help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gilleran, J. P.
Right arrow Articles by Prins, G. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gilleran, J. P.
Right arrow Articles by Prins, G. S.
Endocrinology Vol. 144, No. 5 2046-2054
Copyright © 2003 by The Endocrine Society

The Role of Prolactin in the Prostatic Inflammatory Response to Neonatal Estrogen

Jason P. Gilleran1, Oliver Putz1, Megan DeJong, Samuel DeJong, Lynn Birch, Yongbing Pu, Liwei Huang and Gail S. Prins

Department of Urology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612

Address all correspondence and requests for reprints to: Gail S. Prins, Ph.D., Department of Urology, M/C 955, University of Illinois at Chicago, 820 South Wood, Chicago, Illinois 60612. E-mail: gprins{at}uic.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen exposure in the neonatal rat has been shown to disrupt the normal morphology and development of the prostate gland. The response to this exposure is manifest in adulthood as epithelial dysplasia and chronic inflammation. This inflammatory response consists of infiltrating T-lymphocytes and macrophages, which is typically observed in chronic prostatitis in both rodents and humans. In our rat model, the developmental hormonal milieu is altered following estrogenization, resulting in transient hyperprolactinemia, which begins prepubertally (postnatal d 21) and persists throughout puberty. The purpose of this experiment was to determine the role of prolactin (PRL) in the altered phenotype of the adult rat prostate exposed to neonatal estrogen. Male Sprague Dawley rat pups (n = 104) were randomized at birth to receive oil or estradiol benzoate on postnatal d 1, 3, and 5. They were further randomized to receive bromocriptine (BrC) pellets or placebo at d 15. Animals were killed at d 90. Serum PRL and testosterone levels, prostate lobe, and hormone-dependent and immune-related tissue weights and histology were examined. Animals receiving BrC had significantly lower PRL levels at d 90, regardless of estrogen status. Prostate lobe and testicular weights were significantly reduced in estrogenized animals vs. controls, and BrC did not abate this response, indicating that growth inhibition is not mediated through hyperprolactinemia. Splenic and thymus weights were greater in estrogenized animals, and this was partially reversed with BrC. Neonatal estrogen exposure resulted in a marked infiltration of CD4+ and CD8a+ lymphocytes in the prostate gland, and this was partially reversed by concomitant BrC treatment. In contrast, the estrogen-induced macrophage infiltration of the prostate was not affected by PRL suppression. These findings indicate that prostatic inflammation and immune cell infiltration in the prostate gland of neonatally estrogenized rats is mediated through a PRL-dependent as well as a PRL-independent mechanism. As prostatic inflammation or prostatitis in humans is associated with benign prostatic hyperplasia and prostatic carcinoma, this animal model may provide mechanistic insight with regards to age-associated prostatic lesions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NORMAL DEVELOPMENT, GROWTH, and function of the prostate gland throughout life are dependent on androgens that act in synergy with other modulating hormones such as estrogen and prolactin (PRL; Refs.1, 2, 3). In the rat model, prostate development is initiated late in fetal life and undergoes extensive branching morphogenesis and cellular differentiation during the neonatal period (4, 5). It is during this early developmental period that hormonal modulation can have a permanent and irreversible effect on the gland’s morphology, cellular organization, and function. Administration of exogenous estrogen between postnatal day (PND) 1–5 has been shown to permanently imprint the prostate resulting in reduced growth, compromised secretory function, and decreased responsiveness to androgens in adulthood (6, 7, 8). This developmental estrogenization leads to prostatic epithelial hyperplasia and moderate-to-severe dysplasia (prostatic intraepithelial neoplasia) with aging and, consequently, is considered to be a predisposing factor for prostatic tumor formation later in life (9, 10, 11). The effects are lobe specific, with the ventral lobe being most prominently affected with regards to growth inhibition, secretory dysfunction, and dysplasia (8, 11). In addition, the neonatally estrogenized prostate exhibits significant infiltration of immune cells commencing with puberty which results in chronic inflammation of that organ (11, 12, 13, 14). Thus, the young adult and aged estrogenized prostates are characterized by the presence of luminal granulocytes, marked stromal macrophage infiltration, and mild-to-severe lymphocytic aggregation within the stromal compartment with diapedisis across the epithelium as the animals age. This is noteworthy in that persistent inflammation has been observed in diseases of the human prostate including benign prostatic hyperplasia and adenocarcinoma and a causal relationship has been suggested (15, 16). The basis for the chronic inflammatory response in the neonatally estrogenized prostate is not understood. However, it is known that both estrogens and PRL can have marked effects on the immune system in general and prostatic inflammation in particular.

It is established that estrogens stimulate anterior pituitary PRL secretion through inhibition of hypothalamic dopaminergic suppression pathways (17). Thus, exogenous estrogenic exposures result in relative hyperprolactinemia for variable lengths of time (18, 19). Importantly, estrogen-induced hyperprolactinemia in the adult rat has been shown to induce prostatitis (20). Recently, Stoker and colleagues (21, 22) have shown that elevations in circulating PRL during the neonatal or prepubertal period can increase the incidence of prostate inflammation in the adult rat. These findings are particularly significant in that neonatal estrogen exposure transiently increases prepubertal circulating PRL levels in male rats (19) by altering development of dopaminergic neurons in the arcuate nucleus (23), although adult PRL levels appear unaltered. Thus, the possibility exists that the prostatic inflammation induced by neonatal estrogens is indirectly mediated through transient prepubertal hyperprolactinemia.

In addition to induction of inflammation, it is well established that PRL augments the growth and function of the immature and adult rat prostate gland (24, 25, 26, 27, 28, 29, 30). This effect has been shown to be mediated in part through up-regulation of androgen receptor expression and augmentation of androgen action (31). Importantly, estrogen-induced effects on prostate growth and induction of prostatic dysplasia have both been blocked by PRL suppression with bromocriptine (32, 33), which further implicates a critical role for PRL in mediating certain aspects of estrogenic effects on the prostate gland.

