64 USING PORCINE GRANULOSA CELLS AS FEEDERS FOR PORCINE AND BOVINE TROPHECTODERM CELL CULTURE

2012 ◽  
Vol 24 (1) ◽  
pp. 144
Author(s):  
I. M. Saadeldin ◽  
A. Elsayed ◽  
J. T. Kang ◽  
S. J. Park ◽  
S. J. Kim ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Doubling Time ◽  
Good Alternative ◽  
Blastocyst Stage ◽  
Outer Side ◽  
Embryonic Cells ◽  
Reverse Transcription Pcr ◽  
Porcine Granulosa Cells ◽  
Trophectoderm Cells

The trophectoderm cells, arising from the outer side of the blastomere in the blastocyst stage, are the first differentiated embryonic cells with specific potential as stem cells. The physiology of trophectoderm cells has been studied; however, their functions still remain unclear, because the lack of definitive information of cell lineages. Here, we aimed to establish in culture different feeder-dependent trophectoderm cell lines from 9-day, preimplantation, in vitro-produced porcine and bovine embryos. We used 2 different feeders: porcine granulosa cells (PGC) and mouse embryonic fibroblasts (MEF). Both cells were mitotically inactivated by mitomycin-C and then cultured with a density of 5 × 104 mL–1 on 0.1% (wt/vol) gelatin coated 4-well dishes in DMEM-199 medium supplemented with 10% (vol/vol) fetal bovine serum (FBS), nonessential amino acids (NEAA), β-mercaptoethanol and nucleosides (Talbot et al. 2000 Biol. Reprod. 62, 235–247). Trophectoderm cells were observed by light microscopy and characterised by reverse transcription-PCR using specific primers for both species. Different feeders and trophectoderm cells growth rates were compared after trypsinization using a hemocytometer. Data were analysed using 1-way ANOVA. In results, trophectoderm cells display epithelial characteristics, cuboidal morphology and express mRNA of homebox protein CDX2, cytokeratin 8 (KRT8) and interferon (IFN) gamma or tau for porcine or bovine cells, respectively. Moreover, oestrogen receptor (ESR1) and progesterone receptor (PGR) were expressed in trophectoderm cells of both species. Porcine granulosa cells were highly proliferative with doubling time of 24 h when compared to MEF (P ≤ 0.5), easy to recover and provided a reasonable source of steroids, 17β-oestradiol (E2; 31.21 ± 3.1 ng mL–1) and progesterone (P4; 6.36 ± 0.4 ng mL–1). Moreover, trophectoderm cell colonies of both species that cultured on PGC grew faster, with a doubling time of 48 h when compared to those cultured on MEF (P ≤ 0.5). We speculate that the continuous supplement of steroids and other cytokines during the co-culture of trophoblasts with granulosa cells might help the trophectoderm cells growth more than that of MEF. Further investigations are required in this regard. In conclusion, porcine granulosa cells can be good alternative feeders to culture porcine and bovine trophectoderm. This research was supported by MKE (Grant # 10033839-2011-13) and IPET.

scholarly journals Feeder Cell Type Affects the Growth of In Vitro Cultured Bovine Trophoblast Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-6 ◽  
Author(s):  
Islam M. Saadeldin ◽  
Ahmed Abdelfattah-Hassan ◽  
Ayman Abdel-Aziz Swelum
Keyword(s):  
Granulosa Cells ◽  
Current Approach ◽  
Embryonic Cells ◽  
Interferon Tau ◽  
Porcine Granulosa Cells ◽  
Trophectoderm Cells ◽  
Homeobox Protein ◽  
First Time

