QC check, filtration and alignment
The differential gene expression in the right ovary of chicken on E6, E12 and D1 were studied using data (SRR4029458, SRR4029457, SRR4029464, SRR4029463, SRR4029460, SRR4029459) downloaded from NCBI. The obtained raw data was found to have a total of 121.177678 million reads. The FastQC report was found to have satisfactory quality parameters and do not have any adapter sequences. A total of 108.506697 million processed reads were obtained after filtration for its quality (Q30) using Trimmomatic. When the processed reads were aligned to the
Gallus gallus reference genome (GRCg7b), 76.836692 million reads were mapped resulting in a mapping percentage of 70.81. The mapped reads with SRA processing summary was given in Table 1.
Quantification of differentially expressed genes
There were 373 and 520 differentially expressed significant up-regulated and down-regulated genes respectively from E6 to E12. Similar to this, 708 and 1136 genes, respectively, were shown to be significantly up-regulated and down-regulated during E12 to D1. The genes that were significantly up-regulated during E12 were FGG, APOH, AHSG, HSD17B1, NME7, PROCA1, MLKL and down-regulated during E12 were NANOG, STEAP3, CALML3. The genes DMRT1, HINTW, FOXL2, CYP19A1 , PIWIL1, TDRD5, DND1, FGG, APOH, AHSG, HSD17B1, NME7, CALML3, PROCA1, MLKL
etc. were down-regulated during D1 and STEAP3, NANOG, TRAF5 etc were up-regulated during D1. These DEGs genes were discovered to be involved in pathways that inhibit fibrinolysis, thrombosis, lipase activity, regulation of insulin signalling, programmed cell death, ferroptosis, phosphorylation reactions, tumour necrosis factor (TNF)-induced necroptosis
etc. A volcano plot demonstrating all differentially expressed genes between E6 to E12 and E12 to D1 are shown in Fig 2a and Fig 2b.
Enrichment analysis
HSD17B1 gene was upregulated (0.685248) during early embryonic stage (E6 to E12) and down related (-5.58248) in the late embryonic stages (E12 to D1). The HSD17B1 is involved in estradiol and testosterone synthesis in mouse
(Mindnich et al., 2004) 11 beta hydroxytestosterone and estradiol 17 beta hormonal production is decreased in the right ovary during later embryonic stages suggesting the regression of the right ovary (KEGG:00140). STEAP3 gene was down regulated (-4.76293) in the early embryonic stage and is then upregulated (1.387534) in the late embryonic stages. STEAP3 involved in fenton reaction triggering ferroptosis suggesting its role in the oxidative damage to the cell membrane which further leads to the cell death in the right ovary in the late embryonic stages (KEGG:04216).
Ye et al., 2022 reported higher expression of STEAP3 gene increases sensitivity to ferroptosis. The NME7 gene was upregulated (7.082978) in the early embryonic stage and is then down related (-1.75833) in the late embryonic stages. NME7 gene is involved in phosphorylation ofdADP to dATP, dGDP to dGTPdIDP to dIT, ADP to ATP, GDP to GTP, IDP to ITP dTDP to dTTP, dUDP to dUTP, dCDP to dCTP and vice versa, suggesting apoptosis due to reduced purine and pyrimidine metabolisim for the DNA synthesis in the later embryonic stages (KEGG:00240, KEGG:00230). CALML3 gene was down regulated (-4.57281) in the early embryonic stage (E6 to E12) and upregulated in late embryonic stages (E12 to D1) (-2.41755). Studies reported overexpression of CALML3 significantly prevents the apoptosis
(Jia et al., 2018). Downregulation of CALML3 is involved in inhibition of glycogen synthesis leading to disruption of glucose homeostasis leading to apoptosis (KEGG: 04910). It also leads to the reduced gonadotropin gene expression and secretion which in turn influences the release of hormones from the ovary (KEGG: 04912). CALML3 is highly down regulated preventing the contraction of vascular smooth muscles, thereby enabling better blood circulation to the ovarian tissue in early embryonic stages than the later embryonic stage (KEGG:04270). PROCA1 gene was upregulated (4.013282) in the early embryonic stage and is then down related (-3.83398) in the late embryonic stages. Downregulation of PROCA1 leading to Ca
2+ influx into vascular smooth muscle cells enhancing its contraction which may further prevent the blood supply to the ovarian tissue in later embryonic stages (KEGG:04270). The up regulation (4.902271) of MLKL gene during the early embryonic stage resulted in dephosphorylation of Drpl gene leading to tumor necrosis factor (TNF) induced necroptosis through mitochondrial fission
(Remijsen et al., 2014) (KEGG:04217). The differentially expressed genes and their upregulation and down regulation was given in Fig 3 and Table 2.