It has been determined through the use of organ culture studies that several aspects of estrogenic alterations on prostate morphogenesis and differentiation are direct effects of estrogens at the level of the prostate gland (3, 34). However, these studies are limited to the developmental period due to the inherent time limitations of organ culture studies. The purpose of the present study was to investigate the role of elevated PRL in mediating particular aspects of the neonatally estrogenized phenotype including growth defects and the ensuing hyperplasia, dysplasia and chronic inflammation of the prostate gland observed in adulthood. Specifically, we sought to determine whether PRL suppression with bromocriptine, a dopamine agonist, could reverse any or all of these phenomenon and thereby delineate to what degree estrogen directly or indirectly mediates these effects.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and housing
All animals were handled in accordance with the principles and procedures of the Guiding Principles for the Care and Use of Animal Research. Timed-pregnant Sprague Dawley rats (Zivic-Miller Laboratories, Pittsburgh, PA) were housed individually in polypropylene solid-bottom cages with steel covers on corncob bedding (Bed-o’-corn, The Andersons, Maumee, OH). Rooms were kept at 21 C and 60% humidity, with a 14-h light, 10-h dark schedule. Animals had free access to Harlan Teklad 22/5 rodent diet (Harlan Inc., Madison, WI) and to tap water ad libitum. All animals were given food from the same lot. Pregnant dams were monitored daily for delivery of the pups, and the day of birth was designated postnatal d 0 (PND 0). One day after parturition (PND 1), total numbers of male and female pups were recorded for each litter.

Dosing and treatment
The experiment was performed between July and November 2001. Litters of five females were randomly assigned to one of four treatment groups, so that control and treated animals were not monitored consecutively but simultaneously. A total of 20 gravid females were used in the study. On PND 1, neonatal pups were sexed according to ano-genital distance and the litter size was culled to 10 through the removal of female pups. Newborn male rats were treated on PNDs 1, 3, and 5 with sc injections of 25 µg ß-estradiol-3-benzoate (EB; Sigma, St. Louis, MO) in 25 µl of peanut oil (Arachis sp.) as vehicle or with oil alone. On PND 15, males were randomized and under ether anesthesia, given sc pellets containing either 35 mg bromocriptine mesylate (BrC) or placebo (Plb). An earlier application of BrC was not considered necessary because the mechanism for an estrogen-induced surge of PRL is not intact before PND 12 (35). This resulted in four treatment groups: group 1 = Oil + Plb; group 2 = EB + Plb; group 3 = Oil + BrC; group 4 = EB + BrC. The 90-d release BrC and Plb pellets (Innovative Research of America, Sarasota, FL) provided a steady state release rate of 0.39 mg/d, which is adequate to suppress PRL in adult rats. Animals were killed by decapitation on PND 90, trunk blood was collected and the prostate gland, testes, adrenal glands, spleen, and thymus were removed and weighed. Serum was separated and frozen in multiple aliquots at -70 C for RIA.

Tissue removal and preparation
Individual prostatic lobes (dorsal, lateral 1 and 2, and ventral lobes) were microdissected in PBS at 4 C under an Olympus zoom stereo SZ4045 microscope (Optical Analysis Corp., Nashua, NH). After removal of adipose tissue and sequential removal of the ampullary glands, vas deferens, bladder, seminal vesicles, and coagulating glands, individual prostate lobes were separated. The lateral lobe was further divided into lateral prostate 1 and lateral prostate 2, whereas the ventral lobe was further dissected into smaller ductal arrays.

In half of the animals of each litter, one dorsal, lateral 1, lateral 2, and ventral were embedded in OCT mounting medium (Sakura Finetek USA, Torrance, CA) along their longitudinal orientation, snap frozen in liquid propane, and subsequently stored at -80 C, whereas the other lobes were fixed in Bouin’s solution for 48 h and transferred into 70% ethanol for storage. Prostate glands of the remaining half of the animals from the same litter were snap frozen in liquid nitrogen and stored at -80 C. Testes, adrenal glands, spleen, and thymus were fixed in Bouin’s solution for 48 h and subsequently stored in 70% ethanol. All Bouin’s-fixed tissues were dehydrated and embedded in paraffin.

Serum hormone titers
Serum PRL levels were determined for all animals treated in the present study as well as control and neonatally estrogenized rats killed in previous studies at PND 10, 15, 21, and 35. Serum PRL levels were quantitated using an 125I-rat PRL RIA kit (ALPCO Diagnostics, Windham, NH) with values compared with rat PRL standards. Serum testosterone was quantitated by RIA using a 125I-total testosterone Coat-a-Count kit (Diagnostic Products Corp., Los Angeles, CA) as previously described (36).

Immunocytochemistry
Prostate lobes were examined for androgen receptor (AR) and immune cells by indirect immunocytochemistry as previously described (37). Tissue specimens from five to seven animals were examined for each treatment group. Briefly, individual prostate lobes were mounted on precooled chucks (-24 C) in a CM3050 cryostat (Leica Corp., Deerfield, IL), and 5-µm sections were thaw-mounted onto gelatin-coated glass slides. Individual lobes were sectioned along their longitudinal axis to reveal the proximal-distal orientation. Sections were fixed in either 2% paraformaldehyde with 0.2% picric acid or acetone and treated for both endogenous peroxidase and nonspecific binding with 3% hydrogen peroxide and 2% (vol/vol) normal host serum, respectively. Samples were incubated with primary antibody overnight in a humidified box at 4 C and appropriate biotinylated secondary antibody for 30 min at room temperature (Vector Laboratories, Inc., Burlingame, CA). Biotin was detected with an avidin-biotin peroxidase kit (ABC-Elite, Vector Laboratories, Inc.) using diaminobenzidine tetrachloride as a chromagen. Specimens were counterstained with Gill’s no. 3 hematoxylin (1:4), dehydrated in alcohol, mounted and coverslipped with Permount (Fisher Scientific, Itasca, IL). Images of immunostained specimens were digitalized with an Axioskop 20 microscope (Carl Zeiss, Inc., Thornwood, NY) and a digital AxioCam camera scanner (Carl Zeiss), and were processed and analyzed using AxioVision 2.05 software (Carl Zeiss).

