- Email: [email protected]
Neuropeptide Y directly affects ovarian cell proliferation and apoptosis
reproductive biology 15 (2015) 257–260
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/repbio
Short Communication
Neuropeptide Y directly affects ovarian cell proliferation and apoptosis Alexander V. Sirotkin a,b,c,*, Diana Kardošová a, Saleh Hamad Alwasel c, Abdel Halim Harrath c a
Constantine The Philosopher University in Nitra, Nitra, Slovakia Research Institute of Animal Production Nitra, Lužianky, Slovakia c King Saud University, Department of Zoology, College of Science, Riyadh, Saudi Arabia b
article info
abstract
Article history:
The effects of neuropeptide Y (NPY; 0, 10, 100 and 1000 ng/mL) on the expression of PCNA,
Received 24 February 2015
bax and p53 were examined by immunocytochemistry in porcine luteinized granulosa cells.
Received in revised form
NPY inhibited proliferation as well as promoted apoptosis and accumulation of p53 in the
20 July 2015
cells. This is the first report to demonstrate the direct action of NPY on ovarian cell
Accepted 23 July 2015
proliferation and apoptosis. The results of the study suggest that the effect is mediated
Available online 4 August 2015
by transcription factor p53.
Keywords:
Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Sp. z o.o. All
# 2015 Society for Biology of Reproduction & the Institute of Animal Reproduction and Neuropeptide Y
rights reserved.
Ovary Proliferation Apoptosis Transcription factor p53
1.
Introduction
Neuropeptide Y (NPY) is an important but still not wellunderstood signalling molecule. It was initially discovered as a sympathetic neurotransmitter, but recently, the abundant expression of NPY and its receptors throughout the body has been reported). NPY was found to be involved in the control of a wide array of physiological processes including stress response, development, metabolism, thermogenesis, CNS, adipose tissue status, bone functions, inflammation, tissue repair [1,2] and reproduction [3].
There is a growing body of evidence indicating that NPY is implicated in the control of female reproduction. The action of NPY on rat GnRH neuron function, LH release and fecundity in vivo [3] as well as the fact that the intra-arterial NPY infusion promoted ovine ovarian oxytocin (but not progesterone [P4]) release [4] indicate that NPY can control reproductive processes at the hypothalamus level. In contrast, NPY was not able to alter gonadotropin release in rat [5] or bovine [6] cultured pituitary cells, suggesting that it does not affect reproduction at the pituitary level. On the other hand, Evans et al. [7] demonstrated the ability of NPY to augment basal, GnRH- and oxytocin-induced LH release by cultured rat
* Corresponding author at: Research Institute of Animal Production Nitra, Hlohovecka 2, 951 41 Lužianky, Slovakia. Tel.: +421 903561120. E-mail addresses: [email protected], [email protected] (A.V. Sirotkin). http://dx.doi.org/10.1016/j.repbio.2015.07.004 1642-431X/# 2015 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Sp. z o.o. All rights reserved.
258
reproductive biology 15 (2015) 257–260
pituitary cells. The presence of NPY in bison [8] and ovine [4] ovarian nerve fibres as well as in ovine blood vessels, follicular theca, granulosa cells and corpus luteum [4] may indicate direct autocrine–paracrine effects of NPY on ovarian function. This hypothesis was supported by finding NPY receptors in human ovarian cells [9]. Furthermore, the ability of NPY: 1/to promote P4 (but not testosterone [T] or estradiol [E2]) release by cultured rat ovarian cells [5], 2/to alter basal and gonadotropininduced P4, androstenedione and E2 release by cultured porcine granulosa and luteal cells [10] and 3/to promote basal and gonadotropin-induced E2 (but not P4) release by cultured human granulosa cells [11] has been reported. Nevertheless, no evidence for the direct influence of NPY on ovarian cell function has been reported. The aim of the current study was to examine the direct action of NPY on the presence of markers and/or regulators of ovarian cell proliferation and apoptosis in granulosa cells [12–15]. We have studied the effects of several NPY doses on the expression of PCNA (proliferation marker), bax (proapoptotic protein) and transcription factor p53 (an inhibitor of cell proliferation and promoter of apoptosis) proteins in cultured porcine granulosa cells.
2.
