The Yolk Sac’s Essential Role in Embryonic Development
2023 Volume 11 Pages 243-258
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2023 Volume 11 Pages 243-258
The yolk sac is a pouch that envelopes the yolk. In birds and reptiles, it is a large extraembryonic membrane throughout the embryonic period. It supports all stages of embryonic development by supplying the embryo with nutrients stored in the yolk. The yolk is absorbed by the embryo before it hatches, so that in birds, for example, it cannot be observed unless the egg is artificially cracked. In many mammalian species, the yolk sac is a temporary structure, which in humans, for example, regresses by about 15 weeks of gestation. For these reasons, the yolk sac may be considered a mere nutrient-filled sac in birds and an empty, vestigial sac in mammals and has been received less attention than other organs such as the placenta and liver. However, the yolk sac plays a crucial role in development as an extraembryonic organ that absorbs, metabolizes and distributes nutrients essential for embryonic development, contributes to early hematopoiesis and secretes proteins and growth factors necessary for embryonic growth. In this review, we summarize the studies to date and provide perspective on the function of the yolk sac, mainly focusing on avian and mammalian species.
Terrestrial animals such as reptiles, birds and mammals evolved from ancestors common to amphibians about 370 million years ago [1]. These animals have developed extraembryonic membranes that may allow them to lay fewer eggs than fishes and amphibians, to tolerate some dry environments and to take longer to develop their precious embryos [2]. Reptiles, birds, and mammals all have four extraembryonic membranes: amnion, chorion, yolk sac and allantois. These animals are called amniotes because their embryos grow in the amniotic fluid secreted by the amnion. The extraembryonic membranes provide an aqueous environment, oxygen supply, access to nutrition and waste storage for the developing embryo. Fig.1 illustrates the extraembryonic membranes of birds, where the chorionic and the allantoic membranes are fused to form the chorioallantoic membrane. Above all, the yolk sac is the pouch that envelopes the yolk. In birds and reptiles, it is a large extraembryonic membrane throughout the embryonic period, supporting all stages of embryonic development [3].
The primary function of the yolk sac is to supply the embryo with nutrients stored in the yolk. The yolk sac is an essential organ in embryonic development in animals where the embryo is independent of the mother, such as birds and reptiles. In fish and amphibians, the eggs are laid in aqueous environment, so there is no need for an amnion or hard eggshell. Waste products are diffused into the water via the egg membrane, so an allantoic membrane is also unnecessary. However, even for these animals, the yolk is the only source of nutrition for embryonic development, thus being the yolk sac referred to as the oldest extraembryonic membrane in evolution [4].
While, in mammals, where the mother and fetus are directly connected through the placenta, the significance of the yolk sac has been undervalued. The human’s secondary yolk sac (the de facto yolk sac in primates) is present from the end of the fourth week of gestation, shows signs of degeneration by around the ninth week, and is no longer visible around the 15th week [5, 6]. In ruminants, the yolk sac has also been reported to degenerate early and shrink with the development of the placenta [7]. However, a relationship between yolk sac development and successful embryogenesis has been reported in mammals. In humans, small (or absent) yolk sacs relates to miscarriage [8], with yolk sacs >6 mm in diameter or outside the range of 6 mm ± 2 mm being associated with diabetes, miscarriage or chromosomal abnormalities [9, 10, 11, 12]. It has been reported that 70% of human spontaneous abortions have secondary yolk sac morphological abnormalities [13], suggesting that yolk sac size may also reflect maternal health status [14].
In other words, the yolk sac is not merely a temporary organ until the placenta is completed but an essential organ for mammalian embryonic development. The elucidation of the role of the yolk sac provides crucial basic knowledge for breeding, productivity improvement, and arrangement of breeding conditions according to the physiological status of the animals, not only in poultry such as broilers and layers but also in livestock such as ruminants and non-ruminants. This review summarizes the structure and function of the yolk sac in vertebrates, focusing mainly on mammals and birds.
Figure 1: Schematic diagram of a chick embryo and the extraembryonic membranes at around day 10 of incubation. The allantoic membrane and chorionic membrane have fused to form the chorioallantoic membrane. The egg white will be eventually absorbed into the yolk. The air chamber increases in volume as the embryo breathes.
