Tissue-specific and minor inter-individual variation in imprinting of <i>IGF2R</i> is a common feature of <i>Bos taurus</i> concepti and not correlated with fetal weight

The insulin-like growth factor 2 receptor (IGF2R) is essential for prenatal growth regulation and shows gene dosage effects on fetal weight that can be affected by in-vitro embryo culture. Imprinted maternal expression of murine Igf2r is well documented for all fetal tissues excluding brain, but polymorphic imprinting and biallelic expression were reported for IGF2R in human. These differences have been attributed to evolutionary changes correlated with specific reproductive strategies. However, data from species suitable for testing this hypothesis are lacking. The domestic cow (Bos taurus) carries a single conceptus with a similar gestation length as human. We identified 12 heterozygous concepti informative for imprinting studies among 68 Bos taurus fetuses at Day 80 of gestation (28% term) and found predominantly maternal IGF2R expression in all fetal tissues but brain, which escapes imprinting. Inter-individual variation in allelic expression bias, i.e. expression of the repressed paternal allele relative to the maternal allele, ranged from 4.6−8.9% in heart, 4.3−10.2% in kidney, 6.1−11.2% in liver, 4.6−15.8% in lung and 3.2−12.2% in skeletal muscle. Allelic bias for mesodermal tissues (heart, skeletal muscle) differed significantly (P&lt;0.05) from endodermal tissues (liver, lung). The placenta showed partial imprinting with allelic bias of 22.9−34.7% and differed significantly (P&lt;0.001) from all other tissues. Four informative fetuses were generated by in-vitro fertilization (IVF) with embryo culture and two individuals displayed fetal overgrowth. However, there was no evidence for changes in imprinting or DNA methylation after IVF, or correlations between allelic bias and fetal weight. In conclusion, imprinting of Bos taurus IGF2R is similar to mouse except in placenta, which could indicate an effect of reproductive strategy. Common minor inter-individual variation in allelic bias and absence of imprinting abnormalities in IVF fetuses suggest changes in IGF2R expression in overgrown fetuses could be modulated through other mechanisms than changes in imprinting

3D Liquid Marble Microbioreactors Support In Vitro Maturation of Prepubertal Ovine Oocytes and Affect Expression of Oocyte-Specific Factors

In vitro oocyte maturation (IVM) is a well-established technique. Despite the high IVM rates obtained in most mammalian species, the developmental competence of IVM oocytes is suboptimal. The aim of this work was to evaluate the potential beneficial effects of a liquid marble microbioreactor (LM) as a 3D culture system to mature in vitro prepubertal ovine oocytes, as models of oocytes with intrinsic low competence. Cumulus–oocyte complexes of prepubertal sheep ovaries were in vitro matured in a LM system with hydrophobic fumed-silica-nanoparticles (LM group) or in standard conditions (4W control group). We evaluated: (a) maturation and (b) developmental rates following in vitro fertilization (IVF) and embryo culture; (c) expression of a panel of genes. LM and 4W groups showed similar IVM and IVF rates, while in vitro development to blastocyst stage approached significance (4W: 14.1% vs. LM: 28.3%; p = 0.066). The expression of GDF9, of enzymes involved in DNA methylation reprogramming and of the subcortical maternal complex was affected by the IVM system, while no difference was observed in terms of cell-stress-response. LM microbioreactors provide a suitable microenvironment to induce prepubertal sheep oocyte IVM and should be considered to enhance the developmental competence of oocytes with reduced potential also in other species, including humans

New Challenges in Cryopreservation: A Reproductive Perspective

Cryopreservation is a fundamental procedure to preserve the structure and function of cells and tissues by storing them at low temperatures for long periods [...

Characterization, isolation and culture of primordial germ cells in domestic animals: recent progress and insights from the ovine species

