Chemical Adaptation in Viviparous Birthing and Oviparous Hatching

Serena Kim, Massimiliano Garzia, Lila Oualim, Mark Tchinov

Abstract

One of the first stages of life in any animal is time spent as a developing embryo. Every embryo must undergo complex development aided by its parent for a certain period of time before their birth or hatching, and this process varies greatly depending on the type of species. Specifically, the way in which animal young develop can be divided into three categories of animal type: viviparous, oviparous, and ovoviviparous. However, for the purpose of this paper, only viviparity and oviparity will be discussed. This research paper will delve into the specific biochemical processes and structures involved in the development, nurturing, and protection of embryos for viviparous and oviparous animals. First, the function and properties of placentation in viviparous ruminants will be explored to illustrate its significance in the growth and maturing of the embryo. Next, the composition of the inner egg and egg membrane will be discussed to demonstrate the contributions of each component to the chemical activities responsible for the development of embryos in oviparous animals. Finally, the mineralization of eggshells will be examined in the context of birds and reptiles, specifically looking at the examples of hen and gecko eggs, in order to expand on the role of the eggshell past simply acting as a physical barrier between the embryo and the outside world.

Introduction

The growth and nurturing of an embryo require the joint function of numerous different complex biochemical processes and structures, which vary depending on whether the animal in question is viviparous or oviparous. Viviparous animals are defined as animals that develop their young embryonically within their uterus, and that subsequently give birth to live young. Oviparous animals lay eggs which develop for a short period of time inside the parent, and most of the development occurs externally in the egg until they hatch (Panawala, 2017).

This paper will first discuss placentation in viviparous animals while looking specifically at ruminants, which are mammals including cows, sheep, goats, etc. (Parish et al., 2017). Placentation refers to the formation and arrangement of the placenta as a way to establish physical contact between the embryo and the parent for the transfer of nutrients, hormones, and waste (Wagner, 2018). The development of the placenta allows the mother to embody the majority of the body systems for the embryo so that it can develop healthily, while providing nutrients for the embryo as well as facilitating placental gas transfer. These functions occur through simple and facilitated diffusion across the placenta, which will be further discussed.

The eggs laid by oviparous animals contain numerous structures which are uniquely chemically composed to provide the necessary conditions for development. The egg yolk provides a supply of nutrients for the embryo, the albumen regulates pH, and the eggshell membrane and cuticle are chemically adapted to protect the embryo (Yves et al., 2011).

Eggshells of oviparous animals even differ on a chemical level based on whether or not they are mineralized. Mineralization refers to the calcification of the eggshell, which results in a more rigid shell in avian eggs versus more membrane-like or pliable shells in non-mineralized eggs (Hincke, 2012). This process occurs for almost all avian eggs, while it is very rare for other animals such as snakes, lizards, turtles, and many more, although exceptions exist.

Placentation

Embryonic development in nonmammalian vertebrates depends entirely on nutritional reserves that are predominantly derived from vitellogenin proteins and stored in the yolk of the egg (Brawand et al., 2008). Conversely, mammals have evolved a new resource to nourish their developing and early offspring: placentation. The mammalian placenta is a temporary organ that develops during pregnancy by an intricate fusion of maternal and fetal tissues. The establishment of physical contact between the fetus and the mother inside the mother’s body is responsible for the chemical exchanges between the mother and fetus for optimal fetus survival, growth, and development (Furukawa et al., 2014). More precisely, the purpose of placentation is maternal provisioning of a selective membrane filter, blocking or diffusing the transfer of specific molecules, as well as the removal of waste products from the fetus (Wagner, 2018).

Placental Development in Ruminants

Ruminants are eutherian species that develop placentas during pregnancy. Following fertilization, the embryo goes through stages of placental implantation. First, the ‘blastocoel,’ composed of early embryonic cells called blastomeres, undergoes a blastocyst stage where the fluid-filled central cavity becomes surrounded by a layer of trophoblast cells which form the ‘trophectoderm’ (Fig. 1a) (Schlafer et al., 2000). The outer trophectoderm layer combines with the subjacent embryonic mesoderm and forms the double layered membrane, the ‘chorion.’ Meanwhile, the inner surface of the chorion becomes vascularized by direct apposition to a bulging fluid-filled ‘allantoic cavity’ that develops from the growth of the embryo. This layer, fused by the allantois and chorion, becomes the ‘chorioallantois’ in the placenta (Fig. 1b) (Peter, 2013).

