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F3

ABJ‘RACT

THE EFFEdTE OF AVTI-EG? JELLY REFUF
ON COLL”TIC TFTG E323

by Gary Reynond Poirier

Treating normal uterine frog ears with anti-err
Jelly serum will reduce the percentare of eges that
will cleave (ihaver and Berch 1960). This is
similar to the effecta noted by Tyler and Brookbenk
(1956) using sea urchin eyes treated with a serum
made acainst fertilizin (the Jelly coat).

The present eXperiments are concerned w4th the
effects on the fertfilizability and cleavare of
Eggg niniens Echreber coeloxic er~3 treated with
anti-eat Jelly serum before the deposltion of the
Jelly coat.

Appropriate doaee of pituitary glands were
injected into ferales which previously had had thefir
oviduct; reroved. de coeloric eves thus obtained
were treated with either e5? Jelly ant13erur, non-
inrune rabbit serum, or full strenrth Holtfreter's
solution. The treated ewes were traneferred into

overectorized, pituitary stimulated hoeta for trans-

port down the oviducte and the deposition of the
Jelly coat. The treated egfs were renoved and
inseminated. The results were recorded as percen-
tagea of ears which cleaved.
The data suggest an effect like that obtained
when normal uterine egos were treated with eye
Jelly antiserum. That is, a significantly lower
percentage of cleavage was obtained from the coeloric
eggs when treated with the antiuegé Jelly serum than
with either non-immune serum or full strength Holtfreter's

solution.

TH? EF?ECT1 or AVTI-Rfifi JTLry
’IT??’!WJ cofiLovTc

FT“G HE“?

A THETI?
Tubmitted to
Nichican State University

in partial fulfillncnt of the requirements
for the degree of

FA7TER OF ECTENCJ
Departnent of Zoology

196A

A Prim?" Tum 211*“? T"
3‘. ‘ " I-J,..'., .. . .

. ‘7
. .
wot". . . . _. - .4...‘ . 1-1.5: J

I would like to thank Dr. Jehn R. Thever for
his tu‘dance and encvurajenrnt. Theiks ar- also due
to Dr. *tethanie H. Barch, Dr. Arron F. Yandera and
Dr. willie? L. Frentz for their conztructive criticism
and 101, during this study. I would also like to thank

A

YT. Ronald Tholf for his aid during the statistical

n

O" .. r1 2" 4L". 4 .J" A
analyai» oi the meta.

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III—"T C‘? TABLE"?

TABLE PAQE

1.

2.

3.

Table showing preliminary data

which involved one donoruone

host transfer experiments.

Seven different treatments are

shown. 17

Table showing the total of all
experiments using the one donorb

three heat transfer method.

Three different treatrents are

shown. 18

Table showing the one donor-three
host transfer experiments used

in the statistical analysis.

Three different treatments are

shown. 19

iv

LI KT ”‘3? CT???”

C RAP H

1.

PAGE
Graph showing the percentares of
cleavace of eras subjected to
various treatments before the
deposition of the Jelly coat in
the host animal. 23

1.

2.

Diagrar of an Ouchterlony

plate showinc at leazt three

lines between two of the anti-

egc Jelly sera used and whole

Jelly material. 21

Diagram of an Ouchterlony

plate showing at leatt one line

between two of the anti-err

Jelly sera used and body cavity

eg+ . 21

4
M

LI 7T 0"? APPENDICES
AT? “’ ENDIX P AGE
1. Surrary of Q test and

corperisen of various
treatments. 33

I NT??? DU C TI ’lN

ne Jelly coat around Amphibian eggs plays a
maJor role in the fertilization process. Jellyless
eggs, either before the Jelly coat has been put on
or after the Jelly has been removed, either by
cherical or mechanical means, are incapable of
being fertilized (Bataillon, 1919: Hugh, 1935:
Kanbara, 1933; Tchow.3u and Wang, 1956).

Kanbara (1953). using the toad.§g§ghvulearis
forwosus, found that the second layer of Jelly
from the egg surface, the C layer. is neces ary for
fertilization. Glick and:3haver (1963) demonstrated
in.§aga niniene that the farther the egr proceeds
down the oviduct, and thus the more Jelly the ens
has collected, the more likely it is able to be
fertilised. They have interpreted this result as
indicating that Species-specific components, put down
in the lower oviduct, play a maJor role in the
fertilization reaction.

However, Jellyless egés will develop partheno~
genetically after inoculation with a cellular constit-
uent (Bataillion, 1919) or by the introduction of a
blastula nucleus (inbtelny and Bradt, 1961). The

later investigators observed that coelomic e373 developed

1.

2.

more norrally, after the nuclear transfer, when they
were inserted into a jelly envelope taken from a
uterine e57. Also, Thivers (personal conrunication)
has deronstrated hat body cavity eggs of‘fi. niniena
can be fertilized if inseminated with a Sperm suspen-
sion that was in previous contact with norna jellied
eats. He obtained about 20 percent cleavafie.

Fambara (1953). working with 2,3. ferroana,
has snarested that the cherical corpocition of the
jelly may not be an important factor; he claims
to have obtained fertilization after inseminating
dejellied e533 re-enveloped in a coating of 6 percent
gelatin, indicating that at least one of the fanctiona
of the jelly may be to act as a substrate for a
thigrotatic reopense for the sperm. There is no evidence
of this in any other areoies. Fatagiri (personal
cornunication) has not been able to repeat Karbara's
results even with the same species of toad. He has
however obtained some data indicating that fertilization
is possible when only a small arount of the Jelly coat
is present around the ears.