Primordial germ cell (PGC) allocation, characterization, lineage restriction, and differentiation have been extensively studied in the mouse. Murine PGC can be easily identified using markers as alkaline phosphatase content or the expression of pluripotent markers such as Pou5f1, Nanog, Sox2, Kit, SSEA1, and SSEA4. These tools allowed us to clarify certain aspects of the complex interactions of somatic and germinal cells in the establishment of the germ cell lineage, its segregation from the neighbouring somatic tissue, and the guidance mechanisms during migration that direct most of the germ cells into the genital ridges. Few data are available from other domestic animals and here we reported our preliminary studies on the isolation, characterization, and in vitro culture of sheep PGCs. Sheep PGCs can be identified with the markers previously used in mouse, but, in some cases, these markers are not coherently expressed in the same cell depending on the grade of differentiation and on technical problems related to commercial antibodies used. Pluripotency of PGCs in culture (EGCs) from domestic animals also needs further evaluation even though the derivation of embryonic pluripotent cell lines from large mammals may be an advantage as they are more physiologically similar to the human and perhaps more relevant for clinical translation studies. Comprehensive epigenetic reprogramming of the genome in early germ cells, and derived EGCs including extensive erasure of epigenetic modifications, may be relevant for gaining insight into events that lead to reprogramming and establishment of totipotency. EGCs can differentiate in vitro in a various range of tissues, form embryonic bodies, but in many cases failed to generate tumours when transplanted into immunodeficient mice and are not able to generate germline chimeric animals after their transfer. Such incomplete information clearly indicates the urge to improve the studies on derivation of stem cells in farm animals and shows the need for a multidisciplinary investigation in order to create farm animal models to set up suitable ethical and technical systems for cell regenerative therapies in humans

Microtubular Assessment of C6 Rat Glioma Cell Spheroids Developed in Transparent Liquid Marbles or Hanging Drops

Glioblastoma is a brain tumour frequently used as an experimental model to exploit innovative therapeutic approaches due to its high lethality and refractoriness to therapies. Part of these innovative anticancer therapies address cytoskeletal microtubules (MTs) since specific tubulin post-translational modifications (PTMs) are considered markers of tumour plasticity. In vitro studies, which traditionally employ two-dimensional (2D) culture systems, are now being replaced by three-dimensional (3D) systems that more closely mimic in vivo physiological conditions and allow a better understanding of the signalling between cells. In this work, we compared 2 liquid base 3D methods for the generation of spheroids from C6 rat glioma cells (RGCs) using 30 &micro;L of liquid marble (LM) or the hanging drops (HDs), which contained 2 different cell numbers (5000 or 15,000). After 24 or 48 h of in vitro culture (IVC), the morphology of the spheroids was observed and the behaviour of the two main tubulin PTMs, tyrosinated &alpha;-tubulin (Tyr-T) and acetylated &alpha;-tubulin (Ac-T), was evaluated by fluorescence and Western blot (WB). RGCs spontaneously formed spherical agglomerates more rapidly in the LM than in the HD system. Cell density influenced the size of the spheroids, which reached a larger size (&gt; of 300 &micro;m &Oslash;), with 15,000 cells compared to 5000 cells (150 &micro;m &Oslash;). Moreover, an increase in Tyr-T and Ac-T was observed in both the HD and LM system from 24 to 48 h, with the highest values shown in the 48 h/LM spheroids of 5000 cells (p &lt; 0.05). In conclusion, by comparing the morphology and microtubular architecture of spheroids from C6 rat glioma cells developed by LM or HD methodology, our findings demonstrate that the use of a fumed silica microbioreactor boosts the induction and maintenance of a high plasticity state in glioma cells. RGCs cultured in LM express levels of tubulin PTMs that can be used to evaluate the efficacy of new anticancer therapies

Roscovitina para o atraso da progressão meiótica em oócitos de ovelhas pré-púberes

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.O objetivo deste trabalho foi avaliar a eficiência da roscovitina na inibição reversível de oócitos de ovelhas pré-púberes, no estádio de vesícula germinativa (VG), e investigar a cinética da progressão da meiose após a remoção do inibidor. Complexos cumulus-oócito, recuperados de cordeiras da raça Sarda com 30–40 dias, foram cultivados por 6 horas em meio de maturação (controle) contendo 75 µmol L-1 de roscovitina (Rosco) a 38,5°C e 5% de CO2. Em seguida, os complexos foram submetidos à maturação in vitro (MIV) por 18 ou 23 horas, em meio isento de inibidor, suplementado com gonadotrofinas. A avaliação da configuração nuclear em coloração Hoescht, sob microscópio invertido de fluorescência, revelou que 88,7% dos oócitos tratados permaneceram no estágio VG, conforme observado para os imaturos (97,3%) corados após a coleta. Essa inibição foi reversível; contudo, a proporção de oócitos (83,3%) em metáfase-II, após 23 horas de MIV, foi significativamente maior do que a observada após 18 horas (29,5%), em que a meiose ainda estava em progressão com 34,2% de oócitos em metáfase-I, 11,6% de oócitos em anáfase-I e 18,5% de oócitos em telófase-I. A roscovitina é eficiente no bloqueio da maturação nuclear em oócitos de ovelhas pré-púberes; no entanto, apesar da reversibilidade, a progressão da meiose é retardada e requer mais tempo para ser concluída

Maternal-fetal transplacental leakage of mitochondrial dna in bovine nuclear transfer pregnancies: potential implications for offspring and recipients

The synepitheliochorial placenta of ruminants is constructed of multiple tissue layers that separate maternal and fetal blood. In nuclear transfer cloned ruminants, however, placental anomalies such as abnormal vascular development and hemorrhagic cotyledons have been reported. We have investigated the possible exchange of genetic material between somatic cell nuclear transfer cloned (SCNT) bovine fetuses and recipients at day 80 of gestation using mitochondrial DNA (mtDNA) as a marker. Twenty-three recovered SCNT-fetuses and their recipients were screened for divergent and thus informative mtDNA combinations. Twenty-one fetuses generated by in vitro fertilization (IVF) or multiple ovulation embryo transfer (MOET) and the corresponding recipients served as controls. A search for recipient mtDNA haplotype in DNA extracts from fetal blood by PCR-RFLP analysis revealed three cases of chimerism (two SCNT, one IVF) among a total of 19 informative fetus–recipient pairs (eight SCNT, seven IVF, four MOET). Placental anomalies have also been observed in some IVF fetuses and the present data therefore suggests transplacental leakage of cell components or cells from the recipient into some fetuses generated by in vitro techniques. Further studies are necessary to determine (i) the nature of leaked material, (ii) whether there is bi-directional leakage, and (iii) whether leaked material is present in recipients and calves after parturition, i.e. whether leakage takes place in vivo. If recipients were chimeric for DNA or cells derived from genetically modified SCNT (or IVF) embryos, their subsequent utilization might be affected

Recovery of COCs from ovaries with high follicle numbers enhances in vitro embryo yield in sheep

The aim of the present study was to investigate the correlation between the number of ovarian follicles and in vitro embryo development and quality in sheep. Sarda ewe ovaries were classified according to the number of follicles: ≤4 (Low), 5–7 (Intermediate), and ≥8 (High). IVM, IVF and IVC were performed under standard conditions. Cleavage rate and blastocyst development were assessed 48 h after fertilization and on Days 6, 7 and 8 of culture, respectively. Expanded blastocysts were vitrified; blastocoel re-expansion and hatching rates were assessed at 8, 16 and 72 h post-thawing and hatched blastocysts were analyzed with the TUNEL assay. In a subset of thawed blastocysts the incorporation of amino acids was evaluated. The proportion of ovaries varied significantly among the three groups (ANOVA F = 12.20, P = 0), and more ovaries (59%) were assigned to the Low group than to the Intermediate (28%; ANOVA F = 8.19, P = 0.009) and High group (13%; ANOVA F = 18.63, P = 0), (High vs. Intermediate F = 6.31, P = 0.020). The three groups statistically differed in the proportion of total blastocysts ( χ22= 22.616, P = 0.00), of blastocysts produced on Days 6 ( χ22= 6.829, P = 0.033) and 7 ( χ22= 6.810, P = 0.033), while no difference was found in the proportion of blastocysts obtained on Day 8 ( χ22= 3.874, P = 0.144) of culture after fertilization. A higher proportion of total blastocysts was obtained from the High (44%) compared with the other two groups (Low: 28%, χ22 = 22.629, P = 0; Intermediate: 33%, χ22= 7.266, P = 0.007), while the Low and Intermediate groups did not statistically differ either in the total blastocyst output ( χ22= 3.384, P = 0.066), nor in the number of blastocysts produced on Days 6 (Low: 7%, Intermediate: 9%; χ22= 0.874, P = 0.35), 7 (Low: 14%, Intermediate: 16%, χ22= 1.256, P = 0.26) and 8 (Low: 6%, Intermediate: 7% χ22= 0.554, P = 0.45) of culture. The High group produced a significantly higher percentage of embryos on Days 6 (High: 13%, Low: 7%; χ22= 6.840, P = 0.009) and 7 (High: 21%, Low: 14%; χ22= 6.806, P = 0.009) of culture post-insemination than the Low group. The three categories did not differ in the blastocoel re-expansion ( = 0.095, P = 0.95) and hatching rates ( χ22= 0.754, P = 0.68) after 72 h post-warming, in the total number of cells per blastocyst (ANOVA F = 1.12, P = 0.337) and in the (F = 0.46, P = 0.639) incorporation of amino acids. The number of TUNEL-positive cells per embryo was higher (ANOVA F = 4.32, P = 0.022) in the Low group compared to the other groups. In conclusion, high ovarian follicle number enhances in vitro embryo output in sheep, but has no effect on blastocyst quality