CALIFORNIA STATE UNIVERSITY, NORTHRIDGE

THE EFFECTS OF SUGAR ALCOHOLS ON SEA URCHIN GASTRULATION IN LOW

CALCIUM SEA WATER

A Thesis submitted in partial fulfillment of the requirements

For the degree of Master of Science

In Biology

By

Edward Holmes

May 2015

Copyright 2015, Edward Holmes

ii The thesis of Edward Holmes is approved:

______

Lisa Banner, Ph.D. Date

______

Stan Metzenberg, Ph.D. Date

______

Steven B. Oppenheimer, Ph.D., Chair Date

California State University, Northridge

iii DEDICATION

This research and thesis project has been dedicated to the Holy Trinity.

To my heavenly Abba Father who has adopted me as His son

To my Lord and Savior Jesus Christ

To the Holy Spirit who is my Comforter and Counselor

For their perfect love, grace and mercy

For their eternal honor and glory

iv ACKNOWLEDGEMENTS

Thank you:

To Dr. Steven Oppenheimer as a mentor and adviser for your patience, encouragement, guidance, and understanding throughout my research and thesis project.

To Dr. Stan Metzenberg for your time and constructive criticism as a thesis committee member.

To Dr. Lisa Banner for your time and constructive criticism as a thesis committee member.

To my parents Roger and Phyllis and my brothers Jeff and Dwayne for your love, support, generousity, and patience which enabled me to complete my research and thesis project.

To my brothers and sisters in Jesus Christ from United Campus Ministry (UCM), the Campus

Outreach Response Team (CORT), MT28, and Intervarsity Christian Fellowship for your love, encouragement and prayer support.

To my laboratory partners Kathy Fernando and Tiffany Smith for their teamwork.

To Jouliana Davoudi for her assistance with the statistical analysis of my research and thesis project.

v To Samantha Arvizu and Victoria Arvizu for their assistance with the photography of my research and thesis project.

This research was supported by NIH, NIGMS SCORE SO648680, MARC, RISE, the Joseph Drown

Foundation, and the Sidney Stern Memorial Trust.

vi TABLE OF CONTENTS

Signature Page ……………………………………………………………………………………………………………………..iii

Dedication ……………………………………………………………………………………………………………………………iv

Acknowledgements ………………………………………………………………………………………………………………v

List of Figures ………………………………………………………………………………………………………………………viii

List of Tables ………………………………………………………………………………………………………………………..xii

Abstract ……………………………………………………………………………………………………………………………..xvii

Introduction ………………………………………………………………………………………………………………………….1

Materials and Methods ……………………………………………………………………………………………………….15

Results ………………………………………………………………………………………………………………………………..26

Discussion …………………………………………………………………………………………………………………………..85

References ………………………………………………………………………………………………………………………….96

vii

LIST OF FIGURES

Figure 1: Set of photographs showing each L. pictus sea urchin morphological

type including: complete archenteron, incomplete archenteron, not

invaginated, exogastrulated and dead………………………………………………………………36

Figure 2: Set of photographs showing L. pictus sea urchin embryos at 24-26 hours

after fertilization in LCASW controls and in 0.1M, 0.05M, 0.01M, 0.005M

and 0.001M adonitol…………………………………………………………………………………………37

Figure 3: Set of photographs showing L. pictus sea urchin embryos at 32-34 hours

after fertilization in LCASW controls and in 0.1M, 0.05M, 0.01M, 0.005M

and 0.001M adonitol………………………………………………………………………………………..38

Figure 4: Set of photographs showing L. pictus sea urchin embryos at 24-26 hours

after fertilization in LCASW controls and in 0.1M, 0.05M, 0.01M, 0.005M

and 0.001M L-(-) arabitol………………………………………………………………………………….39

Figure 5: Set of photographs showing L. pictus sea urchin embryos at 32-34 hours

after fertilization in LCASW controls and in 0.1M, 0.05M, 0.01M, 0.005M

and 0.001M L-(-) arabitol………………………………………………………………………………….40

viii Figure 6: Set of photographs showing L. pictus sea urchin embryos at 24-26 hours

after fertilization in LCASW controls and in 0.1M, 0.05M, 0.01M, 0.005M

and 0.001M dulcitol………………………………………………………………………………………….41

Figure 7: Set of photographs showing L. pictus sea urchin embryos at 32-34 hours

after fertilization in LCASW controls and in 0.1M, 0.05M, 0.01M, 0.005M

and 0.001M dulcitol………………………………………………………………………………………….42

Figure 8: Set of photographs showing L. pictus sea urchin embryos at 24-26 hours

after fertilization in LCASW controls and in 0.1M, 0.05M, 0.01M, 0.005M

and 0.001M D-mannitol……………………………………………………………………………………43

Figure 9: Set of photographs showing L. pictus sea urchin embryos at 32-34 hours

after fertilization in LCASW controls and in 0.1M, 0.05M, 0.01M, 0.005M

and 0.001M D-mannitol…………………………………………………………………………………..44

Figure 10: Set of photographs showing L. pictus sea urchin embryos at 24-26 hours

after fertilization in LCASW controls and in 0.1M, 0.05M, 0.01M. 0.005M

and 0.001M D-sorbitol…………………………………………………………………………………….45

Figure 11: Set of photographs showing L. pictus sea urchin embryos at 32-34 hours

after fertilization in LCASW controls and 0.1M, 0.05M, 0.01M, 0.005M

and 0.001M D-sorbitol…………………………………………………………………………………….46

ix Figure 12: Set of photographs showing L. pictus sea urchin embryos at 24-26 hours

after fertilization in LCASW controls and 0.1M, 0.05M, 0.01M, 0.005M

and 0.001M xylitol…………………………………………………………………………………………….47

Figure 13: Set of photographs showing L. pictus sea urchin embryos at 32-34 hours

after fertilization in LCASW controls and 0.1M, 0.05M, 0.01M, 0.005M

and 0.001M xylitol…………………………………………………………………………………………….48

Figure 14: The effects on L. pictus embryos incubated in adonitol and low calcium

artificial sea water at the 24-26 hour stage of gastrulation………………………………49

Figure 15: The effects on L. pictus embryos incubated in adonitol and low calcium

artificial sea water at the 32-34 hour stage of gastrulation…………………………….50

Figure 16: The effects on L. pictus embryos incubated in L-(-) arabitol and low calcium

artificial sea water at the 24-26 hour stage of gastrulation…………………………….51

Figure 17: The effects on L. pictus embryos incubated in L-(-) arabitol and low calcium

artificial sea water at the 32-34 hour stage of gastrulation…………………………….52

Figure 18: The effects on L. pictus embryos incubated in dulcitol and low calcium

artificial sea water at the 24-26 hour stage of gastrulation…………………………….53

x Figure 19: The effects on L. pictus embryos incubated in dulcitol and low calcium

artificial sea water at the 32-34 hour stage of gastrulation…………………………….54

Figure 20: The effects on L. pictus embryos incubated in D-mannitol and low calcium

artificial sea water at the 24-26 hour stage of gastrulation…………………………….55

Figure 21: The effects on L. pictus embryos incubated in D-mannitol and low calcium

artificial sea water at the 32-34 hour stage of gastrulation…………………………….56

Figure 22: The effects on L. pictus embryos incubated in D-sorbitol and low calcium

artificial sea water at the 24-26 hour stage of gastrulation……………………………..57

Figure 23: The effects on L. pictus embryos incubated in D-sorbitol and low calcium

artificial sea water at the 32-34 hour stage of gastrulation…………………………….58

Figure 24: The effects on L. pictus embryos incubated in xylitol and low calcium

artificial sea water at the 24-26 hour stage of gastrulation……………………………59

Figure 25: The effects on L. pictus embryos incubated in xylitol and low calcium

artificial sea water at the 32-34 hour stage of gastrulation……………………………60

xi LIST OF TABLES

Table 1: P-value results for each observed morphological characteristic between low

calcium artificial sea water (LCASW) control and each of the five adonitol

concentrations in LCASW at 24-26 hours after sea urchin fertilization…………………61

Table 2: P-value results for each observed morphological characteristic between low

calcium artificial sea water (LCASW) control and each of the five adonitol

concentrations in LCASW at 32-34 hours after sea urchin fertilization………………..62

Table 3: P-value results for each observed morphological characteristic between low

calcium artificial sea water (LCASW) control and each of the five L-(-) arabitol

concentrations in LCASW at 32-34 hours after sea urchin fertilization………………..63

Table 4: P-value results for each observed morphological characteristic between low

calcium artificial sea water (LCASW) control and each of the five L-(-) arabitol

concentrations in LCASW at 32-34 hours after sea urchin fertilization………………..64

Table 5: P-value results for each observed morphological characteristic between low

calcium artificial sea water (LCASW) control and each of the five dulcitol

concentrations in LCASW at 24-26 hours after sea urchin fertilization………………..65

xii Table 6: P-value results for each observed morphological characteristic between low

calcium artificial sea water (LCASW) control and each of the five dulcitol

concentrations in LCASW at 32-34 hours after sea urchin fertilization…………………66

Table 7: P-value results for each observed morphological characteristic between low

calcium artificial sea water (LCASW) control and each of the five D-mannitol

concentrations in LCASW at 24-26 hours after sea urchin fertilization……………….67

Table 8: P-value results for each observed morphological characteristic between low

calcium artificial sea water (LCASW) control and each of the five D-mannitol

concentrations in LCASW at 32-34 hours after sea urchin fertilization……………….68

Table 9: P-value results for each observed morphological characteristic between low

calcium artificial sea water (LCASW) control and each of the five D-sorbitol

concentrations in LCASW at 24-26 hours after sea urchin fertilization……………….69

Table 10: P-value results for each observed morphological characteristic between low

calcium artificial sea water (LCASW) control and each of the five D-sorbitol

concentrations in LCASW at 32-34 hours after sea urchin fertilization……………….70

xiii Table 11: P-value results for each observed morphological characteristic between low

calcium artificial sea water (LCASW) control and each of the five xylitol

concentrations in LCASW at 24-26 hours after sea urchin fertilization………………71

Table 12: P-value results for each observed morphological characteristic between low

calcium artificial sea water (LCASW) control and each of the five xylitol

concentrations in LCASW at 32-34 hours after sea urchin fertilization………………72

Table 13: Percentage morphology and total sample size for each of the five

concentrations of adonitol in low calcium artificial sea water (LCASW) and

LCASW controls at 24-26 hours after fertilization…………………………………………..73

Table 14: Percentage morphology and total sample size for each of the five

concentrations of adonitol in low calcium artificial sea water (LCASW) and

LCASW controls at 32-34 hours after fertilization…………………………………………..74

Table 15: Percentage morphology and total sample size for each of the five

concentrations of L-(-) arabitol in low calcium artificial sea water (LCASW) and

LCASW controls at 24-26 hours after fertilization…………………………………………..75

xiv Table 16: Percentage morphology and total sample size for each of the five

concentrations of L-(-) arabitol in low calcium artificial sea water (LCASW) and

LCASW controls at 32-34 hours after fertilization…………………………………………….76

Table 17: Percentage morphology and total sample size for each of the five

concentrations of dulcitol in low calcium artificial sea water (LCASW) and

LCASW controls at 24-26 hours after fertilization……………………………………………77

Table 18: Percentage morphology and total sample size for each of the five

concentrations of dulcitol in low calcium artificial sea water (LCASW) and

LCASW controls at 32-34 hours after fertilization……………………………………………78

Table 19: Percentage morphology and total sample size for each of the five

concentrations of D-mannitol in low calcium artificial sea water (LCASW)

and LCASW controls at 24-26 hours after fertilization…………………………………..79

Table 20: Percentage morphology and total sample size for each of the five

concentrations of D-mannitol in low calcium artificial sea water (LCASW)

and LCASW controls at 32-34 hours after fertilization………………………………….80

xv Table 21: Percentage morphology and total sample size for each of the five

concentrations of D-sorbitol in low calcium artificial sea water (LCASW)

and LCASW controls at 24-26 hours after fertilization……………………………………..81

Table 22: Percentage morphology and total sample size for each of the five

concentrations of D-sorbitol in low calcium artificial sea water(LCASW)

and LCASW controls at 32-34 houirs after fertilization……………………………………82

Table 23: Percentage morphology and total sample size for each of the five

concentrations of xylitol in low calcium artificial sea water (LCASW)

and LCASW controls at 24-26 hours after fertilization……………………………………83

Table 24: Percentage morphology and total sample size for each of the five

concentrations of xylitol in low calcium artificial sea water (LCASW)

and LCASW controls at 32-34 hours after fertilization………………………………….84

xvi ABSTRACT

THE EFFECTS OF SUGAR ALCOHOLS ON SEA URCHIN GASTRULATION IN

LOW CALCIUM SEAWATER

By

Edward Holmes

Master of Science in Biology

The sea urchin embryo has been designated by the National Institutes of Health as a model system for studying cellular interactions. Over 25 physiological discoveries were made in sea urchins crucial to understanding their function in many taxonomic groups including humans.

