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Endocrinology Vol. 144, No. 7 3196-3205
Copyright © 2003 by The Endocrine Society

Prostate Development and Carcinogenesis in Prolactin Receptor Knockout Mice

Fiona G. Robertson, Jessica Harris, Matthew J. Naylor, Samantha R. Oakes, Jon Kindblom, Karin Dillner, Håkan Wennbo, Jan Törnell, Paul A. Kelly, Jeff Green and Christopher J. Ormandy

Cancer Research Program (F.G.R., J.H., M.J.N., S.R.O., C.J.O.), Garvan Institute of Medical Research, St. Vincent’s Hospital, Darlinghurst, 2010 Sydney, Australia; Department of Physiology (J.K., K.D., H.W., J.T.), Research Center for Endocrinology and Metabolism, Göteborg University, S-405 30 Göteborg, Sweden; Institut National de la Santé et de la Recherche Médicale Unité 584 (P.A.K.), Faculté de Médecine Necker-Enfants Malades, 75730 Paris, France; and Laboratory of Cell Regulation and Carcinogenesis (J.G.), National Cancer Institute, Bethesda, Maryland 20892-1402

Address all correspondence and requests for reprints to: Dr. Christopher Ormandy, Garvan Institute of Medical Research, 384 Victoria Street Darlinghurst, New South Wales 2010, Australia. E-mail: c.ormandy{at}garvan.org.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hyperprolactinemia results in prostatic hypertrophy and hyperplasia, but it is not known whether prolactin plays an essential role in these processes in the prostate. To address this question, we investigated prostate development, gene expression, and simian virus 40 (SV40)T-induced prostate carcinogenesis in prolactin receptor knockout mice. These animals showed a small increase in dorsolateral and ventral prostate weight but no change in the weight of the anterior prostate. The dorsal but not ventral or lateral lobes showed a 12% loss of epithelial cells; all other morphological parameters were normal. The area of SV40T-induced prostate intraepithelial neoplasia was reduced by 28% in the ventral lobe but not the dorsal lobe, and no tumors were seen in 20 prolactin receptor knockout animals, compared with 1 of 11 detected in wild-type and 4 of 21 found in heterozygous animals. Oligonucleotide microarrays were used to identify essential transcriptional roles of prolactin and revealed a small set of genes with decreased expression involved in sperm/oocyte interaction and copulatory plug formation. Infertility or reduced fertility was apparent in these animals. These findings establish essential though subtle roles for prolactin in the regulation of prostate morphology, gene expression, SV40T-induced neoplasia, and reproductive function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CRITICAL ROLE of androgens in the development and maintenance of the prostate is demonstrated by the dramatic prostate regression that follows castration (1). Early work showed that other endocrine factors are also involved in prostate development, particularly those of the pituitary. Greater prostate regression occurs with additional hypophysectomy (2), androgen replacement after castration restores prostate weight less effectively with hypophysectomy (3, 4), and pituitary grafts reduce the rate and extent of prostate regression induced by castration (5) via androgen-independent mechanisms (6). Prolactin may be the pituitary factor responsible for these effects because studies to date have shown that treatment of intact and castrated animals with prolactin is growth stimulatory for the normal prostate, both in synergy and independently of androgen. Prolactin is capable of producing stromal hyperplasia and intraepithelial dysplastic features with long exposure (7).

The mechanism of prolactin action on the prostate is complex. Prolactin can influence the prostate indirectly via regulation of the level of testicular luteinizing hormone receptors and steroidogenic enzymes to increase testicular testosterone production. In humans (but not rodents) the adrenal glands produce androgen precursors DHEA and dehydroepiandrosterone sulfate in large quantities in response to prolactin (8). Prolactin can also operate directly; prolactin and the prolactin receptor are expressed in human and rodent prostate (9) by the epithelial cells with a weak signal in the fibromuscular stroma (10). In vitro prolactin is mitogenic for cultured prostate epithelial cells (11), and in organ cultures normal morphology was best maintained by the addition of androgen and prolactin (12). Expression in the prostate of both prolactin and the prolactin receptor is increased by androgen treatment in vivo, and prolactin receptor level is also increased by prolactin (9, 13). Prolactin has been viewed as an autocrine/paracrine growth factor (9, 14) or a survival factor (15) for prostate epithelial cells.