Around days 15-20 post-fertilization, the placental implantation process begins in ruminants when the chorionic epithelium of the fetus become cotyledons by developing folds and villi that extend into crypts of raised projections of the caruncle epithelium, also known as the uterine wall (Fig. 2a) (Pastor-Fernández et al., 2021).The combination of the fetal cotyledon and the maternal caruncle is termed placentome (Fig. 2b), which is considered the functional units where the vital placental functions take place (Sebire & Talbert, 2004).

Structure and Function of the Placenta in Ruminants

The ruminant placenta forms an interface between the maternal and fetal circulation, thus fulfilling the role of the digestive, respiratory, excretion, metabolic, and endocrine system of the fetus. Trophoblast cells in the cotyledonary are key components of the ruminant placenta where two different populations are described: the uninucleated trophoblast cells (which form most of the fetal interface and are primarily involved in nutrient exchange) and the binucleated trophoblast cells (that fuse with caruncular cells and are involved in the synthesis of hormones) (Fig. 3). The extracellular matrix (ECM) is another key component of placenta that mediates trophoblast attachment to the uterine wall, induces cell differentiation, acts as a reservoir of growth factors, and serves as a track on which cells can migrate along. The ECM consists of a mixture of proteoglycans, glycosaminoglycans, and structural components such as various types of collagens, fibronectin, and laminin (Pastor-Fernández et al., 2021).

Nutrients

Low molecular weight substances (water, electrolytes, urea, uric acid, creatine) can cross the placenta by simple diffusion. The waste products (urea, uric acid, creatine) are easily excreted from the fetus and transferred to the maternal blood. Other metabolites (glucose, amino acids, fatty acids) are major energy substrates that associate to specific carriers to pass the placenta. These molecules are transported across the placenta by facilitated diffusion via protein transporters. Although in the case of glucose, while the fetus does receive amounts of intact glucose, a large amount is oxidized within the placenta to lactate for fetal energy production. Similarly, more complex molecules (proteins, phospholipids, neutral fats) are modified to other simpler compounds before crossing the placental membrane, and then resynthesized (Pastor-Fernández et al., 2021).

Transport of Gases

The placental gas transfer to the ruminant fetus depends on the diffusing capacity of the placenta based on the blood oxygen and flow rate in the uterine and umbilical arteries. Gases, such as oxygen (O₂) and carbon dioxide (CO₂), diffuse through and across placental tissues in response to differences in partial pressure (Fig. 4) (Pastor-Fernández et al., 2021).

During pregnancy, the partial pressure of oxygen in maternal blood is considerably higher than that in fetal blood (P_O2 maternal > P_O2 fetal). Because the mother’s stream is richer in oxygen than that of the fetus, some O₂ molecules naturally flow towards the fetal circulation by simple diffusion. Other times, fetal hemoglobin needs to pick up O₂ in the placenta by stealing it from the hemoglobin in the bloodstream of the mother’s uterus. The fetal hemoglobin has high affinity for O₂, thus binds to oxygen more strongly ensuring that sufficient O₂ is trapped in the fetus respiratory system (Amberg et al., 2021).

Meanwhile, CO₂ is produced abundantly in the fetus from the uptake O₂ from the maternal blood. The oxygenation of fetal hemoglobin results in decreased affinity for carbon dioxide, so the partial pressure of carbon dioxide (P_CO2) in fetal blood tends to rise (the Haldane Effect). Therefore, CO₂ diffuses from fetal blood, through the placenta, into the maternal circulation, and is disposed of by expiration from the mother’s lungs. Essentially, gas interchange between the mother and the fetus is favored by the high P_O2 in the maternal circulation, as well as by the high affinity of fetal hemoglobin for O₂ (Carter, 2015).

Similar to viviparous animals, those who lay their eggs externally also have many chemical adaptations in place, to make sure the embryo is safely transitioning through the different stages leading up to birth. These happen in the very sophisticated components in the eggs themselves.

