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BOR - Papers in Press, published online ahead of print September 1, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.032144
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BIOLOGY OF REPRODUCTION 72, 200–205 (2005)
DOI: 10.1095/biolreprod.104.032144
© 2005 by the Society for the Study of Reproduction, Inc.

Involvement of the Cytoskeleton in Oxytocin Secretion by Cultured Bovine Luteal Cells1

Masami Shibaya3, Katarzyna M. Deptula4, Anna Korzekwa4, Kiyoshi Okuda3, and Dariusz J. Skarzynski2,4

Laboratory of Reproductive Endocrinology,3 Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan Division of Reproductive Endocrinology and Pathophysiology,4 Institute of Animal Reproduction & Food Research, PAS, Olsztyn 10-747, Poland


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A number of substances have been implicated in the regulation of oxytocin (OT) secretion from bovine corpus luteum in vivo. However, isolated bovine luteal cells cultured in a monolayer lose the ability to secrete OT in response to stimulatory substances. The present study investigated how cell-to-cell contact and the cytoskeleton affect OT secretion by isolated bovine luteal cells. In experiment 1, bovine midluteal cells (Days 8–12 of the estrous cycle) were stimulated with prostaglandin F2{alpha} (PGF2{alpha}; 1 µM), noradrenaline (NA; 10 µM), or growth hormone (GH; 5 nM) in two culture systems: In one system, cell monolayers were incubated in 24-well culture plates, and in the other system, aggregates of cells were incubated in glass tubes in a shaking water bath. The cells cultured in a monolayer underwent considerable spreading and showed a variety of shapes, whereas the cells cultured in glass tubes remained fully rounded during the experimental period and soon formed aggregates of cells. Although PGF2{alpha}, NA, and GH did not stimulate OT secretion by the monolayer cells, all tested substances stimulated OT secretion by the aggregated cells (P < 0.01). In experiment 2, the monolayer cells were pre-exposed for 1 h to an antimicrofilament agent (cytochalasin B; 1 µM) or two antimicrotubule agents (colchicine or vinblastine; 1 µM) before stimulation with PGF2{alpha}, NA, or GH. Although PGF2{alpha}, NA, and GH did not stimulate OT secretion by the monolayer cells in the presence of colchicine or vinblastine, they all stimulated OT secretion in the presence of cytochalasin B (P < 0.001). The overall results show that OT secretion by bovine luteal cells depends on microfilament function and cell shape. Moreover, the aggregate culture system that allows three-dimensional, cell-to-cell contact seems to be a good model for studying OT secretion by isolated bovine luteal cells.

corpus luteum, corpus luteum function, ovary, oxytocin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bovine corpora lutea (CL) synthesize oxytocin (OT) [1, 2]. In bovine CL, the highest concentrations of OT mRNA and OT were found in the early luteal phase and the midluteal phase, respectively [1, 3]. Prostaglandin F2{alpha} (PGF2{alpha}) and catecholamines, including noradrenaline (NA), stimulate OT secretion from the bovine CL in vivo throughout the whole luteal phase [4–6]. Growth hormone (GH) also was found to enhance OT secretion from bovine CL with an in vitro microdialysis system [7]. It is interesting that neither PGF2{alpha} nor NA stimulates OT secretion in cultured bovine luteal cells (monolayer culture systems) [8, 9], even though PGF2{alpha} [10] and NA [11] stimulate the secretion of OT from incubated bovine luteal slices. Therefore, it has been assumed that isolated midluteal cells lose most of their OT in the process of cell separation and/or during long-term monolayer culture [8, 10].

