A novel set of structures within the elasmobranch, ovarian follicle.

Elasmobranch fishes produce some of the largest oocytes known, exceeding 10 cm in diameter. Using various microscopy techniques we investigated the structural adaptations which facilitate the production of these large egg cells in three species of shark: the Atlantic sharpnose shark, Rhizoprionodon terraenovae, dusky smoothound, Mustelus canis and the little gulper shark, Centrophorus uyato. The ovarian follicle of elasmobranchs follows the typical vertebrate pattern, with one notable exception; the zona pellucida reaches extreme widths, over 70 µm, during early oogenesis. Contact between the follicle cells and the oocyte across the zona pellucida is necessary for oogenesis. We describe here a novel set of large, tube-like structures, which we named follicle cell processes that bridge this gap. The follicle cell processes are more robust than the microvilli associated with the follicle cells and the oocyte plasma membrane and much longer. During early oogenesis the follicle increases in size relatively quickly resulting in a wide zona pellucida. At this stage the follicle cell processes appear taut, uniform and radially oriented. As oogenesis continues the zona pellucida narrows and the follicle cell processes change their orientation, appearing to wrap around the oocyte. The presence of the contractile protein actin within the follicle cell processes and their change in orientation may well be an adaptation for maintaining the integrity of these large oocytes. The follicle cell processes also contain electron dense material, identical to material found within the follicle cells, suggesting a role in the transport of metabolites to the developing oocyte.

Immunoglobulins in the eggs of the nurse shark, Ginglymostoma cirratum.

Elasmobranchs, which include the sharks, skates, and rays, emerged over 450 million years ago and are the oldest vertebrates to possess an adaptive immune system. They have evolved diverse reproductive modes, with a variety of physiological adaptations that enhance reproductive success. The nurse shark, Ginglymostoma cirratum, is an aplacental, viviparous elasmobranch in which the egg and its associated vitelline vasculature are the primary route for maternal-embryonic interactions. During gestation, nurse shark embryos hatch from their eggcases and develop free in the uterus, which is flushed regularly with seawater. Similar to higher vertebrates, embryonic and neonatal nurse sharks possess an immune system that is not fully competent. In birds and bony fishes, maternal immunoglobulins (Ig) stored in the egg during oogenesis confer protective immunity to embryos during gestation. However, early research suggested that such transfer of passive immunity does not occur in sharks. To better understand how elasmobranch embryos are protected from waterborne pathogens during this potentially vulnerable time, we have re-examined the existence of Igs in elasmobranch eggs. Using monoclonal antibodies, we establish the presence of two classes of Igs in nurse shark eggs: 7S IgM and IgNAR. The potential transfer of immunoglobulins from elasmobranch eggs is discussed.

Follicular placenta of the viviparous fish, Heterandria formosa: II. Ultrastructure and development of the follicular epithelium.

Embryos of the viviparous poeciliid fish, Heterandria formosa, develop to term in the ovarian follicle where they undergo a 3,900% increase in embryonic dry weight. Maternal-embryonic nutrient transfer occurs across a follicular placenta that is formed by close apposition of the embryonic surface (i.e., the entire body surface during early gestation and the pericardial amnionserosa during mid-late gestation) to the follicular epithelium. To complement our recent study of the embryonic component of the follicular placenta, we now describe the development and fine structure of the maternal component of the follicular placenta. Transmission electron microscopy reveals that the ultrastructure of the egg envelope and the follicular epithelium that invests vitellogenic oocytes is typical of that described for teleosts. The egg envelope is a dense matrix, penetrated by microvilli of the oocyte. The follicular epithelium consists of a single layer of cuboidal cells that lack apical microvilli, basal surface specializations, and junctional complexes. Follicle cells investing the youngest embryonic stage examined (Tavolga's and Rugh's stage 5-7 for Xiphophorus maculatus) also lack apical microvilli and basal specializations, but possess junctional complexes. In contrast, follicle cells that invest embryos at stage 10 and later display ultrastructural features characteristic of transporting epithelial cells. Apical microvilli and surface invaginations are present. The basal surface is extensively folded. Apical and basal coated pits are present. The cytoplasm contains a rough endoplasmic reticulum, Golgi complexes, and dense staining vesicles that appear to be lysosomes. The presence of numerous apically located electron-lucent vesicles that appear to be derived from the apical surface further suggests that these follicle cells may absorb and process follicular fluid. The egg envelope, which remains intact throughout gestation and lacks perforations, becomes progressively thinner and less dense as gestation proceeds. We postulate that these ultrastructural features, which are not present in the follicles of the lecithotrophic poeciliid, Poecilia reticulata, are specializations for maternal-embryonic nutrient transfer and that the egg envelope, follicular epithelium, and underlying capillary network form the maternal component of the follicular placenta. © 1994 Wiley-Liss, Inc.

