The efficacy of human recombinant luteinizing hormone for in vitro embryo production in sheep

Year 2025, Volume: 9 Issue: 1, 19 - 24, 28.03.2025

Ramazan Arıcı , Kamber Demir , Selin Yağcioğlu , Gül Bakirer Ozturk , Ahmet Eser , Nur Ersoy , Israa Faris , Andaç Kılıçkap , Mithat Evecen , Sema Birler , Serhat Pabuccuoğlu

Abstract

Objective: This study was carried out to assess the ability of recombinant human luteinizing hormone (r-LH) to be used instead of sheep hypophyseal luteinizing hormone (h-LH) in the maturation of sheep oocytes and its influence on embryonic development and quality.
Materials and Methods: The oocytes were obtained from slaughtered sheep ovaries. For maturation, oocytes (grade 1) were incubated at 38.5°C, 5% CO2 for 24 hours. 0.1 IU/ml LH (from sheep Piturity LH, Sigma®) was added to the culture media of the hypophyseal LH (h -LH) group and 0.1 IU/ml LH (Luveris® 75 IU, Serono) to the Recombinant LH (r-LH) group. In vitro fertilization (20h) and embryo culture were performed at 5% CO2, 5% O2 and 38.5°C incubation conditions. The maturation rates were reported based on the MII stage chromosomal formation and the existence of polar body by bisBenzimide (Hoechst 33342). Embryonic developments were controlled at 3rd and 8th day of in vitro culture. For the embryonic cell count and determination of inner cell mass (ICM) and trophectoderm cell (TC), the differential staining technique was used with Hoechst 33342 + Propidium Iodide (PI).
Results: The proportion of cleavage (%), the rate of embryos developing the morula (%) and blastocyst stage (%), and the ICM, TC, and total cell numbers of the embryos were found to be statistically similar in the h-LH and r-LH groups.
Conclusion: It was concluded that r-LH could be used as an alternative LH source instead of hypophyseal LH.

Keywords

Embryo development, hypophyseal LH, in vitro maturation, recombinant human LH, sheep oocyte

Ethical Statement

This study was approved by the Experimental Animals Local Ethics Committee at the İstanbul University with decision number 2017-295820. The authors confirm that they have adhered to ARRIVE Guidelines to protect animals used for scientific purposes.

Supporting Institution

The Scientific Research Projects Coordination Unit of Istanbul University- Cerrahpasa

