Structures Associated with Oogenesis and Embryonic Development during Intraovarian Gestation in Viviparous Teleosts (Poeciliidae)

Abstract

Viviparity in teleosts involves, invariably, the ovary in a gestational role. This type of viviparity is due to the combination of unique aspects, different from those found in the rest of vertebrates. These aspects are: The ovary has a saccular structure; the germinal epithelium lines the ovarian lumen; the absence of oviducts; and the intraovarian insemination, fertilization, and gestation. The communication of the germinal zone of the ovary to the exterior is via the caudal zone of the ovary—the gonoduct. The germinal epithelium is composed of oogonia and oocytes scattered individually or in cell nests among somatic epithelial cells. In the ovarian stroma the follicles are included which are formed by the oocyte, which is surrounded by follicular cells and the vascularized theca. The oogenesis comprises three stages: chromatin-nucleolus, previtellogenesis, and vitellogenesis. There is no ovulation, as the oocyte is retained in the follicle. During the insemination, the spermatozoa enter into the ovarian lumen and the intrafollicular fertilization occurs, followed by intrafollicular gestation. The intraovarian gestation of poeciliids involves morphological characteristics associated with the intrafollicular embryogenesis and types of nutrition, such as lecithotrophy and matrotrophy. In lecithotrophy, the nutrients come from the yolk reserves stored during oogenesis, whereas in matrotrophy the nutrients are provided by supplies from maternal tissues to the embryo during gestation. The maternal–embryonic metabolic interchanges converge through the development of the association of maternal and embryonic blood vessels, establishing a follicular placenta.

1. Ovarian Adaptations of Viviparous Teleosts

Among more than 32,672 species of teleost fishes [1], approximately 510 are viviparous [2,3,4]. Viviparity in teleosts has unique aspects compared with the rest of viviparous vertebrates. To compare viviparity of teleosts with other viviparous vertebrates is very useful for defining the morpho-physiological adaptations of reproductive structures, which are unique elements associated with teleost viviparity [5,6,7]. In the evolution of viviparity from oviparity there is a change in the site of embryonic development from the external environment to the internal female reproductive system. Viviparity in fishes is a complex process, which involves several modifications in the reproductive systems of both males and females [8,9]. Some of these aspects are related with the structure of the ovary for gestation, because the viviparity in teleosts involves, invariably, the ovary in a gestational role.

During the adult life of non-teleost vertebrates, the ovarian cortex, which is lined by the germinal epithelium with oogonia for oogenesis, remains in contact with the coelomic cavity where ovulation occurs. In contrast, in the cystovarian type of teleosts, the cortex of the embryonic ovary, also lined by the germinal epithelium, undergoes an invagination that fuses internally becoming the saccular structure of the ovary. This saccular structure has an internal space—the ovarian lumen. As result of this unique development of the ovary of teleosts, the surface of the ovarian lumen contains the germinal epithelium situated internally [10]. Consequently, in teleosts, at ovulation, the eggs are shed into the lumen of the ovary (internal ovulation), in contrast to the rest of vertebrates where ovulation occurs toward the coelomic cavity [11].

Additionally, a distinctive feature of almost all viviparous teleosts occurs when, during the early embryonic development, the right and left ovaries fuse, forming a single ovary [8,11,12]. This fusion establishes a single and saccular ovary, with the germinal epithelium lining the internal lumen.

Another unique characteristic of teleosts among vertebrates is that teleosts do not develop Müllerian ducts during the embryogenesis, as occurs in the rest of vertebrates; consequently, teleosts do not have oviducts. Then, the communication of the germinal zone of the ovary to the exterior occurs via the caudal zone of the ovary—which is called the gonoduct—an ovarian zone that lacks germinal cells [8,13,14]. Therefore, the lumen of the ovarian germinal zone is continuous with that of the gonoduct, where the development of numerous folds of the mucosa forms a limit, similar to a cervix, displayed at the border of the germinal portion of the ovary and the gonoduct [15].

Because of the lack of oviducts in viviparous teleosts, the gestation takes place in the ovary, in contrast to the rest of viviparous vertebrates where the gestation is in the uterus. That is, exclusively in viviparous teleosts, it occurs an intraovarian gestation. Therefore, the ovary of viviparous teleosts is not only the structure where oogenesis occurs, but, also, it receives the spermatozoa during the insemination, maintains the spermatozoa, and it allows the fertilization of oocytes and the development of offspring until birth [16]. The sequence of these processes makes the ovary of viviparous teleosts a very complex organ, developing morpho-physiological adaptations for the insemination, the entrance and movements of spermatozoa into the ovary [15], the storage of spermatozoa until fertilization [17], the fertilization of oocytes [16], and the intraovarian gestation [18].

