Evaluating an in vitro culture system of bovine uterine and oviduct epithelial cells for subsequent embryo co-culture

Three experiments were conducted to evaluate the effects of culture medium and incubation temperature on bovine uterine and oviduct epithelial cell growth, so that the most efficient combination could then be used to develop a co-culture system for bovine embryos. In the first experiment, uterine and oviduct epithelial cells at either the second or third subpassage were incubated for 8 days at 37 degrees C with 5% CO2 in Tissue Culture Medium-199, CMRL-1066, Minimal Essential Medium, Menezo's B2 or Ham's F-12 medium. In addition to plotting growth curves of cell populations, the cell cycle was monitored for 8 days by flow cytometry. Uterine and oviduct epithelial cells incubated in CMRL-1066 exhibited the highest growth rates during the 8-day culture period. However, there were no differences in cell cycle analysis among treatment groups during the incubation period. In the second experiment, CMRL-1066 medium was used to evaluate growth and proliferation of uterine and oviduct epithelial cells incubated at 37 degrees C or 39 degrees C; temperature had no significant effect on growth rates or proliferation rates for either uterine or oviduct cells during the 8-day incubation. In the third experiment, the more promising culture media for epithelial cell culture studies were chosen for in vitro maturation and subsequent in vitro fertilization (IVF) of bovine oocytes. Early cleavage-stage embryos produced by IVF procedures were subsequently cultured in vitro for 7 days in medium alone or with oviduct epithelial cells. In this study, the culture medium did not influence fertilization or cleavage rates. However, more embryos co-cultured with oviduct epithelial cells were considered viable after 7 days of incubation compared with embryos incubated in medium alone. These results indicate that various incubation conditions can influence the growth of bovine uterine and oviduct epithelial cells in vitro. However, in spite of changes in cell growth patterns, there does not appear to be a change in their embryotropic capabilities in vitro.

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The interaction of fresh and frozen-thawed ram spermatozoa with oviducal epithelial cells in vitro

Reproduction Fertility and Development ◽

10.1071/rd00032 ◽

2000 ◽

Vol 12 (6) ◽

pp. 237 ◽

Cited By ~ 11

Author(s):

L. Gillan ◽

G. Evans ◽

W. M. C. Maxwell

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Small Proportion ◽

Oestrous Cycle ◽

Cell Monolayers ◽

Oviduct Epithelial Cell ◽

Epithelial Cell Monolayers ◽

Oviduct Epithelial Cells

In order to investigate the interaction of fresh and frozen–thawed spermatozoa with oviduct epithelial cells, spermatozoa were co-incubated with ovine oviduct epithelial cell monolayers (OECM) derived from either complete oviducts, at any stage of the oestrous cycle (Experiments 1 and 2), or from different regions of the oviduct at different stages of the cycle (Experiment 3). Fresh and frozen—thawed spermatozoa displayed different patterns of binding to, and release from, the OECM. Frozen—thawed spermatozoa immediately bound to the complete oviduct OECM and were released after 2 h. A small proportion of fresh spermatozoa bound immediately, increasing to a maximum after 2 h, and were gradually released thereafter. When only the cells that were released from the OECM were observed by chlortetracycline staining in Experiment 2, it was found that the presence of an OECM increased the number of capacitated fresh spermatozoa while decreasing the number of capacitated frozen–thawed spermatozoa. Overall, the OECM advanced the membrane state of both types of spermatozoa from uncapacitated to acrosome-reacted. Fresh and frozen—thawed spermatozoa bound to OECM derived from the cells of the isthmus and the ampulla in similar proportions. However, more spermatozoa were capacitated when incubated with OECM derived from isthmic rather than ampullary cells. Higher proportions of fresh spermatozoa bound to, and were acrosome-reacted following incubation with OECM derived from post- rather than pre-ovulatory tracts. Such differences were not observed for frozen—thawed spermatozoa. The findings reported in this study show that fresh and frozen—thawed spermatozoa behave differently when in contact with oviduct cells in vitro. This may be a consequence of the more advanced membrane state of the frozen spermatozoa upon thawing.

