Differential role of r-met-hu G-CSF on male reproductive function and development in prepubertal domestic mammals

Autoři: Pedro M. Aponte aff001;  Miguel A. Gutierrez-Reinoso aff004;  Edison G. Sanchez-Cepeda aff005;  Manuel Garcia-Herreros aff006
Působiště autorů: Colegio de Ciencias Biológicas y Ambientales, Universidad San Francisco de Quito (USFQ), Campus Cumbayá, Quito, Ecuador aff001;  Colegio de Ciencias de la Salud, Escuela de Medicina Veterinaria, Universidad San Francisco de Quito (USFQ), Campus Cumbayá, Quito, Ecuador aff002;  Instituto de Investigaciones en Biomedicina “One-health”, Universidad San Francisco de Quito (USFQ), Campus Cumbayá, Quito, Ecuador aff003;  Facultad de Ciencias Agropecuarias y Recursos Naturales, Carrera de Medicina Veterinaria, Universidad Técnica de Cotopaxi (UTC), Latacunga, Ecuador aff004;  AGROCALIDAD, Ambato, Ecuador aff005;  National Secretariat Higher Education, Science, Technology and Innovation (SENESCYT), Quito, Ecuador aff006;  Instituto Nacional de Investigação Agrária e Veterinária, I. P. (INIAV, I.P.), Polo de Santarém, Santarém, Portugal aff007
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
doi: 10.1371/journal.pone.0222871


The understanding of mammalian spermatogenesis niche factors active during sexual development may be leveraged to impact reproduction in farm animals. The aim of this study was to evaluate the effects of r-met-hu/G-CSF (filgrastim) on prepubertal sexual development of guinea pigs (Cavia porcellus) and ram lambs (Ovis aries). Individuals of both species were administered r-met-hu/G-CSF daily for 4 days. During and after administration protocols, testicular function and development were assessed through hematological responses, hormonal profiles (gonadotropins, testosterone and cortisol) testicular morphometry and germ cell kinetics. As expected, r-met-hu/G-CSF acutely mobilized white-lineage blood cells in both species. LH was increased by r-met-hu/G-CSF in guinea pigs (P<0.01) but T remained unchanged. In ram lambs gonadotropins and T increased in dose-response fashion (P<0.01) while cortisol values were stable and similar in treated and control animals (P>0.05). In guinea pigs there were no differences in testicular weights and volumes 2-mo after r-met-hu/G-CSF application (P>0.05). However, ram lambs showed a dose-response effect regarding testis weight (P<0.05). 66.66% of ram lambs had initial testes not yet in meiosis or starting the first spermatogenic wave. After 60-days only 25% of control animals were pubertal while all treated animals (1140-μg) had reached puberty. We propose an integrated hypothesis that G-CSF can stimulate spermatogenesis through two possible ways. 1) r-met-hu/G-CSF may go through the brain blood barrier and once there it can stimulate GnRH-neurons to release GnRH with the subsequent release of gonadotrophins. 2) a local testicular effect through stimulation of steroidogenesis that enhances spermiogenesis via testosterone production and a direct stimulation over spermatogonial stem cells self-renewal. In conclusion, this study shows that r-met-hu/G-CSF differentially affects prepubertal sexual development in hystricomorpha and ovine species, a relevant fact to consider when designing methods to hasten sexual developmental in mammalian species.

Klíčová slova:

Germ cells – Guinea pigs – Sperm – Spermatogenesis – Testosterone – Seminiferous tubules – Spermatids – Sexual differentiation


1. Chocu S, Calvel P, Rolland AD, Pineau C. Spermatogenesis in mammals: proteomic insights. Syst Biol Reprod Med. 2012;58: 179–190. doi: 10.3109/19396368.2012.691943 22788530

2. Fuller MT. Differentiation in Stem Cell Lineages and in Life: Explorations in the Male Germ Line Stem Cell Lineage. Curr Top Dev Biol. 2016;116: 375–390. doi: 10.1016/bs.ctdb.2015.11.041 26970629

3. de Rooij DG. The nature and dynamics of spermatogonial stem cells. Development. 2017;144: 3022–3030. doi: 10.1242/dev.146571 28851723

4. Oatley JM, Brinster RL. The germline stem cell niche unit in mammalian testes. Physiol Rev. 2012;92: 577–595. doi: 10.1152/physrev.00025.2011 22535892

