ZOOLOGICAL 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. 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