According to the nutrients used by the embryos during their gestation, there are two nutritional patterns in viviparous teleosts, both involving ovarian structure and egg morphology. These patterns are lecithotrophy and matrotrophy [7,9,19,20]. In lecithotrophy, the nutrients for the embryo come from the yolk reserves stored in the egg during oogenesis, prior to fertilization, similar to that which occurs in oviparous fishes. In matrotrophy the nutrients are provided not only by those stored in the egg during oogenesis, but also by supplies from the maternal tissues during gestation, consequently, after fertilization [5,6,9,13,14,18]. Pires and Reznick [21] mention that matrotrophic species invest fewer resources prior to fertilization and, instead, provide resources to offspring throughout development. Thus, the nutritional pattern in species with small eggs involves the reduction of lecithotrophy and the increase of matrotrophy. Therefore, the reproductive strategy of viviparous species is significantly connected with the nutrition of the embryos during gestation. Consequently, in accordance with the amount of yolk deposited in the oocyte during oogenesis, the nutrition of the embryo requires the transfer of nutrients from the maternal tissue, developing different levels of matrotrophy: incipient, middle, or high [19]. The understanding of this complex process of nutrition during gestation requires the analysis of the specialized structures related with this process. Then, as it is considered by Blackburn and Starck [20], morphology has a central role in helping to explain the function and evolution of patterns of fetal nutrition in viviparous teleosts.

2. The Family Poeciliidae

The family Poeciliidae includes species of freshwater teleosts of the order Cyprinodontiformes. Poeciliid reproduction involves viviparity. This family contains the most numerous species of viviparous teleosts, with approximately 337 species [2]. In the context of viviparity of this family, there is an interesting species long considered essential in understanding the evolution of viviparity, this species is Tomeurus gracilis, which is the unique zygoparous or embryoparous poecilid. characterized because, after the intraovarian fertilization, the females let the zygotes into the exterior prematurely, this is fertilized eggs or embryos in early stage of development which are attached to plants where the development continues [22].

Poeciliids occur from the southeastern United States to South America, Africa, including Madagascar [3,23,24,25], with a wide distribution in lands of Central Mexico, the Caribbean islands, and around the Gulf of Mexico. Actually, due to the release of specimens from home aquaria (ornamental fishes), poeciliids can be found in all tropical and subtropical freshwater ecosystems [24,26]. Poeciliids have been used in evolutionary and ecological studies analyzing important questions in the field of conservation biology. Related to the viviparous reproduction in teleosts, poeciliids have been of great importance in the definition and understanding of this unique type of gestation [7,9].

3. The Ovary of Poeciliids

The ovary of poeciliids is of cystovarian type, as a single and saccular structure with a central lumen (Figure 1A,B,C). Histologically, the ovarian wall consists of four tissue layers. From the interior to the exterior, these layers are: (1) Germinal epithelium integrated by germ cells, such as oogonia, and oocytes scattered individually or in cell nests among somatic epithelial cells (Figure 2A). The germinal epithelium borders the ovarian lumen and it is separated from the stroma by a basement membrane [10]; (2) stroma, formed by loose and vascularized connective tissue, enclosing the ovarian follicles. The follicles are integrated by oocytes in different stages of development as previtellogenesis or vitellogenesis; (3) smooth muscle layers; and (4) serosa, formed by thin connective tissue and externally lined by mesothelium [7,11,27,28]. The caudal zone of the ovary forms the gonoduct, which lacks germinal cells (Figure 1C and Figure 2A).

The gonoduct acquires special interest in viviparous species, where during birth the offspring goes to the exterior through the gonoduct. Consequently, the gonoduct forms a barrier between the germinal zone of the ovary and the exterior. Campuzano-Caballero and Uribe [15,29] analyzed the gonoduct of Poecilia reticulata [15] and Poeciliopsis gracilis [29], describing this structure as a muscular tube that includes multiple longitudinal folds of the mucosa extended into the gonoductal lumen. The gonoduct is lined internally by single cuboidal or columnar epithelium with ciliated and non-ciliated cells, plus stroma of connective tissue, smooth muscle, and serosa (Figure 2A–E). Melano-macrophage centers are irregularly located in the connective tissue, adjacent to the epithelium; they are round or oval aggregates of cells such as macrophages, lymphocytes, and melanocytes (Figure 2A,B). The presence of these centers suggests its involvement in the protection of the ovary and embryos by immunological functions. Macrophages and lymphocytes may be also located subjacent to the epithelium or in the lumen of the gonoduct (Figure 2C). The presence of ciliated and non-ciliated epithelial cells (Figure 2D,E) may facilitate the transport of spermatozoa during the insemination and embryos during birth.

