Shiroma, Estelle

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MOP & BML The Effects of Mercury on Fertilization and Cleavage of Trivneustes nratilla By Estelle Shiroma Submitted to John McMahon May 3, 1976 ABSTRACT Tripneustes g r a t i l l a  - ova were f e r t i l i z e d  i n  500 ppb, 200 ppb, 100 ppb, 70 ppb, 50 ppb, and 25 ppb HgC12. F e r t i l i z e d  ova were observed f o r  t h e  presence of  f e r t i l i z a t i o n  membranes 15  minutes a f t e r  insemination.  Cleavage was observed 2.5 hours a f t e r  f e r t i l i z a t i o n  and again  a f t e r  18  hours a s  a check on t h e  development of t h e  embryos. With increas ing concentra t ions  of H ~ ~ + ,  t h e  number of f e r t i l i z a t i o n  membranes decreased. Cleavage, 2.5 hours a f t e r  insemination a l s o  followed t h i s  t r end ,  markedly decreasing a t  500 ppb H ~ ~ +  with 4.5% cleavage. Abnormalities i n  t h e  developing embryos were a l s o  observed. Cy to lys i s  was predominant a t  concentrat ions of 100 ppb, 200 ppb and 500 ppb H~*+. Development of  t h e  embryos was a r r e s t e d  a t  f i r s t  cleavage i n  500 ppb and 2t a t  second cleavage i n  200 ppb Hg Abnormalities cons is ted  o f  wrinkled cleavage p lanes ,  l ack  of  cleavage p lanes  d e s p i t e  mul t ip le  n u c l e i  i n  t h e  embryos, assymetric  cleavage and l y s i n g  o f  t h e  f e r t i l i z a t i o n  membrane with ext rus ion of cleaving c e l l s ,  The number o f  abnormal i t ies  decl ined with decreasing concentra t ions  of  ~g*'. INTRODUCTION Mercury t o x i c i t y  has concerned i n v e s t i g a t o r s  s ince  t h e  e a r l y  1900's. I t  i s  known t h a t  c y c l i c  changes i n  t h e  d i s t r i b u t i o n  of  marine i n v e r t e b r a t e s  a r e  o f t en  r e l a t e d  t o  changes i n  t h e  chemical composition o f  sea  water (Waterman, 1937). Extensive i n d u s t r i a l  and a g r i c u l t u r a l  usage can s i g n i f i c a n t l y  a f f e c t  t h e  r e d i s t r i b u t i o n  of heavy metals  such a s  mercury i n  t h e  ocean, posing a t h r e a t  t o  marine f l o r a  and fauna. Kobayashi ( 2 )  proposed t h e  use of  s e a  urchin eggs a s  a s u i t a b l e  m a t e r i a l  f o r  a  bioassay f o r  marine po l lu t ion .  Dif ferent  anomalies induced by po l lu ted '  waters  containing var ious  heavy metals  a t  c e r t a i n  developmental s t a g e s  o f  s e a  urchin eggs and embryos may be a v a i l a b l e  a s  r e l i a b l e  i n d i c a t o r s  i n  bioassay. Kobayashi's prel iminary experiments were i n s p i r a t i o n a l  i n  genera t ing  some ideas  f o r  the  present  study. A previous s tudent  i n  Zoology 420 (Crudele, 1973) s tud ied  t h e  effects o f  var ious  concentra t ions  of  HgCl vs. f e r t i l i z a t i o n ,  based on cleavage 2 r a t e s .  I have attempted t o  expand h i s  work by inc luding q u a l i t a t i v e  observat ions  t o  i l l u s t r a t e  t h e  de t r imen ta l  e f f e c t s  of H~~~ on t h e  develop- ment o f  t h e  sea  urchin embryo. MATERIALS AND METHODS Sea-water was f i l t e r e d  with a 2 micron Mil l ipore  f i l t e r  t o  screen our  b a c t e r i a  t h a t  might be capable of convert ing inorganic mercury t o  m ~ t h y l  mercury.  dilutions .of 500 ppb, 200 ppb, 100 ppb, 70 ppb, 50 ppb and 25 ppb HgC12 were prepared i n  sea  water from a s tock s o l u t i o n  o f  10 ppm HgC12. (See Appendix,) I t  was e s s e n t i a l  t o  prepare t h e s e  s o l u t i o n s  twice during t h e  experiment because H ~ ~ +  ions  a r e  r e a d i l y  adsorbed by glassware . Tripneustes g r a t i l l a  were c o l l e c t e d  from Kaneohe Bay and spawned by i n j e c t i n g  0.55 M K C 1  i n t o  t h e  coelomic c a v i t i e s  of t h e  male and female sea  urchins. The optimum sperm concentra t ion  used was 1 drop of sperm i n  50 m l  of  f i l t e r e d  sea  water. 