Histochem Cell Biol (1996) 105:7-15 �9 Springer-Verlag 1996

Julie A. Johnson. Joseph P. Grande Patrick C. Roche. Rajiv Kumar

Immunohistochemical detection and distribution of the 1,25-dihydroxyvitamin D 3 receptor in rat reproductive tissues

Accepted: 23 June 1995

Abstract Vitamin D 3, via its active metabolite 1,25-di- hydroxyvitamin D 3, plays a critical part in male and fe- male reproduction in the rat. 1,25-Dihydroxyvitamin D 3 activity is mediated by an intracellular receptor (VDR). VDR distribution in reproductive tissue has not been studied using antibodies against the receptor. We devel- oped a polyclonal antibody against the VDR and used it to examine VDR distribution in male and female rat re- productive tissues. In rat testes, VDR epitopes were ob- served in seminiferous tubules, specifically in spermato- gonia, Sertoli cells and spermatocytes. Spermatozoa stained faintly. Epithelial cells of the epididymis, semi- nal vesicles and prostate also expressed VDR epitopes. In the female rat reproductive tract, immunostaining for VDR was seen in ovarian follicles, specifically in granu- losa cells. Weaker VDR immunostaining was observed in follicular thecal cells and in the ovarian stroma and germinal epithelium. Corpus luteal cells stained intense- ly for VDR. Epithelium of fallopian tubes and the uterus also contained VDR epitopes. Both nuclear and cytoplas- mic VDR immunostaining was observed in male and fe- male rat reproductive tissues. We conclude that the VDR is widely distributed in male and female reproductive tis- sues and that it is likely to mediate actions of 1,25-dihy- droxyvitamin D 3 in the tissues.

J.A. Johnson - J.R Grande �9 R. Kumar Nephrology Research Unit, Mayo Foundation, Rochester, MN 55905, USA

J.R Grande �9 RC. Roche Department of Laboratory Medicine, Mayo Clinic, Rochester, MN 55905, USA

R. Kumar (~) Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 First St., SW, 911A Guggenheim Bldg., Rochester, MN 55905 USA Tel. 507-284-0020; Fax 507-284-8566 e-mail: rkumar@mayo, edu

Introduction

Vitamin D 3 has an important role in reproduction. Vita- min D-deficient female rats have reduced fertility and a reduction in the numbers of fetuses delivered per litter compared with normal vitamin D-replete rats (Halloran and Deluca 1980; Hickie et al. 1983; Kwiecinski et al. 1989b). This reduction in fertility is due specifically to the deficiency of vitamin D, and not to a deficiency in calcium, because supplementation of a vitamin D-defi- cient diet with calcium sufficient to raise serum calcium concentrations to normal levels does not correct the de- fect in fertility (Kwiecinski et al. 1989b). Vitamin D is also critical for the maintenance of normal reproduction in male rats. Reduced fertility is observed in male rats given a vitamin D-deficient diet (Kwiecinski et al. 1989a). Vitamin D-deficient male rats have incomplete spermatogenesis and degenerative testicular changes (Osmundsen etal. 1989). The number of 1,25-dihy- droxyvitamin D 3 [1,25(OH)2D3] binding sites in rat tes- tes increases at puberty (Levy et al. 1985a; Walters 1984). The importance of vitamin D 3 for egg hatchability and viability (Narbaitz et al. 1987) also supports the key role of the vitamin in reproductive biology.

Vitamin D 3 is metabolized via an intermediate, 25-hy- dr0xyvitamin D 3, to i t s biologically active form 1,25(OH)2D 3 (Audran et al. 1986; Johnson and Kumar 1994a, b; Henry et al. 1992). Most of the effects of 1,25(OH)2D 3 are mediated by its intracellular receptor (commonly referred to as the vitamin D receptor, VDR) (Walters 1992). All vitamin D responsive tissues express the receptor, and abnormalities in receptor structure and function are associated with severe rickets (Audran et al. 1986). Ligand binding assays, in which homogenates of tissues are tested for their ability to bind to radiolabelled 1,25(OH)2D 3, have demonstrated the presence of the VDR in ovarian (Dokoh et al. 1983) and uterine epitheli- um homogenates (Walters 1981; Walters et al. 1983) and in testicular (Walters 1984; Walters et al. 1983; Levy et al. 1985a, b; Merke et al. 1985) and prostate homoge- nates (Peehl et al. 1994). Obviously, the specific cell

