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BOR - Papers in Press, published online ahead of print October 6, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.029769
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BIOLOGY OF REPRODUCTION 72, 423–435 (2005)
DOI: 10.1095/biolreprod.104.029769
© 2005 by the Society for the Study of Reproduction, Inc.

Effects of Neonatal Exposure to Diethylstilbestrol, Tamoxifen, and Toremifene on the BALB/c Mouse Mammary Gland1

Russell C. Hovey2, Mikiko Asai-Sato3, Anni Warri4, Barbara Terry-Koroma5, Nira Colyn, Erika Ginsburg, and Barbara K. Vonderhaar

Molecular and Cellular Endocrinology Section, Mammary Biology and Tumorigenesis Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-1402


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we compared the long-term effects of neonatal exposure to diethylstilbestrol (DES, 0.0125–50 µg), tamoxifen (TAM, 0.0125–50 µg), and toremifene (TOR, 53 µg) on mammary gland development and differentiation. Allometric growth of the mammary ducts was stimulated by neonatal DES exposure (12.5 µg) and impaired by exposure to TAM (25 µg). Neonatal treatment with high doses of DES resulted in mammary ducts that displayed extensive dilatation and precocious lactogenesis in postpubertal, nulliparous females. Initiation of this precocious differentiation coincided with the absence of corpora lutea, increased levels of serum prolactin (PRL), and the induction of Prl mRNA expression within the mammary glands. Neonatal exposure to 1.25 µg TAM increased alveolar development in postpubertal, nulliparous females similar to that recorded in females treated with low doses of DES. Lower doses of TAM did not affect alveolar development, whereas branching morphogenesis and alveolar development were impaired by higher doses. Increased alveolar development in females exposed to 1.25 µg TAM was associated with elevated serum progesterone (P) and increased alveolar development in response to exogenous P. Taken together, our findings demonstrate that neonatal exposure to both DES and TAM exerts long-lasting effects on the proliferation and differentiation of the mammary glands in female BALB/c, primarily as the result of endocrine disruption

estradiol, lactation, mammary glands, prolactin, puberty


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Growth, morphogenesis, and differentiation of the mammary gland during postnatal development is regulated by a fine balance of various hormones [1]. Ovarian steroid hormones play a pivotal role in these processes and are a primary influence on the progression to and development of mammary neoplasia [2].

The effect of estrogens (E) on the mammary gland is exemplified by the long-term effects of exposure to E in utero or during the neonatal period. A collection of studies, including the work of Bern and colleagues [3–5], has demonstrated that exposure of rodents to steroidal agents within the first 5 days of life predisposes the reproductive tract and mammary glands to a variety of developmental abnormalities. This window represents a period for investigating the consequences of in utero exposure given that the mammary gland anlage [6] of the neonatal mouse resembles that of the human fetus.

The use of diethylstilbestrol (DES) as an antiabortion therapy resulted in transplacental exposure of numerous individuals to this potent synthetic E analogue in utero [7]. These individuals are predisposed to a wide array of reproductive abnormalities, in particular, an increased incidence of vaginal clear-cell adenocarcinoma. The rodent model for neonatal sex steroid exposure has been widely used to assess the effects of DES exposure on the reproductive tract [8], where tumorigenic susceptibility of rodents is increased following DES exposure both in utero and postnatally [9, 10]. Only in the present day are women who were exposed to DES in utero reaching an age of increased breast cancer risk. Although little evidence exists concerning the epidemiology of breast cancer risk in these individuals [11], a recent epidemiological analysis suggests elevated risk in DES-exposed women older than 40 yr [12].

Tamoxifen (TAM) and its analogue, toremifene (TOR), are selective estrogen receptor (ER) modulators with antiestrogenic activity in a variety of tissues. TAM is currently the most commonly used first-line therapy for the treatment of hormone-dependent breast cancer [13–15] and has substantial potential as a prophylactic treatment for women with an elevated risk of developing breast cancer [16]. TAM reduces the proliferation of normal and neoplastic rodent and human breast cells by several mechanisms that lead to apoptosis [17, 18]. Despite its antiestrogenic effects, TAM also has estrogenic activity, particularly in the endometrium [19, 20]. Certain lines of evidence also suggest that TAM may exert an estrogen effect on breast cancer cells [21, 22] and that the development of TAM resistance in breast tumors may reflect increased sensitivity of cells to these agonistic actions [23].

The chronic adverse effects of DES exposure in utero suggest the potential for estrogenic effects of TAM on the fetus in women during the early stages of pregnancy if it is concurrently administered as a prophylactic treatment for breast cancer. In this study, we compared the effects of exposing the mammary glands of neonatal female mice with the estrogenic effects of DES and the antagonist/agonists TAM and TOR. We demonstrate that neonatal exposure to DES, in addition to inducing mammary abnormalities, promotes the long-term functional differentiation of the mammary gland in nulliparous female mice. By contrast, neonatal exposure to TAM can impart dose-dependant estrogenic or antiestrogenic effects on the mammary glands. Such findings bear consideration for the potential risk to the fetus of pregnant women who may be exposed to prophylactically administered TAM.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Treatments

BALB/c mice were maintained in 12L:12D conditions at 23–25°C with ad libitum access to laboratory chow (Purina 5001 lab chow) and tap water. While soy compounds in the lab chow diet used have been implicated as being estrogenic [24, 25], any such exposure was received by all animals in this study and still allowed for the observation of notable treatment differences beyond this background. Newborn pups were given a single s.c. injection of either 0.0125, 0.125, 1.25, 12.5, 25, or 50 µg DES (Sigma, St. Louis, MO); TAM (Sigma); or 53 µg TOR (equimolar to 50 µg TAM; a gift from Orion Corp., Turku, Finland) in 25 µl of sesame oil (SES) or SES alone, within 36 h of birth. Similar alterations in mammary gland function have been previously recorded when DES was administered either in utero or immediately after birth [26]. Animals were weaned at 21–23 days of age and the female offspring retained for subsequent analyses. At least two pups from each dam were analyzed in all treatment groups. Animal experiments were conducted in accordance with National Research Council guidelines.

