified at either early blastocyst stage (EB, n = 65, 6 days post-IVF) or fully expanded blastocyst stage (FB, n = 168, 7 days post-IVF). The vitrification procedure using Sarah constituted a 0.25-mL straw with a capsule having 50-µm pores inserted at one end. Embryos at each stage (EB and FB) were divided into 2 subgroups and vitrified by 1 of 2 methods: (1) multi-step (MS) group-a straw containing 2 embryos was sequentially loaded vertically into 1.5-mL tubes containing 6 different vitrification solutions: 10, 20, 40, 60, 80, or 100% ES (with 100% ES being 7.5% DMSO +7.5% EG + 20% FCS in TCM-199; 90 s each step) followed by 30 s each in 75 and 100% VS (100% VS being 18% DMSO +18% EG + 0.5 M trehalose + BSA in TCM-199); and (2) two-step (TS) group-the straw (2 embryos/straw) was loaded with 100% of ES (5 min), followed by 100% VS solution for 30 s. For both methods, at the end of the preparation steps, the straws were plunged directly into liquid N2. Non-vitrified embryos were maintained in in vitro culture as a control group (n = 102). The warming procedure consisted of placing the straws directly into 5-mL tubes containing 100, 50, 25% WS (WS = 1 M sucrose in TCM-199+ 20% FCS) at 38.6°C (for first solution) and at room temperature for all the rest (5 min each), before being placed into the holding medium. Embryos were recovered from the straws, incubated at 38.6 C in 5% CO2 in air in TCM 199 + 5% FCS, and evaluated for blastocoel re-expansion, embryo survival, and hatching rate at 2, 14, 48 h post-warming. Blastocyst re-expansion (2 h) after warming increased as the developmental stage progressed and was not affected by the vitrification method. In fact, it was significantly (P &amp;lt; 0.05) higher for FB vitrified in the MS and TS methods (77.90% and 71.25%, respectively) compared with the EB method (62.5% and 48.50%, respectively). At 24 h, survival rate of vitrified FB was significantly higher (P &amp;lt; 0.05) in the MS system (95.35%) compared with those in TS (86.25%). Survival rates of FB embryos for both methods (MS and TS) were significantly higher (P &amp;lt; 0.001) than EB embryos vitrified in MS (56.25%) and TS (56.55) methods. After 48 h of culture, the hatching rate for FB vitrified in the MS system (87.21%) was comparable with TS (77.5%) and control (85.3%) groups but significantly higher (P &amp;lt; 0.001) than vitrified EB in MS (43.75%) and TS (36.36%). In conclusion, we showed that a high survival rate of IVP embryos can be achieved by this new in-straw vitrification and warming device (“Sarah”), with hatching rates in vitro comparable with that of control fresh embryos. This method has the potential for use in direct embryo transfer in field conditions.
Ultrasonography has become the method of choice for monitoring gestation in veterinary obstetric.... more Ultrasonography has become the method of choice for monitoring gestation in veterinary obstetric. We provided a chronological description of the development of the ovine conceptus between day 30 and 100 , based on 3 different ultrasonographic morphometric foetal parameters: CRL (Crown-rump length), th th BPD (Biparietal diameter) and TL (Tibia length) and compared these data with ones registered during foetal growth of vitrified/warmed IVP embryos transferred into recipients ewes. Our results showed a regular increasing of the CRL and BPD whereas TL showed a regular increasing trend from day 54 to 89 , when it th th starts a faster raise until 100 day. No significant differences were observed between the parameters of NM th (natural mating) group and V/W (Vitrified/Warmed embryos) group. On the other hand we could highlight an increase of the gestation period in the vitrified/warmed group and the absence of spontaneous lambing in 35%. Newborns derived after transfer of IVP vitrified...
Development and cell differentiation are driven by complex epigenetic mechanisms that regulate ch... more Development and cell differentiation are driven by complex epigenetic mechanisms that regulate chromatin structure and specific gene transcription programs. We recently demonstrated that it is possible to modify the epigenetic signature of terminally differentiated cells, switching their phenotype into one of higher plasticity, through the use of molecules that remove epigenetic marks from DNA and histones (Pennarossa et al. 2013 Proc. Natl. Acad. Sci. 110, 8948–8953; Brevini et al. 2014 Stem Cell Rev. 10, 633–642). Here we drive mammalian fibroblasts into a high plasticity state using the epigenetic eraser, 5-aza-cytidine (5-aza-CR), and investigate whether the simultaneous use of a micro-bioreactor culture system is able to promote three-dimensional (3D) cell rearrangement, boost the induction of high plasticity, and stably maintain it. To this purpose, fibroblasts were either plated on plastic dishes (Group A) or encapsulated in a liquid marble micro-bioreactor (polytetrafluoroet...