Good and Daniel (19h7) obtained a Shall percentare
of cleavage when they treated jellyleas newt (Taricha

torosns) e3"3 with a very dense sperm suspension;

 

“one investigators have sr~*e ted the pos fbility

that th ere ray be in -terectind substances of 0fi”3 and
perm necessary for fertilization. Tyler (193?)

has sh? ested models for the fertilization reaction
involving cornlene ntary sites on the gametes tlflt
not much the sane a1 enti"eo~;nt'bo .y reactions.

It should be noted that any interference with these
"active sites" or neighborinf sites would affect
fertilization and / or devoloorent.

Probably the first to enz"e3t that fertilization
may be similar to a serological interaction wax Lillie
(1919). On the basis of his Sperm-acclutination
experiments, he introduced the concept of fertilizin
(of he era) and antifertilizin (of the snore)
interacting in :.ert 1i zation. Other worhers have
shown sirilar results in neeies of the Phyla Annelida,
Vollusca, and 110 pita (retz 19 37).

Glaser (as cited in lhaver and Barch 1950)
reported arrlutination of'g. nioienc aberrato zoo in
egg water of the sane enecies. anaerona attornts to
repeat thin were nnsacce*ial (P ornate n.193.-:
thaver (personal corrnnication). Hewever Bernstein
(1'5 2) ha is renorted irrever :Bible aficlntination of

gene claritane Sperm in as? water of the rare :peeies.

It is thought that this is due to the fact that

‘E. claritano jelly is more soluble than‘fi. n‘nfiens.
No agglutination was noted however in "egg water"
free jellyless eggs._ Thus it would seem that the

agfilutineting molecule in.§. clarfltane is present in

 

the Jelly corponents. In sea urchins, the mafin
source of fertilizln is the hifihly soluble jelly
coat (Tyler, 1957). However, Fotonura (1950) hes
deronstrated that the eve produces some fertillzln,
which 1e calls cytofertilizin.

It has been deronatrated (Tyler and Brookbank
1956) in the sea urchin that antisera produced against
fertilizin will inhibit fertilization and cleavare.
It may be incorrect, however, to say that fertilizln
antiboaies are indeed blocking Specific sites
necessary for fertilization. They may, in fact, be
attached to adjacent sites and be physically blocking
the operative ones.

Thaver and Barch (1960) noted similar effects
when they treated uterine ages with anti-jelly serum
prior to fertilization. They postulated a barrier
to Sperm penetration produced by the reectfion between
the Jelly coat and the serum. Later lhivers and

hetz (1962) rendered the antibody unlvalent by treatment

5;

zith papain, which eliminated the precfipitation
reaction on the Jelly coat noted by fihaver and

Barch. However making the antibody univalent did

not affect the inhibitory properties of the anti-
serum. 3hivere and Fetz conclude that the inhibition
is due to blocking a receptor site in or on the

Jelly. Whether the blocking is due to an irrulofically
Specific entiren—antihody reaction is still a question.

The locatiOn of the primary receptor site for
the sperr, affected by the antiserum, may be on
the outer layer of the Jelly. qhivers (1962) has
shown by fluorescent-antibody staining that cornonents
shared in cormon with other SpeClGB are located in
the innermost layer of jelly, and that the antisera
most effective in inhibiting fertilization are the
species-specific ones rad acainat the outer layers
of Jelly.

From later worg by ixaver, Barch, and Thivers
(1962) there is some evidence that at leaxt one
component f and in the Jelly coat is similar to one
in the erg. Also, it appears that cherieally dejellied,
fertilized ecca when treated with antijolly serum

will cease cleavage (Corny, 1963). The question aroee

6..

whether the treatment of‘g. minions eggs with anti-
egg Jelly serum, before the jelly coat is depoxited
on them, would inhibit fertilization and cleavafie,
either by occupying a site on the eye surface cornic-
mentary to the antibody directed afainat the era

corponent of the jelly, or by reacting with sites

on the 03: surface complementary to jelly confi,:ration.

FATESTAL LTD FTT"“D3

Two techr iqres have been uJed to a
intorferale trcntfer of cooletic ores. La” n
(1L.r; 3) injected into ho3t forale: vitally eta nod
coelor’e 9&33 whwch hafi been treotei w‘th ver‘.n3
core (e.r. anti-lnr”e oocyto, non—ir'nne rcb.:it
:erur, etc.) before be‘n: tran ferred.

The preccflnro uzel in this :tnflr wax fieveloped

by irnol:1 313 ”hover (1962). This tecnniqne COflSthS

l
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{-5

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of animulatin: an oviiactectoi.

e e¢“3 into the eoelene,

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into a host oxare ctorized ferale.

Althovfih the technioae flevisefl by irnola end
hcver takes lcn"€r, one to the sorcery required,
and i3 a bit rore diffi czlt, th e neri.d of tire in
whiéh effis are outuifie the body cavity of the chiral
i3 103 3 than in Lavin'3 techniqae, wk iciz recuire3
Atafninr aha xm = ..... nM of the OfTS. The -xtra manicu-
letione, reqzir ed for t e19 nroccflnre , are 91 o
el‘r inoteo.

' . . 0,; fl 1 ‘~
pecirene of Pena ziciene ~cnr0oer ett inod from

 

dealers in Wisconein anfl Vernont were u efl;

7.

8.

