Simultaneous effects of IGF1 and Fadrozole on parthenogenesis and pluripotency markers in chicken embryo
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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2019, GeneCitation Excerpt :The positive effect of an antiaromatase (fadrozole) on the weight gain was also reported in chicken (Mohammadrezaei et al., 2014). In addition, it has been verified that antiaromatases have synergistic interaction with growth factors, particularly IGF1 (Hajihoseini and Eghbalsaied, 2018). Higher levels of estradiol was detected in granulosa cells and follicular fluid of low-AFC compared to high-AFC ovaries in cows experiencing follicular phase (Ireland et al., 2009) whereas Downregulation of Cyp19A1 (the gene which encodes aromatase) was detected in high-AFC (antral follicle count) ovaries (Foroughinia et al., 2017).
Cotreatment of igf1 and fadrozole upregulates the expression of rspo1, sox9, and amh in chicken embryos
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