Extraembryonic membranes provide an aqueous environment, oxygen supply, access to nutrition and waste storage for the developing embryo. Concerning access to nutrients by the embryo, mammals have few yolks, and most maternal nutrients pass through the placenta (chorioallantoic placenta) to the embryo. In primates such as humans, the yolk sac is not in contact with the chorion and is very small and transient. Birds, on the other hand, have a well-developed yolk sac that encloses a huge yolk. Other amniotes fall between these extremes [15]. In this section, we will first discuss the yolk sacs of birds and reptiles and then the yolk sacs of mammals.
2.1 Yolk sacs of birds and reptilesThe development and structure of yolk sacs had already been studied in the 1800s, especially using the embryos of domestic birds such as chickens, because embryos can be easily observed by cracking the eggshell. There are excellent books on the development of avian yolk sacs (see [16, 17] for example). In the following, we will present an overview of birds (for reviews and excellent original papers, see [18, 19, 20]) and reptiles.
The avian yolk sac is a sizeable extraembryonic membrane that ultimately encapsulates the whole yolk (Fig 2). Blastoderm refers to the very early embryo, corresponding, as for the chicken, approximately to the time of oviposition (or egg-laying: about 24 hours after ovulation). The blastoderm is present in a floating form on the surface of the yolk. By 80 hours of development, the embryo develops along the yolk, while the yolk stalk, a structure connecting the midgut of the embryo to the yolk sac, is formed. The yolk sac is composed initially of ectodermal and endodermal cells, with the endodermal cells facing the yolk. In the yolk sac can be identified area vitellina, which lacks blood vessels, and area vasculosa, which has well-developed blood vessels; in the latter, there is a mesodermal area between the ectodermal and endodermal layers. In other words, the complete yolk sac comprises three germ layers. The yolk sac does not cover the entire yolk at the beginning of development. The chicken yolk sac develops, crosses the yolk equator at day 3 of incubation (E3), and almost covers the yolk at E5. The area vasculosa, in turn, crosses the equator at E5–E7 and reaches, almost covering the yolk at E14–E15. During the first week of incubation, the absorbed yolk is mainly used to develop the yolk sac. According to our measurements [21], the weight of the yolk sac membrane (wet weight without yolk) was 0.67 g on E4, increased gradually, especially on and after E11 (1.79 g), and peaked on E17 (4.67 g). The yolk sac membrane shrunk after that, reaching 0.68 g on the third day of hatch, almost the same weight as on E4. Yadgary et al. [22] stated that this weight change correlated with an increase in the area of the absorbing portion of the yolk sac.
Figure 2: Schematic diagram of the placentas of birds and mammals. Examples of mammals, marsupials, and eutherians (human and mouse) are shown. The dotted line in the yolk sac of the mouse embryo shows a parietal yolk sac membrane that may be degraded. This degradation can lead to the structure called the inverted yolk sac placenta. See text for more details.
In the completed yolk sac, the surface in contact with the yolk is folded, and the villi are well-developed, forming the absorptive area, while the non-absorptive area does not have the fold (Fig. 3). Like the folded structure of the small intestine, this structure is thought to help nutrient absorption from the yolk by increasing its surface area. The absorbing area of this well-developed fold structure increases more than 10-fold from E5 to E17 in chickens [22]. The height of the folds eventually reaches 3–7 mm. In addition, the yolk sac membrane epithelial cells (i.e., endodermal cells) have microvilli that are randomly arranged and relatively sparse (compared to the small intestinal epithelium, which is also an absorptive epithelium). Microvilli may contribute to increased yolk absorption efficiency as a pathway for yolk uptake other than by endocytosis. In addition, yolk sac contents (i.e., yolk) cannot be transferred to the embryonic side without passing through the cytoplasm of the epithelial cells due to structures like tight junctions between the epithelial cells. This means yolk uptake can be controlled according to physiological requirements during development [3, 23].
Figure 3: Yolk sac membrane of a 10-day-old Japanese quail embryo (in 18-day incubation period). The yolk stalk is cut, and the yolk-facing side (endodermal epithelium) of the yolk sac is placed upside, with the yolk washed away. The vitelline vessels (marked “*”) have been cut at their embryo sides. The picture shows that the yolk sac membrane consists of absorptive and non-absorptive areas [22]. The yolk-contacting surfaces are well-developed folds.
Although the yolk sac is connected to the intestinal tract by the yolk stalk, it is believed that the yolk is not taken directly into the intestinal tract during the incubation period, at least not until just the end of the incubation period [16]. As mentioned above, the yolk sac gradually shrinks with a peak at E17 [21]. By the time of hatching, the yolk sac is retracted into the abdominal cavity of the chick. The remaining yolk is absorbed into the intestinal tract and disappears 5–7 days after hatching [16], but the absorption mechanism is not fully understood.