Primary antibodies used were rabbit polyclonal anti-AR (PG21, 2 µg/ml; Ref.37), mouse monoclonal antirat CD8a lymphocyte (no. 22071D, 1 µg/ml; PharMingen Inc., San Diego, CA), mouse monoclonal antirat CD4 lymphocyte (no. W3–25; 1 µg/ml; Cedar Lane Laboratories; Hornby, Ontario, Canada), mouse monoclonal antirat macrophage (ED2; 1:3000 dilution; Serotec Laboratories, Raleigh, NC). Normal rabbit IgG (Vector Laboratories) or normal mouse ascites fluid (Sigma) were substituted for primary antibodies as negative controls. For immune cells, 5-µm rat spleen tissue sections were used as a positive control.

Grading of immunostaining
All tissue sections stained for CD4 and CD8a were examined by the primary investigator (G.S.P.) and secondary investigator (J.P.G.). Tissue sections from a total of 28 animals from 20 different litters were examined at x100 and x400 using a BH-2 microscope (Olympus Optical Ltd.). The grading system employed to quantify the amount of inflammatory infiltrate was scored on a 0–4 scale, based on the number of cells positively stained per constant area of tissue (0 = none; 1 = mild; 2 = moderate; 3 = heavy; 4 = massive). Two numerical scores were assigned to each tissue section; 1) the primary or most prevalent grade of immunostaining present, and 2) the secondary most common grade of immunostaining present, similar to the Gleason grading system for prostate adenocarcinoma. For example, if a tissue section showed predominantly heavy lymphocytic infiltrate throughout the central ducts but only mild infiltrate in the distal tips, the prostate was graded as 3 + 2, then added to create a final infiltrate score of 5. Values for each animal were obtained and entered for statistical analysis.

The quantitation of macrophage stromal infiltrate was made by manual cell counts in two noncontiguous x100 fields of individual tissue sections for each animal examined. Area of each tissue section was measured in mm2, using a x10 hemocytometer grid for spatial calibration on Zeiss Image software (Carl Zeiss). Values were expressed as density (cells/mm2 area).

Statistics
Parametric data were analyzed by one-way ANOVA after establishing homogeneity of groups followed by post hoc Tukey or Student-Newman-Keuls multiple comparison test. If necessary, a nonparametric Kruskal-Wallis ANOVA followed by the Dunn multiple comparison test were used to compare medians of groups. P < 0.05 was accepted as significant. To avoid variation due to genetic predispositions in animals from different litters (litter effect), the numbers per group are the numbers of litters, not the number of pups. Unless stated otherwise, all values are expressed as means ± SEM with indication of the number of separate determinations corresponding to numbers of pooled litters. All statistical analyses were performed using InStat version 3.01 for Windows 95 (GraphPad Software, Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serum hormone titers
Mean serum PRL levels are shown in Fig. 1Go. As has been previously established in the literature (38), circulating PRL was low in oil-control developing rats at PND 10 and 21 and increased significantly by d 35 (P < 0.01) with adult levels observed at d 90. Rats given neonatal estradiol injections maintained low PRL levels at PND 10 as predicted but by d 21, there was a significant (P < 0.01) precocious increase to adult levels of circulating PRL. This hyperprolactinemia persisted through d 35 but did not further increase so that by d 90, circulating PRL levels were similar between the control and estrogenized rats. Treatment with BrC from PND 15–90 resulted in significantly lower PRL levels (P < 0.05) in both oil-control and neonatally estrogenized rats. These findings demonstrate that neonatal estrogen exposure leads to hyperprolactinemia throughout puberty which dissipates in the adult rat and that the BrC dosage used in the present study was sufficient to suppress circulating PRL levels from PND 15 until the rats were killed at PND 90.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 1. Serum PRL levels (ng/ml) in male rats treated neonatally with oil or EB and d 1, 3, and 5 of life and killed on d 10, 21, 35, or d 90. The d 90 rats were divided into four groups treated with Oil or EB with bromocriptine mesylate (BrC) or Plb pellets from d 15–90. Bars represent the mean ± SEM. a, P < 0.05 vs. d 10 Oil; b, P < 0.01 vs. d 10 Oil; c, P < 0.01 vs. d 21 Oil; d, P < 0.05 vs. d 35 Oil; e, P < 0.05 vs. d 10 EB; f, P < 0.01 vs. d 10 EB; g, P < 0.01 vs. d 35 EB; and h, P < 0.001 vs. d 90 Oil-Plb and d 90 EB-Plb.

 
As has been previously described (36), serum testosterone levels were reduced to 1.51 ± 0.26 ng/ml in the EB-Plb rats from control levels of 3.06 ± 0.33 ng/ml in Oil-Plb rats (P < 0.05). Treatment with BrC did not effect these values in the Oil-BrC rats (3.55 ± 0.47) or the EB-BrC rats (1.76 ± 0.26; P < 0.05 vs. Oil-BrC).

Organ weights
As has been previously established, neonatal exposure to estradiol benzoate in the present study significantly reduced the absolute and relative weights of all three prostate lobes in adulthood (P < 0.01; Fig. 2Go, A–D). Bromocriptine mesylate treatment by itself resulted in a modest and nonsignificant decrease in prostate lobe weights in the adult control rats that most likely reflects the known growth stimulatory effects of PRL on the prostate gland (31). When PRL levels were reduced throughout development, the extent of estrogen-induced prostatic growth inhibition at adulthood was unaffected which indicates that the growth suppressive actions of estrogens are not mediated through estrogen-induced hyperprolactinemia (Fig. 2Go, A–D). Similar responses were observed for the epididymis, seminal vesicles, and coagulating gland (data not shown).