Prolactin may also play a role in disease of the prostate, again both indirectly and directly. Human prostate hyperplasia and early cancer continue to express the prolactin receptor (9, 16). Prolactin alone and in synergy with testosterone causes cell proliferation in benign hyperplastic human prostate (17) and human prostate cancer cell lines (18). Clinical investigation has shown that serum prolactin levels in men with prostate disease are generally not different from age-matched controls when measured at presentation (19) or up to 13 yr before diagnosis (20). During estrogen, antiestrogen, antiandrogen, or GnRH analog therapy for prostate cancer, prolactin levels can increase and are predictive of poor prognosis (21). This has led to 11 reported small uncontrolled clinical trials of adjuvant bromocriptine during antiandrogen therapy, some of which report increased response rates when bromocriptine is used (22), despite the enrollment of late-stage patients in whom tumor prolactin receptor levels can be diminished or lost (23). In addition, a recent study reported the use of a molecular mimic of phosphorylated prolactin in vivo to successfully inhibit tumor initiation and the growth of DU145-derived tumors in nude mice (24).

Together this body of work suggests a role for prolactin in the growth of the normal and cancerous prostate; however, a number of fundamental questions remain unanswered. Chiefly, does prolactin at physiological levels have an essential role in prostate development and function, or is prolactin active only during the abnormal hyperprolactinemic states produced by either pathophysiological conditions or experimental or therapeutic manipulation? We have used histomorphological and transcript profiling techniques to compare wild-type (PRLR+/+) prostates with the prostates from prolactin receptor knockout mice (PRLR-/-). To search for essential roles for prolactin in prostate carcinogenesis, we produced PRLR-/- mice that carry the simian virus 40 (SV40)T oncogene under the C3 promoter.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
All animals were rederived by embryo transfer and SPF barrier housed. All animal use was approved and supervised by the Garvan Institute/St. Vincent’s Hospital Animal Experimentation Ethics Committee. The PRLR-/- mouse was generated by replacement of exon 5 of the PRLR gene, which encodes cysteine residues essential for ligand binding and receptor activation, with the NEO cassette driven by the Tk promoter in the opposite direction to PRLR transcription (25). PRLR-/- and PRLR+/+ mice were derived from E14 ES cells (129/OlaHsd) bred to 129/Sv Pas mice and then subsequently intercrossed. The core colony was maintained by heterozygous matings to produce all genotypes. Castration surgery was via the abdominal route. The C3-SV40T animals (26, 27) were on an inbred FVB/N background, and the core colony was maintained by homozygous matings. The required mixed genotypes were produced by mating 10 PRLR-/- males with 10 homozygous C3-SV40T females and then mating the resultant females with the PRLR-/- males to produce animals that were heterozygous or wild type for C3-SV40T and PRLR+/- or PRLR-/-. Control PRLR+/+ animals were produced by the use of 10 PRLR+/+ in an identical but separate scheme to ensure similar genetic diversity between groups. Animals were aged to 50 wk before prostate collection. Significant numbers could not be aged past this point because of the onset of tumors in the submaxillary glands and sebaceous glands of the paws.

Microdissection and analysis of ductal morphogenesis
The genital tract was removed en bloc to a Petri dish and cleared of periprostatic fat and connective tissue, then anterior prostate and seminal vesicles. A section of the urethra containing the connections of the prostate ducts was then removed and photographed. Individual lobes were removed by cutting the ducts at their urethral connection, and microdissected in depression slides as previously described (28).

Histology and morphometric analysis
Hematoxylin-eosin (H&E)-stained sections were taken from throughout the prostate and photographed digitally at x10 magnification. Genotype information was not recorded so that the subsequent analysis was blind to genotype. Tissue areas were manually defined and areas measured using calibrated software. Epithelial cell nuclei in defined areas were manually counted using images captured at x20 magnification. Areas of prostatic intraepithelial neoplasia (PIN) were identified by their hyperchromatic, enlarged, and elongated nuclei (27). We did not attempt to split the PIN lesions into low and high grades.