It has been reported that the yolk sac membrane also functions in reptiles for yolk absorption, but the morphology of the yolk sac’s inner surface is very different from that of birds [24]. In snakes, lizards, turtles, and crocodilians, endodermal cells from the yolk sac membrane invade and proliferate into the yolk sac cavity and phagocytose the yolk. Eventually, the yolk sac is filled with yolk-phagocytosed endodermal cells. Further, blood vessels invade, and endodermal cells rearrange around the vessels, forming elongated “spaghetti-like (or yarn-like) strands” composed of endoderm-coated blood vessels inside the yolk sac cavity. These structures allow efficient yolk absorption into the yolk sac and nutrient supply to the reptile embryo through the blood vessels. Phylogenetically, Blackburn [24] considers these reptilian yolk sacs to be the ancestral form of sauropsids (non-mammalian members of the amniotes) and that it is unclear whether avian-type yolk sacs arose before or after the divergence of birds from non-avian dinosaurs. Yoshizaki (a Professor Emeritus of Gifu University) and his colleagues [18] found that yolk proteins are somewhat degraded by the coexisting enzyme cathepsin D before being absorbed by the epithelial cells of the yolk sac membrane. Based on this finding, Blackburn [24] suggested that the extra-epithelial digestion of egg yolk occurred in the avian ancestor and that the way “endodermal cells extending into the yolk phagocytose the yolk and digest it intracellularly” as seen in reptiles was finally abolished in the course of evolution. These findings provide insight into how the embryo’s method of absorbing nutrients from the yolk, which contained all the nutrients necessary for embryonic development when our vertebrate ancestors expanded on land, changed throughout evolution. However, as will be discussed in Sections 3 and 5, a large part of avian egg yolk contents is a complex of protein and lipids in the form of lipoproteins. How these components are digested in the yolk sac cavity (outside the epithelial cells), absorbed by the epithelial cells, and processed intracellularly must be investigated more precisely.
2.2 Yolk sac of mammalsThere are excellent original papers and reviews on the development and structure of the mammalian yolk sac [4, 25, 26, 27, 28, 29, 30]. We summarize critical points based on these papers below.
The mammalian yolk sac is a structure that arises from a fertilized egg that has developed into the blastocyst stage. The yolk sac is a two-layered structure and arises from the trophectoderm (trophoblast) and extraembryonic endoderm. In addition, in some or all areas of the yolk sac, the extraembryonic mesoderm (with blood vessels connecting to the vitelline vessel) extends between the ectoderm and the endoderm, resulting in a three-layered structure [27, 28]. In many mammalian species, the yolk sac is in contact with the chorion and forms a choriovitelline placenta. This placenta is transient and eventually transitions to a chorioallantoic placenta (Fig. 2) occur, resulting from the contact of the allantois with the chorion. However, there is significant variation among species [29].
Most marsupials form only choriovitelline placentas (Fig. 2). The ancestral stem species of marsupials possibly had both choriovitelline and chorioallantoic placentas. However, it is thought that the latter became less important during the evolution of marsupial placentas, and choriovitelline placentas predominated [26, 27]. In some rodents and lagomorphs (rabbits), the choriovitelline placenta persists until birth (Fig. 2). In these animals, the yolk sac parallels the chorioallantoic placenta, absorbing nutrients from the mothers’ uterus during pregnancy, digesting them, and transporting them to the fetus [29, 30]. Another essential function is the transport of immunoglobulins to the fetus [28, 29]. During the formation of the choriovitelline placenta in these animals, a part or all of the yolk sac is inverted, forming an absorptive epithelium with the yolk sac endoderm facing the mother (uterus) side (called the inverted yolk sac placenta) [25, 30]. The significant morphological difference from the marsupial yolk sac placenta is the inversion of the yolk sac with degeneration of the parietal yolk sac (the yolk sac located on the mother’s side). In marsupials, on the other hand, the yolk sac is not inverted.
In humans and other primates, the yolk sac is not physically attached to the chorion, and thus the yolk sac exists as a free structure within the extraembryonic coelom (Fig. 2). In these species, only the chorioallantoic placenta is formed [4]. Since the human yolk sac begins to regress around ten weeks of gestation, the mammalian yolk sac was considered a vestigial organ and received little attention until about 50 years ago. A PubMed search for “human (or mouse, rat) yolk sac” reveals very few articles before 1970.
