Reproduction of Marine Life, Birth of New Life! Investigating the Mysteries of Reproduction

Aspects of placentation systems in the scalloped hammerhead shark,Sphyrna lewini, and the manta ray, Manta birostrisSoma, H.1, N. Murai1, K. Tanaka 1, H. Kokuba2, I. Yoshihama2, K. Fujita2, S. Mineo2, M. Kudo2,
M. Toda3 and S. Uchida3
1) Saitama Medical School, Department of Obstetrics & Gynecology and Section of Electron Microscopy, Japan;2) Tokyo Medical University, Section of Electron Microscopy, and Department of Pathology, Japan;
3) Okinawa Churaumi Aquarium, Japan.
Contact e-mail: somahr99@bj8.so-net.ne.jp

Abstract
We investigated yolk sac placentations with umbilical cord specimens of pregnant scalloped hammerhead
sharks (Sphyrna lewini) from Okinawa and Taiwan histologically and immunohistochemically. The
placental tissues were positive for PAS, cytokeratin, hCG, hPL, SPI, PLAP and AFP. Moreover, electron
microscopic study of the umbilical cord revealed that the endothelium of ductus vitellointestinalis had ciliar
filaments arising from the surface of cylindrical, mucin-positive epithelial cells. In the gravid uterus of amanta ray (Manta birostris) specimen with an unborn pup from Okinawa, vascularized villous strings withfilaments showing crypt formation termed trophonemata were observed. The stroma had secretory vacuoles
and granules of protein and lipid in the uterine gland, in addition to Golgi apparatus, mitochondria andrough rER. Thus, we hypothesize that secretions of lipid or protein granules might be released from theepithelial surface.

Introduction
This paper describes the immunohistochemical and electronmicroscopic findings of yolk sac placenta withumbilical cord in the scalloped hammerhead sharks and structures of the gravid uterus in the manta ray.
Although most sharks are oviparous, some species are viviparous including members of Sphyrna andCarcharhinidae. We investigate placentas of two specimens of pregnant scalloped hammerhead sharks(Sphyrna lewini) obtained by one Okinawa Churaumi Aquarium and one from Suao, Taiwan, and one
specimen of a pregnant manta ray (Manta birostris) unborn premature pup obtained by Okinawa ChuraumiAquarium. There are over 400 species known most sharks are oviparous, and the remainder, viviparous.
Viviparous placental sharks utilize three methods of fetal nutrition to nourish their developing young:villogenesis, histotroph secretion and placentation (Hamlett, 1989). Viviparity can be divided into
aplacental viviparity including yolk dependency, oophagy and placental analogues and yolk sac
placentation (Wourms, 1981). The yolk sac placentation consists of a modified embryonic yolk sac which
is closely appeared to the uterine epithelium. The length of gestation for scalloped hammerhead sharks isabout 1 year long, and they typically bear about 20 pups. Until now, very little is known about theimmunologic aspects of shark placenta. Only one immunological study has been made on yolk sac placentain shark, a study by Haines (2006) on the Atlantic sharpnose shark, Rhizoprionodon terraenovae. In someSoma et al.: Placentation systems in scalloped hammerhead shark and manta ray

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Reproduction of Marine Life, Birth of New Life! Investigating the Mysteries of Reproduction

rays, there is a large yolk sac, but no specialized yolk sac attachment. The uterine wall in highly villous andglandular, and its secretion is supposed to be absorbed by the greatly hypertrophied external gill filamentsof the fetus (Mossman, 1987).

Materials and Methods
Placental tissue of two pregnant scalloped hammerhead sharks one from Okinawa and one from Taiwan
were examined. The shark from Okinawa was provided by Okinawa Churaumi Aquarium. It was caught ina set-net, but died before giving birth. The another hammerhead shark sample was obtained by courtesy of
Suao fishing market, Taiwan. Histopathology: The shark placental tissues were stained with PAS (Periodicacid Schiff), cytokeratin, hCG (Human chorionic gonadotrophin), hPL (Human placental lactogen), SP1(Specific pregnancy glycoprotein 1), PL.Alphos (Placental alkaliphosphatase) and AFP (alpha-feto proten).
In October, 2008, a female manta ray (Manta birostris) weighing 963.5kg was captured by dry-net. She
died 3 days and the immature baby was removed from uterus by dissection. The uterine epithelium of themanta ray was investigated by immunohistochemical and electronmicroscopic observations. The gravid
uterine tissues from the manta were stained with human placental proteins, Sudan III, Prolactin and Mucin.
Scanning electron microscopy (SEM): The frozen dehydrated specimens were observed with a Hitachi
S-4200 under accelerator voltage at 10KV following osimium coating.
Transmission electron microscopy (TEM): The sections were made after double staining with uranyl
acetate solution and lead nitrate and observed with a JEM1200 EXII (JEOL) electron microscope at 80KV.

