Table Of ContentZOOLOGICAL SCIENCE 11: 83-87 (1994) 1994Zoological SocietyofJapan
Immunohistochemical Localization of Epidermal Growth Factor
in the Developing Rat Gonads
Yasuhiko Kanno, Satoshi Koike1 and Tetsuo Noumura2
Department ofRegulation Biology, Faculty ofScience, Saitama University,
Urawa, Saitama 338, and 1Upjohn Pharmaceuticals Limited,
Wadai, Tsukuba, Ibaraki300-42, Japan
—
ABSTRACT Epidermalgrowthfactor(EGF)isknowntohavevariousendocrine,paracrineandautocrinerolesinadult
mammalian tissues. In order to clarify the participation of EGF in rat gonadal differentiation, immunohistochemical
localizationofEGFaschronologicallystudiedin perinatal ratgonads. Sprague-Dawleyratgonadsfromgestationalday
(GD) 13 to postnatal day (PD) 21 were fixed in acetic acid-free Bouin's solution and stained with a polyclonal antibody
againstmouseEGFbyusingavidin-biotincomplextechnique. Immunohistochemicalreactivitywaspositiveinalmostall
celltypesintheprenatalmalegonads. MalegermandLeydig/interstitialcellsshowedapositivereactivityfromGD15to
21. Slight and moderate staining were seen in the Sertoli/supporting cells from GD 13 to 21. After birth, positive
expression was not seen in any types ofcells in male gonads except for germ cells on PD 21. On the other hand, in
prenatalfemalegonadspositivesignswerefoundintheinterstitialcellsfromGD 14to21andinthegranulosacellsonGD
21. Duringthepostnatal periodfrom PD5 to21, thegranulosa andthecacellswere slightlypositive andthe interstitial
cells moderately positive. Wolffian ducts in males and Miillerian ducts in females were stained during the prenatal
period. These results indicate that EGFshows stage-specific patterns ofexpression in the developing rat gonadal cells
and may be involved in the rat gonadal development and diffferentiation.
INTRODUCTION MATERIALS AND METHODS
Epidermal growth factor (EGF), a single-chain poly- Aminals
peptide of53 amino acid residues, was first isolated from the Crj: CD (Sprague-Dawley)ratsfrom 13to20weeksofagewere
submandibular gland of male mice [8]. This peptide is a housed in constant temperature (23±1°C), relative humidity (55+
potent mitogen for a wide variety ofcell types in vivo and in 10%) and light-dark cycle (light on 7:00-19:00). Animals were
vitro and distributes in various tissues and fluids in mammals given a commercial diet (CRF-1, Charles RiverJapan Co.) and tap
[7]. EGF is mainly synthesized in the submandibular gland wateradlibitum. Cohabitation was done in the evening ofvaginal
proestrus in the 1:1 basis of male: female. In the next morning,
ofmice [5] and rats [22], andits synthesisisunderthe control copulation was checked by the presence of sperm in the vaginal
of androgens [6] and thyroid hormones [9]. smear. The day when sperm-positive smear was found was desig-
EGF is involved in many physiological functions in the nated asGD0, andthe daywhenlitterwasfoundwasdesignated as
adult gonads [24]. EGF reduced follicle-stimulating hor- PD0.
mone (FSH)-stimulatedtestosteroneproductioninratLeydig
cells in vitro [15], which was due to a regulation in 17/3- Preparation oftissuesforimmunostaining
hydroxylase activity [25]. EGF also attenuated FSH- DamsweresacrificedfromGD 13to21 andneonatesonPDs5,
stimulated aromatase activity in the Sertoli cells [2] and the 11 and21 bydiethyletheranesthesia. Thegonadsandgenitalducts
granulosa cells [15], but stimulated Sertoli cell proliferation dissected from more than three fetuses and pups in different litters
[16]. FSH-stimulated inhibin production in the Sertoli cells werefixedinaceticacid-freeBouin'ssolutionforafewhoursat4°C.
ThesexesoffetusesweredeterminedasdescribedbyAgelopoulouet
[20] and plasminogen activator production in the granulosa
cellswereincreasedbyEGF[10]. Althoughbasicproperties calo.nc[1e]n.traTthieonnsthoefteitsshuaensolweraenddexhyyldernae,tedemtbhreodudgehdaisneripeasraofffignraadnedd
and functions of EGF have been reported in adults, little is sectionedin5//mthickness. Themaleratsubmandibularglandson
known about its contribution to the fetal gonadal develop- PD35werealsodissectedandfixedinthesamefixative,forchecking
ment and diffferentiation. the specificity ofthe antibody used.
InordertoclarifytheparticipationofEGFindeveloping
rat gonads, immunohistochemical expression of EGF was Immunohistochemistry
chronologically studied in the fetal and prepubertal rat Sections were deparaffinized with xylene and hydrated in de-
gonads from gestational day (GD) 13 to postnatal day (PD) creasing concentrations of ethanol. The sections were incubated
mM
21. with 0.5% periodic acid (Sigma Chemical Co.) in 50 Tris-
bufferedsaline(TBS,pH7.6,WakoPureChemicalIndustries,Ltd.)
at room temperature for 30min to block endogenous peroxidase.
