The magnum is highly glandular tissue, and molecules secreted and/or transported from the luminal and glandular epithelium contribute to the egg albumen. The egg remains in the magnum for 1–3 h to complete the deposition of albumen around the yolk. In the first few hours of the ovulation cycle (1–3 h p.o.), the egg is in the magnum, during which the stored proteins from the magnum epithelium are secreted in the lumen [6]. In the later period of the ovulation cycle (4–23 h p.o.), immediately after the egg has left the magnum, the protein synthesis process begins and continues until the next egg reaches the magnum [6,7,8]. Using RNA-Sequencing and qPCR, we identified several novel genes and biological pathways associated with egg albumen formation.
In the present study, RNA-Seq data revealed a total of 540 genes differentially expressed between laying (at 3 h p.o.) and non-laying hens. As previously reported, we observed increased expression of genes encoding the common egg-white proteins such as ovalbumin, lysozyme, and avidin in the magnum when the egg was present in this segment of the oviduct [11, 17, 18]. Amongst the DEGs, several proteases (TMPRSS9, ACE, REN, MMP1, MMP9, MMP10, CAPN2, and PROC) and enzymes for biosynthesis (PHGDH, PSPH, PSAT1, and ASNS) were of particular interest. Some of these genes were detected in the microarray and RNA-Seq studies in the magnum [11, 19]. However, their potential role in the formation of egg-white was not reported. In this study, we validated and assessed the specificity of these identified novel genes and pathways in the laying (3 h and 15–20 h p.o), molting, and non-laying hens using qPCR. Then, we used their expression profile to extrapolate their novel role in the synthesis and/or secretion of egg-white proteins based on existing literature. The newly identified genes were involved in antimicrobial defense, matrix remodeling, albumen synthesis and/or secretion, and egg transport (Fig. 6).
Proteases associated with the albumen synthesis and secretions
Proteases are enzymes having catalytic activity on proteins. There are seven different classes (based on catalytic residue) of proteases, including serine proteases and metalloproteases [20]. Both the serine- and metallo-proteases actively regulate the protein turnover of the extracellular matrix (ECM), influencing various cellular functions [21]. Our RNA-Seq data showed that TMPRSS9 mRNA was the second most upregulated DEG (FC = 33) in laying hens when the egg was inside the magnum. TMPRSS9, also known as polyserase-I, is a transmembrane type II serine protease that uniquely produces three other proteases, including 2 active ones [22]. TMPRSS9 facilitates the formation of urokinase plasminogen activator that converts plasminogen to plasmin responsible for the degradation of ECM components [23]. Higher expression of TMPRSS9 uniquely in laying hens suggests that it potentially participates in the degradation of the ECM to release the stored proteins making them available for receptor binding and signaling action, as proposed by ten Dijke et al. [24]. This process is indeed relevant in laying hens when the egg is present in the magnum or the shell gland to keep up with the enormous amount of protein synthesis and secretion.
Matrix metalloproteases (MMPs) are the primary regulators of ECM remodeling. There are 24 members of the MMPs family in vertebrates, including MMP-1, − 9, and − 10, which are secreted proteins involved in a wide range of physiological activities such as cellular migration and angiogenesis, and inflammation [25, 26]. In this study, the expressions of MMP-1, MMP-9, and MMP-10 were upregulated (FC = 16, 7.5, and 3.5, respectively) in the laying hens with the presence of egg in the magnum, compared to the non-laying hens. MMP1 is down-regulated in magnum when the hen transitions from laying to molting stage [11]. MMP1, also known as collagenase, can degrade the most highly abundant ECM; collagen, in several tissues, including the chicken ovary [27]. MMP-9 (gelatinase) is known to degrade the gelatin matrix [28], provokes angiogenesis [29], and also regulates the laying process in hen [15]. MMP10 breaks down several collagen-related connective tissues [30]. There is no report of MMP10 mRNA expression in the chicken oviduct; however, a metastatic study has confirmed its association in angiogenesis [31]. Several proteins need to be synthesized and transported into the lumen for deposition around the egg yolk for albumen formation. The required proteins are synthesized in the tubular gland cells of the magnum, which require the rapid transport of amino acids from the blood circulation [6]. We speculate that the higher expression of MMP-1, MMP-9, and MMP-10 in the magnum of laying hens are associated with the tissue remodeling and formation of new vasculatures to support the expeditious conveyance of precursor molecules for the biosynthesis of egg-white proteins.
