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Erythropoietin receptor-like immunostaining on human spermatozoa
Reproductive BioMedicine Online (2010) 21, 718– 720
www.sciencedirect.com www.rbmonline.com
SHORT COMMUNICATION
Erythropoietin receptor-like immunostaining on human spermatozoa Niyazi Tug a,b,*, Ulkan Kilic a, Ates Karateke Milas Ugur c, Ertugrul Kilic c
a,b
, Bayram Yilmaz c,
a Yeditepe University Faculty of Medicine, Istanbul, Turkey; b Zeynep Kamil Hospital, Obstetrics and Gynecology Department, Istanbul, Turkey; c Yeditepe University, Faculty of Medicine, Department of Medical Physiology, Istanbul, Turkey
* Corresponding author. E-mail addresses: [email protected], [email protected] (N Tug). All of the authors equally contributed to this work. Niyazi Tug, MD is a graduate of Marmara University, Faculty of Medicine (1995)and served his residency at Firat University, Faculty of Medicine, Obstetrics and Gynecology Department (2002). His current position is gynaecologist in Zeynep Kamil Hospital.
Abstract This study aimed to demonstrate the presence of erythropoietin (EPO) receptor on spermatozoa. Whole ejaculates of four
healthy volunteers were incubated with polyclonal rabbit anti-EPO receptor and subsequently stained with a Cy-3 labelled secondary antibody. Four slides per subject were analysed, no staining was observed in slides incubated with either primary or secondary antibody alone. EPO receptor staining was positive in 92 ± 8% of EPO pre-treated and 91 ± 4% of non-treated sperm cells. The results suggest that spermatozoa express EPO receptor on plasma membrane, which might act to protect these cells from damage after ejaculation. RBMOnline ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: EPO, EPO-receptor, receptor, spermatozoa
Introduction As the principal regulator of erythropoiesis, erythropoietin (EPO) acts synergistically with other cytokines to promote the proliferation, differentiation and survival of erythroid progenitor cells (Konstantinopoulos et al., 2007.). However, the biological activity of EPO extends beyond erythropoiesis including cardiovascular, neural, gastrointestinal and
female and male reproductive tissues and malignancies (Fisher, 2003). Synthesis of EPO principally takes place in the kidneys. In chronic renal failure, the synthesis of EPO does not suffice to maintain erythrocyte mass in circulation and sexual functions also deteriorate which were restored by EPO supplementation. In addition, production of EPO by a human testicular germ cell line suggested that EPO plays role in
1472-6483/$ - see front matter ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rbmo.2010.05.022
Erythropoietin receptor on human spermatozoa male reproductive functions (Fisher, 2003). Epididymal EPO synthesis and secretion was also demonstrated in a mouse model (Kobayashi et al., 2002). EPO was shown to stimulate steroidogenesis from Leydig cells through protein kinase-C system. By its anti-apoptotic properties, EPO was also suggested to reduce germ cell loss in boys with cryptorchidism. It has been shown that EPO is present in human seminal plasma and does not differ between fertile and infertile subjects. The EPO concentrations ranged from 1.5 to 45.0 mIU/ml (Temma et al., 2004). In a recent study, supplementation of washed human sperm suspension with recombinant EPO was shown to be protective against the loss of sperm motility over time (Tug et al., 2009). The present study aimed to demonstrate the presence of EPO receptor on spermatozoa.
719
Results Age of the volunteers was 26.5 ± 5.2 years. Spermiogram parameters according to WHO criteria were normal (volume 3.95 ± 0.42 ml, concentration 96.13 ± 32.6 · 106, fast forward motility 29 ± 8%, slow forward motility: 34 ± 9%, in-situ motility 9 ± 4%, no motility 29 ± 12%). Microscopic analysis of the slides incubated either with primary or secondary antibody only revealed no image. Analysis of slides incubated with primary and secondary antibodies (n = 4, four slides for each sample) revealed robust EPO receptor expression on spermatozoa (Figure 1). Almost all DAPI-positive spermatozoa on the slides (DAPI-positive cells) were EPO receptor positive (91 ± 4% in non-treated and 92 ± 8% in epoitin-treated samples).
