Nutrition During Gestation in 2 Species of Viviparous Fishes (Poeciliidae): Poecilia latipinna (Lecithotrophic) and Heterandria formosa (Matrotrophic)

Abstract

In viviparous teleosts, the lack of oviducts defines intraovarian gestation, with the ovary being the site for oogenesis but also the site for insemination, fertilization, and gestation. Consequently, intraovarian gestation is a complex and exceptional type of reproduction among vertebrates. The analysis of the morphological and physiological components of intraovarian gestation documents the evolutionary process of nutrition in viviparous species. Two types of embryonic nutrition may occur during gestation: (a) lecithotrophy, when most nutrients for the embryo come from the abundant yolk stored during oogenesis, and (b) matrotrophy, when nutrients for the embryo with scarce yolk must be obtained during gestation by additional maternal provisioning, developing a placenta. Then, investment of maternal nutrients for the embryo is greater during oogenesis in lecithotrophic species, and investment of maternal resources for the nutrition of the embryo is greater during gestation in matrotrophic species. Microscopic techniques allow for proper observation of maternal and embryonic structures involved in both types of nutrition during the development of embryos. Specifically, we focused on the morphology of placental structures associated with embryonic nutrition at different stages of development, which are the yolk sac and the pericardial sac. The oocytes of Poecilia latipinna contain a large amount of yolk (an average oocyte diameter of 1.9 mm); in contrast, the oocytes of Heterandria formosa contain extremely reduced amounts of yolk (an average oocyte diameter of 0.4 mm). Therefore, these species represent appropriate models for studying the strategy of two different types of embryonic nutrition, lecithotrophy and matrotrophy, in viviparous teleosts.

1. Introduction

Viviparity, the process of reproduction where the mother retains the embryos during gestation until birth, occurs in all vertebrates except in birds. In viviparity, maternal tissues may provide the embryos with the requirements for the development. Consequently, the embryos in viviparous gestation become progressively dependent on the maternal organism including trophic transfer, osmoregulatory, excretory, respiratory, endocrinological, and immunological procedures [1,2,3,4]. Viviparity among vertebrates makes the evolutionary first appearance in fishes, becoming essential in the understanding of this reproductive strategy [5,6]. Among more than 32,000 species of teleosts, only 510 species are viviparous [7,8]. There are specific morpho-physiological aspects occurring in the viviparity in teleosts which differ to the other vertebrates. These characteristics are related to the embryogenesis of the female reproductive system of teleosts, where there is no development of Müllerian ducts, the structures which form the oviducts where the uterus, the site of gestation in the rest of vertebrates, is developed. As a result of the lack of Müllerian ducts in teleosts, there are not oviducts; therefore, the ovary courses directly to the exterior of the body. The posterior region of the ovary, called the gonoduct, lacks germinal cells and connects the anterior germinal region of the ovary to the exterior at the genital pore [9,10]. Consequently, in viviparous teleosts, the development of the embryos occurs in the ovary, instead of the uterus; this is intraovarian gestation, unique in vertebrates [8,11,12,13]. Given that the ovary of teleosts is the site of gestation, the physiological dynamic of this organ during the reproductive cycle involves great complexity, because, additionally to oogenesis, insemination, reception and maintenance of spermatozoa, fertilization of oocytes, and gestation and birth of the embryos also occur in the ovary. Intraovarian gestation in viviparous teleosts is characterized by intimate contact between maternal and embryonic tissues, a process involved in metabolic interchanges between the two organisms. Intraovarian gestation in different species has numerous morphological variations as the result of adaptations associated with their viviparity.

During oogenesis of different species, the ovary forms different types of oocytes according to diverse amounts of yolk storage. Then, there are species that develop large oocytes with abundant yolk (macrolecithal), in contrast to other species that develop small oocytes with scarce yolk (microlecithal). Therefore, two types of embryonic nutrition may occur during gestation: (a) lecithotrophy, when nutrients for the embryo come from the abundant yolk stored during oogenesis, and (b) matrotrophy, when nutrients for the embryo with scarce yolk must be completed during gestation by additional provisioning from maternal tissues, developing a placenta for maternal–embryonic transfer of nutrients.

The viviparous species of the family Poeciliidae, order Cyprinodontiformes, are the most numerous of viviparous teleosts with approximately 237 species [6,14,15] out of a total of 510, previously mentioned. Most poeciliids occur in freshwater ecosystems, from the southeast of the United States to South America, with the main distribution in Central Mexico, the southeast of México, the Caribbean Islands, and the Gulf of Mexico [7,15,16,17].