5 drops of t h i s  s o l u t i o n  was added t o  5 drops  of  ova (approximately 1000 eggs) i n  5 m l  o f  sea  water conta in ing t h e  spec i f i ed  concentra t ion  of  H ~ ~ $ .  Eggs were f e r t i l i z e d  and observed a f t e r  15  minutes, 2.5 hours,  and a t  18  hours. The first observation was made t o  count f e r t i l i z a t i o n  membranes; t h e  2.5 hour observation measured success  of cleavage; t h e  18 hour observat ion  was used a s  a check t o  observe how f a r  development had proceeded. The ex- periment was repeated f i v e  times over a :four! week period. The c o n t r o l  f o r  t h i s  experiment was f e r t i l i z e d  eggs i n  s e a  water conta in ing no mercury. An a d d i t i o n a l  c o n t r o l  cons i s t ing  o f  u n f e r t i l i z e d  ova i n  t h e  var ious  H ~ ~ +  d i l u t i o n s  was suggested dur ing t h e  Prel iminary Report t o  a s su re  t h a t  H ~ ~ +  d id  not  induce parthenogenesis.  I incorporated t h i s  suggest ion i n t o  t h e  experiment and found t h a t  H ~ ~ +  d i d  no t  induce parthenogensis i n  u n f e r t i l i z e d  eggs. The pH of  t h e  s e a  water was a l s o  checked. The pH of  t h e  s e a  water used was 8.1. No pH change was observed i n  any o f  t h e  d i l u t i o n s  made with HgC12. RESULTS Resul ts  of t h e  experiment r e v e a l  some genera l  t rends .  With inc reas ing  concentra t ions  of H ~ ~ + ,  t h e appearance o f  f e r t i l i z a t i o n  membranes decreased. See Fig, 1. It  should a l s o  be noted t h a t  d e s p i t e  t h e  decreasing t r e n d  i n  t h e  number of f e r t i l i z a t i o n  membranes', t h e r e  was s t i l l  a r e l a t i v e l y  high 'percentage o f  membranes t h a t  appeared even a t  concentra t ions  o f  500 ppb and 200 ppb H ~ * + .  A t  2,5 hours following f e r t i l i z a t i o n ,  t h e  number o f  eggs which success fu l ly  underwent cleavage decreased, There was a sharp ' dec l ine  a t  500 ppb HgC12. See Fig, 2. Certain abnormal i t ies  of t h e  developing embryos were more f r equen t  a t  a p a r t i c u l a r  concentra t ion  of than o thers .  See Table I. 2.5 hours a f t e r  f e r t i l i z a t i o n  c o n t r o l  eggs were i n  first and second cleavage, Cleavage sp ind les  were observed i n  eggs t r e a t e d  with 500 ppb, 200 ppb, and 100 ppb H ~ ~ ~ .  No cleavage furrow was seen i n  eggs t r e a t e d  with 500 ppb H ~ ~ ~ .  In- complete cleavage p lanes  o r  no cleavage furrows were seen i n  eggs t r e a t e d  with 200 ppb H ~ * + ,  d e s p i t e  t h e  presence of mul t ip le  nucle i .  Ova i n  100 ppb HgC12 showed wrinkled cleavage p lanes  o r  no p lanes  formed with t h e  presence o f  mul t ip le  nucle i .  Cleavage occurred without de lay  i n  70 ppb, 50 ppb and 25 ppb. Abnormalities were present  i n  a l l  concentra t ions ,  dec l in ing  i n  frequency r. with decreasing amounts o f  mercury. Membrane damage followed by c y t o l y s i s  was prominent i n  concentrat ions of 500 ppb, 200 ppb,,and 100 ppb. Assymetric cleavage was commonly observed a t  70 ppb Membrane damage occurred a t  70 ppb and 50 ppb with c e l l s  c leaving ou t s ide  of  t h e  boundaries o f  t h e :  f e r t i l i z a t i o n  membrane. See Fig. 3. A small  percentage o f  abnormal i t i e s  occurred i n  eggs t r e a t e d  with 25 ppb H ~ ~ ' ,  Of t h e  concentra t ions  used, 25 ppb H ~ ~ +  a f fec ted  development o f  t h e  embryos minimally. Observations made 18 hours a f t e r  f e r t i l i z a t i o n  showed 500 ppb H ~ * +  t o  be l e t h a l  t o  t h e  development of  t h e  embryos. Development proceeded through first cleavage and was a r r e s t e d  a t  t h i s  stage. 