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Endocrinology Vol. 143, No. 10 4084-4095
Copyright © 2002 by The Endocrine Society

ARTICLE

Reproductive Disturbances, Pituitary Lactotrope Adenomas, and Mammary Gland Tumors in Transgenic Female Mice Producing High Levels of Human Chorionic Gonadotropin

Susana B. Rulli, Aino Kuorelahti, Öznur Karaer, Lauri J. Pelliniemi, Matti Poutanen and Ilpo Huhtaniemi

Department of Physiology (S.B.R., A.K., M.P., I.H.) and Laboratory of Electron Microscopy (O.K., L.J.P.), University of Turku, FIN-20520 Turku, Finland

Address all correspondence and requests for reprints to: Professor Ilpo Huhtaniemi, Department of Physiology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. E-mail: ilpo.huhtaniemi{at}utu.fi.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To assess the consequences of prolonged exposure to elevated levels of LH/human chorionic gonadotropin (hCG) in the female, we developed a transgenic (TG) mouse model (hCGß+) that overexpresses the hCGß-subunit cDNA. Because of the promoter used, ubiquitin C, the transgene is expressed in multiple tissues, including the pituitary gland, in which coupling with the endogenous common {alpha}-subunit results in synthesis of high levels of bioactive hCG. The TG females presented with precocious puberty, infertility, enhanced ovarian steroidogenesis, and abnormal uterine structure. Pituitary enlargement was evident from the age of 2 months, which progressed to adenomas by the age of 10–12 months. Immunohistochemical studies and electron microscopy demonstrated lactotrope origin for the adenomas, associated with severe hyperprolactinemia. The mammary glands of TG females showed marked lobuloalveolar development followed by mammary tumors with characteristics of adenocarcinoma at the age of 9–12 months. More than 90% of penetrance and high frequency of metastasis (47%) was observed. Formation of the pituitary and mammary gland tumors was totally abolished by ovariectomy despite persistently elevated hCG levels. Taken together, these findings suggest that the hCG-induced aberrations of ovarian function are clearly responsible for the extragonadal tumors observed in these TG mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HUMAN CHORIONIC GONADOTROPIN (hCG), secreted normally by the placenta, belongs to the family of glycoprotein hormones, together with pituitary LH, FSH, and TSH. Human CG is a heterodimer of two noncovalently associated subunits, the common {alpha}- and the hormone-specific ß-subunit. Only LH/hCG {alpha}/ß-dimers are biologically active (1). The hCGßprotein has 83% homology with LHß and contains an additional C-terminal 24-amino-acid extension with four additional O-linked glycosylation sites. This structural difference explains the longer circulatory half-life and higher biopotency of hCG over LH (1). LH and hCG interact with the same LH/hCG receptor, promoting ovarian steroidogenesis and triggering ovulation. When administered to rodents, hCG functions as an LH agonist, evoking strong LH-like functional responses (1). Recently LH/hCG receptor expression and LH actions have been reported in extragonadal tissues (2, 3). However, their physiological role still remains unclear.

Inappropriately elevated gonadotropin secretion has been considered a cause of both female infertility and ovarian tumorigenesis (4, 5). Polycystic ovary syndrome in humans is associated with elevated serum LH, which, in conjunction with insulin resistance, may be responsible for increased ovarian androgen production, formation of multiple ovarian cysts, infertility, and chronic anovulation (6). On the other hand, prolonged exposure to female hormones throughout the reproductive years has been associated with diverse pathologies in advanced age, such as breast (7) and ovarian cancer (8). Transgenic (TG) mouse models have provided evidence for the role of gonadotropins as contributing factors to reproductive dysfunction and gonadal tumorigenesis. Mice overexpressing FSH and LH were previously characterized, and the females were infertile with highly hemorrhagic and polycystic ovaries and elevated serum testosterone, estradiol, and progesterone (9, 10, 11, 12). A tumor promoter role for LH has also been proposed on the basis of another TG mouse model, one expressing the Simian virus 40 T-antigen under inhibin {alpha}-subunit promoter (13, 14). On the other hand, the female phenotype arising from the absence of LH action has been well documented on LH receptor knockout mice (15, 16), in which the last step of follicular maturation and ovulation are prevented. This model is a close phenocopy of completely inactivating human LH receptor mutations (4).