Transcript profiling and data analysis
RNA from five to eight prostate lobes of each genotype was pooled in equimolar ratios before probe preparation, chip hybridization (Affymetrix U74A/U74Av2), and analysis (Affymetrix Inc., Santa Clara, CA; MicroArray Suite 4 and 5) according to the manufacturer’s instructions. The experiment was replicated three (ventral) times and two (dorsal) times using a different set of animals for each replication. Identified were genes that showed consistent change in expression level across the replicates, the significance of which was tested using paired t test. Alterations were confirmed by quantitative PCR using the LightCycler (Roche, Basel, Switzerland) and TaqMan (Applied Biosystems, Foster City, CA) instruments according to the manufacturer’s protocols. DChip software (29) was a gift of Dr. Wong, and the JMP statistical software used for principal component analysis was from SAS Institute (Cary, NC). The BioNavigator platform was used for bulk blastx searches of SwissProt+TrEMBL and blastn searches of GenBank, UniGene, and HomoloGene were also used. Medline was used to identify publications relating to gene function.

Statistical analysis
Comparisons were made using an unpaired, two-tailed t test; Kaplan-Meier survival analysis; and simple regression using Statview 4.5. Array data were sorted and graphically analyzed using Excel (Microsoft Corp., Mountain View, CA) and the JMP statistical package.

Quantitative PCR
RNA (1 µg) from the prostates was reverse transcribed using AMV reverse transcriptase (Promega Corp., Madison, WI). PCR primers were designed using MacVector to span an intron. The PCR were performed in a LightCycler (Roche) using 1 µl cDNA diluted 1:2, 5 pmol primers, and the FastStart DNA master SYBR Green I enzyme mix (Roche) in a 10-µl reaction volume. Relative quantification of the product was performed by comparing the crossing points of different samples above background.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gross prostate morphology
In the PRLR-/- animals, the seminal vesicles, coagulating gland (anterior prostate), ventral prostate, dorsal prostate, and lateral prostate were all present and of normal size and appearance, at both 2 wk of age (not shown) and in mature animals of 16–36 wk of age (Fig. 1Go, top panel). The lobes were dissected and weighed. The seminal vesicle and ventral prostate were 25% heavier in PRLR-/- animals (P < 0.0001), corrected for body weight or not. The dorsolateral lobes, dissected as one for this measurement, were 10% heavier with borderline (P = 0.07) statistical significance. In contrast, the coagulating gland (anterior prostate) showed no difference in weight (P = 0.57). The weights of these organs were also examined as a function of age, and an identical pattern was observed for the seminal vesicle and the ventral and dorsolateral prostates. Young animals (10–11 wk) showed no weight differences, but mature animals (16–30 wk) showed a highly significant (P < 0.001) increase in weight in the PRLR-/- animals. As the animals aged, the strength of the P values for this weight difference weakened and became nonsignificant around 1 yr of age. The coagulating gland showed no difference in weight at any age (data not shown). Thus, prolactin plays an essential role in maintaining correct prostate weight during sexually mature life, but its effect is lost in old animals.



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  		Figure 1. Prostate morphology. The ventral (V), lateral (L), and dorsal (D) lobes of the prostate were dissected free of periprostatic fat and connective tissue but left attached to a small section of the urethra (U). Seminal vesicles (SV) and coagulating gland (CG) were removed and are not shown because of their much larger size. Lobes are from mature animals (16–20 wk). The gross morphology of PRLR-/- prostates are indistinguishable from controls. Lobe weights (milligrams ± SE) for the ventral (VP) and combined dorsal-lateral (DLP) prostate are indicated, and P values (t test) for comparison between PRLR+/+ and PRLR-/- are shown. Individual lobes from 16- to 36-wk animals were microdissected further in collagenase under a dissecting microscope to desegregate the interductal stoma, allowing the ducts to be disentangled and arranged in two dimensions, revealing ductal branching patterns. Quantification of ducts (D) attached to the urethra, ductal tips (T), and duct branch points (B) are given with number of lobes (n) analyzed and P value from t test vs. control.

 
Ductal morphogenesis
Microdissection was used to examine branching morphogenesis of individual prostate lobes and quantified by counting urethral ducts and ductal tips and branch points (Fig. 1Go, lower panels). This analysis clearly defined the distinctive ductal branching patterns of each lobe. The ventral and lateral prostate lobes were attached to the urethra by two or three main ducts that showed extensive oak tree branching morphology, and the dorsal prostate consisted of many ducts attached to the urethra that showed less extensive palm tree branching morphology (28). There was no difference between PRLR+/+ and PRLR-/- animals in the number of ducts, number of branch points per duct, or number of ductal tips present in each lobe. The more simply branched coagulating gland also showed no differences between genotypes (data not shown).

