Free Standard AU & NZ Shipping For All Book Orders Over $80!
Register      Login
Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology
RESEARCH ARTICLE

Caspase-3-mediated apoptosis and cell proliferation in the equine endometrium during the oestrous cycle

R. P. Roberto da Costa A , P. M. Serrão B , S. Monteiro B , P. Pessa C , J. Robalo Silva B and G. Ferreira-Dias B D
+ Author Affiliations
- Author Affiliations

A Escola Superior Agrária, Instituto Politécnico de Coimbra, Bencanta, 3040-316 Coimbra, Portugal.

B CIISA, Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa (TULisbon), 1300-477 Lisbon, Portugal.

C Hospitais da Universidade de Coimbra, 3000-075 Coimbra, Portugal.

D Corresponding author. Email: gmlfdias@fmv.utl.pt

Reproduction, Fertility and Development 19(8) 925-932 https://doi.org/10.1071/RD06159
Submitted: 5 December 2006  Accepted: 22 July 2007   Published: 13 September 2007

Abstract

Cell proliferation and apoptosis are hormone-dependent physiological processes involved in endometrial growth and regression. The aims of the present study were: (1) to evaluate endometrial cell proliferation using proliferating cell nuclear antigen (PCNA) expression; (2) to evaluate the induction of endometrial cell death by the expression of active caspase-3 and the apoptotic phenotype visualised by DNA fragmentation; and (3) to relate these observations to endometrial tissue dynamics in the equine endometrium throughout the oestrous cycle. Endometria were assigned to follicular and luteal phases based on ovarian structures and plasma progesterone. Cell proliferation and active caspase-3-mediated apoptosis were expressed in both phases of the oestrous cycle. In the luteal phase, PCNA expression was higher than in the follicular phase. Highest PCNA activity was noted in the luminal and glandular structures. Active caspase-3 staining was increased in luminal epithelium and deep glandular cells during the luteal phase. However, in the follicular phase, stromal cells showed greater active caspase-3 expression. Only a few apoptotic endometrial cells were detected by terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling (TUNEL) and these cells were mostly present in luminal and glandular structures. A simultaneous increase in DNA, cell proliferation and protein synthesis was observed in the endometrium during the mid-luteal phase. This suggests that cell hyperplasia occurs at the time the histotroph is needed for eventual embryo nourishment.

Additional keywords: apoptosis, mare, proliferating cell nuclear antigen, terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling (TUNEL).


Acknowledgements

This work was supported by grants ‘CIISA 45 Corpo Lúteo’, from CIISA, and POCTI/CVT/39519/2001, from Fundação para a Ciência e Tecnologia (FCT), Portugal. The authors thank Mrs Maria do Rosário Luís for technical assistance.


References

Aupperle, H. , Ozgen, S. H. A. , Schoon, D. , Hoppen, H. O. , Sieme, H. , and Tannapfel, A. (2000). Cyclical endometrial steroid hormone receptor expression and proliferation intensity in the mare. Equine Vet. J. 32, 228–232.
Crossref | GoogleScholarGoogle Scholar | PubMed | Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., and Struhl K. (1992). ‘Current Protocols in Molecular Biology.’ (Greene Publishing Association, Wiley-Interscience: New York.)

Baserga R. (1985). Tissue growth. In ‘The Biology of Cell Reproduction’. (Ed. R. Baserga.) pp. 46–58. (Harvard University Press: Cambridge.)

Berkkanoglu, M. , Guzeloglu-Kayisli, O. , Kayisli, U. A. , Selam, B. F. , and Arici, A. (2004). Regulation of Fas ligand expression by vascular endothelial growth factor in endometrial stromal cells in vitro. Mol. Hum. Reprod. 10, 393–398.
Crossref | GoogleScholarGoogle Scholar | PubMed | Brenner R. M., and Slayden O. D. (1994). Cyclic changes in the primate oviduct and endometrium. In ‘The Physiology of Reproduction’, vol. 1. (Eds E. Knobil and J. Neill.) pp. 541–569. (Raven Press: New York.)

Bursch, W. , Paffe, S. , Putz, B. , Barthel, G. , and Schulte-Hermann, R. (1990). Determination of the length of the histological stages of apoptosis in normal liver and in altered hepatic foci of rats. Carcinogenesis 11, 847–853.
Crossref | GoogleScholarGoogle Scholar | PubMed | Chu P. Y., Lee C. S., and Wright P. J. (2006). Degeneration and apoptosis of endometrial cells in the bitch. Theriogenology 66, 1545–1549.