In this study, we aimed to analyze in female mice the physiological and pathological consequences of prolonged exposure to elevated hCG levels. We followed up, besides direct ovarian effects, the eventual extragonadal effects brought about by the aberrant ovarian stimulation. For this purpose, we generated TG mice overexpressing ubiquitously the hCGß-subunit. The promoter used for transgene expression, ubiquitin C, does not respond to negative feedback of gonadal hormones. We therefore expected, and indeed demonstrated, that the strong hCGßtransgene expression would bring about a different phenotype from that of an earlier LH-overexpressing TG model, in which the transgene was expressed under the common {alpha}-subunit promoter (10). The spatiotemporal evolution of the hCG-induced phenotype was followed up throughout the life span of the TG mice. Because LH/hCG action is the major regulator of ovarian steroidogenesis, we hypothesized that chronic hCG hypersecretion would influence ovarian steroidogenesis and consequently determine the development and progression of hormone-dependent tumors in older ages, as occurs in the human. The model also allowed us to address the possible significance of extragonadal LH actions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Transgene construct
A 579-bp cDNA fragment of the ß-subunit of hCG, corresponding to gene 5 (provided by Dr. J. C. Fiddes, California Biotechnology Inc.) (17) was subcloned downstream of the 1.2-kb human ubiquitin C promoter, into the HindIII site of pUB6/V5-HisA plasmid (Invitrogen, Carlsbad, CA). The vector included the ubiquitin C promoter consisting of the nontranslated first exon and intron and the bovine growth hormone polyadenylation signal (Fig. 1AGo). A 2.8-kb ubiquitin C/hCGß fragment (UbiC-hCGß) was released from the vector by digesting with BglII and NsiI enzymes. The fragment was resolved in 1% agarose gel, isolated by electroelution, and purified with Elutip-D columns (Schleicher \|[amp ]\|Schuell, Inc., Keene, NH). Finally, the fragment was diluted in TE buffer (10 mM Tris-HCl; 5 mM EDTA, pH 7.5) at a concentration of 2 ng/µl for microinjection.



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  		Figure 1. Expression of UbiC-hCGß transgene. A, Schematic diagram of the UbiC-hCGß DNA construct used to generate TG hCGß+mice. B, Expression of transgene mRNA in different organs, as demonstrated by RT-PCR. C, Immunostaining for hCGß in the pituitary gland at 6 months of age: (a) wt female; (b) hCGß+ female. Positive immunoreaction in wt pituitary is due to cross-reactivity of the antibody with LHß. Scale bar, 20 µm. D, Secretion of hCGß-subunit to circulation was confirmed by IFMA. The formation of the dimer with endogenous {alpha}-subunit was measured with an in vitro LH/hCG bioassay in serum samples. ND, Not detected. n = 6/group. *, P < 0.05 vs. wt.

 
Before microinjection, the activity of the UbiC-hCGß vector was confirmed in vitro. For this purpose, HEK-293 cells (300,000 cells/35-mm2 culture plate) were transiently transfected with the UbiC-hCGß plasmid using Lipofectamine (Life Technologies, Inc., Grand Island, NY). The cells were incubated for 30 h, and the medium was tested for the presence of hCGß by a specific immunofluorometric assay (IFMA), using time-resolved fluorometry for signal detection (Delfia, Wallac, Inc., Oy, Turku, Finland). Microtitration wells from the hCG Delfia kit containing an immobilized antibody against hCGß were used in combination with an europium-labeled mouse monoclonal antibody against the ß-subunit of hCG from the hLH Delfia kit. The reference preparation from the National Institues of Diabetes and Digestive and Kidney Disorders (NIDDK) (NIH, Bethesda, MD); hCGß CR-121 was used as standard. The detection limit of the assay was 40 µg/liter. The transfection assay demonstrated that the transgene was capable of encoding the production of high amounts of hCGß in vitro.

Generation of transgenic mice
Founder TG mice were generated by microinjecting the transgene into pronuclei of fertilized oocytes from FVB/N mice, and the microinjected embryos were implanted into oviducts of pseudopregnant female mice of the NMRI strain. PCR analyses of genomic DNA from tail biopsies were used to identify TG animals. One microgram genomic DNA was added to the 50 µl PCR containing 10 mM Tris-HCl (pH 8.8), 1.5 mM MgCl2, 50 mM KCl, 200 µM deoxynucleotide triphosphate mix, 0.2 µM primers, and 2.5 U DNA polymerase. An 830-bp DNA fragment of the transgene was amplified using primers specific for the ubiquitin promoter (5'-CGCGCCCTCGTCGTGTC-3') and the hCGß-cDNA (5'-AAGCGGGGGTCATCACAGGTC-3'). The DNA was denatured at 97 C for 4 min, followed by PCR at 96 C for 0.5 min, 58 C for 1 min, and 72 C for 1.5 min for 32 cycles. The resulting PCR products were analyzed by electrophoresis on 2% agarose gel, and the fragments were UV visualized with ethidium bromide.

Establishment of transgenic lines
Five founders were obtained after pronuclear microinjection of the transgene, four males and one female. Two of the four males were fertile, whereas two males and the female were infertile. Consequently, two independent mouse lines overexpressing the hCGß subunit (hCGß+mice) were established by breeding the fertile males with wild-type (wt) FVB/N female mice. Transmission of the transgene was followed by PCR analysis, as described for the TG founder mice. The animals were housed in a specific pathogen-free environment under controlled conditions of temperature and light, and tap water and commercial mouse chow were provided ad libitum. All mice were handled in accordance to the institutional animal care policies of the University of Turku and with appropriate permissions. Animals from F1 to F4 generations were used in this study. Female TG mice derived from both lines presented with identical phenotypes, and, hence, the data obtained were pooled.

Fertility studies
For testing the fertility, female TG mice were bred with wt FVB/N mice for up to 6 months. For analyzing the onset of puberty, females were examined daily for vaginal opening from 20 d of age. Puberty was defined as the day of vaginal opening. The duration of estrous cycle was determined in two groups of six hCGß+ and wt mice, by analyzing the vaginal smears from the onset of vaginal opening until the age of 8 wk.

Measurement of serum hormone levels
Animals were killed in the morning (1000–1200 h) by cervical dislocation, and blood was collected from the heart. Serum samples were separated by centrifugation and stored at -20 C until hormone measurements. Serum hCGß concentration was measured by IFMA, as described above. FSH levels were measured by IFMA, the sensitivity of the assay was 50 ng/liter (18). Prolactin (PRL) was measured by RIA, as described previously (19), using a mouse PRL antibody and mouse reference preparation AFP-6476C, provided by NIDDK. The sensitivity of the assay was 200 ng/liter. Estradiol levels were measured by IFMA after diethylether extraction, using the human estradiol Delfia kit (Wallac, Inc.) adapted for mouse samples. The sensitivity of the assay was 7 pmol/liter. Serum testosterone and progesterone levels were measured by conventional RIAs after diethylether extraction, and the sensitivity assays were 10–20 pmol/liter and 50 pmol/liter, respectively (20, 21).

The bioactivity of circulating hCG was determined by the mouse interstitial cell in vitro bioassay (22). Testosterone production, determined by RIA, was used as an index of the hCG response. Recombinant hCG (specific activity: 14,800 IU/mg, Organon, Oss, The Netherlands) was used as standard. The sensitivity of the bioassay was 0.5 IU/liter, and the intra- and interassay coefficients of variation were less than 5% and 10%, respectively.

RNA isolation and RT-PCR for the transgene
Total RNA was isolated by the single-step acid guanidinium thiocyanate-phenol-chloroform extraction method, as described previously (23). One microgram total RNA was reverse transcribed using the avian myeloma virus reverse transcriptase, Promega Corp., Madison, WI) and the resulting cDNAs were PCR amplified in the same tube. The sense primer corresponded to the first exon of ubiquitin C (5'-TTGGGTCGCGGTTCTTGTTTGTGG-3'), and the antisense primer corresponded to the hCGß cDNA fragment (5'-ACGCGGGTCATGGTGGGGCAGTAG-3'). First, the reverse transcriptase reaction was carried out (50 C for 10 min), followed by a denaturation period of 3 min at 96 C. Thereafter, a PCR of 32 cycles (96 C for 1 min, 62 C for 1.5 min, and 72 C for 1 min, with a final extension period of 10 min at 72 C) was performed. As a negative control tissue, pituitary RNA from a wt female mouse was used. After RT-PCR, 15 µl reaction mixture were loaded onto a 2% agarose gel, electrophoresed, and the ethidium bromide-stained fragments were visualized by UV.

Histological analysis and immunohistochemistry
Tissues were fixed overnight in 4% paraformaldehyde, dehydrated, embedded in paraffin, and 5-µm-thick sections were prepared. For histological studies, sections were stained with hematoxylin and eosin.

Immunohistochemical localization of the pituitary hormones was performed using the streptavidin-biotin-peroxidase technique. Primary antisera against mouse PRL, mouse GH, rat TSHß, human adrenocorticotropin (ACTH), and hCGß (NIDDK) were used at dilutions 1:10,000–1:15,000. After overnight incubation at 4 C, the reactions were visualized using the Vectastain Elite ABC kit (Vector Laboratories, Inc., Burlingame, CA), and 3,3'-diaminobenzidine (Zymed Laboratories, Inc. Corp., San Francisco, CA) as the electron donor.

Electron microscopy
Tissue pieces of 1-mm3 size were cut from the pituitaries of wt and TG mice. The pieces were fixed first in 5% glutaraldehyde (Merck, Darmstadt, Germany) in 0.16 mol/liter s-collidine buffer (pH 7.4) and postfixed in potassium ferrocyanide-osmium tetroxide, embedded in epoxy resin (Glycidether 100, Merck) and cut into sections as described earlier (24). For light microscopic survey, 1-µm-thick sections were stained with 0.5% toluidine blue, and the thin sections for electron microscopy were stained with 5% uranyl acetate and 5% lead citrate in Ultrostainer (Leica Corp., Wien, Austria) and examined in JEM-100SX transmission electron microscope (JEOL, Tokyo, Japan).

Mammary gland whole mounts
The inguinal mammary glands were dissected out, spread onto a glass slide, and fixed overnight in a 1:3 mixture of glacial acetic acid: 100% ethanol at room temperature. The tissues were washed in 70% ethanol, rinsed in water, and stained overnight in 0.2% carmine (Sigma, St. Louis, MO) and 0.5% AlK(SO4)2 at room temperature. The stained tissue was dehydrated through graded series of ethanol, cleared in toluene, and mounted.

Statistical analysis
SigmaStat for Windows 2.03 was used for t test and Student-Newman-Keuls tests. The limit of statistical significance was set at P < 0.05. The values are presented as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
hCGß+ mice display high transgene expression and produce bioactive hCG
The UbiC-hCGß transgene (Fig. 1AGo) was expressed in a number of tissues of hCGß+ mice, including the pituitary gland, as shown by RT-PCR analysis (Fig. 1BGo) and immunohistochemistry (Fig. 1CGo). In addition, the processing and secretion of the transgenic protein appeared correct because of the highly elevated levels of the hCGßsubunit (7.8 ± 2.0 mg/liter) in serum. This, in turn, resulted in marked increase in the dimeric bioactive hormone, consisting of the endogenously expressed mouse {alpha}-subunit associated with the TG human ß-subunit. This was demonstrated by the 40-fold increase in hCG/LH bioactivity in the serum of female hCGß+ mice, compared with wt females (Fig. 1DGo).

Female hCGß+ mice are obese and infertile with severe disturbances in their reproductive system
The body weight gain of the female hCGß+ mice and wt controls was similar up to 2 months of age, but thereafter the weight gain of the TG mice greatly exceeded that of the wt mice (Fig. 2AGo). At the age of 6 months, the TG mice weighed about twice as much as the wt controls, without significant difference in the body lengths (9.3 ± 0.1 cm, n = 6) (Fig. 2BGo). It was found on autopsies that the obesity of the hCG+ mice was mainly due to fat accumulation in the abdominal region.



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  		Figure 2. Body weight increase with age in hCGß+ females mice. A, The weights of mice were measured over a period of 6 months. Each time point represents the average of at least six mice. *, P < 0.05 vs. wt. B, A representative picture of a wt and hCGß+ female mouse at 6 months of age. The hCGß+ female mice are obese with abdominal fat deposition, whereas no difference in length was detected in comparison with wt mice.

 
The hCGß+ females developed precocious puberty as manifested by vaginal opening at 21–22 d of age, whereas it normally occurs at the age of 26–28 d in wt mice of the same strain in our colony. At the age of 21–22 d, the uterus was swollen, with significantly increased weight (hCGß+: 75 ± 9; wt: 11 ± 1 mg, n = 4;P < 0.05). Both observations are clear indicators of marked alteration in ovarian steroid production at this early stage of sexual maturation. None of the hCGß+ mice showed evidence of normal estrous cycle. From the age of vaginal opening until the age of 42–45 d, a constant estrus-type pattern with cornified epithelial cells was present. Starting from 45 d of age, a diestrus-type pattern with persistent leukocyte infiltration was observed, reminiscent of a repetitive pseudopregnant status.

To determine the causes of infertility in the hCGß+ females, histological analysis of the ovaries and uterus were performed (Fig. 3Go). At the age of 6 months, the ovaries were significantly enlarged in the hCGß+ mice (12.5 ± 0.9 mg, n = 8), compared with those of wt mice (7.3 ± 0.7 mg, n = 8, P < 0.001). Massive luteinization was evident, with presence of few follicles at various stages of maturation. Multiple large corpora lutea as well as hemorrhagic cysts (Fig. 3BGo), surrounded by luteinized granulosa cells filled with lipid droplets were present, suggesting active steroid synthesis. The adult uterus did not present changes in weight, compared with wt females (hCGß+: 124 ± 5 mg; wt: 129 ± 14 mg; n = 10). The wt uterine architecture presents a normal endometrial and myometrial compartment (Fig. 3CGo). In contrast, histological evaluation of transverse sections from the uterine horn of hCGß+ mice revealed a thin endometrium, with cystically dilated endometrial glands lined by a flattened epithelium filled with secretory fluid (Fig. 3DGo). The dilated glands displace the surrounding endometrial stroma.



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  		Figure 3. Histology of ovaries and uterus of hCGß+ and wt mice at 6 months of age. A, A wt ovary showing corpora lutea and follicles at all maturational stages. B, Ovary from hCGß+ mice showing multiple corpora lutea (CL) throughout the whole ovary. Hemorrhagic cysts are also present (asterisks). C, Uterus from wt with normal architecture. D, Uterus from hCGß+ mice showing an abnormal endometrium, with cystically dilated endometrial glands lined by flattened epithelium. Hematoxylin and eosin staining. Scale bar, 100 µm.

 
Enhanced ovarian steroidogenesis of female hCGß+ mice
To analyze the direct consequences of constantly high levels of hCG on ovarian function and steroidogenesis, the steroid hormone profile at different ages was measured (Fig. 4Go). At 1 month of age, 3- to 4-fold increase in serum concentrations of estradiol (hCGß+: 198 ± 28;wt: 53.2 ± 8.3 pmol/liter, P < 0.05, n = 4;Fig. 4AGo), progesterone (hCGß+: 13.7 ± 3.3; wt: 4.6 ± 1.4 nmol/liter, P < 0.05; Fig. 4BGo), and testosterone (hCGß+: 0.92 ± 0.21; wt: 0.29 ± 0.03 nmol/liter, P < 0.05; Fig. 4CGo) were detected in the hCGß+ mice, compared with wt females. After 1 month of age, serum progesterone rapidly increased in the hCGß+ mice (Fig. 4BGo), reaching a 20- to 40-fold increase by 6 months of age, with a further increase to about 100-fold levels at 9–12 months of age. Because estradiol showed normal levels after 2 months of age, the ratio of progesterone to estradiol was dramatically increased and could be the reason for many of the reproductive dysfunctions observed in hCGß+ females during adulthood. Serum testosterone levels were significantly increased at 6 months, with an 8-fold elevation at 12 months of age in the hCGß+ females (Fig. 4CGo).



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  		Figure 4. Serum concentrations for sex steroid hormones in hCGß+ (open circles) and wt (filled circles) female mice at different ages. A, Estradiol levels. B, Progesterone levels. C, Testosterone levels. Note the different fold-change in each case. n = 4–8 per time point. *, P < 0.05; **, P < 0.01 vs. wt random-cycling females.

 
At 6 months of age, serum FSH levels in the hCGß+ females (1.6 ± 0.2 ng/ml, n = 11) were comparable with those in diestrous wt females (2.8 ± 0.6 ng/ml, n = 8).

Female hCGß+ mice develop lactotrope adenomas and hyperprolactinemia
The pituitary glands of hCGß+ mice were enlarged after 1 month of age, compared with wt mice, and persistent growth occurred thereafter (Fig. 5AGo). Histological examination at 6 months of age suggested a hyperplastic growth of the pituitary glands. By the age of 10–12 months, the hCGß+ females developed pituitary tumors, ranging up to 100-fold in size. In parallel, serum PRL was markedly elevated, reaching up to 600-fold increases in the mice with the largest adenomas (Fig. 5BGo). Hence, a direct correlation between the pituitary weight and serum PRL was evident. Ovariectomy at 6 wk of age totally prevented the pituitary gland enlargement (Fig. 5AGo) and abolished hyperprolactinemia (Fig. 5BGo). At the same time, the bioactive levels of circulating hCG remained high after ovariectomy (hCGß+: 76 ± 4; wt: 4 ± 0.1 IU/liter, n = 4).



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  		Figure 5. Age-related changes in weights of the pituitary glands (A) or concentration of serum PRL (B) in wt (filled circles) and hCGß+ females (open circles). The effects of ovariectomy on hCGß+ females are indicated by a diamond-shaped symbol at 6 months of age in both figures. n = 6/group. *, P < 0.05; **, P < 0.01 vs. wt.

 
Macroscopically, the pituitary tumors presented with suprasellar expansion and fragile and hemorrhagic appearance. This phenotype was found in 100% of the cases (n = 34). Compression of the brain mass against the skull with a clear cranial deformation was evident at older ages. Typically, the tumors were nodular and irregular in shape. Histological examination showed distorted architecture of the gland, with dilated blood-filled spaces and multifocal nodules compressing the adjacent tissue. Mitotic figures, occasional binucleated cells, or nuclear inclusions were readily identified. In some areas, densely packed, small round cells were present, and some areas showed large giant cells with abundant cytoplasm. The pituitary neoplastic foci showed breakdown of the reticulin fiber network as a diagnostic for adenoma (data not shown).

All the adenomas analyzed showed strong immunocytochemical reaction for PRL. The majority of the cells had a juxtanuclear immunocytochemical reaction at the Golgi region, which is characteristic for sparsely granulated lactotroph adenomas, indicating active secretion (Fig. 6Go, A and B). Immunocytochemical reactions for GH, ACTH, and TSHß are also shown in Fig. 6Go (C–H). Cells stained for these hormones were scattered in areas surrounding the nodules, but no reaction for these hormones was identified within the nodules. These areas apparently represent the remnants of the functional pituitary gland. The specific immunopositive staining for PRL in the tumorous areas of the pituitary gland together with the excess of serum PRL levels confirmed the diagnosis of prolactinoma.



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  		Figure 6. Immunohistochemical analysis of pituitary hormones in pituitaries of the hCGß+ female mice. Sections from pituitary glands from 11-month-old wt (A, C, E, and G), and hCGß+ mice (B, D, F, and H) were analyzed by immunohistochemistry for the expression of PRL (A and B), GH (C and D), TSHß (E and F), and ACTH (G and H). In B, strong immunocytochemical reactivity for PRL is present in the focal nodule (asterisk) and surrounding the nodule. The arrows in D, F, and H indicate cells positive for the corresponding hormone in the area surrounding the neoplastic nodule. Note the absence of positive staining for GH, ACTH, and TSHß in the area of the focal nodule (asterisk). Arrowheads indicate the position of a capillary next to the nodule. The pictures correspond to the same area of a representative specimen from eight pituitaries analyzed. Scale bars, 100 µm.

 
By electron microscopy, the anterior pituitary of female wt mice consisted of ultrastructurally normal somatotropes, lactotropes, corticotropes, and other tropic hormone-producing cells. The lactotropes were readily identified by their pleomorphic dark PRL vesicles of 150–400 nm in size (Fig. 7AGo). Electron microscopy confirmed that the nodules in the enlarged anterior pituitaries of hCGß+ mice consisted of lactotropes (Fig. 7BGo). Their cytoplasm contained several secretory vesicles and the granular endoplasmic reticulum was more extensive than that in the wt cells. As a whole, the cells looked structurally normal and the large size of their nucleolus was in concert with their active PRL production. The interstitial space between the loosely organized cells contained free erythrocytes, and lack of endothelial lining of the dilated blood-filled spaces confirmed the histological observations. The cellular organization and ultrastructure in nonnodular parts of the TG pituitaries was similar to that in wt, and the different types of tropic cells with their respectively characteristic vesicles were present (data not shown).



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  		Figure 7. Electron micrograph of the female anterior pituitary gland. A, In wt, the tissue consists of different endocrine cells, of which shown here are lactotropes (L), somatotropes (S), and corticotropes (C). One red blood cell (R) is seen in a capillary. The PRL granules in the lactotropes (L) are polymorphic from small globules to elongated and curved darkly staining structures in the cytoplasm. All cells have ample granular endoplasmic reticulum and other typical organelles of a protein secretory cell. GH granules in somatotropes (S) are typically large darkly staining globules, whereas the ACTH in corticotropes (C) are small and typically organized along the cell membrane (age 8 months). Scale bar, 2 µm. B, In the female hCGß+ pituitary tumor, the histologically described adenoma nodules consisted exclusively of lactotropes (L) arranged at places loosely leaving empty intercellular spaces in which free erythrocytes (R) are often observed as a sign of hemorrhage. Ultrastructurally, the cells are similar to those in the control pituitaries except that the amount of granular endoplasmic reticulum (G) is usually more extensive (age 11 months). Scale bar, 2 µm.

 
Development of mammary tumors in the hCGß+ female mice
Whole-mount analysis of the mature mammary glands from virgin wt mice demonstrated a finely branched ductal network with multiple alveolar buds associated with each subordinate branch (Fig. 8AGo). In contrast, 6-month-old hCGß+mice showed substantial lobuloalveolar development (Fig. 8BGo), reminiscent of the phenotype of the pregnant mammary gland. When ovariectomy was performed at 6 wk of age and the mammary glands were examined 5 months later, no apparent alveolar buds were associated with the ductal network in wt mice (Fig. 8CGo). Interestingly, in hCGß+mice, the lobuloalveolar development was completely prevented (Fig. 8DGo).



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  		Figure 8. Whole-mount images of mammary glands of wt and hCGß+ female mice at 6 months of age. A, wt virgin female with typical ductal structure of the mammary gland. B, An hCGß+ female showing prominent lobuloalveolar development, resembling a gland of a pregnant female. C, Mammary gland of an ovariectomized wt virgin mouse. D, Gland from an ovariectomized hCGß+ female, with no lobuloalveolar development. The lymph node is present in A–C. Scale bar, 400 µm.

 
Histological examination of mammary glands of the hCGß+ mice at 3–6 months revealed alveolar hyperplasia throughout the gland, with a large number of cytoplasmic lipid vacuoles resembling prelactating mammary gland epithelium (Fig. 9BGo). At the age of 9–12 months, the hyperplastic mammary glands developed into palpable tumors. By 12 months of age, 91% (31 of 34) of the females had developed multiple palpable tumors of up to 2 cm in diameter, originating both from the inguinal and thoracic glands. Because of the dramatically grown pituitary tumors, the animals did not survive more than 12 months and failed to develop palpable tumors in 9% of the cases. However, histological examination of the mammary gland in those specimens without palpable tumors confirmed the development of mammary hyperplasia and adenocarcinoma. The mammary tumors presented with characteristics of adenocarcinoma and high level of malignancy. Figure 9CGo shows the variety of histological organizations in one tumorous area. Dilated and hyperplastic alveolar-type structures filled with secretion were frequently seen (Fig. 9DGo). These structures were associated with epithelial proliferation forming papillary projections that penetrated into the lumen, possibly representing an early stage of tumorigenesis. A papillary pattern was the most common feature found in mammary tumors of the hCGß+ females (Fig. 9EGo), consisting of fingerlike papillae containing a central core of fibrous tissue surrounding the epithelium. Areas of solid nondifferentiated tumor were also detected (Fig. 9FGo). Abundant mitotic figures were found in both histotypes.



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  		Figure 9. Histology of the mammary gland and mammary tumors in hCGß+ female mice. A, Mammary gland of a wt female with typical ductules. B, Mammary gland of a hCG ß+ female at 6 months of age, showing epithelial hyperplasia and cytoplasmic lipid vacuoles. C, Mammary tumor from an 11-month-old hCGß+ female mouse, showing a range of histological types at various stages of differentiation. D, A detailed view of C, showing dilated and hyperplastic alveolar structures filled with secretions, associated with papillary projections proliferating toward the lumen. E, A detailed view of C, showing a tumorous area characterized as adenocarcinoma with papillary growth pattern, consisting of fingerlike papillae with a thin central core of fibrous tissue surrounding the epithelium. F, A detailed view of C, showing a solid tumor with nondifferentiated pattern located in the inner area of the tumor, with multiple mitotic figures (arrows). The pictures correspond to a representative specimen from 10 samples analyzed. Hematoxylin and eosin staining. Scale bars, 100 µm.

