Prostaglandin F2 Alpha Triggers the Disruption of Cell Adhesion with Cytokeratin and Vimentin in Bovine Luteal Theca Cells

Simple Summary Luteolysis is an important event in the control of the corpus luteum function in bovines. However, some aspects of the luteolytic mechanism remain unclear. We evaluated changes in cell adhesion in luteal cells during regression of corpus luteum. Bovine luteal theca cells (LTCs) were treated in vitro with Prostaglandin F2 alpha (PGF2α). Cytokeratin, vimentin and desmoplakin proteins in LTCs were disrupted by PGF2α, affecting cell adhesion. These results suggest that PGF2α plays an important function in cell adhesion during the regression of corpus luteum. Abstract Intermediate filaments (IFs) maintain cell–cell adhesions and are involved in diverse cellular processes such as cytokinesis, cell migration and the maintenance of cell structure. In this study, we investigated the influence of prostaglandin F2 alpha (PGF2α) on cytokeratin and vimentin IFs, Rho-associated protein kinase (ROCK), and cell-cell adhesion in bovine luteal theca cells (LTCs). The luteal cells were isolated from bovine corpus luteum (CL), and the LTCs were treated with 0, 0.01, 0.1 and 1.0 mM PGF2α. Cytokeratin, vimentin and desmoplakin proteins were disrupted and the ROCK protein was significantly increased in PGF2α-treated LTCs. In addition, cell–cell adhesion was significantly (p < 0.05) decreased in the PGF2α-induced LTCs compared to control group (0 mM PGF2α). In conclusion, PGF2α affected the adhesion of cell to cell via disruption of desmoplakin, cytokeratin and vimentin, additionally increasing ROCK in bovine LTCs. These results may provide a better understanding of the mechanism of bovine CL regression.


Introduction
The corpus luteum (CL) is a temporary endocrine organ that synthesizes progesterone (P4) to establish and maintain pregnancy, and it repeatedly undergoes formation (luteogenesis) and regression (luteolysis) during the estrous cycle [1]. Formation of the CL is initiated by the ovulatory surge of luteinizing hormone (LH) and proliferation and adhesion of ovary cells are actively accompanied by angiogenesis [1]. The CL is composed of a heterogeneous populations of cells that are largely classified into two categories according to function: luteal steroidogenic cells (LSCs) and non-LSCs such as luteal endothelial cells (LECs), pericytes, fibroblasts and lymphocytes [2]. Furthermore, the interaction of these cells is not only responsible for the steroidogenic functions, but also for maintaining tissue architecture by tight junctions, gap junctions, adherens junctions, and desmosomes [3][4][5][6]. Therefore, it is necessary to study cell-cell adhesion to understand maintenance and degradation in CL structure.
In general, LSCs are classified as large and small according to their size, with large LSCs originating from granulosa cells (GCs), whereas thecal cells (TCs) differentiate into PGF2α on caspase activation in bovine LSCs and the relation between apoptosis and IF disruption in various mammalian cells have been widely reported; however, the role of PGF2α on IFs and desmosomes has not yet been clearly demonstrated in bovine LSCs. Moreover, even though cytoskeleton disruption is closely associated with Rho/ROCK signaling, most studies regarding cytoskeleton disruption in bovine LSCs have focused on extrinsic and intrinsic apoptotic signaling cascades. From a histological perspective, the CL is composed of a heterogeneous cell mixture, and the LTCs are closely associated with adhesion of heterogeneous cells and connections between heterogeneous cells in the CL [37]. Therefore, this study investigated the influence of PGF2α on Rho/ROCK activities, IFs, desmosomes and cell-cell adhesion in bovine LTCs.

Experimental Design
To investigate influences of PGF2α on Rho/ROCK activities, IFs, desmosomes and cell-cell adhesion in bovine CLs, firstly, we observed the luteal cells in the secretionand regression-phases of CLs using histological assay. Secondly, the LTCs were isolated from secretion-phase CLs and thirdly we investigated mRNA regarding surface marker, steroidogenic, and cytoskeleton in the LTCs. Fourthly, we observed influence of the PGF2α on IFs (cytoskeleton and vimentin) using Immunofluorescence methods and, fifthly, we investigated change of the Rho and ROCK proteins in the PGF2α-induced LTCs to understand the Rho/ROCK relationship and factors regarding cell-cell adhesion. Next, we detected protein regarding desmosome (desmoplakin) in the PGF2α-induced LTCs, and finally investigated the capacity of the cell-cell adhesion of the PGF2α-induced LTCs.

