Effect of Prenatal Vitamin D (Calcitriol)Exposure on the Growth and Development of

the Prostate

Badrinath R. Konety,1,2 Ajay K. Nangia,1 Thu-Song T. Nguyen,2Angela Thomas,2 and Robert H. Getzenberg1,2,3,4,5*

1Division of Urology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania2University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania

3Department of Pathology, University of Pittsburgh School of Medicine,Pittsburgh, Pennsylvania

4Department of Medicine, University of Pittsburgh School of Medicine,Pittsburgh, Pennsylvania

5Department of Pharmacology, University of Pittsburgh School of Medicine,Pittsburgh, Pennsylvania

BACKGROUND. We previously found that in the absence of testosterone (T), calcitriolpromotes proliferation of normal prostatic stroma, while in the presence of T, it has a differ-entiating effect on prostatic epithelium. The present study was conducted to determine theeffect of calcitriol exposure in utero on the postnatal development of the normal prostate.METHODS. Pregnant rats were injected subcutaneously with either 1.25 mg of calcitriol orvehicle alone on alternate days till delivery. Calcitriol-exposed and control pups were sacri-ficed at age 25 days (prepuberty), 63 days (postpuberty), or 102 days (adults), and theirprostates and seminal vesicles were harvested and weighed.RESULTS. Pups prenatally exposed to calcitriol and sacrificed before puberty (25 days) hada 35% greater mean prostatic weight than controls (0.0314 vs. 0.0422 g, P < 0.007), andcalcitriol-exposed adult rats (102 days) had a 68% greater mean prostatic weight than controls(0.1365 vs. 0.2304 g, P < 0.005). No differences were observed in seminal vesicle weights, andin serum calcium and testosterone levels. A disproportionately high mortality rate fromsudden death (71%) was observed at puberty in uncastrated male rats prenatally exposed tocalcitriol.CONCLUSIONS. These findings suggest that high-dose calcitriol exposure in utero mayuniquely influence subsequent prostatic growth. Nonandrogenic steroids such as calcitriolmay also be involved in genetic imprinting of the prostate. Prostate 41:181–189, 1999.© 1999 Wiley-Liss, Inc.

KEY WORDS: vitamin D; prostate development; fetal exposure; animal experiments

INTRODUCTION

Alteration of the sex steroid hormonal milieu in thefetal and neonatal male rat has been found to affectsubsequent prostatic growth [1–4]. Initial studies byRajfer and Coffey [2] suggested that exposure of neo-natal male rats to estrogens resulted in inhibition ofprostatic growth in the postpubertal period as well asa decrease in ability to respond to exogenous andro-gens administered after puberty. However, neonatal

Grant sponsor: NIH; Grant number: DK52697-01; Grant sponsor:American Foundation for Urologic Disease; Grant sponsor: BarryLoveday; Grant sponsor: Ferdinand Valentine Fellowship of theNew York Academy of Medicine; Grant sponsor: FrederickSchwentker Endowment Award from the Department of Urology,University of Pittsburgh.*Correspondence to: Robert H. Getzenberg, Ph.D., University ofPittsburgh Cancer Institute, E1056 BST, 200 Lothrop St., Pittsburgh,PA 15213-2582. E-mail: getzenbergrh@msx.upmc.eduReceived 1 March 1999; Accepted 18 May 1999

The Prostate 41:181–189 (1999)

© 1999 Wiley-Liss, Inc.

androgen exposure, while resulting in smaller pros-tate gland size after puberty, does enhance the respon-siveness of the gland to exogenous androgens admin-istered after puberty. The postpubertal effects of neo-natal androgen administration on the ventral prostateare diminished if the rats are castrated in the neonatalperiod [1]. This suggests the presence of a nonandro-genic testicular factor which may influence subse-quent prostatic growth [1,5,6]. Exposure to low dosesof estrogen in the neonatal period results in increasedresponsiveness of the smaller-than-normal postpuber-tal prostate to exogenous androgen administration [2].Vom Saal et al. [7] demonstrated that exposure of malerat fetuses in utero to low doses of estrogens resultedin increased prostatic growth after birth. This effectwas reversed with the administration of higher dosesof estrogen to the pregnant dams. Data from all ofthese studies suggest that various sex steroids andother hormones may be involved in “genetic imprint-ing” of the prostate as well as other sex accessory tis-sues, which profoundly influences their subsequentgrowth and response to similar hormonal factors.

Calcitriol (1,25-dihydroxyvitamin D3) is the activeform of vitamin D. It is known to be a steroid hormonewhich affects the growth and differentiation of varioustissues, including the prostate [8]. We previously ob-served that administration of exogenous calcitriol tocastrated postpubertal male rats results in prostaticstromal proliferation, with a significant increase inoverall gland weight. However, in the presence of tes-tosterone, calcitriol appears to promote normal pros-tatic differentiation without affecting proliferation [9].Epidemiological data suggest that endogenous vita-min D levels may be related to the incidence of andmortality from prostate cancer [10]. Previous studiesdemonstrated the antiproliferative effects of calcitrioland its analogues on prostate cancer cells in vitro andin vivo [11–13]. Hence it appears that calcitriol mayhave a role in normal prostatic growth and differen-tiation and that it can control neoplastic prostatic pro-liferation.

Since prenatal and neonatal exposure to sex steroidhormones has been found to influence subsequentprostatic growth, we theorized that a similar exposureto calcitriol, a steroid hormone, may have an impactupon prostatic growth after birth. In this preliminaryreport, we present the results of our study on the ef-fects of prenatal exposure to calcitriol (vitamin D) onthe subsequent growth and development of the pre-pubertal and adult rat prostate. We found that fetalexposure to high doses of calcitriol led to at least a 35%increase in mean prostatic weight in the prepubertalrat and a 68% increase in mean prostatic weight in theadult rat. This suggests that nonandrogenic steroidssuch as vitamin D may have a direct role in prostatic

growth, and that “imprinting” of the prostate mayoccur by hormones other than androgens.

MATERIALS AND METHODS

Sixteen pregnant female Sprague-Dawley rats(dams) (Harlan Sprague-Dawley, Indianapolis, IN) at13 days of gestation were divided into three groupsand treated with subcutaneous injections of vehiclealone (group 1, controls) or 1.25 mg of calcitriol inethanol (a kind gift of Dr. Milan R. Uskokovic, Hoff-man-LaRoche, Nutley, NJ) (groups 2 and 3), on alter-nate days, till delivery. Pups born to dams in group 1served as controls for the entire experiment. Pups bornto dams in group 2 were maintained for 25 days afterbirth, and pups born to dams in group 3 were main-tained till either 63 days or 102 days of age. All dams(groups 2 and 3) received four injections of calcitriol orvehicle by the time of natural delivery at 21 days ofgestation. Dams and pups were maintained in thedark on a vitamin D-deficient diet supplemented with0.3% calcium and 0.4% phosphorus (Harlan Teklad,Madison, WI) throughout the study. Once the pupswere delivered they were maintained with the damsand weaned at day 21 into separate cages housing twoanimals per cage and marked according to the desig-nated group. Twenty-three male pups were deliveredfrom dams in group 1, 26 male pups from dams ingroup 2, and 52 male pups from dams in group 3.Dams and most of the female pups were sacrificed bycarbon dioxide overdose after the surviving pups hadbeen weaned. Ten female pups from the control groupand the calcitriol-exposed group were maintained forcomparison with their male counterparts from thesame dams. They were sacrificed with the male pupsat 102 days of age. Seven of the 23 pups from dams ingroup 1 (controls) and all the pups from dams ingroup 2 (26/26 treated) were sacrificed before pubertyat 25 days of age, and their prostates and seminalvesicles were harvested and weighed. The seminalvesicles were blotted to remove secretions before ob-taining their weight. The remaining pups in group 1(15/23 controls) and those in group 3 (52/52 treated)were maintained until after puberty either to 63 days(29 pups) or 102 days (23 pups). These animals werethen sacrificed by carbon dioxide overdose, and theirprostates and seminal vesicles were harvested andweighed. Of the 52 animals in group 3, 10 were cas-trated on day 21, as were 4 of the controls in group 1.All the castrated animals were maintained until 102days of age. Thirty of 42 (71%) of the uncastrated ratsin group 3 died unexpectedly during puberty (33–44days). Of the 22 surviving calcitriol-exposed pups (12uncastrated and 10 castrated), 10 (all uncastrated)were sacrificed on day 63, along with 50% of the un-

182 Konety et al.

castrated control pups (7/15); their prostates andseminal vesicles were harvested. In an effort to deter-mine the cause for the sudden deaths of the calcitriol-exposed intact male rats at time of puberty, two sur-viving but ill-appearing rats from group 3 were sacri-ficed earlier than 63 days. A specialized veterinarysurgeon and pathologist conducted autopsy studieson animals that died suddenly. The remaining animalsin the control (8) and treated (10) groups were sacri-ficed at 102 days. The time course of the experimentsis depicted in Figure 1. Animal weights were deter-mined at the time of each injection and at sacrifice.Serum was obtained from each animal by cardiacpuncture at the time of sacrifice and stored at −70°C.Calcium levels were measured in the serum obtainedprior to sacrifice from all animals, by the Arsenzo IIIdry-slide method [14]. Corresponding female rats(controls) were maintained for each of the threetreated sets of animals (prepubertal, 63-day-old, and102-day-old) and were sacrificed along with the malerats. The operator sacrificing the animals and harvest-ing the prostate and seminal vesicles was blinded tothe treatment group of the animals. The animals andexperiments described in these studies were approvedby the Institutional Animal Care Committee of theUniversity of Pittsburgh according to public healthservice guidelines.

All statistical analyses were performed using Stu-dent’s t-test for independent samples (two-tailed),with Microsoft (Redmond, WA) Excel® software. P <0.05 was considered significant.

RESULTS

All the pregnant dams included in the study wereable to deliver pups. No calcitriol was administered tothe pups after birth. The initial mean body weight ofpregnant dams in group 1 (controls) was 246.25 ± 12.2

g, and the initial mean body weight was 245.44 ± 7.5 gfor the dams in the treatment groups (group 2 and 3).Pregnant dams treated with calcitriol gained signifi-cantly less weight during pregnancy than controldams (Table I). The pups in group 2 which were ex-posed to calcitriol in utero had a lower mean bodyweight as compared to the control pups (group 1)which were not exposed to calcitriol (Table I). Thisdifference in mean total body weight between the cal-citriol-exposed and control animals was more pro-nounced in the animals sacrificed at age 63 days (TableI). A similar difference in mean total body weight wasalso evident in female rats exposed to calcitriol (130 ±8 g) as compared to control females (165 ± 21 g), butthe difference was not statistically significant. In theanimals maintained for 102 days, total body weighttended to be lower in the castrated, calcitriol-exposedpups compared to the castrated control pups (Table I).There was no significant difference in the total bodyweights of the noncastrated, calcitriol-exposed, andcontrol pups.

The mean total weights of the prostate and seminalvesicles obtained from the calcitriol-exposed and con-trol animals were compared at three different timepoints encompassing different hormonal stages of ratdevelopment (prepuberty, postpuberty, and adult). Atage 25 days (prepuberty), the mean total prostaticweight of the calcitriol-exposed pups was 35% greaterthan that of control pups (Fig. 2). At age 63 days (im-mediate postpuberty) there were no statistically sig-nificant differences in the mean total prostate or semi-nal vesicle weights between the calcitriol-exposed orcontrol pups (Fig. 3). However, at age 102 days (adult),mean total weight of the prostates obtained from non-castrated, calcitriol-exposed pups was 68% higherthan that obtained from noncastrated control pupssacrificed postpuberty at age 102 days (Fig. 4). While a

Fig. 1. Time course of various events during the study. Calcitriol was administered to the pregnant animals starting at 13 days of gestation.

Prenatal Calcitriol and Prostatic Development 183

similar difference was observed in mean seminalvesicle weights between the control and study groups,this difference was not statistically significant. We alsocompared the ratio of the prostate and seminal vesicleweights to the total body weights in each of the ani-mals. The results of these comparisons are shown inTable II. Statistically significant differences were onlyobserved between the following groups: 1) prostatesof control and calcitriol-exposed animals sacrificed atage 25 days, and 2) prostates of uncastrated controland calcitriol-exposed animals sacrificed at age 102days.

A significant and unexpected finding was that 30/42 (71%) of the uncastrated pups which were exposed

to calcitriol died suddenly during puberty (age 33–44days) within a relatively short time period. The com-parable mortality was significantly lower in controlmales (2/19 or 10.5%), in castrated, calcitriol-exposedmales (2/10 or 20%), and in females (1/18 or 5.5%).Autopsies of the pups which died suddenly revealedno obvious gross or microscopic visceral abnormali-ties. Specifically, there were no pathologic changes ob-served in the kidneys to suggest the presence of neph-rocalcinosis or other such renal diseases which couldbe attributed to increased calcium deposition. Therewere no characteristic abnormalities in serum levels ofvarious electrolytes except for calcium. The calciumlevel in these animals was low but approximated

TABLE I. Differences in Weight and Serum Parameters Between Control and Treated Animals*

Parameter Controls (± SD) Calcitriol-exposed (± SD) P value

Weight gain by pregnant dams 100.0 (± 7.5) g 87.78 (± 7.8) g <0.01Mean total body wt. (25 days) 75.04 (± 3.7) g 71.64 (± 6.16) g NSMean total body wt. (63 days) 192.0 (± 18.71) g 161.77 (± 19.26) g <0.005Mean total body wt. (C, 102 days) 230.0 (± 22.7) g 208.12 (± 23) g NSTotal body wt. (NC, 102 days) 225.25 (± 17.4) g 221.25 (± 12.5) g NSSerum calcium (25 days) 10.9 (± 2.2) mg/dl 11.25 (± 1.36) mg/dl NSSerum calcium (63 days) 4.25 (± 0.58) mg/dl 4.14 (± 0.53) mg/dl NSSerum calcium (102 days) 5.83 (± 1.1) mg/dlSerum calcium (intact) (102 days) 5.75 (± 0.85) mg/dl NSSerum calcium (C) (102 days) 5.37 (± 0.45) mg/dl NS

*NS, not significant; wt., weight; C, castrated; NC, noncastrated.

Fig. 2. Comparison of prostate and seminal vesicle (Sem. Ves.) weights (±1 SD) in controls (group 1) and treated rat pups at age 25 days(group 2). Note that there was a significant difference in prostatic weights which was not observed in seminal vesicle weights.

184 Konety et al.

those obtained in controls and other surviving ani-mals. Animals maintained up to 63 or 102 days hadlower serum calcium levels than those sacrificed be-fore puberty at 25 days (Table I). There were no sig-nificant differences in serum calcium levels betweencalcitriol-exposed and control pups sacrificed beforeor after puberty.

Microscopic examination of hematoxylin-eosin(H&E)-stained sections of prostates from the animalssacrificed at age 25 days revealed that the control pros-tates appeared less differentiated than those from ani-mals exposed to calcitriol (Fig. 5a,b). The prostates ofthe calcitriol-exposed animals were composed of com-pact acini lined by columnar cells, with increasednumbers of secretory vesicles being visible in the cells.The control prostates were made up of a larger pro-portion of dilated acini and tubules lined by morecuboidal cells. Overall, there were varying degrees ofdifferentiation evident throughout the gland, with theperipheral acini appearing more mature than the cen-tral portions of the gland. Prostates from the survivinganimals sacrificed at 63 days exhibited a more com-pletely matured architecture (Fig. 5c,d). In both con-trol and calcitriol-exposed prostates, the acini werelined by tall columnar cells with the presence of se-cretory “snouts” which are characteristic of apocrineglands. There were no significant histologic differ-ences between control and calcitriol-exposed prostates

in this age group. Prostates from the older survivinganimals (102 days) appeared to be somewhat less dif-ferentiated, with dilated acini and scattered evidenceof squamoid differentiation in both control and cal-citriol-exposed groups (Fig. 5e,f).

DISCUSSION

Calcitriol (1,25-dihydroxyvitamin vitamin D3) hasbeen found to induce differentiation in a variety oftissues including skin, intestinal epithelium, skeletalmuscle, hematopoietic cells, endothelium, and bone[15]. The intranuclear receptors for vitamin D, a mem-ber of the steroid hormone receptor superfamily, havebeen detected in a host of human tissues [16]. In themale genitourinary system, vitamin D receptors havebeen demonstrated in the kidney, epididymis, testes,and prostate [12,16]. Miller et al. [12] first describedthe presence of the vitamin D receptor (VDR) in thehuman prostate cancer cell line LNCaP. Other studieshave since demonstrated the presence of VDR in otherprostate cancer cell lines in culture and in normalprostate cells [17]. The active form of vitamin D (cal-citriol) has been found to inhibit the proliferation ofneoplastic human prostate cells in vitro and in vivo[12,17,18]. We have also been able to demonstrate theantiproliferative effects of calcitriol in vitro and in vivo

Fig. 3. Comparison of prostate and seminal vesicle (Sem. Ves.) weights (±1 SD) in 63-day-old postpubertal rats from the control group(group 1) and rats of the same age exposed to calcitriol in utero (group 3).

Prenatal Calcitriol and Prostatic Development 185

in the Dunning rat model of prostate cancer [13]. Wealso previously found that calcitriol enhances growthof the normal prostate in the absence of testosterone[9]. Castrated adult rats treated with calcitriol exhibitincreased prostatic growth, mainly due to stromal pro-liferation. Conversely, increased normal prostaticglandular differentiation in the absence of stromalproliferation is seen on treatment with calcitriol in thepresence of testosterone.

Increased stromal proliferation with alteration ofthe normal epithelial:stromal ratio (5:1) has been ob-served in male rats treated with estrogen in the neo-natal period and subsequently exposed to exogenousandrogens in adulthood [2]. A similar histologic alter-

ation was found in the prostates of castrated ratstreated with calcitriol in our study. Based on theirfindings, Rajfer and Coffey [2] proposed that “im-printing” of the prostate occurred in male rats duringthe neonatal period. Prenatal low-dose estrogen expo-sure has been shown to exert a growth-promoting ef-fect on the prostate, while the converse is seen whenhigher doses of estrogen are employed [7]. In male ratsexposed to low dose estrogens in utero, subsequentadult prostatic weight was increased by 30%, with anincrease in epithelial budding. A twofold increase inthe number of androgen receptors per cell and a 40%increase in cellular DNA content accompanied thesechanges. These findings are consistent with a hyper-

Fig. 4. Comparison of prostate and seminal vesicle (Sem. Ves.) weights (±1 SD) in 102-day-old adult rats from the control group (group1) and rats of the same age which were exposed to calcitriol in utero (group 3). c, castrate animals; nc, intact animals.

TABLE II. Comparison of Ratio of Sex Accessory Tissue (Prostate and Seminal Vesicle) Weights (± SD) to Total BodyWeight in the Calcitriol-Exposed and Control Groups*

Group

Control prostatewt./total

body wt. %

Calcitriol prostatewt./total

body wt. % P value

Control SVwt./total

body wt. %

Calcitriol SVwt./total

body wt. % P value

25 days 0.041 (± 0.007) 0.058 (± 0.013) 0.0005 0.016 (± 0.004) 0.02 (± 0.005) NS63 days 0.081 (± 0.016) 0.09 (± 0.021) NS 0.102 (± 0.024) 0.108 (± 0.023) NS102 days (C) 0.005 (± 0.003) 0.004 (± 0.002) NS 0.007 (± 0.006) 0.003 (± 0.001) NS102 days (NC) 0.061 (± 0.02) 0.1 (± 0.02) 0.04 0.075 (± 0.029) 0.1 (± 0.02) NS

*wt., weight; SV, seminal vesicle; NS, not significant; C, castrated; NC, noncastrated.

186 Konety et al.

Fig. 5. Photomicrographs show H&E-stained sections of pros-tates from control rats in group 1 (a) and calcitriol-exposed rats ingroup 2 (b), both sacrificed at age 25 days (×10). Also shown aresections of prostates from control rats in group 1 (c) and calcitriol-exposed rats in group 3 (d), both sacrificed at age 63 days (×10).Note the characteristic secretory “snouts” (arrows in c and d).Shown as well are sections of prostates from control rats in group1 (e) and calcitriol-exposed rats in group 3 (f), both sacrificed atage 102 days (×10).

plastic change in the prostate, apparently induced byexposure to low circulating levels of estrogen duringgestation. These results suggest that “imprinting” ofthe prostate may occur prior to birth and may be me-diated by other steroids in addition to androgens. Thisfact is further substantiated by the results of thisstudy. In addition to estrogens, calcitriol also appearsto have an effect on prostatic growth long after theperiod of initial exposure.

In humans, pregnancy is associated with high cir-culating levels of calcitriol [19]. Previous reports sug-gested that circulating levels of calcitriol in the ratfetus are undetectable prior to 14 days of gestation andincrease significantly thereafter [15]. Clements andFraser [20] demonstrated preferential transplacentaltransfer of calcitriol (and other forms of vitamin D)from mother to fetus in rats, and its accumulation infetal muscle. Higher circulating levels of calcitriol inpregnant female rats (which may occur normally dur-ing gestation, as in humans) could lead to increasedaccumulation of this hormone in the fetus. This couldmimic the conditions of our study, with the prenatalrat prostate being exposed to fairly high levels of cal-citriol, resulting in “imprinting” of the prostate inmale fetuses. This evokes the possibility of calcitriolphysiologically influencing subsequent prostaticgrowth and possibly benign prostatic hyperplasia dur-ing late adult life. However, we did not observe anysignificant differences in prostatic weight betweencastrated, control and calcitriol-exposed rats. This mayin part be due to the extreme reduction in prostaticsize in response to androgen withdrawal in bothgroups, which may render it difficult to appreciatesize differences between the two groups in response tocalcitriol alone.

While we have found that calcitriol promotes nor-mal prostatic growth, the exact mechanism by whichthis action is elicited is still unclear. One possibility isthat calcitriol may act through the androgen receptor.However, we have observed calcitriol-promoted pros-tatic stromal proliferation even in animals treated withthe androgen receptor blocking agent flutamide (un-published data). Akin to our previous experiments inadult rats, we were not able to demonstrate any effectof calcitriol on prostatic weight in animals immedi-ately after puberty, when the effect of testosterone canbe expected to be the most significant.

The phenomenon of sudden death which occurredin a majority of uncastrated rats was remarkable andconsistently reproducible. We were not able to detectany unique anatomic or biochemical abnormalities inthese animals to explain the cause of death. Specifi-cally, we were not able to detect any histologic abnor-malities in the heart as examined at autopsy. Therewere also no electrolyte abnormalities which were

unique to the affected animals, and which would havesuggested possible cardiac dysfunctions. The timingof the deaths during puberty suggests that the testos-terone surge at puberty probably contributed to thedeaths of these animals, especially since similar resultswere not observed in identically treated castrated rats.Since these animals were placed on a vitamin D-deficient diet in the dark during the study, low circu-lating levels of calcitriol accompanied by low calciumlevels may have also played a role. Low maternal lev-els of circulating calcitriol have been correlated withdefective development of the contractile proteins inthe neonatal rat heart [21]. It is conceivable that ani-mals exposed to high levels of calcitriol for a shortduration during fetal life as in this study may be moresensitive to subsequent deficiencies of this hormone.Such a defect may render the affected animals lesscapable of coping with the stress of normal puberty,and therefore they may succumb to sudden death. Fi-nally, the premature deaths may not be a result of thetreatment but due to other as yet unknown causes.

CONCLUSIONS

In conclusion, we have investigated the effect ofprenatal calcitriol exposure on the growth and devel-opment of the normal rat prostate. We found that fetalexposure to high doses of calcitriol resulted in a sig-nificant increase in prostatic weight after birth, in theprepubertal period, and well into adulthood. Calcitriolexposure does not appear to affect prostatic growth atthe time of puberty, presumably in the presence ofhigh circulating levels of testosterone. An unusuallyhigh mortality was observed in the calcitriol-exposed,uncastrated animals at the time of puberty which maybe related to increased sensitivity to calcitriol defi-ciency in these animals. It is also possible that thedeaths were not a result of the treatment but were dueto other, unrelated causes.

ACKNOWLEDGMENTS

B.R.K. is a recipient of the Ferdinand Valentine Fel-lowship of the New York Academy of Medicine andthe Frederick Schwentker Endowment Award fromthe Department of Urology, University of Pittsburgh.

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