Accepted December 17, 1993 Sections were subsequently rinsed with 50mM TBS for 20min,
Received October21, 1993 blockednon-specificstainingwith 1.5%normalgoatserumin50mM
2 To whom reprint requests should be addressed.
84 Y. Kanno, S. Koike and T. Noumura
TBSfor20min. Then, sectionsincubatedovernightat4°Cwiththe ized antibody did not stain any cells. Therefore, these
polyclonal antibody against mouse EGF raised in the rabbit (Col- results showed that this polyclonal antibody specifically
laborative Research Inc.) at 0.02mg/ml in 10mM PBS. Dose stained EGF-containing cells, because the rat submandibular
response studyindicated thatthisconcentrationofthe antibodygave gland was confirmed to contain EGF-positive cells [22].
optimal labellingresults. Follwingthisincubationthe sectionswere
rinsed with TBS and then treated with 0.5% biotinylated goat
daintliu-treadbbiint5s0emcoMndaTrBySanftoirbo3d0ymi(nVecattorroLoamb,teImnpce.ratAurBeC. kiStecEtliiotne)s ImmunIomhmiusntoohcihsetmoicchaelmilcoaclaleixzaptrieosnsionofEGFinthedevelop-
were again washed in TBS and subsequently incubated with 2% ing rat gonads was summarized in Table 1. In male gonads,
avidin-biotin complex (Vector Lab, Inc, ABC kit Elite) in 50mM many germ cells were moderately stained from GD 15 to 21
TBS for 40min at room temperature. Avidin and biotin were and PD 21 (Fig. 2.A-C). A fewSertoli/supportingcells and
prepared at least 30min before applied to the sections to allow the almost all Leydig/interstitial cells showed positive reactivity;
complexto form. The sectionswere againwashedinTBS, andthe the staining intensity was moderate or slight in Sertoli/
bound antibody was visualized with 0.05% 3,3'-diaminobenzidine supporting cells from GD 13, the time when the supporting
tetrachloride (Dojindo Laboratories) in 50mM TBS (pH7.2) and cells were firstly recognized by histological examination, to
0.02% hydrogen peroxide for 5min. These sections were counter- GD 21 and marked in Leydig/interstitial cellsfrom GD 15 to
stained with hematoxylin. 21 (Fig. 2.A-C). Ontheotherhand, infemalegonads, afew
Controls included (a) replacing the primary antibody with nor- GD
interstitial cells expressed slight reactivity from 14 to 21
pmrael-irnacbubbiattesderouvmer,ni(gbh)tuasti4n°gCtwhiethp0r.i0m4armyg/amnltimbooudysethEaGtFha(dChbeemie-n and moderate from GD 21 to PD 21 (Fig. 2.E-G). A few
conInternational,Inc.)beforethismixturewasappliedtothesection theca cells slightly stained from PD 5 to 21 and a few
to check specificity of the primary antibody, and (c) omitting the granulosa cells slightly stained from GD 21 to PD 21 (Fig.
primary antibody to check the specificity ofthe secondary antibody. 2.E-G). But the germ cells in females were not shown any
positive reactivity during the experimental period (Fig. 2.E-
G).
RESULTS
The epithelial cells of the genital ducts were stained in
Specificity ofantibody sex-specific manner during the prenatal period: the Wolffian
Preparations which were stained with the antibody to ductsinmaleswere moderatelyormarkedlystainedfromGD
EGF, and with the immunoneutralized antibody by pre- 16 to 21 (Fig. 2.D) and the Mullelrian ducts in females were
incubated with the antigen were shown in Fig. 1. EGF slightly or moderately stained from GD 16 to PD 21 (Fig.
antibody stained the granular convoluted tubule cells of 2.H).
submandibular gland in male rats on PD 35, but the neutral-
3S&S
A
Fig. 1. Demonstration ofstaining specificity. Male rat submandibular glands on PD 35 incubated with the EGF antibody
(A), or with the neutralized antibody (B). Staining is seen in the granularconvoluted tubule cellsofA, but those ofB.
Bar: 50/*m.
Fig. 2. Immunohistochemical localization ofEGFin the gonadofperinatal rats. (A) Malegonadon GD 14. TheSertoli/
supportingcells (arrow) show slight staining. (B) Male gonad on GD 17. In the Leydig/interstitial cells(arrow head),
and the moderate stainings in the germ (thick arrow) and the Sertoli/supporting cells (arrow) express marked staining.
(C) Malegonadon PD5. Gonadalcellsdonotexpressanypositivesign. (D) Male Wolffianduct andon GD 17. The
epithelial cells (arrow) show marked staining. (E) and (F) Female gonads on GDs 14 and 17. The interstitial cells
(arrow) express slight staining. (G) Female gonad on PD 5. The interstitial cells (arrow head) are stained moderately
and the granulosa (arrow) and theca (thich arrow) cellsslightly. (H) Female Miillerian duct on GD 17. The epithelial
cells (arrow) are moderately stained. All sections are shown in the same magnification. Bar: 50//m.