Egg and Egg-Membrane Composition

Many chemical factors are present to ensure the embryo’s safe development within the egg. Properties of matter and behaviors of molecules contribute to the process leading up to the hatching of these complex structures that make up the foundation of oviparous evolution. Female animals that lay eggs must rely on natural chemical changes that are fundamental aspects of external embryonic development. The egg contains several distinct parts with different functions that are important to introduce. The yolk, also known as the vitellus, is responsible for supplying the embryo with the necessary nutrients required for development (Yves et al., 2011). The albumen, otherwise called the egg-white, plays a defensive role against microbes and bacteria while also providing the embryo with water and proteins (Willems et al., 2014). While the outermost shell structure is different amongst different species, these internal components are similar between avian and reptilian eggs (Badham, 1971). Finally, the eggshell and its membrane counterpart serve as a protective shield for the embryo (Fig. 5).

The yolk in the egg is mainly composed of lipids, water and protein which all nourish the embryo until emersion (Meng et al., 2019).  Electrophoresis, a common biological technique for separating proteins and genetic material, allows for a proteomic analysis of the yolk in hen eggs during different stages of development (Meng et al., 2019). There is a clear modification of protein abundance during late-stage embryonic development, including Vitellogenin (VTG), an essential protein for embryogenesis. After proteomic comparison between fertilized and unfertilized hen eggs, only those who carried a viable embryo demonstrated such abundance alteration of VTG and other proteins (Meng et al., 2019). VTG’s importance in embryogenesis stems from its ability to be a precursor to the formation of proteins and lipids needed for the embryo to grow into a matured animal (Sugawara, 2011). This process, known as vitellogenesis, occurs in the formation of the egg in most oviparous animals. It begins in the mothers’ liver, where the corresponding gene synthesizes the proteins (Hara et al., 2016). These essential polypeptides travel through the bloodstream of the mother to eventually make their way through the outermost membrane of the egg (the chorion). These proteins reside in the vitellus during embryogenesis (Fig. 6).

The albumen, which surrounds the yolk, contains the thick egg white, which is more viscous than the thin egg white portion. Its chemical nature adapts to the egg’s surrounding environment and defends the embryo from any harmful substances that venture into the egg (Willems et al., 2014). It does this by regulating its pH. After an avian egg is laid, the pH of the albumen goes from 7.6 to 9.5 in the span of one day. This trend towards a more alkaline nature of the albumen along with enzymes present within it, such as lysozyme, allow for a protective chemical mechanism of incoming bacteria (Willems et al., 2014). Lysozyme, whose chemical composition is shown in Figure 7, goes through an antimicrobial mechanism which facilitates the degradation of the cell wall in bacteria via hydrolysis, a water induced bond breaking chemical reaction (Guyot et al., 2013). Moreover, the albumen is also responsible for the transfer of water into the yolk during storage, done via osmosis (Willems et al., 2014). The main protein found in the egg white is ovalbumin. It is a large protein containing 385 amino acids, and has a molecular weight of 44.5 kDa. Its great size is relevant to how it is able to transport itself through different membranes and passages within and on the border of the egg (Strixner, 2011). Its secondary chemical structure is composed of 30% α and 32% β-sheet, which is important for any possible thermal denaturation during the egg’s incubation period (Chay Pak Ting et al., 2013).

The egg also has a very intricately designed eggshell membrane (ESM). The ESM is a component of the eggshell along with the cuticle (Fig. 8). The chemical compounds that compose this layered organic shell are extremely crucial to the safety of the developing embryo.

In eggs, the shell membrane is composed of multiple layers of fibrous material, with the presence of proteins like collagen (Richards et al., 2000). Some of the proteins detected in the ESM are derived from the previously mentioned components of the egg, like the yolk and the egg white. A significant amount of clusterin was found in the membrane of the eggshell. This peptide plays a cytoprotective role in the maturing of the embryo (Makkar et al., 2015). Cytoprotection in the ESM is a process in which clusterin, a chemical compound, protects the embryo from its surroundings. Whether it be bacterial, environmental or oxidative stress applied on the egg, nature provides chemical adaptations that can keep the embryo safe within this organic vessel (Makkar et al., 2015). When it comes to the structure, the fibres and components of the ESM are interwoven in a network like structure to better interfere with any negatively impacting agents coming into the egg through the pores of the eggshell (Fig. 9) (Sabu et al., 2018). The intricate structure is due to disulfide bonds that are present between components of the ESM (Kim et al., 2019).