Materials and methods Discussion This study was approved by local ethics committee and carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans (http:www.ohsr.od.nih.gov/guidelines/helsinki.html). Experiments were performed in Yeditepe University, Faculty of Medicine, Physiology Department, Istanbul, Turkey. The four subjects were between 25 and 35 years, non-smoking male volunteers who were asked to abstain from ejaculation 3 to 5 days before collection. Ejaculates were obtained by masturbation and collected into sterile cups. After liquefaction at 37C, sperm parameters were examined according to World Health Organization guidelines and normal ones were included in the study. Samples were divided into four equal tubes one of which were incubated for 30 min with recombinant human erythropoietin (Epoitin beta, Neorecormon; Roche Istanbul, Turkey) at a concentration of 100 IU/ml for receptor saturation. A sperm count of 10 · 106/ml and total motility of 50% were accepted as inclusion criteria. Sperm samples were smeared on microscope slides (n = 4, four slides for each tube) and dried at 39C for 30 min. Slides were fixed in 4% paraformaldehyde/0.1 mol/l phosphate-buffered saline (PBS), washed and immersed for 1 h in 0.1 mol/l PBS containing 0.3% triton (PBS-T)/10% normal goat serum. Smears of the study group and those pre-incubated with EPO were incubated overnight at 4C with polyclonal rabbit anti-EPO receptor (sc-5624; Santa Cruz), diluted 1:100 in PBS-T and subsequently stained with a Cy-3 labelled secondary antibody. Smears were finally incubated with DAPI, cover slipped and evaluated under a fluorescence microscope using appropriate filters. Results were given as mean ± standard deviation. Additional smears from the samples of each subject were either incubated with either first or second antibody alone in order to serve as controls. DAPI and EPO receptor expression were microscopically evaluated by counting positive cells in predefined arrays consisting of four regions of interest on the object slides. Data analysis was performed by two investigators blinded to the experimental conditions. The density of the EPO-positive cells were expressed as a percentage of DAPI positive cells. Results were given as mean ± standard deviation.
As far as is known, this study shows for the first time spermatozoa expressing EPO receptors. Immunostaining of the unprepared sperm samples obtained from healthy volunteers revealed that over 90% of the spermatozoa showed positive fluorescence for EPO receptor staining (Figure 1). Absence of any images on the control smears incubated with either primary or secondary antibodies suggests that the positive fluorescence obtained from the smears are indicative of the presence of EPO receptors on the cell membrane of the spermatozoa. Binding of the EPO molecule to the EPO receptors on the plasma membrane leads a conformational change which starts the activation of a couple of pathways (i.e. JAK-STAT, erk-1, 2) resulting in protection of the cell from ischemia, reactive oxygen species and apoptosis (Fisher 2003; Tug et al., 2009). In the present study, receptor saturation of the sperm cells by epoitin pre-treatment at a considerably high dose (100 IU/ml) did not change the rates of the EPO receptor-positive cells on the smears indicating that EPO receptor saturation neither leads to a loss of EPO receptors from plasma membrane nor prevents the binding of the primary antibody used in this study. In a recent study, EPO was reported to improve the motility of spermatozoa (Tug et al., 2009). In this study, recombinant human EPO beta at doses of 1, 10 and 100 mIU/ml was added to human sperm suspensions and shown to improve the motility scores of the samples following an incubation period of 4 h. It is not known whether this effect resulted from a protection of cells from death or improvement of motility per se. Protective effects of EPO in erythroid and non-erythroid tissues are well known but the effect on sperm cells remains to be investigated. Mature sperm cells are relatively resistant to damage (i.e. secondary to reactive oxygen species, pH changes) as a result of their compact chromatin structure but DNA damage and apoptosis are still limiting issues for the success of infertility treatment. Demonstration of the activation of pathways which are known to be linked to the EPO receptor would not only help to illuminate the physiology of EPO receptors expressed by the sperm cells but may also contribute to a better understanding of male infertility.