A possible reason for the failure of cells in a monolayer to secrete OT in response to stimulants is the absence of cell-to-cell contact [12, 13]. Moreover, the possible relationship between cytoskeletal function and steroidogenesis has been reported in cultured bovine granulosa cells [14] and bovine and porcine luteal cells [15, 16]. The cytoskeleton consists of a number of proteins that polymerize and connect with other proteins to form a complex, three-dimensional network that runs from the plasma membrane to the chromosomes in the nucleus [17]. The major functions of the cytoskeleton are to keep a variety of cellular components in place, to transport secretory granules, and to stabilize the shape of the cell [17]. The cytoplasmic skeleton consists of microfilaments, microtubules, and intermediate filaments. Microfilaments participate in biosynthetic processes and also have been implicated in the secretion of protein and steroid hormones [17–19]. Exposure of luteal tissue [15] and luteal cells [16] to an antimicrofilament agent (cytochalasin B) has been shown to suppress basal and gonadotropin-induced progesterone (P4) secretion. Microtubules also are present in ovarian cells and have been implicated in hormone production [19, 20]. Inhibitors of microtubules (vimentin and colchicine) have been demonstrated to increase steroidogenesis in porcine theca cells [21], rat granulosa cells [14], and porcine CL cells [16]. Thus, OT production in cultured bovine luteal cells may depend on the function of the cytoskeleton as well.

The main objective of the present study was to determine an appropriate culture system for studying OT secretion by isolated bovine luteal cells. We also determined whether cell-to-cell contact between luteal cells is involved in OT secretion by the cells in response to PGF2{alpha}, NA, and GH, and we examined the possible relationship between cytoskeletal function and OT secretion in cultured bovine luteal cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Experiments

Ovaries with CL were collected from Holstein cows at a slaughterhouse within 5 min after exsanguination, immediately submerged in ice-cold physiological saline, and transported to the laboratory. The luteal stage of the estrous cycle was defined by macroscopic observation of the ovaries and the uterus [22]. Enzymatic dissociation of the luteal tissue and culture of luteal cells (Days 8–12 of the estrous cycle) were performed as previously described [23]. Cell viability was higher than 90% as assessed by trypan blue exclusion. The cells were adjusted to 2.5 x 105 viable cells/ml of culture medium: Dulbecco modified Eagle medium and Ham F-12 medium (D/F medium; 1:1 [v:v]; catalog no. D-8900; Sigma) containing 5% calf serum (catalog no. C-6278; Sigma) and 20 µg/ml of gentamicin (catalog no. 600-5750AD; Gibco Laboratories). The obtained cell suspension contained few endothelial cells and fibrocytes (5–10%), no erythrocytes, and approximately 20% large and 70% small luteal cells [24]. The cells were stimulated simultaneously with various reagents as described below.

Experiment 1 The purpose of experiment 1 was to evaluate the influence of PGF2{alpha}, NA, and GH on OT secretion by luteal cells incubated in two different culture systems. Four separate experiments were conducted in triplicate. In each experiment, dispersed luteal cells were obtained from two to three CL. Midluteal cells (2.5 x 105 cells/ml) were preincubated in glass tubes in 2 ml of culture medium or in 24-well culture plates (1 ml) for 20 h. After preincubation, the medium was replaced by fresh medium: D/F medium supplemented with 0.1% bovine serum albumin (catalog no. 735078; Boehringer Mannheim GmbH, Mannheim, Germany), 0.5 mM ascorbic acid (catalog no. 013-12061; Wako Pure Chemical Industries Ltd., Osaka, Japan), 5 ng/ml of sodium selenite (catalog no. S-5261; Sigma), 5 µg/ml of holo-transferrin (catalog no. T-3400; Sigma), and 20 µg/ml of gentamicin (the glass tubes ware centrifuged for 5 min at 50 x g, and the medium was changed). The cells were then stimulated for 4 h with PGF2{alpha} (1 µM), NA (10 µM), GH (5 nM), or bovine LH (USDA-bLH-B-6; 100 ng/ml) as a control to confirm proper cell functionality. All treatments were conducted in triplicate. The cell number and the doses of reagents chosen in the present study were based on data from previous experiments [24, 25]. After sampling, the cultured medium was stored at –30°C until the concentrations of OT and P4 were determined. The viability of the cells after culture in the glass tubes and in the plates was assessed by trypan blue exclusion.