4. Oogenesis

The oocyte matures during the process of oogenesis in the follicle (Figure 3A–H). The follicle is composed of the oocyte surrounded by a single layer of follicular cells supported by a thin vascularized theca [28]. The oogenesis includes three stages: chromatin-nucleolus, previtellogenesis, and vitellogenesis [28]. When oogonia initiate meiosis, they form oocytes in the chromatin-nucleolus stage (Figure 3B), where the germinal vesicle (nucleus) displays the lampbrush chromosomes. During previtellogenesis the oocyte acquires the ooplasm components and organelles. During vitellogenesis the oocyte acquires the diverse and complex nutrients stored in the yolk [4,7,23,28,30,31]. Previtellogenesis is marked by several features: The ooplasm is basophilic (Figure 3C), the germinal vesicle (nucleus) has a single nucleolus that proliferates to multiple nucleoli (Figure 3C), numerous lipid droplets (Figure 3C–H) and cortical alveoli appear in the ooplasm, and the oocyte diameter increases. Vitellogenesis is seen when yolk globules are deposited in the ooplasm (Figure 4A–D); the yolk becomes fluid and some lipid droplets may be seen around the oocyte periphery (Figure 4D). The follicular epithelium becomes columnar, indicating intense secretion of nutrients for the oocyte [32]. At the end of vitellogenesis, the oocyte reaches its maximum size and the germinal vesicle migrates to the periphery of the ooplasm at the animal pole [28,32].

Most poeciliids develop large oocytes with abundant yolk. Then, oogenesis forms large oocytes which grow from a diameter of 10 µm of the oogonial stage to 2.0 mm of mature eggs. Some examples of poecilid species and the mean diameter of their mature oocytes are: Girardinus denticulatus, 2.6 mm; Girardinus uninotatus, 2.3 mm [33]; Poecilia reticulata, 2.1 mm [34]; Gambusia puncticulata, 2.6 mm [33]; Poeciliopsis occidentalis, 2.02 mm [34], Poeciliopsis monacha, 2.0 mm; Limia vittata, 2.3 mm [31]; but other species develop smaller oocytes containing less amount of yolk such as Gambusia affinis, 1.7 mm [31], Quintana atrizona, 1.6 mm [33]; Poeciliopsis lucida, 1.4 mm [35] and species with even smaller oocytes, such as Poeciliopsis turneri, 1.0 mm and Poeciliopsis prolifica, 0.8 mm [35]. However, the species with the smallest oocyte, considered a microlecithal oocyte, with a mean diameter of 400 µm, is Heterandria formosa [36]. Turner [37] considered that the diminution of yolk is an important and specialized feature involved in teleost viviparity. Later, Turner [13] commented that the most advanced type of viviparity occurred in species where the yolk has become greatly reduced, as in H. formosa. This is an evolutionary change involved from lecithotrophy to matrotrophy.

5. The Intraovarian Gestation in Poeciliids

The intraovarian gestation in poeciliids is initiated when the oocytes are fertilized in the ovarian follicle. The embryos remain into the follicle throughout all their development, until birth. That is, fertilization and gestation in poeciliids are intrafollicular [7,27,31]. All the morpho-physiological adaptations of the ovary are features essentially related with this reproductive strategy of viviparity: the intraovarian and intrafollicular embryogenesis.

It is common in poeciliids that, after insemination, the ovary stores spermatozoa. In species in which sperm storage occurs, the germinal epithelium is involved in relationships with the spermatozoa as observed in Heterandria formosa [13,16], Poecilia reticulata [38], and Xiphophorus maculatus [17].

The intrafollicular fertilization is possible by a specific structure at the periphery of each oocyte where the spermatozoa make contact with the oocyte. This structure penetrates into each oocyte as a funnel-like invagination of the ovarian lining to the follicular epithelium, opening a duct from the ovarian lumen to the oocyte membrane [16]. This invagination is called delle by Stuhlmann and Philippi [30,31]. Therefore, the delle is the only possible way where the spermatozoa may have access to the oocyte (Figure 5A,B). When mature oocytes are fertilized by the spermatozoa into the follicle, the intrafollicular embryonic development is initiated.

The intrafollicular embryogenesis in poeciliids continues (Figure 5C,D and Figure 6A,B) through the development of peripheral blood vessels, adjacent to the maternal tissue. These vessels permit the exchange of essential supplies for the embryos, such as the transfer of nutrients and gas for respiration, elimination of waste products of the metabolism, and fulfilling hormonal and immunological requirements. For these functionfs, the development of adjacent embryonic and maternal vascularization is essential (Figure 6C,D). The follicular cells become squamous, simplifying the pass of metabolites between maternal and offspring blood vessels.

In early embryogenesis, the amount of yolk is abundant, but it diminishes progressively as the embryo takes the nutrients during the advance of gestation (Figure 7A–C).