View larger version (30K):
[in this window]
[in a new window]
  		Figure 2. Relative weights (mg/kg BW) of the individual prostate lobes reveals that neonatal estrogen exposure significantly lowers prostate size and that PRL deprivation throughout development does not reverse this effect. In estrogenized animals, the dorsal lobe (A), lateral prostate 1 (B), lateral prostate 2 (C), and ventral lobe (D) were reduced by neonatal estrogen (EB + Plb = estradiol benzoate plus placebo pellets) when compared with control (Oil + Plb) weights. Treatment with bromocriptine mesylate (BrC) from d 15–90 did not reverse the estrogen-induced weight decrease (EB + BrC). Bars represent the mean ± SEM. a, P < 0.01 vs. Oil + Plb; b, P < 0.001 vs. EB + Plb; c, P < 0.001 vs. Oil + BrC; and d, P < 0.001 EB + BrC.

 
Estrogen treatment markedly reduced relative testis weights to 81% of control levels (Fig. 3AGo). Bromocriptine mesylate treatment alone did not affect testis weights, but when given with neonatal estrogen exposure, a synergistic effect was observed resulting in a further significant reduction to 56% of control weights. Relative adrenal weights (Fig. 3BGo) were not influenced by estrogen or BrC treatments alone but they were significantly increased in the EB + BrC animals compared with the Oil + BrC rats (P < 0.001). Spleen and thymus weights were measured as potential markers of immune system alterations. Splenic weights were significantly increased by 42% in estrogen-exposed animals compared with controls (P < 0.05; Fig. 3CGo). This estrogenic stimulatory effect abated to a nonsignificant 21% increase with the addition of BrC treatment. Thymus weights were significantly increased by 47% and 30% in estrogenized rats given Plb or BrC, respectively (Fig. 3DGo). Importantly, the increased thymus weight in the EB-BrC-treated animals was significantly lower than the increased thymic weights in the EB-Plb rats (P < 0.05). Absolute organ weight values reflected those for relative organ weights.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 3. Relative testicular weights (A) were significantly reduced by estrogen treatment (EB + Plb) compared with control weights (Oil + Plb). This suppressive effect was greater in the presence of BrC (EB + BrC), where values were significantly smaller compared with EB + Plb rats. Relative adrenal weights (B) were increased in the EB + BrC group compared with Oil + BrC but were not affected by EB exposure alone (EB + Plb vs. Oil + Plb). Relative spleen weights (C) were increase in response to neonatal estrogen exposure (EB + Plb vs. Oil + Plb), and this effect was abrogated by PRL suppression (EB + BrC vs. Oil + BrC). Thymus weights (D) were increased in response to estrogen exposure in both Plb and BrC-treated animals; however, the elevation was significantly lower in the BrC group (P < 0.05; EB + Plb vs. EB +BrC). Bars represent the mean ± SEM. a, P < 0.05 vs. Oil + Plb; b, P < 0.01 vs. EB + Plb; c, P < 0.01 vs. Oil + BrC; and d, P < 0.05 EB + BrC.

 
Immunocytochemistry
Similar to our previous findings, young adult ventral prostates of neonatally estrogenized rats in the present investigation revealed characteristic histologic lesions including epithelial disorganization with loss of basal/apical orientation in many acini, epithelial hyperplasia, relative increase in stromal mass and inflammatory cell infiltrate. Suppression of circulating PRL levels did not affect the development of this phenotype to any obvious degree. To more closely analyze immune cell infiltration in the estrogenized prostates treated with and without BrC, tissue sections were immunostained for CD4+ and CD8a+ T-lymphocytes and macrophages and results were semiquantitated. Lymphocytic infiltration of prostatic tissue by both CD4+ and CD8a+ cells was significantly elevated in the EB + Plb animals compared with oil-treated controls (P < 0.001, Table 1Go and Fig. 4Go, A and B, and E and F). Treatment of estrogenized rats with BrC partially reversed the level of infiltration by these immune cells (Table 1Go and Fig. 4Go, C and D, and G and H), although the decreased response was significant only for the CD8a+ cell population (P < 0.01, EB-BrC vs. EB-Plb). Prostates from estrogenized rats treated with BrC had higher levels of CD4+ cells than Oil + BrC rats (P < 0.05), whereas there was no statistical difference in CD8a+ cells between those two treatment groups.


View this table:
[in this window]
[in a new window]
  		Table 1. Mean grade CD4+ and CD8a+ cell infiltration and mean stromal macrophage density in ventral prostates

 


View larger version (120K):
[in this window]
[in a new window]
  		Figure 4. Immunocytochemical localization of CD4+ and CD8a+ T lymphocytes, macrophages and androgen receptor in the ventral prostate lobes of d 90 estrogenized and control rats. Images demonstrate degree of CD4+ (A–D) and CD8+ (E–H) cellular infiltrate in all four treatment groups at 40x (bar represents 50 µM). In Oil-treated rats, occasional lymphocytes were observed in the interstitial space of the ventral lobes (A, C, E, and G). In the EB-Plb rats, there was a marked increase in infiltration of CD4+ (B) and CD8a+ (F) lymphocytes with some observed crossing into the epithelial layers. Treatment with BrC partially reversed the infiltration in EB-exposed rats and this was most notable for CD8a+ cells (H). Representative prostate sections stained for ED2, an antimacrophage antibody (I–L), demonstrate increased macrophage infiltration in estrogen-exposed rats (J) that is not reversed with BrC (L). The bar for I–L represents 200 µm. Representative prostate sections immunostained for AR are shown in M–P (bar, 50 µm). Regional loss of AR in epithelial nuclei of EB-treated animals (N) was not affected by the administration of BrC as observed in P, where a well-organized epithelium expressing AR (below) and a disorganized epithelium with minimal AR immunostain (top) are observed in two separate acini.

 
The mean stromal macrophage density (cells/mm2) in the ventral prostates of treated animals is shown in Table 1Go. Fixed macrophage density markedly (P < 0.001) increased in the adult prostates of estrogenized rats compared with oil-controls (Fig. 4Go, I and J). Treatment of rats with BrC did not lower the resident macrophage density in either the oil-controls or estrogenized rats (Fig. 4Go, K and L), indicating that PRL had no effect on this immune cell population.