One of the most essential functions of the yolk sac is to supply the embryo with nutrients accumulated in the yolk. The yolk sac, as the machinery of transporting nutrients stored in the yolk, is particularly significant for reptiles and birds, whose embryos are independent of the mother, and the materials stored in the yolk are the only nutrient and energy source for embryonic development. In mammals, which obtain nutrients from the mother via the placenta, the yolk sac plays a temporary but crucial role in nutrient supply until the placenta is completed (e.g., around the 15th week of gestation in humans).
3.1 Transport of maternal nutrients in birdsThe epithelial cells of the yolk sac membrane have one side (apical side) in contact with the yolk and the other (basolateral side) facing the blood vessels across the basement membrane. This bipolarity gives the yolk sac membrane two interrelated functions: to take up nutrients and release them into circulation [15]. The avian yolk sac membrane expresses a wide variety of nutrient transporters and related molecules. The timing of their expression, however, varies. For example, aminopeptidase N (APN), peptide transporter 1 (PepT1), cationic amino acid transporter 1 (CAT1) and glucose transporter 5 (GLUT5) are highly expressed during early embryonic development and tend to decrease towards hatching [31]. The expression levels of sodium/glucose cotransporter 1 (SGLT1) and excitatory amino acid transporter 3 (EAAT3) have been reported to increase toward hatching [32].
Above all the egg yolk components, lipids are the highest-energy substances, and hence the efficient supply of lipids to the embryo is essential for normal embryonic development. A variety of proteins responsible for the process of lipid transfer across the yolk sac membrane including endocytosis receptors, fatty acid transporters, lipases and apolipoproteins, have been detected by biochemical and transcriptome analyses of chicken yolk sac epithelial cells [19, 33]. Enzyme activity of lipase (as assessed by the hydrolysis of 1,2-diacylglycerol) has also been found [22]. These findings provide evidence that, in the epithelial cells of the avian yolk sac, lipids are taken up from the yolk by receptor-mediated endocytosis, hydrolyzed in the epithelial cells and then re-esterified to construct lipoprotein particles that are supplied to the embryo through the bloodstream [3, 19, 33, 34].
In birds, lipophilic hormones such as sex steroids, adrenocorticosteroids and thyroid hormones produced in the maternal organs are known to be transferred from maternal blood to the egg yolk during follicle formation in the mother hen. These hormones promote normal embryonic development and influence embryo and chick development until the embryo’s endocrine organs are fully functional [35, 36].
Our research group investigated the metabolism of thyroid hormones in the yolk sac of the laying hen [21]. Thyroid hormones (like triiodothyronine and thyroxine) are essential for the proper development of the avian embryo. Thyroid hormones accumulate in the yolk from the maternal blood when the mother hen forms the ovarian follicle. Thyroid hormones in the yolk are likely to be supplied to the embryo through the yolk sac membrane and may play an important role until the embryo’s thyroid gland is fully functional. We found the expression of three enzyme genes in the yolk sac membrane of developing chick embryos: iodothyronine deiodinase D2, which activates thyroid hormones; deiodinase D3, which inactivates them; and deiodinase D1, which is involved in iodine recycling. We also found gene expression of organic anion transporter1 C1 (OATP1C1), monocarboxylate transporter 8 (MCT8) and MCT10, which are transporters of small molecules including thyroid hormones, and transthyretin (TTR) and albumin, carrier proteins of thyroid hormones, in the yolk sac membrane of developing chick embryos. Based on these findings, we hypothesize a model of thyroid hormone metabolism in the yolk sac (Fig. 4). The thyroid hormones are taken up from the yolk into the epithelial cells of the yolk sac membrane by receptor-mediated endocytosis, activated or inactivated in the epithelial cells according to embryonic demand during development and released into the bloodstream either alone through membrane transporters or in a protein (TTR or albumin)-bound form by exocytosis. We postulate that they are released into the blood and delivered to the embryo.
Figure 4: Postulated pathway of thyroid hormone (TH) uptake, metabolism, and secretion of the chicken yolk sac epithelial cell. 1) Protein-bound THs are endocytosed from the yolk. 2) Free THs are liberated in the cell possibly by lysosomal digestion of proteins. 3) THs are catalyzed by three deiodinases (D1, D2 and D3) in developmental stage-specific way. 4) Catalyzed THs are bound to newly synthesized TH distributor proteins and released out of the cell and further into circulation. 5) Free THs and iodide (I-) can pass through their specific transporters out of the cell. Protein names are underlined whose expressions in the yolk sac epithelial cell are reported [21]. TTR: transthyretin. ALB: albumin. VLDL: very low-density lipoprotein. R: endocytosis receptor complex (cubilin-megalin-amnionless [34]). T4: thyroxine. T3: triiodothyronine. rT3: reverse T3 (inactive). T2: diiodothyronine (inactive). T3R: nuclear T3 receptor.
