INTRODUCTION

Steroid hormones possess various physiological activities suchas maintenance of ion balance and glucose concentration in theblood and sexual differentiation. It is well known that sixdistinct forms of P-450s are involved in the synthetic pathwayof steroid hormones from cholesterol. These steroidogenic P-450 genes, unlike the other P-450s, are expressed specificallyin the steroidogenic tissues and are under the control of tropicpeptide hormones secreted from the pituitary (Dufau, 1988;Richards and Hedin, 1988; Simpson and Waterman, 1988).Efforts have been made to elucidate the mechanisms of cAMP-dependent and the steroidogenic cell-specific gene regulationof the steroidogenic P-450 genes (CYP genes (Nelson et al.,1991)). Several studies revealed that CRE (cAMP responsiveelement (Montiminy et al., 1986) on the promoters of

CYP11B(P-450(11

β) gene) (Rice et al., 1989; Honda et al., 1990),

CYP11A (P-450(SCC) gene) (Inoue et al., 1991; Takayama etal., unpublished data) and CYP21 (P-450(C21) gene)(Watanabe et al., 1993) is necessary for their responsivenessto cAMP. Although cAMP-responsive regions were identifiedwithin the promoters of the other steroidogenic P-450 genes,no CRE has been found (Kagawa and Waterman, 1991; Zangeret al., 1991; Toda et al., 1992; Wilson et al., 1993; Fitzpatrickand Richards, 1993). It has remained unclear whether thesteroidogenic P-450 genes are controlled by CRE or by otherunknown regulatory sequences. However, interesting resultswere obtained regarding the tissue-specific transcriptional reg-ulation by the investigation of a steroidogenic tissue-specifictranscription factor. The transcription factor, Ad4BP (also des-ignated SF-1 (Ikeda et al., 1993)), was purified from bovineadrenal cortex (Morohashi et al., 1992), followed by theisolation of the corresponding cDNA (Honda et al., 1993). Thenucleotide sequence revealed that Ad4BP, carrying a Zn-finger

2787Development 120, 2787-2797 (1994)Printed in Great Britain © The Company of Biologists Limited 1994

We investigated the expression of Ad4BP (also known asSF-1), a transcription factor regulating steroidogenic P-450genes, in the steroidogenic tissues such as adrenal glands,testes and ovaries through the prenatal and postnatal lifeof rats. Ad4BP was detected in the primordial adrenalglands and gonads of the 13.5 day postcoitum (d.p.c.) fetus.After the appearance of Ad4BP, a steroidogenic P-450 (P-450(SCC)) was also detected in the adrenal glands and itsamount increased gradually. In the fetal gonads of 14.5d.p.c., a significant amount of Ad4BP was detected in thesomatic cells of the testes, whereas only a trace amount waspresent in the ovaries. The sexually dimorphic expressionof Ad4BP continued throughout the neonatal age. Drasticalterations occurred during the first to third week ofpostnatal age accompanied by functional and structuralchanges of the gonads. The expression of Ad4BP in thetestes attained a maximal level one week after birth anddecreased markedly thereafter. By contrast, increase of

Ad4BP in the ovary was detected after the first postnatalweek. Expression of P-450c17 showed a good correlationwith the proliferation of Leydig cells in the testes and thecacells in the ovaries. Immunohistochemical studies revealedthe presence of Ad4BP in Sertoli cells as well as Leydig cellsup to the pubertal age. In the adult rat testis, however,staining of Sertoli cells decreased significantly. Ad4BP wasdetected in granulosa, theca, corpus luteum and interstitialgland cells in the ovary although the expression levels ingranulosa cells varied among follicles. It is suggested thatthe

Müllerian inhibitory substance gene may be a target ofAd4BP since this gene has a conserved Ad4-binding sitewithin the promoter, which is recognized by Ad4BPexpressed in the fetal testes.

Key words: Ad4BP, steroidogenic cell, P-450, sex differentiation,Müllerian inhibitory substance

SUMMARY

Sex-dependent expression of a transcription factor, Ad4BP, regulating

steroidogenic P-450 genes in the gonads during prenatal and postnatal rat

development

Osamu Hatano1, Koichi Takayama2, Tsuneo Imai3, Michael R. Waterman4, Akira Takakusu1, Tsuneo Omura2

and Ken-ichirou Morohashi2,* 1Department of Anatomy, Nara Medical University, Kashihara, Nara 634, Japan2Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Higashi-ku, Fukuoka 812, Japan3Department of Surgery II, Nagoya University School of Medicine, Tsurumai-cho, Showa-ku, Nagoya 466, Japan4Department of Biochemistry, Vanderbilt University School of Medicine, 607 Light Hall, Nashville, Tennessee 37232-0146, USA

*Author for correspondence

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structure as a DNA-binding domain, is a member of thesteroid/thyroid hormone receptor supergene family (Evans,1988). The presence of Ad4BP mRNA was confirmed in thetestis, ovary, placenta, brain and adipose tissue as well as theadrenal gland, all of which express the steroidogenic P-450s(Honda et al., 1993). Moreover, in situ hybridization (Ikeda etal., 1993) and immunostaining (Morohashi et al., 1994) withthe adrenal gland, testis and ovary reconfirmed the rigid speci-ficity of the expression of Ad4BP only in adrenocortical cellsin the adrenal gland, Leydig cells in the testis, and granulosacells and theca cells in the ovary. Functional analyses ofAd4BP were also performed by the identification of thenucleotide sequences recognized by Ad4BP. Interestingly, thenucleotide sequences recognized by Ad4BP were present in allthe promoters of the steroidogenic P-450 genes, whichsuggested the functional significance of the transcription factor(Rice et al., 1991; Morohashi et al., 1992). A recent study, inwhich both the CYP11A and CYP11B gene promoters wereactivated even in the non-steroidogenic cells such as CV-1 andPC-12 cells by the transfection of an Ad4BP expression vector,finally elucidated the function of this factor as being involvedin the transcriptional mechanisms governing the steroidogenictissue-specific transcription of the P-450 genes (Morohashi etal., 1993).

Sex steroids synthesized in gonads are necessary for the sexdifferentiation of the fetus and for sexual maturation duringprepubertal to pubertal ages. The sex steroids also regulate thespermatogenesis and oogenesis in adult gonads in the copres-ence of the peptide hormones secreted from pituitary. In thesesteps of sexual maturation, somatic cells in the gonads, Sertolicells and Leydig cells in testis, and granulosa cells and thecacells in ovary, play important roles in the development of thegonadal tissues as well as steroidogenesis (Byskov, 1988). Itis now widely accepted that sexual differentiation (testiculardifferentiation) is triggered by the transient expression of SRYin the somatic cells but not in germ cells of the urogenital ridge(Sinclir et al., 1993). Namely, the somatic cells in which SRYis expressed differentiate into Sertoli cells and Leydig cells,whereas the somatic cells in which SRY is not expressed dif-ferentiate into granulosa cells and theca cells. The two groupsof somatic cells successively acquire sex-dependent functionsincluding synthesis of sex steroids, which should occur distalto SRY expression.

In this study, we examined the expression of Ad4BP and thesteroidogenic P-450s in rat tissues from prenatal to postnatalperiods. Sex-dependent expression of Ad4BP observed in thegonads just after sex differentiation suggested that the Ad4BPgene might be one of the target genes of SRY. Similarly,important functions of Ad4BP were also suggested in thepubertal period of rats.

MATERIALS AND METHODS

Immunoblot analysisTissues were homogenized with lysis buffer containing 50 mM Tris-HCl buffer (pH 7.5) and 2% SDS, followed by sonication to disruptcellular DNA. Protein concentration was determined as described(Kalb and Bernlohr, 1977). Protein samples separated by SDS-PAGEwere blotted onto a nitrocellulose filter, which then was used fordetection of Ad4BP with the antiserum to bovine Ad4BP (Morohashi

et al., 1994). Before the filter was reused for detection of P-450(SCC)or P450c17, it was incubated with a solution containing 2% SDS, 100mM 2-mercaptoethanol and 50 mM Tris-HCl buffer (pH 6.8) for 30minutes at 55°C to denature the antibodies (Morohashi et al., 1994).ECL western blot reagents (Amersham, UK) were used for detectionof the signals. Antibody against bovine P-450(SCC) was kindlysupplied by Dr Takayuki Hara (Nakamura Gakuen College, Fukuoka).Antibody against human P450c17 was described previously (Imai etal., 1993).

ImmunohistochemistryTissues of prenatal and postnatal rats were frozen in Tissue Tek(Miles; Elkhart, IN, USA). 6 µm sections were fixed with a solutionof acetone/methanol (1:1) at −20°C for 15 minutes and used forimmunostaining with the antiserum to Ad4BP. Signals were detectedby the use of FITC-labeled secondary antibodies (Amersham, UK) asdescribed previously (Morohashi et al., 1994). For the detection ofalkaline phosphatase-positive cells, tissue sections were incubated inthe following solution for 5 minutes prior to incubation with theantiserum; 50 mM sodium bicarbonate buffer (pH 9.8) containing 10mM MgCl2, 28 mM NaCl, 0.4 mM nitro blue tetrazolium and 0.38mM 5-bromo-4-chloro-3-indoylphosphate. In an immunoadsorptionexperiment, antiserum to Ad4BP (1 µl) was incubated with thepurified Ad4BP (6 µg) which had been used for immunization(Morohashi et al., 1994), followed by the staining procedure describedabove.

Gel shift analysisEnd-labeled oligonucleotides, corresponding to −51/−69 bp on themouse Müllerian inhibitory substance gene upstream (Münsterbergand Lovell-Badge, 1991) and −337/−318 bp on the bovine CYP11Bgene promoter (Honda et al., 1990), were used as the probes for gelshift analyses with nuclear extracts prepared from fetal rat testes (19.5d.p.c.) and bovine adrenal cortex. The procedure of the gel shiftanalysis and the preparation of nuclear extract were as described(Morohashi et al., 1990).

RESULTS

Expression of Ad4BP in adrenal glands of fetal andpostnatal animalsIn the developing rat embryos around 14.5 d.p.c., adrenalglands appear in the peritoneal cavity. They are located in closeproximity to the upper poles of the kidney and the sexually dif-ferentiated gonad. Subsequently, the adrenal glands continueto develop keeping the positional relationship with the kidney(Kaufman, 1992). To assess the expression of Ad4BP in theadrenal glands, immunoblot analyses were performed withprenatal and postnatal animals. As shown in Fig. 1 (upperpanel), the adrenal glands isolated from the fetus of 14.5 d.p.c.already expressed significant amounts of Ad4BP and theexpression was slightly higher at 16.5 d.p.c. The level of theexpression was maintained until three weeks after birth andslightly decreased in adult animals. As we described previ-ously, steroidogenic P-450 genes have Ad4 sites in their regu-latory regions (Morohashi et al., 1992) and the genes areactivated by the presence of Ad4BP in a cAMP-dependentmanner (Morohashi et al., 1993). The expression of one of thesteroidogenic P-450s, P-450(SCC), was examined with theadrenal glands of the same time points (lower panel in Fig. 1).Although Ad4BP was detected at 14.5 d.p.c., only a traceamount of P-450(SCC) was found, followed by a gradualincrease during the fetal life. The expression of P-450(SCC)

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2789Ad4BP expression in gonads and adrenal gland

preceded by Ad4BP supported our finding that the P-450 genesare under the control of Ad4BP (Morohashi et al., 1994).Immediately after birth, the expression of P-450(SCC) wasreduced. This was probably caused by transient decrease of thecortical cells just after birth (Josimovich et al., 1954). P-450(SCC) increased again to the maximal level at three weeksof age when the cortex became thicker and was maintainedthereafter. No difference in the expression of Ad4BP and P-450(SCC) was detected between male and female animalsduring the fetal and the postnatal life.

Immunohistochemical studies with adrenal glandsof fetal and postnatal animalsThe expression of Ad4BP in the adrenal glands was examinedfurther immunohistochemically. The nuclei of the 14.5 d.p.c.fetal adrenal stained clearly, whereas those of other surround-ing tissues did not stain (Fig. 2A). On successive days, the cellsunstained with Ad4BP antiserum appeared in the medial area

(Fig. 2B,C), probably due to migration of neural cells (Roglerand Pintar, 1993). These two masses of the cells formed twodivided layers, stained cortex and unstained medulla after birth(Fig. 2D,E), although such clear layers were not found in thefetal adrenal glands. From two to eight weeks after birth, thecortical cells formed three distinct zonae, the zona glomeru-losa, the zona fasciculata and the zona reticularis, accompaniedby an increase in cell volume (Fig. 2E). During the period, thecontent of Ad4BP observed by the immunoblot analysisdecreased as described above. The increase in the amount oftotal cellular proteins was possibly responsible for the apparentdecrease of Ad4BP content. Immunohistochemical observa-tions revealed no significant differences in the expression ofAd4BP among adrenocortical cells at various developmentalstages. To confirm the specificity of the antiserum to Ad4BP,adsorption experiments using the purified Ad4BP wereperformed. As shown in Fig. 2F, all the signals completely dis-appeared, indicating the rigid specificity of the antiserum usedin this study.

Expression of Ad4BP in gonads of fetal andpostnatal animalsAs reported previously, Ad4BP was also detected in thesteroidogenic cells of the adult testis and ovary (Ikeda et al.,1993; Morohashi et al., 1994). In this study, we investigatedthe expression of Ad4BP during gonadal development.Gonadal sex of rats could be distinguished clearly at 14.5 d.p.c.by the presence or absence of testicular cords. Immediatelyafter the appearance of the testicular cords, the male gonad in14.5 d.p.c. fetus already showed significantly higherexpression of Ad4BP than female as shown in Fig. 3 (upperpanel). The level of Ad4BP in the testis increased at 16.5 d.p.c.and was maintained during fetal life, while that in the ovaryremained low. The sexually dimorphic expression continuedthroughout the neonatal period, followed by drastic changesduring sexual maturation. Although the testes of neonatalanimals sustained a high level of Ad4BP expression up to thefirst week, a marked decrease was observed at the third weekafter birth. Only a very small amount of Ad4BP was detectedwith the adult testis. In contrast, the ovaries became active inAd4BP synthesis on the seventh day after birth and continuedto be active until the third week. The expression of Ad4BP waslower in the adult ovary than in the 3-week-old animals. In bothtestis and ovary, sex-dependent alteration in Ad4BP expressionoccurred between the first and third weeks, accompanied bythe morphological changes as will be described below.

With the same samples of the gonadal tissues, we examinedthe expression of P450c17, which is involved in the synthesisof both androgens and estrogens. As shown in Fig. 3 (lowerpanel), P450c17 was expressed abundantly in the fetal testesfrom 16.5 to 20.5 d.p.c. After birth, however, the content ofP450c17 decreased gradually until one week although Ad4BPcontinued to be expressed at a high level. At the third weekafter birth, the content increased slightly again and was main-tained thereafter. Although P450c17 was not detected in theovaries of the prenatal and neonatal animals, a large amountwas detected in the ovaries of 3-week-old animals and a smallamount in the adult ovaries (data not shown). Since the CYP17gene could be the target gene of Ad4BP (Morohashi et al.,1992), the expression profile of the P-450 in the ovaries mightbe expected to show a good correlation with that of Ad4BP.

Fig. 1. Expression of Ad4BP and P-450(SCC) in rat adrenal glandsof various stages of prenatal and postnatal animals. Adrenal glandswere separated from various stages of prenatal (days postcoitum) andpostnatal (days after birth) rats of both sexes. Animals older thaneight weeks were used as adults. The same amounts of total tissuelysates (20 µg) were used for the immunoblot analyses using Ad4BPand P-450(SCC) antisera. After the detection of Ad4BP (upperpanel), the filter was incubated with the SDS-containing buffer todenature the antibodies as described in Materials and methods andreused for the detection of P-450(SCC) (lower panel). Ad4BP and P-450(SCC) are indicated by arrows. Prestained molecular weightmarkers (Bio-Rad, Laboratories, Richmond) were used.

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Immunohistochemical studies with gonads of fetaland postnatal animalsIn the previous report (Morohashi et al., 1994), we showed thatAd4BP was highly enriched in Leydig cells, which coincideswith the limited localization of the steroidogenic capacity inthe testis. However, the continued high expression level ofAd4BP before pubertal age described above did not seem tocorrelate with its localization in Leydig cells, since the numberof the cells reduces around birth (Bortolussi et al., 1990). In

the rat testis of 14.5 d.p.c. fetus, testicular cords are formed bythe alignment of somatic cells surrounding the germ cell aggre-gates. At this gestational age, Ad4BP is detected in the inter-stitial cells and the cells surrounding the germ cells (Fig. 4A).Some of the interstitial cells showed more intense signals thanthe others, while the germ cells were not stained. In the testesfrom 16.5 to 20.5 d.p.c. fetuses, seminiferous tubules becameorganized by alignment of Sertoli cells into a cylinder-likestructure (Kaufman, 1992). At these time points of gestation,

O. Hatano and others

Fig. 2. Expression and localization of Ad4BP in the adrenal glands of prenatal and postnatal animals. Frozen sections of the adrenal glandswere stained with the antiserum to Ad4BP, followed by FITC-labeled secondary antibodies. The adrenal glands in A-C were prepared from themale fetuses of 14.5, 16.5 and 20.5 d.p.c., while those of D and E were prepared from 2- and 8-week-old male animals. Antiserum preincubatedwith the purified Ad4BP was used for the staining of 14.5 d.p.c. adrenal gland (F) as described in Materials and methods. g, zona glomerulosa;f, zona fasciculata; r, zona reticularis; m, medulla. Bars, 80 µm.

2791Ad4BP expression in gonads and adrenal gland

two types of Ad4BP-positive cells stained with higher andlower intensity were clearly segregated (Fig. 4B,C,G), whichjudging from their distribution, seemed to be Leydig cells andSertoli cells, respectively. Since such different levels ofstaining were not observed in the genital ridge before 14.5d.p.c., the differentiation of the two cell types occurred during13.5 to 14.5 d.p.c. Immunoblot analysis indicated that theexpression of Ad4BP attained maximal level at the first weekafter birth, probably due in part to the increase in the numberof Sertoli cells, since that of Leydig cells decreased (Fig.4D,H). Regarding the function of the testis, the first cycle ofspermatogenesis takes place from the first week after birth,accompanied by a morphological alteration (Gondos andBerndtson, 1993). Judging from the intensity of the staining,the content of Ad4BP started to decrease in Sertoli cells in thesame period (compare Fig. 4D and E). The staining intensityof Leydig cells was apparently constant while the number ofLeydig cells increased again from three to eight weeks. These

alterations in cell numbers are consistent with observationsreported previously (Bortolussi et al., 1990). As describedabove, we observed a decrease of P450c17 content aroundbirth. The testosterone concentration in serum has beenreported to show a similar decrease around birth (Weisz andWard, 1980). The alteration of Leydig cell numbers seems tocorrelate well with the content of P450c17 in the testis and thatof testosterone in the serum.

Localization of Ad4BP in the ovaries was investigated at thesame time points as shown in Fig. 5. In the ovaries isolatedfrom 14.5 to 20.5 d.p.c. fetal rats, when the follicles were notyet formed, low intensity Ad4BP signals were detected (Fig.5A,B,C,G). For comparison, we prepared sections containingboth the adrenal gland and the gonad from 14.5 d.p.c. fetusesof the two sexes. The staining intensity of the ovary was sig-nificantly lower than that of the adrenal gland, whereas that ofthe testis was almost identical with the adrenal gland (comparethe staining intensities in Fig. 6A,B). Morphological changesstarted at around one week after birth with the appearance ofgrowing follicles. All the granulosa cells in preantral follicleswere stained, while those in the follicles at the earlier stageswere less intensely stained. Staining was also detected in theinterstitial theca cells located outside of the follicles (Fig.5D,H). The staining of the theca cells was more intense thanthe granulosa cells, which was similar to the observation of the14.5 d.p.c. testis. Interesting staining patterns were observedwith the ovary of 3-week-old animals, in which the Ad4BPexpression attained the maximal level as described above.Multiple layers of theca cells, which are a feature of 3-week-old animals but not adult animals, were stained clearly (Fig.5E). The high level of expression of P450c17 is possibly dueto the well-developed theca cells in that period of the ovariesbecause expression of P-450 is restricted to theca cells asreported (Rodgers et al., 1986; Sasano et al., 1989). Synthesisof estradiol was reported with the immature rat ovaries fromthe first to the third week of neonatal age (Meijs-Roelfs et al.,1975). The expression of P450c17 at the third week correlateswell with the presence of the synthetic activity of estradiol inthe immature rat ovaries. A characteristic staining was alsodetected in the granulosa cells of the 3-week-old animal. Whenthe staining of the granulosa cells was compared, the stainingintensities were not uniform among the follicles. Such a non-uniform expression of Ad4BP among the granulosa cells wasalso detected among the adult ovary. The corpus luteum cellsand the interstitial gland cells were also stained in the adultovary (Fig. 5F). To confirm the specificity of the antiserum toAd4BP, adsorption experiments were performed on the gonadsat several time points. None of the sections examined showedany signal (data not shown).

Expression of Ad4BP in the somatic cells of theearly gonadal tissuesUsing immunoblot analysis, sex-dependent expression ofAd4BP was already detected with gonads of 14.5 d.p.c. It iswidely accepted that the somatic cells in the gonads, in partic-ular the Sertoli cells in the testis, are necessary for early stepsof sex differentiation (Pelliniemi et al., 1993). To confirm theabove observation that Ad4BP was expressed in the somaticcells but not in the germ cells, histochemical staining wasperformed to detect alkaline phosphatase activity, which ishighly expressed in the plasma membrane of the primordial

Fig. 3. Expression of Ad4BP and P450c17 in the rat gonads ofvarious stages of prenatal and postnatal animals. Gonadal tissues,testes and ovaries, were separated from various stages of prenatal(days postcoitum) and postnatal (days after birth) rats. Animals olderthan eight weeks were used as adults. The same amounts of totaltissue lysates (20 µg) were used for the immunoblot analyses usingAd4BP and P450c17 antisera. After the detection of Ad4BP (upperpanel), the filter was incubated with the SDS-containing buffer todenature the antibodies as described in Materials and methods andreused for the detection of P450c17 (lower panel). Ad4BP andP450c17 are indicated by arrows. Prestained molecular weightmarker (Bio-Rad, Laboratories, Richmond) was used.

2792 O. Hatano and others

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2793Ad4BP expression in gonads and adrenal gland

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germ cells. In 14.5 d.p.c. fetal testis, germ cells were identifiedas alkaline phosphatase-positive cell aggregates. No stainingwith the Ad4BP antiserum was detected on the germ cells (Fig.7A). Staining by the antiserum was also not observed with thecells of mesonephric area as shown in Figs 4A and 5A. Thesame result was obtained with the gonads of a 13.5 d.p.c. fetus,in which gonadal primordium protrudes into peritoneal cavityfrom the dorsal wall as a genital ridge. The primordial germcells showing alkaline phosphatase activity and the somaticcells expressing Ad4BP were present in the genital ridge. Thetwo stainings were mutually exclusive. At this time point,adrenal primordium could also be detected in the dorsal wallas a cell mass stained with the antiserum to Ad4BP. Alkalinephosphatase activity was not detected, however, with the cellsin the area (Fig. 7B).

Müllerian inhibitory substance gene as one ofcandidates of target genes of Ad4BP in the fetalSertoli cellsIn previous (Ikeda et al., 1993; Morohashi et al., 1994) and thepresent studies, Ad4BP was detected in the steroidogenic cellsin adult animals, Leydig cells in the testis, and granulosa,theca, corpus luteum and interstitial gland cells in the ovary.

The expression of Ad4BP in such steroidogenic cells wasanticipated since Ad4BP had been identified as a transcriptionfactor regulating the steroidogenic P-450 genes (Honda et al.,1993; Ikeda et al., 1993; Morohashi et al., 1993). In this study,however, we observed a significant expression of Ad4BP inSertoli cells of testes of fetal and neonatal animals. Sertoli cellsdo not produce steroid hormones except for a limited period ofneonatal age (Dorrington and Armstrong, 1975). Accordingly,the Ad4BP in the Sertoli cells of the prenatal testes wasexpected to regulate genes other than the steroidogenic P-450genes. Judging from the spatial and temporal profiles of theAd4BP expression, the Müllerian inhibitory substance (MIS)gene could be one plausible candidate. MIS genes have beenisolated from four animal species (Cate et al., 1985; Münster-berg and Lovell-Badge, 1991; Haqq et al., 1992). Comparisonof the nucleotide sequences of the promoter regions revealedthat a possible Ad4BP-binding site was present approximately60 bp upstream from the TATA box in all the genes and thebinding site was highly conserved among the animal species(Fig. 8, upper panel). To characterize the binding factor(s) tothe putative Ad4 site, we performed gel shift analyses with thenuclear extract prepared from fetal testes. The original Ad4sequence identified in the promoter of the bovine CYP11B gene

O. Hatano and others

Fig. 6. Comparison of the staining intensities of Ad4BP between thegonads of the two sexes using adrenal gland as the control. A sectioncontaining both the gonadal tissue and the adrenal gland wasprepared from male (A) and female (B) fetuses of 14.5 d.p.c. andstained as described. ad, adrenal gland; te, testis; ov, ovary. Bars, 80µm.

Fig. 7. Relationship between the cells expressing Ad4BP and theprimordial germ cells. The testis of a 14.5 d.p.c. fetus (A) and thegenital ridge of a 13.5 d.p.c. fetus (B) were used for the staining withAd4BP antiserum and for alkaline phosphatase activity, whichmarked the primordial germ cells. ad, adrenal primordium; gr,genital ridge. Bars, 40 µm.

2795Ad4BP expression in gonads and adrenal gland

(Honda et al., 1990) and the nuclear extract prepared frombovine adrenal cortex were used as controls. The Ad4sequences in the CYP11B and the mouse MIS genes bothshowed retarded signals at the same position with the fetaltestes and the adrenal cortex nuclear extracts. The signals dis-appeared by the addition of the unlabeled probes as competi-tors. Ad4BP antiserum also inhibited the formation of theprotein-DNA complex with both nuclear extracts (Fig. 8, lowerpanel). These observations revealed that the Ad4BP detectedin the fetal testes could bind with the Ad4 site in the MIS gene.

DISCUSSION

Relationship between the expression of Ad4BP andsex differentiationAd4BP was originally identified as a steroidogenic cell-specific transcription factor able to activate the transcription ofthe steroidogenic P-450 genes, such as the CYP11A andCYP11B (Honda et al., 1993; Morohashi et al., 1993). In thisstudy, we investigated the spatial and temporal profiles of theexpression of Ad4BP in the rat gonads and adrenal glands ofboth sexes during the prenatal and postnatal periods. Sex-dependent expression of Ad4BP was observed from the 14.5d.p.c. fetal gonads, when the genital ridges start to differenti-ate depending on their genotypes. It is generally accepted thatmammalian sex differentiation is triggered by the expressionof SRY in the somatic cells of the undifferentiated genital ridge(Gubbay et al., 1990). In the case of mice, significant mor-phological changes from the undifferentiated gonad to the

testis are observed immediately after the transient expressionof Sry during 10.5 to 11.5 days of gestation (Koopman et al.,1990). Although the period of sexual differentiation of the twoanimals is not identical, it is likely that SRY is expressed inthe genital ridge of the rat fetus before 13.5 d.p.c. (Kaufman,1992). Accordingly, the sexually dimorphic expression ofAd4BP in fetal gonads suggested that the Ad4BP gene islocated downstream of SRY in the cascade of gene activationsnecessary for sex differentiation. This assumption is supportedby the observation that both genes, Ad4BP and SRY, wereexpressed in the identical lineage of the somatic cells in thegenital ridge.

In a recent study, Haqq et al. (1993) suggested that SRYregulates MIS and aromatase P-450 genes through directbinding to the gene promoters. We found the presence of anAd4 site in the promoters of the MIS genes. Although we havenot examined whether the Ad4 site is functional in vivo, thefollowing observations support the functional importance ofthe Ad4 site in MIS gene. (i) The Ad4 site is highly conservedin the gene promoters among various species. (ii) Ad4BPexpressed in the fetal testes has an ability to bind with the Ad4site of the MIS gene. (iii) The expression profile of Ad4BP issimilar to that of MIS even in the ovarian granulosa cells. (iv)It was demonstrated by transgenic animals that 2 kb upstreamregion of the human MIS gene, which contains the Ad4 site,was enough to drive the transcription of a reporter gene in aSertoli cell-specific manner (Peschon et al., 1992). Althoughthese observations do not contradict the assumption that SRYregulates the downstream genes such as those encoding MISand aromatase directly, the observations indicate another pos-

* * ***** ** ******* * * **Human (-87/-45) TGGGGGCGCCGGGCACTGTCCCCCAAGGTCGCGGCA---GAGGAGABovine (-85/-40) GGCTCACAGCAGGCACCAGCCTTCAAGGTCATGTCCCAGGAGGAGARat (-80/-38) AGCGCA-GCCAGGCACTGTCC-CCAAGGTCACCTC--AGGGGTTGAMouse (-83/-44) AGTGC----CAGGCACTGTCCCCCAAGGTCACCTT--TGGTGTTGA

CYP11B (-331/-323) CCAAGGTCCCYP11A (-37/-45) TCAAGGTCA

Fig. 8. The presence of an Ad4 site on the upstream region of theMüllerian inhibitory substance (MIS) gene. (Upper panel) Alignmentof the human (Cate et al., 1986), bovine (Cate et al., 1986), rat (Haqqet al., 1992) and mouse (Münsterberg and Lovell-Badge, 1991) MIS geneupstream regions. The possible Ad4 sites on the gene promoters areenclosed. The conserved nucleotides in all the MIS genes are indicated byasterisks. The Ad4 sites on the promoters of the bovine CYP11B and thehuman CYP11A gene are also shown in the figure. Numbers in theparentheses indicate the location upstream from the TATA box for MISgenes and the location upstream from the transcription initiation site for theCYP genes (Morohashi et al., 1992). (Lower panel) Gel-shift analysis withuse of the Ad4-containing oligonucleotide (−51 to −69 bp) in the mouse MISgene (MIS-Ad4) and the nuclear extract prepared from the 19.5 d.p.c. ratfetal testes. The original Ad4 site on the promoter of the bovine CYP11Bgene (11β-Ad4) (Honda et al., 1990) and the nuclear extract prepared frombovine adrenal cortex were used as controls. Unlabeled probes and antiserumto Ad4BP (Ab) were used for competition.