Results
In hammerhead sharks, numerous pups each with an umbilical cord and thin membraneous placenta can beseen (Fig.1). Each placenta was connected to the uterine wall. An egg-case membrane exists between
maternal and fetal sides.
A) A pregnant scalloped hammerhead sharks with unborn pups at Okinawa Churaumi Aquarium: The
unborn pups measured about 30cm long and umbilical cord were 90cm long with two braided vessels. Theyolk sac placental epithelium had clear cylindrical cells containing blood capillaries and included a mass of
nucleated red cells encircled by a red-colored egg-case membrane (Fig.2 A, B). These epithelial cells werestained positively with PAS, cytokeratin and PL.alphos (Fig3). Furthermore, the epithelial cells showed
positive staining with hCG,hPL and SP1 just as human placental proteins do (Fig.4). In addition, AFP also
showed positive stain in the epithelial cells. From these results showing epithelial cells of scalloped
hammerhead shark placenta react to human placental proteins, it can be postulated that the shark placenta
produces glycoproteins just as human trophoblasts do (Soma,2007).
B) The pregnant scalloped hammerhead shark examined at Suao, Taiwan: A pregnant scalloped
hammerhead shark measuring 150cm long had 20 pups. Each pup measured 42cm long.
The thin yolk sac placenta was 10x10x0.9cm and the umbilical cords were 25cm long. The umbilical cords
had one artery, one vein, a ductus vitellointestinalis and an extraembryonic coelom (Fig.5). The endothelial
Soma et al.: Placentation systems in scalloped hammerhead shark and manta ray14

Reproduction of Marine Life, Birth of New Life! Investigating the Mysteries of Reproduction

surface of the ductus vitellointestinalis was covered with seaweed-like ciliated branches and positively
stained with mucin (Fig.6, 7). In the ductus vitellointestnalis, a bundle of ciliar filaments protruded fromthe surface of cylindrical epithelial cells as dynein-forming microtubules (Fig. 8). In chorionic epithelial
tissue, columnar epithelial cells with dilated blood capillaries including nuclear erythrocytes were observed.
These epithelial cells have nuclei and organellae similar to human cytotrophoblasts, although thedevelopment is poor compared with human trophpblasts (Fig. 9).
C) A pregnant manta ray (Manta birostris) at Okinawa Churaumi Aquarium: Manta ray reproduction
features a uterus with thick villous strings called trophonemata which nourish the unborn pup (Fig. 10).
Proliferation of villous epithelium containing mononuclear cells and uterine vessels including a mass of
nucleated red cells was observed (Fig. 11). In immunohistochemical studies, these epithelial cells stained
positively with PAS and cytokeratin. Also, these epithelial cells stained positively with hCG,hPL,
SP1,PL.alphos and AFP (Fig. 12). In addition, Sudan III, Nile blue and prolactin stains were also positivein these cells (Fig. 13). SEM observations showed villous strings with flat surfaces include crypt formation,

Figure 1. Nine pups dissected from apregnant scalloped hammerhead shark,
showing umbilical cords (U) and thinplacental membrane (P).

Figure 2. A) Scalloped hammerhead placental epithelium (PE) with clear cylinder cells containing blood capillaries(BC) including many nucleated red cells. HE stain (x100); B) Chorionic villi (PE) and uterine tissue (UT) wereintervened by egg-case membranes (Eg). HE stain (x200).

Soma et al.: Placentation systems in scalloped hammerhead shark and manta ray

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Reproduction of Marine Life, Birth of New Life! Investigating the Mysteries of Reproduction

Figure3.

Scalloped

hammerhead

chorionic epithelial cells positively
stained with PAS ( ↑ ). PAS stain(x100).

Figure4.

Scalloped

hammerheadepithelial cells showed positive stainingwith hCG (↓). hCG stain (x100).

Figure 5. Scanning electron-micrographof the umbilical cord of a scalloped
hammerhead shark: A:Umbilical artery,
D:Ductus

vitelloinguinalis,

V:

Umbilical vein, E: Extraem bryoniccoelom. (x120).

Soma et al.: Placentation systems in scalloped hammerhead shark and manta ray

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Reproduction of Marine Life, Birth of New Life! Investigating the Mysteries of Reproduction

Figure 6. The endothelial surface ofductus vitellointestinalis covered with
seaweed-like ciliated branches ( ↑ ).
(x7200).

Figure7.

Endotheliumof

ductusvitellointestinalis positively stained withmucin2. Mucin stain. (x200).

Figure8.

Invitellointestinalis,(CF)thefinestructurebundles ofof

ciliarductusfilaments

protrudefrom

thesurfaceofcylinderepithelial cells(CY)asmicrotubules. Nucleus(N), Mitochondria (M). (x5000).
Soma et al.: Placentation systems in scalloped hammerhead shark and manta ray17

Reproduction of Marine Life, Birth of New Life! Investigating the Mysteries of Reproduction

Figure 9. Ultrastructure of columnar
epithelial cells, adjacent to dilated
blood capillary (BC), showing thesecells contain large nucleus (N) withmitochondria (M) and organellae likehuman trophoblasts. (x5000).