The objective of this work was to evaluate the efficiency of roscovitine on reversibly inhibiting... more 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% CO 2 . 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.
Journal of Assisted Reproduction and Genetics, Jul 1, 2020
PurposeThe age-associated decline in female fertility is largely ascribable to the decrease in oo... more PurposeThe age-associated decline in female fertility is largely ascribable to the decrease in oocyte quality. The subcortical maternal complex (SCMC) is a multiprotein complex essential for early embryogenesis and female fertility and functionally conserved across mammals. The present work evaluated expression dynamics of its components during folliculogenesis in relation to maternal age in sheep.MethodsThe expression of the SCMC components (KHDC3/FILIA, NLRP2, NLRP5/MATER, OOEP/FLOPED, PADI6, TLE6 and ZBED3) was analyzed by real-time PCR in pools of growing oocytes (GO) of different diameters (70–90 μm (S), 90–110 μm (M), or 110–130 μm (L)) derived from non-hormonally treated adult (Ad; age < 4 years), prepubertal (Pr; age 40 days), or aged ewes (age > 6 years).ResultsSpecific expression patterns associated with donor age were observed during folliculogenesis for all genes, except ZBED3. In oocytes of adult donors, the synthesis of NLRP2, NLRP5, PADI6, and ZBED3 mRNAs was complete in S GO, while FILIA, TLE6, and OOEP were actively transcribed at this stage. Conversely, Pr GO showed active transcription of all mRNAs, except for ZBED3, during the entire window of oocyte growth. Notably, aged GO showed a completely inverse pattern, with a decrease of NLRP2, TLE6, FILIA, and PADI6 mRNA abundance during the latest stage of oocyte growth (L GO). Interestingly, MATER showed high expression variability, suggesting large inter-oocyte differences.ConclusionOur study describes the SCMC expression dynamics during sheep oogenesis and reports age-specific patterns that are likely involved in the age-related decline of oocyte quality.
The efficiency of in vitro sheep embryo production is still low compared to that observed in vivo... more The efficiency of in vitro sheep embryo production is still low compared to that observed in vivo and in other species. In this context, meiotic inhibition strategies emerged as a promising alternative to improve this biotechnology. So, this study aimed to evaluate, for the first time, the effects of roscovitine on in vitro maturation of sheep oocytes and their subsequent embryo development. For this, cumulus-oocyte complexes (COCs) were cultured for 6 h in the presence (Rosco) or absence (Control) of 75 μm roscovitine and, subsequently, in vitro matured (IVM) for 18 h with gonadotropins. At 0 (Immature), 6 and 24 h of culture, the nuclear status of oocytes was evaluated by Hoechst staining. Embryo cleavage and blastocyst formation were recorded 30 h after in vitro fertilization and on day 7 of culture, respectively. Blastocyst quality was evaluated by differential staining. At 6 h, the GV rate in the Rosco treatment (93.8%) was similar to that observed in the Immature oocytes (94.9%) and significantly higher compared to Control (41.3%). After IVM for 18 h, a high and similar proportion of oocytes from Rosco (93.6%) and Control (88.4%) reached the MII stage. In both treatments, approximately 70% of oocytes cleaved and 50% of them developed up to blastocyst. The mean percentage of blastocyst cells, embryoblast, trophoblast and pyknosis did also not differ between Control and Rosco. In conclusion, roscovitine, at the studied experimental conditions, was efficient to reversibly inhibit the meiosis of adult sheep oocytes without detrimental effect on development and quality of the in vitro produced embryos.