Calpains, on the other hand, are ubiquitous intracellular cysteine proteases having very low specificity for recognition of amino acid sequence. Calpains have a wide range of functions in various tissues, including membrane repair, cell adhesion and motility, cell death, protein cleavage, and activation [32]. Our study reports an increased expression of CAPN2 in the laying hens during 15–20 h p.o. compared to either molting or non-laying hens. Similarly, the expression of CAPN2 is higher in hens at the laying stage than in the molting stage [11]. Therefore, we posit that CAPN2 is responsible for the maturation and activation of the synthesized egg-white proteins.
Also, we observed that serine protease inhibitor family B member 2 (SERPINB2) was higher (> 3-fold) in the magnum, and similar up-regulation of SERPINB3 expression in the magnum of laying hens was reported by Jeong et al. [11]. Recently, Zhang et al. [33] also reported the upregulation of SERPINF1 and SEPRINH1 when an egg was present in the magnum of duck. This suggests that the SERPIN family of protease inhibitors has an important role in regulating the secretory activity of magnum for egg-white formation. Indeed, proteomic analysis of the egg white has shown that the SERPIN proteins are incorporated in the egg-white [34].
Transporters of proteins in the magnum epithelium
Cingulin is a protein localized at the tight junction of epithelial and endothelial cells, first discovered in the chicken intestine, and creates a barrier for molecular transport across cells [35]. In the present study, CGN mRNA was 5.4 fold higher in laying as compared to the non-laying hens. Cingulin is involved in the organization of the tight junctions, but simultaneously, it inhibits RhoA (Ras homolog gene family member A) activation and suppresses epithelial cell proliferation and gene expression [36]. However, it is also implicated that CGN regulates cell growth and morphology and creates a single layer of small, tightly packed cells [37]. To the best of the available literature on CGN function, we postulate that CGN mRNA is involved in the cellular organization and integrity of the magnum epithelium in laying hens regulating molecular transport across the epithelial barrier.
The solute carriers (SLCs) are exclusive membrane transporters that carry several solutes such as amino acids, organic and inorganic ions, and sugars. Several SLC members, including SLC7A9, SLC1A4, SLC7A11, SLC7A7, and SLC6A17, were increased by 29.9, 5.6, 5.3, 4.5, and 4.4-folds, respectively in the laying hens. The upregulation of these genes in the magnum of laying hens suggests that they actively participate in the transporter of precursor molecules to synthesize egg-white proteins.
Molecules involved in the biosynthesis
Several enzymes such as PHDGH, PSPH, PSAT1, ASNS, ASPG, GALNT6, PDE3A, and PHYKPL were increased in laying hens as shown by our RNA-Seq data. GO enrichment analysis revealed that PHDGH, PSPH, PSAT1, and ASNS were involved in amino-acid biosynthesis. The biosynthesis of L-serine from 3-phosphoglyceraldehyde is mediated by three enzymes PHGDH, PSAT1, and PSPH at each successive step, respectively [38]. Interestingly, the mRNA of PHGDH had higher expression in laying hens at 15–20 h p.o. (during the albumen synthesis period), and PSAT1 and PSPH mRNAs were also relatively higher in those hens. The upregulated expression of PHGDH, PSAT1, and PSPH in laying hens in this study strongly indicates the biosynthesis of serine in magnum, which may be required to synthesize egg-white proteins. Besides, microarray analysis of the magnum has shown that the expression of ASNS and PSPH is higher at the laying stage compared to the molting stage [11]. A report by Li et al. [39] suggests an additional role of these enzymes (PHGDH, PSAT1, and PSPH) in protection from reactive oxygen species (ROS) by providing the substrate-serine for glutathione synthesis. The antioxidative function of serine biosynthesis enzymes in the magnum is plausible since cells of the magnum are involved in the production of a massive amount of proteins, and concurrently ROS as by-products.
Genes involved in albumen secretion and/or oviductal transport of egg
Relaxin hormone produced from the ovary and placenta in mammals helps to ease the parturition process by relaxing the ligaments and dilating the cervix. The relaxin-like family peptide has seven peptides, including relaxin-3, which belong to the insulin superfamily. However, a phylogenetic study showed that the chicken genome had lost all the relaxin family peptides, but relaxin-3 having high homology to the human analog [40]. The relaxin-like peptide is produced in granulosa cells of the post-ovulatory follicles, localized in the uterus of laying hens, and influences the oviduct and uterus to aid in oviposition [41]. Also, loss in functionality of this avian relaxin has been shown to cause a drastic delay in oviposition timing [42, 43]. Studies of Brackett [41] and Wilkinson [40] suggest that the hormonal action of relaxin-3 from ovaries help in egg-laying. This study also detected a significant expression of RLN3 mRNA in the magnum of laying hens (7.5-fold higher) both during albumen synthesis and secretion period. This is a novel report on RLN3 expression suggesting its synthesis in the magnum, and we hypothesize that its over-expression at 3 h p.o. in the oviduct may be related to the mechanical distention of the magnum to ease the passage of the developing egg and/or secretion of the stored egg-white proteins. Since the mechanical pressure on the walls of the magnum provokes the secretion of the synthesized albumen proteins [9], RLN3 potentially is one of the markers of mechanical stimulus for the secretion of albumen from the goblet cells of the magnum.