Histology and quantitative morphometric analysis
H&E-stained sections (Fig. 2Go) were compared by measurement of tissue areas using manual tracing of epithelium, periductal stroma (including the duct basement membrane), interductal stroma, and lumen using calibrated image analysis software. Results are presented as pie charts (Fig. 2Go). Cell nuclei in these areas were also counted manually. The sensitivity of this technique is demonstrated by its ability to unequivocally distinguish the distinctive morphology of the various prostate lobes, in which the differences in epithelial, lumen, and periductal stromal areas showed P values less than 0.0001.



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  		Figure 2. Prostate lobe histology. Representative histology for each lobe and genotype from intact 16- to 36-wk-old animals is shown. Tissue ratios were measured by tissue area measurements using image analysis of H&E-stained histology. Ratios are presented as pie charts from intact mature animals or mature animals 21 d following castration (histology not shown, pie charts denoted C). Measurements (n = 4–5 animals per group) were compared with wild-type control or castrated wild-type control using t test. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.0001.

 
In PRLR+/+ and PRLR-/- animals, there were no differences in tissue areas of the ventral prostate. A small but significant difference in the dorsal prostate was detected. Epithelial and interductal stromal area decreased and lumenal area increased. When the total ductal cross-sectional area was calculated by adding the areas of lumen, epithelium, and periductal stroma, no difference was seen between genotypes, indicating that the changes were not due to an increase in duct size. Epithelial content was examined by a separate analysis of the relative lumen and epithelial areas of 200 ductal cross-sections of each genotype. PRLR+/+ epithelium occupied 67.1% of the ductal cross-sectional area and the PRLR-/- epithelium occupied 58.7% (P < 0.0001), a loss of 12.5% of the epithelial content. Epithelial nuclei were counted and the area they occupied measured in a further 50 random ductal cross-sections; no difference in epithelial cell density was seen, demonstrating that no change in cell size had occurred. Thus, epithelial cells were lost.

Role of androgens
Castration was used to examine the role androgens play in the altered tissue ratios observed in the dorsal lobe of PRLR-/- animals (Fig. 2Go). The loss of PRLR has no effect on testosterone levels (30). Castration resulted in the loss of epithelium and a gain in lumenal area that was accompanied by an overall increase in the area of the stroma. This was due to an increase in periductal stroma despite the loss of interductal stroma so that the most visually striking change was the ratio of epithelium to periductal stroma. In the ventral lobe, these changes were identical between PRLR+/+ and PRLR-/- animals. In the dorsal lobe, however, significant differences were detected between PRLR+/+ and PRLR-/- animals. Importantly, the difference in epithelial area seen in intact animals was lost in castrated animals. In addition, the amount of lumenal area showed a greater decrease, and the area of stroma (especially periductal stroma) showed a greater increase in PRLR-/- animals. These measurements demonstrate that castration-induced regression of the dorsal lobe was greater in PRLR-/- animals than PRLR+/+ controls. Prolactin and androgen cooperate to regulate dorsal lobe composition.

Carcinogenesis
We crossed the mouse line carrying the null mutation of the PRLR with the mouse line carrying a transgenic insert that placed the SV40T antigen under the control of the C3 promoter. Animals were intercrossed using a scheme designed to ensure equivalent background genetic diversity of the test groups (see Materials and Methods). Animals were aged to 50 wk and then killed and their prostates analyzed by H&E-stained serial sections for PIN and tumors. The area of PIN was measured using manual tracing of H&E images in ImageQuant software (Leica Corp.) using the descriptors of PIN used by Shibata et al. (27) (Fig. 3Go, top two panels). As described by Shibata et al., the amount of PIN was higher in the PRLR+/+ ventral lobe than the PRLR+/+ dorsal lobe (Fig. 3Go, panel 3). Loss of PRLR resulted in a 28% decrease (P = 0.026) in the area of PIN in the ventral lobe but was without detectable effect in the dorsal lobe. The level of SV40T antigen expression in the ventral lobe was measure by quantitative RT-PCR (Fig. 3Go, bottom panel). There was no difference (P = 0.21) between PRLR genotypes in SV40T expression level, consistent with identical androgen levels in these animals and indicating that the C3 promoter fragment used is insensitive to prolactin signaling. Only five tumors were detected in the cohort and all occurred in the ventral lobe. One tumor was found in the 11 PRLR+/+ animals examined and four in 21 PRLR+/- animals. None of the 20 PRLR-/- animals showed a detectable prostate tumor.