200 ppb H ~ ~ +  was a l s o  l e t h a l ;  t h e  embryos d ied  a f t e r  second cleavage. In  100 ppb, develop- ment was a r r e s t e d  i n  over 50% of t h e  embryos a f t e r  second cleavage. 12% were i n  t h e  e a r l y  g a s t r u l a  s t age  b u t  moved s luggishly .  I n  70 ppb H~*+, about 50% of  t h e  embryos were v i a b l e  i n  t h e  b l a s t u l a  and e a r l y  g a s t r u l a  s tages .  70 ppb was approximately t h e  LD f o r  t h i s  experiment. A ma jo r i ty  5 0 of  t h e  embryos ( 81%) were v i a b l e  i n  50 zppb -H~?*. Ddvelopment .proceeded through t h e  b l a s t u l a  and g a s t r u l a  s t a g e s ,  Normal development of embryos through t h e  b l a s t u l a  and g a s t r u l a  s t a g e s  was observed i n  25 ppb tlg2+. TABLE: I. SUMMARY OF OBSERVATIONS Fertilization % Cleavage OBSERVATIONS OBSERVATIONS Membranes 2.5 Hours After 2.5 Hours After 18 Hours After (%> Fertilization Fertilization Fertilization CONTROL 500 ppb 100 ppb Normal first and Late blastula/early second cleavage gastrula Cleavage spindle formed but no furrow present Lethal; development up to first cleavage and arrested Incomplete cleavage After second cleavage planes; nuclei pre- embryos die; the rest do not sent but without , cleave cleavage plane form- ation; 32.8% abnormal Rucl8i' present but Development arrested after withoat--cleav%ge'p'kane second cleavage in over formation; 27.3% 50%: 12% in early gastrula abnormal stage; sluggish movements 16.2% Abnormal About 50% viable Assymetric, cleavage Blastula/early gastrula stage; sluggish movements 81% viable Blastuia/gastrula stages Sluggish movements .63% Abnormal Appear to be developing normally ~lastula/gastrula stages 'VExtensive cytolysis observed in 500 ppb, 20@pb, and 100 ppb Hg DISCUSSION AND CONCLUSIONS LI Several  experimental outcomes should be noted, There was a decreas ing number of  f e r t i l i z a t i o n  membranes a s  t h e  Hg2+ concentrat ion increased.  The success  of cleavage a l s o  followed t h i s  t rend.  A s  H ~ ~ *  increased,  t h e r e  was a dec l ine  i n  success fu l  cleavage. I n  a l l  concentra t ions  o f  Hg2*, anomalies i n  development occurred. The p o s i t i v e  c o r r e l a t i o n  between decreasing numbers o f  f e r t i l i z a t i o n  membranes and t h e  dec l ine  i n  successful  cleavage a s   concentrations increased might be a t t r i b u t e d  tp  t h e  a c t i o n  of  mercury a s  a metabolic  poison. Mercury shows a s t rong  a f f i n i t y  f o r  l igands  suchs a s  phosphates, c y s t e i n y l  and h i s t i d y l  s i d e  chains o f  p r o t e i n s ,  pur ines ,  p t e r i d i n e s  and porphyrins. Hence, it can a c t  a t  a l a r g e  number of  biochemical s i t e s ,  4 The p a r t i c u l a r  s i t e  of  a c t i o n  o f  mercury would thus  be d i f f i c u l t  t o  p inpoint  i n  an inves t iga t ion  such a s  t h i s  one. A poss ib le  i n i t i a l  e f f e c t  2+ of Hg would be a change i n  membrane permeability. Hg2* i ons  r e a d i l y  change cel l  membrane permeabil i ty accordinq t o  Vallee and Ulmer, Even small  amounts of heavy metals produce appreciable  changes of su r face  t ens ion  and su r face  of  l i p i d  f i l m  o f  c e l l  membranes, This may lead  t o  marked changes of  permeabi l i ty  and metabolic a c t i v i t i e s  of  enzymes. Hg2+ i s  a l s o  known t o  i n t e r a c t  with su l fhydry l  and d i s u l f i d e  groups of p ro te ins .  