Roza Helmita, Ita Djuwita, Bambang Purwantara


Implantation is the most critical stage in the establishment of pregnancy. In mammals, it has been estimated that between 25% and 60% of conceptuses are lost before or at the time of implantation. The objectives of  this study were to investigate  the ability of blastocyst hatching and attachment of trophoblast cells in in vitro, the outgrowth and differentiation of trophoblast cells in in vitro culture of hatched and non hatched blastocyst, the activity of mitochondria NADH-CoQ reductase and the pattern of mitochondrial distribution. Blastocysts were collected from mice cornua utery at day-4 of pregnancy and were divided into 3 groups: blastocysts undergo hatching within 24 hours, 48 hours and non hatching. Embryos were cultured in DMEM medium in 5% CO2 incubator at 37°C for 10 days. The trophoblasts monolayer were processed for Giemsa staining and histochemistry analysis of NADH-tetrazolium reductase activity. The outgrowth of trophoblast cells were measured using eyespiece micrometer. The results showed the ability of blastocyst hatching in in vitro were different. The trophoblast outgrowth diameter of 24 h hatched blastocyst was significantly different with the 48 h hatched and non hatched blastocyst. The activity of NADH-CoQ reductase of 24 h and 48 h hatched blastocysts showed higher intensity than the non hatched blastocysts (P<0,05) and the distribution of mitochondrial in trophoblast cell cytoplasma of 24 h and  48 h hatched blastocyst were homogen around nucleus whereas thoes of non hatched blastocyst were cluster and heterogen. In conclusion, in in vitro study the failure of blastocysts hatching and implantation was due to the failure of outgrowth and differentiation of the trofoblast cells and the impairment of NADH-CoQ reductase activity in complex I mitochondria to produce energy and the pattern of mitochondrial distribution.

Key words: mitochondrial distribution, NADH- tetrazolium reductase, trophoblast, implantation 


Acton BM, Jurisicova A, Jurisica I, Casper RF. 2004. Alterations in mitochondrial membrane potential during preimplantation stages of mouse and human embryo development. Mol Hum Reprod. 10 (1) : 23-32.

Aplin JD, Kimber SJ. 2004. Trophoblast-uterine interactions at implantantion. Reprod Biol Endocrinol. 2: 48 doi: 10.1186/1477-7827-2-48.

Babawale MO, Mobberley MA, Ryder TA, Elder MG, Sullivan MHF.2002. Ultrastructure of the early human feto-maternal interface co-cultured in vitro. Hum Reprod. 17(5): 1351-1357.

Blerkom JV. 2004. Mitochondria in human oogenesis and preimplantation embryogenesis: engines of metabolism, ion regulation and developmental competence. Reproduction. 128: 269-280.

Blerkom JV, Cox H, Davis P. 2006. Regulatory roles for mitochondria in the peri-implantation mouse blastocyst: possible origins and developmental significance of differential. Reproduction 131: 961–976.

Brookes PS et al. 2004. Calcium, ATP, and ROS: a mitochondrial love-hate triangle. Am J Physiol Cell Physiol. 287: C817-C833.

Cartwright JE et al. 2002. Trophoblast invasion of spiral arteries: A novel in vitro model. Placenta. 23: 232–235.

Collins et al. 2005. ESHRE capri workshop group: Fertility and ageing. Hum Reprod. 11(3): 261–276.

Cummins JM.2001. Mitochondria: Potential roles in embryogenesis and nucleocytoplasmic transfer. Hum Reprod. 7 (2): 217-228.

Davidson M W.2006. Mitochondria. Molecular expression. http://www.microscopy.fsu.edu/cells/animals/mitochondria.html. 26 november 2006.

Dey SK et al. 2004. Molecular cues to implantation. Endocrine Reviews. 25(3): 341-373.

Dimauro S, Schon EA. 2003. Mitochondrial respiratory-chain desease. N Engl J Med. 348 (26): 2656-2668.

Dominguez F, Ya´n˜ez-Mo M, Madrid SF, Simo´n C. 2005. Embryonic implantation and leukocyte transendothelial migration: different processes with similar players?. The FASEB Journal. 19.

Fong CY et al. 2001. Ultrastructural observations of enzymatically treated human blastocysts: zona-free blastocyst transfer and rescue of blastocysts with hatching difficulties. Hum Reprod.16(3): 540-546.

Gardner DK, Lane M, Schoolcraft WB. 2002.Physiology and culture of the human blastocyst. J Reprod Immun. 55:85-100.

Goetz MZ. 2002. Patterning of the embryo: the first spatial decisions in the life of a mouse. Development. 129:815-829.

Grivennikova VG, Kapustin AN, Vinogradov AD. 2001. Catalytic activity of NADH-ubiquinone oxidoreductase (Complex I) in intact mitochondria. J. Biol Chemistry. 276 (12) : 9038–9044.

Harun R. 2006. Cytotrophoblast stem cell lines derived from human embryonic stem cells and their capacity to mimic invasive implantation events. Hum Reprod. 21(6): 1349-1358.

