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Departments of Anatomy (X.L., T.S., N.S., A.W., R.S., S.M.) and Physiology (E.N., W.Y., I.H., M.P.), Institute of Biomedicine, University of Turku, FIN-20520 Turku, Finland; and Unit for Preventive Nutrition (S.M.), Karolinska Institute NOVUM, S-14157 Huddinge, Sweden
Address all correspondence and requests for reprints to: Dr. Matti Poutanen, Department of Physiology, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. E-mail: matti.poutanen{at}utu.fi
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß, have been shown to be widely distributed in male
reproductive organs, including the testis, efferent ducts, epididymis,
vas deferens, bulbo-urethral glands, prostate, and seminal vesicles
(4, 5, 6). This suggests a direct role for estrogens in male
urogenital systems. Recently, the generation of knockout mice as
regards ER (7), ERß (8) and P450
aromatase (P450arom; Ref. 9) have expanded our
understanding of estrogen action, and provided new insights into the
role of estrogens in the male. For example, ER-deficient male mice
are infertile, presenting reduced mating frequency and low sperm number
with abnormal testicular morphology (10). Studies on
ER-deficient male mice further showed that the males have a failure
in the reabsorption of seminiferous tubule fluid in the efferent
ductules, resulting in a defect in posttesticular sperm maturation,
leading to infertility (11). Male mice deficient in
P450arom are initially fertile but show disrupted spermatogenesis and
infertility at an older age (12). Furthermore,
ER-/- males, but not
ERß-/- males, exhibit
abnormal sexual behavior (13, 14). Therefore, the data
show an essential role, direct or indirect, for estrogens in male
reproductive functions.
The P450arom enzyme is the product of the cyp19 gene
(15) and it catalyzes aromatization of the A-ring of
androgens such as testosterone (T) and androstenedione, resulting in
the formation of a phenolic A ring characteristic of estrogens,
E2 and estrone, respectively (16, 17). Aromatization of androgens by P450arom is one of the final
steps in ovarian E2 biosynthesis, but the enzyme
is also widely expressed in female and male extragonadal tissues.
However, extragonadal tissues lack the capacity to synthesize
androgenic precursors, and estrogen production is dependent on these
precursors produced in the gonads. Interestingly, androgen receptor,
ERß and ER, as well as P450arom, are colocalized in several
tissues of male reproductive organs, often with a cell-specific
expression pattern in the tissues (6, 18). This suggests
that there is a delicate balance between estrogen and androgen action
in male reproductive organs. Most importantly, several studies on both
humans and rodents indicate that prenatal or early postnatal exposure
to exogenous estrogens induce severe persistent changes in the
structure and function of male reproductive organs, such as atrophic
and small testes, epididymal cysts, and abnormalities in the rete
testis (19, 20, 21). The results of some studies also suggest
that estrogens might have a central role in the mechanisms leading to
male reproductive tract malformations such as cryptorchidism and
enlarged prostatic utricle, and diseases such as testicular (21, 22) and prostatic tumors (23).
To study further the role of estrogen/androgen balance in male reproductive functions, we have generated a transgenic mouse model with an imbalance in sex hormone metabolism by expressing P450arom under the human ubiquitin C promoter. The promoter is known to be activated on embryonic day 15 in mice (24), and ubiquitous and permanent transgene expression in a large variety of tissues is expected. Hence, in this mouse model, there is increased conversion of androgens to estrogens at all phases of testicular androgen production, in perinatal life, during puberty and in adults. Thus, the mouse lines developed provide a novel model to study the physiological consequences of estrogenization of males during various phases of their life span.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 4.0-kb-long pUbC-AROM fragment was released from the vector backbone by digestion with BglII and DraI enzymes. Before microinjection, the fragment was resolved in 1% agarose gel and isolated by electroelution followed by purification by means of Elutip-D columns (Schleicher & Schuell, Inc., Keene, NH). Finally, the fragment was diluted in TE buffer (10 mM Tris-HCl, 0.5 mM EDTA, pH 7.5) at a concentration of 2 ng/µl.
AROM+ transgenic founder mice
Using the purified pUbC-AROM expression vector (Fig. 1A
), transgenic mice were produced by a
standard technique. The pronuclei of fertilized eggs from the
FVB/N strain were microinjected with the DNA (2 ng/µl).
Microinjected oocytes were implanted into oviducts of pseudopregnant
female mice (NMRI strain) and carried to term. Positive founders
for the transgene (AROM+) were identified by
Southern blot analysis of DNA obtained from tail biopsies. Genomic DNA
(10 µg) was digested with EcoRI and resolved by
electrophoresis in 0.8% agarose gel. The DNA was then blotted onto
nylon membrane, the membrane was cross-linked by UV, prehybridized for
1 h at 64 C in hybridization buffer (5 x SSC, 5x
Denhardt’s solution, 0.5% wt/vol SDS), and with
[-32P]dCTP (Amersham Pharmacia Biotech, Aylesbury, UK) -labeled P450arom cDNA overnight at 64 C
in the hybridization buffer. The membranes were then washed in 2x SSC,
0.1% SDS once at 64 C for 15 min, once in 0.5x SSC, 0.1% SDS at 64 C
for 15 min, and then exposed to x-ray films for 1–3 days at -80
C.
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For routine genotyping of the AROM+ mice, PCR analyses were carried out using DNA extracted from tail biopsies. The sequences of the PCR primers were as follows: 5'-TGGCGAGTGTGTTTTGTG-3' (forward primer) and 5'-CCATCTTGTGTTCCTTGACC- 3' (reverse primer). The PCR (30 cycles) were carried out in a 50-µl volume using the following program: denaturation at 94 C for 1 min, annealing at 50 C for 1 min, and extension at 72 C for 1 min. The resulting PCR products were analyzed by electrophoresis on 2.0% agarose gel and the 200-bp-long PCR products were visualized with ethidium bromide.
