Egg formation in avian reproductive tract is strictly regulated by the hormons and gene expression [20, 22], and the cuticle layer is the last process of egg formation in most avian species , however, the genetic regulation of cuticle deposition is still poorly understood. Considering the importance of eggshell cuticle and the genetic improvement of this trait in poultry production, present study were conducted by analyzing the DEGs and DEPs of the uterus between GC and PC group hens to elucidate potential genes and networks that regulate cuticle deposition. Besides, results of qPCR and a series of phenotypic measurements validated the RNA-Seq data. The current work not only picturised the differential expression profile of the uterus of GC vs PC group hens during eggshell cuticle formation, but also revealed important genes affecting cuticle deposition.
DEGs obtained in the transcriptome analysis of the uterus of GC and PC group hens during cuticle formation (1 h before oviposition) are very likely to be genes that regulate cuticle formation. Based on the results of DEGs, multiple genes (such as PTGDS, PLCG2, ADM and PRLR) related to uterine functions and reproductive hormones were found. The qPCR results further validated the DEGs that the gene expression levels of PTGDS, PLCG2, ADM and PRLR in the uterus of PC group hens were all higher than those of GC group, suggesting their vital roles during cuticle formation.
PTGDS is one of the prostaglandin synthases , and it’s also an important catalytic enzyme for the synthesis of prostaglandins [32, 33]. Studies on humans and mice have demonstrated that prostaglandins are involved in sleep induction [34, 35], intake behavior  and reproduction processes . Prostaglandins are mostly used to induce parturition in clinical practice. In poultry, injection of prostaglandins can be used to control the uterine contractility and then induce premature oviposition [12, 38, 39].
PLCG2, which is a type of the phospholipase C (PLC), is an important mediator of oxytocin to regulate uterine contractions . One of the important functions of oxytocin is to stimulate the contractions of myometrium during parturition in mammals and birds [41–43]. Oxytocin directly binds to the oxytocin receptor, which is subsquent activated and coupled to the G protein, to activate the PLC on the intracellular membranes, activating the influx of calcium and finally regulating contractions of the myometrium by voltage [40, 43–46]. In addition, another function of oxytocin is to increase the synthesis of prostaglandin by stimulating the activity of endometrial prostaglandin synthetase, and then the synergetic effect of prostaglandin and oxytocin enhances the activity of myometrium, thereby inducing labor or oviposition [44–51].
ADM is a peptide with several functions, including vasodilation, regulation of hormone secretion, promotion of angiogenesis, regulation of follicular growth and luteinization of ovarian follicles . In the labor activities of human and mice, ADM in the uterus binds to the receptor and enters the cell to participate in the relaxation of myometrium . Though the relaxation function of ADM is opposite to the contractile effects of oxytocin and prostaglandin in myometrium, ADM could cooperate with oxytocin and prostaglandin to participate in the rhythmical contraction and relaxation of the myometrium [43, 48, 53, 54].
Prolactin (PRL), secreted from the anterior pituitary, plays a series of roles in osmoregulation, corpus luteum formation and maintenance of broody behaviour in laying hens, and its receptor, PRLR, plays an important role in the PRL signal transduction cascade and cell growth and differentiation . The PLR and PRLR genes are expressed in many tissues including the hypothalamus, ovary and oviduct [56, 57], and mediate the formation of egg quality . PRL is generally considered to be a key factor in the onset and maintenance of broody behaviour in birds, and it has been well established that the elevated plasma PRL inhibits gonadotropin release, ovum development and ovulation, resulting poor laying performance and even complete cessation of egg production in laying hens [59–62]. Transcriptome studies have also revealed PRL is associated with egg production performance in chicken hypothalamic-pituitary-ovarian axis [63, 64]. Therefore, the significant difference in eggshell quality (EST, ESS, eggshell weight, and cuticle opacity) and egg production performance between GC and PC group hens may be related to the different expression patterns of PRLR gene in the uterus. Previous study has shown the inhibitory effects of excessive PRL on eggshell formation . Gonadotropin-releasing hormone (GnRH) is considered to be an important factor in promoting the cuticle deposition , however, the elevation of PRL can significantly inhibit the secretion and function of GnRH . Besides, environmental stressors would negatively affect the cuticle deposition with an increased PRL secretion [12, 65]. Follicle maturation, ovulation and egg formation are physiological events involving multiple organizations and processes, thus, the physiological difference of the hen uterus alone seems to be insufficient to explain its impact on egg production performance. Considering the inhibitory effects of PRL on eggshell formation and egg production, it’s speculated that the PRL level and PRLR gene expression level in the hypothalamo-pituitary-gonadal-oviduct axis are higher in the PC group hens. However, the effects of PRL and PRLR on hen uterine function are still unclear.