With the perspective of analyzing the complex features of the maintenance of embryos during intraovarian gestation, we selected two species of the family Poeciliidae (Poecilia latipinna and Heterandria formosa) to document this process and compare morphological analysis of the structures associated with the embryonic nutrition, the yolk sac and the pericardial sac; of these selected species at different stages of development, in particular, we compared the morphological features associated with lecithotrophy and matrotrophy to infer evolutionary adaptations in intraovarian gestation.

The ovary of poeciliids is longitudinally situated, and it is supported to the dorsal wall of the body by a mesentery. The ovary contains a central lumen, corresponding to the cystovarian or saccular type of ovary [18,19,20]. This type of ovary is developed during embryogenesis as an invagination of the genital ridges, which close ventrally, forming the ovarian lumen. As a result, the coelomic epithelium that lines the germinal ridges and becomes the germinal epithelium is located internally along the ovarian lumen. This embryological process establishes the saccular structure or cystovarian type of ovaries. In poeciliids, during early embryogenesis, the two ovaries fuse in a single organ. The saccular ovary provides ample space for the growth of oocytes and embryos during the reproductive cycle.

As it was described in other vertebrates, oogenesis in poeciliids comprises a sequence of oocyte changes during their differentiation to mature oocytes. Oogenesis occurs into the ovarian follicle. Oogenesis is initiated when the oogonium is surrounded by a single layer of squamous follicular cells. The follicular cells form a single follicular epithelium that is based on a basal lamina. Follicular cells have underlying theca formed by vascularized connective tissue. This structure of the follicle maintains close relationships between the follicular cells and the oocyte during all the oogenesis [21]. The follicle cells, as they occur in other epithelia, form a barrier of permeability to control the transport of substances between the blood vessels and the oocyte, an essential function that is very active during all of oogenesis [19,22].

Similarly to the oogenesis of other teleosts, oogenesis in poeciliids involves two main stages: (a) primary growth or previtellogenesis, when ooplasmic components increase as ribosomes, endoplasmic reticulum, and mitochondria, and (b) secondary growth or vitellogenesis when the oocyte grows considerably in diameter by the storage of lipid globules and yolk granules [13,21,23,24]. Yolk consists of vitellogenin, which contains proteins and lipoproteins, being the major material stored in the oocyte for the nutrition of the embryo. The yolk will be used for all the metabolic activities required during the embryogenesis [13,18,22]. Throughout the advance of vitellogenesis in poeciliids, the yolk granules fuse progressively, becoming a fluid and homogeneous single drop [19,20,25]. In mature oocytes the nucleus or germinal vesicle of the oocyte migrates to the periphery of the ooplasm forming the animal pole [19]. In poeciliids there is no ovulation because the oocyte is fertilized inside the follicle and, during all the gestation, the embryo remains inside the follicle, moving out from the follicle to the ovarian lumen before birth; consequently, intraovarian gestation is intrafollicular [1,5,8,18]. In recognition of embryological development during gestation, we follow the suggestion of Blackburn & Starck [26], using the term “embryo”, including all stages of gestation.

The intrafollicular gestation implies that the embryo is surrounded by maternal follicular epithelium and vascularized connective tissue during all the gestation, establishing contact with the growing embryo [1,4,8,27]. In this type of gestation, due to the abundant yolk stored in the oocyte, the yolk provides the most nutrients for the embryo, and respiration and excretion occur through the maternal tissues surrounding the embryo. The intrafollicular gestation in poeciliids has been extensively analyzed by several authors [3,4,5,6,8,18,27,28,29,30,31,32,33,34,35].

The differences in the oocyte size of poeciliids involve differences in the supply of nutrients for the embryos during gestation. If the yolk is enough for the nutrition of the embryo during all gestation, the transfer of nutrients from the mother to the embryo are null or very scarce, so nutrition occurs by lecithotrophy. But if the yolk of the oocyte is scarce, the embryo develops structural adaptations for obtaining nutrients from the mother; then, nutrition occurs by matrotrophy [4,34,36,37,38,39]. Differences in the amount of yolk of the oocyte may occur in different species; consequently, these species display different types of embryonic nutrition. These different strategies of nutrition require modifications in the timing of maternal resources offered to the embryos, and these differences arise during oogenesis, before fertilization, in lecithotrophic species, or after fertilization in matrotrophic species [8,35,38].