Figure 10. Ciliar villous strings(VS)(↑) called trophonemata from thegravid uterus of the manta ray.

Figure 11.

Proliferation of villousepithelium(↑) containing mononuclear
cells and uterine vessels within thegravid uterus of a manta ray. Azanstain. (x100).

Soma et al.: Placentation systems in scalloped hammerhead shark and manta ray18

Reproduction of Marine Life, Birth of New Life! Investigating the Mysteries of Reproduction

Figure 12. Villous epithelium(VE)(↑)
stained with hPL within the gravid
uterus of a manta ray. hPL stain.
(x100).

Figure 13. Villous epithelium stainedwith Sudan III(↑) within the gravid
uterus of a manta ray. Sudan III stain.
(x200).

Figure 14. SEM findings: A) Cut surface of a villous string with flat surface showing crypt formation (↑) (x100); B)
Nucleated red cells below crypts (NKC)(↑). (x5000).

Soma et al.: Placentation systems in scalloped hammerhead shark and manta ray

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Reproduction of Marine Life, Birth of New Life! Investigating the Mysteries of Reproduction

Figure 15. Transmission EM findings: A)Villous strings not only have vacuoles (V) and granules (g) showing secretionof protein or lipid by the uterine gland, but also Golgi apparatus (G), rER and secretory granules (SG) as well as
desmosome (DM), (x21000); B)Secretory granules (SG) such as lipids or proteins are excreted from the surface of theepithelium. (x105000).

where blood capillaries including nuclear red cells were found (Fig. 14 A, B). By TEN, they had vacuoles
and granules showing secretion of lipids within the uterine gland, which contained Golgi apparatus,
mitochondria and rER (Fig. 15 A, B). This demonstrates excretion of lipid or protein granules from thesurface of the epithelium.

Discussion
This is the first detailed description of placenta of Sphyrna lewini and uterine secretion of Manta birostris.
This hammerhead shark placenta is found to utilize yolk and nourish many pups during the relatively longgestation period.
Marsupial reproduction is characterized by a relatively short gestation and production of an extremely
undeveloped new-born (Freyer, 2007). The placenta is formed of a yolk sac that is attached to theendometrium until birth. Unlike eutherian mammals, in most marsupials and monotremes, the allantois
does not contact the chorion to form a placenta.
Scalloped hammerhead shark placenta has unique umbilical cord with appendiculae, chorionic tissue
including blood capillaries and uterine tissue even though there is loose contact between chorionic tissue
and uterine endometrium. The shark placenta is known to be specialized to have characteristics of steroid－
producing tissues (Hamlett,1990).
Chorionic epithelial cells of scalloped hammerhead sharks showed positive staining with human placental
proteins such as hCG, hPL, SP1 and PL.alphos. The trophoblast like cells with large nuclei adjacent todilated capillaries including nucleated red cells were found, though the organelles were not so rich as
human trophoblast. The umbilical cord and appendiculae in the Atlantic sharpnose shark have previously
been studied (Hamlett, 1993, 2005), but this is the first study of ultrastructure of the umbilical cord ofscalloped hammerhead sharks. In addition to typical findings of an umbilical cord consisting of to vessels,
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Reproduction of Marine Life, Birth of New Life! Investigating the Mysteries of Reproduction

the ductus vitellointestinalis and extraembronic coelom, the ductus vitellointestinalis was covered with
seaweed-like ciliated brushes in which ciliar filaments were protruded densely as microtubles forming
dynein. The endothelium stained with mucin-like yolk products. Yolk granules are reportedly transported to
the fetal gut by the ciliated cell lining the ductus vitellointestinalis (Hamlett, 1989). Ultrastructuralobservations suggest that yolk metabolic products are transported across the vittelline epithelium throughthe yolk sac endoderm. The southern stingray, Dasyatis americana has placental analogues called
trophonemata for uterolactation (Hamlett, 1985, 1996, 2005).
This is the first report of placental analogues in manta ray (Manta birostris). The uterus of manta ray with
an immature pop included thick villous strings called trophonemata which were stained positive withhuman placental proteins such as hCG,hPL,SP1,PL.Alphos and AFP, in addition to Sudan III and prolactin.
In ultrastructural findings of villous strings, villous stroma with vacuoles and secretory granules suggest
secretion of protein and lipid, and in organelles, Golgi apparatus, mitochondria and rough ER wereobserved. Thus, it can be concluded that lipid or protein granules are excreted from the surface of theuterine epithelium to nourish the baby inside the mother manta.

Acknowledgements
We wish to express our thanks to Dr. Katherine Muzic for kind inspection of paper manuscript.

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