COMPARISON OF ANTIGENS
IN THE
OVARY,
OVIDUCT
AND
UTERUS OF
THE RABBIT AND
OTHER
MAMMALIAN
SPECIES
A. G. SACCO and C. A. SHIVERS
Department
of Zoology, University of
Tennessee,
Knoxville,
Tennessee37916,
U.S.A.{Received
thJanuary 1972)
Summary.
Sheep
antiseraprepared against
saline extracts of rabbit(Californian)
ovary,oviduct anduterus wereusedtodetect the presenceof
homologous antigens
in the ovaries andreproductive
tracts of the Dutchrabbit,
New Zealand White(NZW)
rabbit,
guinea-pig,
hamster,
mouse and rat.
Absorption
of each antiserum with Californian rabbitplasma, kidney
andspleen
and thereaction of the absorbed antisera in immunodiffusionplates against
thehomologous antigen
from eachspecies produced
thefollowing
results:(1)
absorbedanti-ovary
serumformed
precipitin
bands with twoantigens
in theovaries ofeach of the breeds of rabbit whileforming
no bands whenallowed to reactwith the ovaries ofthe other mammalstested;
(2)
absorbed anti-oviduct serumformed bands with six
antigens
in the oviducts of each of the rabbitbreeds,
twoantigens
in the mouseoviduct,
one in the rat oviduct and formed no bands with the oviducal material from theguinea-pig
andhamster;
(3)
absorbed anti-uterus serum formed bands with twoanti-gens in the uterus of the NZW rabbit and one band with the uterine
material from the Dutch rabbit and other
species.
None of the three absorbed antisera reacted with the sixteennon-reproductive
tissues ortestis
preparations
from each of the differentspecies.
These results demonstrated thatantigens
present
inreproductive
tissues of the Cali-fornian rabbit were alsopresent
in several other mammalianspecies.
INTRODUCTION
Immunological
analysis
revealed that the ovary andreproductive
tract ofthefemale
(Californian)
rabbit containedantigens
notpresent
in other tissues(Sacco
&Shivers,
1973a).
The twoovarian-specific antigens
were localized inthezona
pellucida,
thecainternaand atretic follicles; the sixoviducalantigens,
in themucosal
lining;
and the two uterineantigens,
in the endometrialstromaand cervical lamina
propria (Sacco
&Shivers, 1973b).
Thetissue-specificity
of theseantigens
and their localization withinspecific
areasofthereproductive
* Present address: The Institute for Cancer
Research, 7701 Burholme Avenue, Philadelphia,
Pa. 19111,U.S.A.
tissues
suggests
thepossibility
of the existence of similarantigens
in other mammals. Thepresent
investigation
was undertaken in anattempt
to detectreproductive tissue-specific antigens
in the Dutch and New Zealand White(NZW)
rabbit,
guinea-pig,
hamster,
mouse and rat with antiseraprepared
against reproductive
tissues ofthe female(Californian)
rabbit.MATERIALS AND METHODS
Preparation of
antiseraSheep
antisera wereproduced against
saline extractsof rabbit(Californian)
ovary, oviduct and uterus and
absorbed,
following procedures
describedby
Sacco & Shivers(1973a).
Immunodiffusionanalyses
demonstrated thatabsorp¬
tion of each antiserum with rabbit(Californian) plasma, kidney
andspleen
(p,
k,
s)
rendered the antiserumspecific
toreproductive
tissue.Preparation of
tissuehomogenatesfor
immunodiffusion plates
Tissues were obtained from mature rabbits
(Californian,
Dutch,
NZW),
guinea-pigs
(American),
hamsters(Golden Syrian),
mice(random-bred albino)
and rats(random-bred albino).
The non-rabbitspecies
used were in variousstages
of theoestrouscycle.
All tissues werefreed ofadhering
connective tissueand washed in cold
(4° C) phosphate-buffered
saline(PBS,
0-1m-P04, 0-85%
NaCl,
pH 7-3).
For eachspecies,
identical tissues werepooled, homogenized
in cold PBS to a final concentration of
25%
(w/v),
centrifuged
for 10 min at2500 gto remove tissue débris and the
supernatants
used in immunodiffusionplates.
