William C. Hamlett

Uterine specializations in elasmobranchs

The uterus of the elasmobranch demonstrates a variety of structural and physiological specializations associated with the mode of reproduction utilized by a particular species. Reproductive modes include (1) oviparity (Raja erinacea and Raja eglanteria), (2) aplacental viviparity, yolk sac variety (Squalus acanthias), with oophagy and intrauterine cannibalism (Carcharias taurus), and with trophonemata (Urolophus jamaicensis), and (3) placental viviparity (Rhizoprionodon terraenovae and Carcharhinus plumbeus). In oviparous species, the uterus harbors the egg capsule during capsule sclerotization and thereafter until oviposition, which in skates can be several days. The uterus is specialized for synthesis and secretion of materials that may contribute to capsule surface structure or chemistry or that may facilitate biochemical events associated with capsule polymerization, including provision of oxygen and elimination of water generated by the tanning reaction. In aplacental yolk sac species, as well as those species with oophagy and intrauterine cannibalism, the uterus functions as a respiratory membrane and a regulator of the ionic and osmotic environment (Squalus) but provides no supplemental nutrients to augment the yolk sac contents. In stingrays and placental sharks, however, the uterus assumes the additional function of providing nutrients to the developing embryos after the yolk stores have been depleted. Nutrient provision varies from uterine secretions of epithelial origin in the form of histotroph or uterine milk in stingrays to the transfer of material from the maternal vascular system in some placental sharks. Common features shared by all elasmobranchs include (1) increased vascularity of the uterine mucosa, (2) physiological maintenance of specific intrauterine ionic and osmotic environments in some species, (3) provision of secretory products, (4) increase in surface area for respiratory and metabolic exchange, (5) structural specializations of junctional complexes and intercellular spaces for fluid transport in some species, (6) thinning of the uterine epithelium to reduce the diffusion distance for respiratory exchange in stingrays, and (7) provision of nutrients in some viviparous species. J. Exp. Zool. 282:438–459, 1998.

Ultrastructural analysis of folliculogenesis in the ovary of the yellow spotted stingray, Urolophus jamaicensis

The ovary of the yellow spotted ray, Urolophus jamaicensis, is embedded in the epigonal gland, a lymphomyeloid organ. The covering of the ovary is composed of a germinal epithelium that is cuboidal and dome-shaped with microvilli. Adjacent cells have elaborate intercellular folds that create dilated intercellular spaces. In previtellogenic follicles, the follicle cells are simple cuboidal and contain modest amounts of synthetic or transport organelles. As vitellogenesis proceeds, the epithelium becomes multilaminar. Follicle cells are columnar as yolk precursors are transported from the maternal circulation, through the follicle cell cytoplasm, to the oocyte. Large, round cells occur in the follicle wall that contain lipid-like substances. These cells decrease in size and number as folliculogenesis proceeds and eventually disappear prior to ovulation. Columnar follicular cells and the oocyte have cellular extensions that impinge upon the zona pellucida. Transosomes are follicle cell extensions that indent the oocyte membrane. Tips of transosmes become enclosed by a layer of oocyte plasmalemma. The tips of transosomes pinch off and become resident in the ooplasm. Dense staining material occurs on the inner surface of the transosome membrane derived from the follicle cell. In Other animals, this material has been described as ribosome-like. This study is the first to document the presence of transosomes in a group other than Aves or reptiles. Follicle cells are supported by an extremely thick basal lamina. Subjacent to the lamina is the vascularized theca with fibroblasts embedded in a collagenous network. There is no differentiation into definitive theca interna and externa. In vitellogenic eggs, extensive inward folding of the follicular epithelium occur thereby generating more surface area for the transport of yolk precursors to the oocyte. Atretic follicles are common.

Microscopic organization of the sperm storage tubules in the oviducal gland of the female gummy shark (Mustelus antarcticus), with observations on sperm distribution and storage

Oviducal gland morphology, the microscopic organization of the terminal zone, and sperm storage were described in the female gummy shark (Mustelus antarcticus). Mustelus antarcticus is a nonplacental viviparous hound shark, which displays minimal histotrophy during embryonic development. The animals examined represented all stages of maturity and gestation. The oviducal gland was found to have the same fundamental zonation as in most chondrichthyans. Using recent terminology, the oviducal gland of chondrichthyans has an anterior club zone, followed by a papillary zone, both of which produce jelly that surrounds the egg, a baffle zone that elaborates the tertiary egg envelope and a terminal zone, where sperm storage occurs. Each zone is composed of simple tubular glands that connect to transverse grooves, which extend the full width of the gland. The exception is the terminal zone, which does not have transverse grooves but consists of individual tubules. The microscopic organization and histochemical nature of the zones display similar patterns to those of other chondrichthyan genera. Tubules of the terminal zone contain four types of cell: ciliated cells, alcian blue‐positive secretory cells, periodic acid‐Schiff and alcian blue‐negative secretory cells, and secretory columnar cells. These tubules end in recesses, the sperm storage tubules, which extend beyond the periphery of the baffle zone. Sperm were stored in the sperm storage tubules of all maturing and mature animals examined. Of note is the observation of stored sperm in an animal 1 year prior to first ovulation. Sperm were also observed throughout the uterine sphincter, body of the uterus, isthmus, and oviduct of maturing and mature animals, and in the uterine sphincter of an immature animal. These sperm represent immediately postcopulation aggregations of sperm and sperm in the process of migrating to the site of storage or to the site of fertilization. J. Morphol., 2008.