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Acidified Pepsin Promotes Laryngeal Precancerosis by Upregulating H+/K+-ATPase and Activating Mitophagy in Laryngeal Epithelial Cells

10.21203/rs.3.rs-132708/v1 ◽

2020 ◽

Author(s):

Ke-Jia Cheng ◽

Qiong Xu ◽

Zhi-Mei Li ◽

Shui-Hong Zhou ◽

Yang-Yang Bao ◽

...

Keyword(s):

Cell Growth ◽

Epithelial Cell ◽

Epithelial Cells ◽

Laryngopharyngeal Reflux ◽

Upper Aerodigestive Tract ◽

Promoting Effect ◽

Growth And Survival ◽

Laryngeal Mucosa

Abstract Background: Although laryngopharyngeal reflux (LPR) has been implicated in various upper aerodigestive tract and laryngeal diseases, the underlying mechanisms remain elusive. In this study, we investigated the role of gastric acidified pepsin in laryngeal precancerosis. Results: Acidified pepsin (pH=3) enhanced the growth and survival of mouse laryngeal epithelial cells in vitro and promoted laryngeal mucosal thickening and laryngeal epithelial cell growth in vivo. Furthermore, acidified pepsin promoted autophagy/mitophagy induction, accompanied by a significant decrease in mitochondrial membrane potential (MMP). Inhibition of autophagy by chloroquine abolished the ability of acidified pepsin to promote mitophagy and cell growth in laryngeal epithelial cells. Additionally, chloroquine promoted cell apoptosis and further reduced MMP in laryngeal epithelial cells treated with acidified pepsin. The expression levels of pepsin and H+/K+-ATPase α and β subunits in 31 human laryngeal mucosa specimens were 51.6%, 48.4%, and 48.4%, respectively. Importantly, the pepsin level was correlated with the H+/K+-ATPase β subunit level. H+/K+-ATPase upregulation in laryngeal epithelial cells in response to acidified pepsin was essential for the mitophagy-promoting effect of acidified pepsin. H+/K+-ATPase knockout or inhibition further reduced MMP in the presence of acidified pepsin. Conclusions: Our findings suggest that in an acidic environment, pepsin promotes laryngeal epithelial cell growth and survival by upregulating H+/K+-ATPase and activating mitophagy, potentially leading to laryngeal precancerosis.

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Acidified Pepsin Promotes Laryngeal Precancerosis by Upregulating H+/K+-ATPase and Activating Mitophagy in Laryngeal Epithelial Cells

10.21203/rs.3.rs-117704/v1 ◽

2020 ◽

Author(s):

Ke-Jia Cheng ◽

Qiong Xu ◽

Zhi-Mei Li ◽

Shui hong Zhou ◽

Yang-Yang Bao ◽

...

Keyword(s):

Cell Growth ◽

Epithelial Cell ◽

Epithelial Cells ◽

Laryngopharyngeal Reflux ◽

Upper Aerodigestive Tract ◽

Growth And Survival ◽

Laryngeal Mucosa ◽

Α And Β Subunits

Abstract Background: Although laryngopharyngeal reflux (LPR) has been implicated in various upper aerodigestive tract and laryngeal diseases, the underlying mechanisms remain elusive. In this study, we investigated the role of gastric acidified pepsin in laryngeal precancerosis. Methods: The in vitro and in vivo effects of acidified pepsin on H+/K+-ATPase expression and autophagy/mitophagy induction in mouse laryngeal epithelial cells were assessed by hematoxylin and eosin staining, immunohistochemistry, CCK-8 assay, flow cytometry, Western blotting, and quantitative real-time PCR. Additionally, the levels of pepsin and H+/K+-ATPase α and β subunits in 31 human laryngeal mucosal specimens were assessed by immunohistochemical staining. Results: Acidified pepsin (pH=3) enhanced the growth and survival of mouse laryngeal epithelial cells in vitro and promoted laryngeal mucosal thickening and laryngeal epithelial cell growth in vivo. Furthermore, acidified pepsin promoted autophagy/mitophagy induction, accompanied by a significant decrease in mitochondrial membrane potential (MMP). Inhibition of autophagy by chloroquine abolished the ability of acidified pepsin to promote mitophagy and cell growth in laryngeal epithelial cells. Additionally, chloroquine promoted cell apoptosis and further reduced MMP in laryngeal epithelial cells treated with acidified pepsin. The expression levels of pepsin and H+/K+-ATPase α and β subunits in 31 human laryngeal mucosa specimens were 51.6%, 48.4%, and 48.4%, respectively. Importantly, the pepsin level was correlated with the H+/K+-ATPase β subunit level. H+/K+-ATPase upregulation in laryngeal epithelial cells in response to acidified pepsin was essential for the mitophagy-promoting effect of acidified pepsin. H+/K+-ATPase knockout or inhibition further reduced MMP in the presence of acidified pepsin. Conclusions: Our findings suggest that in an acidic environment, pepsin promotes laryngeal epithelial cell growth and survival by upregulating H+/K+-ATPase and activating mitophagy, potentially leading to laryngeal precancerosis.