Summarizing the functions of PLCG2, PTGDS, ADM and PRLR expressed in the uterus, their express patterns in the uterus regulate the muscular activities and secretion function, which may lead to the difference in egg-laying rhythm between GC and PC group hens. The relatively high expressions of PLCG2, PTGDS and ADM genes could increase the frequency and intensity of contraction and relaxation activities of the myometrium that might negatively affect uterine functions and eventually facilitate oviposition. Combined the results of RNA-seq and qPCR analysis, in other words, hens with good cuticle deposition are more likely to oviposition accompanied by a extended duration for the egg staying in the uterus. This point of view was confirmed by the observations during the experiment, that is, in order to more accurately collect the uterine tissue samples during the cuticle secretion period, in the process of estimating the oviposition time of the hens, it was found that the laying interval between two consecutive ovipositions of the GC group was longer. The difference in laying interval between GC and PC group hens was further confirmed by the larger experimental flock, namely the laying interval between two consecutive ovipositions of GC group hens was significantly longer than that of PC group by 0.7 h (Fig. 3). At the same time, the concentrations of prostaglandin D2 and oxytocin of PC hen serums at 1 h before oviposition were higher than those of GC group hens though the statistical differences were not significant (P > 0.05). There was also a tendency that the concentrations of prostaglandin D2 and oxytocin of hen serum after oviposition were decreased, implying their important role in regulating uterine functions. The statistically insignificant differences may be due to the large difference in physiological status between individuals and the short biological half-life of oxytocin and prostaglandin [66, 67], however, the serum hormone levels was consistent with the RNA-Seq and qPCR results. The cuticle only begins to secrete 2–1.5 h before oviposition, and the secretion stops after the egg enters the vagina, so the secretion duration is relatively short . The results above indicated that during the cuticle secretion period in the uterus, the frequency and intensity of myometrium contraction and relaxation in GC group hens might be lower than that of PC group hens, which might extend the duration of the cuticle formation, and then lead to the good cuticle quality.
There were no DEPs between the uterus samples of GC vs PC group hens analyzed by the proteomic analysis, implying there was no significant difference in the protein composition and content to a large extent. Alternative hypothesis is that undetected DEPs may be due to the extensive posttranslational modification regulation in the biological process [68, 69]. On the other hand, the extremely short half-life of the hormones (e.g. oxytocin) may be the reason why the DEPs were not found [66, 67, 70]. The expression characteristics of the transcriptome and proteome between GC and PC group are highly similar (53 DEGs and no DEPs), suggesting that the biological processes of cuticle formation of the two groups may be the same, but mainly the difference in the duration of cuticle secretion.
Therefore, the duration of cuticle secretion in the uterus may be a major factor affecting cuticle quality. However, from ovulation to oviposition, it takes about 24 hours for the formation of a complete egg in chicken [10, 20]. A complete egg includes egg yolk, egg white, eggshell membranes, calcified eggshell and cuticle formation. The forming egg stays in different segments of the oviduct (i.e., infundibulum, magnum, isthmus and uterus) for different duration, and the egg remains in the uterus for the longest period during shell and cuticle formation, for a duration over 18 h in laying hens [10, 20, 71]. Thus, it is not clear whether the longer laying interval in GC group hens is entirely derived from the extended cuticle secretion time. The egg quality measurement results showed that there were no significantly differences in the egg weight, yolk weight, yolk color, albumen height, haugh unit and egg shape index between GC and PC group. Surprisingly, the eggshell quality (EST, ESS, eggshell weight, and cuticle opacity) of GC group were significantly increased compared with PC group, further suggesting that the the longer laying interval in GC group was mainly due to the extended formation duration of the eggshell in the uterus. The eggshell comprises four morphologically distinct layers that are formed sequentially, starting with the innermost mammillary layer, followed by the palisade, the vertical crystal layer and the cuticle . Eggshell ultrastructure of GC and PC group eggs by SEM shows the significantly increased EST of GC group is largely due to the increase in the thickness of the effective layer (palisade, vertical crystal layer and cuticle), demonstrating the extended laying interval of GC group hens both positively affects the calcified shell and cuticle formation. EST and ESS traits are highly correlated in phenotype and genetic [58, 72]. The effective layer thickness is the major ultrastructural characteristic compromising ESS . Eggshell formation takes place in the uterus over an 18 h period and the laying interval between ovipositions were positively correlated with the duration of eggshell formation, eggshell deposition rate and eggshell quality, and in detail, a difference of 0.7 hours between egg laying intervals could significantly affect eggshell weight and eggshell deposition rate by about 30% . Consequently, in present study, the prolonged laying interval by 0.7 h of GC group hens could explain the significant increase in the effective layer and cuticle layer thickness.