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  		Figure 3. Formation of PIN lesions in the prostate by expression of SV40T. The upper two panels show examples of PIN lesions (p) in the dorsal and ventral lobe induced by expression of SV40T. The area of these lesions was measured in 50-wk-old animals and expressed as a percentage of total tissue area. No difference between PRLR genotypes was detected in the dorsal lobe; however, a significant difference occurred in the ventral lobe. Levels of SV40T expression were measured in the ventral lobe by quantitative RT-PCR and are shown for individual animals of the indicated genotypes, expressed as the crossing point for each corrected for a ß-actin control reaction. A t test was used to test for a significant difference, which was not found.

 
Transcript profiling
To determine whether loss of the PRLR resulted in altered prostate gene expression, we used the Affymetrix GeneChip system to measure the expression of approximately 12,000 genes. The ventral lobe showed no alteration in relative tissue areas and the dorsal lobe a 12% loss of epithelium (Fig. 2Go). Thus, altered tissue ratios will have no effect on ventral gene expression patterns and a small effect on dorsal gene expression patterns. The experiment was replicated three times for the ventral prostate. Analysis showed that duplication resulted in a very large decrease in false positives and a further replication did not dramatically improve the false positive rate. Consequentially, the dorsal lobe was analyzed in duplicate. RNA was pooled from five to eight different animals per replicate. Results were analyzed by GeneChip 3.1 and 5.1 software, and the sources of variation in gene expression levels were explored using principal components analysis (Fig. 4Go). The greatest cause of variance in the data set was the relative abundance of different genes, for example, the level of one of the most abundant genes such as ubiquitin, compared with the level of one of the least abundant genes such as transcription factor NF-ATca. Examination of the next two (dorsal) or three (ventral) principal components showed that the experimental replication provided the next most influential source of variance in gene expression. Examination of the higher principal components identified the third most influential factor as PRLR genotype (Fig. 4Go), revealing a small cluster of genes differentially expressed between genotypes.



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  		Figure 4. Transcript profiles of ventral prostate. RNA from five to eight PRLR+/+ or PRLR-/- ventral or dorsal prostates was pooled in equal ratios and labeled before hybridization to Affymetrix U74A or U74A1 chips. The experiment was replicated three times for the ventral lobe and twice for the dorsal lobe. Analysis was undertaken using Affymetrix GeneChip 3.1/5.1 and principal component analysis. The original axis positions are indicated, light gray behind the plane of the page, black in front. Genes called present in at least one of three PRLR+/+ replicates but without a change in expression level are shown as black squares. Genes called decreasing in two of three replicates (ventral A) or two of two replicates (dorsal, B) are red diamonds. The only two genes called increasing are green diamonds. These genes are labeled in the lower panels (ventral C, dorsal D) in which their average fold change across all replicates is greater than 2. x-axis, P.C. 6; y-axis, P.C. 5; z-axis P.C. 4. All plots are mean centered.

 
Functional annotation of these differentially expressed genes (Fig. 4Go, lower panels) revealed that the common genes comprising these clusters are principally involved in sperm/oocyte interaction or copulatory plug formation (see Discussion for details). These results were confirmed by quantitative PCR using TaqMan and LightCycler methodologies by two independent laboratories (Sydney and Göteborg) using different sets of mouse tissues. Figure 5Go demonstrates the method used on the LightCycler instrument and compares the results with those obtained by 30 cycles of RT-PCR (Fig. 5AGo) and from the Affymetrix chips for the gene AEG-1 (Fig. 5CGo). All methods show diminished AEG-1 expression. The results for the mRNA levels of other genes obtained in this way are given in Fig. 5DGo.



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  		Figure 5. Confirmation of chip data by quantitative PCR. Gene expression changes detected by the oligonucleotide chips were examined using quantitative PCR. The method used is demonstrated for AEG-1. A, RT-PCR for AEG-1 is analyzed after 30 cycles by ethidium bromide staining of an agarose gel. B, Quantitative PCR using the LightCycler instrument. Upper panel shows increase in the concentration of the AEG-1 amplicon with cycle number for samples derived from PRLR+/+ (purple lines) or PRLR-/- (red lines) ventral or dorsal prostates. Amplification of standard curve samples is shown using dashed blue lines, and the standard curve is plotted in the lower panel. A ß-actin standard curve is used to control the reverse transcription step and is not shown. C, Quantitative PCR results expressed as AEG-1 mRNA copies per microliter in the original samples, compared with their AEG-1 signal intensity measured by the oligonucleotide chip. Fold change is indicated for the bars linked by lines. D, Results using quantitative PCR for a subset of functionally related genes with reduced expression in PRLR-/- prostate.

