Animal Reproduction (AR)
Animal Reproduction (AR)
Original Article

Leptin and IGF-I improve bovine embryo quality in vitro

Abdullah Kaya, Hakan Sağirkaya, Müge Misirlioğlu, Ahmet Gümen, John J. Parrish, Erdoğan Memili

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The in vitro embryo culture systems need further improvement to enhance the efficiency of bovine embryo production. Growth factors play key roles in embryo production and quality. The objective of this study was to define the effects of leptin, insulin-like growth factor-1 (IGF-1), and their combination on embryonic development, apoptosis, and expression profiles of a panel of developmentally important genes during 8-day embryo culture. The oocytes were aspirated from slaughterhouse ovaries of mixed breed cows. Following IVM/IVF presumptive zygotes were obtained. To accomplish this objective, presumptive zygotes (16-18 h post-insemination) were cultured in vitro as control (no supplementation, n = 349), 5 ng/ml leptin (Group I, n = 322), 100 ng/ml IGF-1 (Group II, n = 347), and 5 ng/ml leptin and 100 ng/ml IGF-1 (Group III, n = 360). All groups were supplemented with 10% fetal calf serum (FCS) on Day 4, and blastocysts were harvested on day 8. The DNAfragmented nuclei of blastocyst were determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and expression profiles of a panel of developmentally important genes were assayed by real-time polymerase chain reaction (RT-PCR). The cleavage rate and embryo development to 8-16 cell stage were higher in groups II and III as compared to control (P < 0.05), respectively. Percentage of blastocyst and mean cell numbers per blastocyst did not differ among the groups. Addition of IGF-I and/or combination with leptin decreased the number of nuclei with fragmented DNA (P < 0.01) as compared to the control group. Although the expression of glucose transporter 1 (Glut1), desmosomal glycoprotein desmocollin III (DcIII), and insulin like growth factor 2 receptor (Igf2r) transcripts did not change among the groups, interferon-tau (IF-tau) and DNA methyltransferase 3A (Dnmt3a) were down-regulated in group II while heat shock protein-70 (Hsp70) and IF-tau were up regulated in group III. Results indicate that addition of IGF-I in culture media improved the cleavage rate; combination with leptin also improved the development rates to 8–16-cell-stage embryos, decreased the TUNEL-positive nuclei, and caused alterations in the amounts of transcripts for the developmentally important genes assayed.


bovine, preimplantation development, apoptosis, leptin, IGF-I.


Arias-Alvarez M, Bermejo-Alvarez P, GutierrezAdan A, Rizos D, Lorenzo P, Lonergan P. 2011. Effect of leptin supplementation during in vitro oocyte maturation and embryo culture on bovine embryo development and gene expression patterns. Theriogenology, 75:887-896.

Barb C. 1999. The brain-pituitary-adipocyte axis: role of leptin in modulating neuroendocrine function. J Anim Sci, 77:1249-1257.

Block J, Drost M, Monson R, Rutledge J, Rivera R, Paula-Lopes F, Ocon O, Krininger C, Liu J, Hansen P. 2003. Use of insulin-like growth factor-I during embryo culture and treatment of recipients with gonadotropin-releasing hormone to increase pregnancy rates following the transfer of in vitro-produced embryos to heat-stressed, lactating cows. J Anim Sci, 81:1590-1602.

Boelhauve M, Sinowatz F, Wolf E, Paula-Lopes FF. 2005. Maturation of bovine oocytes in the presence of leptin improves development and reduces apoptosis of in vitro-produced blastocysts. Biol Reprod, 73:737-744.

Brill A, Torchinsky A, Carp H, Toder V. 1999. The role of apoptosis in normal and abnormal embryonic development. J Assist Reprod Genet, 16:512-519.

Byrne A., Southgate J, Brison D, Leese H. 1999. Analysis of apoptosis in the preimplantation bovine embryo using TUNEL. J Reprod Fertil, 117:97-105.

Christians E, Campion E, Thompson EM, Renard JP. 1995. Expression of the HSP 70.1 gene, a landmark of early zygotic activity in the mouse embryo, is restricted to the first burst of transcription. Dev Cambridge 121:113-113.

Cioffi JA, Van Blerkom J, Antczak M, Shafer A, Wittmer S, Snodgrass HR. 1997. The expression of leptin and its receptors in pre-ovulatory human follicles. Mol Hum Reprod, 3:467-472.

Craig JA, Zhu H, Dyce PW, Wen L, Li J. 2005. Leptin enhances porcine preimplantation embryo development in vitro. Mol Cell Endocrinol, 229:141-147.

Edwards JL, Ealy AD, Monterroso VH, Hansen PJ. 1997. Ontogeny of temperature-regulated heat shock protein 70 synthesis in preimplantation bovine embryos. Mol Reprod Dev, 48:25-33.

Fedorcsák P, Storeng R. 2003. Effects of leptin and leukemia inhibitory factor on preimplantation development and STAT3 signaling of mouse embryos in vitro. Biol Reprod, 69:1531-1538.

Garcia M, Amstalden M, Williams S, Stanko R, Morrison C, Keisler D, Nizielski S, Williams G. 2002. Serum leptin and its adipose gene expression during pubertal development, the estrous cycle, and different seasons in cattle. J Anim Sci, 80:2158-2167.

Herrid M, Nguyen VL, Hinch G, McFarlane JR. 2006. Leptin has concentration and stage-dependent effects on embryonic development in vitro. Reproduction, 132:247-256.

Jia Z, Zhang J, Wu Z, Tian J. 2012. Leptin enhances maturation and development of calf oocytes in vitro. Reprod Domest Anim, 47:718-723.