During f e r t i l i z a t i o n ,  H ~ ~ +  i ons  might have i n t e r a c t e d  with t h e  s u l f a t e d  mucopolysaccharides extruded from t h e  c o r t i c a l  granules  during t h e  formation of  t h e  f e r t i l i z a t i o n  membrane. Once within t h e  membrane, c e l l  funct ions  would have been af fec ted .  Mercury can bind t o  and a f f e c t  t h e  conformation o f  nuc le ic  a c i d s  and d i s r u p t  metabolic pathways such a s  oxidat ive  phosphorylation. 2+ 2+ The e f f e c t  of  Hg on t h e  spermatozoa must no t  be overlooked. Hg , i f  permeable t o  t h e  membrane, could bind t o  enzymes i n  t h e  acrosomal granule  responsib le  f o r  d iges t ing  t h e  egg membrane dur ing f e r t i l i z a t i o n .  Oxidative phosphorylation i n  t h e  mitochondria might a l s o  be af fec ted .  Many v i s i b l e  abnormali t ies  i n  t h e  embryos appeared t o  be r e l a t e d  t o  membrane damage. F e r t i l i z a t i o n  membranes were t o r n  i n  some eggs wi th  c e l l s  cleaving ou t s ide  of t h e  membrane. This  dccurred a t  a s i n g l e  l o c a l i z e d  a r e a  of t h e  egg. Hoadley (1930) observed t h i s  phenomenon i n  Arbacia punctula ta  and suggested t h i s  a s  a  mechanism f o r  t h e  egg t o  extrude mercury-avid pig- ments from t h e  embryo. Pigments were not  c l e a r l y  d i s t ingu i shab le  i n  t h e  sec t ions  of eggs cleaving ou t s ide  of  t h e  membrane, however, t h i s  i n t e r p r e t a -  t i o n  may be p laus ib le ,  Another abnormality observed were embryos wi th  assymetric  mul t i - ce l l s  during cleavage. Since membrane damage was evident ,  t h e s e  embryos may suggest polyspermy. Recommendations f o r  f u t u r e  s tudy would include s h o r t  term exposures of t h e  embryos t o  mercury so lu t ions  followed by t r a n s f e r  t o  normal s e a  water t o  s tudy recovery, - . (  1..1 Mercuric c h l o r i d e  'Baker Analyzed' Reagent Lot # 411684 Assay ( ~ ~ 6 1 ~ )  99.48% Residue a f t e r  i g n i t i o n  0.012% S o l u t i o n  i n  e t h e r  PACST Iron (Fe) 0.001% The 10 ppm s t o c k  s o l u t i o n  o f  HgCl, w a s  prepared by d i s s o l v i n g  1 g 2 HgC12 i n  1 l i t e r  of f i l t e r e d  s e a  water.  1 m l  o f  t h i s  s o l u t i o n  was d i l u t e d  i n  100 m l  o f  s e a  water r e s u l t i n g  i n  a f i n a l  d i l u t i o n  o f  1 0  ppm. REFERENCES - 1. CrudeLe, Robert. Spring,  1973, Mercury vs. F e r t i l i z a t i o n .  Zoo1 420. 2. Kobayashi, N ,  1971. F e r t i l i z e d  Sea Urchin Eggs as an I n d i c a t o r y  Mate r i a l  f o r  Marine P o l l u t i o n  Bioassay, Pre l iminary  Experiments. Publ,  of t h e  S e t o  Marine R i o l ,  Lab, X V I I I  ( 6 ) ,  379-406. 3. Passow, H., Roths te in  A. and Clarkson,  T. W ,  1961. The General  Pharmacology o f  t h e  Heavy Metals,  armoc,olo~gi~cal Review. 13: 185-221. 4. Val lee ,  B. L. and ULmer, D. D. 1972, Biochemical E f f e c t s  of Mercury, Cadmium, and Lead. 1 ;  91-113. 5. Waterman, A. J. 1937. E f f e c t s  o f  S a l t s  of Heavy Metals on Development o f  t h e  Sea Urchin, Arbacia punctu la ta .  Bio l .  Bull .  73: 401-420. 

The Effects of Mercury on Fertilization and Cleavage of Tripneustes gratilla

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The Effects of Mercury on Fertilization and Cleavage of Tripneustes gratilla

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