Age-associated decline in female fertility is largely attributable to decrease in oocyte quality.... more Age-associated decline in female fertility is largely attributable to decrease in oocyte quality. However, the molecular mechanisms that shape oocyte developmental competence, and that may be involved in reproductive aging, are yet to be elucidated. The subcortical maternal complex (SCMC) is a multiprotein complex located in the subcortex of oocytes that is essential for early embryogenesis and female fertility. It appears to be functionally conserved across mammals; aberrant expression of its members was observed in several animal models of differential competence, and mutations in human SCMC genes were associated with certain human reproductive disorders. At least seven proteins contribute to the complex: KH domain-containing 3 like (KHDC3/FILIA), NLR family pyrin domain-containing 2 (NLRP2), NLRP5 (MATER), oocyte expressed protein (OOEP), peptidyl arginine deiminase 6 (PADI6), transducin-like enhancer of split 6 (TLE6), and zinc finger BED-type-containing 3 (ZBED3), all encoded by maternal effect genes (MEGs). The aim of the present work was to evaluate expression dynamics of the SCMC components during folliculogenesis in relation to maternal age in sheep. Total RNA was isolated and reverse-transcribed from pools of denuded growing oocytes (GO) of different diameters (70-90μm (small, S), 90-110μm (medium, M), or 110-130μm (large, L)) derived from nonhormonally treated prepubertal (Pr, age 40 days), adult (Ad, age &amp;amp;amp;lt;4 years), or aged (Aged, age &amp;amp;amp;gt;6 years) animals (5 pools of 30 oocytes per experimental group). The SCMC expression was assessed by real-time PCR (PCR efficiency of 90-110% and correlation coefficient r2&amp;amp;amp;gt;0.99). Data were normalized against oocyte number and an exogenous spike-in mRNA, Luciferase, as reference gene. Expression dynamics were analyzed within each age group (general linear model ANOVA). Strikingly, patterns specifically associated with donor age were observed during folliculogenesis for six of the seven SCMC components. The Pr group showed active transcription of all mRNA, except ZBED3, during the entire window of oocyte growth (P&amp;amp;amp;lt;0.05). On the contrary, the similar abundance of NLRP2, NLRP5, PADI6, and ZBED3 in Ad S, M, and L GO suggests earlier storage during folliculogenesis; FILIA, OOEP, and TLE6 showed an increase between Ad S and M GO (P&amp;amp;amp;lt;0.05), indicating that the synthesis of these transcripts is complete at this stage (M GOs). Notably, oocytes derived from Aged donors showed a completely inverse expression pattern, with a decrease in abundance of NLRP2, TLE6, FILIA, and PADI6 mRNAs during the last stage of oocyte growth (L GO; P&amp;amp;amp;lt;0.05). Interestingly, MATER showed very high variability in expression (standard error (SE) ranging from 0.79 to 1.13 quantitation cycles (Cq)) in Aged GO, compared to Ad GO (SE 0.16-0.24 Cq) or Pr GO (SE 0.16-0.26 Cq), suggesting large inter-oocyte differences. In conclusion, age affects the storage of the MEGs encoding the SCMC during folliculogenesis. The observed depletion in SCMC transcripts in GO of aged donors is likely to be involved in the age-related decline in oocyte quality.
Abstract A complete assessment of morphological and functional characteristics of ram semen durin... more Abstract A complete assessment of morphological and functional characteristics of ram semen during refrigeration is necessary to optimize the process of semen manipulation and storage. The aim of this study was to describe changes in main predictive parameters of ram semen diluted in a commercial soy lecithin-based extender (OVIXcell), during long term liquid storage at 4 °C. Ejaculates of 5 Sarda rams were collected, pooled and diluted in OVIXcell. Samples were cooled at 4 °C and stored at this temperature until 96 h. At 0-24-48-72-96 h semen samples were analysed for the following parameters: motility [computer assisted sperm analysis (CASA)]; integrity of cytoplasm membrane and acrosome (PI/PSA staining); DNA fragmentation index [DFI(%); sperm chromatin structure assay (SCSA)]; levels of reactive oxygen species (ROS; H2DCFDA staining). Effect of time of storage on main parameters and correlations among them were assessed respectively by ANOVA and Pearson's correlation coefficient. Primary and secondary motility parameters were significantly affected by time of storage (P 0.05). Long term storage did not affect the levels of DFI(%) (P > 0.05) while ROS production significantly increased from 48 to 96 h (P
Journal of Assisted Reproduction and Genetics, Aug 14, 2019