The egg components and their chemical compositions work together to protect the embryo throughout the different stages leading up to hatching. The biochemical adaptations in oviparous animals such as birds, reptiles, fish and others all play the main roles of providing the embryo with a source of nutrition and a complete defense system.

Eggshell Mineralization

The eggshell itself is integral to the reproduction of birds and various species of reptiles. It is often thought to be just a physical barrier to protect the developing young animal within the egg, but this is not necessarily the case. The eggshell fulfills multiple roles in many species; it can enable gas exchange through pores on the eggshell, be used as a calcium reserve in many species, and of course be a barrier used as protection against the outside environment (Hallman, 2015). However, the composition of the eggshell itself is often very different between species. In reptiles, most squamates (scaled reptiles) lay parchment type eggs, meaning the eggs consist of a boundary layer and a fibrous shell membrane (Hallman, 2015). This makes the egg softer and more deformable. In contrast to this, the avian eggshell is a highly ordered structure created by certain minerals (Nys et al., 1999). The key difference between these two is the mineralization of the eggshells. While reptiles have mostly non-mineralized eggs, birds have very mineralized eggshells leading to a less deformable and more rigid structure.

Avian Eggshell Mineralization

The mineralization of the avian eggshell occurs incredibly quickly. It is the fastest calcification process found in nature to date (Rodriguez-Navarro et al., 2015). For example, in hens, this process takes about 18 hours and occurs daily (Nys et al., 1999). The way this process can lead to a very ordered structure has not been well studied, but the chemistry behind the mineralization is well known. In hens, calcium carbonate is the main material used to create the eggshell (Rodriguez-Navarro et al., 2015). Calcium carbonate is an essential compound for many organisms as it is used in the formation of shells of varying types (seashells, eggshells, etc.).

The mineralization of the hen’s eggshell takes place over a period of less than a day, and has three stages: initiation, linear growth, and termination (Fig. 10) (Rodriguez-Navarro et al., 2015). During the first phase, initiation, organic sites are secreted onto the eggshell membrane (Rodriguez-Navarro et al., 2015). These sites are nucleation sites at which the mineralization will occur. The organic-rich sites are secreted before the egg enters the uterus and are used to direct the calcium carbonate to where precipitation and the creation of the mineral structure will occur. They are known as mamillary knobs (or cores) (Rodriguez-Navarro et al., 2015). Once the egg is in the uterus, mineralization is initiated. This first step takes place a few hours after ovulation. In the next stage, linear growth, the rest of the eggshell is mineralized over the course of 10-22 hours (Rodriguez-Navarro et al., 2015). The mineralization of the egg, however, occurs due to the uterine fluid bathing it. The fluid contains all necessary ingredients in order for the egg to be mineralized, including a wide array of organic compounds to aid in eggshell formation, as well as large amounts of calcium ions and carbonate ions (Rodriguez-Navarro et al., 2015). For the step to actually occur, there must be an abundance of Ca²⁺ and HCO₃^– (bicarbonate) ions present in the uterine fluid (Brionne et al., 2012). These ions are derived from the blood and require ion pumps in order to reach the uterus. The bicarbonate ions needed to form the calcium carbonate and mineralize the eggshell actually come from CO₂ in the hen’s blood. It is then transformed into bicarbonate with the help of carbonic anhydrase (Fig. 11.) (Brionne et al., 2012).

The calcium carbonate ions then precipitate onto the membrane to mineralize it at specific nucleation sites (mamillary knobs) (Rodriguez-Navarro et al., 2008). These mamillary knobs are where the organic-rich compounds originally bind to direct this precipitation of the calcium carbonate to mineralize the shell. This step is completely halted in the termination phase a few hours before the egg is expelled by the hen (Rodriguez-Navarro et al., 2015). The precipitated calcium carbonate method of mineralization produces a very precise and ordered structure of large columnar calcite (calcium carbonate) crystals as can be viewed through the cross-sectional view of a segment of the eggshell (Fig. 12.) (Rodriguez-Navarro et al., 2015). The columns of calcite are formed perpendicular to the surface of the shell itself (Rodriguez-Navarro et al., 2008). These particles of calcium carbonate are bound together through intermolecular forces, namely electrostatic forces between them as they are ionic compounds (The Pharmacology Education Partnership, n.d.). The negative and positive charges interact with each other to create very strong bonds. This results in a somewhat smooth eggshell with small pores for gas exchange (Fig. 13.).