The sea urchin embryo was used in this study to examine the effects of sugar alcohols on cell-cell interactions during gastrulation. This study examined six different sugar alcohols in

1.5mM low calcium artificial seawater (LCASW) on 24-26 hour Lytechinus pictus sea urchin embryos and 32-34 hour Lytechinus pictus sea urchin embryos. The embryos were incubated in

1.5mM low calcium artificial sea water with and without (controls) 0.1M, 0.05M, 0.01M,

0.005M, and 0.001M adonitol, L-(-) arabitol, dulcitol, D-mannitol, D-sorbitol, and xylitol in 96 well microplates and were fixed at 48-50 hours, observed, and photographed. Three to six

xvii separate experiments were conducted for each sugar alcohol concentration at each of the two times. The embryo morphologies observed were complete archenteron, incomplete archenteron, not invaginated, exogastrulated, and dead. Sugar alcohol concentrations for each experiment were tested in 12 replicate wells yielding hundreds of sea urchin embryos in each experiment. Low calcium artificial sea water was utilized in this study because it was found to loosen septate junctions found in the blastula epithelium, accelerating the molecular entry into the blastocoel of the embryos without microinjection. An unpaired t-test was performed for each sugar alcohol concentration and morphology compared to the LCASW control. P-values less than 0.05 (p<0.05) suggested that the percentages of morphologies in the sugar alcohol concentration were significantly different than in the LCASW control. The sugar alcohols uniformly had little effect on embryo morphologies. The sugar alcohols causing slight effects on archenteron morphology when added at 24-26 hrs: (most effects) adonitol > L- (-) arabitol > xylitol > dulcitol = D-sorbitol > D-mannitol (least effects). The sugar alcohols causing slight effects on archenteron morphology when added at 32-34 hours (most effects) D-mannitol > dulcitol > adonitol = xylitol > D-sorbitol = L-(-) arabitol (least effects). While effects were minimal, this study indicates that very small differences can be identified quantitatively using this assay system.

xviii INTRODUCTION

Classic System of Sea Urchins

The classic system of the sea urchin was used to investigate molecular and cellular interactions, because of its simple, rapid, and synchronous embryogenesis, (Horstadius, 1973;

Ernst, 1997) transparent embryos, (Horstadius, 1973; Epel, 1975; Ernst, 1997) and using quantitative assays to probe living embryos (Idoni, 2010). Low calcium artificial seawater was found to loosen septate junctions in the blastula epithelium, accelerating molecular entry into the blastocoel of the embryos without microinjection. Other advantages of using the sea urchin model system includes: in vitro fertilization, millions of male and female gametes, (Horstadius,

1973; Epel, 1975; Ernst, 1997) and the seasonal availability of different species of sea urchins

(Horstadius, 1973; Epel, 1975). Sea urchins are echinoderms which means “spiny skin” in Greek

(Campbell & Reece, 2002). The name echinoderm comes from the bumps and spines on the skin of the sea urchin. The bumps and spines on the skin are extensions of its endoskeleton

(Auderisk et al., 2002). The endoskeleton is composed of closely joined calcareous plates forming a rigid wall around the vital organs. This round skeleton surrounding the vital organs of the sea urchin is called a test (Pearse & Buchsbaum, 1987). The physiology of the sea urchins includes a water vascular system with hydraulic canals bifurcating from its tube feet (Campbell

& Reece, 2002). The tube feet are the main organs for respiratory exchange. There are also thin walled projections found on the soft membrane surrounding the mouth, which provide additional respiratory exchange for the jaw muscles. The complex set of jaws of the sea urchin are composed of five sharp pointed teeth, thirty-five ossicles and powerful muscles (Pearse &

1 Buchsbaum, 1987). Tube feet also function in locomotion and capturing food. Sea urchins are found in marine environments (Auderisk et al., 2002) with hard rocky surfaces in crevices and holes which they create themselves with their spines and teeth (Pearse & Buchsbaum, 1987).

The sea urchin classic system is also important because of its phylogenetic position relative to humans. Sea urchins belong to the phylum Echinodermata as well as the class Echinoidea

(Davidson et al. 2002). Echinoderms, hemichordates and chordates are found in the deuterstome subgroup of the Animal Kingdom. Chordates (which include humans and other vertebrates) share a common ancestor with echinoderms (which includes sea urchins)

(Davidson et al. 2002). In 2006, scientists sequenced the genome of the Strongylocentrotus purpuratus sea urchin which contained over 814 million nucleotide bases in the chromosomes of this sea urchin. The letters from the nucleotide bases spelled out 23,300 genes. (Tomlin,

2006).

Sea urchins have some unique characteristics such as having a very complex innate immune system. The innate immune system of the sea urchin is based on proteins rather than antibodies. These proteins detect and signal the cells of the sea urchin that there is a bacterial intruder. Sea urchins also use defensomes. Defensomes are a group of genes which sense, transform, and eliminate chemical toxins. Further study of the protein based innate immune system and defensomes of the sea urchins would provide valuable tools in fighting infectious diseases and toxins (Bowers, 2006).

2 Sea Urchin Development

Fertilization

The time line for the fertilization for the Strongylocentroyus purpuratus sea urchin begins one second after sperm addition to the eggs in which the acrosomal process of the sperm attaches to the vitelline layer and fuses with the plasma membrane of the egg. The acrosome reaction occurs when bindin (Kominami et al., 2008; Vacquire et al., 1977) of the acrosomal process of the sperm attaches to the vitelline layer of the egg (Dan, J.C., 1970; Epel, 1977).

Receptors on the vitelline layer of the egg can only recognize bindin sperm proteins of the same species. (Miller, 1985; Oppenheimer and Lefevre, 1989) Lillie fertilizin which is an acid mucopolysaccharide can be found on the jelly coat of sea urchin eggs which clumps and activates the sperm, causing the acrosomal reaction to occur (Vacquier and Moy, 1977;

Oppenheimer and Lefevre, 1989). Scanning electron micrographs showed that as many as 1500 sperm could attach to a single egg under saturation conditions (Epel, 1977). Microvilli with interconnected ridges appears in the vitelline layer of the unfertilized egg (Tegner and

Epel, 1973; Humphreys, W. J. and Lindsay, D. T., 1971). Fine filaments of the egg surface are seen binding the sperm to its surface (Tegner and Epel, 1973). A progressive increase of sperm binding to the surface of the egg can be seen at 5 seconds and 15 seconds (Tegner and Epel,

1973). Thousands of sperm bind to the egg’s vitelline layer 25 seconds after insemination

(Tegner and Epel, 1973). The cortical reaction (also known as the slow block to polyspermy

(Gilbert, S. F., 2003)) is the first morphological response 25 seconds after insemination which shows the elevation of the vitelline envelope being elevated

3 and becoming the fertilization membrane (Tegner and Epel, 1973; Allen, R. D. and Griffin, J. L.,

1958; Vacquier, V. D. et al., 1972). The vitelline layer which becomes the fertilization envelope detaches from the plasma membrane at 25 seconds also (Tegner and Epel, 1973; Runnstrom, J.,

1966). Mucopolysaccharides are released from the cortical granules, (Gilbert, S. F., 2003) and at

30 seconds, a flat, circular area of the vitelline layer elevates from the plasma membrane of the fertilized egg detaching supernumerary sperm (Tegner and Epel, 1973). The cortical reaction has covered half of the egg at 45 seconds (Tegner and Epel, 1973). At 55 seconds, the vitelline layer continues to elevate until all supernumerary sperm are detached from the vitelline layer

of the fertilized egg (Tegner and Epel, 1973). A peroxidase enzyme called ovoperoxidase is released from the cortical granules crosslinks tyrosine residues on adjacent proteins (Katsura and Tominga, 1974; Foerder and Shapiro, 1977; Klebanoff, et al., 1979; Lafleur et al., 1998;

Gilbert, 2003) which leads to the hardening of the vitelline envelope, and becoming the fertilization membrane at 3 minutes (Tegner and Epel, 1973; Epel, 1977). The cortical granule serine protease (Gilbert, S. F., 2003; Vacquier et al., 1973; Glabe and Vacquier, 1978;

Haley and Wessel, 1999) engages in the detachment of the vitelline layer from the plasma membrane as well as in establishing the slow block to polyspermy (Tegner and Epel, 1973;

Vacquier, V. D. et al., 1972). The cortical granules accumulate within 1-2 micrometers of the vitelline envelope of the egg in small sacs called mosaic globules as the egg matures (Kopf, et al., 1982). The cortical granules are released via calcium-mediated exocytosis after fertilization

(Kopf, et al., 1982; Wessel, 1989). Also cortical granules contain many proteins such as beta-1,3 glucosonase whose hypothesized function is a hydrolase, (Epel et al., 1969; Wessel et al., 1989) ovostatin, which inhibits proteinases that would otherwise digest collagen and other

4 proteins in the fertilization membrane and extracellular matrix, (Nagase and Harris, 1983;

Yamada and Aketa, 1988) and synaptotagmin is a calcium sensor involved in the secretion of other cortical granules (Leguia, et al., 2006). The cortical granules toposome, N- acetylglucosaminidase, glycosaminoglycans and a small amount of hyaline protein works together and in cell adhesion and signaling pathways between the embryo, the fertilization envelope, hyaline layers and the extracellular matrix (Bal, 1970 ; Schuel, et al., 1974; Hylander and Summers, 1982; Noll, et al., 1985; Miller, et al., 1993; Noll, et al., 2007). Structural components of the fertilization envelope such as collagen, sulfated acidic polysaccharides and calcium can be found in the cortical granules (Anderson, 1968; Bryan, 1970; Cardasis, et al.,

1978). Also, the cortical granules block polyspermy and modify the cell surface of the zygote

(Anderson, 1968; Kay and Shapiro, 1985; Schuel, 1985). Immediately following the cortical reaction, the hyaline layer is formed. (Spiegel and Spiegel, 1979; Adelson et al., 1992; Razinia,

2006). The interconnected ridges of the microvilli on the fertilization membrane can no longer be seen. The sperm which fertilized the egg rotates 180 degrees and decondenses within the egg cytoplasm to form the male pronucleus (Epel, 1975). Fusion between the male pronucleus and the female pronucleus occurs when the centrioles pull the female pronucleus towards the male pronucleus, and they make contact (Kominami, et al., 2008; Hamaguchi, et al., 1980) 20 minutes after insemination (Epel, 1975). Initiation of DNA synthesis occurs 20-40 minutes post insemination, followed by mitosis and the first cleavage which occurs at 60-80 minutes and 85-

95 minutes after insemination respectively (Gilbert, 2003).

A fast block to polyspermy occurs simueltaneously as the slow block to polyspermy (cortical reaction). The standard membrane potential of an unfertilized egg is -70mV. The fast block to

5 polyspermy occurs within 1-3 seconds after the first sperm binds to the egg. Once the sperm binds to an unfertilized egg, the membrane potential increases (depolarizes) to +20 mV. This change in membrane potential is due to an influx of sodium ions (Na+) into the egg. This positive membrane potential leads to the supernumerary sperm being unable to bind to the egg (Gilbert, 2003).

Cleavage and Blastula

Synchronous, radial, holoblastic cleavage can be found in sea urchins. The first two cleavages are holoblastic which produces two cells and four cells of equal size (Wolpert, L. et al., 1998).

The third cleavage, which creates eight cells, is equatorial and arranged perpendicularly to the holoblastic cleavages, which divides the animal and vegetal regions (Wolpert, L., et al., 1998).

During the fourth cleavage, the four cells of the animal tier divide meridionally into eight mesomeres (Wolpert, L. et al., 1998). Also during the fourth cleavage, the vegetal tier undergoes an unequal equatorial cleavage to produce four large macromeres and four small micromeres (Scott, 2003; Hardin, 1994). As the sixteen cells cleave, the mesomeres divide equatorially to produce two animal tiers one on top of the other. The macromeres divide meridionally to form eight cells below the lower tier of the mesomeres. The micromeres divide asymmetrically producing a small cluster of cells below the larger macromeres at the furthest end of the vegetal tier. At the sixty-four cell stage, the animal cells divide meridionally while the vegetal cells divide equatorially (Wolpert, L. et al., 1998). At the 128 cell embryo called the blastula, the animal cells divide equatorially while the vegetal cells divide meridionally (Gilbert,

2003). The blastula is a hollow group of cells with a fluid-filled area known as the

6 blastocoel that forms within the morula (Wolpert, L. et al., 1998). The blastula is also covered with several types of fibropellin proteins. Fibropellins become part of the inner surface of the hyaline layer after their secretion. Fibropellins organize themselves into fibers and cover the entire embryo at the blastula stage. The fibropellin network becomes more complex as the sea urchin embryo continues to develop (Bisgrove, et al., 1991). The formation of the central cavity is caused by an influx of water into the blastula that causes the cells to push outward. Surface tension and cell adhesion to the hyaline layer may be contributing factors that causes the cells in the blastula to push outward (Dan, 1960; Wolpert and Gustafson, 1961). The blastula cells develop cilia which allows the blastula to move within the fertilization membrane. Blastula cells found at the animal pole manufactures hatching enzymes that digest the fertilization envelope resulting in a free-swimming hatched blastula (Lepage et al., 1992; Gustafson and Wolpert,

1967).

Gastrulation

Gastrulation follows cleavage and blastula formation. Gastrulation is an organized rearrangements of the cells within the embryo. Gastrulation produces a sea urchin embryo with three germ layers: ectoderm, mesoderm and endoderm. The ectoderm produces the larval skin and neurons. The mesoderm gives rise to the primary and secondary mesenchyme cells, and the circulatory system. The endoderm forms the inner lining and the archenteron (DerHartonian, 2011). Gastrulation begins when the primary mesenchyme cells separate from the blastula wall. The fifty primary mesenchyme cells develop projections called filopodia (Wolpert, L. et al., 1998). Filopodia migrate along the basal

7 lamina which lines the blastocoel to test for specific properties of the substrate which leads to the formation of the syncytial ring near the base of the invaginating archenteron. The long filopodia of the primary mesenchyme cells form cable-like structures (Wolpert. L. et al., 1998).

Calcified spicules form on these cables which serve as the internal skeleton of the sea urchin.

Katow and Solursh (1980) revealed through their ultrastructural studies that the first step of ingression involves the loss of the basal lamina from the vegetal plate as well as the loss of cilia from these cells. Katow and Solursh (1980) also observed that the blastodermal cells protruded into the blastocoel, lost their apical junctions, rounded up and entered the blastocoel.

There is a decrease in the cell division rate just prior to gastrulation in which the embryo comprises 1000 cells. The animal pole of the embryo is composed of a thick epithelium in which the cilia of the apical plate appear longer than on the rest of the embryo. The vegetal pole of the embryo is composed of an epithelium which flattens and forms the vegetal plate.

Primary mesenchyme cells become flask-shaped cells in the epithelial layer during gastrulation.