Fig. 1A-D Immunostaining of rat testes and epididymis for VDR. Bars: 2.1 ~tm. A, B tes- tes. White arrowhead Sertoli cells; black arrow spermatocy- tes; small white arrow sper- matogonia; SP spermatozoa; I interstitial Leydig and connec- tive tissue cells. C, D Epididy- mis. Small white arrow epithe- lium; SP spermatozoa; I inter- stitium

types that contain the receptor cannot be localized by these methods. Stumpf and his group have used [3H]l,25(OH)2D 3 to examine the uptake of radioactivity in various male and female reproductive tissues (Stumpf et al. 1987; Stumpf and Denny 1989; Stumpf 1988). The underlying assumption of these studies is that uptake of [3H]l,25(OH)2D 3 into tissues is dependent upon the presence of the VDR. While this assumption is reason- able, the technique, by its very nature, cannot confirm the presence of receptors in tissues of interest, as uptake may be mediated by other processes and by proteins that may bind to the ligand with low affinity. These corn-

ments notwithstanding, uptake of [3H]1,25(OH)2D3 has been observed in germinal epithelial cells and corpus lu- tea of the ovary (Stumpf and Denny 1989), in cells of uterine and fallopian tube epithelium (Stumpf et al. 1987), and in Sertoli cells of testes, in epididymis and in prostate (Stumpf and Denny 1989; Schleicher etal. 1989).

As outlined above, the importance of 1,25(OH)2D 3 in the maintenance of reproduction is clear. In order to gain further insights into the action of 1,25(OH)2D 3 in repro- ductive tissues, and to assess in which cells VDR is lo- cated, we studied male and female rat tissues with a spe-

Fig. 2A-D Immunostaining of rat seminal vesicles and pros- tate for VDR. Bars: 2.1 ~m. A, B Seminal vesicles. C, D Pros- tate. Small white arrow epithe- lium; S stroma; L central lumen (seminal vesicles); C prostatic concretions (prostate)

cific and sensitive polyclonal antibody directed againSt VDR. We found widespread distribution of VDR in both male and female rat reproductive tissues.

Materials and methods

Animals and tissue collection

For immunohistochemical studies, two 9-month-old male and four 6-month-old female Holtzman rats (Harlan Sprague-Dawley, Indi- anapolis, Ind.) were used. Rats were supplied with standard labo- ratory rat chow (Ca 1%, Pi 0.61%, vitamin D 4.5 IU/g) ad libitum. Animals were euthanized using CO 2 and exsanguinated. Testes.

epididymis, seminal vesicles and prostate were removed from males. Uterus. fallopian tubes and ovaries were removed from fe- males. Tissues were fixed for 18 h in 10% formalin in phosphate- buffered saline, pH 7.4. followed by embedding in paraffin. Paraf- fin-embedded tissues were then cut in 4-pm-thick sections and placed on silanized slides.

Immunohistochemistry

Immunohistochemistry was performed using a polyclonal anti- body to VDR (antibody 1-165). Preparation o f this antibody has been described previously. Briefly, the cDNA for human VDR was expressed in bacteria, and the expressed protein was purified (Ku- mar et al. 1992) and used to produce polyclonal antibodies in rab- bits (Kumar et al. 1994).

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Fig. 5A, B Immunostaining of positive and negative control tissues for VDR. Tissues are from adult rats. Bars: 2.1 ~[m. A Liver (hepatocytes are a neg- ative control for VDR); B duo- denum (positive control tissue for VDR)

Slides of rat reproductive tissues were deparaffinized in xylene, rehydrated in ethanol, rinsed in tap water; endogenous per- oxidase activity was blocked using 0.6% H~O 2 in methanol. After a tap water rinse, sections were placed in 10 mM acetic acid, pH 6.0, heated for 2 rain in a 780 W microwave oven set on high, al- lowed tO cool for 20 rain, and rinsed in tap water. Sections then were treated with 5% normal goat serum in PBS-Tween (phos- phate-buffered saline, pH 7.4, containing 0.05% Tween 20) for 10 min, followed by incubation with primary antibody for 60 rain (rabbit-anti-VDR, 1:1000) at room temperature. After thorough rinsing, all sections were treated with biotinylated goat anti-rabbit IgG (Dako Corporation, Carpinteria, Calif., 1:400), followed by peridoxase-labeled streptavidin (Dako, 1:500) for 30 rain at room temperature. Sections were developed for 15 min by adding 0.1 M sodium acetate, pH 5.2, containii~g the red chromogen, aminoethyl carbazole, and H202. Sections were counterstained with hematox- ylin, and c0verslips were attached using aqUeous mounting media. Negative controls for non-specific staining were done on tissue

sections using 1% non-immune rabbit serum instead of primary antibody. VDR antibody specificity was verified by using VDR antiserum adsorbed with excess expressed human VDR to stain tissue sections. Photomicrographs were taken using an Olympus VANOX AHBS3 research photomicrographic microscope system (Olympus, Lake Success, N.Y.).