Animal Ethics and Tissue Collection
All procedures involving animals were approved by the Kangwon National University Institutional Animal Care and Use Committee (KIACUC-09-0139). The CLs were collected from five slaughtered Korean Native Cattle heifers (28.4 ± 1.1 month) in the slaughterhouse (Jeail Industry, Hongcheon, Korea), ant transferred to the laboratory within 2 h at 4 • C. The CL samples were classified as 12-15 days (secretion-phase) and 18-20 days (regressionphase) after ovulation according to previous study [38]. Five secretion-phase CLs and five regression-phase CLs were used for the histological experiment, and another five secretion-phase CLs were only used to isolate LTCs.

Histological Analysis
The secretion-CLs (n = 5) and regression-phase CLs (n = 5) were fixed in 10% formalin (Sigma, St. Louis, MO, USA) for 24 h at room temperature (RT) after samples were dehydrated by ethanol and xylene, embedded in paraffin. Samples were sliced in 4 µm sections using a microtome, then were deparaffinized in xylene and rehydrated in ethanol solutions (100%, 90%, 80%, and 70%) for 5 min per each step. The samples were stained in hematoxylin solution (Sigma) for 5 min, washed with distilled water for 10 min, and stained in EosinY solution (Sigma) for 1 min. Then samples were washed with distilled water, and dehydrated in ethanol (70%, 80%, 90% and 100%) and 100% xylene for 5 min per each step. The slides were mounted with Histomount Mounting Solution (Thermo Fisher, Waltham, MA, USA), then evaluated using microscope (BX-50, Olympus, Tokyo, Japan).

Isolation of LTCs
The GCs were isolated to compare steroidogenic function with LTCs from large follicles of ovaries (n = 5) and LSCs and LECs were collected from secretion-phase CLs (n = 5) [39,40]. The secretion-phase CL tissues were chopped and enzymatically dissociated to isolate mixed luteal cells (LCs) in Dulbecco Modified Eagle medium (DMEM; Sigma) containing 0.65 mg/mL collagenase A (Thermo Fisher), 50 U/mL DNase I (MGmed, Seoul, Republic of Korea) and 0.1% BSA at 30 • C for 90 min, then 10% (v/v) DMEM/Nutrient Mixture F12 (D/F12; Sigma) containing 10% fetal bovine serum (FBS; Welgene, Seoul, Republic of Korea) was mixed for 5 min. The suspensions were filtered through a cell strainer (100 µm pore size, Sigma), and washed three times using the D/F12 containing 10% FBS and 1% penicillin/streptomycin solution (P/S; Sigma) at 630 g, 440 g, and 330 g for 10 min at 4 • C. The LCs pellet was seeded in a culture dish (Nunc, Roskilde, Denmark) and cultured in D/F12 containing 10% FBS and 1% P/S (culture media) at 38.5 • C, 5% CO 2 . After 24 h seeding, attached LCs were used for experiment, then to isolate LTCs the LCs were seeded at 1.0 × 10 4 cells/mL in culture media at 38.5 • C, 5% CO 2 . After 24 h, LTCs colonies were separately collected using cloning cylinders (Thermo Fisher) according to mesenchymal cell morphology under microscope (TS-100, Nikon, Tokyo, Japan), then centrifuged at 400× g for 5 min; then cultivated LTCs were used for experimental PGF2α treatment.

Flow Cytometric Analysis
The LCs and LTCs were dissociated to single cells using 0.25% Trypsin-EDTA and the samples were centrifuged at 400× g for 5 min. A total of 1.0 × 10 6 cells was resuspended in 1 mL PBS containing 4% formaldehyde (Sigma) and incubated for 10 min at 38.5 • C, then samples were incubated for 1 min at 4 • C. The suspension was centrifuged at 400× g for 5 min, resuspended in 1 mL 90% methanol, incubated for 30 min at 4 • C, and we removed the 90% methanol at 400× g for 5 min. Then 1 mL PBS containing 1% BSA (PBS/BSA) was added in samples, and incubated for 30 min at 4 • C. Samples were centrifuged at 400× g for 5 min, then samples were incubated in 1 mL 3% BSA/PBS containing β-actin mouse monoclonal IgG (1:250, sc-47778, SCBT) for 1 h at RT, after being washed twice (400× g and 5 min) using 3% BSA/PBS solution. The samples were incubated in 1 mL 3% BSA/PBS containing mouse-IgGκ binding protein-conjugated to fluorescein (BP-FITC) (1:250, sc-516140, SCBT) for 30 min at RT in the dark room, after being washed twice using 1% BSA/PBS solution. The cells were resuspended in 1 mL PBS, and analyzed via flow cytometry (FACSCalibur, BD Bioscience, San Jose, CA, USA) using argon laser tuned to 488 nm. The morphological categorization in LCs was conducted according to subpopulation area via FSC and SSC dot-plot data only in β-actin positive cells. Then comparison of relative cellular size between red blood cells, LGCs, LTCs, LECs and others was analyzed using X-axis values peak in FSC histogram data, and all flowcytometric data were analyzed using CELLQuest software (BD Bioscience). The detecting methods of the LCs populations were as referred to in previous studies [9,10].