5. Aponte PM, de Rooij DG, Bastidas P. Testicular development in Brahman bulls. Theriogenology. 2005;64: 1440–1455. doi: 10.1016/j.theriogenology.2005.03.016 16139617

6. Koskenniemi JJ, Virtanen HE, Toppari J. Testicular growth and development in puberty. Curr Opin Endocrinol Diabetes Obes. 2017;24: 215–224. doi: 10.1097/MED.0000000000000339 28248755

7. Bollwein H, Janett F, Kaske M. Impact of nutritional programming on the growth, health, and sexual development of bull calves. Domest Anim Endocrinol. 2016;56 Suppl: S180–190. doi: 10.1016/j.domaniend.2016.02.006 27345315

8. Aponte PM, Gutierrez-Reinoso MA, Sanchez-Cepeda EG, Garcia-Herreros M. Active immunization against GnRH in pre-pubertal domestic mammals: testicular morphometry, histopathology and endocrine responses in rabbits, guinea pigs and ram lambs. Animal. 2018;12: 784–793. doi: 10.1017/S1751731117002129 28835304

9. Anderlini P, Champlin R. Use of filgrastim for stem cell mobilisation and transplantation in high-dose cancer chemotherapy. Drugs. 2002;62 Suppl 1: 79–88.

10. Welte K. G-CSF: filgrastim, lenograstim and biosimilars. Expert Opin Biol Ther. 2014;14: 983–993. doi: 10.1517/14712598.2014.905537 24707817

11. Dale DC, Crawford J, Klippel Z, Reiner M, Osslund T, Fan E, et al. A systematic literature review of the efficacy, effectiveness, and safety of filgrastim. Support Care Cancer. 2018;26: 7–20. doi: 10.1007/s00520-017-3854-x 28939926

12. Niven RW, Lott FD, Cribbs JM. Pulmonary absorption of recombinant methionyl human granulocyte colony stimulating factor (r-huG-CSF) after intratracheal instillation to the hamster. Pharm Res. 1993;10: 1604–1610. doi: 10.1023/a:1018920619424 7507241

13. Miyamoto M, Natsume H, Satoh I, Ohtake K, Yamaguchi M, Kobayashi D, et al. Effect of poly-L-arginine on the nasal absorption of FITC-dextran of different molecular weights and recombinant human granulocyte colony-stimulating factor (rhG-CSF) in rats. Int J Pharm. 2001;226: 127–138. doi: 10.1016/s0378-5173(01)00797-9 11532576

14. Eftekhar M, Naghshineh E, Khani P. Role of granulocyte colony-stimulating factor in human reproduction. J Res Med Sci. 2018;23: 7. doi: 10.4103/jrms.JRMS_628_17 29456564

15. Zambrano A, Noli C, Rauch MC, Werner E, Brito M, Amthauer R, et al. Expression of GM-CSF receptors in male germ cells and their role in signaling for increased glucose and vitamin C transport. J Cell Biochem. 2001;80: 625–634. 11169747

16. Vilanova LT, Rauch MC, Mansilla A, Zambrano A, Brito M, Werner E, et al. Expression of granulocyte-macrophage colony stimulating factor (GM-CSF) in male germ cells: GM-CSF enhances sperm motility. Theriogenology. 2003;60: 1083–1095. doi: 10.1016/s0093-691x(03)00106-7 12935848

17. Rodríguez-Gil JE, Silvers G, Flores E, Jesús Palomo M, Ramírez A, Montserrat Rivera M, et al. Expression of the GM-CSF receptor in ovine spermatozoa: GM-CSF effect on sperm viability and motility of sperm subpopulations after the freezing-thawing process. Theriogenology. 2007;67: 1359–1370. doi: 10.1016/j.theriogenology.2007.02.008 17408732

18. Koruji M, Movahedin M, Mowla SJ, Gourabi H, Arfaee AJ. Efficiency of adult mouse spermatogonial stem cell colony formation under several culture conditions. In Vitro Cell Dev Biol Anim. 2009;45: 281–289. doi: 10.1007/s11626-008-9169-y 19221844

19. Benavides-Garcia R, Joachim R, Pina NA, Mutoji KN, Reilly MA, Hermann BP. Granulocyte colony-stimulating factor prevents loss of spermatogenesis after sterilizing busulfan chemotherapy. Fertil Steril. 2015;103: 270–280.e8. doi: 10.1016/j.fertnstert.2014.09.023 25439845