To permit exchange between the mother and the embryo in poeciliids a follicular placenta has been developed. The placenta contains maternal components: the follicular wall (follicle epithelium and subjacent vascularized connective tissue), and the embryonic components: the yolk sac around the yolk, and the pericardial sac, which expands over the anterior of one-third of the embryo. Both embryonic components make contact with the follicular wall and then near the maternal blood vessels [30]. The yolk sac, surrounding the yolk situated ventrally to the embryo, forms an essential structure in the development of the embryos. The epidermal layer of the yolk sac produces cells that break down the yolk into small elements that circulate through the blood vessels toward the embryo. In this reflection, the yolk sac can be considered an essential organ due to its importance providing nutrients to the embryo [40,41].

2. Material and Methods

In the perspective to compare the morphology of two species of poeciliids with different types of oocytes according to the amount of yolk, we selected two species, Poecilia latipinna, a species with large oocytes (2.0–2.2 mm in diameter) [42], and Heterandria formosa, a species with small oocytes (0.40–0.45 mm. in diameter). The oocytes of H. formosa are the smallest described in poeciliids. They are called microlecithal and represent an extreme in the reduction in yolk deposition in poeciliids [43,44].

Ovaries of the two species presented in this article, P. latipinna and H. formosa, were donated by Dr. Harry Grier in 2014, from his aquaria. Five stages of reproduction were considered: ovaries were selected during non-gestation (in previtellogenesis and vitellogenesis stages) and, during gestation (early, middle, and late stages). Ovaries were fixed in Bouin (8–15 h). After fixation, the ovaries were dehydrated in a series of graded ethyl alcohols (30, 50, 70, and 100%). The dehydrated ovaries were embedded in glycol methacrylate plastic resin (JB-4 embedding kit), (Sigma-Aldrich, Waltham, MA, USA). The embedded ovaries were sectioned at 5 μm thickness. After microtomy, slides were stained with hematoxylin and eosin (H&E). Digital photomicrographs were taken using an OLYMPUS digital camera model C5050Z (Olympus, Tokyo, Japan) coupled to an OLYMPUS CX31 microscope (Olympus, Tokyo, Japan).

3. Results

The oocytes of P. latipinna contain abundant yolk (Figure 1A–C), which occupies most of the oocyte; consequently, the embryonic nutrition develops high degree of lecithotrophy. The embryo depends on nutrients included in the oocyte, which will be consumed progressively during gestation. The embryo remains in the follicle during all gestation (intrafollicular gestation), with the follicular cells in close apposition to the embryo during early gestation (Figure 2A–C), middle gestation Figure 3A–C), and late gestation (Figure 4A–C). This relationship forms a follicular placenta where respiration and excretion occur between the maternal and embryonic tissues, but the nutrients are obtained from the abundant yolk stored during the vitellogenesis. Through this process the embryo develops the yolk sac situated in the ventral region. The yolk sac is involved in the utilization of yolk. The yolk sac is formed from early stages of embryogenesis, when cells around the embryonic disk (Figure 2A–C), as extensions of endoderm and mesoderm, grow covering the surface of the yolk; therefore, the yolk sac is provided by a well-developed absorbent epithelium and loose connective tissue with a vascular network (Figure 2C). Soon, in the early stage of gestation, during neurula, the yolk is surrounded completely by the yolk sac (Figure 2A). This condition will be maintained during all gestation (Figure 3A–C and Figure 4A–C). Then, the epithelium of the yolk sac breaks the yolk into small elements that flow through the abundant capillaries to the whole embryo (Figure 3C and Figure 4C). During gestation the vascularization of the yolk sac increases, the blood vessels being more abundant (Figure 2C, Figure 3C, and Figure 4C). The surface of the yolk sac contacts the maternal follicular epithelium, developing a placental relationship. During the embryogenesis of P. latipinna, the yolk sac progressively diminishes as the yolk is used for the nutrition of the embryo (Figure 3A–C and Figure 4A–C). This reduction is evident in late embryos (Figure 4A–C), and after birth, the offspring may still contain remaining yolk.