Material and methods
Ovaries of non-cycling prepubertal gilts (180 days-old) were obtained at a local slaughterhouse and processed as described previously [10,15]. Cell suspensions in 16-well chamber slides (Nunc Inc., International, Naperville, IL, USA, 200 mL/well, 106 cells/mL medium) were incubated at 37.5 8C in 5% CO2 in humidified air until a 60–75% confluent monolayer was formed (3–5 days), at which point the medium was renewed. Next, the cells were cultured in the absence or presence of NPY (Sigma– Aldrich, St. Louis, MO, USA) at concentrations of 10, 100 or 1000 ng/mL. After two days of culture, the medium was removed and the cells designated for immunocytochemistry (ICH) were washed in ice-cold PBS (pH 7.5), fixed in paraformaldehyde (4% in PBS, pH 7.2–7.4; 60 min) and kept at 4 8C. The cell viability was determined using Trypan blue (Sigma– Aldrich) staining and the number of cells were counted in a hemocytometer. Markers of proliferation (proliferating cell nuclear antigen, PCNA) and apoptosis (B cell lymphoma-associated X protein, bax and p53) were detected by ICH. After washing and fixation, the cells were incubated in the blocking solution (1% goat serum [Santa Cruz Biotechnology, Inc., Santa Cruz, USA] in phosphate-buffered saline [PBS]) at room temperature (RT) for 1 h to block the nonspecific binding of antiserum. Afterwards, the cells were incubated with monoclonal antibodies against either PCNA, bax or p53 (all from Santa Cruz Biotechnology, Inc., 1:500) for 2 h at RT. Thereafter the cells were incubated with secondary goat antibody against mouse IgG, labelled with horseradish peroxidase (Sevac, Prague, Czech Republic, 1:1000) for 1 h. Positive signals were visualized by staining with DABsubstrate (Roche Diagnostics GmbH, Manheim, Germany). Cells treated with secondary antibody and DAB but not with the primary antibody were used as negative controls. The quantification of molecules expression (percentage of cells containing visible PCNA, bax and p53) was performed by light microscopy. At least 10 optical fields and 1000 cells were
analyzed in each chamber. The percentage of cells containing antigen in the different groups of cells was calculated. Each experiment was repeated three times (8–14 animals per experiment). Each experimental group was represented by four chamber-slide wells. The program Sigma Stat/Sigma Plot 11.0 (Systat Software, GmbH, Erkhart, Germany) was used to examine the normal distribution of data. Differences between groups were evaluated using a one-way ANOVA followed by a paired Wilcoxon– Mann–Whitney test using the statistics software Sigma Stat/ Sigma Plot 11.0 (Systat Software, GmbH). Values represent the mean SEM. Differences were reported as statistically significant at p level less than 0.05 ( p < 0.05).
3.
Results and discussion
Immunocytochemical analysis revealed the presence of PCNA, bax and p53 in porcine luteinized ovarian granulosa cells. Trypan blue staining demonstrated high (70–80%) cell viability. No statistically significant differences in cell number and viability between control and experimental groups were observed. The relatively long incubation enabled the cells to undergo a spontaneous luteinization. Furthermore, it was found that NPY affected the number of cells expressing PCNA, bax and p53 (Fig. 1). In contrast to the highest dose of NPY (1000 ng/mL), the two lower doses (10 and 100 ng/mL) significantly reduced the percentage of cells containing PCNA (Fig. 1A). NPY at 10 and 100 ng/mL, but not at 1000 ng/mL, significantly increased the percentage of bax-positive cells (Fig. 1B). Significant increase in the proportion of cells containing p53 was observed after the treatment of the cells with 10 ng/mL of NPY. Higher doses of NPY (100 or 1000 ng/mL) did not affect the examined parameter (Fig. 1C). These findings suggest that NPY, when given at physiological doses, directly reduces PCNA expression and promotes bax expression in the granulosa cells, although during 2-day exposure, NPY did not affect the cell number yet. Balance between cell proliferation and apoptosis determined the fate of ovarian