According to the differences in the amount of yolk of the oocytes, the species having oocytes with abundant yolk have been considered lecithotrophic, even though some transfer of maternal nutrients may also occur. This is the case of Gambusia geiseri [39], a species with large eggs, which has a high level of lecithotrophy but also demonstrates transfer of nutrients from the mother to the embryo as a small matrotrophic contribution. Similar observations were described in other species: Gambusia clarkhubbsi, Gambusia gaigei, Gambusia holbrooki, Gambusia nobilis, Poecilia formosa, Poecilia latipinna, and Poecilia mexicana [19]. The incipient matrotrophy has potential significance in helping us understand the evolution of matrotrophy. Blackburn [6] considered that ancestral features that function in gas exchange may have been adapted for nutrient transfer, through minor modifications of their components.

The follicular epithelium, the maternal layer surrounding the embryo, is very active in allowing the passage of metabolites in species with matrotrophic nutrition. The follicular cells select, digest, and transport nutrients from the blood vessels of the maternal tissue to the embryo [40]. The proximity of embryonic and maternal tissues also offers conditions favoring the evolution of matrotrophy [5,20]. This nutrient transfer involves the association of embryonic and maternal tissues, which form the follicular placenta [14,41].

The components of the follicular placenta are: endothelium of maternal capillaries, follicular epithelium, embryonic surface epithelium, and endothelium of embryonic capillaries. Consequently, a placenta allows the mother to feed embryos during gestation by matrotrophic nutrition, instead of the deposition of abundant nutrients into the oocyte during oogenesis. Extensive matrotrophy is associated with more placental complexity. Analysis of the structure of the placenta in several species of poeciliids allowed the identification of specialized morphological characteristics of the follicular cells that increased the active transfer of nutrients between mother and embryos, such as increase in the number of microvilli and microvilli length [41,42,43]. Thus, extreme matrotrophy is associated with placental characteristics that allow the transference of nutrients between the mother and the embryo. The development and function of the placenta in matrotrophic species are associated with stable environmental conditions, because it requires a constant transfer of resources from the mother to the embryo during gestation [35,41,44,45]. In contrast to this situation, the lecithotrophy of species such as Poecilia vivipara may be related to environmental conditions of a semiarid region, with intermittent rivers, and where food supply is variable [32].

In this diversity of embryonic nutrition, there are several species with extreme matrotrophy such as: Heterandria formosa [13]; Poeciliopsis prolifica, Poeciliopsis turneri, Poeciliopsis retropinna [21,46]; Xenodexia ctenolepis [21]; Poecilia branneri, Poecilia bifurca [45]; Phalloptychus januarius [42]. Consequently, in the context of the analysis of the evolution of poecilid placenta, species with extreme matrotrophic nutrition may reveal essential aspects, taking into account that the evolution of the placenta is one mechanism of matrotrophy [43,47]. As it is considered by Pollux et al. [43], viviparity in fishes have evolved the development of the placenta, when the reduction in pre-fertilization nutrients are associated with trophic specializations for the post-fertilization transfer of nutrients from the mother to the developing young.

Wourms [18] suggested that the trophic relationships in the evolution of viviparity involve the transition from lecithotrophy to matrotrophy, a shift from embryonic nutritional autonomy to maternal nutritional dependency. Blackburn [5,6], comparing lecithotrophy and matrotrophy, discussed the evolution from lecithotrophic oviparity to lecithotrophic viviparity to matrotrophic viviparity. In this context, the small oocytes developed in H. formosa represent an extreme in the reduction of yolk deposition, developing the smallest egg described in poeciliids [36], along with the high level of matrotrophy. H. formosa is considered the species with extreme matrotrophy, with very little yolk available at fertilization and young increasing in dry mass during development [44]. Therefore, this species is an excellent model among poeciliids for the study of the formation of microlecithal oocytes during oogenesis, as well as the structural complexity of the placenta and the complementary evolution to matrotrophy [43,47].

6. Conclusions

Viviparity of teleosts is a specialized and derived mode of reproduction that has evolved from oviparity. The type of viviparity in teleosts is due to the combination of unique reproductive aspects, different from the rest of vertebrates: The saccular ovary, the internal germinal epithelium lining the ovarian lumen, the absence of oviducts, the development of intraovarian fertilization, and the gestation in the ovary. This strategy of reproduction has morphological variations as the result of adaptations associated with viviparity. The intraovarian gestation of poeciliids involves several morphological characteristics: The intrafollicular fertilization, the intrafollicular embryogenesis, and diverse levels of matrotrophy that converge in maternal–embryo interchanges developing a follicular placenta. In order to attain a better understanding of the biology of reproduction in viviparous teleosts, several fields of investigation need to be integrated with the morpho-physiological adaptations, such as ontogenesis, endocrinology, reproductive cycles, and evolutionary trends [5,6]. Additionally, comparative studies at species, genus, and family levels [3,4] will reveal essential aspects related to the viviparity, this unique and extraordinary reproductive strategy developed in viviparous teleosts.