Previous studies in our laboratory have shown that ventral prostate AR levels are down regulated by neonatal estrogen exposure (8, 36) and also that PRL can augment AR expression in the rat prostate gland (39). To determine if PRL suppression affected prostatic AR levels in neonatally estrogenized prostates, AR was evaluated by immunocytochemistry. Rats treated with estrogen neonatally exhibited regional loss of nuclear AR immunostain in the ventral lobes as previously described (Fig. 4NGo). Suppression of circulating PRL had no effect on this response because decreased AR immunoreactivity was routinely observed in EB + BrC prostates, particularly in regions where cellular architecture was compromised (Fig. 4PGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The neonatal estrogen treatment regime given to rats in the present study markedly elevated circulating PRL levels during the prepubertal/pubertal period as has been previously documented (19). By comparing PRL levels over time, the data can be interpreted in two ways: 1) that neonatal estrogen induced transient hyperprolactinemia, which abates with maturation; or 2) that neonatal estrogenic exposure precociously elevated PRL secretion to adult levels by d 21 of life, where it remained thereafter. The net result, nonetheless, is that circulating PRL levels were elevated during an active period of prostate gland maturation. Bromocriptine pellets applied from PND 15 to adulthood were able to suppress the elevations in circulating PRL. Despite this suppression, the present results show that the neonatally estrogenized phenotype within the prostate gland remained intact. The effects of estrogen on prostatic growth retardation, AR down-regulation, and epithelial disorganization that precedes aging-associated ventral lobe dysplasia were not reversed by PRL blockade, suggesting that these aspects of estrogenization are not mediated through transient estrogen-induced hyperprolactinemia. This contrasts with the results of Ho and colleagues (33), who reported that estradiol-induced dorsolateral prostate lobe dysplasia was abolished by concomitant bromocriptine treatment. However, in that study, adult animals were exposed to chronic estrogen-induced hyperprolactinemia. The differential lobe- and time-specific responses to estrogenic exposures suggest that two separate mechanistic pathways are involved in neonatal and adult estrogen-induced prostatic lesions.

Because transient prepubertal hyperprolactinemia was previously shown to result in adult prostatitis (22), we examined the immune response of the estrogenized rats in greater detail. A partial suppression of the immune response was observed when circulating PRL levels were suppressed throughout maturation. Specifically, helper T cells (CD4+) and cytotoxic T cells (CD8+) were more prevalent in the adult ventral prostate when PRL levels were elevated following estrogen exposure. Similarly, the estrogen-induced increase in splenic weight was reversed by bromocriptine treatment, whereas the increased thymus weight was partially reversed with PRL suppression. Collectively, these findings suggest that a component of the estrogen-induced immune response is mediated through hyperprolactinemia. Lymphocytes contain PRL receptors (40) and PRL is a comitogen for T cell proliferation as well as an inducer of cytokine and antibody production (41, 42). Thus, these are potential pathways for the prostatic responses to hyperprolactinemia in estrogenized rats. Notable in the present study is that the immune response persists in the prostate after normal PRL levels are established in the adult rat which suggests that transient elevations in PRL imprint either immune or prostatic biology in some specific long-lasting manner. Elevated serum PRL has been associated with accelerated autoimmune diseases in a wide variety of models, and it has also been considered to have an immunomodulatory effect on estrogen-induction of the same (42). It is noteworthy that in the present study, bromocriptine treatment appeared to have a greater effect on suppressing prostatic CD8+ T cells than on CD4+ lymphocytes. Alternatively, this could be interpreted as a specific estrogenic effect on CD4+ T cells. Estrogen treatment has been reported to increase the proportion of CD4+ cells in the thymus (43). A similar phenotypic shift in mature thymic T cells has been observed upon castration-induced androgen withdrawal, where CD8+ cells decrease and CD4+ cells are unaffected and this may be representative of hormonal influences on T cell maturation (44).

It is equally important to note that the estrogen-induced immune response was not entirely abrogated by bromocriptine treatment. Thus, CD4+ helper T cells were significantly higher in the EB + BrC prostates compared with Oil + BrC and macrophage infiltration was entirely unaffected by PRL suppression. Additionally, the estrogen-induced elevation in thymus weight was only partially reversed by hyperprolactinemia blockade. Together, these findings indicate that certain aspects of immune system estrogenization are PRL-independent and are mediated directly through estrogens or other currently unidentified mediators. Estrogens are known to augment autoimmune diseases in humans and a variety of animal models (42, 45), and this is best represented by the fact that females are more prone to autoimmune diseases such as multiple sclerosis, lupus erythematosis, and rheumatoid arthritis. Additionally, the thymus is known to be extremely estrogen sensitive (46, 47). In our animal model, thymus size at d 10 is markedly decreased in estrogen-exposed rats (unpublished data), which is before the onset of hyperprolactinemia and may represent a thymolytic effect of estrogens. In contrast, at d 90, the thymus gland weights of estrogen-exposed rats were significantly elevated. Such a delayed hypersensitivity response of the thymus to neonatal diethylstilbestrol exposure has also been reported for female mice (48) rats (49) and humans (50). These findings suggest that early estrogenic exposure may have affected immune cell education early in life before the onset of hyperprolactinemia. Normal development of the thymus and thymocyte maturation in male mice is known to require estrogen receptor {alpha} (ER{alpha}), whereas ERß is required for estrogen-induced phenotypic alterations in the thymic T cell repertoire (47, 51, 52). In aged ERß knockout mice, we have recently observed a marked increase in prostatic T cell aggregation with associated reactive epithelium that increases in severity as the males age, thus suggesting that ERß may play an immunoprotective role in the prostate gland (53). This is highly significant because we have also observed that ERß expression is down-regulated in the adult prostate glands of neonatally estrogenized rats (54). Together, these findings suggest that the PRL-independent component of estrogenization-induced prostatic inflammation may involve ER{alpha} at the level of the thymus as well as loss of ERß expression at the level of the prostate gland.