Reproduction, placentation, and embryonic development of the Atlantic sharpnose shark, Rhizoprionodon terraenovae.

The Atlantic sharpnose shark Rhizoprionodon terraenovae (Richardson) is a small carcharhinid that is a common year-round resident along the southeast coast of the United States. It is viviparous and its embryos develop an epithelio-vitelline placenta. Females enter shallow water to give birth in late May and early June. Mating occurs shortly after parturition, and four to seven eggs are ovulated. Fertilized eggs attain the blastoderm stage in early June to early July. Separate compartments for each egg are formed in the uterus when the embryos reach 3-30 mm. Embryos depend on yolk for the first 8 weeks of development. When embryos reach 72 mm their yolk supply is nearly depleted and they shift to matrotrophic nutrition. When the embryos reach 40-55 mm, placental development begins with the vascularization of the yolk sac where it contacts the uterine wall. Implantation occurs at an age of 8-10 weeks by which time the embryos reach 70-85 mm. The expanding yolk sac engulfs the maternal placental villi, and its surface interdigitates with the villi to form the placenta. The rest of the lumenal surface of the uterus is covered by non-placental villi that appear shortly after implantation. Histotrophe production by the non-placental villi begins just after their formation. The placenta grows continuously during gestation. The egg envelope is present throughout gestation, separating maternal and fetal tissues. Embryos develop numerous appendiculae on the umbilical cord. Young sharks are born at 290-320 mm after a gestation period of 11 to 12 months. © 1993 Wiley-Liss, Inc.

The follicular placenta of the viviparous fish, Heterandria formosa. I. Ultrastructure and development of the embryonic absorptive surface.

Embryos of the poeciliid Heterandria formosa develop to term in the ovarian follicle in which they establish a placental association with the follicle wall (follicular placenta) and undergo a 3,900% increase in embryonic dry weight. This study does not confirm the belief that the embryonic component of the follicular placenta is formed only by the surfaces of the pericardial and yolk sacs; early in development the entire embryonic surface functions in absorption. The pericardial sac expands to form a hood-like structure that covers the head of the embryo and together with the yolk sac is extensively vascularized by a portal plexus derived from the vitelline circulation. The hood-like pericardial sac is considered to be a pericardial amnion-serosa. Scanning and transmission electron microscopy reveal that during the early and middle phases of development (Tavolga's stages 10-18 for Xiphophorus maculatus) the entire embryo is covered by a bilaminar epithelium whose apical surface is characterized by numerous, elongate microvilli and coated pits and vesicles. Electron-lucent vesicles in the apical cytoplasm appear to be endosomes while a heterogeneous group of dense-staining vesicles display many features characteristic of lysosomes. As in the larvae of other teleosts, cells resembling chloride cells are also present in the surface epithelium. Endothelial cells of the portal plexus lie directly beneath the surface epithelium of the pericardial and yolk sacs and possess numerous transcytotic vesicles. The microvillous surface epithelium becomes restricted to the pericardial and yolk sacs late in development when elsewhere on the embryo the non-absorptive epidermis differentiates. We postulate that before the definitive epidermis differentiates, the entire embryonic surface constitutes the embryonic component of the follicular placenta. The absorptive surface epithelium appears to be the principle embryonic adaptation for maternal-embryonic nutrient uptake in H. formosa, suggesting that a change in the normal differentiation of the surface epithelium was of primary importance to the acquisition of matrotrophy in this species. In other species of viviparous poeciliid fishes in which there is little or no transfer of maternal nutrients, the embryonic surface epithelium is of the non-absorptive type.

Embryonic growth and trophotaenial development in goodeid fishes (Teleostei: Atheriniformes).