Experiment 2 The purpose of experiment 2 was to determine the influence of cell shape and cytoskeleton-disrupting drugs on OT secretion by monolayer-cultured bovine luteal cells. Dispersed luteal cells from Days 8–12 of the estrous cycle were incubated for 19 h in 48-well culture plates. After cell attachment, culture media were replaced by fresh culture medium (the same medium as used in experiment 1), and the cells were exposed or not exposed to an antimicrofilament agent (cytochalasin B; 1µM; Cytochalasin B Helminthosporium dematioideum; catalog no. C-6762; Sigma) or two antimicrotubule agents, colchicine (1 µM; catalog no. C-9754; Sigma) and vinblastine (1 µM; catalog no. C-1377; Sigma). The dose of reagents, dissolving vehicle (dimethyl sulfoxide), and manner of cell stimulation were based on previous studies [15, 16, 21]. After a 1-h incubation, the cells were additionally stimulated with PGF2{alpha} (1 µM), NA (10 µM), or GH (5 nM). All treatments were conducted in triplicate. After an additional 4-h incubation, culture media were collected and stored at –30°C until the concentration of OT was determined. The viability of the cells after treatment with antimicrofilament or antimicrotubule agents in a monolayer-cultured cell was assessed by 3-[4, 5-dimethylthiazol-2yl]2,5-diphenyltetrazolium bromide (MTT) assay.

MTT Assay

The viability of the cells was determined by TOX-1 Kit including MTT (catalog no. TOX-1; Sigma). The MTT is a yellow tetrazolium salt that is reduced to formazan crystals, which are insoluble in aqueous solutions by live cells containing active mitochondria. The crystals are dissolved in acidified isopropanol. The culture medium was replaced with 100 µl of D/F medium without phenol red, and 10 µl of MTT were added to each well. The cells were then incubated for 4 h at 37°C. Next, MTT Solubilization Solution (100 µl; Tox-1, Sigma) was added to each well, and the absorbance (A) was read at 450 nm using a microplate reader (model 450; Bio-Rad, Hercules, CA). Cell viability was determined by dividing the mean A of the treated wells (Atest) by the mean A of the nontreated wells (Acontrol). The mean A of wells without the cells was subtracted from the mean A of all experimental wells. The cell viability (%) was calculated as follows:

Hormone Determination

The P4 in the culture media was measured with a direct enzyme immunoassay (EIA) as described previously [26]. Antiserum of P4 (IFP-4; kindly donated by Dr. S. Okrasa of the University of Warmia and Mazury, Olsztyn, Poland) was used at a final dilution of 1:100 000. The standard curve ranged from 0.39 to 100 ng/ml, and the effective dose for 50% inhibition (ED50) of the assay was 4.5 ng/ml. The intra- and interassay coefficients of variation were 5.5% and 8.5%, respectively.

The EIA for OT was based on the second antibody method using the biotin-streptavidin-peroxidase technique as described previously [24]. Anti-rabbit OT serum (R-1; kindly donated by Dr. G. Kotwica of the University of Warmia and Mazury, Olsztyn, Poland) was used at a final dilution of 1:150 000. The standard curve ranged from 3.91 to 1000 pg/ ml, and the ED50 of the assay was 39.2 pg/ml. The intra- and interassay coefficients of variation were 6.9% and 10.4%, respectively.

Statistical Analysis

Data are presented as the mean ± SEM of four separate experiments, each performed in triplicate. The statistical significance of differences between controls and treated groups was assessed by one-way ANOVA followed by the Bonferroni multiple-comparison test (GraphPad Prism software; GraphPad Software, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1: Secretion of OT and P4 by Bovine Luteal Cells Incubated in Two Different Culture Systems

The PGF2{alpha}, NA, and GH did not stimulate OT secretion (P > 0.05) from monolayer-cultured bovine luteal cells (Fig. 1a). However, when aggregates of cells were incubated in glass tubes, PGF2{alpha}, NA, and GH augmented OT secretion (P < 0.001) to approximately 280%, 320%, and 370% of the baseline, respectively (Fig. 1a). On the other hand, PGF2{alpha}, NA, and GH stimulated P4 production by the cells cultured in both systems (P < 0.001) (Fig. 1b). The viabilities of the cells after culture in the glass tubes and in the plates were similar (~97.6%).



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  		FIG. 1. Secretion of oxytocin (a) and progesterone (b) by bovine luteal cells obtained from the midluteal stage (Days 8–12) of the estrous cycle (mean ± SEM; n = 4 experiments). The cells were preincubated for 20 h in monolayer culture with 24-well culture plates (white bars) or in the aggregate culture in a shaking water bath (gray bars). Finally, the cells were exposed for 4 h to PGF2{alpha} (1 µM), NA (10 µM), and GH (5 nM). Different superscript letters indicate significant differences (P < 0.05)

The cells cultured in a monolayer underwent considerable spreading and showed a variety of shapes (Fig. 2a). The cytoplasm of these cells often extended two or more projections. In contrast, the cells cultured in glass tubes remained fully rounded during the experimental period and soon formed aggregates of cells (Fig. 2b). These cells were regular in outline, and cytoplasm extension was short and inconspicuous.



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  		FIG. 2. Bovine midluteal cells cultured for total 24 h (a) in 48-well culture plates (monolayer culture) and (b) in glass tubes in a shaking water bath (aggregates of cells). Bars = 100 µm

Experiment 2: Influence of Cell Shape and Cytoskeleton-Modulating Drugs on OT Secretion by Monolayer-Cultured Bovine Luteal Cells

Although OT secretion was augmented by antimicrofilament and antimicrotubule agents compared with that in the nontreated groups, no response to any stimulant was observed in the presence of colchicine or vinblastine (P > 0.05) (Fig. 3, a and b). However, the stimulating effect of PGF2{alpha}, NA, and GH on OT secretion by monolayer-cultured cells was observed in the presence of an antimicrofilament agent, cytochalasin B (P < 0.001) (Fig. 3c).



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  		FIG. 3. Effects of the antimicrotubule agents (a) colchicine, (b) vinblastine, or an antimicrofilament agent (cytochalasin B; c) on oxytocin secretion by cultured midluteal cells. The cells were preincubated for 19 h in 48-well culture plates (monolayer culture system) and after a medium change were exposed for 1 h to anticytoskeletal drugs (1 µM). The cells were then exposed for an additional 4 h to PGF2{alpha} (1 µM), NA (10 µM), or GH (5 nM). The data were ascertained based on the cell viability (Control = 100% = 2.5 x 105 cells). Different superscript letters indicate significant differences (P < 0.05)

Colchicine and vinblastine did not influence cell viability compared to that in the control (P > 0.05). However, cytochalasin B significantly decreased cell viability (P < 0.05) to 75% of the control value.

As shown in Figure 4, the control, colchicine-treated, and vinblastine-treated cells cultured in a monolayer underwent considerable spreading and showed a variety of shapes (Fig. 4, a–c). However, the cells treated with cytochalasin B remained fully regular and rounded during the experimental period (Fig. 4, day).