A recent study that characterized the induction of pro-inflammatory genes in adult Wistar rat prostate lobes following estrogen exposure reported an immediate (within 4 d) induction of IL-1ß, IL-6, macrophage inflammatory protein-2, and inducible nitric oxide synthase within the lateral lobe before the histologically observed inflammation which occurs primarily in the lateral lobe (55). After 4 wk, there was a marked increase in lateral lobe mRNA levels for IL-4, IL-5, IL-6, macrophage inflammatory protein-2, and inducible nitric oxide synthase, whereas cox-2, interferon-{gamma}, IL-2 and IL-12 were unaffected. Although these investigators did not determine the role of PRL in proinflammatory gene induction, previous studies have shown that estrogen-induced lateral prostate inflammation in adult Wistar rats can be entirely blocked by bromocriptine (20). Collectively, these data suggest that the immune response in the lateral prostate following adult estrogen-induced hyperprolactinemia resembles a T helper-cell 2-response of T cells that is characterized by IL-4, IL-5, and IL-13 production, mast cell activation, eosinophil influx, and an immediate hypersensitivity (allergic) response (56). This contrasts with a T helper-cell 1-response that is observed in cell-mediated inflammatory conditions and autoimmune disorders and is characterized by production of interferon-{gamma}, IL-2 and macrophage activation. Although these cytokine genes were not examined in the present study, it is interesting to note that, in addition to helper and cytotoxic T cells, a chronic infiltration of macrophages was observed in the ventral lobes of neonatally estrogenized rats, and this was not reversed by bromocriptine treatment. Chronic tissue infiltration with T cells and macrophages is a hallmark indicator of a tissue-specific autoimmune response, and it is possible that neonatal estrogenization induces a type of autoimmune reaction in the prostate gland, although this awaits confirmation with functional studies. This response differs from the relatively nonspecific granulocytic infiltration (neutrophils, eosinophils) with moderate lymphoid aggregation observed in lateral prostates of hyperprolactinemic rats (20, 21). Because the adult vs. neonatal estrogen-induced prostate immune responses differ with regards to lobe specificity, PRL dependency and immune cell types involved, it appears that different mechanisms may be involved in these phenomenon.

In conclusion, the present study provides evidence for a PRL-dependent and a PRL-independent estrogen-mediated effect on prostatic inflammation and immune cell infiltration in the prostate gland of neonatally estrogenized rats. The implications for these findings are important with regard to an animal model for human prostate pathology. Estrogenized rats present with a relative increase in fibromuscular mass in the adult prostate which may be related, in part, to immune cell cytokines that are well known to stimulate fibroblast proliferation (57). Because human prostate inflammation has been associated with fibromuscular growth and is highly associated with benign prostatic hyperplasia (57, 58) as well as prostate cancer (16), it is possible that the early endocrinologic insults that manifest in adulthood as prostate inflammation may predispose the prostate gland to aging-associated lesions.


   Footnotes
 
Supported by grants from the NIH (NIDDK 40780) and the Environmental Protection Agency (STAR R826299).

1 J.P.G. and O.P. made equal contributions to this study. Back

Abbreviations: AR, Androgen receptor; BrC, bromocriptine; EB, estradiol benzoate; ER, estrogen receptor; Plb, placebo; PND, postnatal day; PRL, prolactin.

Received November 13, 2002.