follicle – its selection, atresia, development and ovulation [15]. The anti-proliferative and pro-apoptotic action of NPY observed in our experiments is the first demonstration of the direct inhibitory action of NPY on ovarian follicular cells. The inhibitory action of NPY on reproduction at the level of hypothalamic gonadotropin-releasing hormone (GnRH) was reported previously. It has been proposed that NPY, the orexigenic peptide, is a mediator of the calorie intake/ metabolic state effect on reproduction at the level of the hypothalamus, but not at the level of the pituitary [3]. The presence of NPY not only in the hypothalamus but also in the ovary [4,8], the existence of NPY receptors in the ovary [9], as well as the ability of NPY to affect ovarian steroid hormone [5,10,11] and oxytocin [4] secretion suggest an direct effect of NPY on ovarian cell function. Our observations are the first to evidence the direct influence of NPY on ovarian cell proliferation and apoptosis. The data demonstrated that NPY can potentially inhibit reproductive functions not only at the central nervous system, but also at the gonadal level. The mechanism of direct NPY action on ovarian cells is almost unknown. The present data demonstrated that NPY
reproductive biology 15 (2015) 257–260
Percentage of cells expressing PCNA
A
demonstrated the up-regulation of p53 by NPY. Furthermore, the association between hormone- or transfection-induced p53 accumulation and expression of apoptosis markers in porcine ovaries has been observed in our previous [15] and present studies. Taken together, these data suggest that hormonal stimuli can suppress ovarian cell functions via promotion of p53, which in turn inhibits proliferation and promotes apoptosis. Recognition of the role of NPY in reproductive processes as well as the site and mechanism of NPY action requires further study. Our observations are the first to demonstrate the direct action of NPY on ovarian cell proliferation and apoptosis. In addition, we showed that this effect may be mediated by transcription factor p53.
80
70
60
50
* *
40
30
20
0
10
259
100
1000
NPY dose added (ng/mL medium)
Conflict of interest
Percentage of cells expressing bax
B 120
None declared.
* 100
*
80
Acknowledgments 60
40
20
0
0
10
100
1000
NPY dose added (ng/mL medium) Percentage of cells expressing p53
C 30
*
We would like to thank Mrs. Katarína Tóthová and Ing. Žofia Kuklová (Research Institute of Animal Production in Nitra, Lužianky, Slovakia) for technical assistance. This study was supported by the Operational Programme Research and Development funded from the European Regional Development Fund, project ‘‘ZDRAVIE’’ no. 26220220176. The authors would also like to extend their sincere appreciation to the Dean of Scientific Research at King Saud University for funding this Research NO (RG#164).
references
28 26 24 22 20 18 16 14 12
0
10
100
1000
NPY dose added (ng/mL medium) Fig. 1 – The effect of neuropeptide Y (NPY) on immunoexpression of PCNA (A), bax (B) and p53 (C) in porcine luteinized granulosa cells (n = 3). Data (mean W SEM) are presented as percentages of cells expressing PCNA, bax and p53. *Significant differences between the treatment groups (NPY: 0, 10, 100 or 1000 ng/mL); p < 0.05.
increased the number of cells expressing p53 protein. The transcription factor p53 is considered to be a suppressor of cell proliferation, a promoter of DNA repair and an inducer of apoptosis of non-ovarian [14] and ovarian [15] cells. We have
[1] Malva JO, Xapelli S, Baptista S, Valero J, Agasse F, Ferreira R, et al. Multifaces of neuropeptide Y in the brain– neuroprotection, neurogenesis and neuroinflammation. Neuropeptides 2012;46:299–308. [2] Shi YC, Baldock PA. Central and peripheral mechanisms of the NPY system in the regulation of bone and adipose tissue. Bone 2012;50:430–6. [3] Roa J. Role of GnRH neurons and their neuronal afferents as key integrators between food intake regulatory signals and the control of reproduction. Int J Endocrinol 2013;2013:518046. http://dx.doi.org/10.1155/2013/518046 [4] Keator CS, Custer EE, Hoagland TA, Schreiber DT, Mah