Expression of androgen receptors in upper human fetal reproductive tract

Y. Sajjad1, S.M. Quenby2,4, P. Nickson2, D.I. Lewis-Jones1,2 and G. Vince3

1 Reproductive Medicine Unit, Liverpool Womens Hospital, Crown Street, Liverpool L8 7SS, 2 Department of Obstetrics and Gynaecology, and 3 Department of Immunology, University of Liverpool, Liverpool L69 3BX, UK

4 To whom correspondence to be addressed. e-mail: squenby{at}liv.ac.uk


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Androgens play a key role in human fetal development. All androgens act through a single intracellular androgen receptor (AR), which is encoded by a single copy gene on the X chromosome. ARs are expressed as early as 9 weeks in the epithelium and mesenchyme of the urogenital sinus, paramesonephric (Müllerian) and mesonephric (Wolffian) ducts. METHODS: Using immunohistochemistry, we investigated the distribution of ARs in the gonads and lower genital tracts of 54 human fetuses at 8–11 weeks of gestation. Gender was determined by PCR. RESULTS: The AR was expressed in a similar pattern in both male and female fetuses. There appears to be no difference in expression in the mesonephros or the mesonephric ducts when male and female pelvises were compared. Expression in the female paramesonephric duct was within the epithelium, whereas, in the male pelvises, expression was in the mesenchyme of the paramesonephric duct. When AR expression was compared in the ovary and testes, both gonads seem to express AR at 9 weeks, but this expression was extended into the 10th week of gestation in the male. CONCLUSION: The specific pattern of AR expression implies a key role in gonadal development. However, the pattern of staining was similar in the gonads at 8 and 9 weeks in both sexes, although staining persisted longer in the testis until the 10th week. AR expression, therefore, is not a key determinant of human gonadal differentiation.

Key words: androgen receptor/fetus/gonad/immunohistochemistry/mesonephros


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Androgens are essential for masculinization of the reproductive tract and external genitalia during development in utero (George and Wilson, 1988Go). Androgens play a key role in human fetal development (Ellsworth and Harris, 1995Go) and have a characteristic role during male sexual differentiation, development and maintenance of secondary male characteristics, as well as during the initiation and maintenance of spermatogenesis (Brinkmann, 2001Go). Androgen synthesis in the Leydig cells of the testis leads to secretion of the main male androgen steroid, testosterone (Hiort et al., 1998Go). Testosterone is converted in peripheral target organs to dihydrotestosterone (DHT) by 5{alpha}-reductase (Zhou et al., 1995Go). The actions of androgens are mediated by androgen receptors (ARs) (Brinkmann, 2001Go) encoded by a single copy gene in the X chromosome (Yong et al., 2000Go).

The AR is a ligand-activated intracellular transcription factor that belongs to the steroid/nuclear receptor superfamily, members of which include receptors to estrogen, adrenal hormones and thyroid hormones (Tsai and O’Malley, 1994Go). In common with other steroid receptors, when activated by androgens, the AR translocates to the nucleus and binds to specific genomic DNA sequences (androgen response elements) in the regulatory regions (promoters/enhancers) of AR-regulated genes (Yong et al., 2000Go). Testosterone and DHT bind to the same receptor and require a functional AR to induce the transcriptional regulation necessary for internal and external genital development (Quigley et al., 1995Go). Testosterone promotes the differentiation of the mesonephric (Wolffian) structures into the epididymis, the vas deferens and the seminal vesicles, while DHT is essential for the development of the penis, scrotum and prostate (Hiort et al., 1998Go). The mesonephric duct (MD) in the male develops into the epididymis, vas deferens, seminal vesicle and the ejaculatory ducts, whilst in the female it gives rise to rudimentary structures such as the duct of the epoöphoron and Gärtners duct (Williams and Warwick, 1980Go). The paramesonephric duct (PMD) develops into the Fallopian tubes, uterus, cervix and upper part of the vagina (Sadler, 1995Go). In the male, most of this duct disappears, leaving only a rudimentary structure, the appendix testis.

ARs have been detected using immunohistochemistry in the external genitalia of both male and female human fetuses at 18–22 weeks of gestation after the time of androgen-dependent differentiation of the external genitalia (Kalloo et al., 1993Go). Most of the studies to date at earlier gestations have been performed on animals (Cooke et al., 1991Go; Bentvelson et al., 1995Go; Majdic et al., 1995Go).