Accepted for publication January 16, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Price D 1963 Comparative aspects of development and structure in the prostate. In: Vollmer E, ed. Biology of the prostate and related tissues. Washington, DC: National Cancer Institute; vol 12:1–27
  2. Grayhack J 1963 Pituitary factors influencing growth of the prostate. In: Vollmer E, Kauffmann G, ed. Biology of the prostate and related tissues. Washington, DC: U.S. Government Printing Office; vol 12:189–199
  3. Putz O, Prins GS 2002 Prostate gland development and estrogenic imprinting. In: Burnstein KL, ed. Steroid hormones and cell cycle regulation. Boston: Kluwer Academic Publishers; 73–89
  4. Hayashi N, Sugimura Y, Kawamura J, Donjacour AA, Cunha GR 1991 Morphological and functional heterogeneity in the rat prostatic gland. Biol Reprod 45:308–321[Abstract]
  5. Prins GS, Birch L 1995 The developmental pattern of androgen receptor expression in rat prostate lobes is altered after neonatal exposure to estrogen. Endocrinology 136:1303–1314[Abstract]
  6. Rajfer J, Coffey DS 1978 Sex steroid imprinting of the immature prostate. Invest Urol 16:186–190[Medline]
  7. Naslund MJ, Coffey DS 1986 The differential effects of neonatal androgen, estrogen and progesterone on adult rat prostate growth. J Urol 136:1136–1140[Medline]
  8. Prins GS, Woodham C, Lepinske M, Birch L 1993 Effects of neonatal estrogen exposure on prostatic secretory genes and their correlation with androgen receptor expression in the separate prostate lobes of the adult rat. Endocrinology 132:2387–2398[Abstract/Free Full Text]
  9. Arai Y, Mori T, Suzuki Y, Bern HA 1983 Long-term effects of perinatal exposure to sex steroids and diethylstilbestrol on the reproductive system of male mammals. In: Bourne GH, Danielle JF, eds. International review of cytology. New York: Academic Press, Inc.; vol 84:235–268
  10. Santti R, Newbold RR, Makela S, Pylkkanen L, McLachlan JA 1994 Developmental estrogenization and prostatic neoplasia. Prostate 24:67–78[Medline]
  11. Prins GS 1997 Developmental estrogenization of the prostate gland. In: Naz RK, ed. Prostate: basic and clinical aspects. Boca Raton, FL: CRC Press; Chapter 10:247–265
  12. Naslund MJ, Strandberg JD, Coffey DS 1988 The role of androgens and estrogens in the pathogenesis of experimental nonbacterial prostatitis. J Urol 140:1049–1053[Medline]
  13. Stoker TE, Robinette CL, Cooper RL 1999 Perinatal exposure to estrogenic compounds and the subsequent effects on the prostate of the adult rat: evaluation of inflammation in the ventral and lateral lobes. Reprod Toxicol 13:463–472[CrossRef][Medline]
  14. Prins GS, Chang WY, Wang Y, van Breemen RB 2002 Retinoic acid receptors and retinoids are up-regulated in the developing and adult rat prostate by neonatal estrogen exposure. Endocrinology 143:3628–3640[Abstract/Free Full Text]
  15. Lloyd S, McClinton S, Brown I, Miller I, Kirk D, Eremin O, Moffat L, Leake R 1990 Epidermal growth factor receptor, nuclear proliferation antigen and interleukin-2 receptor expression in prostatic cancer. J Clin Lab Immunol 32:55–58[Medline]
  16. DeMarzo AM, Marchi VL, Epstein JI, Nelson WG 1999 Proliferative inflammatory atrophy of the prostate implications for prostatic carcinogenesis. Am J Pathol 155:1985–1992[Abstract/Free Full Text]
  17. Meites J, Lu KH, Wuttke W, Welsch CW, Nagasawa H, Quadri SK 1972 Recent studies on functions and controls of prolactin secretion in rats. Recent Prog Horm Res 28:471–526
  18. Oldereid NB, Thomassen Y, Rui H, Gordeladze JO, Haug E, Purvis K 1986 Effects of oestrogen-induced hyperprolactinemia on the rat prostate. Androlgia 18:516–522
  19. Bellido C, Gaytan F, Aguilar R, Pinilla L, Aguilar E 1985 Prepuberal reproductive defects in neonatal estrogenized male rats. Biol Reprod 33:381–387[Abstract]
  20. Tangbanluekal L, Robinette CL 1993 Prolactin mediates estradiol-induced inflammation in the lateral prostate of Wistar rats. Endocrinology 132:2407–2416[Abstract/Free Full Text]
  21. Stoker TE, Robinette CL, Britt BH, Laws SC, Cooper RL 1999 Prepubertal exposure to compounds that increase prolactin secretion in the male rat: effects on the adult prostate. Biol Reprod 61:1636–1643[Abstract/Free Full Text]
  22. Stoker TE, Robinette C, Cooper RL 1999 Maternal exposure to Atrazine during lactation suppresses suckling-induced prolactin release and results in prostatitis in the adult offspring. Toxicol Sci 52:68–79[Abstract/Free Full Text]
  23. Simerly R, Zee M, Pendleton J, Lubahn D, Korach K 1997 Estrogen receptor-dependent sexual differentiation of dopaminergic neurons in the preoptic region of the mouse. Proc Natl Acad Sci USA 94:14077–14082[Abstract/Free Full Text]
  24. Grayhack JT, Bunce PL, Kearns JW, Scott WW 1955 Influence of the pituitary on prostatic response to androgen in the rat. Bull Johns Hopkins Hosp 154–163
  25. Grayhack J, Lebowitz J 1967 Effect of prolactin on citric acid of lateral lobe of prostate of Sprague-Dawley rat. Invest Urol 5:87–94
  26. Lasnitzki I 1972 The effect of prolactin on rat prostate glands in organ culture. In: Boynes AR, Griffiths K, eds. Prolactin and carcinogenesis. Cardiff, Wales: Alpha Omega Alpha Publishers; 200–206
  27. Negro-Vilar A, Saad W, McCann S 1977 Evidence for a role of prolactin in prostate and seminal vesicle growth in immature male rats. Endocrinology 100:729–737[Abstract/Free Full Text]
  28. Keenan E, Klase P, Thomas J 1981 Effects of prolactin on DNA synthesis and growth of the accessory sex organs in male mice. Endocrinology 109:170–175[Abstract/Free Full Text]
  29. Prins GS, Lee C 1983 Biphasic response of the rat lateral prostate to increasing levels of serum prolactin. Biol Reprod 29:938–945[Abstract]
  30. Costello LC, Liu Y, Franklin RB 1995 Prolactin specifically increases pyruvate dehydrogenase El{alpha} in rat lateral prostate epithelial cells. Prostate 26:189–193[Medline]
  31. Prins GS 1987 Prolactin influence on cytosol and nuclear androgen receptors in the ventral, dorsal, and lateral lobes of the rat prostate. Endocrinology 120:1457–1464[Abstract/Free Full Text]
  32. Lee C, Prins GS, Henneberry MO, Grayhack JT 1981 Effect of estradiol on the rat prostate in the presence and absence of testosterone and pituitary. J Androl 2:293–299[Abstract]
  33. Lane K, Leav I, Ziar J, Bridges R, Rand W, Ho SM 1997 Suppression of testosterone and estradiol-17ß-induced dysplasia in the dorsolateral prostate of Noble rats by bromocriptine. Carcinogenesis 18:1505–1520[Abstract/Free Full Text]
  34. Jarred R, Cancilla B, Prins GS, Thayer K, Cunha GR, Risbridger G 2000 Evidence that estrogens directly alter androgen-regulated prostate development. Endocrinology 141:3471–3477[Abstract/Free Full Text]
  35. Puig-Duran E, MacKinnon CB 1978 Ontogeny of prolactin and luteinizing hormone responses to oestrogen and progesterone in immature rats. J Endocr 76:321–331[Abstract/Free Full Text]
  36. Prins GS 1992 Neonatal estrogen exposure induces lobe-specific alterations in adult rat prostate androgen receptor expression. Endocrinology 130:3703–3714[Abstract/Free Full Text]
  37. Prins GS, Birch L, Greene GL 1991 Androgen receptor localization in different cell types of the adult rat prostate. Endocrinology 129:3187–3199[Abstract/Free Full Text]
  38. Negro-Vilar A, Krulich L, McCann SM 1973 Changes in serum prolactin and gonadotropins during sexual development of the male rat. Endocrinology 93:660–664[Abstract/Free Full Text]
  39. Theveny B, Bailly A, Rauch C, Rauch M, Delain E, Milgrom E 1987 Association of DNA-bound progesterone receptors. Nature 329:79–81[CrossRef][Medline]
  40. Pellegrini I, Lebrun J-J, Ali S, Kelly PA 1992 Expression of prolactin and its receptor in human lymphoid cells. Mol Endocrinol 6:1023–1031[Abstract/Free Full Text]
  41. Clevenger CV, Freier DO, Kline JB 1998 Prolactin receptor signal transduction in cells of the immune system. J Endocr 157:187–197[CrossRef][Medline]
  42. McMurray RW 2001 Estrogen, prolactin, and autoimmunity: actions and interactions. Int Immunopharm 1:995–1008
  43. Screpanti I, Morrone S, Meco D, Santoni A, Grulino A, Paolini R, Crisanti A, Mathieson BJ, Frati L 1989 Steroid sensitivity of thymocyte subpopulations during intrathymic differentiation. Effects of 17ß-estradiol and dexamethasone on subsets expressing T cell antigen receptor or IL-2 receptor. J Immunol 142:3378–3383[Abstract]
  44. Olsen NJ, Kovacs WJ 1996 Gonadal steroids and immunity. Endocr Rev 17:369–384[Abstract/Free Full Text]
  45. Paavonen T 1994 Hormonal regulation of immune responses. Ann Med 26:255–258[Medline]
  46. Rijhsinghani AG, Thompson K, Bhatia SK, Waldschmidt TJ 1996 Estrogen blocks early T cell development in the thymus. Am J Reprod Immunol 36:269–277
  47. Staples JE, Gasiewicz TA, Fiore NC, Lubahn DB, Korach KS, Silverstone AE 1999 Estrogen receptor alpha is necessary in thymic development and estradiol-induced thymic alterations. J Immunol 163:4168–4174[Abstract/Free Full Text]
  48. Forsberg JG 1996 The different responses of the female mouse thymus to estrogen after treatment of neonatal, prepubertal, and adult animals. Acta Anat 157:275–290[Medline]
  49. Leceta J, Fernandez-Galaz C, Sanchez C, Navarro R, Gomariz RP, Zapata A, Tejero A 1988 Effects of neonatal treatment with estrogens in the development of the thymus in rats. Dev Comp Immunol 12:175–383
  50. Ways SC, Mortola JF, Zvaifler NJ, Weiss RJ, Yen SSC 1987 Alterations in immune responsiveness in women exposed to diethylstilbestrol in utero. Fertil Steril 48:193–197[Medline]
  51. Yellayi S, Teuscher C, Woods J, Welsh TJ, Tung K, Nakai M, Rosenfeld C, Lubahn D, Cooke P 2000 Normal development of thymus in male and female mice requires estrogen/estrogen receptor-{alpha} signaling pathway. Endocrine 12:207–213[CrossRef][Medline]
  52. Erlandsson MC, Ohlsson C, Gustafsson JA, Carlsten H 2001 Role of oestrogen receptors {alpha} and ß in immune organ development and in oestrogen-mediated effects on thymus. Immunology 103:17–25[CrossRef][Medline]
  53. Prins GS, Birch L, Shappell S, Couse JF, Korach KS, Aging males lacking estrogen receptor-ß exhibit increased prostatic lymphocytic infiltration but normal indices of prostatic epithelial proliferation in a double-blinded analysis. Program of the 84th Annual Meeting of The Endocrine Society, San Francisco, CA, 2002, p 500 (Abstract P2-207)
  54. Prins GS, Marmer M, Woodham C, Chang WY, Kuiper G, Gustafsson JA, Birch L 1998 Estrogen receptor-ß messenger ribonucleic acid ontogeny in the prostate of normal and neonatally estrogenized rats. Endocrinology 139:874–883[Abstract/Free Full Text]
  55. Harris MT, Feldberg RS, Lau K-M, Lazarus NH, Cochrane DE 2000 Expression of proinflammatory genes during estrogen-induced inflammation of the rat prostate. Prostate 44:19–25[CrossRef][Medline]
  56. Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL 1986 Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 136:2348–2357[Abstract]
  57. Kramer G, Steiner GE, Handisurya A, Stix U, Haitel A, Knerer B, Gessl A, Lee C, Marberger M 2002 Increased expression of lymphocyte-derived cytokines in benign hyperplastic prostate tissue, identification of the producing cell types, and effect of differentially expressed cytokines on stromal cell proliferation. Prostate 52:43–58[CrossRef][Medline]
  58. Robinette C 1988 Sex-hormone-induced inflammation and fibromuscular proliferation in the rat lateral prostate. Prostate 12:271–286[Medline]