As mentioned above, the uptake of nutrients in the yolk sac membrane of birds is carried out by endodermal epithelial cells. Also in human yolk sac membranes, well-developed microvilli and endocytic vesicles have been identified in the epithelial cells at the microstructural level [37, 38]. In other words, nutrients in mammals are likely to be absorbed via the yolk sac endodermal epithelial cells, taken up by the blood and supplied to the embryo.
Once the placenta has developed, nutrients supplied by the mother (e.g., amino acids, carbohydrates, and lipids) are transferred from the maternal circulation to the fetal circulation via the placenta. However, in early pregnancy, the blood vessels in the chorionic part of the placenta are poor and contain only fragile capillaries. While the vasculature of the yolk sac is well developed [39], and thus the possibility of fetal circulation through the yolk sac has been suggested [40].
Some mammalian species (as discussed in Section 1) differ in the “orientation” of the epithelial cells of the yolk sac, resulting in different procedures for the supply of nutrients [29]. The yolk sacs of some eutherian species, including humans, are not attached to the chorionic membrane. Thus, after being released into the intervillous space, nutrients from the uterus endometrium are phagocytosed by syncytiotrophoblasts of the chorionic villi and digested by their intracellular lysosomes. The resulting products (e.g., amino acids) are translocated via transporters into the coelomic fluid. Then, yolk sac mesothelial cells take them up and they enter the fetal circulation. Alternatively, the nutrients diffuse into the yolk sac cavity and are taken up by the yolk sac epithelial cells. In addition, some proteins are released into the coelomic fluid and then phagocytosed by the mesothelial cells. Unlike humans and other mammals, rodent yolk sacs are attached to the chorionic membrane. Accordingly, nutrient secretions from the uterus endometrium are phagocytosed by yolk sac epithelial cells and digested intracellularly by lysosomes before entering fetal circulation (see [29]). Experiments using cultured rat embryos have demonstrated that proteins are absorbed by the yolk sac cells, degraded by lysosomes into amino acids and transported to the embryo [41]. In all cases, nutrient absorption occurs in the endodermal, epithelial cell layer of the yolk sac membrane [40, 42].
In human yolk sacs, transcripts involved in transporting cholesterol and other lipids, including transcripts of apolipoprotein and ATP-binding cassette (ABC) transporter genes, have been found [29]. It has long been known that cholesterol is synthesized de novo in rat yolk sacs [43]. In mice, apolipoprotein B (apoB) gene expression starts soon after the yolk sac is formed (between embryonic days 6 and 7) [44]. The yolk sac is thus thought to supply the embryo with lipids of maternal origin and lipids synthesized in the yolk sac in the form of lipoprotein packages. In addition, the yolk sacs of humans and other mammals express various transporters belonging to the solute carrier (SLC) transporter superfamily, which are involved in the transport of amino acids, carbohydrates and vitamins such as vitamins A, B12, E, folic acid and retinoic acid [29, 45, 46, 47, 48], as well as transport of ions such as calcium [49]. Immunoglobulins of maternal origin are also supplied to the embryo through the yolk sac membrane [50].
It has long been known that the yolk sac is a hematopoietic organ during the embryonic period. Chick embryos have often been used in embryonic development studies, including hematopoiesis during embryogenesis. This is because, among other reasons, refrigeration of the eggs after laying can halt the developmental process for at least a week without affecting later embryogenesis; the development of all eggs can be synchronized by placing them in incubators at the same time; and development can be easily observed by partially breaking the shell [51]. Hematopoiesis occurs in ‘waves’ throughout the avian and mammalian embryonic stages, with the first being termed “primitive” and the subsequent ones “definitive” hematopoiesis. These hematopoietic sites shift during development [52]. All the hematopoietic stem cells in these sites do not originate from the yolk sac [53]. Studies using chick-and-quail chimeras have suggested that the stem cells can originate from the aorta-gonad-mesonephros (AGM) region of the chick, derived from the lateral plate mesoderm [54]. In addition, the period during which the yolk sac is involved in hemopoiesis appears to differ between avian species, in which the yolk sac persists throughout development, and mammals, particularly humans, in which it retracts by the time the placenta is formed [55]. Schematic diagrams of hematopoietic locations of bird and mammal embryos are shown in Fig. 5.