Mammary Gland Whole Mounts and Morphology

The age-dependent appearance of morphological abnormalities within the mammary glands was examined in mice (n ≥ 6 from each treatment group) killed between 3 and 28 wk of age. Mammary gland morphology was assessed from whole-mount preparations. Either the #4 abdominal or the combined #2 and #3 thoracic mammary glands were removed from the subcutis, spread onto glass slides, and fixed overnight in Carnoy fixative. The two thoracic glands were then separated from the pectoralis muscles under a dissecting microscope and stained with alum carmine [27]. Mammary glands were destained in water, dehydrated to xylene, and mounted with Permount (Fisher, Pittsburgh, PA). Whole mounts were assessed and scored blind for morphological development by two experienced independent investigators. Ductal elongation within the mammary glands of 33-day-old female mice exposed to DES or TAM was measured as the distance between the nipple and the extremity of the ductal tree in #4 mammary glands using a calibrated eye piece on a dissecting microscope. Abnormal mammary glands were classified as having at least 50% of the mammary gland occupied by ducts with a diameter greater than that in any control (or normal) female mouse. The extent of ductal dilatation was assessed using a subjective scoring scale between 1 and 5, where 1 = no ductal abnormalities and 5 = extensive dilatation of the mammary ducts accompanied by alveolar hypertrophy. Mammary glands were also scored for alveolar development using a subjective scale of 1– 8. The scoring system used was: 1 = mature ducts with few side branches and no alveolar buds, 2 = ducts with side branches and no alveolar buds, 3 = ducts with few alveolar buds, 4 = ducts with end buds and few alveoli, 5 = ducts with limited alveolar development, 6 = ducts with moderate alveolar development, 7 = ducts with extensive alveolar development, and 8 = ducts with alveolar development equivalent to that in glands from a midpregnant female mouse.

Ovariectomy and Hormone Priming

A subset of mice were bilaterally ovariectomized at 35–40 days of age and subsequently killed at 12 wk of age. In experiments where hormone priming was performed, ovariectomized animals were administered a s.c. cholesterol-based pellet prepared according to the method described by us previously [28] containing either cholesterol only, 17ß-estradiol (E:cholesterol = 1:200), P (P:cholesterol = 1:1), or E and P (E:P:cholesterol = 1:1000:2002) 1 wk after surgery. Hormone-primed mice were killed at 8.5 wk of age following 2 wk of hormone treatment.

Mammary and Reproductive Tract Histology

Thoracic mammary glands and reproductive tracts for histological analysis were fixed in 4% paraformaldehyde in PBS overnight at 4°C, dehydrated, and processed into paraffin. Tissue sections (4 µm) were stained with hematoxylin-eosin. Samples from at least n = 4 animals per treatment group were examined.

In Vivo Tissue Recombination Experiments

Three experiments were conducted to determine the contribution of epithelial and stromal fractions to the phenotypes observed following neonatal exposure to DES. In the first experiment, female BALB/c mice were treated with DES within the first 36 h after birth. Between 22 and 24 days of age, the inguinal mammary fat pads of these mice were cleared of endogenous epithelium according to the method of Faulkin and DeOme [29]. At this time, a 1-mm3 explant of mammary tissue from an 8-wk-old untreated BALB/c female was inserted directly into the residual cleared fat pad. The opposite tissue recombination experiment was similarly performed whereby an explant of epithelial tissue from a DES-exposed female mouse (8 wk of age) was inserted into the cleared fat pad of an untreated mouse. Each transplant group contained six mice that were killed 10–12 wk after transplantation.

In a second experiment, the contribution of systemic factors was determined by suturing glands from 28-wk-old neonatally treated mice onto the peritoneum of untreated 8-wk-old female mice using 5-0 chromic gut sutures (Johnson and Johnson, Somerville, NJ). Mice were allowed to recover from surgery and killed 10–12 wk later to enable the mammary glands to be prepared as whole mounts for morphologic examination. Each transplant group contained six mice.

A third tissue recombination experiment tested whether morphological abnormalities within the mammary gland were influenced by local stromal factors. At 3 wk of age, the epithelium-free mammary fat pad of one #4 gland was surgically removed from DES-exposed mice. The excised epithelium-free fat pad was then sutured onto the peritoneum of an untreated 8-wk-old female BALB/c mouse using 5-0 chromic gut sutures. A small pocket was then made in the transplanted mammary fat pad, into which a 1-mm3 explant of mammary tissue from an untreated 8-wk-old female mouse was inserted. Mice were allowed to recover, and all mice (donors and transplant recipients) were killed at 10–12 wk of age to allow analysis of mammary glands as whole mounts.

Whole-Organ Culture

Whole-organ culture was performed to determine whether neonatal exposure to DES or TAM impacted the response by the mammary epithelium to defined and direct mammogenic stimulation. Successful whole-mammary-gland culture requires that intact mice first be primed with E+P [28]. Female mice at 24 days of age (at least n = 6 per group) were primed with either a pellet containing E+P (2002:1001:1 cholesterol:P:E) or remained unprimed. Mice were killed 9 days later and the #4 mammary glands placed in whole-organ culture according to previously described methods [28]. Mammary glands were cultured in Waymouth serum- and E-free medium supplemented with insulin (5 µg/ml), aldosterone (100 ng/ ml), hydrocortisone (100 ng/ml), ovine prolactin (PRL; 1 µg/ml), and epidermal growth factor (60 ng/ml). The presence of phenol red in these cultures is not estrogenic in mammary gland whole-organ culture (K. Plaut, personal communication). Glands were incubated in a 37°C humidified atmosphere with 50% O2 and 5% CO2 and the media changed every 2 days. After 6 days in culture, the glands were prepared as whole mounts for morphological assessment or were snap frozen in liquid nitrogen for RNA extraction.