Dallenbach-Hellweg, G. (1988). The endometrium in natural and artificial luteal phases. Hum. Reprod. 3, 165–168.
PubMed | Ginther O. J. (Ed.) (1992a). Characteristics of the ovulatory season. In ‘Reproductive Biology of the Mare: Basic and Applied Aspects’, 2nd edn. pp. 173–232. (Equiservices: Cross Plains, WI.)

Ginther O. J. (Ed.) (1992b). Endocrinology of the ovulatory season. In ‘Reproductive Biology of the Mare: Basic and Applied Aspects’, 2nd edn. pp. 233–290. (Equiservices: Cross Plains, WI.)

Harada, T. , Kaponis, A. , Iwabe, T. , Taniguchi, F. , Makrydimas, G. , Sofikiti, N. , Paschopoulo, M. , Paraskevaidis, E. , and Terakawa, N. (2004). Apoptosis in human endometrium and endometriosis. Hum. Reprod. Update 10, 29–38.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Hayashi, K. , Carpenter, K. D. , and Spencer, T. E. (2004). Neonatal estrogen exposure disrupts uterine development in the postnatal sheep. Endocrinology 145, 3247–3257.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Hengartner, M. O. (2000). The biochemistry of apoptosis. Nature 407, 770–776.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Heryanto, B. , and Rogers, P. A. (2002). Regulation of endometrial endothelial cell proliferation by oestrogen and progesterone in the ovariectomized mouse. Reproduction 123, 107–113.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Ito, K. , Sasano, H. , Watanabe, K. , Ozawa, N. , Sato, S. , and Yajima, A. (1993). Immunohistochemical study of PCNA (proliferating cell nuclear antigen) in normal and abnormal endometrium. Int. J. Gynecol. Cancer 3, 122–127.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Johnson, M. L. , Redmer, D. A. , and Reynolds, L. P. (1997). Uterine growth, cell proliferation, and c-fos proto-oncogene expression throughout the estrous cycle in ewes Biol. Reprod. 56, 393–401.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Jones, R. K. , Searle, R. F. , Stewart, J. A. , Turner, S. , and Bulmer, J. N. (1998). Apoptosis, bcl-2 expression, and proliferative activity in human endometrial stroma and endometrial granulated lymphocytes. Biol. Reprod. 58, 995–1002.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Kaeoket, K. , Persson, E. , and Dalin, A. M. (2001). The sow endometrium at different stages of the oestrous cycle: studies on morphological changes and infiltration by cells of the immune system. Anim. Reprod. Sci. 65, 95–114.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Kayisli, U. A. , Mahutte, N. G. , and Arici, A. (2002). Uterine chemokines in reproductive physiology and pathology. Am. J. Reprod. Immunol. 47, 213–221.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Kokawa, K. , Shikone, T. , and Nakano, R. (1996). Apoptosis in the human uterine endometrium during the menstrual cycle. J. Clin. Endocrinol. Metab. 81, 4144–4147.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Lai, M. D. , Lee, L. R. , Cheng, K. S. , and Wing, L. Y. (2000). Expression of proliferating cell nuclear antigen in luminal epithelium during the growth and regression of rat uterus. J. Endocrinol. 166, 87–93.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Lakhani, S. A. , Masud, A. , Kuida, K. , Porter, G. A. , Booth, C. J. , Mehal, W. Z. , Inayat, I. , and Flavell, R. A. (2006). Caspases 3 and 7: key mediators of mitochondrial events of apoptosis. Science 311, 847–851.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Lynch, M. P. , Nawaz, S. , and Gerschenson, L. E. (1986). Evidence for soluble factors regulating cell death and cell proliferation in primary cultures of rabbit endometrial cells grown on collagen. Proc. Natl Acad. Sci. USA 83, 4784–4788.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Martin, L. , Das, R. M. , and Finn, C. A. (1973). The inhibition by progesterone of uterine epithelial proliferation in the mouse. J. Endocrinol. 57, 549–554.
PubMed |

Masters, R. A. , Crean, B. D. , Yan, W. , Moss, A. G. , Ryan, P. L. , Wiley, A. A. , Bagnell, C. A. , and Bartol, F. F. (2006). Neonatal porcine endometrial development and epithelial proliferation affected by age and exposure to estrogen and relaxin. Domest. Anim. Endocrinol. ,, In press.
PubMed |