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Electron Microscopic Study of the Epithelium of the Seminal Vesicle of Aging Rats

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100050706 ◽

1974 ◽

Vol 32 ◽

pp. 138-139

Author(s):

V. F. Allison ◽

G. C. Fink ◽

G. W. Cearley

Keyword(s):

Cell Growth ◽

Epithelial Cell ◽

Epithelial Cells ◽

Seminal Vesicle ◽

Seminal Vesicles ◽

Epithelial Layer ◽

Epithelial Hyperplasia ◽

Electron Microscopic ◽

Aging Rats ◽

Pseudostratified Epithelium

It is well known that epithelial hyperplasia (benign hypertrophy) is common in the aging prostate of dogs and man. In contrast, little evidence is available for abnormal epithelial cell growth in seminal vesicles of aging animals. Recently, enlarged seminal vesicles were reported in senescent mice, however, that enlargement resulted from increased storage of secretion in the lumen and occurred concomitant to epithelial hypoplasia in that species.The present study is concerned with electron microscopic observations of changes occurring in the pseudostratified epithelium of the seminal vescles of aging rats. Special attention is given to certain non-epithelial cells which have entered the epithelial layer.

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Optimized culture system to maximize ovarian cell growth and functionality in vitro

Cell and Tissue Research ◽

10.1007/s00441-021-03415-w ◽

2021 ◽

Author(s):

Myung Jae Jeon ◽

Young Sik Choi ◽

James J. Yoo ◽

Anthony Atala ◽

John D. Jackson

Keyword(s):

Cell Growth ◽

Culture System ◽

Ovarian Cell

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c-Src inactivation reduces renal epithelial cell-matrix adhesion, proliferation, and cyst formation

AJP Cell Physiology ◽

10.1152/ajpcell.00163.2010 ◽

2011 ◽

Vol 301 (2) ◽

pp. C522-C529 ◽

Cited By ~ 38

Author(s):

Justine Elliott ◽

Nadezhda N. Zheleznova ◽

Patricia D. Wilson

Keyword(s):

Cell Proliferation ◽

Epithelial Cell ◽

Epithelial Cells ◽

Collecting Duct ◽

Specific Inhibitor ◽

Cell Phenotype ◽

Epithelial Cell Proliferation ◽

Matrix Adhesion ◽

Cyst Lining

c-Src is a non-receptor tyrosine kinase whose activity is induced by phosphorylation at Y418 and translocation from the cytoplasm to the cell membrane. Increased activity of c-Src has been associated with cell proliferation, matrix adhesion, motility, and apoptosis in tumors. Immunohistochemistry suggested that activated (pY418)-Src activity is increased in cyst-lining autosomal dominant polycystic kidney disease (ADPKD) epithelial cells in human and mouse ADPKD. Western blot analysis showed that SKI-606 (Wyeth) is a specific inhibitor of pY418-Src without demonstrable effects on epidermal growth factor receptor or ErbB2 activity in renal epithelia. In vitro studies on mouse inner medullary collecting duct (mIMCD) cells and human ADPKD cyst-lining epithelial cells showed that SKI-606 inhibited epithelial cell proliferation over a 24-h time frame. In addition, SKI-606 treatment caused a striking statistically significant decrease in adhesion of mIMCD and human ADPKD to extracellular collagen matrix. Retained viability of unattached cells was consistent with a primary effect on epithelial cell anchorage dependence mediated by the loss of extracellular matrix (ECM)-attachment due to α2β1-integrin function. SKI-606-mediated attenuation of the human ADPKD hyperproliferative and hyper-ECM-adhesive epithelial cell phenotype in vitro was paralleled by retardation of the renal cystic phenotype of Pkd1 orthologous ADPKD heterozygous mice in vivo. This suggests that SKI-606 has dual effects on cystic epithelial cell proliferation and ECM adhesion and may have therapeutic potential for ADPKD patients.

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Cell cycle regulation of mammary epithelial cell detachment byStaphylococcus aureus

Journal of Dairy Research ◽

10.1017/s0022029900032088 ◽

1996 ◽

Vol 63 (4) ◽

pp. 543-553 ◽

Cited By ~ 1

Author(s):

Boris Zavizion ◽

Andrew J. Bramley ◽

Ioannis Politis

Keyword(s):

Cell Cycle ◽

Epithelial Cells ◽

Culture Medium ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Cell Detachment ◽

Staph Aureus ◽

Bovine Mammary Epithelial Cells ◽

Isogenic Mutants ◽

Cycle Regulation

SummaryThe effect ofStaphylococcus aureuson detachment of bovine mammary epithelial cells in culture was examined. Mammary epithelial cells became detached from fresh monolayers following a 3 h incubation in the presence ofStaph. aureusM60. Two different procedures indicated that cell detachment coincided with the S-phase of the cell cycle. The roles of proteinases, toxins and Ca availability in inducing cell detachment were examined. Addition of the proteinase inhibitor phenyl-methylsulphonyl fluoride (1 mM) to the culture medium prevented cell detachment. Addition of a combination of purified staphylococcal proteinases XVI and XVII-B to the culture medium of mammary epithelial cells induced cell detachment in the absence ofStaph. aureus. Cell detachment may be caused by a staphylococcal proteinase. However, addition of Ca (10 mM) to the culture medium abolishedStaph. aureus-induced cell detachment, despite the fact that proteinase activity was still apparently present. Isogenic mutants ofStaph. aureusM60, expressing either ± or β toxins but not both, induced cell detachment, but to a lesser extent than the wild type. Thus, Ca and toxins play some role during cell detachment. Clones established from detached cells that were washed and replated showed the same susceptibility toStaph. aureus-induced cell detachment as the parental cells. This indicated that there is no subclone of mammary epithelial cells more sensitive to this effect.

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A Dual Role for Oncostatin M Signaling in the Differentiation and Death of Mammary Epithelial Cells in Vivo

Molecular Endocrinology ◽

10.1210/me.2008-0097 ◽

2008 ◽

Vol 22 (12) ◽

pp. 2677-2688 ◽

Cited By ~ 45

Author(s):

Paul G. Tiffen ◽

Nader Omidvar ◽

Nuria Marquez-Almuina ◽

Dawn Croston ◽

Christine J. Watson ◽

...

Keyword(s):

Gene Expression ◽

Epithelial Cell ◽

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Oncostatin M ◽

Specific Gene ◽

Mammary Involution

Abstract Recent studies in breast cancer cell lines have shown that oncostatin M (OSM) not only inhibits proliferation but also promotes cell detachment and enhances cell motility. In this study, we have looked at the role of OSM signaling in nontransformed mouse mammary epithelial cells in vitro using the KIM-2 mammary epithelial cell line and in vivo using OSM receptor (OSMR)-deficient mice. OSM and its receptor were up-regulated approximately 2 d after the onset of postlactational mammary regression, in response to leukemia inhibitory factor (LIF)-induced signal transducer and activator of transcription-3 (STAT3). This resulted in sustained STAT3 activity, increased epithelial apoptosis, and enhanced clearance of epithelial structures during the remodeling phase of mammary involution. Concurrently, OSM signaling precipitated the dephosphorylation of STAT5 and repressed expression of the milk protein genes β-casein and whey acidic protein (WAP). Similarly, during pregnancy, OSM signaling suppressed β-casein and WAP gene expression. In vitro, OSM but not LIF persistently down-regulated phosphorylated (p)-STAT5, even in the continued presence of prolactin. OSM also promoted the expression of metalloproteinases MMP3, MMP12, and MMP14, which, in vitro, were responsible for OSM-specific apoptosis. Thus, the sequential activation of IL-6-related cytokines during mammary involution culminates in an OSM-dependent repression of epithelial-specific gene expression and the potentiation of epithelial cell extinction mediated, at least in part, by the reciprocal regulation of p-STAT5 and p-STAT3.

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