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  		FIG. 4. a) Bovine luteal cells cultured for a total of 24 h (a) in 48-well culture plates (monolayer culture system). b–d) Effects of two antimicrotubule agents, (b) colchicine and (c) vinblastine, or an antimicrofilament agent, (d) cytochalasin B, on the cell shape of midluteal cells (Days 8–12 of the estrous cycle). The cells were preincubated for 19 h in a monolayer culture system and after a medium change were exposed to the anticytoskeletal drugs (1 µM) for 5 h. Bar = 100 µm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although the role, locus, and temporal pattern for secretion of ovarian OT have been extensively studied during the last two decades, the cellular mechanisms regulating the production and output of this peptide from luteal cells are still unclear. Luteal OT has been shown to have some roles in luteal development and maintenance [2, 23, 27] and in regression of bovine CL [28]. In addition, OT appears to have a systemic role in the control of bovine luteal function, in which a positive-feedback loop exists at luteolysis between luteal OT and endometrial PGF2{alpha} [28–31]. In fact, exogenous PGF2{alpha} has been shown to stimulate OT secretion from the bovine CL both in vivo [4, 6] and in vitro [10]. However, we did not find any changes in OT secretion after PGF2{alpha} treatment for monolayer-cultured luteal cells (Fig. 1a). This result is in agreement with those of Barrett and Wathes [8], in which PGF2{alpha} had no effect on OT secretion in bovine luteal cells cultured in a routine monolayer culture system. Therefore, it was concluded that isolated bovine luteal cells lose most of their OT in the process of separation and during long-term culture [10, 30].

We showed in the present study that PGF2{alpha} as well as NA and GH stimulated OT secretion from aggregates of bovine luteal cells cultured in a glass tube, although the effects of these substances were not observed in the monolayer culture system. In a previous study, Swann et al. [32] showed that isolated bovine and ovine luteal cells incorporated [S35]methionine into OT following a 7-h incubation. These findings demonstrate that isolated luteal cells are able to synthesize OT and that the lack of secretory response in the monolayer system is not caused by a loss of OT synthesis. The non-attached cells, cultured in glass tubes in a shaking water bath after 12–18 h, were confirmed to aggregate and keep contact with other cells. Therefore, one possibility for the lack of success of the monolayer culture model in the earlier studies of OT secretion may be the absence of three-dimensional, cell-to-cell contact. Cell-to-cell contact was shown to be necessary for regulating the secretory function of luteal cells (steroidogenesis) [12, 13], and contact between large and small bovine luteal cells during culture was found to promote P4 production [33]. Thus, the formation of cell aggregates in a glass tube allows three-dimensional contact between cells, which creates more physiological conditions for their function [34] and seems to be a good model for studying OT secretion by bovine luteal cells.

The stimulatory effects of PGF2{alpha}, NA, and GH on OT secretion were observed only in the cells cultured in glass tubes. These cells appeared rounded and almost normal. Based on these findings, we assume that the ability of bovine luteal cells to secrete OT is influenced mainly by the change of cell shape in vitro. In support of this idea, the possible relationship between cell shape, cytoskeleton, and hormone production has been reported in cultured bovine granulosa cells [14] and in bovine and porcine luteal cells [15, 16, 21, 34]. Carnegie and Tsang [14] have shown that bovine granulosa cells cultured in collagen-gel culture retained their physiological shape (rounded) compared to monolayer-cultured cells. Moreover, cells cultured in a collagen-gel system produced threefold more P4 than did cells cultured in a monolayer. However, in the present study, no differences were observed in P4 production (secretion) between the cells cultured in a monolayer system and those cultured in glass tubes. Our aggregate culture system may not be as effective in supporting steroidogenesis as a collagen-gel culture [14]. Taken together, these results suggest that an aggregate cell culture, which maintains natural cell shape, provides more physiological conditions for assessing the secretory response of luteal cells to stimulatory substances, although the steroidogenic ability does not differ between the cells in the two culture systems.

When luteal cells cultured in a monolayer were treated with two antimicrotubule agents (colchicine or vinblastine), the cells increased their OT secretion compared with that secretion of untreated controls, but OT secretion was not affected by PGF2{alpha}, NA, or GH treatment. In contrast, the cells cultured in the presence of cytochalasin B (an antimicrofilament agent) increased OT secretion in response to PGF2{alpha}, NA, and GH stimulation. These findings suggest that microfilaments, but not microtubules, play an important role in the response of OT secretion to stimulants. Cytochalasin B preferentially blocks elongation of F-actin [17, 35]. Thus, monolayer cells cultured in the presence of this antimicrofilament drug may retain a more physiological shape and cytoskeletal function by rearrangement of microfilaments in the monolayer culture [35]. The cytoskeleton is a dynamic, three-dimensional network, and each of the components is thought to associate with other components in an orderly fashion [17–19]. Therefore, the above results suggest that the regulation of OT secretion in bovine luteal cells depends on the proper function of microfilaments and the establishment of three-dimensional contacts.