(Katow and Solursh, 1980) Primary mesenchyme cells move (Solursh, 1986) and also utilize sulphated proteoglycans on their surfaces and/or in the blastocoel for their migration (Lane and

Solursh, 1988; Lane and Solursh, 1991; Solursh et al., 1986). Primary mesenchyme cells initially move from the vegetal plate to form a syncytial ring in the vegetal pole. The vegetal plate moves into the blastocoel to form a cylinder called the archenteron. Primary invagination of the archenteron occurs when the archenteron extends 1/4-1/2 way across the blastocoel (Wolpert,

L. et al., 1998). During secondary invagination of the archenteron, the secondary mesenchyme cells use filopodia to reach the blastocoel roof and walls (Gilbert, S.F., 2000).

8 The archenteron tip binds near the animal pole. After the archenteron tip reaches the blastocoel roof the primary mesenchyme cells form calcareous skeletal rods. Coelomic pouches or two bilateral outpocketings appear at the tip of the archenteron. The tip of the archenteron binds to the blastocoel to form the mouth. The archenteron becomes tripartite as the pluteus larva develops (Bisgrove and Burke, 1987).

Low Calcium Sea Water Effects

Cell-cell interactions and cell adhesion in the blastula and gastrula have been shown to be very strong. These cells have a high affinity for neighboring cells as well as to the hyaline layer.

Cellular tight junctions made it difficult for scientists to experiment on embryos (Kiehart, 1982).

The sea urchin blastula epithelium contains septate junctions, which are only found in invertebrates. Sea urchin septates are made up of pleated, single-layered septa in an anastomosing pattern. Starting at the four-cell stage, two to four septa can be observed and as many as twenty septa can be seen in the pluteus larva stage (Spiegel and Howard, 1983).

Presumably, these intercellular junctions act as a permeability barrier (Green and Bergquist,

1982; Gilula, 1973; Andreuccetti et al., 1987). A previous study has shown that macromolecules can penetrate the blastula epithelium before the tenth cleavage. Treatment after the tenth cleavage resulted in blastcoel shrinking, which indicated that the permeability barrier was in place at this stage (Moore and Burt, 1939; Moore, 1940). Previous studies required exogenous macromolecules to be injected into the embryo blastocoel (Kiehart, 1982). The microinjection method can only be performed on a few embryos at one time, but can’t be used to access the interior of thousands of embryos at a single time. Microinjection is difficult to perform as well

9 as causing injury and trauma to the injected embryos, which may lead to distorted results

(Kaneko et al., 1995).

Dan-Sohkawa et. al. demonstrated through their study that incubating starfish embryos in a hypertonic solution allowed macromolecules to enter the blastocoel of these embryos. The results of their experiments suggested that septate junctions were not completely sealed or easily loosened (Dan-Sohkawa et al., 1995). Latham et al. demonstrated that incubating sea urchin embryos in low calcium artificial sea water would allow macromolecules to penetrate the embryo blastocoel. Latham’s experiment labeled specific lectins known to atatch to primary mesenchyme cells with fluorochrome. The sea urchin embryos were suspended in low calcium artificial sea water and the fluorochrome stained lectins were introduced to the external environment. The results that were found without harming the developing embryo were that macromolecular access was accelerated when the embryos were treated with low calcium artificial sea water (Latham et al., 1998).The adhesion between blastula cells in the sea urchin embryo is dependent upon the calcium concentration found in the sea water. When the calcium concentration in the sea water is lowered, this causes the junctions to loosen between the septas (Herbst, 1900; Balinsky, 1958).

In another study, Itza et. al. demonstrated that treating sea urchin embryos in calcium and magnesium free sea water loosened the septate junctions in the blastula epithelium, which allowed macromolecules to penetrate the embryo blastocoel. These macromolecules were able

to permeate and diffuse into the sea urchin blastocoel during the incubation period (Itza et al.,

2005). The usage of low calcium sea water is a simple and ideal method, which allows thousands of embryos to be treated simultaneously without being damaged.

10 Sugar Alcohols

Adonitol

Adonitol is a pentose alcohol which is found within the family Ranunculaceae from the species Adonis vernalis and Adonis amurensis (Stecher, 1968). Adonitol which is also known as ribitol is produced in a crystalline form through the process of ribose reduction

(Stecher, 1968; Adonitol, 2012). Adonitol is often compared to other cell permeating molecules such as formaldehyde, propanediol, and DMSO as a cryopreservation agent (SIGMA-ALDRICH,

2012).

L (-) Arabitol

L (-) arabitol is obtained by the reduction of L (-) arabinose with sodium amalgam

(Stecher, 1968). L-(-) arabitol is a rare sugar alcohol which has been studied as a food additive that reduces fat deposits in the intestines. L-(-) arabitol is used as an inducer of xylanase expression in Hypocrea jecorina (Trichoderma reesi). L-(-) arabitol is used to identify, differentiate and characterize L-arabitol dehydrogenase(s) (SIGMA-ALDRICH, 2012).

11 Dulcitol

Dulcitol also known as galactitol, is found in dulcite or Madagascar manna known as the Melampyrum nemorosum. Dulcitol is found in within the family of Celastraceae from the species of Melampyrum, Scrophulariaceae, and Evonymus atropurpureus (Stecher, 1968).

Dulcitol is derived through reduction of galactose (SIGMA-ALDRICH, 2012).

D-Mannitol

D-mannitol can be found tree exudates including the Manna ash (Fraxinus ornus) as well as figs, olives, larches, certain species of edible fungi and seaweed (Laminaria species).

The first industrial extraction of D-mannitol was from the Manna ash tree in 1806. Over 95% of the world’s production of D-mannitol comes from the catalytic hydrogenation of D-fructose, which is obtained from invert sugar or starch. This process produces both D-mannitol and D- sorbitol in which D-mannitol is separated using fractional crystallization. D-mannitol exists in the alpha, beta, gamma, delta, and kappa crystalline forms. D-mannitol is an isomer of D- sorbitol with the difference being the orientation of the OH group on the second carbon atom

(Lawson, 2007). D-mannitol is medically used as an osmotic diuretic and in tests to determine kidney function (Stecher, 1968). D-mannitol can also be applied as a dusting agent or an anticaking agent in the confectionary industry. D-mannitol is used to chelate iron, copper, and nickel (Nelson, 2000). D-mannitol generates flammable and/or toxic gases by combining with

12 alkalai metals, nitrides, strong reducing agents and strong oxidizing agents (Chemical Book,

2012).

D-Sorbitol

D-sorbitol is a natural monosaccharide polyhydric sugar alcohol (Lawson, 2007) which was first isolated in 1872 by French chemist Joseph Boussingault (Dwivedi, 1986) from the ripe berries of the mountain ash Pyrus aucuparia (Stecher, 1968). D-sorbitol also occurs in black currents, red currents, rasberries, elderberries (Dwivedi, 1986) and in cherries, plums, pears, apples, seaweed, algae and has also been detected in blackstrap molasses (Stecher,

1968). D-sorbitol is prepared industrially from glucose by high pressure hydrogenation

(Schallenberger and Birch, 1975; Stick, 2001) supported with a nickel or Raney nickel catalyst (Dwivedi, 1986) or by electrolytic reduction. Electrolytic reduction can be performed with sodium amalgam (NaHg) in ethanol (Schallenberger and Birch, 1975; Stick, 2001) and lithium aluminum hydride (Schallenbergeer and Birch, 1975). Sucrose can produce 75% D- sorbitol and 25% mannitol simultaneously through the hydrogenation of invert sugar (Dwivedi,

1986). D-sorbitol can be found in three forms crystalline, liquid, and instant. There are alpha, beta, and gamma crystalline forms of D-sorbitol. The gamma crystalline form is the most stable form (Nelson, 2000). D-sorbitol is used in humectants (moisture conditioner), on printing rolls, in leather, and tobacco (Stecher, 1968) as well as lowering the water activity for baking

(Wilson, 2007) and for foods (Nelson, 2000). D-sorbitol is also found in antifreeze mixtures with glycerol or glycols, candy manufacturing, pharmaceutical

13 compounding as a sugar substitute for diabetics. D-sorbitol is used to increase the absorption of vitamins and other nutrients in pharmaceutical preparations. D-sorbitol is medically used as an osmotic diuretic, cathartic; sweetening agent for diabetics and as a parenteral alimentation

(Stecher, 1968). Further applications of D-sorbitol include: being able to sequester specific multivalent metals, being a stabilizer and as a viscosity control agent (Dwivedi, 1986). D-sorbitol may be applied for washing spheroplasts and in isoelectric focusing to minimize endoosmotic flow in agarose gels, and also may be used to induce osmotic stress (SIGMA-ALDRICH, 2012).

Xylitol

Xylitol is a five carbon monosaccharide polyhydric alcohol (Bond, 2007).

Xylitol is produced from hydrogenation of D-xylose obtained from hemicellulose sources such as birch, (Stecher, 1968) beech, other hardwoods, corn cobs, (Bond, 2007) almond shells, straw, and wastes from the paper and pulp industries. (Bar, 1986) Xylitol is a desirable confectionary ingredient for mint candies and sugarless chewing gum (Bond, 2007).

Xylitol is noncarcinogenic because it is not metabolized by the microflora of the mouth that produce dental plaques (Stecher, 1968). Xylitol inhibits Streptococcus mutans which is associated with dental caries (Bond, 2007). Cataracts has been observed through experiments on rats. Xylose through a series of oxidative and reductive biochemical reactions produces xylitol. Xylitol accumulates in the lens of the eye producing insoluble protein fibers which leads to opaqueness and cataracts of the eye. Also, xylitol by itself can’t cause cataracts because xylitol can’t penetrate the membrane of the lens (Shallenberger and Birch, 1975).

14 Materials and Methods

Sea Urchins

Lytechinus pictus sea urchins were utilized in these experiments. This species of sea urchins is known as the “white sea urchin”. These sea urchins were purchased from Marinus Scientific which is located in Garden Grove, California. The seasonal availability of the white sea urchin goes from May through September. The sea urchins were stored in refrigerated sea water aquariums at temperatures of 16 C – 17 C until they were needed for experimental use.

Artificial sea water (ASW)

ASW was prepared using formula 130 of the Marine Biological Laboratory (Bidwell and Spotte,

1985). Four liters of ASW were prepared by using double distilled Arrowhead Mountain Spring

Water and a 4L Erlenmeyer flask (Pyrex, USA). The flask was placed on a Fisher Scientific magnetic stirrer and a Fisher Scientific stir bar was placed inside the flask. The initial chemical amounts of 98.88g of NaCl, 2.68g of KCl, 5.44g of CaCl2*2H2O, 18.64g of MgCl2*6H2O, and

25.16g of MgSO4*7H2O were carefully measured using a digital, analytical scale and then added to the water filled flask. These chemical ingredients were stirred for 15 minutes at 300 rpm until completely dissolved. After these chemical ingredients were completely dissolved,

0.72g of NaHCO3 was carefully weighed using a digital, analytical scale and added to the solution to stir for an additional 15 minutes until it was fully dissolved. A Beckham pH meter was used to

15 determine the initial pH of the ASW. The standard pH was tested using a pH 7 buffer solution.

The pH electrode was rinsed with double distilled Arrowhead water and wiped with Kim Wipes

(Kimberly-Clark, Roswell, GA). The pH of the ASW was carefully measured. Trizma base (Fisher

Scientific) was added a few granules at a time, then stirred for five minutes and measured the pH again. This step was repeated until the pH reached 8.0. The pH electrode was rinsed, wiped and returned to the buffer solution between reading pH measurements. The ASW Erlenmeyer flask (Pyrex, USA) at pH 8.0 was sealed with Para-film M (American Can. Chicago, IL), labeled, dated and maintained at 16 C. ASW could be used for up to two weeks after preparation.

Low calcium artificial sea water (LCASW)

LCASW was prepared utilizing the ASW formula 130 of the Marine Biological Laboratory

(Bidwell and Spotte, 1985). One half liter of LCASW was prepared using double distilled

Arrowhead Mountain Spring Water was placed into a 1L jar (Pyrex, USA). The jar was placed on a Fisher Scientific magnetic stirrer and a Fisher Scientific stir bar was placed inside the flask. The initial chemical amounts of 12.36g NaCl, 0.335g KCl, 0.0565g CaCl2*2H2O, 2.33g MgCl2*6H2O,

3.145g MgSO4*7H2O were carefully measured on a digital, analytical scale and added to the water filled jar. These chemical ingredients were stirred for 15 minutes at 300 rpm until they were completely dissolved. After these chemical ingredients were completely dissolved, 0.09g of NaHCO3 was carefully weighed a digital, analytical scale and added to the solution until it was fully dissolved. A Beckham pH meter was used to determine the initial pH of the LCASW.

The standard pH was tested by using a pH 7 buffer solution. The pH electrode was rinsed with

16 double distilled Arrowhead water and wiped with Kim Wipes (Kimberly-Clark, Roswell, GA). The pH of the LCASW was carefully measured. Trizma base (Fisher Scientific) was added a few granules at a time, then stirred for five minutes and measured the pH again. This step was repeated until the pH reached 8.0. The pH electrode was rinsed, wiped and returned to the buffer solution between reading measurements. The LCASW jar (Pyrex, USA) at pH 8.0 was sealed with Para-film M (American Can. Chicago, IL), labeled, dated and maintained at 16 C.

LCASW could be used for up to two weeks after preparation.

Potassium chloride (KCl)

The 0.55M KCl was prepared using 8.2005 g of KCl in 200mL of dd water. The potassium chloride was incubated at a temperature of 16 C and used for up to two weeks after preparation.

Formaldehyde

Ten percent formaldehyde was prepared by adding 10.0 mL of 37% formaldehyde to 18.4 mL of freshly prepared ASW at pH 8.0. The 50 mL Falcon tube was covered with aluminum foil to prevent photo-oxidation of the formaldehyde solution. The formaldehyde solution was used to stop and fix the morphological development of the sea urchin embryos,

The freshly prepared formaldehyde was stored under the fume hood for up to two weeks.