The aim of this study was to define the expression pattern and time of appearance of AR (from 8–11 weeks gestation) in male and female fetal gonads, mesonephroi, the proximal end of the PMD and the proximal end of the MD to further our understanding of the role of androgens in human genital development.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient population
The Liverpool Research Ethics Committee granted ethical approval for this study. Women undergoing therapeutic termination of pregnancy were invited to participate. Patients did not have an ultrasound scan prior to the termination and therefore the gestational age of the fetus was determined by the foot length measurements (in mm) as well as the last menstrual period (LMP) in accordance with previously published data (Streeter, 1920Go; Lehtinen, 1984Go; Munsick, 1984Go). The gestational age used throughout the paper is based on the LMP and does not refer to post-ovulatory weeks.

Terminations were performed by surgical evacuation of the uterine cavity. Phosphate-buffered saline [PBS; 100 ml containing 2500 IU of heparin (CP Pharmaceuticals, Wrexham, UK)] was added to the evacuation container before the sample to prevent clotting of the specimen. Samples were then washed in PBS (pH 7.6) to reduce blood contamination, and pelvic parts were removed and identified using a dissecting microscope. The selected fetal tissues were placed in processing cassettes to hold the tissue specimens during embedding (BDH, Poole, UK). The fetal tissues were then fixed in 4% buffered paraformaldehyde (BDH) for 24 h at 4°C. Fifty-four samples of 8–11 weeks gestational age (6–9 weeks post-conception) were used, of which 40 samples were found to contain gonadal tissue. In some samples, only mesonephroi and/or ducts were present.

DNA extraction
In order to determine the sex of the fetal samples used in this study, sex karyotyping was performed on paraffin sections from 54 fetuses, gestational age 8–11 weeks. PCR was used to detect the presence of X and Y chromosome material at the Amelogenin (AMXY-specific for X chromosome) and SRY (for Y chromosome) loci. DNA was extracted using a standard phenol chloroform extraction method. For each set of reactions, two known male and female samples were used as positive controls. The primers used were AMXY-1 (TGACCAGCTTGGTTCTAWCCCA), AMXY-2 (CARATGAGRAAACCAGGGTTCCA), SRY-F (dGTCCAGTTGCACTTCGCTGCCAG) and SRY-R (dAGGCAACGTCCAGGATAGAGTGAAG) (Invitrogen, Paisley, UK). PCR was performed and the products run out on an agarose gel. Molecular weight standards (pBR322, HaeIII digest, Sigma, Poole, UK) were included on each gel to identify the bands obtained. A negative control (water) was also included to rule out contamination. The resultant bands were then photographed.

Immunohistochemistry
After fixation, the tissues were processed in a tissue processing machine (Shandon Scientific, UK) and wax blocks were cast. Section of 5 µm were cut and mounted on microscopy slides which had been coated for 10 min with 10% poly-L-lysine (Sigma).

Sections were dewaxed in xylene and rehydrated in graded ethanols. Pressure cooker antigen retrieval was optimized to 2.5 min in citric acid buffer at pH 6.0. Endogenous peroxidase activity was blocked using 1% aqueous hydrogen peroxide (Sigma) for 10 min. The sections were transferred to Tris-buffered saline (TBS; pH 7.4) prior to staining. Preliminary experiments were performed to determine the optimum dilution for the primary antibody, and a dilution of 1:50 in TBS was selected.

Sections were incubated for 30 min in a humidity chamber with mouse anti-human AR antibody (AR 441, Dako, Cambridgeshire, UK) which was diluted 1:50 in TBS. Following incubation, the sections were washed three times in TBS for 5 min each. The sections were then incubated for 30 min with rabbit anti-mouse immunoglobulins (Z0259, Dako) at a dilution of 1:25. Following three washes in TBS, the sections were incubated for 30 min with mouse monoclonal peroxidase–antiperoxidase (PAP) (P0850, Dako) diluted to 1:100 in TBS. After further washing in TBS, visualization was carried out using 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma). After checking the staining intensity, the sections were washed in water and counterstained with Harris’s haematoxylin (Merck, Poole, UK). Following dehydration and clearing in xylene, the sections were mounted in DPX mountant (Merck).

Slides were examined by two independent observers who were blinded to gestational age.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The sex of 54 fetuses from gestational age 8–11 weeks was analysed using a PCR-based assay using Amelogenin (AMXY) and SRY loci. The Amelogenin loci produces an X-specific band (270 bp) and a Y-specific band (100 bp). The presence of the SRY gene results in a band of 210 bp. A female embryo is characterized by a single band (270 bp) (female control, lanes 2 and 16 in Figure 1), and a male embryo by three bands (270, 210 and 100 bp) (male control, lanes 1 and 15 in Figure 1). Overall, 29 females and 25 male samples were examined.



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Figure 1. PCR-based assay to determine the sex of the sample by amplification of the Amelogenin (AMXY) and SRY loci. The Amelogenin locus produces an X-specific band (270 bp) and a Y-specific band (100 bp). The presence of the SRY gene results in a band of 210 bp. Lanes 1 and 15 are the male controls with three bands. Upper band: X specific at 270 bp. Lower band: Y specific at 100 bp. Middle band: SRY specific at 210 bp. Male samples are shown by the three bands in lanes 3, 5, 7, 8, 9 and 11. Lanes 2 and 16 are the female controls with a single band at 270 bp. Female samples are shown by a single band in lanes 4, 6, 10, 12, 13 and 14. Lane 17 is the water control. pBR322 is used as the molecular weight standard.

 
Gestational ages 8–11 weeks were used because the gonads could be identified in the fetal tissues by 8 weeks but, after 12 weeks, collection of the samples was difficult because the gonads were easily damaged during the suction termination procedure. Not all samples contained all tissues. For all tissues examined using immunohistochemistry, mouse IgG was used as a negative control (Figure 2A).




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Figure 2. Immunostaining for AR in human fetal ovaries (ov), testis (t), rete testis (rt), interstitial cells (ic), testicular cords (tc), mesonephros (m), Bowmans capsule (bc), paramesonephric (->) and mesonephric duct (md) from 8–11 weeks of gestation. The AR staining is the brown coloration. (A) Negative control (IgG) of an 8 week ovary (magnification x100). (B) Staining of AR in an 8 week male mesonephros, but the testis and mesonephric duct lack any staining (x160). (C) AR-positive staining in a 9 week ovary; slight staining in medulla (me) and more prominent staining in the mesonephros (x160). (D) Higher magnification of the mesonephros in (C); the Bowmans capsule is seen to be strongly positive for AR (x400). (E) A 9 week male showing positive staining in rete testis and mesonephros. The Bowmans capsule is also positive. The interstitial cells (Leydig cell precursors) are positively stained whilst the spermatic cords lack staining (x100). (F) High power view of the mesonephros in (E). The Bowmans capsule in the mesonephros shows strongly positive staining with AR (x400). (G) A 10 week testis with positive staining of the interstitial tissues between the testicular cords (x100). (H) High power view of the interstitium between the testicular cords in (G), showing positive AR staining (x400). (I) A 10 week female showing the relationship of the ovary to the mesonephros, mesonephric duct and paramesonephric duct (x100). (J) An 11 week female paramesonephric duct with positive staining in the epithelium of the duct although the stroma is negative. The mesonephric duct is also positive for AR (x400). (K) A 10 week male showing the relationship of the mesonephric duct and the paramesonephric duct. The paramesonephric duct has positive staining in the surrounding mesenchyme with the epithelium free of staining. The mesonephric duct also shows positive (x400). (L) A 10 week male paramesonephric duct with positive staining in the surrounding mesenchyme; epithelium is negative (x400).

 
Female
At 8 weeks, seven female samples were examined, of which five had intact ovaries. The ovaries were all negative for AR (Table I). However, on examination, all mesonephroi showed positive staining for AR, whereas only three out of seven MDs were positive (Table II). At 9 weeks, six samples were examined, with four containing ovaries. All four had isolated areas of positive staining within the medullary region of the ovary (Table I, Figure 2C), whilst five out of six MDs and all of the mesonephroi were shown to be positively stained (Table II and Figure 2C). The Bowmans capsule in the mesonephros showed strong staining throughout, particularly near the medulla of the ovary, while the tissues inside the Bowmans capsule showed some scattered positive staining (Figure 2D). At 10 weeks, staining was absent from all six ovaries examined (Table I) although all the mesonephroi (Table II) and the MDs were positively stained (Table II). At this gestation, the mesonephroi start showing signs of disintegration but were still positively stained. Figure 2I shows the relationship of the gonads to the mesonephroi, MD and PMD in a 10 week female. At 11 weeks, 10 samples were used with seven ovaries, which remained negative for AR staining (Table I). Only two samples contained mesonephroi, which looked dispersed and scanty and were negative (Table II). Three out of six MDs were positive for AR staining (Figure 2J, Table II).


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Table I. ARs in female and male gonads at 8–11 weeks gestation
 

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Table II. ARs in the female pelvis at 8–11 weeks gestation
 
The proximal PMD showed no positive staining in the epithelium or surrounding mesenchyme at 8 weeks of gestation (Table II), but, from 9 to 11 weeks, the epithelium of the PMD showed positive staining, whilst the surrounding mesenchyme remained negative (Table II, Figure 2J).

Male
At 8 weeks, three male samples were examined, of which two contained testes. AR staining was absent in the testes (Table I, Figure 2B). All the mesonephroi and the MDs exhibited strong AR staining (Table III). At 9 weeks, all samples had AR staining in the testes (Table I, Figure 2E), mesonephroi (Fig 2E) and MDs (Table III). Staining in the testes was predominantly in the rete testes (Figure 2E). At 10 weeks, eight samples (six testes) were examined, with four testes (Table I, Figure 2G), seven MDs (Table III, Figure 2K) and all the mesonephroi showing positive staining (Table III). In the testis, the staining was observed to be uniformly distributed within the interstitial tissues (Leydig cell precursors) between the testicular cords (Figure 2G and H). At 11 weeks, seven samples were examined, only four with testes. No staining was observed in the testes (Table I). At this gestation, the mesonephroi had disappeared, but the MD remained strongly positive in the majority of samples examined (Table III).


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Table III. ARs in the male pelvis at 8–11 weeks gestation
 
There was an absence of staining in the epithelium of the PMDs as well as the surrounding mesenchyme at 8 weeks of gestation (Table III), but from 9 to 10 weeks, the epithelium of this duct remained negative, whilst the surrounding mesenchyme was strongly positive (Table III, Figure 2K and L). At 11 weeks, the PMD epithelium, as well as the surrounding mesenchyme, was negative in the majority of samples (Table III).

Our study demonstrates that ARs are expressed in the fetal ovaries only at 9 weeks of gestation. In the fetal testis, ARs are found at the same gestation but persist to 10 weeks before disappearing at 11 weeks. Expression was observed initially in the rete testes, and then spread to the medullary region and the cortex. The interstitial cells (Leydig cell precursors) were positive whilst the peritubular cells remained negative. The pattern of AR expression was very similar in male and female mesonephroi and MDs. In contrast, in the PMD, the pattern of AR expression was different in the male and female. In the female, AR was observed in the epithelium whilst the surrounding mesenchyme was negative. In the male, AR expression was seen in the surrounding mesenchyme of the PMD, whilst the epithelium remained negative.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
AR expression has been described previously in reproductive tissues in the second trimester of human fetal development, within the penis, prostate, testes, epididymus, scrotal skin, labial skin, uterus, cervix and ovary (Wilson and McPhaul, 1996Go). AR expression has also been shown in both the early and midgestation (9.5–23 week) human fetal prostate (Adams et al., 2002Go). Aumuller et al. (1998Go) identified AR in the developing human fetal prostate from the 19th to 36th week of gestation, and suggested that AR expression is required for prostatic development. Although AR involvement has been described in the development of the vagina (Shapiro et al., 2000Go), here we describe the expression of AR in the first trimester fetal gonads, mesonephros, MD and PMD. Human fetal tissues of first trimester gestation (8–11 weeks) have been examined for AR staining by immunohistochemistry. Sex determination was performed by PCR, not simply by morphology.

In the testis, AR was detected at 9 and 10 weeks, while in the ovaries, AR was detected only at 9 weeks. Staining in the gonads differed in that initial expression in the testis was seen in the rete testes at 9 weeks gestation and became more generalized within the interstitial tissues at 10 weeks. The developing Leydig cells were seen to be strongly positive for AR. The interstitial cells of Leydig are derived from the original mesencyhme of the gonadal ridge and are responsible for the production of testosterone (Sadler, 1995Go). In the ovary, AR staining was always within the medullary region. Similar experiments in rats have reported AR to be present earlier in male rats, by 15.5 days gestation (Carnegie stage 19, equivalent to 7–7.5 weeks in human) but disappearing by day 17 (Carnegie stage 22, equivalent to 8.5 weeks in humans) (Majdic, 1995Go) (total gestational length of rats is 21–23 days). Furthermore, contrary to our findings of AR in human ovaries, no AR was observed in rat ovaries (Majdic, 1995Go).

The pattern of AR expression was similar in the male and female mesonephroi and MDs. Human AR expression was detected in both male and female mesonephroi and MDs at 8 and 9 weeks of gestation, later than previously shown in fetal rats, at day 14 (6.5 weeks in human) (Bentvelson et al., 1995Go). This is in agreement with the work of Shapiro et al. (2000Go) who showed AR expression in the PMD (Müllerian) and MD (Wolffian) at 9 weeks gestation. As AR expression was seen in both sexes, AR expression would not appear to be a key determinant of human gonadal differentiation, although, in the female, staining was reduced in the MD at 11 weeks, as the Wolffian structures regress.

The proximal end of the PMD showed a specific and different pattern of AR expression in male and female. No AR staining was observed in either of the sexes at 8 weeks. Considerable expression of AR was seen in the female PMD epithelium at 9, 10 and 11 weeks, although the mesenchyme remained negative. In the male, AR expression was mostly in the mesenchyme, but the epithelium remained negative. This specific difference in AR expression between male and female suggests that AR in the female is in epithelium, and may be involved in growth of the Fallopian tube, whereas in males the AR is in the mesenchyme and may be responsible for the regression of this structure.

The specific expression pattern for AR does suggest that androgens may be an important factor in the growth of the gonads and MDs in early gestation. Previous studies have also suggested that ARs initiate the stimulus for growth of the phallus in the male before 12 weeks, and are important during androgen-mediated growth of this structure in the second trimester (Shapiro et al., 2000Go). ARs can be detected in the fetal prostate at 11.5 weeks gestation (Adams et al., 2002Go)

In the male, the Y chromosome contains the sex-determining SRY gene on its short arm (Pritchard and Goodfellow, 1987Go; Sinclair et al., 1990Go), and it is suggested that this determines the development of fetal Leydig cells which then, through their androgen/testosterone secretion, cause differentiation of the male genitalia (Ellsworth and Harris, 1995Go). The capacity for testosterone synthesis in the human male fetal testis begins at ~8 weeks fetal age and is maximal at 14–16 weeks gestation (Siiteri and Wilson, 1974Go; Jirasek, 1977Go; Winters et al., 1977Go; Cunha, 1986Go). Once differentiated, the testis will secrete Müllerian-inhibiting hormone from Sertoli cells, which will cause the PMD system to regress (Blanchard and Josso, 1974Go; Tran et al., 1977Go; Behringer et al., 1990Go). Without the Y chromosome, and in the presence of a second X chromosome, female sex differentiation occurs (Grumbach and Barr, 1958Go), the gonads develop into ovaries, no Leydig cells are formed, no androgen/testosterone is produced and therefore it has been assumed that ARs within the differentiating ovary are not activated and subsequently the Leydig cells disappear (Moore and Persuad, 1993Go).

In the second trimester, androgen-dependent differentiation of the male MD as well as masculinization of external genitalia occurs (Ortiz et al., 1966Go; Naessany et al., 1980Go; Inomata et al., 1989Go), and it is suggested that ARs in both first and second trimester are markers rather than mediators of differentiation (Kalloo et al., 1993Go).

At 8 weeks gestation, only the mesonephroi and the MDs express ARs which, however, persisted even when gonads became negative for AR staining at 10 weeks. This was even present in the female MD, which will disappear at a later stage as the PMD system develops and the mesonephric system regresses. In the male, it is suggested that AR expression in the MD is required for the differentiation of the duct into the duct of the epididymus, ductus deferens, the ejaculatory ducts and the seminal vesicles. Increasing testosterone production from fetal Leydig cells from 8 to 9 weeks gestation may stimulate the production of a greater number of ARs, as has been demonstrated in the rat as early as day 14 (equivalent to 6 weeks in human) (Bentvelson et al., 1995Go).

Whether testosterone plays a part in the genital differentiation of the fetus in the first trimester and DHT comes into action in the second trimester is not clear, nor whether testosterone and DHT play any part in genital development. However, Ellsworth and Harris (1995Go) state that testosterone is sufficient for the development of internal mesonephric structures, but DHT is required for the differentiation of male external genitalia and fetal prostate.

Further work is required to elucidate the effects of hormone–AR interaction in normal human development.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on March 25, 2003; accepted on April 8, 2004.





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