Plasma,
serum and red blood cell(RBC)
preparations
obtained aspreviously
described(Sacco
&Shivers,
1973a)
were also tested.Detection
of antigens
Because of the
large
numbers oftissues examined and the smallquantity
of material available for sometissues,
a modification of the immunodiffusiontechnique
(see
Mansi,
1958)
was used. Microdiffusionplates
wereprepared
by pouring
1-5 ml ofa1%
solution ofagarose in distilledwateror PBS on to 1x3 in.glass
slides. Thegel
was cut with atemplate
togive
one central welland six smaller
peripheral
wells(9
mm betweenwells). Liquid
levels in wells weremaintainedby
fluidaddition,
giving
a total volume of 0-08to0-12 ml perwell. Plates were allowed to
develop
for 48to 72 hr at roomtemperature
(20
to22°C).
RESULTS Unabsorbed antisera
Antisera
produced
insheep against
theuterus of the Californian rabbitweretested in immunodiffusion
plates
against
saline extracts ofreproductive
andnon-reproductive
tissues fromCalifornian,
Dutch and NZW rabbits and similar tissues fromguinea-pigs,
hamsters,
mice and rats. The number ofpre¬cipitin
bandsproduced
by
such tests(shown
in Table1)
wereinterpreted
asComparison of reproductive
antigens
being
tested and the uterus ofthe Californian rabbit. Ahigh degree
ofcross-reactivity
was seen between the anti-uterus sera and extracts ofreproductive
and
non-reproductive
tissues from thethreebreeds of rabbit(compare
ColumnsA,
and C in Table1).
Alowerdegree
ofcross-reactivity
was observed whentheantiseraweretested
against
reproductive
andnon-reproductive
tissues fromthe othermammalian
species
(compare
ColumnsAtoC with columns D toG,
Table1).
Table 1. Tissue
antigens
from variousspecies
detectedby
unabsorbed anti-uterussera* Antigen Ovary Oviduct Uterus Adrenalgland Brain Duodenum Fat Heart Ileum Kidney Liver Lung Pancreas Plasma Red blood cells Serum Skeletal muscle Spleen Stomach Testis Species Californian rabbit 11 12 10 11 3 10 8 9 8 12 10 10 5 13 3 14 5 9 7 8 Dutch rabbit 11 12 12 8 10 8 12 8 10 11 16 7 15 3 10 9 11 C New Zealand White rabbit 12 10 12 10 4 10 5 11 10 10 9 10 6 15 7 15 5 10 6 9 DGuinea-pig Hamster Mouse
0 0 3 0 0 0 0 0 0 1 0 2 1 0 0 2 0 0 Rat
Each numeral indicates the maximum number ofprecipitin bands observed in im¬
munodiffusionplateswhen theantiserawereallowedto reactagainstaparticulartissue.
Eachreactionwastestedatleasttentimes and involved theuseofdifferentpreparations
ofreactants.
*Prepared
againstCalifornian rabbituterus.
(—) =Notexamined.
To determine whether the low
degree
ofcross-reactivity
observed between anti-uterusseraand thenon-rabbit tissueswasunique
totheuterus, unabsorbedantisera
against
ovary and oviduct were examined. Both theseantisera,
whenallowed to react with the non-rabbit
tissues,
produced
results similar to those observed for the anti-uterus sera, i.e.they
expressed
ahigh degree
ofcross-reactivity
with the rabbit tissues(Tables
2 and3,
Columns AtoC)
and a lowdegree
ofcross-reactivity
with thenon-rabbit tissues(Tables
2 and3,
Columns D toG).
Such results indicated that the tissues ofthe non-rabbitspecies
con¬tained few
antigens
capable
ofcross-reacting
with antiseraprepared
against
these threerabbit tissues.Absorbed antisera
Since the -, k- and s-absorbed antisera have been shown to be
specific
forantigens
present
only
inCalifornianrabbitreproductive
tissue(Sacco
&Shivers,
1973a),
such antisera were allowed to react with tissues from otherspecies
todetermine whether
reproductive
tissue-specific
antigens
were alsopresent
inthese
species.