K, Lawson AM, et al. Evidence for a potential role of neuropeptide Y in ovine corpus luteum function. Domest Anim Endocrinol 2010;38:103–14. [5] Baranowska B, Chmielowska M, Radzikowska M, Borowiec M, Roguski K, Wasilewska-Dziubinska E. Effects of neuropeptide Y (NPY), galanin and vasoactive intestinal peptide (VIP) on pituitary hormone release and on ovarian steroidogenesis. Neuro Endocrinol Lett 1999;20:385–9. [6] Denniston DJ, Thomas MG, Kane KK, Roybal CN, Canales L, Hallford DM, et al. Effect of neuropeptide Y on GnRHinduced LH release from bovine anterior pituitary cell cultures derived from heifers in a follicular, luteal or ovariectomized state. Anim Reprod Sci 2003;78:25–31. [7] Evans JJ, Pragg FL, Mason DR. Release of luteinizing hormone from the anterior pituitary gland in vitro can be
260
reproductive biology 15 (2015) 257–260
concurrently regulated by at least three peptides: gonadotropin-releasing hormone, oxytocin and neuropeptide Y. Neuroendocrinology 2001;73:408–16. [8] Skobowiat C, Panasiewicz G, Gizejewski Z, Szafranska B. Coexpression pattern of dopamine beta-hydroxylase (DbH) and neuropeptide Y (NPY) within sympathetic innervation of ovary and umbilical cord of the European bison (Bison bonasus L.). Tissue Cell 2013;45:402–6. [9] Jørgensen JC. Neuropeptide Y in mammalian genital tract: localization and biological action. Dan Med Bull 1994;41:294–305. [10] Pitzel L, Jarry H, Wuttke W. Effects of substance-P and neuropeptide-Y on in vitro steroid release by porcine granulosa and luteal cells. Endocrinology 1991;129:1059–65.
[11] Barreca A, Valli B, Cesarone A, Arvigo M, Balasini M, Battista La Sala G, et al. Effects of the neuropeptide Y on estradiol and progesterone secretion by human granulosa cells in culture. Fertil Steril 1998;70:320–5. [12] Hübscher U, Maga G. DNA replication and repair bypass machines. Curr Opin Chem Biol 2011;15:627–35. [13] Gillies LA, Kuwana T. Apoptosis regulation at the mitochondrial outer membrane. J Cell Biochem 2014;115:632–40. [14] Jackson JG, Post SM, Lozano G. Regulation of tissue- and stimulus-specific cell fate decisions by p53 in vivo. J Pathol 2011;223:127–36. [15] Sirotkin AV. Regulators of ovarian functions. New York, USA: Nova Science Publishers, Inc.; 2014. p. 194.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/repbio
Short Communication
Neuropeptide Y directly affects ovarian cell proliferation and apoptosis Alexander V. Sirotkin a,b,c,*, Diana Kardošová a, Saleh Hamad Alwasel c, Abdel Halim Harrath c a
Constantine The Philosopher University in Nitra, Nitra, Slovakia Research Institute of Animal Production Nitra, Lužianky, Slovakia c King Saud University, Department of Zoology, College of Science, Riyadh, Saudi Arabia b
article info
abstract
Article history:
The effects of neuropeptide Y (NPY; 0, 10, 100 and 1000 ng/mL) on the expression of PCNA,
Received 24 February 2015
bax and p53 were examined by immunocytochemistry in porcine luteinized granulosa cells.
Received in revised form
NPY inhibited proliferation as well as promoted apoptosis and accumulation of p53 in the
20 July 2015
cells. This is the first report to demonstrate the direct action of NPY on ovarian cell
Accepted 23 July 2015
proliferation and apoptosis. The results of the study suggest that the effect is mediated
Available online 4 August 2015
by transcription factor p53.
Keywords:
Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Sp. z o.o. All
# 2015 Society for Biology of Reproduction & the Institute of Animal Reproduction and Neuropeptide Y
rights reserved.
Ovary Proliferation Apoptosis Transcription factor p53
1.
Introduction
Neuropeptide Y (NPY) is an important but still not wellunderstood signalling molecule. It was initially discovered as a sympathetic neurotransmitter, but recently, the abundant expression of NPY and its receptors throughout the body has been reported). NPY was found to be involved in the control of a wide array of physiological processes including stress response, development, metabolism, thermogenesis, CNS, adipose tissue status, bone functions, inflammation, tissue repair [1,2] and reproduction [3].