The interleukin 1 (IL-1) system in the uteroplacental complex of a cartilaginous fish, the smoothhound shark, Mustelus canis

Cartilaginous fish are the oldest extant jawed vertebrates and the oldest line to have placentae. Their pivotal evolutionary position makes them attractive models to investigate the mechanisms involved in the maternal-fetal interaction. This study describes the tissue expression of the cytokine interlukin-1 (IL-1) α, IL-1 β and its specific membrane receptor, IL-1 receptor type I (IL-1R tI) in a placental cartilaginous fish, the smoothhound shark, Mustelus canis. The presence of this cytokine has been reported in many mammalian placentae, as well as in the placenta of a squamate reptile and this study extends these observations to the cartilaginous fishes. The uteroplacental complex in M. canis consists of a yolk sac modified into a functional yolk sac placenta and complimentary uterine attachment sites. Immunohistochemistry for IL-1 α, IL-1 β and the receptor reveals leucocytes of both the mother and fetus to be positive, as well as the apical aspect of paraplacental cells and the apical vesicles in the umbilical cord epithelium. Yolk sac endoderm is also positive with all the stains while the ectoderm is positive only for IL-1 α. Immunoreactivity in the uterine epithelium was obtained for IL-1 α and the receptor. The egg envelope is always negative.In light of the recent finding of IL-1 β gene in a cartilaginous fish and of the high level of conservation of proteins implicated in IL-1 action, our data suggest that IL-1 system is a key mediator of the materno-fetal interaction since the oldest extant placental vertebrates.

Ultrastructural analysis of sperm storage and morphology of the oviducal gland in the Oman shark, Iago omanensis (Triakidae)

Sperm storage, oviducal gland morphology and ultrastructure are described in pregnant females of the viviparous placental Oman shark, Iago omanensis, that were harbouring uterine eggs, embryos or fetuses. Zonation of the gland from anterior to posterior is similar to those previously described for other elasmobranchs and is designated as club, papillary, baffle and terminal zones. The terminal zone has two regions, the first comprising short tubules adjacent to the baffle zone that are positive by periodic acid–Schiff. Posteriorly, the terminal zone has deep clefts that terminate as dilated recesses harbouring laterally arrayed bundles of spermatozoa surrounded by a flocculent matrix. Sperm were found in the terminal zone of all animals examined. Samples were not available throughout gestation, so it is not known whether the sperm is stored from a single insemination or from multiple matings. The club zone stains positive by the periodic acid–Schiff method, as does the papillary zone. The last lamella of the papillary zone is positive by Alcian blue staining. The secretory product of the baffle zone is the tertiary egg envelope that encloses the developing embryo during much of gestation.

Gestational morphogenesis of the uterine epithelium of the gummy shark (Mustelus antarcticus)

Developing embryos of the non‐placental, viviparous gummy shark (Mustelus antarcticus) are supplied with yolk from external and internal yolk sacs throughout the initial stages of gestation. Yolk supplies are exhausted by the 7th month of an 11–12 month gestation. During embryonic development, there is an approximate 800% gain in dry mass. These factors suggest nutrients are transferred from the mother to the young. The results of the present study provide some insights into how this is occurring. The uteri are paired and both are functional. Using both light and transmission electron microscopy, regions of the uterus were examined throughout maturation and gestation. The layers of the uterine wall throughout the entire length are similar to the uteri of other chondrichthyans previously examined. The uterine epithelium of the body of the uterus is smooth contoured, does not form villi, and undergoes cyclical and secretory changes throughout maturity and gestation. In immature uteri, the epithelium is simple columnar with minimal periodic acid‐Schiff‐positive and Alcian blue‐positive secretory vesicles. In mature uteri, the epithelium is highly stratified with cuboidal cells containing numerous Alcian blue‐positive and periodic acid‐Schiff‐positive vesicles. With pregnancy, prominent changes include a reduction in the number of cell layers, a reduction in cell size, a reduction in the connective tissue intervening between epithelium and blood vessel endothelium, and an increase in blood vessel number and size, so that at term, the uterine compartment consists of a single layer of squamous cells immediately underlain by sinusoidal‐like blood vessels. These features along with a small number of secretory vesicles, dilated intercellular spaces, and clear transport vesicles suggest the transepithelial transfer of water and minerals from the maternal to embryonic environment, supplemented by minimal uterine secretions. This is defined as minimal histotrophy and this article represents the first detailed description of this reproductive mode. J. Morphol., 2009.

Ontogeny of the umbilical cord and placenta in the Atlantic sharpnose shark,Rhizoprionodon terraenovae

SynopsisThe Atlantic sharpnose shark is a viviparous anamniote that develops an epitheliochorial yolksac placenta. Initially, contents of the yolksac nourish the embryos. Yolk is partially digested in the yolk syncytial-endoderm complex and subsequently transferred to the vitelline circulation. Yolk is also transported by ciliary activity of the yolk stalk ductus to the fetal gut for digestion. When embryos are 4.0cm in length, vascular ridges, termed appendiculae, develop on the yolk stalk. As yolk stores are depleted, the yolksac differentiates into the fetal portion of the placenta and the uterus abutting the yolksac differentiates into the maternal portion of the placenta. The yolk stalk differentiates into an elongate umbilical cord. The uterine epithelium produces secretions that are positive by the periodic acid-Schiff and alcian blue methods and metachromatic when stained with toluidine blue. Uterine capillaries are continuous and the surface epithelium is active both in secretion and transport of nutrients. When the embryos are 7–10cm in length, appendiculae are elongate, branched and populated by separate microvillar and granulated cells. Appendiculae may function as a paraplacental nutrient absorptive organ and be involved in the regulation of osmolarity of periembryonic fluids. The fetal placenta has two functional regions: a proximal portion that is presumed to function as a steroid producing organ and a distal portion that effects nutrient and metabolic exchange between the mother and fetus. Characteristics of the fetal placenta include endocytotic activity, crystalline-like cytoplasmic bodies and fenestrated capillaries. Fetal and maternal components of the placenta are separated by the egg envelope.

Microscopic organisation of the oviducal gland of the holocephalan elephant fish, Callorhynchus milii

The study of chondrichthyan reproductive biology has a long history, but the structure and function of the holocephalan oviducal glands (OG) is poorly known; these organs are a vital component in the understanding of chondrichthyan life history. Histochemical techniques revealed that a fundamental zonation was evident in the OG of Callorhynchus milii, similar to most elasmobranchs. In sexually mature females, the following zones occurred (anterior to posterior): (1) club zone, periodic acid–Schiff positive (PAS+), indicating glycoprotein or any mucus substance containing neutral sugars, and Alcian blue positive, pH 2.5 (AB+), indicating the presence of sulfated and unsulfated acid glycosaminoglycans and sialoglycoproteins; (2) papillary zone (AB+); (3) baffle zone (PAS–, AB–); and (4) terminal zone (AB++). Using histological and histochemical techniques not used previously with the holocephalan group, we demonstrated that the structure and function of the OG zones were equivalent between oviparous elasmobranchs and C. milii, even though their final egg capsule morphologies differed. It was also evident that the club and papillary zones produce the egg jelly that surrounds the egg and the baffle zone formed the multilaminate egg capsule. Furthermore, the terminal zone had functions associated with sperm storage and the production of fine hairs that decorate the surface of the egg capsule.

Sperm Aggregations in the Spermatheca of Female Desmognathine Salamanders (Amphibia: Urodela: Plethodontidae)

The alignment of sperm in a cloacal sperm storage gland, the spermatheca, was studied in female desmognathine salamanders by scanning and transmission electron microscopy. Females representing nine species and collected in spring, late summer, and fall in the southern Appalachian Mountains contained abundant sperm in their spermathecae. The spermatheca is a compound tubuloalveolar gland connected by a single common tube to the middorsal wall of the cloaca. Sperm enter the common tube in small groups aligned in parallel along their axes, and continue in a straight course until encountering divisions of the common tube (neck tubules) or luminal borders of distal bulbs, which can act as barriers. Sperm may form tangles, in which small clusters retain their mutual alignment, at the branches of the neck tubules from the common tube, or in the lumen of the distal bulbs, where subsequent waves of sperm collide with sperm already present. The nuclei of some sperm from the initial group to encounter the walls of the distal bulbs appear to become embedded in secretory material on the luminal border or in the apical cytoplasm of the spermathecal epithelial cells. We propose that these sperm become trapped in the spermatheca and are ultimately degraded. J. Morphol. 238:143–155, 1998.

Microscopic structure of the gravid uterus in the little skate, Raja erinacea

The morphology of the uterus in oviparous elasmobranchs has not been systematically examined in any species. The uterus in oviparous sharks and skates houses the eggs during the crucial chemical events that result in the requisite polymerization of the assembled egg capsule materials. Capsule sclerotization involves a quinone tanning mechanism in which catechols are introduced in utero and subsequently oxidized to quinones prior to oviposition. The uterus in Raja erinacea is morphologically specialized for four well-defined functional activities associated with egg capsule formation: regionally distinct structural modifications, both in the uterine wall and the epithelial lining, for active movement of the capsule through the uterine lumen; biosynthesis and secretion of materials into the lumen; vascular facilitation for oxygen transfer to fuel the oxidation process involved in capsule tanning; and intercellular spaces for removal of water from the uterine lumen. The first three activities are qualitatively similar to those operating in viviparous species. The uterus throughout its length has longitudinal folds punctuated by secretory crypts. Proceeding from the anterior to the posterior end of the uterus there is a progressive decrease in the number of cilia and the depth of the lamina propria, and an increase in vascularity and the width of the muscularis. J. Exp. Zool. 282:421–437, 1998.