PurposeTesticular tissue cryopreservation prior to gonadotoxic therapies is a method to preserve ... more PurposeTesticular tissue cryopreservation prior to gonadotoxic therapies is a method to preserve fertility in children. However, the technique still requires development, especially when the tissue is immature and rather susceptible to stress derived from in vitro manipulation. This study aimed to investigate the effects of vitrification with a new cryodevice (E.Vit) on cell membrane integrity and gene expression of prepubertal testicular tissue in the ovine model.MethodsPieces of immature testicular tissue (1 mm3) were inserted into “E.Vit” devices and vitrified with a two-step protocol. After warming, tissues were cultured in vitro and cell membrane integrity was assessed after 0, 2, and 24 h by trypan blue exclusion test. Controls consisted of non-vitrified tissue analyzed after 0, 2, and 24 h in vitro culture (IVC). Expression of genes involved in transcriptional stress response (BAX, SOD1, CIRBP, HSP90AB1), cell proliferation (KIF11), and germ- (ZBDB16, TERT, POU5F1, KIT) and somatic- (AR, FSHR, STAR) cell specific markers was evaluated 2 and 24 h after warming.ResultsPost-warming trypan blue staining showed the survival of most cells, although membrane integrity immediately after warming (66.00% ± 4.73) or after 2 h IVC (59.67% ± 4.18) was significantly lower than controls (C0h 89.67% ± 1.45). Extended post-warming IVC (24 h) caused an additional decrease to 31% ± 3.46 (P < 0.05). Germ- and somatic-cell specific markers showed the survival of both cell types after cryopreservation and IVC. All genes were affected by cryopreservation and/or IVC, and moderate stress conditions were indicated by transcriptional stress response.ConclusionsVitrification with the cryodevice E.Vit is a promising strategy to cryopreserve prepubertal testicular tissue.
The age‐associated decline in female fertility is largely ascribable to a decrease in oocyte qual... more The age‐associated decline in female fertility is largely ascribable to a decrease in oocyte quality. This phenomenon is multifaceted and influenced by numerous interconnected maternal and environmental factors. An increase in the rate of meiotic errors is the major cause of the decline in oocyte developmental competence. However, abnormalities in the ooplasm accumulating with age — including altered metabolism, organelle dysfunction, and aberrant gene regulation — progressively undermine oocyte quality. Stockpiling of maternal macromolecules during folliculogenesis is crucial, as oocyte competence to achieve maturation, fertilization, and the earliest phases of embryo development occur in absence of transcription. At the same time, crucial remodeling of oocyte epigenetics during oogenesis is potentially exposed to interfering factors, such as assisted reproduction technologies (ARTs) or environmental changes, whose impact may be enhanced by reproductive aging. As the effects of mat...
Somatic cell nuclear transfer (SCNT) is a key technology with broad applications that range from ... more Somatic cell nuclear transfer (SCNT) is a key technology with broad applications that range from production of cloned farm animals to derivation of patient-matched stem cells or production of humanized animal organs for xenotransplantation. However, effects of aberrant epigenetic reprogramming on gene expression compromise cell and organ phenotype, resulting in low success rate of SCNT. Standard SCNT procedures include enucleation of recipient oocytes before the nuclear donor cell is introduced. Enucleation removes not only the spindle apparatus and chromosomes of the oocyte but also the perinuclear, mitochondria rich, ooplasm. Here, we use a Bos taurus SCNT model with in vitro fertilized (IVF) and in vivo conceived controls to demonstrate a ∼50% reduction in mitochondrial DNA (mtDNA) in the liver and skeletal muscle, but not the brain, of SCNT fetuses at day 80 of gestation. In the muscle, we also observed significantly reduced transcript abundances of mtDNA-encoded subunits of the...
Cryopreservation is routinely used to preserve cells and tissues; however, long time storage brin... more Cryopreservation is routinely used to preserve cells and tissues; however, long time storage brings many inconveniences including the use of liquid nitrogen. Freeze-drying could enable higher shelf-life stability at ambient temperatures and facilitate transport and storage. Currently, the possibility to freeze-dry reproductive tissues maintaining vitality and functions is still under optimization. Here, we lyophilized sheep ovarian tissue with a novel device named Darya and a new vitrification and drying protocol and assessed effects on tissue integrity and gene expression. The evaluation was performed immediately after lyophilization (Lio), after rehydration (LR0h) or after two hours of in vitro culture (IVC; LR2h). The tissue survived lyophilization procedures and maintained its general structure, including intact follicles at different stages of development, however morphological and cytoplasmic modifications were noticed. Lyophilization, rehydration and further IVC increasingly ...
Novel approach to cell sampling from preimplantation ovine embryos and its potential use in embry... more Novel approach to cell sampling from preimplantation ovine embryos and its potential use in embryonic genome analysis
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