2796

sibility that SRY regulates the genes through the sex-dependentexpression of Ad4BP.

Ad4BP expression during the process of gonadalmaturationWe observed a characteristic expression profile of Ad4BPduring the process of the sexual maturation. In the Sertoli cellsof postnatal animals, higher expression of Ad4BP wasobserved in the limited period of prepubertal age. Testes startthe first cycle of spermatogenesis at around the first week afterbirth, which causes a rapid increase in germ cells at variousstages. As the function of Sertoli cells is different before andafter the first cycle of spermatogenesis (Gondos and Berndston,1993), it seems reasonable to assume that Ad4BP is necessaryfor the transcription of the genes indispensable for the functionof the Sertoli cells before the first cycle. Since the prepubertalSertoli cells have estrogen synthesis activity due to the functionof steroidogenic P-450(arom) (Dorrington and Armstrong,1975), the gene should be one of the target genes of Ad4BP inthis particular period in Sertoli cells. The expression of Ad4BPin Sertoli cells decreased dramatically from the first to the thirdweek of postnatal age. During the same period, aromataseactivity, Müllerian inhibiting substance (MIS) and α inhibin inthe Sertoli cells also decreased (Gondos and Berndston, 1993).The genes showing the temporal coincidence in the expressionwith Ad4BP, including the above three genes, are candidatesof the target genes.

In contrast, the content of Ad4BP increased in the ovariesof the neonatal animals from the first week after birth. Manypreantral follicles of early stages appeared, expressing Ad4BPin both the granulosa cells and the theca cells. As in the caseof fetal and neonatal testes, the MIS gene product appeared inthe granulosa cells in the preantral follicles (Ueno et al., 1989a;Münsterberg and Lovell-Badge, 1991), which also suggeststhat MIS gene is one of the target genes of Ad4BP even in theovary. In the growing ovaries later than three weeks after birth,the content of Ad4BP in granulosa cells varied among thefollicles. Similarly, MIS (Ueno et al., 1989b; Münsterberg andLovell-Badge, 1991), inhibin α, βA, βB-subunits (Meunier etal., 1988) and P-450(arom) (Ishimura et al., 1989) geneexpression varied depending on the estrous cycle or the growthstage of the follicles. Although the expression of Ad4BPduring the estrous cycle and in the whole developing stages offollicles has not been investigated, it may show a pattern thatcorrelates with these putative target genes. Whether any or allof these genes are directly controlled by Ad4BP is the subjectof current investigation.

Regulation of the Ad4BP geneA drastic decrease of Ad4BP in Sertoli cells and the variedlevels of the expression among the granulosa cells of differentfollicles were observed. Since both Sertoli cells andgranulosa cells are under the control of follicle stimulatinghormone secreted from pituitary, the transcription of Ad4BPgene also might be influenced by the hormone. In contrast,such a significant change was not observed in the case of theadrenal cortex. The control mechanism of the Ad4BP geneseems to be dependent on the cell type. Immunohistochemi-cal observations confirmed the expression of Ad4BP as earlyas in the 13.5 d.p.c. fetus. The Ad4BP gene in adrenocorticalprimordium and the gonadal primordium in the fetus of 13.5

d.p.c. might be under the control of another factor when thepituitary is not yet functionally active (Watanabe andDaikoku, 1979).

Ad4BP as a marker protein for the identification ofadrenocortical and gonadal primordiumAdrenal glands appeared in the dorsal coelomic cavity con-tacting the upper pole of the mesonephros at 14.5 d.p.c. asround-shaped tissues. It was reported from morphologicalobservations that the adrenal primordium originated from thecoelomic epithelium embedded in the dorsal wall before pro-truding into the dorsal cavity (Pankratz, 1931). This study isthe first report describing direct identification of the adrenalprimordium embedded in the dorsal wall using Ad4BP as themarker protein. Investigation of the expression of Ad4BP inthe fetus at stages earlier than those used in this study wouldclarify the ontogeny of the adrenal cortex and the gonads.

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(Accepted 23 June 1994)