PGF2α Treatment in LTCs
LTCs were pre-incubated for 24 h, the cells washed twice with PBS containing 0.1% BSA and the medium was replaced with fresh medium containing phenol red free D/F12 (Sigma) supplemented with 0.1% BSA, 1% P/S solution, and treated with 0, 0.01, 0.1 and 1.0 mM PGF2α (sc-203219, SCBT, Santacruz, CA, USA) for 24 h. The minimum concentration of PGF2α to induce regression in bovine LTCs was determined according to previously studies [41,42].

Quantitative PCR
The cultivated GCs from follicles were collected using Trizol (Takara, Shiga, Japan) and LCs (Figure 1b), LTCs (Figure 1g), and LECs ( Figure 1f, yellow line area) from secretionphase CLs were also dissociated using TRIzol (Takara) from culture dishes. The part of the secretion-phase CLs, which were used to isolate LTCs, were gathered into Trizol (Takara) and these samples were used as positive control in the experimental steroidogenic verification. All processes of the extraction mRNA using Trizol (Takara) were followed according to product manual. The mRNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher). The mRNA was extracted using TRIzol, then total 5.0 µg mRNA was transcripted to cDNA using PrimScript 1st strand cDNA synthesis kit (Takara), and reverse transcription was performed at 45 • C for 60 min after 95 • C for 5 min. The 1.0 µL synthesized cDNA were used to conduct PCR according to the primer conditions (Table 1) using PCR premix kit (Bioneer, Seoul, Republic of Korea). The PCR products were separated with 2.0% aga-rose gel electrophoresis at 100 V for 20 min and visualized with ethidium bromide (Sigma) and UV light. The PCR product expression was analyzed with ImageJ software (NCBI, USA).

Western
Blotting 0 and 0.01 mM PGF2α-induced LTCs were washed three times using PBS and Mammalian Protein Extraction Reagent (Thermo Fisher) and resuspended for extraction of the proteins. All processes of protein extraction were followed according to the instructions of the manufacturer. The proteins (25 µg/20 µL) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 30 V for 20 min after 100 V for 90 min, transferred to a polyvinylidene difluoride (PVDF) membrane at 30 V for 180 min at 4 • C, and incubated in tris-buffered saline containing 5% skim milk and 0.5% Tween-20 (TBS-T) at RT for 60 min. The PVDF membranes were incubated with TBS-T with 1% BSA containing Rho (1:1000, sc-418, SCBT), ROCK (1:1000, sc-17794, SCBT), and β-actin (1:1000) mouse monoclonal IgG antibodies at 4 • C for overnight. The membranes were washed three times with TBS-T every 5 min and incubated with goat-anti-mouse IgG-HRP (1:5000, sc-2005, SCBT) for 1 h at RT after washing, then visualized using the West Save Enhanced Chemiluminescence kit (AbFrontier, Austin, TX, USA). Protein expression was measured using the EZ-Capture II system (ATTO, Tokyo, Japan) and protein band intensity was calculated using ImageJ software.