Medh, R. D. , and Thompson, E. B. (2000). Hormonal regulation of physiological cell turnover and apoptosis. Cell Tissue Res. 301, 101–124.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Morris, G. F. , and Mathews, M. B. (1989). Regulation of proliferating cell nuclear antigen during the cell cycle. J. Biol. Chem. 264, 13 856–13 864.
PubMed |

Narkar, M. , Kholkute, S. , Chitlange, S. , and Nandedkar, T. (2006). Expression of steroid hormone receptors, proliferation and apoptotic markers in primate endometrium. Mol. Cell. Endocrinol. 246, 107–113.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Nawaz, S. , Lynch, M. P. , Galand, P. , and Gerschenson, L. E. (1987). Hormonal regulation of cell death in rabbit uterine epithelium. Am. J. Pathol. 127, 51–59.
PubMed |

Otsuki, Y. (2001). Apoptosis in human endometrium: apoptotic detection methods and signaling. Med. Electron Microsc. 34, 166–173.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Reynolds, L. P. , Killilea, S. D. , and Redmer, D. A. (1992). Angiogenesis in the female reproductive system. FASEB J. 6, 886–892.
PubMed |

Roberto da Costa, R. P. , Branco, V. , Pessa, P. , Robalo Silva, J. , and Ferreira-Dias, G. (2005). Progesterone receptors and proliferating cell nuclear antigen expression in the equine luteal tissue. Reprod. Fertil. Dev. 17, 659–666.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Roberto da Costa, R. P. , Ferreira-Dias, G. , Mateus, L. , Korzekwa, A. , Andronowska, A. , Platek, R. , and Skarzynski, D. J. (2007). Endometrial nitric oxide production and nitric oxide synthases in the equine endometrium: relationship with microvascular density during the estrous cycle. Domest. Anim. Endocrinol. 34, 287–302.
Crossref | GoogleScholarGoogle Scholar |

Selam, B. , Kayisli, U. A. , Mulayim, N. , and Arici, A. (2001). Regulation of Fas ligand expression by estradiol and progesterone in human endometrium. Biol. Reprod. 65, 979–985.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Shikone, T. , Kokawa, K. , Yamoto, M. , and Nakano, R. (1997). Apoptosis of human ovary and uterine endometrium during the menstrual cycle. Horm. Res. 48, 27–34.
PubMed |

Sukjumlong, S. , Persson, E. , Kaeoket, K. , and Dalin, A. M. (2004). Immunohistochemical studies on oestrogen receptor alpha (ERalpha) and the proliferative marker Ki-67 in the sow uterus at oestrus and early pregnancy. Reprod. Domest. Anim. 39, 361–369.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Sukjumlong, S. , Dalin, A. M. , Sahlin, L. , and Persson, E. (2005). Immunohistochemical studies on the progesterone receptor (PR) in the sow uterus during the oestrous cycle and in inseminated sows at oestrus and early pregnancy. Reproduction 129, 349–359.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Taylor, K. M. , Gray, C. A. , Joyce, M. M. , Stewart, M. D. , Bazer, F. W. , and Spencer, T. E. (2000). Neonatal ovine uterine development involves alterations in expression of receptors for estrogen, progesterone, and prolactin. Biol. Reprod. 63, 1192–1204.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Van Cruchten, S. , Van den Broeck, W. , Duchateau, L. , and Simoens, P. (2003). Apoptosis in the canine endometrium during the estrous cycle. Theriogenology 60, 1595–1608.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Vaskivuo, T. E. , Stenback, F. , Karhumaa, P. , Risteli, J. , Dunkel, L. , and Tapanainen, J. S. (2000). Apoptosis and apoptosis-related proteins in human endometrium. Mol. Cell. Endocrinol. 165, 75–83.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Vaux, D. L. (1999). Caspases and apoptosis: biology and terminology. Cell Death Differ. 6, 493–494.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Vaux, D. L. , and Korsmeyer, S. J. (1999). Cell death in development. Cell 96, 245–254.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Waga, S. , Hannon, G. J. , Beach, D. , and Stillman, B. (1994). The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature 369, 574–578.
Crossref | GoogleScholarGoogle Scholar | PubMed |

Wasowska, B. , Ludkiewicz, B. , Stefanczyk-Krzymowska, S. , Grzegorzewski, W. , and Skipor, J. (2001). Apoptotic cell death in the porcine endometrium during the oestrous cycle. Acta Vet. Hung. 49, 71–79.
Crossref | GoogleScholarGoogle Scholar | PubMed |