20. Kotzur T, Benavides-Garcia R, Mecklenburg J, Sanchez JR, Reilly M, Hermann BP. Granulocyte colony-stimulating factor (G-CSF) promotes spermatogenic regeneration from surviving spermatogonia after high-dose alkylating chemotherapy. Reprod Biol Endocrinol. 2017;15. doi: 10.1186/s12958-017-0236-7

21. Kwan EM, Beck S, Amir E, Jewett MA, Sturgeon JF, Anson-Cartwright L, et al. Impact of Granulocyte-colony Stimulating Factor on Bleomycin-induced Pneumonitis in Chemotherapy-treated Germ Cell Tumors. Clin Genitourin Cancer. 2017; doi: 10.1016/j.clgc.2017.08.012 28943331

22. Höglund M. Glycosylated and non-glycosylated recombinant human granulocyte colony-stimulating factor (rhG-CSF)—what is the difference? Med Oncol. 1998;15: 229–233. 9951685

23. Anderlini P, Champlin RE. Biologic and molecular effects of granulocyte colony-stimulating factor in healthy individuals: recent findings and current challenges. Blood. 2008;111: 1767–1772. doi: 10.1182/blood-2007-07-097543 18057230

24. Baldo BA. Side effects of cytokines approved for therapy. Drug Saf. 2014;37: 921–943. doi: 10.1007/s40264-014-0226-z 25270293

25. Farese AM, MacVittie TJ. Filgrastim for the treatment of hematopoietic acute radiation syndrome. Drugs Today. 2015;51: 537–548. doi: 10.1358/dot.2015.51.9.2386730 26488033

26. Engelken TJ. The development of beef breeding bulls. Theriogenology. 2008;70: 573–575. doi: 10.1016/j.theriogenology.2008.05.038 18538836

27. Scaramuzzi RJ, Martin GB. The importance of interactions among nutrition, seasonality and socio-sexual factors in the development of hormone-free methods for controlling fertility. Reprod Domest Anim. 2008;43 Suppl 2: 129–136. doi: 10.1111/j.1439-0531.2008.01152.x 18638114

28. Thundathil JC, Dance AL, Kastelic JP. Fertility management of bulls to improve beef cattle productivity. Theriogenology. 2016;86: 397–405. doi: 10.1016/j.theriogenology.2016.04.054 27173954

29. Perry V, Chenoweth PJ, Post TB, Munro RK. Fertility indices for beef bulls. Aust Vet J. 1990;67: 13–16. doi: 10.1111/j.1751-0813.1990.tb07383.x 2334366

30. Geuna S, Herrera-Rincon C. Update on stereology for light microscopy. Cell Tissue Res. 2015;360: 5–12. doi: 10.1007/s00441-015-2143-6 25743692

31. Noller DW, Flickinger CJ, Howards SS. Duration of the cycle of the seminiferous epithelium in the guinea pig determined by tritiated thymidine autoradiography. Biol Reprod. 1977;17: 532–534. doi: 10.1095/biolreprod17.4.532 922088

32. Bilaspuri GS, Guraya SS. The seminiferous epithelial cycle and spermat ogenesis in rams (ovis aries). Theriogenology. 1986;25: 485–505. doi: 10.1016/0093-691x(86)90133-0 16726140

33. Yarney TA, Sanford LM. Pubertal development of ram lambs: Physical and endocrinological traits in combination as indices of postpubertal reproductive function. Theriogenology. 1993;40: 735–744. doi: 10.1016/0093-691x(93)90209-n 16727355

34. Belibasaki null, Kouimtzis null. Sexual activity and body and testis growth in prepubertal ram lambs of Friesland, Chios, Karagouniki and Serres dairy sheep in Greece. Small Rumin Res. 2000;37: 109–113. 10818310

35. Salhab SA, Zarkawi M, Wardeh MF, Al-Masri MR, Kassem R. Development of testicular dimensions and size, and their relationship to age, body weight and parental size in growing Awassi ram lambs. Small Rumin Res. 2001;40: 187–191. 11295401

36. Syrstad O, Ruane J. Prospects and strategies for genetic improvement of the dairy potential of tropical cattle by selection. Trop Anim Health Prod. 1998;30: 257–268. doi: 10.1023/a:1005083430140 9760718

37. Valasi I, Chadio S, Fthenakis GC, Amiridis GS. Management of pre-pubertal small ruminants: physiological basis and clinical approach. Anim Reprod Sci. 2012;130: 126–134. doi: 10.1016/j.anireprosci.2012.01.005 22326612

38. Kim J, Lee S, Jeon B, Jang W, Moon C, Kim S. Protection of spermatogenesis against gamma ray-induced damage by granulocyte colony-stimulating factor in mice. Andrologia. 2011;43: 87–93. doi: 10.1111/j.1439-0272.2009.01023.x 21382061

39. Quirion E. Filgrastim and pegfilgrastim use in patients with neutropenia. Clin J Oncol Nurs. 2009;13: 324–328. doi: 10.1188/09.CJON.324-328 19502191

40. Sanzari JK, Krigsfeld GS, Shuman AL, Diener AK, Lin L, Mai W, et al. Effects of a granulocyte colony stimulating factor, Neulasta, in mini pigs exposed to total body proton irradiation. Life Sci Space Res (Amst). 2015;5: 13–20. doi: 10.1016/j.lssr.2015.03.002 25909052

41. Endo Y, Hobo S, Korosue K, Ootsuka K, Kitauchi A, Kikkawa R, et al. Effects of low-dose G-CSF formulation on hematology in healthy horses after long-distance transportation. J Vet Med Sci. 2015;77: 507–509. doi: 10.1292/jvms.14-0586 25648988

42. Licollari A, Riddle K, Taylor SR, Ledon N, Bolger GT. Safety and Biosimilarity of ior®LeukoCIM Compared to Neupogen® Based on Toxicity, Pharmacodynamic, and Pharmacokinetic Studies in the Sprague-Dawley Rat. J Pharm Sci. 2017;106: 1475–1481. doi: 10.1016/j.xphs.2017.02.009 28238900

43. Satyamitra M, Kumar VP, Biswas S, Cary L, Dickson L, Venkataraman S, et al. Impact of Abbreviated Filgrastim Schedule on Survival and Hematopoietic Recovery after Irradiation in Four Mouse Strains with Different Radiosensitivity. Radiat Res. 2017;187: 659–671. doi: 10.1667/RR14555.1 28362168

44. Hashimoto S, Itoh M, Nishimura M, Asai T. Effect of filgrastim administration for steady-state mobilization of peripheral blood stem cells. Ther Apher. 2002;6: 431–436. 12460406

45. Zylińska K, Mucha S, Komorowski J, Korycka A, Pisarek H, Robak T, et al. Influence of granulocyte-macrophage colony stimulating factor on pituitary-adrenal axis (PAA) in rats in vivo. Pituitary. 1999;2: 211–216. 11081156

46. Barreca T, Gobbi M, Franceschini R, Berisso G, Vallebella E, Corsini G, et al. Effect of granulocyte colony-stimulating factor on secretion of prolactin, growth hormone, thyroid-stimulating hormone, and cortisol in humans. Current Therapeutic Research. 1997;58: 88–92. doi: 10.1016/S0011-393X(97)80117-8

47. Bielli A, Gastel MT, Pedrana G, Moraña A, Castrillejo A, Lundeheim N, et al. Influence of pre- and post-pubertal grazing regimes on adult testicular morphology in extensively reared corriedale rams. Anim Reprod Sci. 2000;58: 73–86. 10700646

48. Lunstra DD, Echternkamp SE. Repetitive testicular biopsy in the ram during pubertal development. Theriogenology. 1988;29: 803–810. doi: 10.1016/0093-691x(88)90217-8 16726401

49. Skinner JD, Booth WD, Rowson LE, Karg H. The post-natal development of the reproductive tract of the Suffolk ram, and changes in the gonadotrophin content of the pituitary. J Reprod Fertil. 1968;16: 463–477. doi: 10.1530/jrf.0.0160463 5673744

50. Dirami G, Ravindranath N, Pursel V, Dym M. Effects of stem cell factor and granulocyte macrophage-colony stimulating factor on survival of porcine type A spermatogonia cultured in KSOM. Biol Reprod. 1999;61: 225–230. doi: 10.1095/biolreprod61.1.225 10377053

51. Salmassi A, Schmutzler AG, Huang L, Hedderich J, Jonat W, Mettler L. Detection of granulocyte colony-stimulating factor and its receptor in human follicular luteinized granulosa cells. Fertil Steril. 2004;81 Suppl 1: 786–791. doi: 10.1016/j.fertnstert.2003.09.039 15019810

52. Engelhardt B. Development of the Blood-Brain Interface. Blood-Brain Barriers. Wiley-Blackwell; 2007. pp. 9–39. doi: 10.1002/9783527611225.ch1

53. McLay RN, Kimura M, Banks WA, Kastin AJ. Granulocyte-macrophage colony-stimulating factor crosses the blood—brain and blood—spinal cord barriers. Brain. 1997;120 (Pt 11): 2083–2091.

54. McLay RN, Banks WA, Kastin AJ. Granulocyte macrophage-colony stimulating factor crosses the blood-testis barrier in mice. Biol Reprod. 1997;57: 822–826. doi: 10.1095/biolreprod57.4.822 9314586

55. Banks WA, Kastin AJ. Human interleukin-1 alpha crosses the blood-testis barriers of the mouse. J Androl. 1992;13: 254–259. 1601745

56. Ryan GR, Dai XM, Dominguez MG, Tong W, Chuan F, Chisholm O, et al. Rescue of the colony-stimulating factor 1 (CSF-1)-nullizygous mouse (Csf1(op)/Csf1(op)) phenotype with a CSF-1 transgene and identification of sites of local CSF-1 synthesis. Blood. 2001;98: 74–84. doi: 10.1182/blood.v98.1.74 11418465

57. Shibata M, Friedman RL, Ramaswamy S, Plant TM. Evidence that down regulation of hypothalamic KiSS-1 expression is involved in the negative feedback action of testosterone to regulate luteinising hormone secretion in the adult male rhesus monkey (Macaca mulatta). J Neuroendocrinol. 2007;19: 432–438. doi: 10.1111/j.1365-2826.2007.01549.x 17504437

58. Oatley JM, Oatley MJ, Avarbock MR, Tobias JW, Brinster RL. Colony stimulating factor 1 is an extrinsic stimulator of mouse spermatogonial stem cell self-renewal. Development. 2009;136: 1191–1199. doi: 10.1242/dev.032243 19270176

59. Cohen PE, Zhu L, Nishimura K, Pollard JW. Colony-stimulating factor 1 regulation of neuroendocrine pathways that control gonadal function in mice. Endocrinology. 2002;143: 1413–1422. doi: 10.1210/endo.143.4.8754 11897698

60. Gaytan F, Bellido C, Morales C, García M, van Rooijen N, Aguilar E. In vivo manipulation (depletion versus activation) of testicular macrophages: central and local effects. J Endocrinol. 1996;150: 57–65. doi: 10.1677/joe.0.1500057 8708563

61. Cohen PE, Nishimura K, Zhu L, Pollard JW. Macrophages: important accessory cells for reproductive function. J Leukoc Biol. 1999;66: 765–772. doi: 10.1002/jlb.66.5.765 10577508

62. Cohen PE, Hardy MP, Pollard JW. Colony-stimulating factor-1 plays a major role in the development of reproductive function in male mice. Mol Endocrinol. 1997;11: 1636–1650. doi: 10.1210/mend.11.11.0009 9328346

63. Kokkinaki M, Lee T-L, He Z, Jiang J, Golestaneh N, Hofmann M-C, et al. The molecular signature of spermatogonial stem/progenitor cells in the 6-day-old mouse testis. Biol Reprod. 2009;80: 707–717. doi: 10.1095/biolreprod.108.073809 19109221

64. Khanlarkhani N, Pasbakhsh P, Mortezaee K, Naji M, Amidi F, Najafi A, et al. Effect of human recombinant granulocyte colony-stimulating factor on rat busulfan-induced testis injury. J Mol Histol. 2016;47: 59–67. doi: 10.1007/s10735-015-9647-y 26714726

65. DeFalco T, Potter SJ, Williams AV, Waller B, Kan MJ, Capel B. Macrophages Contribute to the Spermatogonial Niche in the Adult Testis. Cell Rep. 2015;12: 1107–1119. doi: 10.1016/j.celrep.2015.07.015 26257171

Článek vyšel v časopise


2019 Číslo 9