 
Fertility
In the light of the transcript profiling results, we examined male PRLR-/- fertility in the breeding colony held at the Garvan Institute. PRLR+/+ (n = 31), PRLR+/- (n = 28), and PRLR-/- (n = 52) males were first joined monogamously and continuously at ages between 40 and 120 d. The rates of infertility, defined as no litters in 120 d, were PRLR+/+ 1 of 31, PRLR+/- 0 of 28, and PRLR-/- 5 of 52, confirming an increased rate of total infertility in the PRLR-/- genotype, as previously reported (25).

It is not known whether PRLR-/- males capable of producing litters are fully fertile. Latency to pregnancy was examined using Kaplan-Meier survival analysis (Fig. 6AGo). Latency to first litter was significantly delayed (log rank P = 0.0007) in PRLR-/- matings, with 50% outside the minimal time (20-d gestation + 4 d estrus), compared with less than 20% of either PRLR+/+ or PRLR+/- matings. Analysis using a Cox proportional hazards model indicated the chance of pregnancy for PRLR-/- matings to be 40.9% (P = 0.0005) of PRLR+/+ animals. The latency to second pregnancy (Fig. 6BGo) was indistinguishable among PRLR genotypes, as were the latencies for third, fourth, and fifth pregnancies (data not shown).



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  		Figure 6. Male fertility in PRLRKO breeding colony. Three types of monogamous, continuous pairings were analyzed for their breeding life; PRLR-/- males with PRLR+/- females (n = 52 pairs, squares), PRLR+/- males with PRLR+/- females (n = 28 pairs, circles), and PRLR+/+ males with PRLR+/+ females (n = 31 pairs, triangles). A and B, Kaplan-Meier survival analysis of latency to first pregnancy (A) and second pregnancy (B). Log rank P value for PRLR-/- males with PRLR+/- females vs. PRLR+/- males with PRLR+/- females. C, Correlation between latency at first pregnancy and second pregnancy (R2 correlation coefficient). D, Correlation of male age at pairing with latency to first pregnancy (R2 correlation coefficient).

 
First-pregnancy latency was compared by regression analysis to second-pregnancy latency to determine whether a subpopulation of low fertility PRLR-/- males persisted (Fig. 6CGo). No correlation was found (R2 = 0.003). To determine whether reduced fertility in PRLR-/- males was a function of delayed onset of full fertility, we correlated the age of the male when first housed with a female to first pregnancy latency (Fig. 6DGo), and again no relationship was found (R2 = 0.00005). Fertile PRLR-/- males produced 3.3 ± 1.8 litters with 5.8 ± 0.3 pups, compared with 4.0 ± 1.9 litters (P = 0.190) and 4.8 ± 0.5 pups (P = 0.222) for PRLR+/- males. Thus, the ability of PRLR-/- males to produce a first pregnancy is just 40% of wild-type animals but is 100% for subsequent pregnancies, with no persistent subpopulation of poorly fertile males and no effect of age on fertility.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the experiments detailed in this report, we have used genetically modified mice to search for essential roles of prolactin in the prostate, using morphological, gene transcript, and functional end points. Detailed morphological investigations showed that a loss of prolactin receptor caused a 20% increase in prostate weight, a loss of some epithelial area from the dorsal lobe, but no alteration in branching morphology. Castration of PRLR-/- animals showed that castration-induced regression is more extreme in dorsal lobes without prolactin receptor, indicating independent mechanisms of complementary action of androgen and prolactin. These data show that prolactin plays a subtle but essential role in the control of normal dorsal prostate morphology. In contrast, the effects of hyperprolactinemia are pronounced. A transgenic model of hyperprolactinemia showed a 10- to 20-fold increase in prostate weight, 5-fold increase in cell content, altered relative amounts of epithelium to stroma, and increased cellularity of the interductal stroma. As these animals aged, intraepithelial dysplastic features became apparent (7). A recent transgenic model using prostate-specific prolactin expression showed a similar prostate phenotype (31) in the absence of systemic hormonal effects, conclusively demonstrating the direct effects of hyperprolactinemia on the prostate. These results indicate that many previous experimental investigations, using the general paradigm of prolactin endocrine ablation with prolactin readdition, have measured the effects of hyperprolactinemia but attributed them to an essential role of prolactin. Our results show the essential morphological roles of prolactin contrast to those produced by hyperprolactinemia, limiting the essential actions to maintenance of the dorsal epithelium and fine regulation of secretion.