This article has been cited by other articles:


Home page
 Am. J. Pathol.Home page
N. N.C. Tam, I. Leav, and S.-M. Ho
Sex Hormones Induce Direct Epithelial and Inflammation-Mediated Oxidative/Nitrosative Stress That Favors Prostatic Carcinogenesis in the Noble Rat
Am. J. Pathol., October 1, 2007; 171(4): 1334 - 1341.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
J. J. Bianco, S. J. McPherson, H. Wang, G. S. Prins, and G. P. Risbridger
Transient Neonatal Estrogen Exposure to Estrogen-Deficient Mice (Aromatase Knockout) Reduces Prostate Weight and Induces Inflammation in Late Life
Am. J. Pathol., June 1, 2006; 168(6): 1869 - 1878.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
G. S. Palapattu, S. Sutcliffe, P. J. Bastian, E. A. Platz, A. M. De Marzo, W. B. Isaacs, and W. G. Nelson
Prostate carcinogenesis and inflammation: emerging insights
Carcinogenesis, July 1, 2005; 26(7): 1170 - 1181.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
N. Atanassova, C. McKinnell, J. Fisher, and R. M Sharpe
Neonatal treatment of rats with diethylstilboestrol (DES) induces stromal-epithelial abnormalities of the vas deferens and cauda epididymis in adulthood following delayed basal cell development
Reproduction, May 1, 2005; 129(5): 589 - 601.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
T. Pakarainen, F.-P. Zhang, S. Makela, M. Poutanen, and I. Huhtaniemi
Testosterone Replacement Therapy Induces Spermatogenesis and Partially Restores Fertility in Luteinizing Hormone Receptor Knockout Mice
Endocrinology, February 1, 2005; 146(2): 596 - 606.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gilleran, J. P.
Right arrow Articles by Prins, G. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gilleran, J. P.
Right arrow Articles by Prins, G. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals