1,721,191 research outputs found
Nuclear dynamics and developmental potential of sheep nuclear transfer embryos treated with protein kinase inhibitor 6-dimethylaminopurine
No full textCharacterization, isolation and culture of primordial germ cells in domestic animals: recent progress and insights from the ovine species
No full textPrimordial 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, Nunog, 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. (C) 2010 Elsevier Inc. All rights reserved
The subcortical maternal complex: multiple functions for one biological structure?
No full textThe subcortical maternal complex (SCMC) is a multiprotein complex uniquely expressed in mammalian oocytes and early embryos, essential for zygote progression beyond the first embryonic cell divisions. Similiar to other factors encoded by maternal effect genes, the physiological role of SCMC remains unclear, although recent evidence has provided important molecular insights into different possible functions. Its potential involvement in human fertility is attracting increasing attention; however, the complete story is far from being told. The present mini review provides an overview of recent findings related to the SCMC and discusses its potential physiological role/s with the aim of inspiring new directions for future research
Cell coupling and maturation-promoting factor activity in in vitro-matured prepubertal and adult sheep oocytes
No full textWe examined some differences between prepubertal and adult ovine oocytes; in particular we analyzed the functional status of the cumulus-oocyte complex, protein synthesis during in vitro maturation, and because no information is available on prepubertal and adult sheep, maturation-promoting factor (MPF) fluctuations throughout meiotic progression both in prepubertal and adult sheep oocytes. After 24 h of maturation, percentages of MII oocytes were similar between prepubertal and adult animals. Electron microscopy examinations showed that prepubertal oocytes had fewer transzonal projections than adult oocytes. Methionine uptake was significantly lower in prepubertal cumulus-enclosed oocytes examined through meiotic progression. On the contrary, denuded prepubertal oocytes showed a higher methionine incorporation in the first 4 h of incubation compared with adult oocytes. We also found some differences in MPF activity between prepubertal and adult oocytes at MII stage. In fact, prepubertal MII oocytes had a significantly lower level of MPF activity than adult oocytes did and, after fusion with germinal vesicle oocytes, they were unable to induce nuclear breakdown and chromosome condensation 1-2 h post-fusion, whereas adult MII oocytes could induce these processes. Our findings show that the lesser competence of prepubertal oocytes could be due to morphological anomalies and alterations in physiological activity and that oocytes do not reach full developmental competence until puberty
Novel approach to cell sampling from preimplantation ovine embryos and its potential use in embryonic genome analysis
No full textThe major obstacle in the extensive analysis of the embryonic genome is the small number of cells typically obtained after the embryo biopsy. The object of the present study was to develop a simple approach that would allow the collection of a sufficient number of cells from a single embryo for use in further analyses. A micromanipulator was used to make a hole in the zona pellucida of 28 compacted morulae, 27 early blastocysts and 31 expanded blastocysts. After further culture, the trophoblastic cells, which herniated through this hole, were cut and cultured in vitro for different periods and used for embryo sexing. The results showed that biopsies can be taken successfully from 96.3% of early blastocysts, compared with 67.7% of expanded blastocysts and 71.4% of compacted morulae. The trophoblastic vesicles contained 20.8 +/- 6.7 cells (mean +/- SEM) and, when cultured, formed a confluent monolayer. The sex of cells cultured was assayed by PCR and the 12 lambs born after transfer of biopsied embryos confirmed its 100% accuracy. Moreover, no significant differences were found in the viability rates in vitro among blastocysts vitrified immediately after biopsy (77.8%), blastocysts biopsied and vitrified after 24 h culture (76.9%) and blastocysts vitrified without manipulation (88.5%). In experiments in vivo, the lambing rate of biopsied and vitrified blastocysts was significantly (P < 0.05) lower (40.0%) compared with vitrified control embryos (68.7%). This new approach to the biopsy of preimplantation embryos is a useful good model in the assisted reproductive technologies of domestic, wild and human species
KINETICS OF THE ONTOGENIC AND REVERSIBLE HEMOGLOBIN SWITCHING IN THE MOUFLON (OVIS-MUSIMON) AND SHEEP X MOUFLON HYBRID
No full text1. Hemoglobin (Hb) switching in the perinatal life of wild mouflon (Ovis musimon) was characterized by the replacement of Hb F by 60% levels of Hb C, and subsequently of Hb C by Hb B. 2. The recently discovered Hb M variant was not replaced by Hb C; thus, Hb BM heterozygote newborns synthesized 30% Hb C at the expense of Hb B. 3. Hybrid B mouflon x B sheep synthesized only 5% Hb C at birth but were able to produce 30% Hb C in adult life following induced anemia. 4. Adult BB and BM mouflons, after the same extent of induced anemia, synthesized HB C levels similar to those produced at birth. The results indicate a mouflon beta-globin gene cluster arrangement similar to those of sheep and goat, the beta-c gene having an intermediate expression. Results also suggest a selective disadvantage in hybrid animals
Production and lambing rate of blastocysts derived from in vitro matured oocytes after gonadotropin treatment of prepubertal ewes.