 
High frequency of metastasis was detected in several tissues (16 of 34, 47%), suggesting the aggressive phenotype of the mammary tumors. The most common sites of metastasis were the liver, spleen, and lung. Metastasis in the uterus and adrenal glands was also detected in cases in which the other organs were also invaded. The papillary architecture with abundant mitotic figures was usually displayed in the metastases. Age-matched virgin wt FVB/N females remained free from tumors.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have generated a TG mouse model (hCGß+) with chronically elevated levels of circulating hCG. As expected, the ubiquitin C promoter used (25) directed the transgene expression to a variety of tissues. The transgene was properly translated and secreted because the hCGß-subunit was detected in different tissues, including the pituitary gland and circulation. The transgenic hCGß was also associated with mouse {alpha}-subunit and formed high levels of biologically active dimeric hCG. The dimerization process is expected to occur mainly in gonadotropes and thyrotropes (26) but possibly also in some extrapituitary tissues, such as the ovary, in which {alpha}-subunit has been found to be expressed (27). As a result, serum hCG/LH bioactivity was 40-fold increased in the female hCGß+ mice. The amount of {alpha}-subunit apparently becomes rate limiting in dimerization because free hCGß will be synthesized and secreted in excess. However, the subunit monomers are biologically inactive and not recognized by the LH/hCG receptor (28).

The hCGß+ females developed precocious puberty and altered hormonal profile during sexual maturation. The first evident step in these responses is hyperstimulation of the immature ovary by hCG, resulting in high levels of estradiol, testosterone, and progesterone. In the ovary, estrogen and FSH are able to induce LH receptor expression in granulosa cells of preovulatory follicles (29), facilitating luteinization. Once luteinization starts, granulosa and theca cells become nonmitotic and will be reprogrammed to express luteal specific genes (30). In hCGß+ mice, the high hCG induces massive luteinization of granulosa, theca, and interstitial cells. Hyperandrogenism was accompanied by a transient increase in estradiol levels only during prepuberty, suggesting that the FSH-induced aromatization would decline once luteinization occurs (30). The dominant steroidogenic pathway of the hCGß+ovary was consequently altered, and progesterone became the primary steroid hormone produced, reaching levels far in excess of those of pregnancy. A reason for this could be that the hCG-induced luteinization of the ovaries was so strong that insufficient number of granulosa cells were left to maintain the initially elevated estrogen production. In most rodents, PRL is a luteotrophic hormone maintaining the structural and functional integrity of the corpus luteum (31). In our model, the high levels of circulating PRL and hCG apparently jointly prolong the luteal life span, and excessive progesterone production.

A very clear obese phenotype developed in the TG female mice exposed to chronically elevated levels of hCG. The substantial imbalance in the female hormone profile provides the primary clue for understanding this phenotype. Although not studied in the current study, this mouse model offers a good opportunity to address the multiple endocrine alterations that contribute to the pathogenesis of obesity.

A TG model with chronically elevated LH was previously described by Risma et al. (10), in which the bovine {alpha}-subunit promoter was fused to the coding region of a chimeric bovine (b) LHß-CTP gene. It displays certain similarities with our model. Both present with severe disturbances in the female reproductive system, such as precocious puberty, associated with increased estrogen and androgen levels (32), infertility, and anovulation (33). Remarkable differences also exist between the two models. Firstly, because of different promoters, the human ubiquitin C promoter used in our study is universally and constitutively activated (25), whereas the {alpha}-subunit promoter used in bLHß-CTP mice is specifically gonadotrope directed and responsive to negative feedback regulation (34). This explains the different levels of circulating LH/hCG in the two models, 5- to 10-fold in the bLHß-CTP mice (10), and around 40-fold in the hCGß+mice. Secondly, a different pattern of ovarian steroidogenesis is evident. Adults hCGß+ mice produced 40- to 100-fold excess of progesterone, physiological levels of estradiol, and a 3- to 6-fold increase in androgens. In contrast, 2- to 4-fold increases in testosterone, estradiol, and progesterone were detected in the bLHß-CTP model (12). Thirdly, the bLHß-CTP mice present mainly with a polycystic ovary syndrome-like ovarian phenotype and granulosa cell tumors or luteomas (10, 11). The hCGß+ mice show a predominantly luteinized ovarian phenotype, accompanied by few hemorrhagic cysts derived from follicular or luteal tissue. As suggested by Keri et al. (11), the genetic background of the mice could affect the ovarian phenotype. Depending on strain, chronic elevation of hCG/LH could lead to polycystic ovaries, luteinization, or tumor development. It was proposed that susceptibility to granulosa cell tumors is an oligogenic trait controlled by three unlinked genes (11).

By the age of 10–12 months, all TG females developed pituitary prolactinomas. Hence, chronic hCG hyperstimulation of the ovary leads to lactotrope proliferation and hyperprolactinemia, followed by formation of lactotrope adenomas. The ultrastructure of the anterior pituitary cells in the wt mice was similar to that in earlier publications (35, 36), and that of the tumors confirmed their lactotrope origin. Like the hCGß+ TG mice, those overexpressing transforming growth factor-{alpha} (37), bLH (12), and GHRH (38) as well as mice deficient of dopamine D2 receptors (39) develop pituitary hyperplasia followed by multifocal adenomas. An integrated theory of pituitary tumorigenesis proposes that adenomas develop from transformed cells that are dependent on hormonal and/or growth factor stimulation for tumor progression (40). Most pituitary adenomas arise as monoclonal neoplasms, indicating that one or more somatic mutations underlie the onset of tumorigenesis. However, it is likely that a proliferating stimulus is also needed to create a permissive environment potentiating cell mutations and subsequent clonal expansion of the initially mutated cell.