4.1 The hematopoietic function of the yolk sac in birdsIn avian embryos, the yolk sac is an essential hematopoietic tissue supporting the immature embryo until the bone marrow has a hematopoietic function (Fig. 5). In chickens, primitive erythropoiesis has been observed in the yolk sac on day 1 (E1) of embryonic development [56]. The yolk sac becomes the main hematopoietic site from E6 onwards, and hematopoietic function in the yolk sac continues until it declines from E17 onwards. In the bone marrow, erythropoiesis and granulopoiesis are observed from around E12–E13, but hematopoiesis in the yolk sac also continues [55]. Hematopoietic cells are also found in the liver, spleen, bursa of Fabricius and thymus from mid-incubation onwards. However, unlike in mammals, the function of the embryonic liver as a hematopoietic organ is limited [53, 55].
Figure 5: Hematopoiesis sites (indicated in red) in mammalian and avian embryos. The roles of different organs related to hemopoiesis may differ in birds, where the yolk sac persists throughout development, and in most mammals, where the yolk sac retracts by the time the placenta is formed. Unlike mammals, the embryonic liver has a limited function as a hematopoietic organ in birds.
Studies have been conducted on the hematopoietic function of the yolk sac in mammalian embryos, many using mice. In mouse embryos, erythropoiesis is observed in the mesodermal layer of the yolk sac from day 7.5 (E7.5) of embryonic development. The hemoglobin variant type of erythrocytes at this time differs from that of erythrocytes at later stages of embryogenesis, and they have a higher affinity for oxygen. In mouse embryos, the hemopoietic function of the yolk sac is followed by the placenta at E9, the liver at E12 and the bone marrow after E17.5 [57, 58]. In the yolk sacs of mouse embryos, megakaryocytes, which are responsible for the production of platelets, as well as erythrocytes, are found at E7.25 and E9.5. In contrast, platelets in the peripheral blood are found at E10.5 [59]. Macrophages were observed at E9 and in the yolk sacs of mouse embryos [60]. Signals and transcription factors known to regulate hematopoietic stem cell formation from mesoderm include BMP4, Flk-1, c-kit, Scl, Lmo2, Gata2, Runx1, CBFβ, Mll, c-Myb, Stat5a, Notch and Hedgehog, which act at different timing to generate the stem cells [61].
The developmental process of the human yolk sac differs from that of the mouse in that the primary yolk sac develops first, degenerating to form the secondary yolk sac. The primary yolk sac is transient, and its role remains unclear [4]. Hematopoiesis reportedly begins functioning in the secondary yolk sac at around three weeks of gestation. Hematopoiesis is reduced after eight weeks, and the human yolk sac starts to regress around ten weeks. The liver subsequently functions as the primary site of hematopoiesis at 6–22 weeks, after which the bone marrow becomes the site of lifelong blood cell production [62, 63].
Hematopoietic functions in the yolk sac have also been reported outside of birds and mammals: the yolk sac is known to be the first site of hematopoiesis in the European pond turtle [64]. Hemopoiesis in the yolk sac has also been observed in fish (zebrafish and rainbow trout) [65, 66].
In other words, in the early stages of development, when hematopoietic organs such as the bone marrow are immature, the yolk sac instead undergoes hemopoiesis, and this yolk sac hematopoietic function may be conserved not only in amniotes but also in fish.
Because the avian yolk sac membrane is composed of three distinct cell layers derived from endoderm, ectoderm and mesoderm, Wong and Uni [67] call it not just a membrane but a multifunctional organ that is indispensable during avian development. Various metabolic enzymes are present in the avian yolk sac membrane [67, 68], among which we have focused our attention on carbohydrate metabolism, because, in avian species, carbohydrate content in the yolk sac drastically increases toward the end of incubation [67]. In this section, we concentrate on the avian yolk sac because, in mammals like humans and rodents, the yolk sac as a metabolic organ has not been drawing much attention except for its function in nutrient transfer, such as glucose and lipids [30].
Carbohydrates are necessary for the survival and growth of animals, also during embryonic development. In mammals, carbohydrates are supplied to the fetus continuously (via the placenta). On the other hand, since avian and reptilian embryos in their eggs are independent of the mother, embryonic development must be carried out using only the nutrients stored in the egg.
