John Buckland-Nicks

NaCl transport and ultrastructure of opercular epithelium from a freshwater-adapted euryhaline teleost, Fundulus heteroclitus

We adapted killifish to defined freshwater (FW: 1.0 mM Na, 1.0 mM Cl, + 0.1 mM Ca) and by fluorescence light microscopy and scanning and transmission electron microscopy found that the opercular epithelium retained mitochondria rich (MR) cells that were significantly larger but less numerous than in SW-acclimated tissues. Opercular epithelia mounted in vitro with FW bathing the mucosal surface take up Cl against a large negative inside transepithelial potential (Vt, grand mean –64.1 mV) and concentration gradient; the observed flux ratio was significantly different (P < 0.001) from that predicted for passive ion distribution but the net flux was consistently negative. The Na flux ratio suggested that Na was passively distributed. Vt was largely a Na diffusion potential, based on unilateral manipulations of [Na]. Cl unidirectional uptake was unaffected by mucosally added SITS (0.1 mM) but was inhibited by SCN (1.0 mM) and by anaerobiosis. Killifish transferred from SW to FW for 48 h had reduced Cl secretion by the opercular epithelium compared to SW controls but had not yet developed Cl uptake, indicating a slow adaptive process for development of Cl absorptive transport. Opercular epithelia of FW adapted fish, if bathed with isotonic saline on both sides, has a modest net Na and Cl uptake, unlike SW opercular epithelium that strongly secretes Cl under similar conditions. FW killifish opercular epithelium may provide a model to study ion regulation by euryhaline fish. J. Exp. Zool. 277:23–37, 1997.

Prosobranch parasperm: Sterile germ cells that promote paternity?

Abstract In a number of animal groups, including insects, chilopods and prosobranch snails, serm dimorphism occurs in which sterile parasperm are produced alongside fertile eusperm. This phenomenon has been most extensively studied in insects from the standpoint of paternity assurance through sperm competition. Although genesis and structure of snail parasperm have been researched for over a century, some aspects of paraspermatogenesis are still poorly understood and little is known about the function of snail parasperm or their involvement in sperm competition. Paraspermatogenesis of various snails was examined with light and electron microscopy. Parasperm range from simple flagellated cells typically lacking a nucleus, to complex motile giants that transport hundreds of fertile eusperm. Parasperm generally lack an acrosome, although there are a few exceptions, including the Cerithioidea and Campaniloidea. Tonnoidea, such as the ranellid Fusitriton oregonensis , have polymorphic sperm; a carrier parasperm provides transport for a cohort of fertile eusperm, whereas a lancet parasperm is independent. Plastic embedded sections of testis of Fusitriton stained for polysaccharides and DNA were correlated with thin sections in the electron microscope. Results show that massive production of glycoprotein bodies during paraspermatogenesis stems from activities of the rough ER and Golgi bodies and is largely independent of nuclear degradation. This differs from previous ideas that proposed a direct transfer of material derived from degradation of the nucleus into glycoprotein bodies. Post-translational regulation of gene function must largely account for the complex secretory and morphogenetic events that occur in parasperm after the nucleus is incapacitated. Comparable secretions in some insect parasperm have been shown to act as nuptial gifts, suppress female remating desire, or create a hostile environment for rival ejaculates in the female. A variety of secretion products are released by exocytosis from snail parasperm. For example, in Cerithium dense granules are secreted into the testicular fluid (Fig. 2), whereas in Littorina a fine granular secretion is released. Lysosomes, identified in lancet parasperm using acridine orange, may be involved in creating a hostile pre-fertilization environment for rival sperm. In this context, lancet parasperm form a sperm plug in the bursa copulatrix that binds eusperm in the lumen. These processes are poorly understood in snails but are central to our understanding of the role of parasperm in reproduction. Progress in this area has been limited by our inability to analyse parasperm in isolation. In this study, parasperm of Fusitriton were successfully separated for the first time by centrifugation on Percoll gradients. In conclusion, the establishment of multiple paternity and mixing of ejaculates in the female has created selection pressure favouring sperm competition between rival males. Parasperm of snails, like their insect counterparts, may be gamete ‘eunuchs’ that have evolved to serve their fertile siblings.

Spermatogenesis of a marine snail, Littorina sitkana.