Embryonic growth and trophotaenial development are examined in two species of goodeid fish, Ameca splendens and Goodea atripinnis. During gestation of A. splendens, embryonic dry mass may increase from 0.21 mg at the onset of development to 31.70 mg at term. In G. atripinnis, embryonic dry mass ranges from 0.25 mg at the onset of development to 3.15 mg at term. Increase in mass is primarily due to the uptake of maternally derived nutrients by trophotaeniae, externalized embryonic gut derivatives. Trophotaenial development in both species is divisible into five phases. During the first phase, the anus is formed. The second phase involves dilation of the anus, enlargement of the perianal lips, differentiation of the hindgut absorptive epithelium, and formation of the trophotaenial peduncle. The third phase is characterized by a further marked hypertrophy and lateral expansion of the perianal lips that results in the formation of short trophotaenial processes. During the fourth phase, there is continued outward expansion of the inner mucosal surface of the trophotaenial peduncle that results in its eversion and lobulation. Placental function is established by this phase. Axial elongation and dichotomous branching of trophotaenial processes occurs during the fifth phase. Development of rosette and ribbon trophotaeniae differ in the degree of axial elongation during the fifth and final phase.

Follicular placenta and embryonic growth of the viviparous four-eyed fish (Anableps).

In the four-eyed fish, Anableps (Atheriniformes, Anablepidae), eggs are fertilized and embryos develop to term within the ovarian follicles. Development is highly matrotrophic. During gestation, the largest term embryo of A. anableps examined had grown to a total length of 51 mm and attained a dry weight of 149 mg. The postfertilization weight increase is 298,000%. The largest term embryo of A. dowi examined had grown to a total length of 77 mm and attained a dry weight of 910 mg. The postfertilization weight increase is 843,000%. Embryonic weight increases result from nutrient transfer across the follicular placenta. This structure is formed by apposition of the maternal follicular epithelium to absorptive surface cells of the embryo's pericardial trophoderm. The latter, a ventral ramification of the pericardial somatopleure, replaces the yolk sac during early gestation. The external surface of the pericardial trophoderm develops hemispherical projections, termed vascular bulbs. Within each bulb, the vascular plexus of the trophoderm expands to form a blood sinus. Cells of the external surface of the bulbs possess microplicae. Microvilli are absent. During middle to late gestation, the juxtaembryonic follicular epithelium differentiates into two regions. One region consists of shallow, pitlike depressions within which vascular bulbs interdigitate in a "ball and socket" arrangement. Follicular pits are formed by the curvilinear distortion of the apical surfaces of follicle cells. The second region in contact with the dorsal and lateral surfaces of the embryo, is comprised of villous extensions of the hypertrophied follicular epithelium. In both regions, follicle cells appear to constitute a transporting rather than a secretory epithlium. In terms of percentage of weight increase, the follicular placenta of Anableps appears to be the most efficient adaptation for maternal-embryonic nutrient transfer in teleost fishes and closely approaches the efficiency (1.2 × 106 %) of oophagy and embryonic cannibalism in lamnoid sharks.

Trophotaeniae, embryonic adaptations, in the viviparous ophidioid fish, Oligopus Longhursti: A study of museum specimens.

Prepartum embryos obtained from old museum specimens of the ovo-viviparous fish, Oligopus longhursti, possess external intestinal appendages. They are structurally identical to the trophotaeniae described by Turner ('37) and Mendoza ('37) in goodeid fishes. This is the first report of trophotaeniae in the viviparous ophidioids. Two developmental Stages, A and B, were observed. A is a tailbud stage, 2.0-2.25 mm in length, and B is a finfold embryo, 3.0-3.25 mm in length (Wourms and Bayne, '73). Trophotaeniae occur in the form of a single median anterior process and a pair of median posterior processes. They originate from a conspicuous peduncle formed around the anus. The processes of stage A are 1.5-2.0 mm long, 0.05 mm in diameter at their base and 0.04 mm at their tip. The stage B processes are 2.75-3.00 mm long, 0.075 mm in diameter at their base and 0.050 mm at their tip. Serial sections show that the surface epithelium of the trophotaeniae is continuous with and identical to the surface epithelium of the trophotaeniae is continuous with and identical to the surface epithelium of the embryonic gut. Examination both by transmission and scanning electron microscopy confirms that the apical surface of the trophotaenial epithelium and intestinal epithelium are covered with microvilli. Trophotaeniae are considered to function in the uptake of nutrients since they are structurally identical to intestinal epithelial cells. We suggest that maternal nutrients absorbed by trophotaeniae rather than yolk reserves are the principal source of embryonic metabolites. Trophotaeniae may afford a selective advantage since their existence in O. longhursti maximizes the number of large size embryos which a female can produce at one time. Occurrence of trophotaeniae in ophidioid, goodeid and zoarcid embryos is a remarkable example of convergent evolution.