The renin-angiotensin system (RAS), besides its well-known endocrine role in maintaining extracellular fluid in the body, also regulates ovarian growth dynamics [44]. Renin found in ovarian theca cells [45, 46] and angiotensin-converting enzyme (ACE) localized in the granulosa cells and blood vessels of the ovary [47] are the principal components of the RAS system. Apart from the endocrine function of RAS, the localized action of RAS in the ovary is towards follicular development and ovulation [48]. In this study, REN mRNA had significantly increased expression in the magnum of laying hens during the albumen secretion period as compared to molting and non-laying hens. The ACE mRNA was also higher (14.8 folds) in laying hens relative to non-laying hens. There are some reports on the activity of RAS in the uterus of humans [49], rats [50], rabbits [51], and quail [52]. So far, there is no report on RAS in the chicken oviduct. Verma and Panda [52] reported that ACE is expressed in immature and mature (with exogenous estrogen) quails with the highest expression in magnum, amongst the other oviductal parts. REN and ACE, fundamental molecules of the RAS, are predominantly found in the glandular epithelium of the human uterus, where the RAS had different roles during the menstrual cycle [49]. Collectively, the RAS controls the blood supply to the magnum by altering the vascular smooth muscle tone (through bradykinin), and forming new blood vessels [53]. Also, the RAS system, specifically in the magnum, might aid in relaxing the magnum to retain the egg for sufficient time, allowing optimum deposition of albumen. Concurrently, the expression of the ACE gene in the magnum of pigeon decreases by more than four-fold when the egg has passed through the magnum during the egg-laying cycle [19]. The previous studies in association with the findings of this study suggest that the expression of REN and ACE in the magnum of laying hens is strong evidence that the RAS system is also involved in the oviductal transport of egg in the chicken.
Antimicrobials for the egg defense
Antimicrobial agents are crucial for the livability of the hen’s embryo. The albumen holds the yolk (with ovum) in the center of the egg, without any contact with the eggshell. Albumen acts as a thick protective layer consisting of several antibacterial proteins. One such established protein is avidin, and interestingly in our study, AVD was the most overly expressed (250.7 folds) mRNA in laying hens. Avidin is also abundant in the egg white [1] and has a very high affinity for biotin required for bacterial growth and proliferation, thus preventing the invasion by microbial pathogens [54].
Another newly discovered and widely studied chicken antimicrobial protein is avian beta-defensins (AvBDs). AvBD11 is among the 14 members of the AvBDs whose mRNA expression was increased by 7.5 folds in the magnum of laying hens in our study. Previous studies have also revealed the expression of AvBD11 in the egg vitelline membrane, eggshell membrane, eggshells, and magnum, suggesting that the AvBD11 is an important molecule for innate immunity in hens [17, 55,56,57]. Taken together, AvBD11 incorporation in the albumen protects the developing embryo and might increase the hatchability of the eggs.
Antioxidant for protection of the magnum epithelium
Glutathione peroxidase (GPX) is a well-known enzyme capable of protecting the cells and tissues from ROS, such as hydrogen peroxidases and other lipid hydroperoxides. GPX3 is an isoform of the enzyme GPX class, localized in plasma and extracellular spaces [58]. We observed a 5.6 folds higher expression of GPX3 mRNA in the magnum of laying hens during the albumen synthesis period. These findings are indeed concurrent with the underlying physiological activities in laying hens. In the magnum of laying hens, rapid protein synthesis occurs at 4–23 h p.o. indicating that the cells of magnum have increased metabolism. As a result, simultaneous with protein synthesis, there is the release of ROS and other free radicals. So, the increased GPX3 expression in the magnum is indicative of the protective response against oxidative damage. Also, several other genes differentially expressed in laying hens, such as urotensin 2 and spermine oxidase involved in the production of ROS and hydrogen peroxide [59, 60], respectively, support the fact that oxidative stress is evident in the magnum.