Immunofluorescence
The LTCs (1.0 × 10 4 cells/mL) were incubated in the culture media using a

Cell to Cell Adhesion Assay
The two cultivated dishes containing LTCs were prepared for cell to cell adhesion assay. After 24 h, one monolayer of LTC was washed with twice DMEM supplemented 0.1% BSA and another LTC was dissociated by 0.25% trypsin-EDTA which was incubated with DMEM supplemented 0.1% BSA and 1.0 µM Celltracker Red CMTPX (Thermo Fisher) for 30 min at 37 • C in the dark room. Then cell suspensions were washed twice with DMEM supplemented 0.1% BSA at 400× g for 5 min. Incubated LTCs with Celltracker Red were seeded on monolayer LTCs, then the cells were incubated with phenol free D/F12 supplemented 0.1% BSA, 1% P/S and 0.01 mM PGF2α. After 24 h, the LTCs were gently washed twice with DMEM supplemented 1% BSA and observed by fluorescence microscopy (BX-50, Olympus). The fluorescent LTC images were collected using digital camera system (EOS750D, Canon, Tokyo, Japan), the images were converted to 8 bit and controlled to detect red-stained LTCs by threshold filter. The red fluorescence stained cells were considered as attached LTCs on monolayer cells. The number of attached cells were counted using the analyze particles function of the ImageJ software.

Statistical Analysis
Data were analyzed using SAS ver. 9.4 software (SAS Institute, Cary, NC, USA). Data are presented as mean ± standard error mean. Intensity of the Rho/ROCK protein expression and number of attached LTCs were normalized to 0 mM PGF2α-induced group (control). The values of 0 mM PGF2α-induced group (n = 5) and 0.01 mM PGF2α-induced group (n = 5) were compared using Student's t-test. A p-value < 0.05 was considered statistically significant.

Location of LGCs and LTCs in CL Tissue
The morphology of secretion and regression phase bovine CL sections is shown in

Detection of the LTC Subpopulation by Flow Cytometry
The schema of LTC isolation and flow cytometric analysis are shown in Figure 1. Isolated LCs from secretion-phase CLs (Figure 1a) were attached in the culture dish (Figure 1b). The colony of LTCs (Figure 1f, white line area) was observed when seeding density of LCs was low (Figure 1f) and isolated LTCs, by cloning cylinder, were cultured in the form of a monolayer in the new culture dish (Figure 1g). The β-actin-positive LCs (Figure 1c,d red dots) were divided into three subpopulations: red blood cells (Figure 1d, yellow area), other luteal cells (Figure 1d, green area; LTCs, LECs, and other cells) and LGCs (Figure 1d, blue area). In contrast, β-actin-positive LTCs (Figure 1h,i, red dots) were detected in only one subpopulation (Figure 1i, black area). However, red blood cells also contain β-actin [43], and the ratio of the two, excluding for the red blood cell subpopulation, were 89.4 ± 1.2% (LTCs, LECs and other cells) and 10.6 ± 1.2% (LGCs) in the LC subpopulation (Figure 1e). In addition, the maximum peaks of the x-axis in one of the other luteal cells (Figure 1e, red arrow) and LTCs (Figure 1j, red arrow) were exactly matched in the FSC histogram.

TC Lineage, Steroidogenic and IF Marker Assays
The expression of luteinizing hormone receptor (LHR), fibroblast growth factor 7 (FGF7), FGF10, lysyl oxidase (LOX), and platelet-derived growth factor receptor alpha (PDGFRA) mRNA is shown in Figure 3a. The expression of LHR, FGF7, FGF10, LOX and PDGFRA mRNA were detected in LCs and LTCs, but expression of these mRNAs was not observed in GCs (Figure 3a). Steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNAs were expressed in LCs and LTCs, but not in LECs (Figure 3b). In addition, expression of StAR, P450scc and 3β-HSD mRNA were less in LTCs compared to LCs (Figure 3b). Cytokeratin and vimentin mRNAs were expressed in LCs, LTCs and LECs (Figure 3b).

The Distribution of Cytokeratin and Vimentin Proteins in PGF2α-Induced Bovine LTCs
The expressio of cytokeratin protein is shown in Figure 4. The cytokeratin proteins were disrupted in the 0.01 mM (Figure 4b,f), 0.1 mM (Figure 4c,g) and 1.0 mM (Figure 4d,h) PGF2α-inducedc LTC groups compared with those of the control group (0 mM PGF2α; Figure 4a,e). In addition, aggregated cytokeratin proteins were observed in PGF2α-induced LTCs (Figure 4f-h, yellow arrows). The vimentin proteins were normally expressed in bovine LTCs ( Figure 5); similar to cytokeratin, vimentin proteins in LTCs were also damaged by PGF2α (Figure 5b-d,f-h, white arrows), but aggregated vimentin protins were not detected in the control group (Figure 5a,e). Cytokeratin and vimentin proteins were normally distruibuted in the cytoplasm of bovine LTCs (Figures 4a and 5a).