The physiologic importance of estrogens in regulating the anterior pituitary function, including lactotrope proliferation and PRL gene expression, has been recognized (31). Treatment with estrogens induces development of PRL-producing pituitary tumors in different inbred rat strains sensitive to estrogens (41, 42). In humans, pregnancy induces a coordinated increase in lactotrope proliferation and PRL production, an effect attributed to estrogens (43, 44), and few lactotrope adenomas may grow during gestation (45). In view of the previous observations, the fact that the hCGß+ mice were exposed to elevated estrogens at an early age would partially explain the occurrence of pituitary lactotrope adenomas in adulthood. After the initial process of transformation, normal estrogen levels in concert with local growth factors would appear sufficient to maintain lactotrope proliferation. If elevated estrogen production is responsible for the pituitary tumors, it is curious that a short-lived prepubertal estrogen peak brings about this effect. However, the persistence of high androgen levels may well be the source of locally produced estrogens, because there is aromatase activity in the brain and pituitary (46, 47). The involvement of progesterone or testosterone in lactotrope proliferation is not clearly understood (31), and further investigations are needed to clarify the pathogenesis of the pituitary adenomas in this model.

A remarkable feature of the hCGß+ model is the development of the mammary gland phenotype, culminating in mammary tumor formation in older ages. Several hormones including estrogens, progesterone, PRL, glucocorticoids, and a number of growth factors participate in the normal development and carcinogenesis of the mammary gland. Recently TG and knockout models from various genes have proven useful for elucidating the contribution of different hormones to the regulation of mammary gland growth and differentiation and identifying the signaling pathways involved (48, 49, 50, 51, 52, 53). The present results demonstrate that the abnormal lobuloalveolar development of the mammary gland in hCGß+ female mice is dependent on ovarian hyperfunction. Therefore, indirectly orchestrated by the high levels of hCG, the increased estrogens, progesterone, and PRL stimulate the proliferation and differentiation of the mammary gland and generate the hCGß+ mouse phenotype resembling late pregnancy. Metastatic mammary gland adenocarcinomas developed in the female hCGß+ mice with more than 90% penetrance by the age of 10–12 months.

Because sex hormones are associated with increased breast cancer risk (7), the increased proliferation induced by estradiol at early age could result in accumulation of genetic damage resulting in neoplasia in later life. Nevertheless, numerous reports have suggested a role for progesterone in mammary gland carcinogenesis (51, 54), as a priming factor for the action of growth factors on breast cancer cells (55). The role of PRL in the initiation and progression of rodent mammary carcinomas is also well established. Interestingly, TG mice overexpressing rat PRL spontaneously developed nonmetastatic mammary tumors at 11–15 months of age (56). Hyperprolactinemia induced by pituitary grafts has also been shown to induce mammary tumors (57) and enhance chemically induced tumorigenesis (58). Accordingly, cross-talk between the steroid system and the PRL pathway has been recently proposed to synergize in responses of the mammary epithelium (55). In humans, the role of PRL in the development of breast cancer remains controversial, but recent data indicate that higher plasma levels of PRL are associated with increased cancer risk in postmenopausal women (59). The analysis of the mammary phenotype emerging from chronic exposure to elevated levels of hCG is complicated by the fact that many of the hormones involved in mammary gland development and carcinogenesis may display abnormal secretory features already in early life. A way to dissect out the specific contribution of the different hormones will be to cross the hCGß+ mice individually with knockout mice for the estrogen, progesterone, and PRL receptors.

In summary, we show here that ovarian hyperstimulation induced by chronic and elevated secretion of hCG produces hyperprolactinemia and lactotrope adenomas in older age. Development of the pituitary tumors is associated with appearance of metastatic adenocarcinomas in the mammary gland. We also show that chronically elevated LH/hCG levels alone do not generate these phenotypes. This information is relevant to humans, indicating that the elevated gonadotropin levels in postmenopausal women are unlikely to have major effects in the absence of functional ovaries. The situation is totally different if the elevated gonadotropins stimulate ovarian responses. This TG model provides the opportunity to study the sequence of hormonal effects in a multistep fashion, not only in the ovary and pituitary gland, but also in breast cancer progression from the initial tumor formation to the final metastatic stage. This work paves the way for future studies on the genetic pathways involved in hormone-dependent tumorigenesis of the pituitary and mammary glands.


   Acknowledgments
 
The skillful technical assistance of N. Messner, J. Vesa, and T. Laiho is gratefully acknowledged.


   Footnotes
 
This work was supported by grants from the Sigrid Jusélius Foundation, Finnish Cancer Societies, Academy of Finland, and Center for International Mobility.

Abbreviations: ACTH, Adrenocorticotropin; hCG, human chorionic gonadotropin; IFMA, immunofluorometric assay; PRL, prolactin; TG, transgenic; UbiC-hCGß, ubiquitin C/hCGß fragment; wt, wild-type.

Received May 7, 2002.

Accepted for publication June 26, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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