The Fibrinogen gamma chain (FGG) gene is involved in the regulation of fibrinolysis and thrombosis
(Ma et al., 2007). FGG gene was upregulated (5.75) in the early embryonic stage and is then down related (-0.18) in the late embryonic stages. Upregulation of FGG during E12 day in the right ovary suggests increased vasoconstriction that may lead to reduced blood flow in the right ovary causing its regression. Apolipoprotein H (Apo-H) gene was upregulated (5.2) in the early embryonic stage, than during the late embryonic stages (0.47) leading to fibrinolysis
(Crook et al., 2001). Angiopoietin-like protein 3 (ANGPTL3) regulates the lipid catabolism
(Adam et al., 2020) and was upregulated (6.32) in the early embryonic stage and is then down related (-2.9482) in the late embryonic stages. Overexpression of this gene cause increased activity of lipoprotein lipase that may result in degeneration of the right ovary. Reduced fertility and abnormal follicular development were reported in estrogen receptor- b knockout (BERKO) mice
(Cheng et al., 2002) and found its correlation with ITIH2 expression
(Hamm et al., 2008). ITIH2 gene was upregulated (5.99) in the early embryonic stage resulting in reduced oestrogen receptors leading to regression and reduced fertility of right ovary in chicken. Alpha-2-HS-glycoprotein (AHSG) gene was upregulated (7.2) in the early embryonic stage than late embryonic stages (2.43). This gene is involved in glucose metabolism and the regulation of insulin signalling. AHSG may affect glucose uptake and lipid oxidation in adipocytes
(Zhuo et al., 2015). TRAF5 proteins is involved in programmed cell death (
Muzib, 1998) and its overexpression induced the apoptosis of mouse podocytes
(Wu et al., 2018).
The reduction of the DMRT1 protein expression in-ovo resulted in feminization of the embryonic gonads in genetically male (ZZ) embryos
(Smith et al., 2009). In early-stage chicken embryos, the HINTW probe ubiquitously and robustly labelled all female cells. According to
Nagai et al., (2014), this probe can be employed in developmental research to differentiate intraspecific, inter-sex donor/host tissues. FOXL2 (Fork head box L2) is only expressed in female embryos and is essential for controlling ovarian development
(Luo et al., 2020). A crucial gene for the feminization of the gonad in chicken embryos, Cyp19a1 encodes an aromatase that catalyses the conversion of progesterone to oestrogen
(Ellis et al., 2012). This gene has been identified as being expressed exclusively in females from the earliest stages of gonadal sex differentiation. PIWIL1 plays a crucial part in gametogenesis by inhibiting transposable elements and blocking their mobilisation. PiRNAs plays an important role in the avian ovarian asymmetry
(Shaikat et al., 2018, Zhang et al., 2021). The major differentially expressed ovarian genes are strongly linked to phagosome function, carbon metabolism, neuroactive ligand-receptor interaction and calcium signalling all of which accelerate the degeneration of the right ovary
(Yu et al., 2017). The ageing of the cell and the initiation of the inflammatory response results in degeneration of right ovary in ducks and geese
(Ran et al., 2023).