SummaryThe fine structure of the spermatogonium, spermatocyte and spermatid of a marine snail, Littorina sitkana is described. The ring centriole (annulus) is formed from the distal centriole and it migrates to the base of the mitochondrial region where it lies in a joint-like structure which is formed by an area of invaginated plasma membrane. The distal and proximal centrioles are at first perpendicular to each other but the proximal centriole rotates to a position coaxial with the distal centriole and fuses with it. The peripheral doublet fibers are continuous between the two centrioles but the central fibers originate only in the distal centriole. The acrosome differentiates from the proacrosomal granule which is derived from a Golgi body. Microtubules, present at this stage, may assist acrosomal formation. Chromatin condensation begins with the formation of fibrous strands, then to lamellar plates which become folded and later twisted around the flagellar shaft. In the final stages the lamellae appear in cross section as concentric rings which eventually fuse to form a homogeneously dense nuclear tube.

Fine structure of Sertoli cells in three marine snails with a discussion on the functional morphology of Sertoli cells in general

SummaryThe fine structure of Sertoli cells in three marine prosobranch molluscs has been studied with light- and electron microscopy. Sertoli cells of prosobranchs are modified columnar epithelial cells that maintain continuous contact with the basal lamina and extend from it to the lumen of a testicular tubule. Spermatogenesis takes place between adjacent Sertoli cells, but a continuous layer of cytoplasm separates the spermatogonia from the basal lamina, thus restricting the basal compartment to spermatogonium mother cells. Substances traversing the basal lamina from the interstitial space must pass either through or between the Sertoli cells. However, between the cells, a permeability barrier composed of septate and desmosome-like junctions blocks the passage of substances, such as the tracer lanthanum nitrate. The basally-located nucleus is irregularly shaped with fine granular euchromatin and some peripheral heterochromatin; satellite karyosomes border the nucleolus. There is an extensive intracellular digestive system that is used effectively to phagocytize waste sperm and residual cytoplasm. Cytoplasmic processes of Sertoli cells penetrate throughout the germinal epithelium. In some prosobranchs that exhibit sperm polymorphism these processes must coordinate to bring together a clone of eupyrene sperm and a carrier sperm at a particular time in development. The only cytoskeletal elements available within the processes to generate such movements are microtubules.We propose that the term ‘nurse cell’, which has been used in the past to describe at least three different cell types, including Sertoli cells and apyrene sperm, be restricted to abortive oogonia that contribute to development of an oocyte.

The fine structure of the spermatozoon of Littorina (gastropoda: prosobranchia), with special reference to sperm motility

SummaryThe filiform spermatozoon of Littorina contains a conical acrosome positioned anteriorly to the nucleus. The nucleus is essentially a tube of condensed chromatin surrounding the flagellar shaft and only the apical third is covered by a nuclear envelope. A structure resembling a basal body, which is either a modified proximal centriole, or a derivative of the distal centriole, terminates in a basal plate at the tip of the nucleus. Distally it gives rise to the nine outer flagellar doublets, which are continuous with the outer fibres of the distal centriole located 0.26 μ behind the “basal body”. Thus the “basal body” and distal centriole are lined up with each other and the axis of the flagellum. A supernumerary tubule connects the outer arm of the A subfibre of doublet 5 with the “basal body”. The flagellar shaft extends from the apex of the nucleus to the tip of the tail. In the mitochondrial region the plasmalemma fuses with the outer mitochondrial sheath to form a series of pores that connect the mitochondria directly with the external environment. A distinct joint separates the mitochondrial and tail regions enabling the tail to beat perpendicularly to the main flagellar axis. The last section of this paper is given to a discussion of sperm motility in the light of these structural observations.

Multiple Paternity in Littorina obtusata (Gastropoda, Littorinidae) Revealed by Microsatellite Analyses

Parental identity for juvenile Littorina obtusata was determined from three egg masses by means of microsatellite DNA markers. Results confirm that the attendant adult female in each case was the dam of the offspring and that at least 4–6 males contributed to each brood. This correlates with our behavioral observations that indicated multiple copulations between the female and several males in each experimental aquarium. A significant number of offspring from each brood were sired by non-sampled males (males that had copulated with females before capture) whose sperm had been stored by the female. This is the first direct evidence of multiple paternity in the Littorinidae. Results are discussed in reference to current theories of sperm competition, male precedence, and cryptic female choice.