A. ="-TZ7'TT“AL P'."="‘C7".TTTTTT 7"” "T“"TT'TC‘T "VTTD'TCT".
JMHDCVtTIEZ

To rerove the oviduct, the donore were ether
anesthetized in a pint canning Jar. They were

reroved when reverent coaxed and a folded water-

if]

oahei paper touch, held in place with a ruhher
‘band, ya: wrapped around their hind 1958. This

was done to keep the frofie irrobilized since 3ome
recovered from the other quite rapidly, soretiree
before the operations were corpleted. A small
incision about 6 or in length was then made through
the skin and the rusouleture dorsal to the forearm
and posterior to the head. he oviduct was grasped
with a pair of forceps and gently pulled through
the Opening. he anterior end, including the
ostiue, was pulled from its attachment, the resentery
attaching the oviduct to the body wall and kidney
was out alone the entire length, and he posterior
end was 3eetioned in the roaion of the uterus. The
muscle layers and skin were then separately sewn
using 5-0 silk eurfiical sutures. The procedure

was repeated on the opoeite site. However, hefore
the second side ua3 sutured, whole 3. ninien3

pituitary glands were introduced into the body cavity

9.

to evoke ovulation, the number of Flands being
varied according to the season. Three blends
were injected per day for two days dur'na the
winter ronths and two per day for two days as
normal ovulation tire (sprinz) annroached.

Forty eight hours later the donors were nithed
and their body cavities opened. In the rejority of
female: close to 1001 of the egos had been ovulated.

The hosts were nethethized in the same manner
as he donors. An incision about 10 mm long was
made on the abdominal wall over the area of the
ovaries but Just lateral of the median line, so as
not to injure the ventral abdominal vein. Both
ovaries were then pulled through the same incision;
blood vessels were tied off and out distal to the
ligation.

Post of the host ferales were prepared shortly
prior to the time of transfer. A few were prenared
two to three days in advance to deterrine if the
shock of having ovarectowy at the time of transfer
inhibited the transferred egos from reachino the
ovisacs.

The treated egos from the donor were then injected

into the body cavity of the host, through the were

10.

incision from which the ovaries had been reroved,

with the aid of a syrinre and a blunt number 12
needle. Pituitary glands were then added, the number
varying according to the season, to stimulate the
cilia of the coelovic well and of the oviducta to

move the eyes up to the ostia and down the oviducts.
Twenty four hours later the eggs were strinned onto
glass slides and inserinated with a spore suspension
made up of two macerated testes per 10 ml. of 0.11
Holffreter's solution. The ears were tr,ated with the
sperm suspension for 10 rinutes, after which it was
decanted. The egos were then placed into larre finoer
bowls partly filled with aerated tap water. The eyes
were kept at room torperature and P to 13 hours after
fertilization they were checked for the percentaoe of
cleavafie.

After the ears were reroved, the hosts were
pithed and the body cavities were inspected for
remnants of ovaries. They were found only once and
the results of that experiment were discarded.

B. GETF?AL FETC757?E V“? T33 T”AU7?F? AVE
TIRE; A T1323}: '1‘

The actual transfer process consisted of scooping

11.

the e373 From the eypozed body cavity of the donor
and plec cin: tron in Full stronjtn Vo‘tfretcr
solution. All of the CT”S were reroved Quickly
since, if teren as needed they would be trenned in
the donor's coe"ulnt?nq blood. They were divided

w

into three betcnct, eecn of which received a different

Holtfreter'e solution, 2) non~irrune eerun, and 3)
an ent”:erfir nronfircd 9"" n t e"; 3e
angry erperirente (Table I) anti-oviduct serur,

anti-lower ovifiwct aerur, and on i-unner ovidzct

The e563 were left in the eere, at pH 7.h to
7.5, For F to 10 ninutee. They were then woehed either
with full strength Holtfreter's Solution or 0.11
Holtfreter's eolution and trer-fcrrcd. The controlS,
treeted with Holtfreter's solution alone, were
handle: as tony tires a3 the other two froups.

The order of treatmente was varied to n nir ze en
delet or:ou3 eFFect3 the miér t r03‘11t free beinf in the
Holtfreter's Solution for e nrol onjed period of tire.
Levin (1963) Claire that body cavity core can be held
in this way for up to twelve hours with only minor lose

of viability.

12.

C. (3171777311; 7"73TCIZD'JTTT; I?" 2"}; $7177} £77!) 77‘1"” 5.7772171
Pf"?! "FT“"I-i'

The entieernr was checked for antibodies by
meene of a modified Puchterlony double difF? ion
rcthod (Tbevcr. 1961). The presence of three distinct
con3tituer t3, a3 indicated by precipitot’on lines
betwcfn the anti-eh” Jelly ce:um end its honologous

antiserum of tre u3uel entilodv titre. Free iritetio

Q.

.4

line; were ebeent wFon the no :h*nnc serum was
reacted areinzt jolly entificn3. Three lines were

choien to dercn3trote t c ecti

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serum been! e tln 3re ere et lee 37t tint very eeoerete
corponent3 idontiffaklc in tnc Jelly taken From e333

(Trover end Berch, 196?). (7ee Plate I.)

Q

The err-Jolly entifcn was prepared my blending

10 r:. of lyonrilized or: jelly per r1 of O.F5§ Necl,

\
b

low we m m: 73"» at 957.2. Tlen 1-5 31-

LJ.
I

f e rixterc oF téie erti~cn end Freund adjuvent was

0

1'1". 1‘1) C befl ”Ta" L10 Inn1l5-Ni'1‘t,‘J b3? tile glib :1 cgvrnij‘a'r zaoylte.
Three to Four WCCFS later toe animals were bled for

enti3nrnv. All acre were frozen until no c ed after

'e

"5

being dielzyed are‘n. t Arnoibien ."line (0.65 3) o

‘

A? houre. Fon-inrrne 3eru~ was .. ta nod tel ore toe

injection oF the anti"en.