720
N Tug et al. Epo Pre-treated
Control-A
Control-B
EPOR / DAPI
DAPI
EPOR
Epo Non-treated
Figure 1 Erythropoietin receptor (EPOR) is expressed on spermatozoa (arrows). EPO pre-treated and EPO non-treated samples showed immunofluorescent staining for EPO receptor (red Cy-3 fluorescence) superimposed with DAPI staining of sperm nuclei (blue fluorescence). No receptor image was observed in the control smears A and B, which were incubated either with primary or secondary antibody only. Bar, 200 lm. (For interpretation of references to color in this figure legend, the reader is referred to see the web version of this article.)
In conclusion, this study, by immunohistochemistry, demonstrates the presence of the EPO receptor on spermatozoa for the first time. However, the functionality of this receptor remains to be investigated.
References Fisher, J.W., 2003. Erythropoietin: physiology and pharmacology update. Exp. Biol. Med. 228, 1–14. Kobayashi, T., Yanase, H., Iwanaga, T., Sasaki, R., Nagao, M., 2002. Epididymis is a novel site of erythropoietin production in mouse reproductive organs. Biochem. Biophys. Res. Commun. 296, 145–151. Konstantinopoulos, P.A., Karamouzis, M.V., Papavassiliou, A.G., 2007. Selective modulation of the erythropoietic and tissue-pro-
tective effects of erythropoietin: time to reach the full therapeutic potential of erythropoietin. Biochim. Biophys. Acta 1776, 1–9. Temma, K., Shimoya, K., Hashimoto, K., Zhang, Q., Koyama, M., Murata, Y., 2004. Detection of erythropoietin in human seminal plasma. Fertil. Steril. 81 (Suppl. 1), 798–801. Tug, N., Altunkaynak, M.E., Aktas, R.G., et al., 2009. Does erythropoietin affect motility of spermatozoa? Arch. Gynecol. Obstet. [Epub ahead of print, doi: 10.1007/s00404-009-1289-4]. Declaration: The authors report no financial or commercial conflicts of interest. Received 22 March 2010; refereed 21 May 2010; accepted 26 May 2010.
www.sciencedirect.com www.rbmonline.com
SHORT COMMUNICATION
Erythropoietin receptor-like immunostaining on human spermatozoa Niyazi Tug a,b,*, Ulkan Kilic a, Ates Karateke Milas Ugur c, Ertugrul Kilic c
a,b
, Bayram Yilmaz c,
a Yeditepe University Faculty of Medicine, Istanbul, Turkey; b Zeynep Kamil Hospital, Obstetrics and Gynecology Department, Istanbul, Turkey; c Yeditepe University, Faculty of Medicine, Department of Medical Physiology, Istanbul, Turkey
* Corresponding author. E-mail addresses: [email protected], [email protected] (N Tug). All of the authors equally contributed to this work. Niyazi Tug, MD is a graduate of Marmara University, Faculty of Medicine (1995)and served his residency at Firat University, Faculty of Medicine, Obstetrics and Gynecology Department (2002). His current position is gynaecologist in Zeynep Kamil Hospital.