The results obtained when such absorbed antisera were allowed to reactagainst
the variousreproductive
tissues are summarized in Table 4.Table 2. Tissue
antigens
from variousspecies
detectedby
unabsorbedanti-ovary
sera* Antigen Plasma Kidney Spleen Lung Species Californian rabbit 15 14 10 13 Dutch rabbit 14 12 12 12 C New Zealand White rabbit 14 12 12 11 DGuinea-pig Hamster Mouse Rat
Each numeral indicates the maximum number ofprecipitin bands observedin
im-mudodiffusionplateswhen the antiserawereallowedtoreactagainstaparticulartissue.
Eachreactionwastestedatleasttentimesand involved theuseof differentpreparations
ofreactants.
*Prepared againstCalifornian rabbit
ovary.
Table 3. Tissue
antigens
from variousspecies
detectedby
unabsorbed anti-oviduct sera* Antigen Plasma Kidney Spleen Lung Species Californian rabbit 13 12 10 11 Dutch rabbit 11 II 12 13 C New Zealand White rabbit 10 11 9 13 DGuinea-pig Hamster Mouse Rat
Each numeral indicates the maximum number ofprecipitin bands observed in im¬
munodiffusionplateswhen theantiserawereallowedtoreactagainstaparticulartissue.
Eachreactionwastestedatleasttentimes and involved theuseofdifferentpreparations
ofreactants.
*PreparedagainstCalifornian rabbit oviduct.
Each absorbed antiserum was allowed to react
only against homologous
anti¬gens fromeach of the
species (e.g. anti-ovary
versusovarybutnotversusoviduct oruterus)
andconsequently
no data arepresented
forcross-reactions betweenthe three
reproductive
tissuesforanyparticular
absorbed antiserum. The three absorbed antiseraproduced
noantigen-antibody precipitin
bandswhen allowedComparison of reproductive
antigens
to reactwith the sixteen
non-reproductive
tissues orthe testispreparations
fromeach of the different
species.
Controls
For
controls,
thevarious rabbit and non-rabbit tissueswere allowed to react in immunodiffusionplates
withsheep
and rabbit control sera, PBS and PBSwhich had been
subjected
to the p-, k- ands-absorption
procedure.
Neither of the two controlsera nor the PBSproduced
precipitin
bandsagainst
anyof theTable 4.
Reproductive tissue-specific antigens
presentin differentspecies
Reactants
Anti-ovarysera*versus
ovary
Anti-oviduct sera*versus
oviduct
Anti-uterus sera*versus
uterus Species Californian rabbit Dutch rabbit New Zealand White rabbit Guinea-pig
Hamster Mouse Rat
Numeralsrepresentthe numbers ofantigen-antibody precipitinbands observed in immuno¬ diffusionplateswhen the antiserawereallowed toreact againstthehomologousreproductive tissuefromthe differentspecies.Each reactionwastestedatleast fifteen times and involved the
useof differentpreparationsofreactants.
*Preparedinsheep againstCalifornian rabbitreproductivetissue and absorbed with Califor¬
nian rabbitplasma, kidneyandspleen.
reproductive
ornon-reproductive
tissues.Phosphate-buffered
saline absorbedwithp,
k,
sdidproduce
'non-immuneprecipitin
bands'(Niece
&Barrett,
1963)
against
certain tissues. These non-immuneprecipitin
bands were subtractedfrom the total number of bands
produced by
the absorbed antisera.The
antigens
represented
in Table 4 do not include these non-immuneprecipitin
bands.DISCUSSION
Theresults of this
investigation
have shown that unabsorbed antiseraprepared
against
Californian rabbitovary,oviduct anduterusexpressed
ahigh degree
ofcross-reactivity
for thetissuesof thetwoheterologous
breeds of rabbits testedbut a lowdegree
ofcross-reactivity
forhomologous
tissues of theguinea-pig,
ham¬ster, mouse and rat. The numbers of
precipitin
bands observed between the antisera andanyparticular
rabbit tissueweresimilar,
indicating
the presence of commonantigens
inhomologous
tissues of the three breeds of rabbit. Insomeinstances, however,
moreprecipitin
bands wereobservedagainst
a tissue of theheterologous
breed rather thanagainst
thecorresponding
Californian tissue. It has been established inaprevious
paper(Sacco
&Shivers,
1973a)
that many ofthe
precipitin
bandsproduced by
the unabsorbed antiseraactually
consist offamilies of
bands,
i.e. severalantigen-antibody complexes
present
in the sameprecipitin
bands observed between an antiserum and atissue extract indicatedonly
the minimum number ofprecipitating antigen-antibody
complexes.