It is hypothesized that extended duration of eggshell formation and better secretion function of the uterus simultaneously promote the formation of good eggshell and cuticle quality. The heritability of cuticle trait is 0.27–0.40 [8, 19, 74]. Previous studies have also shown the cuticle quality is weakly positive correlated with the EST/ESS (< 0.2 in phenotypic and genetic correlation) in several commercial laying hen flocks (brown-shelled, white-shelled and tinted-shelled) [17, 74]. The results of present study directly prove that eggs with good or poor cuticle generally embody thicker or thinner EST that the top (good) and tail (poor) of the cuticle deposition distribution amplified the difference in eggshell quality traits. It’s suggested that eggshell calcification is precisely regulated by the interaction processes between mineral and organic precursors under gene expressions, including transcellular and paracellular transport of minerals and the secretion of different matrix proteins [28, 75–78]. The processes establish the eggshell texture and ultrastructure, and then influence eggshell quality traits [78–80]. Eggshell matrix proteins (e.g., ovocleidin-17, ovocleidin-116, ovocalyxin-32, ovocalyxin-21 and ovalbumin) present in both the calcified eggshell and the cuticle, and eggshell matrix proteins constitutes the main components of the cuticle [78, 81, 82]. Previous transcriptomic study in the uterus of laying hens that produce eggs with good or poor eggshell quality (EST/ESS) demonstrated the most of identified DEGs were involved in eggshell calcification (ion transport) and cuticularization (matrix proteins) pathways . It was found that the expression of FGF13 (fibroblast growth factor 13) is up-regulated in the uterus of PC group as compared to GC group (Table 1). FGF13 is a well-known growth factor belonging to the FGF family and is the modulator voltage-gated Na+, Ca2+ and K+ channels [84, 85]. FGF13 could increase voltage-gated Na+ and Ca2+ channel availability [84, 86], and it’s suspect that the up-regulated FGF13 also increased the uterine contractility. Simultaneously, there is also a significant association between ovocleidin-116, ovocalyxin-32 and ovalbumin genes with EST and cuticle deposition [8, 29]. Thus, the poor eggshell and cuticle qualities are likely mediated by dysregulation of both ion channels and matrix proteins during eggshell formation, which may also caused by the high expressions of PTGDS, PLCG2, ADM and PRLR genes that the uterine functions are negatively affected by the muscular activities based on the voltage-gated ion channels. To put it simply, during the eggshell formation period, the uterine secretion environment of GC hens is more stable, which ensures the duration for eggshell formation and thus the eggshell and cuticle quality.
The difference in eggshell quality and laying interval between GC and GC group may result from the difference in the uterine biological clock rhythm. Generally, hens lay eggs at intervals precisely controlled by the hormonal secretion and gene expression of the hypothalamo-pituitary-gonadal-oviduct axis. In laying hens, during the maturation of follicular, ovulation, and formation of the egg, the coincidence of the different physiological processes in time demonstrates that the laying rhythm is regulated by the endogenous circadian clock . The circadian clock is a major regulator of a wide range of behavioral and physiological processes including metabolism, food intake, sleep, body temperature, endocrine, immune, reproductive systems and orchestrates rhythmic gene expression in diverse organisms to maintain consistency between body metabolism and the external environment [87, 88]. In chickens, clock genes include PER, CRY, BMAL and CLOCK genes, which are expressed in several organisms including the central circadian clock (the pineal, the retina and the hypothalamus) and the peripheral circadian clock (almost all tissues throughout the body), is essential for synchronizing gene expression of key metabolic pathways to coordinate the time course of physiological and behavioral processes [89, 90]. In present study, the difference in laying interval of GC and PC group hens is mainly due to the difference in the calcified eggshell and cuticle formation period in the uterus, showing the uterine clock may be a main factor affecting the cuticle deposition as the eggshell formation is directly regulated by the uterine clock . This inference is supported by earlier research, that is, clock gene expression in the uterus during shell formation may be responsible for controlling the cuticle deposition, and PER2, CRY2, CRY1, CLOCK and BMAL1 genes were differentially expressed when cuticle deposition was prevented by arginine vasotocin . The contractile responses of the uterus to prostaglandins and arginine vasotocin play an important role in inducing oviposition . Arginine vasotocin and prostaglandin is thought to mediate the brain to ovary signalling of oviposition timing being involved in cuticle deposition, and the premature oviposition induced by arginine vasotocin and prostaglandin significantly reduce the EST and cuticle deposition , which supports the results of present study. The transcriptome results of present study also captured genes that regulate uterine muscular activities such as PLCG2, PTGDS, and ADM, however, clock genes and proteins were not detected in the transcriptome and proteome analysis, which could be due to the half-life of clock genes and proteins that would not be apparent in the transcriptome and proteome [30, 93]. Therefore, genes related to uterine timing mechanism and muscular events constitute the components of cuticle deposition regulation, but the regulatory networks need further research.