Serum Hormone Measurements

Blood collected by cardiac puncture from mice treated as neonates with DES or TAM was centrifuged to collect serum. Blood was obtained from cycling animals (n = 5–6; 8–10 wk old) killed by cervical dislocation at diestrus (as determined by vaginal appearance and lavage) [30, 31] between 1000 h and 1500 h. Following ether extraction of serum, E and P levels were assayed by RIA (Diagnostic Products Corporation, Los Angeles, CA; E double antibody RIA: sensitivity, 2.5 pg/ml, interassay coefficient of variation [CV] 11.3%, intraassay CV 15%; P antibody-coated tube RIA: sensitivity, 0.2 ng/ml, interassay CV 7%, intraassay CV 5.3%). The RIA for E does not cross-react with TAM and has not been tested for cross-reactivity with DES or TOR. Serum prolactin (PRL) levels were determined on pooled serum samples using a previously described bioassay that utilizes the PRL responsive Nb2 rat lymphoma cell line [32]. Cells were maintained in Fischer leukemia medium supplemented with 2-ß-mercaptoethanol (10–4 M), 10% horse serum, 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 µg/ml). For each assay, 2 x 105 cells in 2 ml were plated into six-well culture dishes in Fischer leukemia medium supplemented with 2-ß-mercaptoethanol (10–4 M), 10% horse serum, penicillin (100 U/ml), and streptomycin (100 µg/ml). Varying levels of human PRL (hPRL; National Hormone and Pituitary Program, NIDDK) or mouse sera were added as indicated. Growth of Nb2 cells was linear in the range of 10–1000 pg/ml. After 3 days in culture, cells were collected by centrifugation and counted using a Coulter counter. Each assay was performed at least twice using duplicate cultures.

In Situ Hybridization

The ß-casein riboprobes were generated from a mouse ß-casein cDNA (corresponding to nucleotides 569–811; GenBank accession NM_009972) cloned into PCRscript (Stratagene, LaJolla, CA). Digoxigenin (DIG)-labeled sense and antisense cRNAs were transcribed from the T7 and T3 promoters, respectively, using the Ambion in vitro transcription kit, T7 or T3 polymerase (Stratagene), and DIG-UTP (Boehringer Mannheim, Indianapolis, IN). Tissues for in situ hybridization were fixed in 4% paraformaldehyde at 4°C for 16 h, embedded in paraffin, sectioned at 4 µm, and placed on silanized slides. Tissue sections were pretreated with 0.2 M HCl, digested with proteinase K (10 µg/ml; Life Technologies, Gaithersburg, MD) for 30 min at 37°C, postfixed in 4% paraformaldehyde, and acetylated. Prehybridized sections were then incubated with cRNA probes (1 µg/ml) in hybridization cocktail (4xSSC, 50% formamide, 1x Denhardt, 10% [w/v] dextran sulfate, 0.4% SDS) for 16 h at 42°C. Following hybridization, sections were washed twice in 2x SSC and twice in 0.2x SSC at 62°C. Hybridized probe was detected using the DIG-Genius detection kit (Boehringer Mannheim) with NBT/BCIP as the chromogen.

Immunohistochemistry

Paraffin sections (4 µm) were dewaxed, rehydrated, then blocked with 10% normal rabbit serum and 0.1% BSA in PBS for 30 min at room temperature. Sections were incubated with a sheep anti-mouse casein antibody (20 µg/ml; 33') in 2% rabbit serum overnight at 4°C. After 2 x 5-min washes in PBS-0.1% Tween (plus 0.1% BSA and 1% rabbit serum), horseradish peroxidase-conjugated secondary antibody (rabbit anti-sheep; Jackson ImmunoResearch, 313036003) was applied (diluted 1:500 in PBS-Tween plus 0.1% BSA and 1% rabbit serum) for 30 min. Sections were then washed 2 x 5 min in the same buffer and then for 5 min in PBS-Tween. Immunoreactivity was detected with diaminobenzidine and the sections counterstained with Mayer hematoxylin.

Western Blotting

Total protein was recovered from tissue homogenized in TRIzol according to the manufacturer's instructions. Protein concentration was determined by the Bradford method using the Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA). Total protein (80 µg) was pooled from n = 5 animals in each treatment group, separated by 4–20% SDS-PAGE gradient gels, and transferred to nitrocellulose. After blocking in Tris-buffered saline/0.01% tween (TBST) with 5% milk at room temperature for 1 h, the blot was incubated with a sheep anti-mouse milk casein antibody (1:500) overnight at 4°C. The blot was washed three times in TBST, then incubated for 1 h with the rabbit anti-sheep horseradish peroxidase-conjugated secondary antibody (1:4000) described under Immunohistochemistry. Immunoreactive proteins were visualized using an ECL kit from Amersham Pharmacia (Arlington Heights, IL).

RNA Extraction, Northern Blotting, and Reverse Transcription-Polymerase Chain Reaction

The #4 inguinal mammary glands from mice killed at either 6 or 12 wk postexposure were snap frozen in liquid nitrogen before extraction of total RNA using TRIzol (Life Technologies). For Northern analysis, total RNA (10 µg) was prepared from pooled tissue (n ≥ 5 animals per treatment), separated by electrophoresis on a 1% agarose/0.07% formaldehyde gel, and then transferred to GeneScreen (NEN Research Products, Boston, MA). The ß-casein, {alpha}-lactalbumin, or whey acidic protein (Wap) cDNA probes were labeled with {alpha}32P-dCTP using Klenow and random primers (Life Technologies). The ß-casein cDNA was generated by reverse transcription-polymerase chain reaction (RT-PCR) using primers that gave a product corresponding to nucleotides 569–811 base pairs (bp) of GenBank accession NM_009972 (Table 1). The murine {alpha}-lactalbumin and Wap cDNAs were kindly provided by Dr. Lothar Hennighausen (NIDDK). Blots were hybridized in Rapid-Hyb buffer (Amersham Pharmacia) at 42°C, then washed at 60°C with increasing stringency from 2x SSC/0.5% SDS to 0.1x SSC/0.5% SDS.


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  		TABLE 1. Forward and reverse primer sequences, amplicon size, and annealing temperatures for RT-PCR

For RT-PCR analyses, total RNA was treated with DNase (1U/µg RNA; Boehringer Mannheim) before reverse transcription. First-strand cDNA was generated from 1 µg total RNA using MMLV-RT (Life Technologies) primed with oligo-dT (Amersham Pharmacia). Amplification by PCR was performed using 2.5 µl of reverse transcription products and Taq polymerase-based PCR Master Mix (Roche Molecular Biochemicals, Indianapolis, IN) with each primer at a concentration of 0.2 µM. PCR conditions for each primer pair were optimized for the set of samples analyzed to ensure amplification was in the linear phase for each primer set. The primer sequences, annealing temperatures used, and the expected product sizes are given in Table 1. Gene expression results were referenced against the respective level of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA amplified using primers from Clontech (Palo Alto, CA).