Abstract This study aimed to demonstrate the presence of erythropoietin (EPO) receptor on spermatozoa. Whole ejaculates of four
healthy volunteers were incubated with polyclonal rabbit anti-EPO receptor and subsequently stained with a Cy-3 labelled secondary antibody. Four slides per subject were analysed, no staining was observed in slides incubated with either primary or secondary antibody alone. EPO receptor staining was positive in 92 ± 8% of EPO pre-treated and 91 ± 4% of non-treated sperm cells. The results suggest that spermatozoa express EPO receptor on plasma membrane, which might act to protect these cells from damage after ejaculation. RBMOnline ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: EPO, EPO-receptor, receptor, spermatozoa
Introduction As the principal regulator of erythropoiesis, erythropoietin (EPO) acts synergistically with other cytokines to promote the proliferation, differentiation and survival of erythroid progenitor cells (Konstantinopoulos et al., 2007.). However, the biological activity of EPO extends beyond erythropoiesis including cardiovascular, neural, gastrointestinal and
female and male reproductive tissues and malignancies (Fisher, 2003). Synthesis of EPO principally takes place in the kidneys. In chronic renal failure, the synthesis of EPO does not suffice to maintain erythrocyte mass in circulation and sexual functions also deteriorate which were restored by EPO supplementation. In addition, production of EPO by a human testicular germ cell line suggested that EPO plays role in
1472-6483/$ - see front matter ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rbmo.2010.05.022
Erythropoietin receptor on human spermatozoa male reproductive functions (Fisher, 2003). Epididymal EPO synthesis and secretion was also demonstrated in a mouse model (Kobayashi et al., 2002). EPO was shown to stimulate steroidogenesis from Leydig cells through protein kinase-C system. By its anti-apoptotic properties, EPO was also suggested to reduce germ cell loss in boys with cryptorchidism. It has been shown that EPO is present in human seminal plasma and does not differ between fertile and infertile subjects. The EPO concentrations ranged from 1.5 to 45.0 mIU/ml (Temma et al., 2004). In a recent study, supplementation of washed human sperm suspension with recombinant EPO was shown to be protective against the loss of sperm motility over time (Tug et al., 2009). The present study aimed to demonstrate the presence of EPO receptor on spermatozoa.
719
Results Age of the volunteers was 26.5 ± 5.2 years. Spermiogram parameters according to WHO criteria were normal (volume 3.95 ± 0.42 ml, concentration 96.13 ± 32.6 · 106, fast forward motility 29 ± 8%, slow forward motility: 34 ± 9%, in-situ motility 9 ± 4%, no motility 29 ± 12%). Microscopic analysis of the slides incubated either with primary or secondary antibody only revealed no image. Analysis of slides incubated with primary and secondary antibodies (n = 4, four slides for each sample) revealed robust EPO receptor expression on spermatozoa (Figure 1). Almost all DAPI-positive spermatozoa on the slides (DAPI-positive cells) were EPO receptor positive (91 ± 4% in non-treated and 92 ± 8% in epoitin-treated samples).
Materials and methods Discussion This study was approved by local ethics committee and carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans (http:www.ohsr.od.nih.gov/guidelines/helsinki.html). Experiments were performed in Yeditepe University, Faculty of Medicine, Physiology Department, Istanbul, Turkey. The four subjects were between 25 and 35 years, non-smoking male volunteers who were asked to abstain from ejaculation 3 to 5 days before collection. Ejaculates were obtained by masturbation and collected into sterile cups. After liquefaction at 37C, sperm parameters were examined according to World Health Organization guidelines and normal ones were included in the study. Samples were divided into four equal tubes one of which were incubated for 30 min with recombinant human erythropoietin (Epoitin beta, Neorecormon; Roche Istanbul, Turkey) at a concentration of 100 IU/ml for receptor saturation. A sperm count of 10 · 106/ml and total motility of 50% were accepted as inclusion criteria. Sperm samples were smeared on microscope slides (n = 4, four slides for each tube) and dried at 39C for 30 min. Slides were fixed in 4% paraformaldehyde/0.1 mol/l phosphate-buffered saline (PBS), washed and immersed for 1 h in 0.1 mol/l PBS containing 0.3% triton (PBS-T)/10% normal goat serum. Smears of the study group and those pre-incubated with EPO were incubated overnight at 4C with polyclonal rabbit anti-EPO receptor (sc-5624; Santa Cruz), diluted 1:100 in PBS-T and subsequently stained with a Cy-3 labelled secondary antibody. Smears were finally incubated with DAPI, cover slipped and evaluated under a fluorescence microscope using appropriate filters. Results were given as mean ± standard deviation. Additional smears from the samples of each subject were either incubated with either first or second antibody alone in order to serve as controls. DAPI and EPO receptor expression were microscopically evaluated by counting positive cells in predefined arrays consisting of four regions of interest on the object slides. Data analysis was performed by two investigators blinded to the experimental conditions. The density of the EPO-positive cells were expressed as a percentage of DAPI positive cells. Results were given as mean ± standard deviation.
