196,087 research outputs found
Granulosa cells & three-dimensional scaffold: towards a follicle-like structure in the domestic cat
No full textThe in vitro development of feline oocytes is still suboptimal, especially in case of low competence gametes, such as cryopreserved or denuded oocytes, valuable resources for fertility preservation purposes. Modifications to media composition alone have not led to truly satisfactory results; therefore, since physical factors can positively influence the chemical conditions (1), a three-dimensional (3D) follicle-like structure could enhance meiosis resumption and further embryo development in vitro of poorly competent oocytes. In the domestic cat, the establishment of granulosa cells (GCs) culture was performed in a bidimensional (2D) system, and steroidogenesis was revealed along culture (2). However, since the follicular environment is three-dimensionally organized, the encapsulation of GCs in a 3D matrix might improve their self-organization, proliferation and endocrine functions. With the final aim of using these follicle-like structures for low competence oocyte IVM, functionality and efficiency of GCs encapsulated in biocompatible 3D matrix were evaluated during culture, and estradiol (E2) and progesterone (P4) concentrations were compared to those obtained from GCs cultured in a 2D monolayer. Cat granulosa cells were collected from isolated ovaries by enzymatic denudation (hyaluronidase 80 IU) of fresh COCs. Cells were processed as in (2) and then re-suspended in culture medium [CM=TCM199 supplemented with 0.1% BSA, 5% FBS, 0.5 IU FSH + 0.5 IU LH, 10 μg/mL 22(R)-hydroxycholesterol, 25 ng/ml epidermal growth factor, 0.1% ITS premix (1.0 μg/mL insulin; 0.55 μg/mL transferrin; 0.5 ng/mL sodium selenite), 10-7 M androstenedione]. Granulosa cells were in vitro cultured in a controlled atmosphere for 6 days in 3D follicle-like structures [obtained by one-step encapsulation in barium alginate (3)], or in 2D system (multiwell plates). Aliquots of conditioned CM were collected on days 2 and 6 and stored at -20°C until hormonal (E2 and P4) determination with an Enzyme Linked Fluorescent Assay (ELFA, MiniVIDAS®, Biomerieux, France). Log transformed hormonal concentrations were analyzed by a two-way ANOVA followed by Tukey test; the level of significance was set at p<0.05. Differences in hormonal production among days and systems of GCs culture were observed. Estradiol and P4 concentrations differed significantly during time, resulting higher on day 6 compared to day 2 (p=0.018) and day 0 (p<0.001) of culture. Steroidogenesis was obtained in 3D follicle-like structures as well as in 2D monolayers, although not to the same extent; the systems differed significantly only on day 6 (P4: 2.44 ± 1.28 ng/ml in 3D and 610 ± 607 ng/ml in 2D, p<0.001; E2: 173 ± 145 pg/ml in 3D and 26,709 ± 17,921 pg/ml in 2D, p=0.02). Present results underline that GCs in 3D microcapsules maintained their physiological features, as hormonal secretion. To test whether these 3D conditions could act as proper follicle-like structures, further experiments will be focused on the assessment of their nursing ability for the in vitro maturation of low competence oocytes.
This research project was funded by EVSSAR Grant 2017.
References
(1) Luvoni GC. The never ending search of an ideal culture system for domestic cat oocytes and embryos. Reprod Domest Anim 2018; accepted.(2) Simsek O, Arikan S. Effects of cholesterol, FSH and LH on steroidogenic activity of cat granulosa cells cultured in vitro. Anim Reprod 2015;12:931-938.
(3) Vigo D, Villani S, Faustini M, et al. Follicle-like model by granulosa cell encapsulation in a barium alginateprotamine membrane. Tissue Eng 2005;11:709-14
Overcoming flat biology: three-dimensional follicle-like structures for domestic cat vitrified oocytes
No full textFollicle-like structures are a combination of living cells and semipermeable 3D matrices, in which physical (3D systems) and chemical (cell-secreted molecules) factors create an in vivolike microenvironment for the in vitro development of female gametes (1, 2). This artificial and viable milieu, successfully applied for fresh oocytes, could be even more useful for stressed gametes, as the low competence vitrified oocytes (VOs). Key players of fertility preservation efforts, VOs poorly achieve full maturation because their inner competence is severely impaired by unavoidable cryodamages also involving their surrounding cumulus cells. The replacement of damaged somatic compartment with species-specific follicle-like structures could restore the developmental competence of VOs. Thus, in this work the creation of a follicle-like structure of granulosa cells (GCs) in 3D barium alginate microcapsules was aimed at improving the nuclear competence of domestic cat VOs. Therefore, a comparison with “flat” GCs culture (monolayer) was performed to test whether 3D spatial organization could be more adequate in preserving GCs nursing ability for stressed gametes. Isolated ovaries (n. 22) from domestic queens were minced to retrieve fresh oocytes that were enzymatically denuded (hyaluronidase 80IU) to collect GCs. After purification (3), GCs were suspended in culture medium [CM=TCM199 supplemented with 0.1% BSA, 5% FBS, 0.5 IU FSH + 0.5 IU LH, 10 μg/mL 22(R)-hydroxycholesterol, 25 ng/ml epidermal growth factor, 0.1% ITS premix (1.0 μg/mL insulin; 0.55 μg/mL transferrin; 0.5 ng/mL sodium selenite), 10- 7 M androstenedione], and the one-step encapsulation technique in barium alginate (2) was performed to obtain the 3D follicle-like structures. Granulosa cells capsules (3D system) or monolayers (2D) were in vitro cultured in CM at controlled atmosphere (38.5°C and 5% CO2 in air) for 2 or 6 days and used as artificial milieu for VOs IVM. Vitrified oocytes were obtained by Cryotop protocol (Kitazato Co., Fujinomiya, Japan) from 127 fresh COCs, and after warming only viable gametes (stained by FDA/PI, n. 112) were in vitro matured (24 h) in 3D and 2D system, either on day 2 or on day 6 of GCs culture. Chromatin configurations were determined by bis-benzimide (Hoechst 33342; Sigma) staining, and data were analyzed by Chi-square test, with the level of significance set at p≤0.05. Very encouraging meiosis resumption rates were obtained in VOs matured in 3D follicle-like structures (45.5%) and monolayer (56.7%) on day 2 of GCs culture. Prolonged culture of GCs for 6 days severely impaired the nursing ability of monolayer (21.7%, p=0.007), but not that of 3D follicle-like structures (26.7%, p=0.07), that supported higher proportions of fullmaturational (TI-MII) stages (17.4%) than GCs cultured for 2 days (0%). Culturing GCs for 2 days allowed lower VOs degeneration rates than 6 days (p=0.001). The ideal culture environment for low competence VOs has to combine physical and chemical factors and 3D follicle-like structures gave promising results for the restoration of VOs fertility potential, but further improvements are needed to promote GCs polarization and self-assembly into tissue-like structures.
This research project was funded by EVSSAR Grant 2017.
(1) Luyckx V, Dolmans MM, Vanacker J et al. A new step towards the artificial ovary: survival and proliferation of isolated murine follicles after autologous transplantation in a fibrin scaffold. Fertil Steril, 2014; 101: 1149–56.
(2) Vigo D, Villani S, Faustini M et al. Follicle-like model by granulosa cell encapsulation in a barium alginateprotamine membrane. Tissue Eng, 2005; 11: 709-14.
(3) Simsek O, Arikan S. Effects of cholesterol, FSH and LH on steroidogenic activity of cat granulosa cells cultured in vitro. Anim Reprod, 2015; 12: 931-38
Oocytes vitribanking in the domestic cat model: developmental competence in 3D culture
No full textIn the domestic cat model, vitrified oocytes (VOs) produced promising results, however their potential to develop into late stage embryos is often impaired.
Instead of culturing VOs in two-dimensional (2D) microdrops of medium (conventional system), the use of three-dimensional (3D) scaffolds could better maintain their functional integrity, contributing to oocyte in vitro outcomes. Therefore, the aim of this study was to investigate survival and embryo development (oocyte competence) of feline VOs cultured in 3D barium alginate microcapsules.
Morphologically selected immature cumulus-oocyte complexes (COCs, n=250) were vitrified by Cryotop method, and viable oocytes after warming were in vitro matured for 24 hours in 2D (2D VOs) or 3D system, with fresh companion COCs in 1:1 ratio [3D VOs(+)] or without [3D VOs(-)]. After in vitro fertilization, presumptive zygotes were cultured for 7 days in 3D or 2D system, according to the maturation conditions. Viability of VOs after warming and number of blastomeres of embryos were assessed by fluorescent stainings (fluorescein diacetate/propidium iodide and bis-benzimide, respectively). Arcsin transformed data were analysed by one-way ANOVA followed by Tukey test, and the level of significance was set at p<0.05.
After warming, 91.2% VOs were viable. Embryos were successfully obtained at similar proportions in each group [mean ± SD: 3D VOs(+) 0.18 ± 0.17; 3D VOs(-) 0.17 ± 0.12; 2D VOs 0.15 ± 0.09; p>0.05], but the use of the 3D system together with companion COCs was determinant for their development, since 3D VOs(+) were the only group to reach the blastocyst stage (mean ± SD: 0.01 ± 0.03, day 7). Degeneration rates did not differ among groups.
These positive results prompt the use of 3D enriched culture systems for VOs to exploit this valuable germplasm and widen the potential of vitribanking
Vitrification of immature feline oocytes with a commercial kit for bovine embryo vitrification
No full textThe aim of this study was to evaluate the suitability of a commercial kit for bovine embryo vitrification for cryopreserving cat oocytes and to evaluate comparatively the effects of its use with slow freezing procedure on cryotolerance in terms of morphology and oocyte resumption of meiosis. Germinal vesicle stage oocytes isolated from cat ovaries were either vitrified (n=72) using a vitrification kit for bovine embryo or slow frozen (n=69) by exposing oocyte to ethylene glycol solution before being transferred to a programmable embryo freezer. After thawing and warming, oocytes were cultured for 48h and then were examined for meiosis resumption using bisbenzimide fluorescent staining (Hoechst 33342). Fresh immature oocytes (n=92) were used as the control group. The proportion of oocytes recovered in a morphologically normal state after thawing/warming was significantly higher in frozen oocytes (94.5%) than in the vitrified ones (75%, p<0.01). Morphological integrity after culture was similar in vitrified (73.6%) and slow frozen oocytes (76.8%); however, only 37.5% of the morphologically normal oocytes resumed meiosis after vitrification compared to 60.9% of those submitted to slow freezing procedure (p<0.01). Fresh oocytes showed higher morphological integrity (91.3%) and meiosis resumption rates (82.6%, p<0.002) than cryopreserved oocytes, irrespective of the procedure used. These results suggest that immature cat oocytes vitrified with a kit for bovine embryos retain their capacity to resume meiosis after warming and culture, albeit at lower rates than slow frozen oocytes. Vitrification and slow freezing methods show similar proportions of oocytes with normal morphology after culture, which demonstrate that thawed and warmed oocytes that resist to cryodamage have the same chances to maintain their integrity after 48h of culture. © 2012 Blackwell Verlag GmbH.Universidade Estadual Paulista (UNESP) Departmento de Medicina Veterinária Preventiva e Reprodução Animal, Jaboticabal campus, Jaboticabal-SPCSU Equine Reproduction Laboratory Colorado State University, Fort Collins, CODipartimento di Scienze Cliniche Veterinarie Sezione di Clinica Ostetrica e Ginecologica Veterinaria Università degli Studi di Milano, MilanoUniversidade Estadual Paulista (UNESP) Departmento de Medicina Veterinária Preventiva e Reprodução Animal, Jaboticabal campus, Jaboticabal-S
Chemical imaging of canine oviducts during the post-ovulatory period
No full textIt is well known that bitches present a peculiar oocyte maturation, characterized by ovulation of immature oocytes and a long period of viability in the oviduct. Thus, the oviduct in this species plays an essential role in oocyte maturation, in addition to fertilization and early stage embryo development. Different approaches have been used in order to identify the factors involved in each step (from resumption of meiosis through development of embryos) and recently a new technique was used for spatial identification of oviductal proteins in the domestic cat (1). With the purpose to contribute to the understanding of these factors, we applied MALDI imaging mass spectrometry (MALDI-IMS) to obtain protein profiling and imaging of canine oviducts. Reproductive tracts were collected from 4 bitches in estrus (cross-breed, 2 to 6 years old) undergoing routine ovariohysterectomy. Post-ovulatory period was confirmed by blood serum progesterone concentrations (P4 mean±SD: 15.5±2.5) and ovarian morphology. The oviducts were carefully dissected, divided into three segments (distal to the ovary-isthmus, proximal to the ovary-infundibulum and the mid-section between the two-ampulla; further confirmed by histology), snap-frozen in liquid nitrogen and stored at -80oC until use. Then, they were sectioned (11 μm) in a cryostat and fixed on ITO (indium tin oxide) conductive glass slides, while serial sections were collected on microscope slides for haematoxylin and eosin staining. For MALDI-IMS, samples were coated with a thin homogeneous layer of CHCA (α-Cyano-4-hydroxycinnamic acid) matrix using a nebulization device. MALDI images were acquired on an Autoflex III Smartbeam instrument (Bruker Daltonics) with 400 shots/spectrum, over the mass range of m/z 2 to 20 kDa in the positive ion mode. The spatial resolution of images was 80 μm. Mass spectra were characterized by abundant ions of m/z 2279, 2401, 3458 and 4976; which have been tentatively attributed to actin cytoplasmic 2, keratin type 1, neutrophil defensin 1 and thymosin β4, respectively. Actin has been detected in oviduct fluid of alpaca (2) and is related to epithelial cell renewal or secretory activity (3). As previously described in queens (1), defensin, keratin and thymosins are defense proteins that integrate the innate immune systems and are involved in the biological response to cellular damage. These data might contribute to piecing together the puzzle of factors that are involved in the peculiar aspects of the domestic dog reproductive physiology that might hamper in vitro embryo production.
1) Apparicio M, Santos VG, Rocha DFO et al. Matrix-assisted laser desorption/ionization imaging mass spectrometry for the spatial location of feline oviductal proteins. Reprod Domest Anim. 2017;52 (Suppl. 2):88–92.
(2) Apichela SA, Argañaraz ME, Zampini R et al. Biochemical composition and protein profile of alpaca (Vicugna pacus) oviductal fluid. Anim Reprod Sci. 2015,154:79-85
(3) Steffl M, Schweiger M, Sugiyama T et al. Review of apoptotic and non-apoptotic events in non-ciliated cells of the mammalian oviduct. Ann Anat. 2008,190: 46-52
Dr. Duane M. Jackson, Morehouse College, July 2011
No full textThis video is a conversation with Dr. Duane M. Jackson. Dr. Jackson talks about his paper, "Recall and the Serial Position Effect: The Role of Primacy and Recency on Accounting Students' Performance." Jackie Daniel, AUC Woodruff Library, is the interviewer
"Reflections on the subject of Emigration from Europe with a view to Settlement in the United States" By M. Carey.
No full text"Reflections on the subject of Emigration from Europe with a view to Settlement in the United States: containing bried sketches of the moral and political character of those states.
By M. Carey, member of the American philosophical, and of the American Antiquarian Society, and author of The Olive Branch, Cindiciae Hibernicae, essays on banking, on political economy, and on internal improvement.
To which are now added the English editor's comments on the subject; together with Important Advice to Emigrants, and Cautions Against Impositions Practiced in the Outports
Dispelling the Myths Behind First-author Citation Counts
No full textWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
Dr. Glendon Swarthout
No full textHosted by Roger M. Busfield, MSU Assistant Professor of Speech and Theater, Meet the Author is designed to introduce a general audience to a contemporary author and their work through in-depth interviews. This episode features a conversation between Dr. Glendon Swarthout, prolific author and English professor at MSU, and assistant professors Sam S. Baskett and Theodore B. Strandness
- …