Statistical Tests

Statistical analyses were performed using one- or two-way analysis of variance (StatView, SAS). Multiple means comparisons were made by Fisher least significant difference test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Neonatal Exposure to DES or TAM Alters Peripubertal Ductal Development

We compared ductal development during the peripubertal period in female mice neonatally exposed to SES, DES, and TAM. Given that ductal elongation in BALB/c mice is isometric until 31 days of age [34], whereafter it becomes allometric, ductal length was measured at 21 and 33 days of age. At 21 days of age, the mammary ductal tree in all treatment groups had developed similarly as a simply branched rudiment (Fig. 1). By contrast, ductal development markedly differed between the groups at 33 days of age. Ductal outgrowth determined by linear measurement from the nipple was increased by 30% in females exposed to 12.5 µg DES relative to SES-treated females (Fig. 1; P < 0.05). Exposure to 25 µg TAM resulted in a 35% reduction in ductal outgrowth relative to SES-exposed mice (Fig. 1; P < 0.05), whereas treatment with 1.25 µg TAM was without effect (P > 0.05).



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  		FIG. 1. Ductal development within the mammary glands of prepubertal mice neonatally treated with sesame oil excipient (SES), diethylstilbestrol (DES, 12.5 µg), or tamoxifen (TAM, 1.25 or 25 µg). A) Representative whole mounts of mammary glands from neonatally treated mice. Bar = 5 mm. B) Length of ductal outgrowth (mean ± SEM, n = 3–5 animals/group) in the mammary glands of the various treatment groups at 21 and 33 days of age. Ductal length was measured as the distance between the nipple and the furthermost point of ductal outgrowth using a calibrated microscope eyepiece. a,b,cMeans with different superscripts are significantly different, P < 0.05

Neonatal Exposure to DES and TAM Results in Abnormal Mammary Gland Development Postpuberty

While neonatal exposure to DES and TAM impacted ductal development at 33 days of age, neither DES- nor TAM-treated females showed any alterations in mammary gland morphology (Fig. 2, A and B) or histology (data not shown) at 6 wk of age. By contrast, pronounced abnormalities were evident at 12 wk of age. Following DES exposure, a significant proportion of females displayed pronounced dilatation of mammary ducts that was frequently accompanied by the presence of cystic alveoli arising from the mammary ducts (Fig. 2E). These abnormalities were histologically characterized by the presence of thin-walled, dilated ducts filled with proteinaceous secretion and lined by cells filled with lipid droplets (Fig. 2, D and G). Compared with mammary glands from mice exposed to DES, those from TAM-treated females did not display the same morphological abnormalities. However, mammary glands from females treated with 12.5–50 µg TAM presented with ducts that were regressed and atrophic both at the morphological and histological level (Fig. 2H).



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  		FIG. 2. Morphological and histological development of #3 thoracic mammary glands from nulliparous female mice at 6 (A and B) or 12 (C–E) wk of age that received a single injection of sesame oil (SES) excipient (A and C) or 25 µg diethylstilbestrol (DES; B, D, and E) within 36 hr of birth. Arrows in D and E indicate dilated ducts and cystic alveolar structures. Bar in A–E = 3 mm. F–H) Typical histological appearance of mammary glands from females at 12 wk of age that were neonatally treated with either SES, DES (25 µg), or tamoxifen (TAM; 25 µg), respectively. Arrows in G indicate lipid droplets within epithelial cells of alveolar structures. Arrows in H indicate regressed epithelial cells in condensed ducts of TAM-treated females. Bar for F, G, and H = 100 µm

Effect of DES, TAM, and TOR on Lobulo-Alveolar Development

Alveolar development in the mammary glands of 12-wk-old females treated as neonates with various levels of DES, TAM, or the TAM analogue, TOR, was quantified from scored whole mounts. Exposure to low doses (≤0.125 µg) of DES increased alveolar development relative to SES-exposed controls (Fig. 3A). By contrast, alveolar development was reduced (P < 0.05) following exposure to high doses of DES (≥12.5 µg; Fig. 3A). This outcome was inversely correlated with the incidence of females presenting with dilated mammary ducts and precocious lactogenesis (Fig. 3B). Alveolar development in 12-wk-old females was unaltered by neonatal exposure to low doses (0.0125 µg and 0.125 µg) of TAM (P > 0.05; Fig. 3C), whereas it was significantly increased in females exposed to 1.25 µg TAM (P < 0.05). Conversely, neonatal exposure to higher doses of TAM (≥12.5 µg) resulted in reduced alveolar development (P < 0.05). Similar to the effect of 50 µg TAM, treatment with an equimolar dose of TOR (53 µg) suppressed alveolar development (P < 0.05).



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  		FIG. 3. Alveolar development and ductal dilatation in mammary glands of mature nulliparous mice exposed to various levels of diethylstilbestrol (DES) or tamoxifen (TAM) within the first 36 hr of birth. A) Whole mounts of thoracic mammary glands from DES-exposed females killed at 12 wk of age were scored for alveolar development (AD) as described in Materials and Methods. Data are means ± SEM (n = 10–20/treatment). *P < 0.05 vs. SES-exposed group. B) Mammary gland whole mounts from DES-treated animals above were assessed for the presence of ductal dilatation and other abnormal ductal structures by two independent investigators, and the number of positive cases expressed as a percentage of the total number examined. C) Mammary gland whole mounts from mice administered various doses of TAM within the first 36 hr of birth were scored for AD by two independent investigators using the scale described in Materials and Methods. Data are means ± SEM (n = 10–20/treatment). *P < 0.05 vs. SES-exposed group. D–J) Representative whole mounts from mature nulliparous females following neonatal exposures. D) Mammary gland from a SES-exposed mouse showing normal ductal structures. E–G) Whole mounts of mammary glands from mice neonatally exposed to (E) 0.0125 µg, (F) 1.25 µg, or (G) 25 µg DES. The arrows in G indicate dilated ducts observed following exposure to a high level of DES. H–J) Whole mounts of mammary glands from mice exposed to (H) 0.0125 µg, (I) 1.25 µg, or (J) 25 µg TAM. Bar = 2 mm

Neonatal Exposure to High Doses of DES Induces Precocious Lactogenesis

We further investigated whether the mammary ducts of nulliparous BALB/c mice exposed to DES (12.5 µg/pup) as neonates were engorged with milk. Total RNA extracted from the mammary glands of SES-, DES-, and TAM-treated mice was analyzed for the expression of the milk protein genes {alpha}-lactalbumin, ß-casein, and Wap by Northern analysis (Fig. 4A). No expression of these mRNAs was detected in any treatment group at 6 wk (data not shown). By comparison, mRNA for {alpha}-lactalbumin and ß-casein was detected in mammary glands from DES-treated but not TAM-treated females at 12 wk of age. No concomitant expression of Wap mRNA could be detected in DES-treated females due either to sensitivity limitations or differential regulation of this gene. Northern analysis of mammary tissue from 6- and 12-wk-old nulliparous females exposed as neonates to TOR also failed to reveal the expression of ß-casein mRNA (data not shown).



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  		FIG. 4. Analysis of milk protein expression in mammary glands of mice exposed to diethylstilbestrol (DES) or tamoxifen (TAM) as neonates. A) Northern analysis of milk protein gene expression in mammary glands of 12-wk-old nulliparous females treated with sesame oil (SES) or various doses of DES or TAM. Electrophoresed total RNA (10 µg) was hybridized with 32P-labeled cDNAs specific for ß-casein, whey acidic protein (Wap), and {alpha}-lactalbumin. Equivalence of loading is indicated by ethidium bromide-stained ribosomal RNA. Lactating mouse mammary gland (Lact). B) Western analysis of caseins within protein extracts from the mammary glands of 12-wk-old nulliparous female mice treated with SES or various doses of DES or TAM. Total mouse milk and proteins extracted from mammary tissue of normal lactating female mice (lact MG) served as positive controls. Identified caseins and the migration of molecular size markers are indicated. C) Localization of ß-casein synthesis in the mammary glands of 12-wk-old nulliparous female mice exposed to DES. Top panels show immunohistochemical localization of caseins in the mammary ducts (left panel, plus antibody; right panel, negative control in which the primary antibody was omitted). No significant immunoreactivity was recorded in the mammary glands of sesame oil-treated control females (not shown). Mayer hematoxylin counterstain. Bottom panels depict localization of ß-casein mRNA by in situ hybridization within the ductal epithelium using a digoxigenin-labeled antisense probe. Left panel is antisense probe, right panel is negative control in the presence of sense probe. Nuclear fast red counterstain. Bar = 50 µm

Western analysis of mammary tissue extracts using an anti-mouse casein antibody confirmed that several caseins, including ß- and {gamma}-casein, were present in the mammary glands of DES-treated but not TAM-treated females at 12 wk of age (Fig. 4B). Similar immunoreactive proteins were present in mammary tissue from lactating mice and in mouse milk. No milk proteins were detected in mammary tissue from mice in any of the groups at 6 wk of age (data not shown). These data were corroborated by immunohistochemical results showing that the mammary luminal epithelial cells and the proteinaceous contents of the mammary ducts in 12-wk-old DES-treated female mice were immunoreactive for milk casein (Fig. 4C). In situ hybridization indicated that the cells of both ducts and alveolar structures were capable of substantial levels of milk protein synthesis as indicated by the presence of ß-casein mRNA (Fig. 4C).

Age-Dependent Onset of DES-Induced Precocious Lactogenesis

The observation that mammary glands from DES-treated BALB/c female mice displayed marked morphological abnormalities at 12 wk of age, yet were morphologically normal at 6 wk, led us to investigate the age dependence of this effect in the mammary glands and reproductive tract. Females exposed to DES (12.5 µg) first displayed ductal dilatation and the formation of cystic alveoli within the mammary glands at 7 wk of age (Fig. 5A). The incidence of these abnormalities increased steadily to 53% by 12 wk of age, and was 100% in mature, nulliparous females at 28 wk. This age-dependent increase in mammary gland abnormalities corresponded to a similar profile for the expression of ß-casein mRNA (Fig. 5B). The onset of mammary ductal dilatation and milk protein gene expression followed the rapid disappearance of corpora lutea in 100% of females at 5 wk of age (data not shown). This disappearance also corresponded to the appearance of uterine squamous metaplasia and persistent keratinization in 100% of DES-exposed females (data not shown). Neonatal exposure to various doses of TAM did not influence the presence of corpora lutea despite the fact that TAM exposure did alter mammary gland morphology. Furthermore, there was no difference in the number of ovarian follicles in the various neonatal treatment groups (P > 0.05, data not shown). However, there was reproductive failure in females treated with 25 µg TAM given that they failed to conceive despite multiple prolonged exposures to fertile male mice.



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  		FIG. 5. Age-dependent onset of mammary gland abnormalities in nulliparous female mice neonatally exposed to 25 µg diethylstilbestrol (DES) within the first 36 hr of birth. A) Incidence of ductal dilatation within the mammary glands of nulliparous, DES-exposed females at various ages. B) Age-dependent expression of ß-casein mRNA in mammary glands of DES-exposed females as determined by Northern analysis using a 32P-labeled mouse ß-casein cDNA. Responders and nonresponders (nonresp.) were classified as animals that did or did not display ductal dilatation within their mammary glands, respectively, at the indicated ages (weeks). 6DES, Mammary glands from DES-exposed females at 6 wk of age that had not yet demonstrated any response

Effect of Neonatal DES or TAM Exposure on Steroid Hormone Levels

Given the effects of neonatal DES or TAM on subsequent mammary gland development, levels of E and P were measured in the serum of mice from various treatment groups killed between 8 and 10 wk of age (Fig. 6). While females treated with 12.5 µg DES, 1.25 µg TAM, or 50 µg TAM tended to have lower serum E, there was no significant difference between treatment groups. Measurement of serum P levels revealed effects of the different treatments. Whereas exposure to 1.25 µg, 25 µg DES or 50 µg TAM had no effect, females treated with 1.25 µg TAM had an elevated level of serum P (P < 0.05).



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  		FIG. 6. A) Serum levels of estrogen (E) and B) progesterone (P) following neonatal exposure to diethylstilbestrol (DES) or tamoxifen (TAM). Mice were treated with the indicated doses of DES or TAM within 36 hr of birth and killed between 8 and 10 wk of age. Data are means ± SEM, n = 5– 6. Serum E levels were not statistically different. a,bProgesterone means with different superscripts are significantly different, P < 0.05

Effect of Neonatal Exposure on Systemic and Local PRL

Given the postpubertal induction of milk protein gene expression within the mammary glands of DES-exposed females, the level of PRL in the serum of SES- and DES-treated females was measured. Compared with SES-treated females, PRL in the serum of DES-exposed females was elevated at 6 wk and even more so at 12 wk (Fig. 7A). We also investigated whether precocious lactogenesis in DES-exposed females was associated with the local synthesis of PRL within the mammary glands. As shown in Figure 7B, the mammary glands of females treated with 12.5, 25, or 50 µg DES or 12.5 µg TAM, expressed detectable Prl mRNA but not those from females treated with SES or 25 or 50 µg TAM.



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  		FIG. 7. Serum and mammary gland PRL levels in mice neonatally exposed to diethylstilbestrol (DES) or tamoxifen (TAM). A) Levels of PRL in pooled serum samples from female mice at 6 and 12 wk of age that had been treated as neonates with either sesame oil (SES) or various doses of DES. B) RT-PCR analysis of Prl gene expression in total RNA extracted from the mammary glands of 6- or 12-wk-old nulliparous females that had been treated with SES or various doses of TAM or DES within the first 36 hr of birth. RT-PCR products were resolved by agarose gel electrophoresis and stained with ethidium bromide. –VE, Negative control; LMG, lactating mammary gland

Effect of Neonatal Exposure on Hormone Responsiveness In Vivo and In Vitro

Given altered hormone profiles, precocious lactogenesis and altered alveolar development in the mammary glands of DES- and TAM-exposed females, we tested the response of mammary glands in these animals to the individual or combined effects of exogenous E or P. Ovariectomy of SES-, DES-, or TAM-treated females led to cessation of mammary growth, leaving a sparsely branched ductal tree lacking end buds (Fig. 8, A–D). After 2 wk of exposure to E, the extent of ductal dilatation reflected the neonatal treatment imposed (Fig. 8, E–H). SES-exposed females treated with E displayed increased end bud development without any aberrant ductal morphology (Fig. 8E). Mice exposed as neonates to 12.5 µg DES, then treated with E, demonstrated more ductal abnormalities, with a 20% higher level of dilatation compared with SES-exposed females (Fig. 8, E, F, and M). Animals treated with 1.25 µg TAM displayed variable responsiveness to E. Responsive animals demonstrated aberrant ductal dilatation similar to that observed in DES-exposed females (Fig. 8G). Despite this, the average extent of E-induced ductal dilatation was similar to that in SES-exposed females (Fig. 8M). By comparison, neonatal exposure to 25 µg TAM suppressed E-induced ductal dilatation by 26% (Fig. 8, H and M).



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  		FIG. 8. Effect of exogenous ovarian steroid hormones on mammary gland morphology in female mice neonatally exposed to sesame oil (A, E, and I), diethylstilbestrol (DES 12.5 µg: B, F, and J), or tamoxifen (TAM 1.25 µg: C, G, K; or 25 µg: D, H, or L) within 36 hr of birth. Mice were ovariectomized at 35–40 days of age, then 1 wk later received a s.c. pellet of cholesterol carrier (A–D), estrogen (E; E–H) or progesterone (P; I–L) for 2 wk before being killed. Mammary glands were prepared as described in Materials and Methods. Bar = 2 mm. M and N) Quantitative analysis of mammary gland development following the administration of exogenous ovarian steroids described above. M) The extent of ductal dilatation in response to E was scored on a scale of 1–5, ranging from 1 = no ductal dilatation to 5 = extensive ductal and alveolar dilatation. N) The degree of alveolar development (AD) in response to P was scored on a scale of 1–8, ranging from 1 = mature ducts with few side branches and no alveolar buds to 8 = ducts with AD equivalent to that in glands from a midpregnant female mouse. Data are means ± SEM. Means with different letters are significantly different, P < 0.05

Ducts within the mammary glands of ovariectomized, SES-exposed mice treated for 2 wk with P were thin with few branches (Fig. 8I), as indicated by a low alveolar development score (Fig. 8N). By contrast, glands from females neonatally exposed to DES (12.5 µg) and subsequently treated with P underwent more extensive ductal branching (Fig. 8, J and N; P < 0.05). Interestingly, females exposed to 1.25 µg TAM also demonstrated increased alveolar development following treatment with P (Fig. 8, K and N; P < 0.05). Mice exposed to 25 µg TAM and treated with P displayed a level of alveolar development that was not different from that in SES-exposed females (Fig. 8, L and N; P > 0.05).

Administration of E plus P to ovariectomized females stimulated extensive alveolar development but did not result in ductal dilatation. Assessment of alveolar development in the various groups following exposure to E plus P indicated no differences between the neonatal treatments (data not shown).

Beyond the clear systemic consequences of neonatal exposure, we also tested the direct responsiveness of the mammary glands from DES- and TAM-exposed females to lactogenic stimulation using a defined, whole-organ culture system (Fig. 9). Prior to culture, and consistent with our previous observations, the extent of ductal outgrowth in the mammary glands of mice exposed as neonates to increasing amounts of TAM was impaired, even after steroid priming (data not shown). Furthermore, only limited alveolar development was observed in all treatment groups after priming with E plus P, consistent with the fact that this regimen has minimal effect on alveolar development at this age in mice [28].



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  		FIG. 9. Effects of neonatal exposure to diethylstilbestrol (DES) or tamoxifen (TAM) on mammary development and differentiation in whole-organ culture. Female mice treated with sesame oil (SES), DES, or TAM within the first 36 hr of birth were primed for 9 days with a cholesterol-based pellet containing estrogen + progesterone, then cultured for 5 days in the presence of lactogenic hormones. A–D) Lobulo-alveolar development of mammary glands from mice neonatally treated with (A) SES, (B) 25 µg DES, (C) 12.5 µg TAM, or (D) 50 µg TAM after culture in lactogenic hormones for 5 days. Reduced ductal outgrowth is evident in TAM-exposed glands. Bar = 5mm. E) Expression of ß-casein mRNA in mammary tissue from SES-, DES-, or TAM-exposed females following culture in lactogenic hormones as analyzed by RT-PCR. Equivalence of mRNA levels is indicated by the abundance of RT-PCR-amplified Gapdh mRNA

When hormone-primed mammary glands from mice exposed to TAM or DES were cultured in the presence of mammogenic + lactogenic hormones, glands from all groups underwent similar lobulo-alveolar development in vitro (Fig. 9, A–D). Mammary glands from females exposed to the various neonatal treatments also expressed ß-casein mRNA in response to lactogenic stimulation in vitro. Glands from TAM-exposed mice expressed ß-casein mRNA at a level similar to that in SES-treated mammary glands, whereas neonatal exposure to DES enhanced the expression of ß-casein mRNA (Fig. 9E).

Tissue Recombination Experiments

Transplant and tissue recombination experiments were performed to elucidate the contribution of systemic and local elements to the phenotype induced by DES exposure. Results of these experiments are summarized in Figure 10. In the first experiment, when mammary epithelium from untreated 3-wk-old normal female mice was transplanted into the cleared mammary fat pad of DES-exposed mice, both the reconstituted mammary gland and the mammary gland of the host demonstrated ductal dilatation (Fig. 10, A and B). By contrast, when mammary epithelium from 3-wk-old DES-exposed mice was transplanted into the cleared mammary fat pad of untreated hosts, neither the reconstituted mammary gland nor the other mammary glands of the host demonstrated any ductal dilatation (not shown). As expected, dilated ducts were absent in age-matched 15-wk-old control females (not shown). The requirement for systemic factors in maintaining aberrant ductal dilatation within the mammary glands of DES-exposed females was established by transplanting mammary glands from 28-wk-old DES-exposed mice onto the peritoneum of normal female hosts. At the time of transplantation, the mammary glands of DES-exposed females contained dilated ducts as evidenced from whole mounts of the contralateral mammary gland (Fig. 10C). After transplantation to normal hosts, the dilated ducts and cystic alveoli regressed to a more normal diameter (Fig. 10D), indicating that systemic alterations within DES-exposed females primarily supported ductal dilatation.



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  		FIG. 10. A, B) Results from mammary tissue recombination experiments. A) Mammary gland from a female mouse treated with 12.5 µg diethylstilbestrol (DES) within the first 36 hr of birth. B) Outgrowth of mammary epithelium from a normal female transplanted to the epithelium-free mammary fat pad of a DES-exposed female host. Arrows in A and B indicate abnormal, dilated ducts. C–E) Results of whole mammary transplantation experiments. C) Mammary gland of a DES-exposed female that was the source of mammary tissue transplanted to a normal host (D). Arrows in C indicate cystic alveolar structures. Arrow in D indicates regressed cystic alveolar structures. Arrowhead indicates a regressed duct adjacent to the more primary ducts that have not yet undergone complete regression (white arrowhead). Mammary gland from the normal host is illustrated in (E). F) Recombination of normal mammary epithelium into the mammary fat pad from a DES-exposed host that was then transplanted to the peritoneum of a normal female mouse. G) Mammary gland of the normal female host showing comparable ductal branching to that observed in the recombined tissue in F. Bar for A and B = 2 mm; C–G = 1 mm

We also tested whether stromal factors regulated ductal dilatation by transplanting cleared mammary fat pads from DES-exposed mice that contained an implant of normal mammary epithelium onto the peritoneum of untreated mice (Fig. 10, F and G). Recombination of these tissues resulted in the normal outgrowth of mammary epithelium within the transplanted, DES-exposed mammary fat pad. Taken together, these data indicate that ductal dilatation resulting from neonatal DES exposure primarily results from an altered hormonal environment.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The objective of this study was to investigate the effects of neonatal exposure to estrogenic and antiestrogenic agents on the subsequent development and function of the mouse mammary gland. Our findings reveal that these agents exert chronic effects on mammary gland development and differentiation that may bear considerable implications for human exposure in utero.

Exposure of neonatal BALB/c females to microgram amounts of DES induced dramatic abnormalities in the mammary glands of a substantial proportion of females at 12 wk of age. Neonatal exposure to other sex steroids, including androgens [4] and progestins [35], also results in the long-term development of dilated ducts and/or precocious alveolar development. While similar morphological alterations following neonatal DES exposure have been reported, their occurrence has generally been described in aged females [26] or in strains of mice carrying mouse mammary tumor virus [4] that is hormone-activated, leading to the transcription of various proto-oncogenes [36, 37]. Here we have identified that the morphological anomalies induced by DES corresponded to terminal differentiation of the mammary epithelium. This aberrant morphology in mice was previously classified as adenosis and/or various forms of dysplasia [26, 38]. While temporal changes in milk protein expression occur within the mammary gland during the estrous cycle [6], these are minor and transient. However, precocious secretion of milk by the mammary epithelium has been observed in other situations. For example, the secretion of witch's milk can occur in neonatal humans, probably due to maternal hormonal influence [39]. Similarly, a mixture of hormones (insulin, PRL, and corticoids) can induce mammary glands from nulliparous female mice to undergo alveolar development and milk protein synthesis in vitro [28], while overexpression of human growth hormone in mice leads to precocious mammary gland development and milk protein synthesis [40]. One question that arises is whether dilatation and distortion of ducts and alveoli in DES-treated females results from the hypersecretion of milk constituents by epithelial cells leading to a subsequent increase in the intraluminal pressure.

Our present results indicate a major systemic component to this phenotype, given that the onset of mammary duct dilatation and milk protein gene expression was tightly coordinated with absence of corpora lutea from the ovaries in 100% of females beyond 5 wk. This proposal concurs with the results from our transplantation studies. Neonatal exposure to DES also markedly increased levels of serum PRL, consistent with the majority of findings made by others [41, 42]. A novel finding in these experiments was that mRNA encoding Prl was also specifically expressed in the mammary glands of DES-treated females at 12 wk of age. The level of Prl mRNA was proportional to the likely estrogenicity of the DES or TAM dose. Expression of Prl mRNA within the mammary glands of females exposed to the lowest dose of TAM (12.5 µg) is in agreement with our finding that neonatal exposure to a low dose of TAM imparted estrogenic effects on the mouse mammary gland, while high doses were antiestrogenic. The high doses of DES that led to elevated Prl mRNA expression in the mammary glands also induced precocious milk production and dilated ducts. Based on the results from our transplantation studies, it is likely that local Prl mRNA expression in the mammary gland was induced by an altered endocrine profile. While the contribution of local PRL synthesis to precocious lactogenesis and increased serum PRL is unclear, Prl mRNA is expressed in the mammary gland of rats, specifically during lactation [43], and has been implicated as an autocrine factor in breast cancer [44].