These observed effects vary a little from those observed in other genetically modified models of prolactin action. Loss of Stat5a results in prostate hypersecretion but with disorganization of the ventral epithelium not seen in PRLR-/- animals (32). Stat5a forms one branch of the prolactin receptor signaling pathway but also transduces signals from other receptors such as the epidermal growth factor receptor. Thus, only a subset of phenotypic abnormalities should be common between PRLR-/- and Stat5a-/- animals and suggests Stat5a as the mediator of prolactin’s control of secretion, but effects on ventral prostate organization may be caused by epidermal growth factor or other activators of Stat5. Loss of the prolactin gene resulted in a reduction in prostate weight (33), not a gain in weight as would be expected from our results. The rodent prostate begins development at d 17 of gestation (1) and has complete architecture (but not size or ductal branching) at 2 wk of age. During this period prolactin knockout animals receive maternal prolactin via the maternal circulation, placental lactogen (a prolactin receptor ligand) from the embryonic placenta, and prolactin from milk until weaning at 3 wk. Thus, the prolactin knockout is not prolactin deficient until after basic prostate development is complete, and this may be responsible for the contrasting observations made in prolactin and prolactin receptor knockouts (34).

The introduction of the SV40T antigen demonstrated that loss of the PRLR produced a small but significant reduction in the level of PIN in the ventral but not dorsal lobe. In our model the ventral lobe showed higher levels of PIN than the dorsal lobe, as does the original C3-SV40T model. Ventral lobe tumor incidence was 16% (PRLR+/- and PRLR+/+ combined), compared with the original model of around 40%, and the dorsal lobes did not produce any tumors, indicating that the alteration in genetic background has reduced the tumor incidence from levels reported in the original model. No prostate tumors were detected in PRLR-/- animals, but only five tumors were detected in other genotypes. Although these numbers are not sufficient to draw conclusions regarding the role of prolactin in tumor formation, the observation is consistent with the reduction in PIN seen in the ventral lobes of PRLR-/- animals.

To determine whether prolactin was exerting essential effects on gene expression that were not expressed as altered growth or morphology, we used high-density oligonucleotide arrays to transcript profile the ventral and dorsal prostates from PRLR+/+ and PRLR-/- males. The ventral lobe showed no changes in ductal branching or relative tissue areas, allowing altered gene expression to be attributed solely to transcriptional regulation. The dorsal lobe showed a 12% loss of epithelium, which must be taken into account when analyzing the resulting expression profiles. The use of replicates from pooled samples allowed a small number of consistently altered genes to be identified. Analysis of the ventral data showed that the third replication offered only small additional discriminatory power, compared with the major effect of duplication, and so the dorsal lobe was profiled in duplicate only.

Literature searches showed the most differentially regulated genes to be involved in fertility. Two subgroups were apparent. The first involves genes mediating sperm-egg interaction; acidic epididymal glycoprotein, HE5, and zonadhesin. Acidic epididymal glycoprotein is also known as AEG1 or protein DE and is the product of the CRISP-1 gene. This androgen-regulated glycoprotein is secreted by the epithelium of the epididymis and associates with the principal piece of the sperm tail, the sperm middle piece, and the postacrosomal region of the sperm head during sperm maturation. It is thought to be involved in sperm-oocyte plasma membrane fusion (35, 36, 37). HE5 is a glycoprotein that is also inserted onto maturing spermatozoa and shows variable surface presentation during sperm capacitation (38). Zonadhesin functions as a sperm-zona pellucida-binding protein, conferring species specificity. It is synthesized by primary spermatocytes and located to the anterior acrosome (39). The second subgroup contains the antiinflammatory and procoagulant seminal vesicle proteins, which function in copulatory plug formation and protection of sperm from female immune response; SVP2/SV-IV, semenoclotin and seminal vesicle F protein/SVP5 (40, 41, 42).

Expression of the prolactin receptor was also reduced, as expected from the targeting strategy employed (25). Small proline-rich protein 2A was detected by two independent probe sets, and small proline-rich proteins 2B and 2E were also found in the cluster revealed by the higher principal components but were not detected by GeneChip 3.1 or 5.1. These proteins are expressed in stratified squamous epithelia as components of the cornified cell envelope providing an epidermal barrier function (43). Their function in prostate, if any, is unknown. Quantitative PCR using oligos shown by blast searches to be specific for 2A demonstrated that 2A expression did not change. The probe sets present on the chip for 2A, 2B, and 2E are directed at regions of these genes that show high sequence homology among family members. Thus, the GeneChip result may be due to the integration of signals from multiple small proline-rich protein genes.

The reduction in expression level of genes required for plug formation and sperm-oocyte interaction may affect fertility, which we checked carefully in records obtained from multiple years of PRLR+/+ and PRLR-/- breeding. Overall infertility of PRLR-/- males was seen in 5 of 52 animals, compared with 1 of 59 of the other genotypes, supporting results of our initial investigation using a small number of animals (25). The potential subfertility of PRLR-/- males has not previously been investigated. In fertile animals latency to first pregnancy was longer in PRLR-/- animals, and overall they had only 40% probability of producing a first pregnancy, compared with PRLR+/+ animals. Intriguingly, these animals show a return to full fertility following the first pregnancy. Mating behavior appears normal in these males, discounting a learning defect that is overcome by experience. Another possibility arises from the breeding method. These results were obtained from a production colony that exploits the efficiency of postpartum mating. In contrast to the first pregnancy, the second and subsequent pregnancies were achieved in the majority of cases by postpartum mating, which may have alleviated the physiological deficit present in PRLR-/- animals. Regardless of mechanism these data establish a clear role for prolactin in male fertility and provide specific candidate genes whose combined loss may underlie the reduction of fertility.

In summary we have shown that the prolactin receptor is essential for full male fertility. Reduced expression of genes involved in sperm-egg recognition, reduction in maternal immune response to sperm, and copulatory plug formation may underlie this effect. Dorsal prostate epithelium is lost and fewer ventral PIN lesions are formed in response to SV40T when PRLR is lost. The subtle nature of the essential developmental roles for prolactin contrast dramatically with the effects produced by transgenic overexpression of prolactin in the prostate, where the prostate is enlarged 10- to 20-fold because of hypersecretion and stromal hyperplasia and exhibits intraepithelial dysplastic features (7). These effects have been shown to be independent of the elevated systemic androgen levels present in the transgenic model (44). Furthermore, generation of a prostate-specific prolactin transgenic mouse has demonstrated a similar prostate phenotype, indicative of a local direct effect of prolactin in the absence of altered systemic hormone levels (31). Thus, prolactin is developmentally very active in the hyperprolactinemic state but has a subtle essential role. Experimental approaches that involved prolactin treatment, with or without prior pituitary ablation, may have mimicked hyperprolactinemia rather than normal prolactin levels and thus may have wrongly attributed the trophic effects of prolactin as an essential function of the hormone. The implications of these findings for the treatment of prostate cancer are significant. Antiandrogen therapy results in hyperprolactinemia in a significant number of patients (21). It is possible that the trophic stimulus provided by hyperprolactinemia during antiandrogen therapy allows some cells in early neoplastic lesions to survive and continue to proliferate following androgen withdrawal. These cells, already androgen insensitive, may then continue to accumulate the genetic mutations that will enable the development of disseminated disease. This may be the mechanism by which preoperative serum prolactin levels have no prognostic value but postoperative serum prolactin levels become prognostic.


   Acknowledgments
 
We thank Karl Peters for assistance with the quantitative PCR.


   Footnotes
 
This work was supported by grants from the National Health and Medical Research Council of Australia (to C.J.O.), Cancer Council of New South Wales, Institut National de la Santé et de la Recherche Médicale (to P.A.K.), the Swedish Cancer Foundation (to J.T. and J.K.) and the Gothenburg Medical Society (to J.K.).

Abbreviations: H&E, Hematoxylin-eosin; PIN, prostatic intraepithelial neoplasia; PRLR+/+, wild-type mice; PRLR-/-, prolactin receptor knockout mice; SV40, simian virus 40.

Received January 16, 2003.

Accepted for publication April 1, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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