The oocytes of H. formosa contain scarce yolk and some lipids (Figure 5A–B); consequently, the embryonic nutrition develops a high degree of matrotrophy from early gestation (Figure 6A–D), middle gestation (Figure 7A,B), and late gestation (Figure 8A–C). This process is associated with the development of placental structures that increase the efficiency of nutrient transfer between mother and embryo [35,44,45]. During early gestation, an extension of the embryo surrounds the scarce yolk and the lipid globules, starting a yolk sac. When the scarce yolk and the lipid globules are reduced during early embryogenesis, the yolk sac is also reduced (Figure 7A). During this process is progressively activated the transfer of maternal nutrients through matrotrophy. Then, an extension of the embryonic disk grows, developing the pericardial sac, which dorsally surrounds the embryo (Figure 7A,B and Figure 8A–C), initiating the development of an ample vascular plexus (Figure 7A,B and Figure 8A–C), which is very efficient in the absorption of nutrients. The pericardial sac continues its growth, developing larger blood vessels around the head. There are portions of the pericardial sac that come in close contact to the maternal tissue, that is, the surface of the pericardial sac contacts the maternal follicular epithelium, allowing a placental relationship to emerge. During middle gestation (Figure 7A,B), the embryonic pericardial sac contains larger blood vessels, some of them in contact to the maternal follicular epithelium, increasing the placental surface. Throughout late gestation (Figure 8A–C), as in the middle stage, there are portions of the pericardial sac in contact with the maternal follicular epithelium (Figure 8C), where larger blood vessels of both the embryo and mother have an active transfer of nutrients through the end of gestation.

4. Discussion

This analysis compares the structural characteristics of the nutrition occurring during intraovarian gestation of the species P. latipinna and H. formosa, showing the contrast in the structure of the oocytes according to the amount of stored yolk before fertilization, during three stages of gestation (early, middle, and late), and the structures developed during gestation for embryonic nutrition according to the availability of nutrients. The ovary of both species analyzed in this study, exhibit a central lumen, corresponding to the cystovarian or saccular type of ovary. As it was described in other vertebrates [21,23,24], the oocytes were surrounded by a layer of follicular cells, and oogenesis presented a sequence of oocyte changes during previtellogenesis and vitellogenesis in developing mature oocytes. The difference between vitellogenesis of both species, where the oocytes of P. latipinna present abundant yolk compared to scarce yolk in the oocytes of H. formosa, define the development of the yolk sac and the pericardial sac. Consequently, the yolk sac is large in P. latipinna, which is gradually reduced as the yolk is consumed during gestation, coinciding with observations of Kuzmina [41]; and the yolk sac is small in H. formosa; meanwhile in this species, the pericardial sac is developed during gestation, containing abundant capillaries similar to that described by Ponce de León and Uribe [35]. The source of nutrients for the embryonic development, from the yolk in P. latipinna, or from the interchange with the maternal tissues in H. formosa, define the lecithotrophic or matrotrophic nutrition observed in these species. Coinciding with Lombardi [37], these changes in maternal physiology, related to the nutrition of developing embryos, have essential ecological and evolutionary significance.

According to Wourms [2], lecithotrophy is the primitive condition because it involves the gestation of embryos with abundant yolk, without the development of specialized structures for transferring maternal nutrients to the embryos. Schrader and Travis [36] consider that matrotrophy is a complex mechanism of parental care that represents a diverse transition in reproduction since it requires a shift in the timing of maternal resource provision from pre- to post-fertilization. In this physiological sequence, Pollux & Reznick [38], and Fleuren et al. [40], suggest that the evolution of the embryonic nutrition is associated with the reduction in size in the oocytes, favoring females to attain better conditions through oogenesis, with small oocytes, and through early gestation, with embryos with scarce yolk making females less heavy during these times of the reproductive cycle, favoring their movements in the habitat. Lecithotrophy has been revealed to be better adapted energetically to seasonally unpredictable habitats, whereas matrotrophy requires a predictable food supply during all gestation for a constant transfer of nutrients from the mother to the embryos. Then, the condition of the habitat of the species determines the diversity of systems for the embryonic nutrition during gestation, and consequently, the convenience of lecithotrophy or matrotrophy [27,29,35,38]. Consequently, the compared analysis of the occurrence of both types of nutrient transfer, lecithotrophy, and matrotrophy, as essential processes during gestation in species of the family Poeciliidae, contributes to the understanding of the evolution of viviparity in teleosts.

5. Conclusions

Differences in the oocyte size of poeciliids involve differences in the supply of nutrients for the embryos during gestation; therefore, these species display different types of embryonic nutrition. If the yolk is enough for the nutrition of the embryo during all the gestation, nutrition occurs via lecithotrophy, but if the yolk is scarce, the embryo develops structural adaptations for obtaining nutrients from the mother, so nutrition occurs via matrotrophy. In the lecithotrophic species P. latipinna, the oocyte is big, containing abundant yolk; consequently, the yolk sac has large development. In the matrotrophic species, H. formosa, the oocyte is small, with scarce yolk, so the yolk sac has low development, and the pericardial sac has abundant blood vessels near the maternal follicular epithelium, developing a high degree of complexity compared to the lecithotrophic species P. latipinna.