Muralidharan C. Pillai

Factors controlling sperm entry into the micropyles of salmonid and herring eggs

When the micropyle area of salmonid (trout and salmon) eggs was observed continuously from the moment of insemination, spermatozoa were seen moving along the surface of the chorion and entering the micropyle one by one in a directed fashion. The ability of spermatozoa to enter the micropyle was reduced after the treatment of chorions with pronase; this reduction in sperm entry was observed even before the outer opening of the micropyle channel was narrowed due to gradual swelling of the chorion by pronase treatment. Herring spermatozoa, unlike spermatozoa of most other marine fishes, were motionless in seawater. However, they became vigorously motile on contact with the micropyle area of the herring egg chorion and entered the micropyle rapidly and efficiently. Motility initiation of herring spermatozoa in the micropyle area was dependent on extracellular calcium and potassium. Sodium also appears to be intricately involved in this process as demonstrated by the initiation of sperm movement in sodium‐free seawater. When herring eggs were treated with acidic seawater, organic solvents, or glutaraldehyde, spermatozoa did not initiate movement in the micropyle area, and sperm entry was not observed. Herring spermatozoa did not initiate movement in the micropyle area of salmonid eggs. These and other observations suggest that the micropyle areas of salmonid and herring eggs possess some sperm guidance factors which facilitate entry of homologous spermatozoa into the micropyle.

Jelly Layer Formation in Penaeoidean Shrimp Eggs

Etude histologique et chronologique de la formation de la couche gelatineuse des œufs de crevettes Penaeoidea par microscopies optique et electronique a transmission

Hatching envelope formation in shrimp (Sicyonia ingentis) ova: Origin and sequential exocytosis of cortical vesicles

The ova of Sicyonia ingentis lack cortical vesicles at the time of spawning. Within 30 min post-spawning, two populations of cortical vesicles are organized in the ooplasm which, during cortical vesicle exocytosis (cortical reaction), successively release two morphologically different exudates. The first type of cortical vesicles (dense vesicles) appears to be derived from the Golgi complexes after spawning. The second type (the ring vesicles) is formed by the fusion of asternal elements which contain loosely packed ring-shaped structures that are present in the unactivated ova. During exocytosis of the dense vesicles an electron dense material is released which coalesces with the surface coat of the ovum to form a thin hatching envelope which eventually lifts from the ovums surface. Subsequent to the formation of the thin hatching envelope, the ring vesicles undergo exocytosis resulting in an accumulation of ring-shaped structures in the perivitelline space. These structures coalesce and form an electron translucent layer on the inner surface of the thin elevated envelope to form the thickened hatching envelope. The formation of the cortical vesicles, their exocytosis and the elaboration of the hatching envelope are normally completed within 40-45 min after spawning.

Inhibition of the sea urchin sperm acrosome reaction by a lignin-derived macromolecule

Abstract The major organic components of effluents from commercial pulping processes are lignin-derived macromolecules (LDMs), which have recently been shown to inhibit fertilization and embryonic development in a variety of marine organisms, as well as to exhibit immunostimulating activity in mammalian cells. We conducted studies on the effects of an isolated LDM from bleached kraft mill effluent (BKME), and its sub-components, at the cellular level utilizing the purple sea urchin ( Strongylocentrotus purpuratus ) sperm acrosome reaction (AR) as an experimental system. The AR is an event that is induced by the eggs jelly coat and is prerequisite for successful fertilization. Sperm were preincubated with increasing concentrations of isolated LDM or electrophoretically purified LDM sub-components, followed by addition of isolated egg jelly to induce the AR in vitro. These LDM preparations significantly inhibited the AR as assessed by fluorescence (utilizing the rhodamine-conjugated phallicidin) and transmission electron microscopy. Preincubation of sperm with LDM did not have any effect on sperm motility. The level of AR inhibition was comparable to that observed in experiments assessing successful fertilization. The ability of LDM to inhibit jelly induced AR was overcome by the calcium ionophores A23187 and ionomycin. In addition, LDM was shown to inhibit the normal increase in intracellular calcium (Ca ++ ) associated with induction of the AR. When eggs were preincubated with LDM prior to addition of unexposed sperm, no effect on fertilization was observed, indicating that LDM specifically affects sperm function during fertilization. Fine structural studies, utilizing biotinylated LDM, confirmed LDMs specificity and revealed that its binding was restricted to the plasma membrane domain of the sperm head. The present observations on the inhibition of the AR by LDM is consistent with our hypothesis that this macromolecule inhibits the AR by blocking egg jelly interaction with the sperm surface, thus inhibiting ionic events such as increases in intracellular calcium. Our present approach also demonstrates that echinoderm sperm functions can be used as a model system for the investigation of the mode of action of toxicants at the sub-cellular level.