Trophectoderm cells are the foremost embryonic cells to differentiate with prospective stem-cell properties. In the current study, we aimed at improving the current approach for trophoblast culture by using granulosa cells as feeders. Porcine granulosa cells (PGCs) compared to the conventional mouse embryonic fibroblasts (MEFs) were used to grow trophectoderm cells from hatched bovine blastocysts. Isolated trophectoderm cells were monitored and displayed characteristic epithelial/cuboidal morphology. The isolated trophectoderm cells expressed mRNA of homeobox protein (CDX2), cytokeratin-8 (KRT8), and interferon tau (IFNT). The expression level was higher on PGCs compared to MEFs throughout the study. In addition, primary trophectoderm cell colonies grew faster on PGCs, with a doubling time of approximately 48 hrs, compared to MEFs. PGCs feeders produced a fair amount of 17β-estradiol and progesterone. We speculated that the supplementation of sex steroids and still-unknown factors during the trophoblasts coculture on PGCs have helped to have better trophectoderm cell’s growth than on MEFs. This is the first time to use PGCs as feeders to culture trophectoderm cells and it proved superior to MEFs. We propose PGCs as alternative feeders for long-term culture of bovine trophectoderm cells. This model will potentially benefit studies on the early trophoblast and embryonic development in bovines.

RGD-mediated adhesion of porcine granulosa cells modulates their differentiation response to FSH in vitro

1995 ◽  
Vol 83 (2-3) ◽  
pp. 169-177 ◽  
Author(s):  
Béatrice Goxe ◽  
Jacques E. Flechon ◽  
Solange Delasalle ◽  
Roland Salesse
Keyword(s):  
Granulosa Cells ◽  
Porcine Granulosa Cells

COMPARISON OF THE STIMULATORY EFFECTS OF OVINE, PORCINE AND HUMAN FOLLICLE-STIMULATING HORMONE AND OF OVINE AND HUMAN LUTEINIZING HORMONE ON THE ACCUMULATION OF CYCLIC AMP BY PORCINE GRANULOSA CELLS

1979 ◽  
Vol 80 (1) ◽  
pp. 9-20 ◽  
Author(s):  
ADA M. LINDSEY ◽  
CORNELIA P. CHANNING
Keyword(s):  
Cyclic Amp ◽  
Granulosa Cells ◽  
Incubation Medium ◽  
Follicle Stimulating Hormone ◽  
Similar Response ◽  
Intracellular Accumulation ◽  
Porcine Granulosa Cells ◽  
Tenfold Increase ◽  
Incubation Periods

The effects of ovine, porcine and human FSH, and ovine and human LH on the accumulation of cyclic AMP by porcine granulosa cells obtained from follicles at various stages of maturation were investigated. During incubation periods of 15 min, 10 μg ovine FSH pretreated with antiserum to LH or 10 μg human FSH resulted in an 11- to 18-fold, five-to ninefold, and less than a twofold increase in intracellular accumulation of cyclic AMP by granulosa cells from small (1–2 mm), medium (3–5 mm) and large (6–12 mm) follicles respectively. Similar patterns of response occurred with addition of porcine FSH. After incubation for 30 and 60 min with ovine, porcine or human FSH, significant accumulation of cyclic AMP in the incubation medium occurred with cells obtained from small and medium-sized follicles. After 60 min of incubation with FSH the accumulation of cyclic AMP in the incubation medium exceeded the intracellular cyclic AMP levels in granulosa cells from small and medium-sized follicles. During incubation periods of 15 min, 1·0 μg ovine LH resulted in less than a twofold, a fourfold and greater than a tenfold increase in intracellular accumulation of cyclic AMP by granulosa cells from small, medium and large follicles respectively. Addition of human LH brought about a similar response. Incubation periods of 30 and 60 min with 1·0 μg ovine or human LH resulted in significant accumulation of cyclic AMP in the incubation medium by granulosa cells from large follicles; cyclic AMP content in the incubation medium was greater after 60 min compared with 30 min of incubation. It was concluded that ovine FSH pretreated with an antiserum to LH had similar effects on cyclic AMP levels as did purified human and porcine FSH, and that the stimulatory effects of the less pure ovine FSH were probably not due to an impurity in the FSH preparation. Porcine granulosa cells obtained from small follicles should be suitable as an in-vitro FSH bioassay while granulosa cells obtained from large follicles should be suitable as an in-vitro LH bioassay.

scholarly journals Does 2-hydroxyflutamide Inhibit Apoptosis in Porcine Granulosa Cells? ^|^mdash; An In Vitro Study