As far as is known, this study shows for the first time spermatozoa expressing EPO receptors. Immunostaining of the unprepared sperm samples obtained from healthy volunteers revealed that over 90% of the spermatozoa showed positive fluorescence for EPO receptor staining (Figure 1). Absence of any images on the control smears incubated with either primary or secondary antibodies suggests that the positive fluorescence obtained from the smears are indicative of the presence of EPO receptors on the cell membrane of the spermatozoa. Binding of the EPO molecule to the EPO receptors on the plasma membrane leads a conformational change which starts the activation of a couple of pathways (i.e. JAK-STAT, erk-1, 2) resulting in protection of the cell from ischemia, reactive oxygen species and apoptosis (Fisher 2003; Tug et al., 2009). In the present study, receptor saturation of the sperm cells by epoitin pre-treatment at a considerably high dose (100 IU/ml) did not change the rates of the EPO receptor-positive cells on the smears indicating that EPO receptor saturation neither leads to a loss of EPO receptors from plasma membrane nor prevents the binding of the primary antibody used in this study. In a recent study, EPO was reported to improve the motility of spermatozoa (Tug et al., 2009). In this study, recombinant human EPO beta at doses of 1, 10 and 100 mIU/ml was added to human sperm suspensions and shown to improve the motility scores of the samples following an incubation period of 4 h. It is not known whether this effect resulted from a protection of cells from death or improvement of motility per se. Protective effects of EPO in erythroid and non-erythroid tissues are well known but the effect on sperm cells remains to be investigated. Mature sperm cells are relatively resistant to damage (i.e. secondary to reactive oxygen species, pH changes) as a result of their compact chromatin structure but DNA damage and apoptosis are still limiting issues for the success of infertility treatment. Demonstration of the activation of pathways which are known to be linked to the EPO receptor would not only help to illuminate the physiology of EPO receptors expressed by the sperm cells but may also contribute to a better understanding of male infertility.
720
N Tug et al. Epo Pre-treated
Control-A
Control-B
EPOR / DAPI
DAPI
EPOR
Epo Non-treated
Figure 1 Erythropoietin receptor (EPOR) is expressed on spermatozoa (arrows). EPO pre-treated and EPO non-treated samples showed immunofluorescent staining for EPO receptor (red Cy-3 fluorescence) superimposed with DAPI staining of sperm nuclei (blue fluorescence). No receptor image was observed in the control smears A and B, which were incubated either with primary or secondary antibody only. Bar, 200 lm. (For interpretation of references to color in this figure legend, the reader is referred to see the web version of this article.)
In conclusion, this study, by immunohistochemistry, demonstrates the presence of the EPO receptor on spermatozoa for the first time. However, the functionality of this receptor remains to be investigated.
References Fisher, J.W., 2003. Erythropoietin: physiology and pharmacology update. Exp. Biol. Med. 228, 1–14. Kobayashi, T., Yanase, H., Iwanaga, T., Sasaki, R., Nagao, M., 2002. Epididymis is a novel site of erythropoietin production in mouse reproductive organs. Biochem. Biophys. Res. Commun. 296, 145–151. Konstantinopoulos, P.A., Karamouzis, M.V., Papavassiliou, A.G., 2007. Selective modulation of the erythropoietic and tissue-pro-
tective effects of erythropoietin: time to reach the full therapeutic potential of erythropoietin. Biochim. Biophys. Acta 1776, 1–9. Temma, K., Shimoya, K., Hashimoto, K., Zhang, Q., Koyama, M., Murata, Y., 2004. Detection of erythropoietin in human seminal plasma. Fertil. Steril. 81 (Suppl. 1), 798–801. Tug, N., Altunkaynak, M.E., Aktas, R.G., et al., 2009. Does erythropoietin affect motility of spermatozoa? Arch. Gynecol. Obstet. [Epub ahead of print, doi: 10.1007/s00404-009-1289-4]. Declaration: The authors report no financial or commercial conflicts of interest. Received 22 March 2010; refereed 21 May 2010; accepted 26 May 2010.