Results

Prominen t nuclear and cy top lasmic immunos ta in ing of V D R was obse rved in spermatocy tes in rat testes (Fig. 1A, B). Spe rma togon i a and Ser tol i cel ls showed less in- tense V D R staining. S p e r m a t o z o a s ta ined very weak ly or not at all for the V D R (Fig. 1 A - D ) . Inters t i t ia l Leyd ig and connect ive t issue cells, basemen t m e m b r a n e and my-

old cells were negative for VDR. Epithelial cells of the epididymis had strong immunostaining for the VDR, but no immunostaining was observed in epididymal stroma (Fig. 1C, D). In seminal vesicles immunostaining for VDR was seen in ciliated epithelium (Fig. 2A, B). Inter- stitial cells of seminal vesicles did not contain VDR epi- topes. VDR immunostaining was prominent in epithelial cells of rat prostate (Fig. 2C, D). Prostate stroma was negative for epitopes of VDR.

VDR epitopes were observed in both nuclei and cyto- plasm of granulosa cells of ovarian follicles (Fig. 3A, B). Some weaker staining was present in thecal cells of folli- cles (Fig. 3A, B). Intense VDR staining was present in corpora lutea (Fig. 3A). Some VDR staining was seen in the ovarian stroma and in the germinal epithelium, but the interstitium of rat ovaries was negative for VDR epi- topes (Fig. 3A). Fallopian tube epithelium displayed VDR epitopes; the muscular stroma, however, showed no VDR epitopes (Fig. 3C, D). Uterine lining tissue con- tained epitopes for VDR in some glandular epithelial cells (endometrium) (Fig. 3E, F). No VDR epitopes were present in endometrial stroma (Fig. 3E, F).

Pre-adsorption of polyclonal antiserum to VDR with excess expressed human VDR eliminated immunostaining in rat reproductive tissues (Fig. 4A-F), verifying the spec- ificity of our antibody. VDR immunostaining of adult rat liver hepatocytes, a tissue negative for VDR, gave negative staining as expected (Fig. 5A). Immunostaining of adult rat duodenum, a tissue known to be positive for VDR, gave appropriate positive immunostaining (Fig. 5B).

Discussion

The important role of 1,25(OH)2D 3 in reproduction in both males and females is clear (Halloran and DeLuca 1980; Hickie et al. 1983; Kwiecinski et al. 1989a, b; Osmundsen et al. 1989; Levy et al. 1985a; Waiters 1984, Narbaitz et al. 1987). The specific mechanisms by which the hormone influences reproductive function, however, are unclear. 1,25-Dihydroxyvitamin D 3 exerts its effects primarily through its receptor (Walters 1992). Knowl- edge of the specific localization of VDR in reproductive tissues, therefore, is important for designing appropriate in vitro experimental models to use to determine the pre- cise mechanisms by which 1,25(OH)2D 3 influences re- production.

Using immunohistochemical methods, we have dem- onstrated the specific localization of VDR in male and female rat reproductive tissues. Previous attempts at spe- cific localization of VDR in testes were performed by physically separating cell types and measuring the num- ber of binding sites for radiolabelled 1,25(OH)2D 3 (Levy etal. 1985a, b; Walters 1984; Merke et al. 1985; Os- mundsen et al. 1989) and by autoradiographic methods in which [3H]1,25(OH)2D3 uptake was examined (Stumpf et al. 1987; Schleicher et al. 1989).

Our demonstration of VDR by immunohistochemistry in Sertoli cells is supported by biochemical and autora-

13

diographic evidence of 1,25(OH)2D3-binding sites in Sertoli cells (Merke et al. 1985; Schleicher et al. 1989; Stumpf et al. 1987). Autoradiographic (Stumpf et al. 1987; Schleicher et al. 1989) and 1,25(OH)2D3-binding studies (Merke et al. 1985) failed to show any evidence of VDR in spermatogonia or spermatocytes, while our immunohistochemical data clearly demonstrate the pres- ence of the receptor in spermatogonia and spermato- cytes. This disparity may be a result of inadequate sensi- tivity of the biochemical or autoradiographic methods; or as stated by Merke et al. (1985), there may be a discrep- ancy in the method used to isolate different seminiferous tubule cell types.

Our immunostaining results show that VDR epitopes are not present in interstitial Leydig or fibroblast ceils of testes. Because 1,25(OH)2D 3 has been shown to affect circulating testosterone levels (Sonnenberg et al. 1986), and Leydig cells are the primary source of testosterone (Geneser 1986), it has been postulated that 1,25(OH)2D 3 regulates testosterone synthesis though VDR in Leydig cells. In light of our specific data, however, it seems that 1,25(OH)2D 3 regulates testosterone synthesis indirectly. Indeed, changes in plasma concentrations of luteinizing hormone (LH), which stimulates testosterone synthesis by Leydig cells, parallel those of testosterone in re- sponse to changes in vitamin D status (Sonnenberg et al. 1986).