No full textThe aim of this study was to evaluate the effect of gonadotropin treatment on the in vitro maturation, blastocyst production, and developmental potential to term of oocytes collected from Sardinian neonatal and prepubertal ewes at 4 to 6 wk of age. Cumulus-oocyte complexes were recovered at 24 h after withdrawal of a 1/6th size progestagenated pessary from the donors, of which each received 120 IU FSH/LH and 400 IU PMSG in a single dose 36 h before sponge removal. Treated donors produced a greater (P<.01) number of oocytes per animal (86.2 +/-7.9) compared with slaughterhouse (untreated) prepubertal ewes (55.5+/-6.1) of the same age or with treated neonatal ewes (6.1+/-0.7) 10 d old. During oocyte maturation, there were no differences in the percentage of germinal vesicle break-down (78.08 vs. 74.24), metaphase I (89.13 vs. 87.18), and metaphase II (77.91 vs. 76.38) when evaluated after 8, 14, and 24 h of maturation, respectively, between oocytes from treated and slaughterhouse (untreated) prepubertal ewes. The embryo cleavage (71.1 vs. 73.7) and blastocyst rates (22.2 vs. 19.8) were similar in the treated and the untreated prepubertal ewes after transfer of in vitro matured oocytes into ligated oviducts of temporary recipients. The in vitro viability rates of vitrified blastocysts (81.2 vs. 76.9) and the in vivo survival rates (46.1 vs. 50.0) of embryos derived from in vitro matured and in vivo fertilized oocytes showed no difference. The data suggest that gonadotropin treatment increases oocyte production per animal but has no effect on oocyte quality because embryo production and lambing rates of blastocysts derived from in vitro matured oocytes were not markedly different from those derived from untreated prepubertal ewes of the same age
Production and lambing rate of blastocysts derived from in vitro matured oocytes after gonadotropin treatment of prepubertal ewes
Full text linkThe aim of this study was to evaluate
the effect of gonadotropin treatment on thein vitromaturation, blastocyst production, and developmental
potential to term of oocytes collected from Sardinian
neonatal and prepubertal ewes at 4 to 6 wk of age.
Cumulus-oocyte complexes were recovered at 24 h
after withdrawal of a 1/6th size progestagenated
pessary from the donors, of which each received 120
IU FSH/LH and 400 IU PMSG in a single dose 36 h
before sponge removal. Treated donors produced a
greater ( P < .01) number of oocytes per animal (86.2
± 7.9) compared with slaughterhouse (untreated)
prepubertal ewes (55.5 ± 6.1) of the same age or with
treated neonatal ewes (6.1 ± 0.7) 10 d old. During
oocyte maturation, there were no differences in the
percentage of germinal vesicle break-down (78.08 vs
74.24), metaphase I (89.13 vs 87.18), and metaphase
II (77.91 vs 76.38) when evaluated after 8, 14, and 24
h of maturation, respectively, between oocytes from
treated and slaughterhouse (untreated) prepubertal
ewes. The embryo cleavage (71.1 vs 73.7) and
blastocyst rates (22.2 vs 19.8) were similar in the
treated and the untreated prepubertal ewes after
transfer ofin vitromatured oocytes into ligated
oviducts of temporary recipients. Thein vitroviability
rates of vitrified blastocysts (81.2 vs 76.9) and thein
vivosurvival rates (46.1 vs 50.0) of embryos derived
fromin vitromatured andin vivofertilized oocytes
showed no difference. The data suggest that
gonadotropin treatment increases oocyte production
per animal but has no effect on oocyte quality because
embryo production and lambing rates of blastocysts
derived fromin vitro maturedoocytes were not
markedly different from those derived from untreated
prepubertal ewes of the same age
Development of parthenogenetic and cloned ovine embryos: Effect of activation protocols
No full textPreliminary experiments carried out on ovine oocytes were designed to establish correlations between activation protocols and subsequent rates of embryonic development. The best activation protocols were thereafter used in studies on ovine parthenogenesis and cloning. The first study established that chemical activators induce pronuclear development at a slightly higher rate than physical activation (ionomycin, 96%; ethanol, 95%; electro activation, 80%). Inhibition of second polar body extrusion and one single pronucleus were observed in the majority of the oocytes (similar to 90%) treated for 3 h with 6-dimethylaminopurine (6-DMAP) following either ionomycin or ethanol activation. While over 80% of these oocytes cleaved after transfer to the oviducts of recipients, progression to the blastocyst stage was higher after ionomycin as compared with ethanol activation (58% vs. 19%). The ionomycin plus 6-DMAP activation protocol was used to produce parthenogenetic blastocysts whose subsequent development was monitored both by ultrasonography and by direct fetal examination. Over 70% of parthenogenotes were viable on Day 21 of pregnancy but dead by Day 25. The effects of 6-DMAP on nuclear remodeling and fetal development of cloned embryos was then investigated. Control cloned embryos underwent nuclear envelope breakdown (NEBD), premature chromatin condensation (PCC), and inhibition of DNA synthesis. By contrast, reconstructed embryos treated with 6-DMAP exhibited intact nuclear membranes, interphase chromatin, and no interference on DNA synthesis. Moreover, cloned embryos developed to blastocyst;stage in higher percentage after 6-DMAP treatment (83% vs. 25%). We conclude that ionomycin followed by 6-DMAP incubation yields high percentages of diploid parthenogenetic embryos that develop to Day 25 before dying. Cloned embryos activated by the ionomycin-6-DMAP protocol develop readily to term
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