There is a growing body of evidence indicating that NPY is implicated in the control of female reproduction. The action of NPY on rat GnRH neuron function, LH release and fecundity in vivo [3] as well as the fact that the intra-arterial NPY infusion promoted ovine ovarian oxytocin (but not progesterone [P4]) release [4] indicate that NPY can control reproductive processes at the hypothalamus level. In contrast, NPY was not able to alter gonadotropin release in rat [5] or bovine [6] cultured pituitary cells, suggesting that it does not affect reproduction at the pituitary level. On the other hand, Evans et al. [7] demonstrated the ability of NPY to augment basal, GnRH- and oxytocin-induced LH release by cultured rat
* Corresponding author at: Research Institute of Animal Production Nitra, Hlohovecka 2, 951 41 Lužianky, Slovakia. Tel.: +421 903561120. E-mail addresses: [email protected], [email protected] (A.V. Sirotkin). http://dx.doi.org/10.1016/j.repbio.2015.07.004 1642-431X/# 2015 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Sp. z o.o. All rights reserved.
258
reproductive biology 15 (2015) 257–260
pituitary cells. The presence of NPY in bison [8] and ovine [4] ovarian nerve fibres as well as in ovine blood vessels, follicular theca, granulosa cells and corpus luteum [4] may indicate direct autocrine–paracrine effects of NPY on ovarian function. This hypothesis was supported by finding NPY receptors in human ovarian cells [9]. Furthermore, the ability of NPY: 1/to promote P4 (but not testosterone [T] or estradiol [E2]) release by cultured rat ovarian cells [5], 2/to alter basal and gonadotropininduced P4, androstenedione and E2 release by cultured porcine granulosa and luteal cells [10] and 3/to promote basal and gonadotropin-induced E2 (but not P4) release by cultured human granulosa cells [11] has been reported. Nevertheless, no evidence for the direct influence of NPY on ovarian cell function has been reported. The aim of the current study was to examine the direct action of NPY on the presence of markers and/or regulators of ovarian cell proliferation and apoptosis in granulosa cells [12–15]. We have studied the effects of several NPY doses on the expression of PCNA (proliferation marker), bax (proapoptotic protein) and transcription factor p53 (an inhibitor of cell proliferation and promoter of apoptosis) proteins in cultured porcine granulosa cells.
2.
Material and methods
Ovaries of non-cycling prepubertal gilts (180 days-old) were obtained at a local slaughterhouse and processed as described previously [10,15]. Cell suspensions in 16-well chamber slides (Nunc Inc., International, Naperville, IL, USA, 200 mL/well, 106 cells/mL medium) were incubated at 37.5 8C in 5% CO2 in humidified air until a 60–75% confluent monolayer was formed (3–5 days), at which point the medium was renewed. Next, the cells were cultured in the absence or presence of NPY (Sigma– Aldrich, St. Louis, MO, USA) at concentrations of 10, 100 or 1000 ng/mL. After two days of culture, the medium was removed and the cells designated for immunocytochemistry (ICH) were washed in ice-cold PBS (pH 7.5), fixed in paraformaldehyde (4% in PBS, pH 7.2–7.4; 60 min) and kept at 4 8C. The cell viability was determined using Trypan blue (Sigma– Aldrich) staining and the number of cells were counted in a hemocytometer. Markers of proliferation (proliferating cell nuclear antigen, PCNA) and apoptosis (B cell lymphoma-associated X protein, bax and p53) were detected by ICH. After washing and fixation, the cells were incubated in the blocking solution (1% goat serum [Santa Cruz Biotechnology, Inc., Santa Cruz, USA] in phosphate-buffered saline [PBS]) at room temperature (RT) for 1 h to block the nonspecific binding of antiserum. Afterwards, the cells were incubated with monoclonal antibodies against either PCNA, bax or p53 (all from Santa Cruz Biotechnology, Inc., 1:500) for 2 h at RT. Thereafter the cells were incubated with secondary goat antibody against mouse IgG, labelled with horseradish peroxidase (Sevac, Prague, Czech Republic, 1:1000) for 1 h. Positive signals were visualized by staining with DABsubstrate (Roche Diagnostics GmbH, Manheim, Germany). Cells treated with secondary antibody and DAB but not with the primary antibody were used as negative controls. The quantification of molecules expression (percentage of cells containing visible PCNA, bax and p53) was performed by light microscopy. At least 10 optical fields and 1000 cells were
analyzed in each chamber. The percentage of cells containing antigen in the different groups of cells was calculated. Each experiment was repeated three times (8–14 animals per experiment). Each experimental group was represented by four chamber-slide wells. The program Sigma Stat/Sigma Plot 11.0 (Systat Software, GmbH, Erkhart, Germany) was used to examine the normal distribution of data. Differences between groups were evaluated using a one-way ANOVA followed by a paired Wilcoxon– Mann–Whitney test using the statistics software Sigma Stat/ Sigma Plot 11.0 (Systat Software, GmbH). Values represent the mean SEM. Differences were reported as statistically significant at p level less than 0.05 ( p < 0.05).