Spermatogonia are the germ cells from which sperma- tozoa ultimately arise (Geneser 1986). Testosterone and follicle stimulating hormone (FSH) are both required fo r spermatogenesis (Geneser 1986). Sertoli ceils, which also contain some VDR epitopes, have androgen recep- tors and metabolize testosterone to the active form, dihy- drotestosterone (Geneser 1986). 1,25-Dihydroxyvitamin D 3, through VDR, may act in concert with testosterone and FSH to stimulate maturation of male germ cells from spermatogonia into spermatocytes and, finally, spermato- zoa.

In the epithelium of the epididymis, VDR probably plays a role in the resorption of testicular fluid for sperm concentration. Interestingly, the epididymis develops from the mesonephros (Geneser 1986), which is a fetal precursor to the kidneys. VDR plays a part in regulating renal fluid resorption in the distal tubule epithelium of the kidneys (Johnson and Kumar 1994a, b; Audran et al. 1986).

VDR and 1,25(OH)2D 3 may play a role in regulating secretion of the prostate fluid, as suggested for the epi- thelium of the epididymis and seminal vesicles. Pub- lished data suggest a role for 1,25(OH)2D 3 and VDR in the regulation of growth and differentiation of normal and malignant prostate cells (Peehl et al. 1994; Miller et al. 1992; Skowronski et al. 1993). Prostate cancer risk correlates with factors that are associated with decreased vitamin D 3 synthesis, such as increasing age, black race and living in Northern latitudes (Schwartz and Hulka 1990).

The role of the VDR that we observed in germinal ep- ithelium of rat ovaries is unknown. VDR may play a part

14

in regulating transport across the epithelium. Primordial follicle epithelial cells, which also contain VDR epi- topes, are derived f rom the germinal epithelium (Geneser 1986). Granulosa cells in the ovarian follicle correspond to Sertoli cells in the testicular tubules. Both contained epitopes of VDR. The actions of both cell types are regu- lated by FSH, and both produce those steroids necessary for germ cell maturation (Geneser 1986).

1,25(OH)2D 3 has been found in human follicular fluid (Potashnik et al. 1992) and may act with the VDR in fol- licle granulosa cells to regulate some of their functions. Granulosa cells have a P450 aromatase activity that con- verts androstenedione, which is synthesized by thecal follicular cells, to estradiol (Erickson et al. 1990). Estra- diol synthesis is necessary for normal folliculogenesis and, thus, ovulation (Geneser 1986). In human skin fi- broblasts, 1,25(OH)2D 3 treatment increases the rate of conversion of androstenedione to estrone by increasing aromatase activity (Hodgins and Murad 1986). 1,25-Di- hydroxyvitamin D 3, through VDR, may exert similar ef- fects in ovarian follicle granulosa cells.

All oocytes observed were negative for VDR epi- topes, yet spermatocytes contained prominent staining for VDR. This is not an unexpected difference because spermatocytes are actively undergoing division, while most oocytes are in an arrested phase of division, until after ovulation and fertilization (Geneser 1986).

The intense staining for VDR seen in the corpus lutea appears to be in granulosa lutein cells, which produce large amounts of progesterone following stimulation by LH (Geneser 1986). Although 1,25(OH)2D 3 has been shown to have systemic effects on FSH (Zofkova et al. 1989), LH (Sonnenberg etal . 1986), and testosterone (Sonnenberg et al. 1986) secretion, the local actions of 1,25(OH)2D 3 and VDR on ovarian cells remain to be de- termined.

The VDR found in luminal epithelial cells of the fal- lopian tubes may regulate Ca 2§ transport across fallopi- an tube epithelium, as it does in intestinal epithelium (Johnson and Kumar 1994a; Audran et al. 1986). Fertili- zation of the ovum, which occurs in the fallopian tube lumen, can only occur within a narrow range of extra- cellular Ca 2+ concentrations (Fujimoto etal . 1994). VDR epitopes were prominent in glandular epithelium of the uterine lining (endometrium). In cultured endo- metrial ceils f rom ovariectomized rats pretreated with estrogen and progesterone, 1,25(OH)2D 3 alters the activ- ity of alkaline phosphatase (Lieberherr et al. 1984). The precise role of the VDR in uterine epithelium remains to be determined.

This study shows, for the first time, the distribution and specific localization of VDR in both male and fe- male rat reproductive tissues using immunohistochemi- cal techniques. This localization is specific as demon- strated by pre-adsorption of VDR antiserum with excess expressed VDR. Several roles for the VDR in these tis- sues are suggested by its particular localization. The pre- cise actions of the VDR, along with 1,25(OH)2D 3, in re- productive tissues remain to be determined. Our data

suggest that 1,25(OH)2D 3 and its receptor are likely to play significant parts in reproductive tissues.

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