Harvey AJ, Kind KL, Thompson JG. 2002. Redox regulation of early embryo development. Reproduction. 123: 479-486.

Hashash AHK El dan Kimber SJ. 2004. Trophoblast differentiation in vitro: establishment and characterisation of a serum-free culture model for murine secondary trophoblast giant cells. Reproduction. 128:53-71.

Haupt S, Berger M, Goldberg Z, Haupt Y. 2003. Apoptosis – the p53 network. J Cell Sci. 166(20): 4077-4085.

Hogan B, Beddington R, Costantini F, Lacy E.1994. Manipulating the mouse embrio: a laboratory manual. 2nd Ed. New York USA. Cold Spring Harbor Laboratory Press. Pp: 49

Horse KR et al. 2004. Trophoblast differentiation during embryo implantation and formation of the maternal-fetal interface J Clin Invest. 114:744-754.

Jauniaux E, Hempstock J, Greenwold N, Burton GJ. 2003. Trophoblastic oxidative stress in relation to temporal and regional differences in maternal placental blood flow in normal and abnormal early pregnancies. Am J Patho.162:115-125.

James JL, Stone PR, Chamley LW. 2005. Cytotrophoblast differentiation in the first trimester of pregnancy: evidence for separate progenitors of extravillous trophoblasts and syncytiotrophoblast. Reproduction 130: 95-103.

John JC. 2002. The trasmission of mitochondrial DNA following assisted reproductive techniques. Theriogenology. 57:109-123.

Joswig A, Gabriel HD, Kibschull M, Winterhager E. 2003. Apoptosis in uterine epithelium and decidua in response to implantation: evidence for two different pathways. Reprod Biol Endocrinol. 1 (44):1477-7827.

Kiernan JA. 1990. Histological and histochemical methods. 2nd Ed. New York USA. Pergamon Press. Pp:108; 345

Kimber SJ dan Spanswick C. 2000. Blastocyst implantation: The adhesion cascade. Cell & Develop Biol. 11: 77-92.

Klobuear NK dan Gorup M. 2004. Histochemical investigations of NADPH-and NADH-tetrazolium reductase Actvity in the Liver of Selenium annd Copper Treated Carp (Cyprinus carpio L.). Veterinarski Arhiv. 74 (5): 331-340.

Ludwig TE, Squirrell JM, Palmenberg AC, Bavister BD. 2001. Relationship between development, metabolism, and mitochondrial organization in 2-cell hamster embryos in the presence of low levels of phosphate1. Biol Reprod. 65: 1648–1654.

Lunghi et al. 2007. Control of human trophoblast function. Reprod Biol Endo. 5:1477-7827.

Malik S, Sudoyo H, Marzuki S. 2000. Microphotometric analysis of NADH-tetrazolium reductase deficiency in fibroblast of patients with leber hereditary optic neuropathy. J Inherit Metab. 23:730-744.

Nadra K et al. 2006. Differentiation of trophoblast giant cells and their metabolic functions are dependent on peroxisome proliferator-activated receptor ß/δ. Mol Cell Biol. 26:3266-3281.

Nagai et al. 2006. Correllation of abnormal mitochondrial distribution in mouse oocytes with reduce developmental competence. J Exp Med. 210:137-144.

Norwitz ER, Chust, JS, Fisher JF. 2001. Implantation and the survival of early pregnancy. N Engl J Med. 345(19).

Summers MC , Biggers JD. 2003. Chemically defined media and the culture of mammalian preimplantation embryos: historical perspective and current issues. Hum Reprod. 9 (6 ): 557-582.

Straszewski-Chavez SL, Abrahams VM, Mor G. 2005. The role of apoptosis in the regulation of trophoblast survival and differentiation during pregnancy. Endocrine Reviews. 26 (7):877–897.

Tamassia et al. 2004. In vitro embryo production effciency in cattle and its association with oocyte adenosine triphosphate content, quantity of mitochondrial DNA, and mitochondrial DNA haplogrup. Biol Reprod. 71:697–704.

Tayade C, Black GP, Fang Y, Croy BA.2005. Differential gene expression in endometrium, endometrial lymphocytes, and trophoblasts during successful and abortive embryo implantation. J Immunology. 0022-1767.

Trimachi RJ et al.2000. Oxidative phosphorylation-dependent and -independent oxygen consumption by individual preimplantation mouse embryos. Biol Reprod. 62: 1866-1874.

Turrens JF. 2003. Mitochondrial formation of reactive oxygen species. J Physiol. 552: 335-344.

Vogel RO et al. 2005. Human mitochondrial complex I assembly is mediated by NDUFAF1. FEBS J. 272 : 5317–5326.

Wang X. 2001. The expanding role of mitochondria in apoptosis. Gen & Develop. 15:2922–2933.

Zess CJ. 2005. The apoptosis-necrosis continuum: Insights from genetically altered mice. Vet Pathol .40:481–495.

DOI: http://dx.doi.org/10.31958/js.v7i1.121


  • There are currently no refbacks.

Copyright (c) 2016 Roza Helmita, Ita Djuwita, Bambang Purwantara

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.


Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.