3.
Results and discussion
Immunocytochemical analysis revealed the presence of PCNA, bax and p53 in porcine luteinized ovarian granulosa cells. Trypan blue staining demonstrated high (70–80%) cell viability. No statistically significant differences in cell number and viability between control and experimental groups were observed. The relatively long incubation enabled the cells to undergo a spontaneous luteinization. Furthermore, it was found that NPY affected the number of cells expressing PCNA, bax and p53 (Fig. 1). In contrast to the highest dose of NPY (1000 ng/mL), the two lower doses (10 and 100 ng/mL) significantly reduced the percentage of cells containing PCNA (Fig. 1A). NPY at 10 and 100 ng/mL, but not at 1000 ng/mL, significantly increased the percentage of bax-positive cells (Fig. 1B). Significant increase in the proportion of cells containing p53 was observed after the treatment of the cells with 10 ng/mL of NPY. Higher doses of NPY (100 or 1000 ng/mL) did not affect the examined parameter (Fig. 1C). These findings suggest that NPY, when given at physiological doses, directly reduces PCNA expression and promotes bax expression in the granulosa cells, although during 2-day exposure, NPY did not affect the cell number yet. Balance between cell proliferation and apoptosis determined the fate of ovarian follicle – its selection, atresia, development and ovulation [15]. The anti-proliferative and pro-apoptotic action of NPY observed in our experiments is the first demonstration of the direct inhibitory action of NPY on ovarian follicular cells. The inhibitory action of NPY on reproduction at the level of hypothalamic gonadotropin-releasing hormone (GnRH) was reported previously. It has been proposed that NPY, the orexigenic peptide, is a mediator of the calorie intake/ metabolic state effect on reproduction at the level of the hypothalamus, but not at the level of the pituitary [3]. The presence of NPY not only in the hypothalamus but also in the ovary [4,8], the existence of NPY receptors in the ovary [9], as well as the ability of NPY to affect ovarian steroid hormone [5,10,11] and oxytocin [4] secretion suggest an direct effect of NPY on ovarian cell function. Our observations are the first to evidence the direct influence of NPY on ovarian cell proliferation and apoptosis. The data demonstrated that NPY can potentially inhibit reproductive functions not only at the central nervous system, but also at the gonadal level. The mechanism of direct NPY action on ovarian cells is almost unknown. The present data demonstrated that NPY
reproductive biology 15 (2015) 257–260
Percentage of cells expressing PCNA
A
demonstrated the up-regulation of p53 by NPY. Furthermore, the association between hormone- or transfection-induced p53 accumulation and expression of apoptosis markers in porcine ovaries has been observed in our previous [15] and present studies. Taken together, these data suggest that hormonal stimuli can suppress ovarian cell functions via promotion of p53, which in turn inhibits proliferation and promotes apoptosis. Recognition of the role of NPY in reproductive processes as well as the site and mechanism of NPY action requires further study. Our observations are the first to demonstrate the direct action of NPY on ovarian cell proliferation and apoptosis. In addition, we showed that this effect may be mediated by transcription factor p53.
80
70
60
50
* *
40
30
20
0
10
259
100
1000
NPY dose added (ng/mL medium)
Conflict of interest
Percentage of cells expressing bax
B 120
None declared.