The egg white, nearly 90% of its weight is water, about 10% protein, and less than 1% carbohydrates. In egg yolk, water accounts for the weight of about 44%, lipids about 36%, protein about 17%, minerals 2%, and carbohydrates less than 1% [69, 70]. In other words, the total carbohydrate content of the egg is less than 1%.
In adult birds (as well as adult mammals), carbohydrate metabolism is carried out in the liver and kidney, with the liver playing a major role in maintaining blood glucose levels. Various metabolic functions of the avian liver, including maintenance of blood glucose levels, glycolysis and gluconeogenesis, glycogen breakdown and synthesis, lipid metabolism, and antioxidant function, have long been investigated using chicken embryos [71, 72, 73, 74, 75, 76, 77, 78, 79].
Birds are well-known hyperglycemic animals, with a blood glucose level of about 300 mg/dl in pigeons, 200 mg/dl in ducks, 330 mg/dl in budgerigars and rising to 740 mg/dl after feeding in hummingbirds [80, 81]. In chickens, blood glucose levels are about twice as high as in humans [82, 83]. High blood glucose levels have also been reported in chick embryos, with blood glucose levels above human levels at day 9 (E9) and equivalent to adult chickens at E18 of 21–day egg incubation [84].
The fact that avian embryos maintain high blood glucose levels despite the low percentage of carbohydrates stored in the egg suggests that gluconeogenesis occurs at the embryonic stage. Gluconeogenesis is a biochemical pathway that produces glucose from non-carbohydrate materials, the substrates being glycerol and amino acids resulting from lipolysis and protein degradation, respectively [85, 86]. Gluconeogenesis is the reverse pathway of glycolysis, where glucose is broken down to produce pyruvate. The gluconeogenesis pathway involves irreversible reactions catalyzed by four different enzymes, respectively. These enzymes are glucose-6-phosphatase, fructose-1, 6-bisphosphatase, cytosolic and mitochondrial isozyme of phosphoenolpyruvate carboxykinase and pyruvate carboxylase. It is well known that gluconeogenesis takes place in the liver and kidney in adults, both in mammals and birds. As a large organ, the liver plays a significant role in maintaining blood glucose levels. However, the liver alone cannot explain the entire carbohydrate metabolism system in the avian embryo.
Yadgary and Uni [87] studied chick embryos after E11 and found that gluconeogenesis enzymes (glucose-6-phosphatase, fructose-1, 6-bisphosphatase, phosphoenolpyruvate carboxykinase) mRNAs were expressed in the liver by E13; Roy et al. [88] reported that mRNAs for glycolytic enzymes were expressed by E11. These enzyme gene expressions varied throughout the subsequent incubation period. However, before these dates, the liver is not large enough (0.3 mg on E4 and 5 mg on E7 compared to 70 mg on E11 [89]) to be a main metabolic organ. However, in the chicken egg, which has a low carbohydrate content (less than 1% as written above) from the start of incubation, gluconeogenesis must be activated, much earlier than the liver starts to function, to maintain and increase blood glucose levels in the embryo and to provide energy for embryonic development. In other words, there should be metabolic organs other than the liver involved in gluconeogenesis before the liver fully develops.
The yolk sac may have a potential role in gluconeogenesis [21, 90]. The yolk sac membrane is the largest of the intra- and extraembryonic organs throughout the incubation period of the chicken egg. At E17, for example, the wet weight of the yolk sac membrane (4.67 g [21]) weighs nearly ten times that of the embryonic liver (0.479 g [89]). Yadgary and Uni [87] reported that mRNA for gluconeogenesis enzymes (glucose-6-phosphatase, fructose-1, 6-bisphosphatase, phosphoenolpyruvate carboxykinase) is expressed in the yolk sac membrane on E11. In addition, microarray analysis of the chicken yolk sac membrane on E1–E4 [68] showed the expression of fructose-1, 6-bisphosphatase and phosphoenolpyruvate carboxykinase. Based on these findings, we hypothesize that glycogenesis in the yolk sac may help maintain high blood glucose levels in embryos during incubation when the liver and kidney are still immature.
Among the metabolic functions of the avian yolk sac, lipid metabolism is essential when one considers the amount of lipids in the egg. Lipids account for about 10% of the egg weight in the chicken egg, including the eggshell [69]. Nutritionally, lipids (9 kcal) have more than 2.2 times the calories per weight (g) of carbohydrates and proteins (4 kcal), and 6.7 times if one considers that lipids are not hydrated in the body.