EGF Expression in Developing Rat Gonads 85
86 Y. Kanno, S. Koike and T. Noumura
Table 1. Immunohistochemical localization of EGF in the developing rat gonad
Male Female
Gonad Duct Gonad Duct
G S P L W M G Gr T I W M
GD 13 - +/- * * - * - * * * - *
14 - +/- * * - - - - * +/- - -
15 ++ +/- * +++ - - - - * +/- - -
16 ++ +/- * +++ ++ - - - * + - ++
17 ++ ++/- * +++ +++ * - - * + - ++
18 ++ ++/- - +++ ++ * - - * + - +
19 ++ ++/- - +++ ++ * - - * + * +
20 ++ ++/- - +++ ++ * - - * + * +
21 ++ +/- - +++ ++ * - +/- * ++ * ++
PD 5 - - - - - * - +/- +/- ++/- * ++
11 - - - - - * - +/- +/- ++ * +
21 ++ +/- - - - * - +/- +/- ++ * +
GD: Gestational day, PD: Postnatal day, G: Germ cell, S: Sertoli/Supporting cell, P: Peritubular cell, L: Leydig cell, W:
Wolffian duct, M: Mullerian duct, Gr: Granulosa/Supportingcell,T: Thecacell, I: Interstitial/Stromalcell, Grade, —: Not
detectable, +: Slightbutabovebackgroundlevels, ++: Moderate, +++: Markedstaining, *:Thecellsortissueswerenot
found in that day.
known to participate in physiological roles on the ovary, at
DISCUSSION
least, during the prenatal and early postnatal period. The
EGF
Differentiation and development ofgonads are complex positive staining in these cells suggests that is likely to
events which involve various cell-cellinteractions. EGFisa mediate the ovarian functions in autocrine and/or paracrine
potent mitogen [12, 13] and affects to gonadal steroid and fashions. EGF stimulates or inhibits the immature rat gra-
peptide syntheses [15, 26]. Male germ cells showed a posi- nulosa cell proliferation depending on the presence or abs-
tive reactivityfrom GD 15. Germ cells in males are encom- ence of FSH [3]. In addition, the theca cells produce an
passed by the somatic cells from GD 14 ([11] and this EGF-like substance, which may regulate the granulosa cell
experiment) and thereafter undergo mitotic division. functions [23]. Togetherwith these results, positive staining
Therefore, positive expression from GD 15 indicates that in the granulosa and theca cells during the perinatal period
EGFmayhaveafunction inthegermcellproliferationduring suggests that EGF contributes to the follicle formation
the fetal period. through the granulosa and theca cell poliferation.
The positive reactivityto anti-EGFantiserumwasshown The immuno-reactivity was seen in the Wolffian ducts in
from GD 13 in the somatic cells of the developing gonads. malesand Mullerian ducts in females. EGFplaysimportant
The supporting and interstitial cells in the gonads proliferate roles in the male and female reproductive-tract development
rapidly during the fetal period [19]. Therefore, the express- duringthe perinatalorcritical period [4, 14]. Therefore, the
ion in the Sertoli/supporting and Leydig/interstitialcellsmay immuno-positive expression in the gonoducts in this stage
indicate that EGFstimulatesthe proliferation ofthesegonad- supports the previousfindings that EGFhas been reported to
al somatic cells as well as the germ cells. EGF decreased beconcerningwiththedevelopment anddifferentiation in the
FSH-stimulated testosterone production by regulating 17/3- gonoducts.
hydroxylase activity in Leydig cells in vitro [15, 25]. Fetal Other growth factors have also been reported to partici-
rat testes have steroidogenic activity during the prenatal pate in the gonadal development in rat fetuses. Transform-
period in vitro [21]. So positive reactivity in the Leydig/ ing growth factor-/? (TGF-/?) localized in many types ofcells
interstitialcellsindicatesthat EGFmaycontribute tothe fetal in developing fetal gonads [18], like EGF. However, in-
Leydig cell steroidogenesis. But the marked staining of the hibin-a, a member ofTGF-/?superfamily, localized in Sertoli
Leydig/interstitial cells during the prenatal period was lost cells at the time ofseminiferous tubule formation and in the
after birth. This may be due to a regression offetal Leydig Leydig cells during the late gestational period [17].
cells which begin shortly before birth and replace to adult- Together with these results, growth factors could play an
type Leydig cells after birth in the rat [11]. important role in the gonadal development during the peri-
The interstitial cells in female gonads have not been natal period. The mechanisms and roles of these growth
EGF Expression in Developing Rat Gonads 87
factors on gonadal development are under investigation. 13 GospodarowiczD,BialeckiH(1979) Fibroblastandepidermal
growth factors are mitogenic agents forcultured granulosacells
of rodent, porcine, and human origin. Endocrinology 104:
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