* 100
*
80
Acknowledgments 60
40
20
0
0
10
100
1000
NPY dose added (ng/mL medium) Percentage of cells expressing p53
C 30
*
We would like to thank Mrs. Katarína Tóthová and Ing. Žofia Kuklová (Research Institute of Animal Production in Nitra, Lužianky, Slovakia) for technical assistance. This study was supported by the Operational Programme Research and Development funded from the European Regional Development Fund, project ‘‘ZDRAVIE’’ no. 26220220176. The authors would also like to extend their sincere appreciation to the Dean of Scientific Research at King Saud University for funding this Research NO (RG#164).
references
28 26 24 22 20 18 16 14 12
0
10
100
1000
NPY dose added (ng/mL medium) Fig. 1 – The effect of neuropeptide Y (NPY) on immunoexpression of PCNA (A), bax (B) and p53 (C) in porcine luteinized granulosa cells (n = 3). Data (mean W SEM) are presented as percentages of cells expressing PCNA, bax and p53. *Significant differences between the treatment groups (NPY: 0, 10, 100 or 1000 ng/mL); p < 0.05.
increased the number of cells expressing p53 protein. The transcription factor p53 is considered to be a suppressor of cell proliferation, a promoter of DNA repair and an inducer of apoptosis of non-ovarian [14] and ovarian [15] cells. We have
[1] Malva JO, Xapelli S, Baptista S, Valero J, Agasse F, Ferreira R, et al. Multifaces of neuropeptide Y in the brain– neuroprotection, neurogenesis and neuroinflammation. Neuropeptides 2012;46:299–308. [2] Shi YC, Baldock PA. Central and peripheral mechanisms of the NPY system in the regulation of bone and adipose tissue. Bone 2012;50:430–6. [3] Roa J. Role of GnRH neurons and their neuronal afferents as key integrators between food intake regulatory signals and the control of reproduction. Int J Endocrinol 2013;2013:518046. http://dx.doi.org/10.1155/2013/518046 [4] Keator CS, Custer EE, Hoagland TA, Schreiber DT, Mah K, Lawson AM, et al. Evidence for a potential role of neuropeptide Y in ovine corpus luteum function. Domest Anim Endocrinol 2010;38:103–14. [5] Baranowska B, Chmielowska M, Radzikowska M, Borowiec M, Roguski K, Wasilewska-Dziubinska E. Effects of neuropeptide Y (NPY), galanin and vasoactive intestinal peptide (VIP) on pituitary hormone release and on ovarian steroidogenesis. Neuro Endocrinol Lett 1999;20:385–9. [6] Denniston DJ, Thomas MG, Kane KK, Roybal CN, Canales L, Hallford DM, et al. Effect of neuropeptide Y on GnRHinduced LH release from bovine anterior pituitary cell cultures derived from heifers in a follicular, luteal or ovariectomized state. Anim Reprod Sci 2003;78:25–31. [7] Evans JJ, Pragg FL, Mason DR. Release of luteinizing hormone from the anterior pituitary gland in vitro can be
260
reproductive biology 15 (2015) 257–260
concurrently regulated by at least three peptides: gonadotropin-releasing hormone, oxytocin and neuropeptide Y. Neuroendocrinology 2001;73:408–16. [8] Skobowiat C, Panasiewicz G, Gizejewski Z, Szafranska B. Coexpression pattern of dopamine beta-hydroxylase (DbH) and neuropeptide Y (NPY) within sympathetic innervation of ovary and umbilical cord of the European bison (Bison bonasus L.). Tissue Cell 2013;45:402–6. [9] Jørgensen JC. Neuropeptide Y in mammalian genital tract: localization and biological action. Dan Med Bull 1994;41:294–305. [10] Pitzel L, Jarry H, Wuttke W. Effects of substance-P and neuropeptide-Y on in vitro steroid release by porcine granulosa and luteal cells. Endocrinology 1991;129:1059–65.
[11] Barreca A, Valli B, Cesarone A, Arvigo M, Balasini M, Battista La Sala G, et al. Effects of the neuropeptide Y on estradiol and progesterone secretion by human granulosa cells in culture. Fertil Steril 1998;70:320–5. [12] Hübscher U, Maga G. DNA replication and repair bypass machines. Curr Opin Chem Biol 2011;15:627–35. [13] Gillies LA, Kuwana T. Apoptosis regulation at the mitochondrial outer membrane. J Cell Biochem 2014;115:632–40. [14] Jackson JG, Post SM, Lozano G. Regulation of tissue- and stimulus-specific cell fate decisions by p53 in vivo. J Pathol 2011;223:127–36. [15] Sirotkin AV. Regulators of ovarian functions. New York, USA: Nova Science Publishers, Inc.; 2014. p. 194.