Early development in an algal gametophyte: role of the cytoskeleton in germination and nuclear translocation

SummaryBiflagellate zoospores from the giant kelpMacrocystis pyrifera underwent germination after adhering to a substrate and produced germ tubes that were approximately 13–15 μm in length. Coincident with the germ tube elongation was organelle (other than the nucleus) translocation along the tube. Shortly after formation of the germ tube, the zoospore nucleus divided and one daughter nucleus translocated along the germ tube. The nucleus did not appear to undergo chromosomal condensation prior to division. The nuclear division and/or translocation of the daughter nucleus did not begin until well after germ tube elongation was complete, demonstrating that these are temporally distinct developmental events. The translocation of one daughter nucleus coincided with differentiation of the distal end of the germ tube into a bulbous structure. Following this, the first gametophytic cross wall was formed and, subsequently, the daughter nucleus remaining in the original zoospore body underwent repositioning, assuming a position in the germ tube near the cross wall. Cytochalasin D inhibited germ tube elongation suggesting that actin microfilaments are probably involved in this developmental process. In addition, when cytochalasin D was added to the culture after the germ tube elongation was complete, it did not affect either nuclear division or translocation, indicating that microfilaments were not directly involved in these nuclear events. Colchicine and the plant specific microtubule disrupting agent, amiprophos methyl blocked nuclear division and translocation without affecting germination or germ tube elongation. These data suggest that actin microfilaments are primarily responsible for complete germination, specifically germ tube elongation, while microtubules are involved in nuclear division and translocation. The present study demonstrates that germination (and germ tube elongation) and nuclear translocation inM. pyrifera gametophytes are temporally and mechanistically distinct developmental events.

Nuclear events during early development in gametophytes of Macrocystis pyrifera, and the temporal effects of a marine contaminant

The time course of DNA synthesis in developing haploid gametophytes of the giant kelp Macrocystis pyrifera was determined, and the effects of arsenic (As) on the temporally distinct nuclear events, DNA synthesis and subsequent nuclear division/translocation, were investigated to establish which of these specific events may be disrupted by this contaminant. Experiments were carried out on material collected from kelp beds near Santa Barbara, California from 1993–1994. Timing of DNA synthesis was determined during development by use of the fluorochrome, DAPI (4′,6-diamidino-2-phenylindole), and single-cell microspectrofluorometry. Zoospores, which result from meiosis, had already undergone two rounds of DNA synthesis at the time of release. The developing gametophytes underwent an additional two rounds of replication of DNA by 16 h of development, and following the first nuclear division/translocation, the gametophyte contained eight times the minimum DNA level throughout subsequent development. Both DNA synthesis and nuclear division/translocation, were found to be inhibited by As. Phosphate enrichment reduced the inhibitory effects of As on division/translocation of the nucleus, supporting the hypothesis that As interferes with phosphorylation. Gametophytes were more severely affected by As under light conditions, as opposed to dark, suggesting that photosynthesis may be more sensitive than dark metabolism.

Response of embryos of the sea urchin Strongylocentrotus purpuratus to aqueous petroleum waste includes the expression of a high molecular weight glycoprotein

The present study investigated the sublethal effects of the largest aqueous waste associated with offshore oil development, produced water (PW), on sea urchin embryo development. PW inhibited normal gastrulation in purple sea urchin (Strongylocentrotus purpuratus) embryos exposed at the hatching stage. The exposed embryos did not form complete archenterons, and secondary mesenchyme cells exhibited an unusual behavior with respect to the wall of the blastocoelic cavity. In addition, an abundance of extracellular matrix was observed in the blastocoelic cavities in the embryos exposed to higher concentrations of PW. An inhibition of the development of embryos through the pluteus stage was observed as a result of PW exposure. This was manifested by inhibition of normal spicule formation with concomitant abnormal pluteus morphology. To determine if a biochemical stress response occurs as a result of PW exposure, embryos at the early gastrula stage were exposed to PW for 2 h, followed by an additional 1 h in 3H-leucine and PW. These embryos expressed a 253 kDa protein as observed by one-dimensional SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis and autoradiography. This protein could occasionally be observed in gels stained for protein, and was glycosylated as demonstrated by staining with periodic acid Schiff (PAS). The 70 to 73 kDa heat-shock proteins previously described in sea urchin embryos (and other organisms) as a result of stress were not synthesized as a result of PW exposure. Heat shock did not induce synthesis of the 253 kDa glycoprotein. Combined PW and heat-shock exposure elicited both the heat-shock response (73 kDa protein) as well as expression of the 253 kDa glycoprotein. These data demonstrate the use of expression of endogenous macromolecular markers to distinguish exposures of embryos to different perturbations. Arsenic (as sodium arsenite), a metal constituent of PW, also induced the 253 kDa glycoprotein in early gastrula-stage embryos. We suggest that this glycoprotein may be a marker for PW (and a metal constituent) exposure in sea urchin embryos, and may be related to morphological abnormalities.