Effect of PGF2α on Rho/ROCK and Desmoplakin Expression in Bovine LTCs
The influence of PGF2α on Rho and ROCK protein expression is shown in Figure 6. Rho protein was not significantly different between 0 and 0.01 mM PGF2α-induced LTCs, whereas ROCK protein was increased (p < 0.05) in bovine LTCs in the 0.01 mM PGF2α treatment group compared to that of the control group (0 mM PGF2α). The distribution of desmoplakin in bovine LTCs is shown in Figure 7. The desmoplakin proteins were normally arranged around the plasma membrane in LTCs (Figure 7c, white arrows). However, protein of desmoplakin was aggregated and irregularly observed in the 0.01 mM PGF2αinduced bovine LTCs (Figure 7d, yellow arrows).

Influence of PGF2α on the Cell-Cell Adhesion Ability in Bovine LTCs
The cell-cell adhesion ability of the PGF2α-induced bovine LTCs is shown in Figure 8. The 0 and 0.01 mM PGF2α-induced LTCs were attached to a monolayer of LTCs (Figure 8a,b,  red). In addition, the number of attached LTCs on monolayer cells was significantly decreased in 0.01 mM PGF2α-induced LTCs compared to that of untreated PGF2α LTCs (p < 0.05).  ((a,b), red dots) on the monolayer cells were counted using the segmentation tools of ImageJ, and the number of attached cells was normalized to 0 M PGF2α treatment (c). * p < 0.05, n = 5.