Ifallantigen-antibody complexes
present
in the immunodiffusionplates
could bedistinguished,
there wouldprobably
be a much closer correlation in bandnumbers
produced against
any onetype
ofrabbit tissue.The occurrence of non-immune
precipitation
in immunodiffusion testsinvolving
tissue extracts has beenreported (Berenbaum,
Kitch &Cope,
1962;
Niece &Barrett,
1963;
Clausen,
1969).
Such non-immuneprecipitin
bandsareprobably
due tolipid,
lipoprotein
orlipopolysaccharide
components
of low watersolubility
or to enzymecomponents
(Clausen, 1969).
All theantigenic
preparations
used in thepresent
investigation
as well as the p-, k- ands-absorbed antisera and controls would include these
types
ofcomponents.
The lowdegree
ofcross-reactivity displayed by
the unabsorbed antisera for theheterologous
tissues was notunexpected. Electrophoretic analysis
has shown(Deutsch
&Goodloe, 1945; Moore,
1945)
thatappreciable
andspecific
characteristic differences occur in the distribution and relative
proportions
ofblood
proteins
amongdifferentspecies
of animals.Furthermore,
immunological
dataindicate that the occurrenceandintensity
of cross-reactionsamong anti¬gens from different
species
parallel
thedegree
ofzoological relationship
between thespecies
(Kabat
&Mayer, 1964). Consequently,
lesscross-reactivity
wouldbe
expected
between aparticular
antiserum and tissues from differentspecies
ascompared
totissues from thehomologous
species.
However,
the observationthat
antigen-antibody
precipitin
bands wereproduced
in reactionsinvolving
plasma-, kidney-
andspleen-absorbed
antisera and several ofthereproductive
tissues from the non-rabbitspecies
is of interest. Such bandswereinterpreted
asindicating
the presence ofantigens specific
to thesereproductive
tissues sincethe absorbed antisera did not
produce antigen-antibody precipitin
bandsagainst
anynon-reproductive
tissue tested. If thesetissue-specific antigens
areinvolved in
reproductive
processes, then antiseraprepared against
these anti¬ gensmight
be useful infertility
control.Indeed,
the results of thepresent
investigation
suggest
that antiseraprepared against tissue-specific
antigens
inone
species
may beeffectively
utilized in tests forcontrolling fertility
in otherspecies.
ACKNOWLEDGMENTS
This work was
supported by
agrant
(HD 04020)
from the National Instituteof Child Health and Human
Development
andby
a National Institutes ofHealth Predoctoral
Fellowship (5
FOI GM46521).
REFERENCES
Berenbaum,M. C, Kitch, G. M. &Cope, W. A. (1962) Spurious'auto-immune' reactions in
gel-diffusionplates.Nature,Lond. 193,81.
Clausen, J. (1969) Immunochemical techniquesfor the identification and estimation ofmacromolecules. In:
LaboratoryTechniquesinBiochemistryand MolecularBiology,Vol. 1,pp.419,499. Eds. T.S.
WorkandE. Work. American ElsevierPublishing Co.,NewYork.
Deutsch,H. F. & Goodloe,M. B. (1945) An electrophoreticsurveyof various animalplasmas. J'.
Comparison of
reproductive
antigens
Kabat,E. A. &Mayer,M. M. (1964) Experimental immunochemistry,2ndedn,p.22.Charles C.Thomas, Springfield,Illinois.
Mansi, W. (1958)Slidegeldiffusionprecipitintest.Nature,Lond. 181, 1289.
Moore,D. H. (1945) Speciesdifferencesinserumproteinpatterns.J.biol. Chem.161,21.
Niece, J.L. &Barrett,J.T.(1963)Non-immunegel precipitintestswithanenzymeantigen.Nature,
Lond. 197, 1021.
Sacco, A. G. &Shivers, C. A. (1973a) Antigensof the rabbitovary, oviduct anduterus.J.Reprod. Fert.32,403.
Sacco, A. G. & Shivers, C. A. (1973b) Localization oftissue-specific antigens in the rabbitovary, oviduct anduterusbydie fluorescentantibody technique.J. Reprod.Fert.32,415.