2012 ◽  
Vol 58 (4) ◽  
pp. 438-444 ◽  
Author(s):  
Malgorzata DUDA ◽  
Malgorzata DURLEJ ◽  
Malgorzata KNET ◽  
Katarzyna KNAPCZYK-STWORA ◽  
Zbigniew TABAROWSKI ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
In Vitro Study ◽  
Vitro Study ◽  
Porcine Granulosa Cells

Localization of cytoskeletal proteins in preimplantation mouse embryos

Development ◽  
1980 ◽  
Vol 55 (1) ◽  
pp. 211-225
Author(s):  
E. Lehtonen ◽  
R. A. Badley
Keyword(s):  
Blastocyst Stage ◽  
Cytoskeletal Proteins ◽  
Mouse Embryos ◽  
Trophectoderm Cells ◽  
Apparent Concentration ◽  
Preimplantation Mouse ◽  
Early Mouse ◽  
Filament Protein

The immunofluorescence technique was used to detect the presence and distribution of actin, alpha-actinin, tubulin and 10 nm filament protein in early mouse embryos. Actin and alpha-actinin stainings showed a distinct concentration to a peripheral layer in the cleavage-stage blastomeres and in trophectoderm cells. Dots of fluorescence appeared in this cortical staining pattern. The distribution of tubulin staining in the blastomere cytoplasm was relatively even with apparent concentration at the perinuclear region and frequently at wide intercellular contact areas. 10 nm filament protein was distributed evenly in the blastomere cytoplasm without cortical concentration of the label. At the blastocyst stage, the trophectoderm cells in blastocyst outgrowths in vitro developed well organized cytoskeletons including both microfilament, microtubule and 10 nm filament elements. Comparable structures were not observed in blastocysts in vivo, or in late hatched blastocysts cultured in suspension. The morphogenetic significance of the observations is discussed.

scholarly journals Centrosomal protein centrin is not detectable during early pre-implantation development but reappears during late blastocyst stage in porcine embryos

Reproduction ◽  
2006 ◽  
Vol 132 (3) ◽  
pp. 423-434 ◽  
Author(s):  
G Manandhar ◽  
D Feng ◽  
Y-J Yi ◽  
L Lai ◽  
J Letko ◽  
...  
Keyword(s):  
Western Blotting ◽  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
De Novo ◽  
Germinal Vesicle ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Embryonic Cells ◽  
Perinuclear Area

Centrin is an evolutionarily conserved 20 kDa, Ca+2-binding, calmodulin-related protein associated with centrioles and basal bodies of phylogenetically diverse eukaryotic cells. Earlier studies have shown that residual centrosomes of non-rodent mammalian spermatozoa retain centrin and, in theory, could contribute this protein for the reconstruction of the zygotic centrosome after fertilization. The present work shows that CEN2 and CEN3 mRNA were detected in germinal vesicle-stage (GV) oocytes, MII oocytes, and pre-implantation embryos from the two-cell through the blastocyst stage, but not in spermatozoa. Boar ejaculated spermatozoa possess centrin as revealed by immunofluorescence microscopy and western blotting. Immature, GV oocytes possess speckles of centrin particles in the perinuclear area, visualized by immunofluorescence microscopy and exhibit a 19 kDa band revealed by western blotting. Mature MII stage oocytes lacked centrin that could be detected by immunofluorescence or western blotting. The sperm centrin was lost in zygotes afterin vitrofertilization. It was not detectable in embryos by immunofluorescence microscopy until the late blastocyst stage. Embryonic centrin first appeared as fine speckles in the perinuclear area of some interphase blastocyst cells and as putative centrosomes of the spindle poles of dividing cells. The cells of the hatched blastocysts developed centrin spots comparable with those of the cultured cells. Some blastomeres displayed undefined curved plate-like centrin-labeled structures. Anti-centrin antibody labeled interphase centrosomes of cultured pig embryonic fibroblast cells as distinct spots in the juxtanuclear area. Enucleated pig oocytes reconstructed by electrofusion with pig fibroblasts displayed centrin of the donor cell during the early stages of nuclear decondensation but became undetectable in the late pronuclear or cleavage stages. These observations suggest that porcine zygotes and pre-blastocyst embryonic cells lack centrin and do not retain exogenously incorporated centrin. The early embryonic centrosomes function without centrin. Centrin in the blastocyst stage embryos is likely a result ofde novosynthesis at the onset of differentiation of the pluripotent blastomeres.

Prolactin, LH, and estradiol-17β in utilization of lipoprotein substrate by porcine granulosa cells in vitro

1988 ◽  
Vol 66 (5) ◽  
pp. 561-566 ◽  
Author(s):  
K. Rajkumar ◽  
P. Klingshorn ◽  
P. J. Chedrese ◽  
B. D. Murphy
Keyword(s):  
Granulosa Cell ◽  
Cell Cultures ◽  
Granulosa Cells ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
Porcine Granulosa Cells ◽  
Progesterone Synthesis ◽  
Serum Free Conditions

Porcine granulosa cells cultured under serum free conditions responded by increased progesterone secretion to the addition of the leuteotropic hormones, LH, prolactin, and estradiol. Provision of extracellular substrate for steroidogenesis in the form of porcine high density lipoprotein or low density lipoprotein enhanced progesterone accumulation by granulosa cell cultures. Estradiol, LH, and prolactin all greatly increased progesterone accumulation in the presence of either high or low density lipoproteins. Increases in progesterone accumulation following addition of prolactin or LH in combination with estradiol suggested the presence of a synergistic interaction among leuteotropins. Pre-exposure of granulosa cell cultures to estradiol increased the subsequent stimulatory effect of prolactin on lipoprotein utilization. It is concluded that all three leuteotropins function to enhance and may interact in the utilization of extracellular lipoprotein substrate for progesterone synthesis.

scholarly journals The Cultivation of Human Granulosa Cells

2008 ◽  
Vol 51 (3) ◽  
pp. 165-172 ◽  
Author(s):  
Lenka Brůčková ◽  
Tomáš Soukup ◽  
Jiří Moos ◽  
Martina Moosová ◽  
Jana Pavelková ◽  
...  
Keyword(s):  
Growth Factors ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Doubling Time ◽  
Ovarian Follicle ◽  
Vitro Fertilization ◽  
Thecal Cells ◽  
Cell Doubling Time

The major functions of granulosa cells (GCs) include the production of steroids, as well as a myriad of growth factors to interact with the oocyte during its development within the ovarian follicle. Also FSH stimulates GCs to convert androgens (coming from the thecal cells) to estradiol by aromatase. However, after ovulation the GCs produce progesterone that may maintain a potential pregnancy. Experiments with human GCs are mainly focused on the purification of GCs from ovarian follicular fluid followed by FACS analysis or short-term cultivation. The aim of our study was to cultivate GCs for a long period, to characterize their morphology and phenotype. Moreover, we have cultivated GCs under gonadotropin stimulation in order to simulate different pathological mechanisms during folliculogenesis (e.g. ovarian hyperstimulation syndrome). GCs were harvested from women undergoing in vitro fertilization. Complex oocyte-cumulus oophorus was dissociated by hyaluronidase. The best condition for transport of GCs was optimized as short transport in follicular fluid at 37 °C. GCs expansion medium consisted of DMEM/F12, 2 % FCS, ascorbic acid, dexamethasone, L-glutamine, gentamycine, penicillin, streptomycin and growth factors (EGF, bFGF). GCs transported in follicular fluid and cultivated in 2 % FCS containing DMEM/F12 medium supplemented with follicular fluid presented increased adhesion, proliferation, viability and decreased doubling time. Cell viability was 92 % and mean cell doubling time was 52 hrs. We have optimized transport and cultivation protocols for long-term cultivation of GCs.

scholarly journals Effects of transferrin on aromatase activity in porcine granulosa cells in vitro.

2009 ◽  
Vol 46 (4) ◽  
Author(s):  
Małgorzata Durlej ◽  
Małgorzata Duda ◽  
Katarzyna Knapczyk ◽  
Maria Słomczyńska
Keyword(s):  
Granulosa Cells ◽  
Aromatase Activity ◽  
Porcine Granulosa Cells
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