17 Sugar Alcohols

The sugar alcohols were stored at room temperature until they were prepared for experimental usage. Fifty milliliter Falcon tubes (Polystyrene Conical Tube (30 x 115mm) style,

Becton Dickinson, Franklin Lakes, New Jersey) were used to prepare 40mL of 0.1M stock solutions for each sugar alcohol using LCASW. Sugar alcohols were precisely weighed using a digital, analytical scale. Sugar alcohol stock solutions were serially diluted using LCASW. Ten mililiters of LCASW was pipetted into the 50mL Falcon tube and ten mililiters of the 0.1M sugar alcohol stock solution was pipetted into the 50mL Falcon tube to produce 0.05M sugar alcohol solution. This Falcon tube was vortexed, dated and labeled. Twenty-seven mililiters of the

LCASW was pipetted into a different 50mL Falcon tube and three mililiters of 0.1M sugar alcohol stock solution was pipetted into the 50mL Falcon tube to produce 0.01M sugar alcohol solution. This Falcon tube was vortexed, dated and labeled. Twenty-seven mililiters of the

LCASW was pipetted into another 50mL Falcon tube and three mililiters of the 0.05M sugar alcohol solution was pipetted into the 50mL Falcon tube to produce 0.005M sugar alcohol solution. This Falcon tube was vortexed, dated and labeled. Twenty-seven mililiters of LCASW was pipetted into a new 50mL Falcon tube and three mililiters of 0.01M sugar alcohol solution was pipetted into the Falcon tube to produce 0.001M sugar alcohol solution. This Falcon tube was vortexed, dated and labeled. This protocol was repeated for each sugar alcohol. All stock solutions of sugar alcohols and serial diluted sugar alcohols were incubated at 16 C for up to one week after preparation.

18 Sugar Alcohol Catalog # Batch #

Adonitol A5502 117K0760

L (-) Arabitol A3506 067K1920

Dulcitol D0256 116K0096

D – Mannitol M9546 057K00042

D – Sorbitol S1876 047K0025

Xylitol X3375 087K0212

Photographs

The Olympus CR2 light microscope (Japan) and the Kodak Easy Share Z 1285 digital camera were used together to take photographs of fixed control embryos and fixed experimental embryos. The photographs were stored on SD cards which could be uploaded to a computer.

Spawning of gametes

Lytechinus pictus sea urchins were randomly selected from the aquariums and placed in plastic tubs lined with paper towels soaked with ASW. Each sea urchin was intracoelomically injected near the mouth region with 3mL of 0.55M KCl using a 5mL syringe in order to induce therelease of gametes. The sucking mouth is located on the underside of the sea urchin.

19 Gametes were released 1-3 minutes after KCl was injected into the sea urchins. The sea urchins

released their gametes from the gonoducts which are located on top of the sea urchin. Sea

urchins which released yellow eggs were female. Sea urchins which released white sperm were

males

Collection and viability of eggs

ASW at pH 8.0 was poured into a 100mL beaker (Pyrex, USA) which was then covered with

Para-film M (American National Can. Chicago, IL). A small central opening was made in the

Para-film M and the female sea urchin was placed securely upside down and partially submerged in the ASW while the female sea urchin continued to release its eggs in to the beaker. The eggs were incubated for about 20 minutes at 16 C while they released their eggs.

Once the sea urchin finished releasing its eggs, an egg sample was taken from each female sea urchin using a plastic transfer pipette (Fisher Scientific, Mexico) and placed on a Petri dish

(Falcon Petri Dish, 60 x 15mm) to test for viability. All egg samples were observed under a

Fisher Scientific microscope. Viable egg morphology was characterized as being fully round and having a pronucleus present. Batches of eggs that were damaged, lysed or not completely round were discarded. All batches of viable eggs from the sea urchins were transferred and combined into a larger beaker filled with ASW. These viable eggs were incubated at 16 C until they settled to the bottom of the beaker.

20 Egg Washing

Once the combined viable eggs settled to the bottom of a large beaker, the

ASW was aspirated to remove contaminants and debris without disturbing the eggs. Fresh ASW was slowly poured and the eggs were incubated at 16 C and resettled. The process of egg washing was repeated three times.

Collection and viability of sperm

Once the male sea urchins began to release their semen, they were placed upside down in a

Petri dish over a small tub of ice until the sea urchin released all of its sperm. The semen remained undiluted until fertilization. The semen were collected from the gonoducts of the male sea urchin and from the surface of the Petri dish, then transferred to a 1.5mL micro- centrifuge tube and stored on ice.. Viability of the sperm was tested by using a toothpick (Diamond flat, Muncie Indiana) sample of sperm on a slide and diluting the sperm with one drop of ASW. The sperm were observed under a Fisher Scientific compound microscope for viability and motility. Sperm were determined to be viable when at least 90% of the sperm were motile.

Fertilization Sample

A small amount of eggs and sperm were combined into a Petri dish (Falcon Petri Dish, 60mm x

15mm) test for fertilization. The eggs were transferred to the Petri dish via transfer pipette.

21 (Fisher Scientific, Mexico) The sperm was then added and mixed in via a toothpick. Fertilization was observed under a Fisher Scientific compound microscope. A fertilization membrane in over

90% of the sample characterized a successful fertilization.

Fertilization

A 1000mL graduated cylinder was filled with 990mL of ASW. The 1.5mL micro-centrifuge tube containing the semen was diluted with ASW using a plastic transfer pipette. The diluted semen was placed in the 1000mL graduated cylinder containing the 990mL of ASW. ASW was added to make a 1L solution of sperm. Para-film M was used to cover the top of the 1L graduated cylinder. The 1L graduated cylinder was gently shaken back and forth until there was a homogeneous sperm solution. Two hundred mililiters of ASW was slowly poured into the

250mL beaker with the washed eggs. The washed eggs were now dispersed homogeneously in the ASW which allowed the sperm to fertilize more eggs. Ten milliliters of the sperm solution were pipetted into the beaker containing the dispersed eggs. The date and time of fertilization was recorded and labeled on the beaker containing the fertilized eggs. Three minutes after fertilization, some fertilized eggs were observed under a light microscope for the fertilization envelope. The fertilized eggs settled. The ASW was aspirated from the settled eggs using a glass pipette to remove any denatured sperm and other contaminants. The embryo washing and settling occurred three times. The settling of the embryos took 20 minutes. A Pyrex lasagna dish was rinsed with ASW at pH 8.0. Five hundred mililiters of ASW at pH 8.0 was added to the Pyrex lasagna dish. The washed and settled embryos in 200mL of ASW were added to the 500mL of

ASW in the lasagna dish. Aluminum foil

22 was dated, labeled and loosely covered the Pyrex lasagna dish containing the embryos. The loosely covered lasagna dish was placed in the incubator at 16 C for 24 hours. The embryos were treated at 24 hours and then returned to the incubator until the embryos were treated at

32 hours.

Microwell Plate Assay

The 96 microwell plates (Polystyrene Microtest Flat Bottom with Low Evaporation Lid, Becton

Dickinson, Franklin Lakes, New Jersey) were rinsed with Arrowhead double distilled water, shaken, and allowed to air dry before plating with the sea urchin embryos. At 24 hours after fertilization, a small sample of swimming sea urchin blastula stage embryos were observed under a Fisher Scientific compound microscope to verify the viability of the sea urchin embryos.

A plastic pipette (Fisher Scientific, Mexico) was cut and used to transfer 40-50mL of the

swimming sea urchin embryos to a 100mL beaker. (Pyrex, USA) Three separate microwell tests were made by pipetting 25 microliters of the swimming sea urchin embryos, 50 microliters of

LCASW, and 10 microliters of 10% formaldehyde into each microwell. The sea urchin embryos were fixed and counted from each microwell using a Fisher Scientific compound microscope and then averaged. The ideal range of sea urchin embryos per microwell was 10-40. If the average number of sea urchin embryos per microwell was greater than 40 sea urchin embryos, then the 40-50mL of swimming sea urchin embryos would be diluted with ASW. The volume of

ASW depended on the average number of sea urchin embryos per microwell. The volume of

ASW ranged from 10-30mL. The microwell tests would be repeated until the average number of sea urchin embryos was between 10-40 sea urchin embryos. Three microwell plates were used

23 for both 24-26 hour sea urchin embryo treatments and 32-34 hour sea urchin treatments. Once the swimming blastula sea urchin embryos were diluted, fifty microliters of LCASW and twenty- five microliters of L. pictus embryos in ASW were pipetted into the first two rows of 12 microwells as the controls. In rows 3-5 of the first microwell plate, fifty microliters of 0.1M sugar alcohol and twenty-five microliters of L. pictus in ASW were pipetted into 36 microwells.

In rows 6-8 of the first microwell plate, fifty microliters of 0.05M sugar alcohol and twenty-five microliters of L. pictus in ASW were pipetted into 36 microwells. This protocol was repeated for the second microplate except the concentration for rows 3-5 was 0.01M sugar alcohol and rows

6-8 was 0.005M. This protocol was also repeated for the third microwell plate except the concentration for rows 3-5 was 0.001M sugar alcohol. The micro assay plates were dated, labeled, and loosely overlayed with aluminum foil and positioned in the incubator at 16 C.

Embryos at forty-eight hours after fertilization in the control wells were observed under the

Fisher Scientific compound microscope for the formation of complete archenterons. When the sea urchin embryo control wells had a vast majority of complete archenterons, all the control embryos and experimental embryos were fixed by pipetting 10 microliters of 10% formaldehyde solution into each well. The embryos were fixed between 48-50 hours after fertilization. Morphological characteristics of each embryo such as complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated, (EX), and dead (D) in each well were recorded and photographed.

24 Statistical Analysis

Counts of all sea urchin morphologies for all trials for each concentration of each sugar alcohol were added together to give a total sample size. Counts of all sea urchin morphologies for all control (LCASW) trials from each sugar alcohol were added together to give a total sample size.

Sea urchin morphologies included: complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead (D).The percentages of each morphological type were calculated. The unpaired t-test was utilized to contrast the sample means between each morphological type in the control (LCASW) and each morphological type of each sugar alcohol concentration. P-values below 0.05 were statistically significant.

25 Results

In this study, a total of 6 free sugar alcohols in low calcium seawater were tested on 24-26 hour and 32-34 hour Lytechinus pictus sea urchin embryos to establish the effects on morphology during gastrulation. The sugar alcohols were analyzed in order to determine their effects on the formation of the archenteron in sea urchin gastrulas. The sugar alcohol concentrations were 0.1M, 0.05M, 0.01M, 0.005M, and 0.001M. The sugar alcohols were introduced to the embryos at the 24-26 hour blastula stage or the 32-34 hour gastrula stage. The Olympus CR2 light microscope (Japan) was used to make observations at the 24-26 hour blastula stage, the 32-34 hour gastrula stage, and at the time of fixation which was 48-50 hours. A 10.0 percent formaldehyde solution was used to fix the Lytechinus pictus embryos at the 48-50 hour gastrula stage. All tables, graphs, and figures contain data that were obtained from fixed embryos at the 48-50 hour gastrula stage. All the morphological characteristics were observed, noted, counted and photographed at the 48-50 hour gastrula stage. Each morphology was characterized as having a complete archenteron (CA), incomplete archenteron

(IA), not invaginated (NI), exogastrulated (EX) or dead (D). One set of photographs shows each

L. pictus sea urchin morphological type including: complete archenteron, incomplete archenteron, not invaginated, exogastrulated and dead. (Figure 1) There are six sets of photographs showing L. pictus sea urchin embryos at 24-26 hours after fertilization in LCASW controls and in 0.1M, 0.05M, 0.01M, 0.005M and 0.001M for adonitol, L-(-) arabitol, dulcitol, D- mannnitol, D-sorbitol and xylitol. (Figures 2, 4, 6, 8, 10 and 12) There are six sets of photographs showing L. pictus sea urchin embryos at 32-34 hours after fertilization in LCASW

26 controls and in 0.1M, 0.05M, 0.01M, 0.005M and 0.001M for adonitol, L-(-) arabitol, dulcitol, D- mannitol, D-sorbitol and xylitol. (Figures 3, 5, 7, 9, 11 and 13) The embryo counts from each sugar alcohol concentration and the low calcium artificial sea water controls were added together to give a total sample size. Each morphology percentage was calculated for each sugar alcohol concentration and low calcium artificial sea water controls. Unpaired t-tests were used to compare the sample means of each sugar alcohol concentration to those of the low calcium artificial sea water controls. P-values in which p < 0.05 were statistically significant.

Photographs were taken utilizing a Kodak Easy Share Z 1285 digital camera after the sea urchin embryos were fixed at the 48-50 hour gastrula stage.

Adonitol treatments in Lytechinus pictus embryos

Table 1 and Table 2 shows the p-values, and Table 13 and Table 14 shows the morphology percents for the adonitol treatments at 24-26 hour blastula stage and 32-34 hour gastrula stage at concentrations 0.1M, 0.05M, 0.01M, 0.005M, and 0.001M, and complete archenteron, incomplete archenteron, not invaginated, exogastrulated, and dead morphologies.

The p-values for adonitol were statistically significant at the 24-26 hour treatments and the

32-34 hour treatments compared to the low calcium artificial sea water controls. There were significant p-values for the 24-26 hour adonitol treatments for the complete archenteron morphology at concentration 0.1M and 0.005M. There were no significant p-values for the 24-

26 hour adonitol treatments for the incomplete archenteron morphology. The p-values were significant for the 24-26 hour adonitol treatments for the not invaginated morphology at

27 concentrations 0.05M, 0.01M and 0.005M. The p-values were significant for the exogastrulated morphology at concentrations 0.1M, 0.05M, 0.01M and 0.001M. The dead morphology had significant p-values at concentrations 0.05M, 0.01M, 0.005M and 0.001M. The p-values were not significant for the 32-34 hour adonitol treatments for the complete archenteron morphology for any of the concentrations.The p-values were significant for the 32-34 hour adonitol treatments for the incomplete archenteron morphology at concentrations 0.05M,

0.01M and 0.005M. The p-values were significant for the 32-34 hour adonitol treatments for the not invaginated morphology at concentration 0.001M. The p-values were not significant for the 32-34 hour adonitol treatments for the exogastrulated morphology for any of the concentrations. The p-values were significant for the 32-34 hour adonitol treatments for the dead morphology at concentration 0.1M.

The greatest effect on archenteron binding to the blastocoel roof during the 24-26 hour adonitol treatment came from the exogastrulated and dead morphologies. The least effect on archenteron binding to the blastocoel roof during the 24-26 hour adonitol treatment came from the incomplete archenteron morphology. The greatest effect on archenteron binding to the blastocoel roof during the 32-34 hour adonitol treatment came from the incomplete archenteron morphology. The least effect on archenteron binding to the blastocoel roof during the 32-34 hour adonitol treatment came from the complete archenteron and exogastrulated morphologies. The 24-26 hour adonitol treatment had a greater overall effect than the 32-34 hour adonitol treatment.

28 L-(-) Arabitol treatments in Lytechinus pictus embryos

Table 3 and Table 4 shows the p-values, and Table 15 and Table 16 shows morphology percents for the L-(-) arabitol treatments at the 24-26 hour blastula stage and the 32-34 hour gastrula stage at concentrations 0.1M, 0.05M, 0.01M, 0.005M, and 0.001M, and complete archenteron, incomplete archenteron, exogatrulated, and dead morphologies.

The p-values for L-(-) arabitol were statistically significant for the 24-26 hour compared to the low calcium artificial sea water controls. There was only one significant p-value for the 24-

26 hour L-(-) arabitol treatments for the complete archenteron morphology at concentration

0.05M. The p-values were significant for the 24-26 hour L-(-) arabitol treatments for the incomplete archenteron morphology at concentrations 0.05M, 0.01M, 0.005M and 0.001M.

The p-values were significant for the 24-26 hour L-(-) arabitol treatments for not invaginated morphology at concentrations 0.1M, 0.005M and 0.001M. The p-values were significant at the

24-26 hour L-(-) arabitol treatments for the exogastrulated morphology at concentrations of

0.1M and 0.05M. The dead morphology had significant p-values for the 24-26 hour treatments at concentrations 0.005M and 0.001M. There was only two significant p-values during the 32-

34 hour L-(-) arabitol treatments for the incomplete archenteron morphology at concentration

0.05M and not invaginated morphology at concentration 0.005M.

The greatest effect on archenteron binding to the blastocoel roof during the 24-26 hour

L-(-) arabitol treatment came from the incomplete archenteron and not invaginated morphologies. The least effect on archenteron binding to the blastocoel roof during the 24-

26 hour L-(-) arabitol came from the complete archenteron, exogastrulated and dead

29 morphologies. There was almost no effect by L-(-) arabitol during the 32-34 hour treatments except for the incomplete archenteron morphology at concentration 0.05M and the not invaginated morphology at concentration 0.005M. The 24-26 hour L-(-) arabitol treatment had a greater overall effect than the 32-34 hour L-(-) arabitol treatment.

Dulcitol treatments in Lytechinus pictus embryos

Table 5 and Table 6 shows the p-values, and Table 17 and Table 18 shows morphology percents of dulcitol treatments at the 24-26 hour blastula stage and the 32-34 hour gastrula stage at 0.1M, 0.05M, 0.01M, 0.005M, and 0.001M, and complete archenteron, incomplete archenteron, not invaginated, exogastrulated, and dead morphologies.

The p-values for dulcitol were statistically significant for the 24-26 hour treatments and the

32-34 hour treatments compared to the low calcium artificial sea water controls. The p-values were not significant for the 24-26 hour dulcitol treatments for the complete archenteron morphology for any concentrations. There was one significant p-value for the 24-26 hour dulcitol treatments for the incomplete archenteron morphology at concentration 0.001M. The p-values were significant for the 24-26 hour dulcitol treatments for the not invaginated morphology at concentrations 0.1M and 0.005M. There was one significant p-value for the 24-

26 hour dulcitol treatments for the exogastrulated morphology at concentration 0.005M. There were no significant p-values for the 24-26 hour dulcitol treatments for the dead morphology at any concentration level. There was one significant p-value for the 32-34 hour dulcitol treatments for the complete archenteron morphology at concentration 0.01M. The p-values

30 were significant for the 32-34 hour dulcitol treatments for the incomplete archenteron morphology at 0.1M, 0.05M, 0.01M and 0.005M. The p-values were significant for the 32-34 hour dulcitol treatments for the not invaginated morphology at concentrations 0.05M, 0.01M,

0.005M, and 0.001M. There was one p-value that was significant for the 32-34 hour dulcitol treatments for the exogastrulated morphology at concentration 0.005M. There were no significant p-values for the 32-34 hour dulcitol treatments for the dead morphology at any concentration level.

The greatest effect on archenteron binding to the blastocoel roof during the 24-26 hour dulcitol treatments came from the not invaginated morphology. The least effect on archenteron binding to the blastocoel roof during the 24-26 hour dulcitol treatments came from the complete archenteron morphology. The greatest effect on archenteron binding to the blastocoel roof during the 32-34 hour dulcitol treatments came from the incomplete archenteron and the not invaginated morphologies. The least effect on archenteron binding to the blastocoel roof during the 32-34 hour dulcitol treatments came from the dead morphology.

The 32-34 hour dulcitol treatment had a greater overall effect than the 24-26 hour dulcitol treatment.

D-Mannitol treatments in Lytechinus pictus embryos

Table 7 and Table 8 shows the p-values, and Table 19 and Table 20 shows morphology percents of D-mannitol treatments at the 24-26 hour blastula stage and the 32-34 hour gastrula stage at concentrations 0.1M, 0.05M, 0.01M, 0.005M, and 0.001M, and complete archenteron,

31 incomplete archenteron, not invaginated, exogastrulated, and dead morphologies.

The p-values for D-mannitol were statistically significant for the 24-26 hour treatments and the 32-34 hour treatments compared to the low calcium artificial sea water controls. There was only one significant p-value during the 24-26 hour D-mannitol treatments for the complete archenteron morphology at concentration 0.05M. There were no significant p-values during the

24-26 hour D-mannitol treatments for the incomplete archenteron, not invaginated, and dead morphologies for any concentrations. There were two significant p-values during the 24-26 hour D-mannitol treatments for the exogastrulated morphology at concentrations 0.1M and

0.05M. The p-values were significant for the 32-34 hour D-mannitol treatments for the complete archenteron morphology at concentrations 0.1M, 0.05M, and 0.005M. The p-values were significant for the 32-34 hour D-mannitol treatments for the incomplete archenteron morphology at molarities 0.1M, 0.01M, 0.005M, and 0.001M. There was one significant p- value for the not invaginated morphology at 0.01M. There were no significant p-values for the exogastrulated morphology. There were three significant p-values for the 32-34 hour D- mannitol treatments for the dead morphology at concentrations 0.05M, 0.01M and 0.001M.

The greatest effect on archenteron binding to the blastocoel roof during the 24-26 hour

D-mannitol treatment came from the exogastrulated morphology. The least effect on archenteron binding to the blastocoel roof during the 24-26 hour D-mannitol treatment came from the incomplete archenteron, not invaginated, and dead morphologies. The greatest effect on archenteron binding to the blastocoel roof during the 32-34 hour D-mannitol treatment came from the incomplete archenteron morphology. The least effect on archenteron binding to the blastocoel roof during the 32-34 hour D-mannitol treatment came from the

32 exogastrulated morphology. The 32-34 hour D-mannitol treatment had a greater overall effect than the 24-26 hour D-mannitol treatment.

D-Sorbitol treatments in Lytechinus pictus embryos

Table 9 and Table 10 shows the p-values, and Table 21 and Table 22 shows morphology percents of the D-sorbitol treatments at the 24-26 hour blastula stage and the 32-34 hour gastrula stage at concentrations 0.1M, 0.05M, 0.01M, 0.005M, and 0.001M, and complete archenteron, incomplete archenteron, not invaginated, exogastrulated, and dead morphologies.

The p-values for D-sorbitol were statistically significant for the 24-26 hour treatments and the 32-34 hour treatments compared to the low calcium artificial sea water controls. The p- values were significant at the 24-26 hour D-sorbitol treatments for the complete archenteron morphology at 0.1M, 0.05M, 0.005M and 0.001M. There were no significant p-values at the 24-

26 hour D-sorbitol treatments for the incomplete archenteron, not invaginated, exogastrulated, and dead morphologies at any of the concentration levels. The p-values were not significant at the 32-34 hour D-sorbitol treatments for the complete archenteron, incomplete archenteron, and not invaginated morphologies at all five concentration levels. There was one significant p- value at the 32-34 hour D-sorbitol for the exogastrulated morphology at concentration 0.005M.

There was one significant p-value at the 32-34 hour D-sorbitol for the dead morphology at concentration 0.01M.

The greatest effect on archenteron binding to the blastocoel roof during the 24-26 hour D-

33 sorbitol treatment came from the complete archenteron morphology. The least effect on archenteron binding to the blastocoel roof during the 24-26 hour D-sorbitol treatment came from the incomplete archenteron, not invaginated, exogastrulated, and dead morphologies.

The greatest effect on archenteron binding to the blastocoel roof during the 32-34 hour D- sorbitol treatment came from the exogastrulated and dead morphologies. The least effect on archenteron binding to the blastocoel roof during the 32-34 hour D-sorbitol treatment came from the complete archenteron, incomplete archenteron, and not invaginated morphologies.

The 24-26 hour D-sorbitol treatment had a greater overall effect than the 32-34 hour D-sorbitol treatment.

Xylitol treatments in Lytechinus pictus embryos

Table 11 and Table 12 shows the p-values, and Table 23 and 24 showed morphology percents of the xylitol treatments at the 24-26 hour blastula stage and the 32-34 hour gastrula stage at concentrations 0.1M, 0.05M, 0.01M, 0.005M, and 0.001M, and complete archenteron, incomplete archenteron, not invaginated, exogastrulated, and dead morphologies.

The p-values for xylitol were statistically significant for the 24-26 hour treatments and the

32-34 hour treatments compared to the low calcium artificial sea water controls. The p-values were significant for the 24-26 hour xylitol treatments for the complete archenteron morphology at molarities 0.05M, 0.01M, 0.005M and 0.001M, and for the incomplete archenteron morphology at concentration 0.005M. The p-values were significant for the 24-26 hour treatments for the not invaginated morphology at concentrations 0.01M and 0.001M. The

34 p-values were not significant for the exogatrulated morphology at any of the concentrations.

The p-values were significant for the 24-26 hour xylitol treatments for the dead morphology at concentrations 0.1M, 0.01M and 0.001M. The p-values were significant for the 32-34 hour xylitol treatments for the complete archenteron morphology at concentrations 0.005M and

0.001M. There was one significant p-value for the incomplete archenteron morphology at concentration 0.005M. There were no significant p-values at the 32-34 hour xylitol treatments for the not invaginated and exogastrulated morphologies at any of the concentration levels.

There were two significant p-values for the 32-34 hour xylitol treatments for the dead morphology at concentrations 0.05M and 0.005M.

The greatest effect on archenteron binding to the blastocoel roof during the 24-26 hour xylitol treatment came from the complete archenteron morphology. The least effect on archenteron binding to the blastocoel roof during the 24-26 hour xylitol treatment came from the exogastrulated morphology. The greatest effect on archenteron binding to the blastocoel roof during the 32-34 hour xylitol treatment came from the complete archenteron and dead morphologies. The least effect on archenteron binding to the blastocoel roof during the 32-34 hour xylitol treatment came from the not invaginated and exogastrulated morphologies. The 24-26 hour xylitol treatment had a greater overall effect than the 32-34 hour xylitol treatment.

35

48

L. pictus Embryos Incubated in Adonitol and Low Ca+2 Artificial Sea Water at the 24-26 Hour stage of Gastrulation

* **

*** * ***

*** ** ** * *

* * * *

Figure 14. Observed percentages of complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead (D) L. pictus embryos at the 24-26 hour stage of gastrulation. Embryos were incubated with different concentrations of adonitol solutions in low calcium artificial sea water. Embryos incubated in low calcium artificial sea water were used as the controls. Error bars were based on standard error. The p-values for the unpaired t-tests of p < 0.05 were marked with (*), p < 0.01 were marked with (**), and p < 0.001 were marked with (***) to indicate that the data from adonitol were significant compared to the low calcium controls.

49 L. pictus Embryos Incubated in Adonitol and Low Ca+2 Artificial Sea Water at the 32-34 Hour Stage of Gastrulation

*** ** ***

*

*

Figure 15. Observed percentages of complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead (D) L. pictus embryos at the 32-34 hour stage of gastrulation. Embryos were incubated with different concentrations of adonitol solutions in low calcium artificial sea water. Embryos incubated in low calcium artificial sea water were used as the controls. Error bars were based on standard error. The p-values for the unpaired t-tests of p < 0.05 were marked with (*), p < 0.01 were marked with (**), and p < 0.001 were marked with (***) to indicate that the data from adonitol were significant compared to the low calcium controls.

50

L. pictus Embryos Incubated in L-(-) Arabitol and Low Ca+2 Artificial

Sea Water at the 24-26 Hour Stage of Gastrulation

**

*** ** *** ***

*** ** **

** ***

* *

Figure 16. Observed percentages of complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead (D) L. pictus embryos at the 24-26 hour stage of gastrulation. Embryos were incubated with different concentrations of L-(-) arabitol solutions in low calcium artificial sea water. Embryos incubated in low calcium artificial sea water were used as the controls. Error bars were based on standard error. The p-values for the unpairedt- tests of p < 0.05 were marked with (*), p < 0.01 were marked with (**), and p < 0.001 were marked with (***) to indicate that the data from L-(-) arabitol were significant compared to the low calcium controls.

51

L. pictus Embryos Incubated in L-(-) Arabitol and Low Ca+2 Artificial

Sea Water at the 32-34 Hour Stage of Gastrulation

* *

Figure 17. Observed percentages of complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead (D) L. pictus embryos at the 32-34 hour stage of gastrulation. Embryos were incubated with different concentrations of L-(-) arabitol solutions in low calcium artificial sea water. Embryos incubated in low calcium artificial sea water were used as the controls. Error bars were based on standard error. The p-values for the unpaired t- tests of p < 0.05 were marked with (*), p < 0.01 were marked with (**), and p < 0.001 were marked with (***) to indicate that the data from L-(-) arabitol was significant compared to the low calcium controls.

52 L. pictus Embryos Incubated in Dulcitol and Low Ca+2 Artificial

Sea Water at the 24-26 Hour Stage of Gastrulation

*

* *

** ** *

Figure 18. Observed percentages of complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead (D) L. pictus embryos at the 24-26 hour stage of gastrulation. Embryos were incubated with different concentrations of dulcitol solutions in low calcium artificial sea water.Embryos incubated in low calcium artificial sea water were used as the controls. Error bars were based on standard error. The p-values for the unpaired t-tests of p < 0.05 were marked with (*), p < 0.01 were marked with (**), and p < 0.001 were marked with (***) to indicate that the data from dulcitol was significant compared to the low calcium controls.

53 L. pictus Embryos Incubated in Dulcitol and Low Ca+2 Artificial

Sea Water at the 32-34 Hour Stage of Gastrulation

**

** ** ** ***

*** *** *** *

*

Figure 19. Observed percentages of complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogatrulated (EX) and dead (D) L. pictus embryos at the 32-34 hour stage of gastrulation. Embryos were incubated with different concentrations of dulcitol solutions in low calcium artificial sea water. Embryos incubated in low calcium artificial sea water were used as the controls. Error bars were based on standard error. The p-values for the unpaired t-tests of p< 0.05 were marked with (*), p < 0.01 were marked with (**), and p < 0.001 were marked with (***) to indicate that the data from dulcitol was significant compared to the low calcium controls.

54 L. pictus Embryos Incubated in D-Mannitol and Low Ca+2 Artificial

Sea Water at the 24-26 Hour Stage of Gastrulation

*** *

* *

Figure 20. Observed percentages of complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead (D) L. pictus embryos at the 24-26 hour stage of gastrulation. Embryos were incubated with different concentrations of D-mannitol solutions in low calcium artificial sea water. Embryos incubated in low calcium artificial sea water used as the controls. Error bars were based on standard error. The p-values for the unpaired t-tests of p < 0.05 were marked with (*), p < 0.01 were marked with (**), and p < 0.001 were marked with (***) to indicate that the data from D-mannitol was significant compared to the low calcium controls.

55 L. pictus Embryos Incubated in D-Mannitol and Low Ca+2 Artificial

Sea Water at the 32-34 Hour Stage of Gastrulation

** ** *

** ** *** ***

*

*** ** *

Figure 21. Observed percentages of complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead (D) L. pictus embryos at the 32-34 hour stage of gastrulation. Embryos were incubated with different concentrations of D-mannitol solutions in low calcium artificial sea water. Embryos incubated in low calcium artificial sea water were used as the controls. Error bars were based on standard error. The p-values for the unpaired t- tests of p < 0.05 were marked with (*), p < 0.01 were marked with (**), and p < 0.001 were marked with (***) to indicate that the data from D-mannitol was significant compared to the low calcium controls.

56 L. pictus Embryos Incubated in D-Sorbitol and Low Ca+2 Artificial

Sea Water at the 24-26 Hour of Gastrulation

*** ** ** ***

Figure 22. Observed percentages of complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead (D) L. pictus embryos at the 24-26 hour stage of gastrulation. Embryos were incubated with different concentrations of D-sorbitol solutions in low calcium artificial sea water. Embryos incubated in low calcium artificial sea water were used as the controls. Error bars were based on standard error. The p-values for the unpaired t-tests of p < 0.05 were marked with (*), p < 0.01 were marked with (**), and p < 0.001 were marked with (***) to indicate that the data from D-sorbitol was significant compared to the low calcium controls.

57 L. pictus Embryos incubated in D-Sorbitol and Low Ca+2 Artificial

Sea Water at the 32-34 Hour Stage of Gastrulation

*

*

Figure 23. Observed percentages of complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead (D) L. pictus embryos at the 32-34 hour stage of gastrulation. Embryos were incubated with different concentrations of D-sorbitol solutions in low calcium artificial sea water. Embryos incubated in low calcium artificial sea water were used as the controls. Error bars were based on standard error. The p-values for the unpaired t-tests of p < 0.05 were marked with (*), p < 0.01 were marked with (**), and p < 0.001 were marked with (***) to indicate that the data from D-sorbitol was significant compared to the low calcium controls.

58 L. pictus Embryos Incubated in Xylitol and Low Ca+2 Artificial

Sea Water at the 24-26 Hour Stage of Gastrulation

*** * *** ***

* * *

* **

**

Figure 24. Observed percentages of complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead (D) L. pictus embryos at the 24-26 hour stage of gastrulation. Embryos were incubated with different concentrations of xylitol solutions in low calcium artificial sea water. Embryos incubated in low calcium artificial sea water used as the controls. Error bars were based on standard error. The p-values for the unpaired t-tests of p < 0.05 were marked with (*), p < 0.01 were marked with (**), and p < 0.001 were marked with (***) to indicate that the data from xylitol was significant compared to the low calcium controls.

59 L. pictus Embryos Incubated in Xylitol and Low Ca+2 Artificial

Sea Water at the 32-34 Hour Stage of Gastrulation

* **

**

** *

Figure 25. Observed percentages of complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead (D) L. pictus embryos at the 32-34 hour stage of gastrulation. Embryos were incubated with different concentrations of xylitol solutions in low calcium artificial sea water. Embryos incubated in low calcium artificial sea water were used as the controls. Error bars are based on standard error. The p-values for the unpaired t-tests of p < 0.05 were marked with (*), p < 0.01 were marked with (**), and p < 0.001 were marked with (***) to indicate that the data from xylitol was significant compared to the low calcium controls.

60

Adonitol Complete Incomplete Not Exogastrulated Dead Concentration Archenteron Archenteron Invaginated

0.1M 0.039642 0.191786 0.050535 9.65E-05 0.065945

0.05M 0.10111 0.064629 3.21E-06 0.002623 0.019809

0.01M 0.440363 0.541686 0.021503 0.003245 0.021605

0.005M 0.007796 0.296481 0.000311 0.138362 0.013692

0.001M 0.160716 0.246537 0.454114 0.039215 0.04233

Table 1. Adonitol p-value results at 24-26 hours after fertilization for complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead (D) morphologies for 0.1M, 0.05M, 0.01M, 0.005M and 0.001M compared to the low calcium artificial sea water (LCASW) control. P-values less than 0.05 were significant and shown in bold print.

61 Adonitol Complete Incomplete Not Exogastrulated Dead Concentration Archenteron Archenteron Invaginated

0.1M 0.343573 0.070242 0.074169 0.346724 0.038835

0.05M 0.608376 8.49E-05 0.235284 0.783431 0.663292

0.01M 0.271262 0.001313 0.218815 0.757937 0.958859

0.005M 0.571834 1.95E-06 0.789863 0.302853 0.316773

0.001M 0.118905 0.700593 0.018829 0.894783 0.877335

Table 2. Adonitol p-value results at 32-34 hours after fertilization for complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead ( D) morphologies for 0.1M, 0.05M, 0.01M, 0.005M and 0.001M compared to the low calcium artificial sea water (LCASW) control. P-values less than 0.05 were significant and shown in bold print.

62 L-(-) Arabitol Complete Incomplete Not Exogastrulated Dead Concentration Archenteron Archenteron Invaginated

0.1M 0.074125 0.155507 2.29E-06 0.003125 0.140647

0.05M 0.002874 9.55E-05 0.121137 7.44E-05 0.707026

0.01M 0.1577 0.023861 0.055269 0.079072 0.933264

0.005M 0.877347 8.74E-10 0.016742 0.739474 0.012167

0.001M 0.877347 8.74E-10 0.016742 0.739474 0.012167

Table 3. L-(-) arabitol p-value results at 24-26 hours after fertilization for complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead (D) morphologies for 0.1M, 0.05M, 0.01M, 0.005M and 0.001M compared to the low calcium artificial sea water (LCASW) control. P-values less than 0.05 were significant and shown in bold print.

63 L-(-) Arabitol Complete Incomplete Not Exogastrulated Dead Concentration Archenteron Archenteron Invaginated

0.1M 0.544441 0.300925 0.553571 N/A 0.792897

0.05M 0.738497 0.040788 0.266014 0.325582 0.050901

0.01M 0.803715 0.36675 0.334462 N/A 0.627933

0.005M 0.384023 0.533765 0.036453 N/A 0.847313

0.001M 0.828698 0.245567 0.470742 N/A 0.315498

Table 4. L-(-) arabitol p-value results at 32-34 hours after fertilization for complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead (D) morphologies for 0.1M, 0.05M, 0.01M, 0.005M and 0.001M compared to the low calcium artificial sea water (LCASW) control. P-values less than 0.05 were significant and shown in bold print.

64 Dulcitol Complete Incomplete Not Exogastrulated Dead Concentration Archenteron Archenteron Invaginated

0.1M 0.765276 0.29262 0.040654 0.101458 0.366183

0.05M 0.542322 0.947479 0.088739 0.493441 0.025931

0.01M 0.72253 0.519718 0.08406 0.146727 0.072788

0.005M 0.290166 0.151847 0.029383 0.043265 0.468565

0.001M 0.636634 0.004963 0.243237 0.126251 0.128489

Table 5. Dulcitol p-value results at 24-26 hours after fertilization for complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead (D) morphologies for 0.1M, 0.05M, 0.01M, 0.005M and 0.001M compared to the low calcium artificial sea water (LCASW) control. P-values less than 0.05 were significant and shown in bold print.

65 Dulcitol Complete Incomplete Not Exogastrulated Dead Concentration Archenteron Archenteron Invaginated

0.1M 0.4177249 0.009731 0.125862 0.31941 0.370787

0.05M 0.747906 0.001961 1.32E-05 0.708666 0.708666

0.01M 0.006708 0.00921 2.16E-05 0.099577 0.09751

0.005M 0.330324 0.000247 0.000391 0.019399 0.099577

0.001M 0.699168 0.050288 0.022477 0.313224 0.313224

Table 6. Dulcitol p-value results at 32-34 hours after fertilization for complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead (D) morphologies for 0.1M, 0.05M, 0.01M, 0.005M and 0.001M compared to the low calcium artificial sea water (LCASW) control. P-values less than 0.05 were significant and shown in bold print.

66 D-Mannitol Complete Incomplete Not Exogastrulated Dead Concentration Archenteron Archenteron Invaginated

0.1M 0.928077 0.083233 0.796996 0.010395 0.605895

0.05M 0.000209 0.706924 0.640032 0.010395 0.30775

0.01M 0.703998 0.083233 0.968827 0.644383 0.834763

0.005M 0.356421 0.758266 0.282372 0.119266 0.416727

0.001M 0.232784 0.406484 0.558461 0.70933 0.188891

Table 7. D-mannitol p-value results at 24-26 hours after fertilization for complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead (D) morphologies for 0.1M, 0.05M, 0.01M, 0.005M and 0.001M compared to the low calcium artificial sea water (LCASW) control. P-values less than 0.05 were significant and shown in bold print.

67 D-Mannitol Complete Incomplete Not Exogastrulated Dead Concentration Archenteron Archenteron Invaginated

0.1M 0.005902 0.004113 0.794325 N/A 0.313892

0.05M 0.00169 0.262451 0.742077 N/A 0.000117

0.01M 0.989587 0.00414 0.017013 0.321854 0.008119

0.005M 0.045505 2.26E-08 0.384268 N/A 0.103569

0.001M 0.774809 0.000901 0.185029 N/A 0.025163

Table 8. D-mannitol p-value results at 32-34 hours after fertilization for complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead (D) morphologies for 0.1M, 0.05M, 0.01M, 0.005M and 0.001M compared to the low calcium artificial sea water (LCASW) control. P-values less than 0.05 were significant and shown in bold print.

68 D-Sorbitol Complete Incomplete Not Exogastrulated Dead Concentration Archenteron Archenteron Invaginated

0.1M 0.000275 N/A 0.148375 N/A N/A

0.05M 0.008082 0.324375 0.92323 N/A N/A

0.01M 0.32488 0.160909 0.551064 N/A N/A

0.005M 0.001009 N/A 0.290764 N/A N/A

0.001M 8.05E-05 N/A 0.915453 N/A N/A

Table 9. D-sorbitol p-value results at 24-26 hours after fertilization for complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead (D) morphologies for 0.1M, 0.05M, 0.01M, 0.005M and 0.001M compared to the low calcium artificial sea water (LCASW) control. P-values less than 0.05 were significant and shown in bold print.

69 D-Sorbitol Complete Incomplete Not Exogastrulated Dead Concentration Archenteron Archenteron Invaginated

0.1M 0.391517 0.489141 0.543439 0.058482 0.818108

0.05M 0.660394 0.752142 0.720219 0.238179 0.356875

0.01M 0.483011 0.275315 0.45365 0.679905 0.038436

0.005M 0.791334 0.560441 0.23216 0.017178 0.249723

0.001M 0.697901 0.515341 0.073882 0.054734 0.175179

Table 10. D-sorbitol p-value results at 32-34 hours after fertilization for complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead (D) morphologies for 0.1M, 0.05M, 0.01M, 0.005M and 0.001M compared to the low calcium artificial sea water (LCASW) control. P-values less than 0.05 were significant and shown in bold print.

70 Xylitol Complete Incomplete Not Exogastrulated Dead Concentration Archenteron Archenteron Invaginated

0.1M 0.194536 0.856311 0.105001 0.07339 0.012934

0.05M 3.71E-07 0.319978 0.03813 0.23599 0.39698

0.01M 0.042282 0.400736 0.017442 0.420104 0.019529

0.005M 4.13E-08 0.008806 0.113877 0.244505 0.624868

0.001M 1.01E-05 0.898655 0.001745 0.731879 0.012934

Table 11. Xylitol p-value results at 24-26 hours after fertilization for complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead (D) morphologies for 0.1M, 0.05M, 0.01M, 0.005M and 0.001M compared to the low calcium artificial sea water control (LCASW) control. P-values less than 0.05 were significant and shown in bold print.

71 Xylitol Complete Incomplete Not Exogastrulated Dead Concentration Archenteron Archenteron Invaginated

0.1M 0.282642 0.884125 0.627682 0.158788 0.614057

0.05M 0.192262 0.693057 0.905947 0.158788 0.007301

0.01M 0.811873 0.217851 0.061153 N/A 0.68043

0.005M 0.012504 0.003117 0.556655 0.320963 0.035556

0.001M 0.003388 0.683143 0.785554 N/A 0.248434

Table 12. Xylitol p-value results at 32-34 hours after fertilization for complete archenteron (CA), incomplete archenteron (IA), not invaginated (NI), exogastrulated (EX) and dead (D) morphologies for 0.1M, 0.05M, 0.01M, 0.005M and 0.001M compared to the low calcium artificial sea water (LCASW) control. P-values less than 0.05 were significant and shown in bold print.

72 Adonitol Complete Incomplete Not Exogastrulated Dead Sample Concentration Archenteron Archenteron invaginated Size

0.1M 88.44% 0.94% 8.67% 0.00% 1.95% 1488

0.05M 84.57% 1.11% 13.34% 0.17% 0.82% 1717

0.01M 86.81% 0.87% 11.00% 0.26% 1.05% 1145

0.005M 82.32% 1.19% 15.05% 0.80% 0.64% 1256

0.001M 87.14% 1.23% 9.05% 0.51% 2.06% 972

LCASW 83.53% 0.58% 6.82% 1.21% 7.85% 2229 Control

Table 13. Data at 24-26 hours after fertilization showing percents for each morphology and the total sample size for each of the five concentrations of adonitol in low calcium artificial sea water (LCASW) and LCASW controls.

73 Adonitol Complete Incomplete Not Exogastrulated Dead Sample Concentration Archenteron Archenteron Invaginated Size

0.1M 84.76% 1.65% 11.21% 0.24% 2.14% 2060

0.05M 83.78% 3.93% 11.09% 0.17% 1.04% 1732

0.01M 85.06% 2.58% 11.39% 0.11% 0.86% 1747

0.005M 84.70% 6.03% 8.71% 0.05% 0.52% 1941

0.001M 85.97% 1.00% 12.09% 0.14% 0.81% 2110

LCASW 88.93% 1.00% 9.06% 0.14% 0.86% 3478 Control

Table 14. Data at 32-34 hours after fertilization showing percents for each morphology and the total sample size for each of the five concentrations of adonitol in low calium artificial sea water (LCASW) and LCASW controls.

74 L-(-) Arabitol Complete Incomplete Not Exogastrulated Dead Sample Concentration Archenteron Archenteron Invaginated Size

0.1M 93.11% 3.33% 1.03% 0.11% 2.41% 1742

0.05M 90.94% 5.36% 2.28% 0.00% 1.42% 1976

0.01M 85.71% 4.63% 7.78% 0.34% 1.54% 1491

0.005M 82.90% 8.07% 7.89% 0.77% 0.36% 1685

0.001M 84.87% 7.47% 5.63% 1.16% 0.86% 1633

LCASW 90.70% 2.69% 4.49% 0.72% 1.41% 3343 Control

Table 15. Data at 24-26 hours after fertilization showing percents for each morphology and the total sample size for each of the five concentrations of L-(-) arabitol in low calcium artificial sea water (LCASW) and LCASW controls.

75 L -(-) Arabitol Complete Incomplete Not Exogastrulated Dead Sample Concentration Archenteron Archenteron Invaginated Size

0.1M 96.52% 2.25% 0.82% 0.00% 0.41% 488

0.05M 93.65% 3.17% 0.60% 0.20% 2.38% 504

0.01M 95.64% 2.09% 1.91% 0.00% 0.35% 574

0.005M 95.72% 0.97% 2.72% 0.00% 0.58% 514

0.001M 97.27% 0.78% 1.75% 0.00% 0.19% 513

LCASW 96.83% 1.42% 1.20% 0.00% 0.55% 916 Control

Table 16. Data at 32-34 hours after fertilization showing percents for each morphology and the total sample size for each of the five concentrations of L-(-) arabitol in low calcium artificial sea water (LCASW) and LCASW controls.

76 Dulcitol Complete Incomplete Not Exogastrulated Dead Sample Concentration Archenteron Archenteron Invaginated Size

0.1M 89.45% 1.34% 7.91% 0.40% 0.90% 2010

0.05M 91.44% 0.93% 7.57% 0.55% 0.00% 1822

0.01M 88.84% 1.17% 8.16% 0.30% 1.52% 1972

0.005M 89.62% 1.42% 7.73% 0.39% 0.83% 2043

0.001M 91.71% 2.71% 4.88% 0.60% 0.11% 1845

LCASW 92.61% 0.97% 5.86% 0.11% 0.45% 3518 Control

Table 17. Data at 24-26 hours after fertilization showing percents for each morphology and the total sample size for each of the five concentrations of dulcitol in low calcium artificial sea water (LCASW) and LCASW controls.

77 Dulcitol Complete Incomplete Not Exogastrulated Dead Sample Concentration Archenteron Archenteron Invaginated Size

0.1M 92.59% 1.59% 5.40% 0.00% 0.42% 1444

0.05M 89.31% 1.35% 9.21% 0.06% 0.06% 1628

0.01M 87.09% 1.41% 10.51% 0.28% 0.71% 1418

0.005M 88.72% 2.20% 8.23% 0.58% 0.26% 1543

0.001M 92.51% 1.01% 6.16% 0.16% 0.16% 1283

LCASW 95.82% 0.38% 3.72% 0.04% 0.04% 2605 Control

Table 18. Data at 32-34 hours after fertilization showing percents for each morphology and the total sample size for each of the five concentrations of dulcitol in low calcium artificial sea water (LCASW) and LCASW controls.

78 D-Mannitol Complete Incomplete Not Exogastrulated Dead Sample Concentration Archenteron Archenteron Invaginated Size

0.1M 96.23% 0.00% 1.36% 0.00% 2.41% 955

0.05M 96.04% 0.17% 1.07% 0.00% 2.73% 1211

0.01M 96.90% 0.00% 1.20% 0.30% 1.60% 1001

0.005M 97.06% 0.10% 1.68% 0.10% 1.04% 954

0.001M 93.47% 0.35% 1.63% 0.35% 4.20% 857

LCASW 96.36% 0.16% 1.25% 0.42% 1.82% 1923 Control

Table 19. Data at 24-26 hours after fertilization showing percents for each morphology and the total sample size for each of the five concentrations of D-mannitol in low calcium artificial sea water (LCASW) and LCASW controls.

79 D-Mannitol Complete Incomplete Not Exogastrulated Dead Sample Concentration Archenteron Archenteron Invaginated Size

0.1M 96.64% 0.00% 1.10% 0.00% 2.26% 1369

0.05M 98.03% 0.76% 0.91% 0.00% 0.30% 1319

0.01M 96.94% 1.68% 0.31% 0.08% 0.99% 1307

0.005M 92.66% 5.17% 0.65% 0.00% 1.53% 1239

0.001M 91.02% 2.03% 1.26% 0.00% 5.69% 1671

LCASW 95.95% 0.35% 0.82% 0.00% 2.88% 3158 Control

Table 20. Data at 32-34 hours after fertilization showing percents for each morphology and the total sample size for each of the five concentrations of D-mannitol in low calcium artificial sea water (LCASW) and LCASW controls.

80 D-Sorbitol Complete Incomplete Not Exogastrulated Dead Sample Concentration Archenteron Archenteron Invaginated Size

0.1M 98.64% 0.00% 1.36% 0.00% 0.00% 1179

0.05M 99.06% 0.09% 0.85% 0.00% 0.00% 1060

0.01M 98.96% 0.26% 0.78% 0.00% 0.00% 771

0.005M 98.74% 0.00% 1.26% 0.00% 0.00% 1115

0.001M 99.22% 0.00% 0.78% 0.00% 0.00% 1412

LCASW 98.90% 0.00% 1.10% 0.00% 0.00% 1733 Control

Table 21. Data at 24-26 hours after fertilization showing percents for each morphology and the total sample size for each of the five concentrations of D-sorbitol in low calcium artificial sea water (LCASW) and LCASW controls.

81 D-Sorbitol Complete Incomplete Not Exogastrulated Dead Sample Concentration Archenteron Archenteron Invaginated Size

0.1M 95.67% 1.01% 1.86% 0.17% 1.29% 1778

0.05M 95.12% 0.72% 2.05% 0.28% 1.83% 1805

0.01M 92.82% 1.12% 1.75% 0.56% 3.75% 1601

0.005M 93.26% 1.03% 3.28% 0.12% 2.31% 1647

0.001M 94.91% 1.10% 1.59% 0.18% 2.21% 1632

LCASW 95.16% 0.83% 2.29% 0.50% 1.22% 3367 Control

Table 22. Data at 32-34 hours after fertilization showing percentsfor each morphology and the total sample size for each of the five concentrations of D-sorbitol in low calcium artificial sea water (LCASW) and LCASW controls.

82 Xylitol Complete Incomplete Not Exogastrulated Dead Sample concentration Archenteron Archenteron Invaginated Size

0.1M 95.49% 2.42% 2.09% 0.00% 0.00% 1242

0.05M 92.34% 2.30% 2.37% 0.070% 2.92% 1436

0.01M 94.70% 2.73% 2.34% 0.16% 0.078% 1284

0.005M 93.54% 3.22% 1.65% 0.072% 1.51% 1395

0.001M 94.96% 2.02% 2.81% 0.22% 0.00% 1389

LCASW 94.12% 2.49% 1.47% 0.36% 1.58% 2212 Control

Table 23. Data at 24-26 hours after fertilization showing percents for each morphology and the total sample size for each of the five concentrations of xylitol in low calcium artifial sea water (LCASW) and LCASW controls.

83

Xylitol Complete Incomplete Not Exogastrulated Dead Sample Concentration Archenteron Archenteron Invaginated Size

0.1M 96.24% 0.58% 2.31% 0.12% 0.75% 1729

0.05M 97.30% 0.52% 2.01% 0.11% 0.057% 1742

0.01M 98.47% 1.18% 1.40% 0.00% 0.96% 1359

0.005M 94.87% 3.26% 1.60% 0.053% 0.21% 1871

0.001M 95.87% 0.94% 2.49% 0.00% 0.70% 1283

LCASW 95.95% 0.65% 2.22% 0.00% 1.18% 3063 Control

Table 24. Data at 32-34 hours after fertilization showing percents for each morphology and the total sample size for each of the five concentrations of xylitol in low calcium artificial sea water (LCASW) and LCASW controls.

84 Discussion

Carbohydrate Structures and Functions

The cell surface consists of sugars, lipids, and proteins. Virtually, all cells found in living organisms are coated with sugars. The sugar chains extends furthest from the surface of the cell which allows them to make first contact with nearby cells and molecules

(Oppenheimer, 1978). Carbohydrates have the formula C subscript n H subscript 2n O subscript n where n is greater or equal to three. Carbohydrates play an important role as markers for specific cellular recognition (Asao and Oppenheimer, 1979). Cell surface carbohydrates are important in morphogenesis through cellular interactions (Oppenheimer, 1977). Carbohydrate complexities and variabilities has led to a very high capacity for carbohydrates to carry information (Sharon and Lis, 1993).

Carbohydrates binding to lipids and proteins is crucial for biological activities (Sharon and

Lis, 1993). Glycoproteins, glycolipids, and polysaccharides cover the cell surface to extend and interact with neighboring cells. Interactions between lectins and carbohydrates are surface specific. Sugars and proteins have a lock and key style receptor sites allowing them to adhere

(Sharon and Lis, 1993; Oppenheimer, 1977). In this study, the focus of cell interactions and adhesion occurs during gastrulation , when sugars on the mesenchyme cells of the archenteron tip atatch to receptors located at the blastocoel roof at the animal pole. External sugar alcohols were introduced to impede, inhibit or promote the development and atatchment of the archenteron tip to the blastocoel roof. Six sugar alcohols were investigated

85 in this study which included: adonitol, L-(-) arabitol, dulcitol, D-mannitol, D-sorbitol, and xylitol.

Carbohydrates and Cancer

Understanding the effects of carbohydrates on cell-cell interactions and cell adhesion plays a critical role in understanding cancer metastasis. Early findings in cancer research have shown that oligosaccharides are common markers for tumor progression. Scientists have used these findings to study glycosylation in the role of cancer formation and cancer metastasis (Kim and

Varki, 1997). Cancer cells have been shown to have complex altered carbohydrates on their cell surfaces, demonstrating that carbohydrates may be important in malignant cell transformation and cancer metastasis. (Gorelik et al., 1995). Tumor cells exhibit major changes in glycosylation which effects the folding, localization and organization of membrane proteins, membrane rigidity, and sensitivity to anti-metastatic defense (Gorelik, 2001).

The malignant transformation of cells is associated with changes in cell surface glycosylation.

