Elsevier

Theriogenology

Volume 114, 1 July 2018, Pages 317-323
Theriogenology

Simultaneous effects of IGF1 and Fadrozole on parthenogenesis and pluripotency markers in chicken embryo

https://doi.org/10.1016/j.theriogenology.2018.04.009Get rights and content

Highlights

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    Co-administration of IGF1 and Fadrozole improved development of parthenogenic embryos.

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    IGF1 and Fadrozole increased SOX2 and NANOG expression parthenogenetic embryos.

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    IGF1 and Fadrozole decreased the TBX3 expression in parthenogenetic embryos.

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    PRDM16, IGF2, NODAL and HDAC2 expressed very low in parthenogenetic embryos.

Abstract

So far, a synergistic effect was detected between insulin-like growth factor-I (IGF1) and anti-aromatase for sex reversal and pre-/post-natal growth of fertilized chicken embryo. Here, we hypothesized whether the growth and sexual female-to-male reversal effects of IGF1 and an anti-aromatase, Fadrozole, could improve the development of unfertilized, parthenogenetic chicken embryos. Simultaneous administration of IGF1 and Fadrozole increased the percentage of grade A embryos from 1.7% (no injection group) to 70.6%. The expression profile of parthenotes and newly laid fertilized embryos showed that IGF1 and Fadrozole increased SOX2 and NANOG expression, while decreased the TBX3 expression in the parthenogenetic embryos. However, a considerably higher expression of PRDM16, IGF2, NODAL and HDAC2 was observed in the fertilized group compared to the parthenogenetic embryos. In conclusion, chicken sexual determination is initiated at the earliest stage of embryonic development before gonadal differentiation. Combined administration of IGF1 and Fadrozole increased the developmental rate of parthenogenetic embryos. Also, simultaneous supplementation of IGF1 and Fadrozole induced the expression of pluripotency genes with no effect on the expression of growth and differentiation factors.

Introduction

Parthenogenesis is an unusual asexual way of reproduction in which an unfertilized oocyte starts cellular division, continues through embryonic development, and eventually gives rise to offspring without male genetic contribution. Notably, parthenogenesis is different from cloning where a diploid cell is transferred into the unfertilized oocyte and embryo development is initiated by chemical activation. Derivation of embryonic stem cells from parthenogenetic embryos, the most straight forward way by which many aspects of the ethical issues can be circumvented, has been a field of study for various applications in regenerative medicine such as cell therapy and tissue repair [1].

Avian species have unique parthenogenesis features that render them one of the most appropriate models to study the phenomenon of parthenogenesis. Parthenogenesis naturally takes place in various bird species, such as Zebra Finch and Chines painted quail [2,3]. More importantly, the parthenogenetic embryo can develop into a live bird by ‘virgin birth’ [2] as observed in at least one domestic bird species, the turkey [4]. However, all parthenogenetic hatchlings of turkeys have been male [4]. Also, a significant bias toward the Z chromosome was detected in developing parthenogenetic chicken embryos [5]. Avian spermatozoa normally contain only Z chromosomes, while the oocyte can carry either Z or W chromosomes. Use of sperm cells which were produced from female-to-male sex-reversed chicks showed that healthy blastocysts can be produced from sperm cells which carried the W chromosome [6]. This suggests that a paternally-inherited Z chromosome is not a strict requirement for the embryonic development in avian. However, it is not completely understood if the Z chromosome dosage or the W chromosome superiority, or multi-optional mechanisms determine the avian sex [7]. On the other side, it has been suggested that sex-determining genes in birds are expressed at the earliest stages of embryonic development, prior to the gonadal differentiation [7,8]. However, it has yet to be determined if male specific factors are indispensable for the development of parthenogenetic embryos before the gonadal differentiation. Therefore, in this study, we investigated whether combined effects of insulin-like growth factor-I (IGF1) and an anti-aromatase, Fadrozole, on growth and sexual female-to-male reversal [9] would improve the development of parthenogenetic chicken embryos. The differential gene expression pattern in the parthenogenetic embryos was also evaluated for a series of paternally imprinted, embryonic stem cell, and growth and differentiation genes.

Section snippets

Injection materials

Iranian native fowl in Isfahan has been subjected to a long term breeding plan which is composed of selection and cross-breeding strategies to meet the national market demands of meat and egg [10]. Unfertilized chicken eggs were purchased from the Iranian Native Fowl Breeding Center, Isfahan, April 2015. The flock was at 30–34 weeks of the production period. The eggs were stored flat end upright for 24 h at 20 °C before the injection process. This step was helpful for the air sac formation as

Combined effect of IGF1 and Fadrozole on chicken parthenogenesis

The growth of the unfertilized embryos was classified into three grades (Fig. 1). The grade A embryos had the highest quality with some similarity to HH1-3 while parthenogenic embryos which were classified as grade B and C did not have embryonic structure compared to the day 0 fertile embryo (HH1). Injection of the sham injection solution increased the grade B embryos compared to the non-injection group (p-value < 0.05). This inducement in embryonic development could be due to either the sham

Discussion

Chickens have a negligible rate of parthenogenesis, however by implementing a long-term breeding plan in Cornish chickens the rate of parthenogenesis was increased up to 3% [14]. In the current study, the occurrence of grade A growth of the unfertilized embryos was very rare in Iranian native fowl. Supplementation of IGF1 led to more than a tenfold increase in the rate of embryonic development. Moreover, a very strong combined effect of IGF1 and Fadrozole was detected on the developmental rate

Acknowledgements

This study was carried out at Isfahan branch of Islamic Azad University, Isfahan, Iran. Authors would like to thank Guojun Sheng from the International Research Center for Medical Sciences (IRCMS) at Kumamoto University, Japan for kindly providing sex determination primers and Stefan Wagner from the Rowett Institute at the University of Aberdeen for editing the manuscript, and Mr. Majid Farahi from Islamic Azad University, Iran for processing the figures.

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