Project Number

TSG-2017-20659

References

• Accardo C, Dattena M, Pilichi S, Mara L, Chessa B, Cappai P. Effect of recombinant human FSH and LH on in vitro maturation of sheep oocytes; embryo development and viability. Anim Reprod Sci. 2004; 81(1-2):77-86.
• Adhikari D, Liu K. The regulation of maturation promoting factor during prophase I arrest and meiotic entry in mammalian oocytes. Mol Cell Endocrinol. 2014; 382(1): 480-487.
• Andresiez C, Ferraretti AP, Magli C, et al. Effect of recombinant human gonadotrophins on human, bovine and murine oocyte meiosis, fertilization and embryonic development in vitro. Hum Reprod. 2000; 15(5):1140-1148.
• Arıcı R, Ak K, Birler S, et al. Effects of melatonin addition to the cold storage medium on cumulus oocyte complex apoptosis, viability and in vitro maturation rates of cat oocytes. Kafkas Univ Vet Fak Derg. 2022; 28(2):193-200.
• Arroyo A, Kim B, Yeh J. Luteinizing hormone action in human oocyte maturation and quality: signaling pathways, regulation, and clinical impact. Reprod Sci. 2020; 27(6): 1223-1252.
• Baruselli PS, Abreu LÂD, Catussi BLC, et al. Use of new recombinant proteins for ovarian stimulation in ruminants. Anim Reprod. 2023; 20(2): e20230092.
• Birler S, Pabuccuoğlu, Atalla H, et al. Transfer of in vitro produced sheep embryos. Turk J Vet Anim Sci. 2002; 26(6):1421-1426.
• Birler S, Pabuccuoğlu S, Alkan S, et al. Effects of supplements to maturation and fertilization media and co-culture with sheep oviductal epithelial cells on in vitro production of sheep embryos. Istanbul Univ Vet Fak Derg. 2003; 29(1):157-165.
• Conti M, Hsieh M, Zamah AM, Oh JS. Novel signaling mechanisms in the ovary during oocyte maturation and ovulation. Mol Cell Endocrinol. 2012; 356(1-2): 65-73.
• De Koning WJ, Walsh GA, Wrynn AS, Headon DR. Recombinant reproduction. Nat Biotechnol. 1994; 12(10):988-992.
• De la Fuente R, King WA. Use of a chemically defined system for the direct comparison of inner cell mass and trophectoderm distribution in murine, porcine and bovine embryos. Zygote. 1997; 5(4):309-320.
• Demir K, Evecen M, Arıcı R, et al. In vitro effect of recombinant human gonadotropins on meiotic competence of dog oocytes. Turk J Vet Anim Sci. 2019; 43(4):469-473.
• Gifre L, Aris A, Bach A, Garcia-Fruitos E. Trends in recombinant protein use in animal production. Microb Cell Factories. 2017; 16(1):1-17.
• Hesser MW, Morris JC, Gibbons JR. Advances in recombinant gonadotropin production for use in bovine superovulation. Reprod Domest Anim. 2011; 46(5):933-942.
• Jaffe LA, Egbert JR. Regulation of mammalian oocyte meiosis by intercellular communication within the ovarian follicle. Annu Rev Physiol. 2017; 79(1): 237-260.
• Kanitz W, Becker F, Schneider F, et al. Superovulation in cattle: practical aspects of gonadotropin treatment and insemination. Reprod Nutr Dev. 2002; 42(6):587-599.
• Krisher RL. In vivo and in vitro environmental effects on mammalian oocyte quality. Annu Rev Anim Biosci. 2013; 1(1):393-417.
• Leppens G, Gardner DK, Sakkas D. Co-culture of 1-cell outbred mouse embryos on bovine kidney epithelial cells: effect on development, glycolytic activity, inner cell mass: trophectoderm ratios and viability. Hum Reprod. 1996; 11(3):598-600.
• Lonergan P, Fair T, Corcoran D, Evans ACO. Effect of culture environment on gene expression and developmental characteristics in IVF-derived embryos. Theriogenology. 2006; 65(1):137-152.
• Mehlmann LM. Stops and starts in mammalian oocytes: recent advances in understanding the regulation of meiotic arrest and oocyte maturation. Reproduction. 2005; 130(6): 791-799.
• Moor RM, Trounson AO. Hormonal and follicular factors affecting maturation of sheep oocytes in vitro and their subsequent developmental capacity. Reprod. 1977; 49(1):101-109.
• Paramio MT, Izquierda D. Recent advances in in vitro embryo production in small ruminants. Theriogenology. 2016; 86(1):152-159. Rizos D, Fair T, Papadopoulos S, Boland M, Lonergan P. Developmental, qualitative, and ultrastructural differences between ovine and bovine embryos produced in vivo or in vitro. Mol Reprod Dev: Incorporating Gamete Research. 2002; 62(3):320-327.
• Rosano LG, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol. 2014; 5:1-17.
• Sha W, Xu B, Li M, Liu D, Feng HL, Sun QY. Effect of gonadotropins on oocyte maturation in vitro: an animal model. Fertil Steril. 2010; 93(5):1650-1661.
• Shirazi A, Shams‐Esfandabadi N, Ahmadi E, Heidari B. Effects of growth hormone on nuclear maturation of ovine oocytes and subsequent embryo development. Reprod Domest Anim. 2010; 45(3): 530-536.
• Tharasanit T, Thongkittidilok C, Sananmuang T, Techakumphu M. Recombinant human follicle stimulating hormone and growth factors improve the meiotic and developmental competence of cat oocytes. Thai J Vet Med. 2014; 44(1):107-115.
• Thouas GA, Korfiatis NA, French AJ, Jones GM, Trounson AO. Simplified technique for differential staining of inner cell mass and trophectoderm cells of mouse and bovine blastocyst. Reprod Biomed Online. 2001; 3(1):25-29.
• Törnell J, Bergh C, Selleskog U, Hillensjö T. Effect of recombinant human gonadotrophins on oocyte meiosis and steroidogenesis in isolated pre-ovulatory rat follicles. Mol Hum Reprod. 1995; 1(4):219-222.
• Vanroose G, Van Soom A, De Kruif AD. From co-culture to defined medium: state of the art and practical considerations. Reprod Domest Anim. 2001; 36(1):25-28.
• Wani NA. In vitro Maturation and in vitro fertilization of sheep oocytes. Small Rumin Res. 2002; 44(2):89-95.
• Zhu J, Moawad AR, Wang CY, Li HF, Ren JY, Dai YF. Advances in in vitro production of sheep embryos. Int J Vet Sci. 2018; 6:15-26.