Jousan F, Hansen P. 2004. Insulin-like growth factor-I as a survival factor for the bovine preimplantation embryo exposed to heat shock. Biol Reprod, 71:1665-1670.

Kamalamma P, Kona S, Chakravarthi VP, Kumar AS, Punyakumari B, Rao V. 2016. Effect of leptin on in vitro development of ovine preantral ovarian follicles. Theriogenology 85: 224-229.

Kaneda M, Okano M, Hata K, Sado T, Tsujimoto N, Li E, Sasaki H. 2004. Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting. Nature, 429: 900-903.

Kawamura K, Sato N, Fukuda J, Kodama H, Kumagai J, Tanikawa H, Nakamura A, Tanaka T. 2002. Leptin promotes the development of mouse preimplantation embryos in vitro. Endocrinology, 143:1922-1931.

Kendall N, Gutierrez C, Scaramuzzi R, Baird D, Webb R, Campbell B. 2004. Direct in vivo effects of leptin on ovarian steroidogenesis in sheep. Reproduction, 128:757-765.

Khaki A, Batavani R, Najafi G, Tahmasbian H, Belbasi A, Mokarizadeh A. 2014. Effect of leptin on in Vitro nuclear maturation and apoptosis of buffalo (Bubalus bubalis) oocyte. Int J Fertil Sterl, 8:43.

Kšiňanová M, Čikoš Š, Babel'ová J, Šefčíková Z, Špirková A, Koppel J, Fabian D. 2017. The Responses of Mouse Preimplantation Embryos to Leptin In Vitro in a Transgenerational Model for Obesity. Front Endocrinol (Lausanne), 8:233.

Latham KE, Schultz RM. 2001. Embryonic genome activation. Front Biosci, 6:D748-759.

Lonergan P, Khatir H, Piumi F, Rieger D, Humblot P, Boland M. 1999. Effect of time interval from insemination to first cleavage on the developmental characteristics, sex ratio and pregnancy rate after transfer of bovine embryos. J Fertil Sterl, 117:159-167.

Memili E, First NL. 1999. Control of gene expression at the onset of bovine embryonic development. Biol Reprod, 61:1198-1207.

Misirlioglu M, Page G, Sagirkaya H, Kaya A, Parrish J, First N, Memili E. 2006. Dynamics of global transcriptome in bovine matured oocytes and preimplantation embryos. Proc Natl Acad Sci, 103:18905-18910.

Neuber E, Powers R. 2000. Is the mouse a clinically relevant model for human fertilization failures? Hum Reprod, 15:171-174.

Panda BSK, Pandey S, Somal A, Parmar MS, Bhat IA, Baiju I, Bharti MK, Sai Kumar G, Chandra V, Sharma GT. 2017. Leptin supplementation in vitro improved developmental competence of buffalo oocytes and embryos. Theriogenology. 98:116-122.

Parrish J, Krogenaes A, Susko-Parrish J. 1995. Effect of bovine sperm separation by either swim-up or Percoll method on success of in vitro fertilization and early embryonic development. Theriogenology, 44:859-869.

Paula-Lopes FF, Boelhauve M, Habermann FA, Sinowatz F, Wolf E. 2007. Leptin promotes meiotic progression and developmental capacity of bovine oocytes via cumulus cell-independent and-dependent mechanisms. Biol Reprod, 76:532-541.

Pfaffl MW. 2001. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res, 29:e45-e45.

Pfaffl MW, Horgan GW, Dempfle L. 2002. Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res, 30:e36-e36.

Prelle K, Stojkovic M, Boxhammer K, Motlik J, Ewald D, Arnold GJ, Wolf E. 2001. Insulin-Like Growth Factor I (IGF-I) and Long R3IGF-I Differently Affect Development and Messenger Ribonucleic Acid Abundance for IGF-Binding Proteins and Type I IGF Receptors in in Vitro Produced Bovine Embryos 1. Endocrinology, 142:1309-1316.

Rizos D, Gutierrez-Adan A, Perez-Garnelo S, De La Fuente J, Boland M, Lonergan P. 2003. Bovine Embryo Culture in the Presence or Absence of Serum: Implications for Blastocyst Development, Cryotolerance, and Messenger RNA Expression 1. Biol Reprod, 68:236-243.

Sagirkaya H, Misirlioglu M, Kaya A, First NL, Parrish JJ, Memili E. 2006. Developmental and molecular correlates of bovine preimplantation embryos. Reproduction, 131:895-904.

Swain JE, Dunn RL, McConnell D, GonzalezMartinez J, Smith GD. 2004. Direct effects of leptin on mouse reproductive function: regulation of follicular, oocyte, and embryo development. Biol Reprod, 71:1446-1452.

van Tol HT, van Eerdenburg FJ, Colenbrander B, Roelen BA. 2008. Enhancement of Bovine oocyte maturation by leptin is accompanied by an upregulation in mRNA expression of leptin receptor isoforms in cumulus cells. Mol Reprod Dev, 75:578-587.

Wrenzycki C, Herrmann D, Carnwath J, Niemann H. 1999. Alterations in the relative abundance of gene transcripts in preimplantation bovine embryos cultured in medium supplemented with either serum or PVA. Mol Reprod Dev, 53:8-18.

Wrenzycki C, Herrmann D, Lucas-Hahn A, Korsawe K, Lemme E, Niemann H. 2004. Messenger RNA expression patterns in bovine embryos derived from in vitro procedures and their implications for development. Reprod Fertil Dev, 17:23-35.

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