Was internal fertilization an innovation of early Bilateria? Evidence from sperm structure of a mollusc

Spermiogenesis and mature sperm ultrastructure has been studied for the first time in the Neomeniomorpha, a group of worm-like aplacophorans considered ‘primitive’ among extant Mollusca. The striking similarity between this sperm and that of some ‘primitive’ annelids, as well as an overall similarity with filiform sperm of some other invertebrate groups including basal flatworms, indicates homology and an ancient link between these groups that includes internal fertilization. This idea is supported by geological, palaeontological and reproductive data, but contradicts the widely accepted theory that early Bilateria were free-spawning, triploblastic organisms with planktotrophic larvae.

On the nurse cell and the spermatozeugma in Littorina sitkana

SummaryNurse cells develop from diploid cells in the testis. Each cell undergoes a reduction division which leaves the nucleus with half the volume of a normal diploid cell. They send out pseudopodia which form desmosomelike junctions with developing spermatids. The nurse cells detach from the testicular wall, their nuclei degenerate and secretion droplets form in the cytoplasm. The pseudopodia are drawn in as the cytoplasmic secretions swell and the nurse cell becomes spherical. The eupyrene sperm become grouped unilaterally and at this stage are attached to the nurse cell by only the tips of their acrosomes. At maturity the nurse cells with their clumps of attached eupyrene sperm (spermatozeugmata) are released from the testis via ducts into the seminal vesicles, where they are stored prior to copulation. Nurse cells serve similar functions to those of apyrene sperm which are common among the Molluscs. We believe that the nurse cell and apyrene sperm are homologous.

Ultrastructure of the male reproductive system and of spermatogenesis in the viviparous brittle-star, Amphipholis squamata

The fine structure of the male reproductive system of the hermaphroditic brittle‐star, Amphipholis squamata, has been studied in specimens from both the Pacific coast (Washington) and the Atlantic coast (New Hampshire). Each testis is a small (100‐μm) sphere and is attached to the internal wall of the bursa by peritoneal suspensor cells. Occasional flagellated cells are found on the external surface of the testis. The testicular wall of A. squamata is a multilayered structure, similar to that of other ophiuroids, but the hemal sinus is PAS negative in this species. Germinal cells are surrounded throughout their development by the filopodia of interstitial cells. Adjacent interstitial cells are interconnected, and thus form a structural network within the testis. Positionally and functionally, the interstitial cells resemble Sertoli cells; however, their origin, behavior and ultrastructure are different in many ways.

The fine structure of the polymorphic spermatozoa of Fusitriton oregonensis (Mollusca: Gastropoda), with notes on the cytochemistry of the internal secretions.

SummaryThe prosobranch Fusitriton oregonensis exhibits an unusual form of sperm polymorphism. The viable, eupyrene sperm are attached in groups of about fifty to worm-shaped, apyrene, carrier sperm. There is a second apyrene sperm, which is lancet-shaped and has a different internal organization than the carrier, but does not transport eupyrene sperm.The eupyrene sperm are filiform (185 μm long), with a conical acrosome, elongate nucleus and midpiece. They contain large stores of glycogen in the principal piece, together with an unusually high proportion of protein. The latter is due to a complex interconnecting system of fibres that supports the tail internally. A distinct annulus is located, characteristically, at the junction between midpiece and principal piece.The carrier sperm has a core of about 112 axonemes that arise from basal bodies in the anterior end and extend through its entire length of 36 μm. The basal bodies have unstriated rootlets that are embedded in a granular cap. Large membrane-bound “yolk bodies” are arranged along the length of the carrier sperm, on either side of the median axonemal core. Dense bodies, which may be indigestible residues formed from the degeneration of the nucleus, are excreted by exocytosis. Individual carrier sperm are capable of “corkscrew” propulsion, resembling that of spirochaetes.The lancet sperm is three times as long as the carrier. The sixteen or so axonemes, which are arranged peripherally like a cage enclosing the cytoplasm, originate from a dense centriolar plate in the anterior end. The cytoplasm is filled with secretions including small yolk granules, dense bodies (also excreted), clear vesicles, and a membranated granular secretion that resembles mucus. The possible functions of the lancet and carrier sperm are discussed.