Chicken egg yolk’s lipid content (w/w) is about 62% triglycerides, 33% phospholipids and less than 5% cholesterol. The major fatty acids comprising egg yolk lipids include oleic acid (about 60%), palmitic acid (about 20%) and linoleic acid (about 7%). However, these vary markedly depending on the feed of the mother hen [69].
As already mentioned in Section 3, in the epithelial cells of the avian yolk sac, lipids are taken up from the yolk by receptor-mediated endocytosis, hydrolyzed and then re-esterified to construct lipoprotein particles that are supplied to the embryo through the bloodstream [3, 19, 33, 34].
Histological observation revealed numerous villi on the apical surface of the epithelial cells of the yolk sac membrane, indicating the uptake of yolk components into the cells. Intracellularly, yolk granules with heterogeneous electron density were observed, suggesting that lysosomes were fused to the incorporated phagosomes. Lipid droplets were also observed in the cells, and the number of lipid droplets gradually decreased from E15 to closer to hatching. In addition, lipid spherules were observed being released by exocytosis from the basolateral surface of the epithelial cells [91].
In the last week of chicken egg incubation, 0.5 g of lipid per day is transported from the yolk sac to the embryo [92]. Epithelial cells of the yolk sac membrane, through which these lipids pass, are active in fatty acid desaturases, monoacylglycerol and diacylglycerol acyltransferases and glycerol phosphate acyltransferases. Therefore, yolk lipids taken up by the epithelial cells of the yolk sac membrane should undergo remodeling such as hydrolysis/re-esterification of yolk lipids and synthesis of unsaturated fatty acids [3, 68].
Intracellular Acyl-CoA:cholesterol acyltransferases (ACAT) quickly esterify cholesterol taken up by the epithelial cells of the yolk sac membrane, and the primary product is reported to be cholesterol oleate [3, 93]. The sphere diameter of VLDL (VLDLy) accumulated in the yolk in the follicle of the mother hen (about 30 nm) is smaller than the diameter of common VLDL (about 70 nm) [94]. Thus, the surface area is relatively large, and correspondingly, a large amount of cholesterol is present on the surface as a non-esterified, polar molecule. This requires cholesterol esterification when cholesterol is reconstructed into new lipoproteins (LDL and HDL [95]), the main components of the embryonic blood lipoproteins. The abundant ACAT activity in the epithelial cells of the yolk sac membrane corresponds to this requirement.
In summary, the avian yolk sac membrane does not simply transfer yolk lipids to the embryo but rather remodels the lipids to create new lipids to be transferred to the embryo.
In addition to the various functions discussed so far, the yolk sac is also the site of plasma protein synthesis. Transcriptome analysis of chick embryos have revealed that surprisingly, many proteins are synthesized in the yolk sac. Genes for plasma proteins such as transthyretin (TTR), albumin and α-fetoprotein are expressed, in addition to various metabolic enzymes, plasma membrane transporters and growth factor receptors [33, 68].
In mammals, mRNA expression of TTR, retinol-binding protein, transferrin and α-fetoprotein in rat embryos was higher in the yolk sac than in the liver throughout the 22-day gestation period until around day 20; these proteins were secreted mainly in the direction of the fetus rather than the mother [96]. Paternal expression (genomic imprinting) of insulin has also been reported in human [97] and mouse [98] yolk sacs. Insulin should thus influence fetal growth in utero.
We would like to draw the following overall conclusions: It can be emphasized again that the following framework of extraembryonic membranes is essential as basic structures for animals to advance onto land in the course of evolution: the amnion as a housing for embryos, the allantois as a bag for discarding waste products, the yolk sac as a bag for storing nutrients and the chorion covering the entire structure and working for the exchange of substances with the outside world. Among these membranes, we focused on the yolk sac in this review. As discussed above, the yolk sac plays an essential role in development as an extra-embryonic organ that absorbs, metabolizes and distributes nutrients indispensable for embryonic development, contributes to early hematopoiesis and secretes proteins and growth factors for the growth of the embryo. In these regards, differential regulation of genes and transcription factors responsible for these varieties of functions in a single yolk sac epithelium cell is fascinating to be elucidated, which is currently underway in our laboratory.
This work was supported in part by the Japan Society for the Promotion of Science KAKENHI, a Grant-in-Aid for Scientific Research, to AI (No. 20K06444).