Within the mammary glands, the epithelium from DES-exposed females demonstrated increased responsiveness to lactogenic stimulation in organ culture. This finding is in accord with that of Warner et al. [45]. In contrast, epithelial cells isolated from DES-exposed female mice were less sensitive to the lactogenic effects of PRL in a collagen gel system [46]. This discrepancy between the response of isolated epithelial cells and organ culture experiments may reflect the absence of stromal cells of the mammary fat pad, a component of the mammary gland that serves an integral role in the hormonal responsiveness of mammary epithelium [47]. However, using various transplantation and tissue recombination approaches in the present study, we were unable to identify any stromal contribution beyond alterations in the systemic hormone profile.

Despite the obvious contribution of systemic factors to these abnormalities, the precise mechanisms dictating their appearance in individual animals remain uncertain, given that, at 12 wk of age, only approximately 60% of females displayed ductal and alveolar dilatation. Previous works have also documented this variable responsiveness by individual animals to in utero and neonatal hormone exposure [4, 26]. While the reason for this variation is unclear, its systemic basis suggests that differential sensitivity and plasticity exists within the ovarian-pituitary axis.

TAM presently represents the most efficacious prophylactic therapy for women at high risk of developing hormone-dependent breast cancer [13, 14]. While it primarily acts as an antiestrogen, TAM also exerts estrogenic effects on various tissues, including bone and the reproductive tract [13]. TOR, an analogue of TAM, also has substantial antiestrogenic properties without the negative effects of TAM on the liver and DNA stability [48]. Within this study, the long-term effects of neonatal exposure to TAM on the mammary gland were dose dependent. This dose dependency coincides with observations in vitro. Low levels of TAM can act as an E agonist to induce progesterone receptor (PR) expression [49] and cell proliferation [50, 51], whereas high concentrations of TAM have antagonistic effects on such parameters [52]. Particularly noteworthy is the finding that a relatively low (1.25 µg) dose of TAM exerted chronic E-like effects on the mammary gland, including increased alveolar development, enhanced P-responsiveness, and local gene expression changes similar to those induced by a low dose of DES. Induction of alveolar development by neonatal exposure to 1.25 µg TAM coincides with the findings of Halakivi-Clarke et al. [53], where in utero exposure to TAM increased susceptibility of the offspring to chemically induced mammary carcinogenesis. In another study, these same authors showed that neonatal exposure to 20 µg ICI 182 780 increased end bud and alveolar development [54]. Our present results indicate that part of the mammogenic effect of 1.25 µg TAM reflects a combined response to increased serum P and increased sensitivity of the mammary gland to P. This proposal is consistent with the demonstration that neonatal exposure to E increases P responsiveness in the mouse vagina and uterus [55, 56] by increasing the level of PR [8]. Indeed, TAM increases ER and PR levels in the mammary glands of macaques, possibly due to an agonistic effect of E [57]. It is well established that P stimulates alveolar development within the mammary gland by promoting branching morphogenesis [34] and alveologenesis [58], as enhanced by PRL [59]. The role of PRL in this relationship is being investigated further. Given our findings for DES, we anticipate that neonatal exposure to 1.25 µg TAM imparts an estrogenic effect on the pituitary to increase systemic and local synthesis of PRL, thereby enhancing the responsiveness of mammary epithelium to P. Of further note, glands from TAM-treated females underwent full development and differentiation in response to PRL in vitro. Therefore, mammary epithelium of TAM-exposed females remains responsive to lactogenic stimulation, a consideration of importance for women being administered TAM for prophylactic purposes.

These findings emphasize that attention must be given to the use of TAM as a prophylactic treatment. It is acknowledged that TAM should not be administered during pregnancy [60]; our results give further credence to this policy, given its possible long-term estrogenic effects on the mammary gland. Along these lines, Tewari et al. [61] reported a case of ambiguous genitalia in a female infant exposed in utero to TAM when it was used as a prophylactic treatment. While a direct comparison is not possible between the pharmacological doses and routes of administration used herein and prophylactic doses administered to women, our present results do emphasize the need for further investigation.

Conversely, high doses of TAM impaired ductal development, branching morphogenesis, and alveolar development. The means by which this exposure imparted its significant, long-lasting antagonistic effect on the mature mammary gland are presently unclear. However, similar morphological and histological responses have been described in the mammary glands of mature female mice exposed to TAM or another antiestrogen, EM-800 [62]. While females exposed to a high neonatal dose of TAM did not conceive, they possessed relatively normal serum E and P levels and their ovaries were histologically normal. One partial explanation may come from a report that neonatal exposure to a high dose (100 µg) of TAM abolishes sexual activity in female rats [63]. Further studies are underway to establish the contribution of PRL to this phenotype. Indeed, a role for PRL is supported by the findings of Aoki Mdel et al. [64], who recently demonstrated that TAM induces apoptosis in lactotrophs, while a separate study showed that administration of the antiestrogen, raloxifene, to postmenopausal women suppressed levels of serum PRL [65]. Taken together, these findings suggest that higher doses of TAM may restrict mammary development through a negative impact on pituitary cell number or function.

Taken together, our results indicate that neonatal exposure to an estrogen agonist or antagonists can have pronounced effects on the mammary gland of the nulliparous female mouse. These responses are mediated by significant changes in endocrine status that are markedly impacted by the level of exposure.


   ACKNOWLEDGMENTS
 
We thank Dr. Josephine Trott for assistance in preparing this manuscript.


   FOOTNOTES
 
1 R.C.H. and M.A.S. contributed equally to this work. Back

2 Correspondence: Russell C. Hovey, Lactation and Mammary Gland Biology Group, Department of Animal Science, University of Vermont, 121 Terrill Hall, 570 Main St., Burlington, VT 05405 Back

3 Current address: Department of Obstetrics, Gynecology and Molecular Reproduction Science, Yokohama City University Graduate School of Medicine, Yokohama, Japan Back

4 Current address: University of Turku, Turku, Finland Back

5 Current address: Congressionally Directed Medical Research Programs, Fort Detrick, MD 21702-5024 Back

Received: 16 March 2004.

First decision: 19 April 2004.

Accepted: 16 September 2004.


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