Glycoproteins present on cancer cells are very characteristic of the degree of malignancy: the more aberrant the glycosylation, the higher the degree of malignancy (Minko, 2003). Increased branching, addition of sialic acid residues and fucose, change in type or number of sugar residues and oxidation of sialic acid describe the changes in glycosylation on tumor cells (Mody et al., 1995). Changes in sialylation have been linked to an increase in metastatic potential of cancer cells because of the negative charge of sialic acid and terminal localization (Litynska et al., 2001). Changes involving an increase in beta 1-6 branching as well as larger more highly branched N-linked oligosaccharides present on metastatic cells were described by (Gorelik et

86 al., 2001; Fernandes et al. 1991). These changes are among the most important associated with metastasis and invasion (Litynska et al., 2001). Changes in glycosylation are considered to be significant in terms of invasive growth and metastisis because of the involvement of cell surface carbohydrates in cell adhesion. Disruption of the adhesive properties of a cell can promote cell detachment contributing to metastasis (Mody et al., 1995).

Medical Applications of Carbohydrates

Carbohydrates on cell surfaces serve as attachment points between cells as well as binding points for infectious bacteria and viruses, harmful toxins and other molecules. Medical research is using sugars to treat and prevent cancer and infections caused by bacteria and viruses (Sharon and Lis, 1993). Carbohydrate based vaccines are being used to treat pneumonia, typhus, and meningitis (Oppenheimer et al., 2008). Advances in glycobiology can provide new treatments for a variety of diseases. Bacterial adhesion is extremely important in infection. These bacterial infections become resistant to antibiotics and carbohydrate based drugs are an excellent alternative to treat and prevent bacterial infections (Sharon and Lis,

1993). Bacterial infections could be blocked by designing a carbohydrate based drug or vaccine that would incorporate similar carbohydrates found on the cell surface of the host. The carbohydrate based drug or vaccine would treat the bacterial infection by selectively inhibiting the bacteria adhering to the host cell by binding to the bacterial lectin (Ghazarian et al., 2010).

Carbohydrate based drugs and vaccines provide a strategic way to fight bacterial diseases.

87 A steady increase of resistance towards antibiotics has sparked a renewed interest in carbohydrate based vaccines. Carbohydrate based vaccines have been developed from cell surface carbohydrates from a variety of pathogenic bacteria which have been used to control

Haemophilis influenza Type B, Neisseria meningitides, Streptococcus pneumonia, Klebsiella pneumonia, Salmonella typhi (Vliegenthart, 2006) as well as carbohydrate based vaccines being developed to treat anthrax, malaria, and leishmaniasis (Oppenheimer et al., 2008).

Cell surface carbohydrates are not under direct genetic control which leads to the true benefit of carbohydrate based drugs and vaccines, which is their inability not to stimulate selective pressures on pathogens that they destroy (Vliegenhart, 2006; Hecht, 2009; Idoni,

2010; DerHartonian, 2011).

Lectins have become another target for glycan drug delivery (Bies, et al., 2004; Gabor et al.,

2004). Lectins are proteins with no enzymatic or immune function which have carbohydrates binding specificity. Lectins are usually oligometric proteins which contain several carbohydrate binding sites on each molecule (Gorelik et al., 2001). The carbohydrates to which they bind can be free sugars or sugar residues on glycoproteins, glycolipids, or polysaccharides which can be free or cell-membrane bound. The lectin-carbohydrate binding is usually specific.

Cells are coated with a carbohydrate coat called the glycocalyx. These carbohydrates on the cell surface function in: cell-cell attachment, immune reactions, maintaining the negative charge on a cell membrane, and as hormone receptors (Minko, 2003).

88 Related Studies

Exogastrulation, in which the primitive gut falls out from the embryo, was observed when the sea urchin embryos were treated with the mannose/glucose specific lectin Pisum sativum agglutinin (Latham et al., 1999). The results from this study suggest that lectins Lens culinaris and Pisum sativum may function in archenteron growth, development, and attatchment to the sea urchin blastocoel roof. Wheat germ agglutinin which selectively recognizes N-acetyl-D- glucosamine-like groups was found to be implicated in the control of primary mesenchyme cell positioning and function (Latham et al., 1999).

Additional research elucidated the roles carbohydrates play in cellular interactions and cell adhesion. Twenty-two sugars were investigated and only alpha cyclodextrin dramatically inhibited cellular interactions in the sea urchin embryo (Sajadi, 2007). Alpha-cyclodextrin is a cyclic polysaccharide which consists of 6 glucosyl residues linked by alpha-1, 4 bonds. At the lowest concentration of 0.003M, alpha-cyclodextrin blocked archenterons from binding to the blastocoel roof of the sea urchin in 67% of the sea urchin embryos, while other sugars that were examined had no effect (Sajadi, 2007). These results show that glucose-binding receptors are important sea urchin gastrulation, and that long sugar chains such as alpha- cyclodextrin may be necessary to inhibit cellular interactions (Cambry et al., 2006; Sajadi et al.,

2007). Alpha-cyclodextrin is a cyclic oligosaccharide composed of six dextrose units joined by an alpha (1-4) D-glucoptranosyl bond. Past studies have shown that alpha-cyclodextrin, which is an oligosaccharide composed of six glucose residues linked by alpha-(1->4)-D-glucoptranosyl units, inhibited archenteron formation or binding to the blastocoel roof (Garcia-Lopez, et al.,

89 1999; Sajadi, 2007). Cyclodextrins may also bind to lipids. (Sajadi et al., 2007).

More recent studies helped to further elucidate the roles of sugars and sugar receptors on archenteron organization, formation, and elongation. Research by Le in 2008 oberved the effects of sugars on sea urchin gastrulation in low calcium artificial sea water using different sugars. The findings from this study were that all sugars inhibited archenterons attachment to the sea urchin blastocoel roof in 60% or more of the sea urchin embryos (Le, 2008). The results from this study suggest that low calcium artificial sea water might be more effective in allowing sugars to enter the sea urchin blastocoel. A further study by Fernando in 2010 demonstrated that low sugar concentrations had a greater effect on archenteron formation and blastocoel attachment in low calcium artificial sea water (Fernando, 2010). Another study by Mokhnatkina in 2011 showed that all ten sugars in low calcium artificial sea water treating sea urchin embryos at 24 hours resulted in 30%-40% of sea urchin embryos developing incomplete archenterons, uninvaginated archenterons or were dead (Mokhnatkina, 2011). A study by

DerHartonian in 2011 showed all six sugars in low calcium artificial sea water at the first holoblastic cleavagel stage of the sea urchin embryo (2 hours) resulted in 1%-!0% incomplete archenterons, uninvaginated gastrulas or dead. These results were significant but small

(DerHartonian, 2011).

Current Study

The objective of this study was to gain a greater understanding for the role of sugar alcohols on the development, elongation, and attachment of the archenteron in Lytechinus pictus sea urchin embryos. A total of 6 free sugar alcohols in low calcium artificial seawater were tested

90 on 24-26 hour blastula stage and 32-34 hour blastula stage sea urchin embryos to establish the effects on morphology during gastrulation. The sugar alcohols were analyzed in order to determine their effects on the formation and growth of the archenteron during gastrulation.

All six free sugar alcohols during both treatment times in this study had very small effects on sea urchin gastrulation. All of the sugar alcohols resulted in at least 1-10% embryos that developed incomplete archenterons to the blastocoel roof, were invaginated, exogastrulated or dead. There have been no previous studies using sugar alcohols. The sea urchin embryos were treated with sugar alcohols ranging in concentrations from 0.1M, 0.05M, 0.01M, 0.005M and

0.001M. The five morphologies observed after the sugar alcohol treatments included complete archenteron, incomplete archenteron uninvaginated, exogastrulated and dead. In this study, disorganized embryos were incorporated into the category of uninvaginated embryos.

The sugar alcohols that had the most effects to the least effects on Lytechinus pictus archenteron morphology when added at 24-26 hours were adonitol > L-(-) arabitol > xylitol > dulcitol = D-sorbitol > D-mannitol. The sugar alcohols that had the most effects to the least effects on Lytechinus pictus archenteron morphology when added at 32-34 hours were D- mannitol > dulcitol > adonitol = xylitol > D-sorbitol = L-(-) arabitol.

Possible Experimental Complications

Experimental errors can be attributed to human error and inherent variables while conducting these experiments for this study. In order to make the stock solutions for each sugar alcohol, weighing paper was used to measure small amounts in grams which were added to empty Falcon tubes before adding the LCASW. Each stock solution was vortexed to make a

91 homogeneous solution. Serial dilutions were used to prepare lower concentrations for each sugar alcohol.

Contamination was another difficulty for this experiment. The laboratory is not a sterile environment. Bacterial contamination can come via adherence to the jellycoat of the female gametes, (Stephens, 1972) ambient air through vents and open doors. Another source of bacterial contamination could come from the endoskeleton, spines, and inner chambers of the sea urchin. Proteinacious sperm can also be a source of contamination. Limiting contamination is critical for working with sea urchins, their gametes and embryos. Sea urchins, their gametes and embryos require a specific temperature and pH for their sea water environment.

Contamination can effect the normal growth and development of sea urchin embryos.

Contamination was limited by autoclaving flasks and beakers, rinsing the glassware with either

ASW or LCASW, and then covering the glassware with Parafilm. Repeated aspirations of proteinacious sperm from beakers containing sea urchin embryos reduced this source of contamination. Furthermore, sea urchins were kept in aquariums within a sealed room in which the temperature was regulated by digital thermometers. Seasonal changes of the sea urchin were important in determining the best time to harvest viable and huge quantities of sperm and eggs which yields millions of normal embryos. The control media and the experimental media are susceptible to evaporation in the microplates. The media and fixed embryos in the microplates are wrapped in Parafilm and stored in the hood. The addition of exogenous materials does not tell the scientific researcher which stages of development and in what manner these stages of development are effected (Coyle-Thompson and Oppenheimer, 2005).

Some fixed embryos are not spatially oriented which leads to improper scoring of the archenteron morphology and skewed data results. Another problem within the microplate

92 wells is embryo overcrowding which results in the inability to retain proper osmotic pressure via an acceptable uptake of salts or an unbalanced amount of experimental material can lead to embryo death or a skewing of experimental significance (Idoni, 2010). The best time was during the middle of the sea urchin season.

A major disadvantage of using whole living sea urchin embryos in a microplate assay is the difficulty to directly assess the effect of a specific exogenous molecular probe on cellular interactions under study while this same molecular probe influences cell adhesion, other cellular interactions or molecular pathways in the sea urchin embryo.

Future Direction and Research

Two-cell stage sea urchin embryos were treated with monosaccharides and disaccharides to investigate the possible role of sugars and sugar receptors on cell surfaces of the archenteron tip and the blastocoel roof during gastrulation (DerHartonian, 2011). This research can be continued by using complex sugars, lectins, and glycosidases to investigate cell-cell interactions and cell adhesion during sea urchin gastrulation. The second holoblasic cleavage of the embryo and every cell division of the sea urchin embryo up to the hatching stage of the sea urchin embryo using simple and complex sugars, lectins, and glycosidases to study cell-cell interactions and cell adhesion during sea urchin gastrulation. The knowledge from these experiments can increase our understanding of cell-cell interactions and cell adhesion during sea urchin gastrulation and how it applies to diseases such as cancer.

From Saldain’s Master’s thesis from CSUN in 2010, the Lytechinus pictus embryos were separated into single cells by using calcium-magnesium-free artificial sea water (CMFASW) at

93 the late blastula-early gastrula stage. The late blastula-early gastrula stage is an important developmental stage in which newly synthesized N-linked glycoproteins are essential for proper development to occur. Live embryos were immersed in CMFASW and the cells detached from one another (McClay and Matranga, 1986; Coyle-Thompson and Oppenheimer, 2005). In future research, calcium free seawater should be used to study the effects on archenteron development, elongation, and blastocoel roof attachment. (DerHartonian, 2011) Future studies should also investigate the minimum concentration of calcium required to dissociate into single cells for each stage of cell division from the first holoblastic cleavage of the embryo up to the hatching stage of the embryo. Also, future studies should investigate the minimum concentration of magnesium required to dissociate into single cells for each stage of cell division from the first holoblastic cleavage of the embryo up to the hatching stage of the sea urchin embryo. The knowledge from these experiments can increase our understanding of cell adhesion and how it applies to diseases such as cancer.

Radiolabeling of simple and complex sugars to determine the adherence location of these sugars in the sea urchin embryo (DerHartonian, 2011). Flow cytometry, fluorescent tagging, proteomic investigation, x-ray crystallography, and many other scientific technologies could provide the scientific community the opportunity to understand the complexities of glycan interactions. Future research could also include the study of specific glycosidases to determine the functions of glycans in cellular interactions during sea urchin gastrulation (Idoni, 2010).

The most significant p-values from all the exogenous materials and their concentrations which have been tested in our lab could be used in our microdissection experiments to determine which exogenous materials have the greatest inhibition on the attachment between the archenteron and the blastocoel roof of the embryo. These microdissection

94 experiments would advance our knowledge regarding cellular interactions and cell adhesion during sea urchin gastrulation.

In addition, the rate of sugars and lectins entering the blastocoel of the sea urchin in low calcium artificial sea water versus artificial sea water could be investigated in future studies.

The effects of those rates on the sea urchin embryo could also be studied (Mokhnatkina, 2011).

The rate of sugars and lectins entering the two-cell stage, four-cell stage, and every cell division stage of the embryo up to the hatching stage of the embryo and their effects could be studied in future research projects. Future studies could also focus on how sugars and lectins effect the growth and development of the ectoderm, endoderm, and mesoderm. Using sugars with different functional groups such as a ketone or an aldehyde, using two different sugars, two different lectins or a sugar and a lectin to investigate the roles and effects on cell-cell interactions and cell adhesion during sea urchin gastrulation as well as applying this information in advancing cancer research.

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