1,2

1‘ . .. . 9. . ~0- ‘ .
129 rer W113 of the exper;fient3 ere n101ented

in 753103 I, 71, aid 11!, 31d 1217reeh I. Table I

. J. .9, 5... 4.7- 9 . g :I . v... -, . V. .M H, - ,_
center}. owe. mete ebte no.1. frm: 131.~£<lfi7..fne.113 stpm-i-

rent3, in w‘icfi efire free one aenwr were tre “ 1erwed

.2"

*0 only one hast, efter one of t? e 3even fiif1erent
trentwep nt3 omelnyefl The3e re331t3 we: e not statis-
t1cally analyzed, becau e it we; 1: elt that 1nd:1%1_ - m,1

V P. r J. - . ,4 ‘ f 7-3 ..\ "I ‘l
wezietien3 0W3 donors, a: well e3 3 on: hL3t3,

U3

Vfifllfl be too great for accurate u1e .l;r;13. Therefore,
t“e next etherlre~tz were de373nefl tc el1efinete the
veriet1en3 arse: fienor er"3 in 32” one experirent.
Th13 we: eccerpliihed by Jab“ viding the eggs from

one donor into three batches and eurjecting each be tch
te e d1fferent treatment, an& treniferr11: ech batch
to e 3eperete heet. Table II nrcacntz the results of
all exper1ren13 perfe :vcd in this renter. Cf the

131.1 .1; eirfht ewes?“ at;

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fir
flyteew cnuld be uwed fer 3tet13t1cel enwlygis, 31nce
in ten en3e , no cleavefie oecured 1n the 07:3 treated

efitker w1th Heltfreter'3 3e Lt1en 03 with memoirfiune

'7'

39‘97 9“? ‘9 twolvp 99793 9t 39° t fine of h» three

rewovefl. Table III

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pr9¢99t3 £99 ratnlt: of tfie afixtcen exhcrfimont? wn ch
were svbjectod to an .nalyzis of vnrflance. This was
per £99794 to fletcrthot9997919ity avcny the rejult:
of the varinut troatront . The ?tati:‘i. °~1 dcsi n
of the analvzi: tndn into account the variat59nc in
the e733.frwn diffeyort £9999». Variatfi9ne bofiwe9n
hozt fer 9 e recefivfint 9771-9? the 3999 1999? could
not be ana’yzed by thin proceflwro. The prabanility
of 993:39197fi991 dflfference3 97977 the 193t3 Javinr
S’pnificant effects on the d9nor 9779 39993 satner
slifiht, however. The F va us obta’nefi fr9m the
amblyiis was J. 57 9 3 tho tam 9 value ( T9bl.e 10.5.3

in #9919999, 19;6) for the 999999“ “t9 fle9roc 0f

freedom (2 an} Sfi) 99* 3.?

. J

5".)

9t.the 51 19% Cl. Tut;
1:51cat03 thot tfi9ro 993 9’79'”;cgnt heterofc: city

579 to the effect? on the 977.3 of the three 6 fferont

In 99 atterbt t9 ”eterfiino whi9h flifforonce was
sirnfificpnt a zeqwe9ti91 C teat waz u 99. The actual
aifforonco between the 99993 of the effect; of the
full “at “9'97“” R911: ”“11““. imlution and the 7:1.9..-fi.:7rrune

serum 99: (.3 (#99 999993iX) unit: 1933 than the

corputefi G'f?9 9799.99. D (D 33 the preeuet of x a? d
a factor F take.n from table 10.6.1 in .nedecor, 1956).
This Gefinitcly stew: that there 13 no significant

‘V

eifferehee between the e two groups. The d‘fierence

between the effe We of the e the? tre pa Dir: of treatments
were, however, s’7nificant (Bee areenfiix) at the five

percent level. The value calculated few the actual
Eiffer nee between the 99993 of the effects of the
full 3tren7th Veltfrete7': Soluti7n and the anti-egg

jelly 90999 wee 1.26 utits more than the 099-»:rnndipr

e
"3
’3
CT
5
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I

Cevbvtel D 97d t3
ir7une eewum 99d tFe ertfl-e77 jelly screw W93 O.F7 unit
were.

In 097999 n7 the perceze 9793 of cleavefe in Tables
I, II, ate III ( 9139 see :reeh I). it can b- seen that
9 7999991 treni occurfi. T et 13 t‘et, PP”Q«G7CJ- 0f

the tranfifcr preceflure 979d (one demer-one host or one

fisher-three heats), 9773 treated with nee—ievune serum

ted hfi7her pernonta7ee of cleava7e than the 6 treated
w‘th the anti-er" 3911 y 9 Tum. ilnce the 999??"13 of
variance cletectcd Fote79‘eeotty 9;' 997 tfiCnbwefltS and
the 0 test showed ttat he a: fferc7'.: between the
control 991 the exeerirentel 39999: were c’gnificent,
the refultn deren¢tr9te that the entiteflies ”reduced
e79? nit 977-39] y, 5_n t’e ceheextrfit 9n Buzed, had a

fiefinite effect 19 lowering tee fert 1*zeb‘lity of the

16.

Levin (1973) ebtafdcé

o 3-.-. 1 ., p. m _ '0 ‘ ‘
n7 etgerttf9e, of mut;.era 99

(D

'1

’ :35
H

' 3

‘4
t .h

327’19? repults

ceele9ic 9773,

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. u11 fitren”th Volt. F3n- fir. ‘er. Anti-Ffi:_Jelly for.
#e¢~s _£pl. 101. “e333 #01. 131. 593:3 #01. 101.
399 50 12.6 550 59 10.? AZ" A 0.8
170 he 99.2 102 16 15.7 }? 5 7.1
21? 163 73.8 290 153 52.8 29 202 69.7
312 56 11.5 377 110 22.3 52 O O
131 131 92.9 83 1 1.2 152 O O
397 211 55.3 169 56 33.1 313 0 O
216 32 13.8 108 7 7.5 18a 23 12.5
53b 11 2.1 305 10 3.1 276 4 1.5
163 40 21.9 330 81 24.6 272 25 8.8
25? 11 4.3 55 ”5 17.7 395 20 5.1
53 173 A9.O 375 216 57.6 343 1*3 41. 6

368 53 15.0 507 205 33.3 :21 170 40.3
2F} 16 6.6 369 183 52.7 167 55& 52.3
170 30 17.6 562 115 20.6 310 O O
273 A6 16.6 108 1 0.9 269 7 2.6
212 9 4.1 92“ A 4.2 56 2 2.5

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DIfiCUB 79H

The ba3?c question that arises in conzidcrinfi
the results of thewe erUeUironts cancevns the 1a are

of tFo‘inhibitnrr effoc t f the ntise1e u: 01‘1, 0n the

1,1
“.3

swrfnce of e¢~1 hefove jelly 1° clejn3 32ted. The surpass

n p~ In. , v‘ -. q ' an. -. a .-
01 the trcmtvcnts of ’cllyflesa 03"» W33 to fieturnlne

inhibit fortilizat‘on nnfl cloavnro of trontca c" 3,
aftC? thny had “flfiffid fawn t“? oviflUcts of the ho:
anifial. TH» antizema ervloyefl in tfiexe fie t3 Cfifltfl7fled
”ntfbnfiies a510ctod arn’nst covpcnen1: imi 2-1031 with

9?? .1unr.3-. a“ well a: a"ain:t Pn"hfirn31; directed

a¢n*n“t 9?? 36111 ( co 111+ 9 II). This can“1nv
the observa ion of 9harnn 20105, and fihivorj (1962),
who have dis CU*‘Ci th" crfiflln Of t‘9 ””t9“i:1 in 083“‘

501’y thvt reacts with ovarfav efir material. A11 ouyh

with err on““mnonu1, in the firmness of refiavinfl he

30113 fmow utcr‘ne Cfié3, is the anvrce of the cf? $.11 eng,
this seem: Unlike1y, in View of the Gaye taken in the
dejollyfinm proces1 ..... Ud of the tho mush w33hfn; of the

Jolly. Shaver, Barch and hivers (1962) conridor it

"a a fl- .‘ u ‘1‘ + ‘F 4‘ ‘r-‘ 5 c n-J' ‘ “. ‘_1 a. '- r ‘l'.“ U ‘ '
F01 O 1.11.6.1}! Lr. '1‘.» -'-.‘ gt”; 1;" {‘4 2‘3 01. L113. C...- l 1‘23"." vi-C' 5.35.3

4 ‘1 ' ‘- ‘ u .’ tn ’5 . . " x‘: .‘1 'rq. "- , ~'» “a J" 9"
1mm he Jail}; 52.1121 U he puma-.1 of 113111‘Ut1-7L1 01 9.19
My 0,11. r. -. -~. w-U rvv" m1 ~. vM'" . a b1 {M
u‘- "‘y C... 2.: L). $51-- J L’J TALLL-LJAIJL' {"11 'It-.: 4‘ - p.,“‘ 8 \JG'..’. J ‘...G

71.: ‘. .J 4». ’ +1. r. 0. ,I. D "’ J— -" -

0.3.1101???) 3.1".1'1 with C‘;_ 1.713e-11 (1:51 or in 134,311 3:31,:
C r. A. (U! ‘3 9' ‘Te 1"" AT '1'
-‘ J:‘.’. J ' 1-399..) -J

h,._ ‘ 1 ,1 . f; , . 1 .v -33 , - 1.0 ‘ - -. .
FC.JU?CPLCM, an1 roacuCL Lit; 011353 ach-je.ig aeer
Li- . 4‘ ’ .‘IU- " ' O... 1 -1 ‘-
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CUL» IPUU qun 391;; £33 firUn use epis. LAOU; LQU gone

1! , . '1. ,. . .L" J. ..1._ ,, \ ,3 - 7- '- . , 3 ._ ,J. g .3 .4. , ‘..
ovxflence irvn 3.0 “91L. Leno oy hue ; LUJO o1rauor=, tLUt
AN... ‘ .. ....z. n 1 ,o . 1-3. , ‘1. . -1 fl ,.
LAC UTUUUL CL or? pUterral L1 U39 2011; cant UL3 in er.

-:~, “#5:, A, ,9 - -- ,3 .14 JJ‘ ,J.-. ..J." 4.... ‘ 4.1-. ...,_. . 1.. ..
r‘t‘,,_.,.. [1 9.”: ‘43:? 127.11,} bf) 1’31} I411 {LL b.1319 2.1.13 0 1 1: 111'}. e

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c

‘- ' ’ ~ Q, '\ 1 . O JO 0 F..- n, 'V, “n ‘ . . h '1
mean 1U‘UQ.QU 1n L7u.3. 1113 Leave? may 09 UUC 93uaon

W33 DUUU U1*’;’rn react Mns on r<3 diffu;1an plates UUe

' ‘ A ’ ‘Q s - u .3 paw-4 ,- -- .q“ ~ ,1 . 0"
qaite vaz-zcb19, 130a UUL1-egb ell; bc-mm gnu 01311an

C...»

‘- l - Ar 4- .fi
Later 3 are flCSCuCu.

:u 4‘ .9. ON! .? . t- -- .N . u ”1.". ’\ ~ 4’ ,3 ' c 4", u ‘i .9 1-0 rm-
p?€u-CZ1 ’u G,.;,IO?3.V(TQ L»; , it: C, L) u;"CQ.bC'-.! “41 ml 3.110.114-0” _ Jelly
zermr beche 3:11" @69031tLfin CUulé onus o» CIUZL nod
’1 J- *1“ I ‘ ~‘O ' p ‘1' .1 "1 v. -'~ 1‘ ‘4‘
1U 33?» on L10 bazi1 01 ”c Lloc U c of 31t93 on no

. A T q fl a. ... 0‘ . - . J. ...-.. A A
1C1 VoaLd Le 03 Llewcnca1~ Lo 81 71'L Ju5103

".I. “ “I" ‘5 " ile- m n— »‘ g . h -- O 0", -.. ‘c ‘ ‘ ., ...-a &
in MAG autidcrng direc ed agaffi.i 95, O‘NSGflwntjo Tnflb
4-? 3: .11.. ,. 'a ‘ .‘ . .I. 4.1 _4.o 4...
u3.3 %"U1 U131 U003 10» expla.n 0&0 aCULQn of anvi-

e"f jelly zero on uter’ne (3011363 ) efifa, hUwevcr,

13 infiicated by. :ome 0M) orvUt30U of ih3vers (1961).
Th 33 inve tirat or noted that anti-eff jelly dorum,
when ab nrbefl w3th ovarian material, prior to
treating uterine er": with it still recs ned
inhibitory effecta on fertilizatiUn. Thus in

afliitiUn to the interaction between the site.

L}
0
L3

F!

the jelT"1ell KC Snrlace aha antibofi.cu directed

‘-

q

ECU n*t egg material, postulated Uvac, which coxld
also Uccur at thn surface of jellicd CUUS, one
we‘ld have to pnstulate interactions between compon-

ents in the jelly coat of utcr‘no eg~3 and antibodies

11
1...:
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:3.)

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zst the%, such 33-10 1d 3m.ve been
CUUtained 3n ghiver's absorbed ti er1330th t"pes
of CUUUonenta Would be contained1’nt.m UUti~ej3 jelly
Bera UUCU in tfie prefient experivents, Una the Que tiUn

ariaos as to the rUle of each of them in the fertlli zap

‘ore obaervationa of Shivers (perSUnal can unicau
ticn) way contribute to an under JtUUding of thfs
problcr. He obta3ned cleavaée of 3611V1933 5, UiUiens
03¢3 w'chh had 3een in CUEIIU.ted with a snarm 3‘:Uon ion
t:*nt ham Ure"10u 3 contact with uto r3 me 9333. This
UUUMCUt: b? at t c Uctu\«refit of the JUUUUUtUUJCU to

the eyw swrface annlvcs a two~gtop proccfia. The

firwt step ray he an Cataraction betwehn the sperm

h.

92a tfie jolly tn "activate" t?e male fio?ct0. it

ray be at thi: point w‘ero fi:-z7b Etfion 0001?: ihon
uterine (jelliod) @353 are tre: bed w th anti-jolly
aerur (”havor aka Bflrch ICED). The second Ptep
ray invclve the inteyzctjmn of the
3901? fiozofin h t1 s‘tes on the e7: Curie ce. This
stem rm: on the one affected when jollV1es3 e333
ran t w tT antihndics in artiaer' 3611

AG 05‘ raterial in the ally.

directed nfainzt t

Additional eviflcnce that two or rare sites on the

“zen roactian, is the fact tha antiovar-
fierun will Shnw Dreclp itatM n lines wb en reacted
afngnflt Sperm antircn3, :Lile jelly antiserum does
not ( hevcr, PaTCh, and khivera, 1962). A334r1n3
tfiqt tfie lines betwocn the antiovnry zerum an& the
Bbcrm rehwn eat cavwnn0n93 int nlvcd in fez u‘liL ation,
nne can fee th.t in t e frfi" two Seuam te litGS, one
on the jelly and one on t to 0" aurface, comla be
postulated.

In tfie sea urchin, based on electron microscope
stvafiea showin” that the plaswa membrane of un art 1-

ized e~¢3 extends throufih the vitalline merorane,

26.

Tyler (1963) Bugiesta that the fertilizin molecules

of the Jelly are an extension of the plasma membrane
fertilizin. A3 a component of the plasma membrane,
POlySDermy after fertilization and removal of the
fertilization membrane (Tyler, Fonroy, and Metz, 1956).
Tyler also states that the vitelline membrane does not
contain fertilizin molecules. It is possible that

in frog egos two sites for the location of Jelly
molecules exist such as eugceeted above for the

sea urchin 6?”, one in the Jelly coat and the other

at the egg surface. Kemp (1956), in an electron micro-
scope study, has described, in developing‘fi. Eioiens
OOCyteS, protopleeric proce33e3 (microvilli) project-
ing through the zone radiate or vitelline membrane
intertwining with microvilli of follicular cells.
Wheter theee egg cell processes in the frog contain
"fertilizin" like molecules is unknown. Unlike the
see-urchin 959, however, where the source of the Jelly-
coet is still in question (Ketz, 1957). all of the
Jelly layers around frog eggs are secreted by oviducel
09113. Thus, analogous functions in fertilization of

sea urchin and frog egfs, may be accomplished by

cub tnnccs of éiffcrcnt orifiTn.

of

ohccfos can be netivntefl no“ hone 011C1£811F by

of htvent W‘bn tyncr on c or hypotonic #313 "339?:
A.

b" *h'Hr‘n‘a. ~1-m on‘h- - Rae-unrw-n‘n’fi'r‘ coma «17“ ‘7 an r
.r’ UV ...-’3‘ ... ‘cna L- s C c (3 ' .5. .5 . Lat». ‘ '.".5 U“. ‘.' $1 ‘34:..1-L k.¢ -'. 233C.» 0

otncr occnts (:ylcr, 1911). forlvnnn (13??) Claire

Entifen “f”, it located in the CC”, lrlcr (1353)
hoe boon rnnElc to activate 0““: of a affterent
*nccfhfi of fee urcVin rite anti are z’zilar to the e
11:03:. 1.)? TC‘e.‘-l.‘=*‘ofif1:1. He 721.1310.th (1963) that we
chcnrcz obicrvcfl by ?erlnnnn not be due to 03 tclytic

J.‘

action of the anti org. Fons of e phenoncna

a . .9 ...; , .-.-.‘ ‘4-3, 4.. ».. I C -- - . "
P~ To : tcfi Xfiuq act Jaclon ouce u; the li fting oi the
-.‘ J— . ‘ 9 . ‘ ‘ _a a ‘_ __ . ..' . '1
VluCLLEflC c"3xr‘o or :ztr L31 Cl the ”econu polar

bony was hotel in thi: to fly eii m1 cr after the eifie

r - 3", '-~ ~"-.--~~ * "'1 r "'o' ‘ Q" ’l
tore trer.t0rl 3 Lu anti are, or bolero chef MCfe fort l-

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me c: Theo vr1n”linf. bligtorinf. and cvt)1y:i‘ w

30°33 333 dchllied Jon uretzln 0"“3 7:ere trc. ted with

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i 2-17.92 §-2°.23
r... - “f : . MWwvw__{- “W_u.-‘1
Treatments - ,
"* yr .91 2) ' 1303'! 3003
F1111 .‘t. SOlto I" D...” . t _(12007) ‘ i {10..09 !
r a :
Eon‘rmm ‘ an 3 on I 1
serum 3__“_ ‘i'23 : (i8:02) i ;
nt 1-1“?" ‘ I '
sally 29mm 1 17.29 g i' I
{ .
( ) indicate calculated J values
d. f. baaed on er; or term (30 d.f.)
- ”f ‘ ”- :\/?. . error
9' *k Q J“ \ no. of exp.
3x = 123.11
16
5% I 3.17
CJJ“”I J n of variéus tr attent
{an ...:i VD(¥..QQ.L.)__-.-____D(._Qb&§_1+~_QC—’¥LN 00-00
F‘” to AEJ‘ _235fi_£,3o“7 ;- 12-07 ._-¥3134. 1- “thagéw
NT3 to ALJi _g:§grm_»3.47 ; 10.02 10.89 i a0.87
FSH t0 T713 2.99 30h? ‘} 9.089 3.03 ' 06.99

F“" full Strength Pfi1l1“9‘€"3 salut‘fin
PT‘3no -4rrune :erum
“rT-’n“ 1~0"j jolly sovy:

341

L TF7“ ‘T’TTTIC CITT‘T‘D

Arioli J.T., and ‘hav 2, J.?. 1961 InfiePfCfiale
trahffo2 .f 6“”? and ovrr‘o: in tfio frofi.
Exfitl. C011 ”03., 27. 133-13“.

n

332 h, '.?., and ’hnvcr, J.“.§1?C7 ficrifinal

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O
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no u 4.: ' fi 1-. .‘-'|" .... ,‘1‘ o f'o " ’1. I, ‘-s. ‘ :- 'l
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of nrfitoir: 8U21flf 1?? fioveiéfircnx of the

‘- . " o- u- '3 h- '- ‘ "‘ ' I“:
arpgro Di? m1 cnblyu. anbl. Cell 104., ~- QQQ-ICQ.

’1‘ ~ - fl ”.f‘ an I—a ' y o .‘
u12~cn,". 'KKT 1-0 “411ifcm c? :ocsotfnn..
“« "Qfi. ' Pf. 7r (,0 , . g. ‘s
ALF”. Ffih ”“1‘ t .y: J ~373 (c1 tel 1n.S;nv92
-.. _,. r I‘f--,
£11 Bagch, 19L , p. 1 1).

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. . n n; :‘r’ .' , | -. . j 'q « f1 .
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05:" 12 ' C . 1‘“ v'“ - 1-2 0.130111. 10*: . 1 01-. 1- .0 1'2"".10
1

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Gm“1, G. T“5 13211, J. 1913 70211113113¢n nf 9001“”"0
c~ : n? mvfit~~-1 “:M¢;g;. UHLV. Calif. Tubl.
Ic11., :1; 179.

.aibcrs, “. 1973 PClo C? 3011? en2elogo of 1021 o~23
12 Cw2tillzétién. flYHEtTt. 2391. Jafinn.,.gg:

l

K;
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o

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of ” 2“a n hfi0n3.c

rye“ 2.
25., 11"... '

. 1919
CAicago Fres.

1""?

w-
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a
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, C*‘-.;.\.)Ouf-

devolohwont.

Yetz, 0.3. 1937 “pocfific eff am:
sad "2*‘LALTQn of the 07“. In:
of T“: n‘fifi'fin‘ C “01,03 f)""‘Tf"(_‘~'}"( 3:5.
(T..;-.L C. I. T (3-1 u 1 Q CG. .3.) EU. 2 ,)-(9
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.‘t -i on TOTA ......

01

LA 10—.-

2001.

1952 Gertie

Annot.

{*rp._ "- 0'3
qu33'94n. '

Itotonuru, I.
tile f fal“ P. _o.

P0131rz‘"=.:’111, I”. 195!!- trxlrr on the.
fertfilizod sea urchin eq"3.
483~w92.

T)

.1.

.1

v. 1‘“

P 0 1°]. “We

'1 1511' . 1Q ’37
30a urchine
Corvarative

antinera.

F313 0?
".3, by 1308.113 of?!
.4.

h.) kl-J-

Fer ca 1 a

?”H~, P. 1959 Iflruvochew’
of L‘s "CW11V“CA_D 0""- p
fertilization.

{In I
~53.

D. 1935
J‘31.L.Liu.. Jad-iJ ”Lil.

.3. 1951

153.

3361., 113

Ehaver, J A Sirblo

{“UCd .‘1;

T'xfle

0“”oct of efitfiz
EthJ—o (1011 330': OI

fly of the efLeet
Exptl. Cell Re3., l};
cnfiroach to f“c
f"UOPiCnLiw‘;:

Follflcular rVDLHWO to fertili

- . .t‘.‘
flcfionfitpatinn o;

tro n rinrr we vy of rrowin: oocytos
J. Dioghyr. Biochem,

6" .1.
Kly “01 . ’

-q . C Q V011
Ahezfimvul

cionce,

O

h’a

3““~Lnn, D. C.

03 in : o 9""3 of
:3“: N‘ 0-2}1.
{3. on

G"
"
.-

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O.
"Q
)

1
-J

gar-fly 130*?

I} 1.314;.

J. n
{‘1'}, L; i 7'61?-

&ntibody refictinnfi. Fetronol. Fetréit "cience
Rev., 2?:- 13-20.

thafez, 3.2. ancl Dfiwc“ *.H. l-—ICO Fvvn“%ro firal 3*"3301
on th e role of jolly coat PAchI 1 in fortilfi Mzatifin
3 tag rm“. 'Ctn Fr’?"nl. eL F'orpiol. Fifie .,

0G
*2.

00:3

190-1

L4!

37.

1AH - .. - ~ .... «a ° .. ...,
419.102.”, QT. ;. , 3232:1C11’.1T00;$(_ 1; ”1431“) ’ C. A. 1962
T13 TAO-.1730: 7.17.03 ’31:? of 1°17 I? _ "to-...]o‘IJy 91“} '03:).

930;}.an 5-91:1. 1‘50... 31., 141:6 95-100.

KniveIt, C.fl 1962 LOCI713a“““ of tIe inhibfitOIy
offset 3 1-30117 AeIum on fnwt‘17°°+7nn in
r.

:5
7-1.
1
b
Fig.
I-)

:
’J

l

I.. V. 1‘ . , ,. f 1 :

gniveII, C... aad Feta 0.8. 19oz IIQIbition of
ferti-1zatia -yg eié3 by univzlont fIaIIonfis
of IaLbit aIti 9,. Frnc. 300. Exp. Biol.
— . . - ‘-‘ 7'" '76
841d 301., 3:22: 5:...‘DIOJ-x.7.

 

:1 .. u r. 1-4. .I.», 1 a ,. . 4.x, ,9" v1.2 a“
10G999P, 0.“. 1995 u£u1.t1:o7 IO -7uy. rIlon
‘4“. .3 Ifi‘a o ‘ o . "‘
13.11.3101,). Loo 1012’». I jL-JL .ii‘)e ‘1‘.) Olive: 9 :‘J: CD'S" J .

Tubtolny, 7. and BIaGt, C. 1951 TIan3p In Mat'wn of

blaitiln nIcc?i in o ct‘“3unfl 0993 f1“? b0 W

ce.v1ty aI:d frém t7 uterus of “Ira :fEIOT“.

II. eVI‘nIIC t o tae I9C1I10I£7b09v Inv’tv
7

kk‘) \J\
0353. Develop. Bio-

.‘_‘_'.-. i

u ‘1‘?! -,.. / ’
Tcnow- u et a. t Hu-lo n 1936 E tude3 exworiIentales
J-

I 19 I019 flu Incus G65 0v 1112b: 5893 in
fennwantiol 0 oz 1e cIa,aud, et la can:1.eGeInt10n
’CIIIIIC 21? la IGSIIECIG ie a pénotzo t1 n
SICIIatiquo. (En Chinese, with FIonch GWIIfiwy.)
Lita Dh-,crircnla1e3 Biolojica ‘inica, §; 75-122.

Tyl P,rfi. 1951 ‘I47f1031 parfihonogefielffi. 9101. Eev.,
1C! 291.

TJler A°.1957 1”“unn7MI7087 3t .Gios of co 11y 90"nlfifin

“'C MDT-71: Twrs finra-S-qa- €mm‘3 AFT”? 13%"51‘7

.4. E

(A. Tyler .c.1v6n 3044t01

 

 

, InG 0.3. Tet , 6G3;T
nw.3"1-)?%.Io“1~nn A: .0013 t‘on nt AdvnIIcIent

of Glance, $93191 t0.n, 9.0.

Tyler, .1. 1939 9on9 innufioleIch 1 0"“CT’WC“ 3
f “*‘1'18qu IIG early dovelo~m33+ ‘n :93

L)

urchins. Irptl. Cell 203. (Zupnl.)‘Z:193-199;

011

Tyler. A. 1963 The Inn49I13t1.n3 of Facwfi“flle””’”r

3nb3ta.aco.3 GnI1nfi fo~t‘lfizfiL%Iq and efi.I1y an elnfi-
mcnt of aniIal of 2. Am. Zoologi3t, E; 109-126.

 

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ROOM USE ONLY

ROOM USE ONLY.

 

MICHIGAN STATE UNIVERSITY I ARIES

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