Both microfilaments and microtubules participate in maintaining cell shape: Microfilaments are close to the plasma membrane, whereas microtubules are located around the centrosome, which is close to the nucleus [14, 17–19]. However, the function of the cytoskeleton is more than simply to maintain cell shape. It also has been implicated in a variety of cellular processes, such as cell motility, cell migration, intracellular transport of particles, spatial distribution of cell organelles, intracellular communication, and cellular responses to membrane events (exocytosis and endocytosis). Oxytocin is secreted by exocytosis, which involves transport of vesicles through a cytoskeletal matrix, including an actin cortex in close apposition with the plasma membrane [36–39]. Thus, to secrete OT in the luteal cells, the proper structure and function of microfilaments are required. The culture of cells in glass tubes may represent a more physiological approach to maintaining the appropriate actin cortex and microfilament structure. The integrity of the actin cortex is maintained by the cross-linking of actin filaments by a number of proteins, including myristoylated alanine-rich C kinase substrate (MARCKS) protein [39, 40]. Phosphorylation of MARCKS protein in response to PGF2{alpha} was shown to be correlated with the exocytosis of luteal OT in bovine CL [38, 39]. This suggested that the PGF2{alpha}-induced phosphorylation and translocation of MARCKS disrupts the F-actin-based cytoskeleton and leads to fusion of secretory vesicles with the plasma membranes, resulting in OT exocytosis [38, 39]. Our data clearly show that the monolayer-cultured cells secreted OT in response to PGF2{alpha}, NA, and GH treatment when they were treated with cytochalasin B. The actin filaments might be rearranged in these cells. Thus, the failure of monolayer cells to secrete OT in response to stimulating substances may be caused by the transformation of an F-actin-based cytoskeleton and disturbance of actin-dependent exocytosis mechanisms.

In conclusion, the present results indicate that OT secretion by the bovine luteal cells depends on proper function of microfilaments that may maintain cell shape. Moreover, the aggregate culture system that ensures a three-dimensional, cell-to-cell contact creates more physiological conditions for cell function and seems to be a good model for studying OT secretion by bovine luteal cells.


   ACKNOWLEDGMENTS
 
We thank Dr. Genowefa Kotwica and Dr. Stanislaw Okrasa of Warmia and Mazury University, Olsztyn, Poland, for OT and P4 antiserum, respectively. We thank Dr. A.F. Parlow of the National Hormone and Pituitary Program, University of Maryland School of Medicine, and the National Institute of Diabetes and Digestive and Kidney Disease for bovine LH (USDA-bLH-B6).


   FOOTNOTES
 
1 Supported by Grants-in-Aid for Scientific Research from the Polish Ministry of Scientific Research and Information Technology (PBZ-KBN-084/ P06/2002), the Japan Society for the Promotion of Sciences (JSPS; 14360168), and the Japanese-Polish Joint Research Project under an agreement between JSPS and the Polish Academy of Sciences. Back

2 Correspondence. FAX: 48 89 524 0347; skadar{at}pan.olsztyn.pl Back

Received: 13 May 2004.

First decision: 2 June 2004.

Accepted: 18 August 2004.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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K. Shirasuna, T. Shimizu, K.-G. Hayashi, K. Nagai, M. Matsui, and A. Miyamoto
Positive Association, in Local Release, of Luteal Oxytocin with Endothelin 1 and Prostaglandin F2alpha During Spontaneous Luteolysis in the Cow: A Possible Intermediatory Role for Luteolytic Cascade Within the Corpus Luteum
Biol Reprod, June 1, 2007; 76(6): 965 - 970.
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