Discussion
It is well known that there are morphologically diverse LSCs and LECs in bovine CL, and the proportion the number of cells is larger for LTCs than LGCs, but the volume of LGCs is approximately 13 times larger than that of LTCs [2]. Based on these morphological characteristics of LCs, it was confirmed that large and small LSC cell populations were clearly divided according to dimeters in LCs isolated from sheep and cows [9,10]. Similarly, the results of flow cytometry in the current study showed that β-actin-positive LCs isolated from secretion-phase CLs were largely divided into three subpopulations. In addition, there were two FSC peaks for other luteal cells (LTCs, LECs and other cells), which suggests that other luteal cell subpopulations contain more than two types of cell. We also successfully isolated LTCs from LCs according to their morphology characteristics and confirmed that the maximum values of X-axis FSC peaks in LTCs and other cell subpopulations containing LTCs matched exactly. In addition, to determine the origins of GCs and TCs, LTC membrane surface markers [44] and TC-specific molecular markers such as FGF7 and FGF10 [45] were analyzed, and there was stronger mRNA expression in TCs and LTCs than in GCs and LGCs of genes such as LOX and PDGFRA [46]. Furthermore, the expression of steroidogenic factors (StAR, P450scc and 3β-HSD) in LTCs was lower than that in LGCs. The results of marker assay in this study shows that the LTCs originated from TCs and participate in less P4 production than LGCs, and the expression of cytokeratin and vimentin in bovine LTCs indicates that the IFs are present in LTCs. In general, the normal expression of vimentin in TCs from mesenchymal stem cells has been well known through other studies [16,47], whereas the study of cytokeratin in bovine LTCs has not been clearly demonstrated. Moreover, studies on cytokeratin protein in bovine LCs reported that cytokeratin 8/18 protein was normally expressed in LCs collected from bovine proliferation-and regression-phase CL tissues, and previous studies used a heterogeneous luteal cell mixture derived from bovine CL tissues [18,48]. Our study confirmed cytokeratin expression in bovine LTCs.
Direct degradation of LSCs is caused by cytokines, nitric oxide (NO), leukotriene C4, and endothelin-1, and mechanisms of bovine LSC degradation by direct PGF2α administration have not been clearly elucidated [33,49]. The reason for the lack of studies on LSC regression by direct PGF2α administration is that PGF2α is known to indirectly activate apoptosis of LSCs via activating tumor necrosis factor-α (TNFα), interferon-γ (IFNγ), NO and endothelin-1 of lymphocytes, macrophages and LECs [50]. In general, studies on the influence of PGF2α in bovine LSCs reported that 0.01 and 0.001 mM PGF2α increased P4 production in bovine LSCs, and 0.01 mM PGF2α is known to increase intracellular Ca 2+ (cellular secondary messenger) [41,42]. In summary, the previous studies of PGF2α-mediated effects on bovine LSCs mainly used PGF2α to induce LSC degeneration with minimum stimulation [41,42,[51][52][53][54]. Interestingly, treatment of bovine LSCs with 1.0 µg/mL synthetic PGF2α (2.8 µM dinoprost, 2.4 µM cloprostenol and 2.2 µM luprostiol) for 24 h resulted in decreased cell viability, increased DNA fragmentation, and caspase-3 activity [42], and a previous study means that a single PGF2α treatment directly affects bovine LTC degeneration. Therefore, based on previous studies regarding single PGF2α treatment of bovine LSCs, 0.01 mM (minimum concentration) was used for the present study to disrupt cell-cell adhesion in bovine LTCs.
To date, there have been no studies on the effects of PGF2α on IFs and desmosomes in bovine LSCs. Some studies on IFs in bovine LCs reported that cytokeratin 8/18 was decreased in the LCs derived from the regression-phase CL; additionally, Fas protein was increased in cytokeratin-positive LCs damaged by acrylamide [18,48]. Although the level of PGF2α is increased in regression-phase CL, this is still not enough to verify the relationship between PGF2α and IFs. Furthermore, changes in IF protein in LTCs were primarily demonstrated in this study, and LTCs also had depolymerized cytokeratin and vimentin IFs at concentrations over 0.01 mM PGF2α. The results showed that direct PGF2α can disrupt IFs in LTCs, even though it is not one of the cytokines, NO, leukotriene C4 and endothelin-1 that are known to cause apoptosis of LCs [33,49]. Thus, at concentrations over 0.01 mM PGF2α could directly regress bovine LTCs via disruption of cytokeratin and vimentin IFs, possibly controlling cellular signaling involved in the cytoskeleton.
Rho, one of the Ras superfamily members, activates ROCK, controls cytoskeletal organization, such as actin filaments and IFs, and it has been reported that PGF2α activates Rho protein signaling through PGF2αR, one of the GPCRs in HEK-293 cells; however, studies on bovine LCs have not yet been done. Our study focused on the Rho/ROCK signals that regulate IFs such as cytokeratin and vimentin in LTCs and confirmed that the IFs were disrupted when ROCK protein was increased. Thus, we determined that single administration of PGF2α (over 0.01 mM) directly increased ROCK and affected the disruption of cytokeratin and vimentin in LTCs, and we hypothesized that PGF2α may disconnect cell-cell desmosome adhesions.
The desmoplakin is an anchoring plaque connecting CAMs (desmocollin and desmoglein) and IFs and is an essential organelle for desmosomal adhesion [55]. Studies on desmoplakinnull cells have reported that desmoplakin plays a significant role in the connection between cytokeratin, vimentin, and cell adhesion in mammals [56,57]. Additionally, studies on the structure and adhesion of tumor cells through suppression of the desmosome system have been actively carried out in the oncology field [58]. Likewise, our study showed that cytokeratin and vimentin IFs were disrupted and that the desmoplakin arrangement was also disturbed by PGF2α in bovine LTCs. In addition, it was confirmed that LTC-LTC adhesion ability was also reduced by PGF2α; PGF2α had a negative influence on the cytoskeleton and cell-cell adhesion in bovine LTCs. Unfortunately, in the present study, we did not investigate the association between apoptotic signals and IF damage caused by PGF2α; however, we will investigate the relationship between Rho/ROCK, IFs, and cell-cell adhesion by PGF2α without apoptotic signals in bovine LTCs.
From a histological perspective, because LTCs exist between LGCs, LECs and other cells, we hypothesized that LTCs may be involved in the adhesion of heterogeneous cells in CL tissues and investigated the distribution of cytokeratin and vimentin IFs and desmoplakin protein by PGF2α-and Rho/ROCK-mediated changes in GPCR activation. Consequently, PGF2α not only activated ROCK but also disrupted IF proteins (cytokeratin and vimentin), desmoplakin and cell-cell adhesion. For several decades, even though apoptosis by cytokines, NO, leukotriene C4 and endothelin-1 have been the focus of structural regression of bovine CL.
Based on our results, we would like to suggest a study on G-protein activation via PGF2α-GPCR stimulation and disruption of cytoskeleton-cell adhesion in bovine CL. It is well known that the extrinsic and intrinsic apoptotic signaling cascades adversely affect small G-proteins and cause cytoskeleton disruption. Nevertheless, if the direct influence of PGF2α-induced small G-protein, cytoskeleton and cell-cell adhesion is more thoroughly studied in bovine LTCs, the results could be helpful in the study of cancer and tumor therapy regarding the disruption of the cytoskeleton and cell adhesion in ovarian tumors using selective cells.