The process of mineralization can also be viewed when looking at eggs that were prematurely expelled. In such cases, the stage of mineralization can be viewed and better understood. As seen in figure 14, the expelled egg is beginning to show mineralization as there is the beginning of calcium carbonate deposits on the egg membrane. The particles are somewhat grouped, but there should be more that form a highly organized structure. In the center of each grouping however, there is a small hole which is used to facilitate gas exchange between the outside world and inside the egg membrane (Nys et al., 1999). Another example shows a soft-shelled egg which has a completely random assortment of mineral deposits, inconsistent with normal development (Nys et al., 1999).

Reptilian Eggshell Mineralization

Reptiles, in contrast to birds, do not often have a mineralized eggshell. This is especially true for squamates (scaled reptiles), for which almost none have mineralized shells (Hallmann & Griebeler, 2015). However, the reptilian eggshells do have similar functions as those of birds. The reptilian non-mineralized egg allows for gas exchange as well as water uptake (permeability), in addition to protecting the embryo. The amounts of calcium in the shell can also be used to nourish the embryo (Hallmann & Griebeler, 2015). The eggshell of these squamates is also quite simple compared to avian eggshells; the squamate eggshell consists of a thick membrane of protein fibrils. These fibrils provide the main structure of the eggshell and allow flexibility, in contrast to the stiff and brittle avian eggshell. They may also contain a superficial layer of calcite (Hallmann & Griebeler, 2015). These shells do not seem as strongly calcified as avian ones.

One large exception to this is the eggshell of the Gekkota (Fig. 15.) (Choi et al., 2018). The gecko species do have hardened shells due to calcification of the outer layer, but this occurs in a very different way than how it forms in birds. The eggshell of geckos lacks the mammillary layer that can be found in bird eggs (such as the hen) (Choi et al., 2018). This layer is one of the calcified layers of the hen egg. The growth off the eggshell begins at the outer surface and travels inward, in direct contrast to how avian eggshells form (Choi et al., 2018). Although the gecko eggshell has very similar mineralization with calcium carbonate to that of a hen, it grows and mineralizes in the opposite direction compared to the hen egg. It also seems to lack the layer in which the calcium carbonate is first deposited in hen eggs (mammillary layer).

The Gekkota is an exception in having a mineralized eggshell when compared to other squamates and is also different when compared to the avian eggshell. Most other reptiles have flexible and non-hardened eggshells with no mineralization. It is possible that most reptiles have non mineralized eggs to facilitate diffusion of water into the inside of the egg (Hallmann & Griebeler, 2015). Bird eggs have developed a mineralized eggshell to further protect against microbial contamination, while also allowing for gas exchange (Gautron et al., 2021). It has also been found that chicken eggshells reflect a large amount of UV light therefore stopping it from passing through, acting as protection for the embryo (Fecheyr-Lippens, 2017).

Conclusion

The contributing factors to the development of an embryo are generally taken for granted due to their inherent significance for every animal, even though in reality these factors are very chemically complex and must work in harmony to nurture and protect the early stages of life. In viviparous animals such as ruminants, placentation is a deeply crucial process as it sets the stage for every aspect of embryonic, and later fetal, development to occur in good health. The formation of the placenta not only acts as a physical protective barrier for the embryo but also as an intimate connection between the parent and the embryo through which all the necessary nutrients and gases for the embryo to grow can be transferred via different levels of diffusion. In contrast to this, the eggs laid by oviparous animals cannot rely on their parent for protection or a steady supply of nutrients due to their development externally from the mother. Thus, eggs have developed unique chemical adaptations and structures to create the conditions necessary to harbor the embryo during its growth. Furthermore, the composition of eggshells differs greatly between different oviparous animals on the basis of whether or not they are mineralized. As explained through the examples of hen and gecko eggs, eggshell mineralization provides a more rigid-bodied egg which is better adapted for avian eggs and certain exceptions of non-avian eggs. The examples discussed in this paper provide a small glimpse into the complex chemical adaptations associated with the beginning of life for every animal, but there is still much more to explore in hopes that they can someday be replicated outside of nature.

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