Identification of transgene expression by RT-PCR
Total RNA was isolated from brain, heart, kidney, liver, spleen,
and testis using the acid phenol method, and RT-PCR was carried out.
Four micrograms of total RNA were incubated with 10 IU of avian
myeloblastosis virus reverse transcriptase (Finnzymes, Espoo, Finland)
at 50 C for 20 min. The cDNAs were then denatured at 95 C for 5 min and
amplified by 25 cycles of PCR using the following conditions: 94 C for
1 min, 50 C for 1 min, and 72 C for 1 min. The primers used were as
follows: 5'-ACCTCTACACCGCTCTTC-3' (forward primer) and 5'-
GCTTCTCTCACCAATAACAGTC-3 (reverse primer). As an internal control, a
200-bp fragment of the L19 ribosomal protein gene was amplified, using
5'-GAAATCGCCAATGCCAACT-3' (forward primer) and
5-TCTTAGACCTGCGAGCCTCA-3' (reverse primer). An aliquot of the RT-PCR
product was subjected to agarose gel electrophoresis and visualized by
ethidium bromide staining.
Measurement of serum hormones and morphological and histological
analyses
WT and AROM+ mice were obtained at similar
ages. They were anesthetized by ip injection of 300–600 µl 2.5%
avertin. Blood was collected by cardiac puncture, and tissues were
dissected out for macroscopic analyses and for obtaining the organ
weights. Serum samples were separated by centrifugation and stored at
-20 C until hormone concentrations were measured. Concentrations of LH
and FSH were measured by using time-resolved immunofluorometric assays
(25, 26). Serum T was measured by RIA after diethyl ether
extraction, as described previously (27). Concentrations
of E2 were measured using a commercial RIA kit
(Immunotech, Beckman Coulter, Inc.,
Marseille, France), according to the manufacturer’s instructions. For
histological evaluation, the tissues were fixed in 4%
paraformaldehyde, except for the testes, which were fixed in Bouin’s
solution. The tissues were then dehydrated, embedded in paraffin, and
sectioned. Two- to three-micrometer-thick sections were deparaffinized
in xylene and then stained with hematoxylin and eosin.
Statistical analysis
Statview software (Statview for Windows, v. 4.57; Abacus
Corporation, Berkeley, CA) was used for ANOVA and Fisher’s
Protected least significient difference post hoc
tests.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) were
generated. A total of five transgenic founder mice (two males and three
females) were identified by Southern hybridization analysis of genomic
DNA obtained from tail biopsies. Three of them (two females and one
male) were infertile as judged by their inability to produce offspring
over a 4-month period. This suggests that a high overexpression of
P450arom may disrupt the reproductive function both in males and
females.
The two fertile founders (one female, no. 21, and one male, no. 33)
transmitted the transgene to subsequent generations in a Mendelian
fashion, and were used to produce AROM+ transgenic
lines. To analyze the distribution of transgene messenger RNA (mRNA)
expression in AROM+ mice, RT-PCR was carried out on mRNA
prepared from a variety of tissues in the line 21. The strongest signal
for transgene mRNA was detected in the testis, and the mRNA was
detected at a lower level in the heart, brain, and liver (Fig. 1B).
Hence, it is evident that the transgene is expressed both in gonadal
and extra-gonadal tissues of the AROM+ male mice.
To analyze the consequences of the transgene expression into
circulating hormone concentrations, serum sex steroid and gonadotropin
concentrations were measured in 4-month-old AROM+
mice of the line 21, and were compared with WT mice. The
AROM+ male mice demonstrated elevated
E2 levels of 98–225 pg/ml (Fig. 2A), whereas in the WT males
E2 concentrations were below the detection limit
of the assay used (10 pg/ml). In accordance with the high
E2 concentration, serum T concentrations in the
AROM+ males were dramatically reduced (Fig. 2B).
However, detectable T concentrations were present (65–323 pg/ml),
showing that T biosynthesis continued, providing an androgenic
precursor for E2 biosynthesis in the
AROM+ males. Because sex steroids are known to
exert strong feedback effects on serum gonadotropins, we also measured
the circulating concentrations of LH and FSH. No significant difference
was found between LH levels in AROM+ and WT males
(Fig. 2C). However, there was less variation in the serum LH values in
the AROM+ males, suggesting that their LH
secretion probably displayed less marked pulsatility. Interestingly,
the mean serum FSH levels were moderately decreased in the
AROM+ males, compared with age-matched WT mice
(Fig. 2D). In addition to the sex hormone imbalance, highly elevated
levels in serum PRL (Fig. 2E) and corticosterone (Fig. 2F)
concentrations were measured in 4-month-old AROM+
males.
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). In contrast to males, the
AROM+ females were not found to have drastic
defects in their reproductive functions. They went through pregnancy,
delivered pups, and nursed the offspring. However, the possible
long-term effects of elevated P450arom expression in the female mice
remain to be analyzed.
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). Furthermore, the testes were
enlarged, with Leydig cell hypertrophy/hyperplasia. However, the
tubular structures appeared normal in light microscopy, and thus the
reason for infertility is not known.
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), and the
weights of the testes, epididymis, seminal vesicles, and prostate lobes
were significantly reduced (Fig. 3C and Table 1). In the seminiferous
epithelium of the testis, there were no germ cells beyond the stage of
pachytene, and numerous degenerating germ cells could be seen near the
lumen (Fig. 4, B and C). Furthermore,
cells with intensively staining nuclei and eosinophilic cytoplasm,
morphologically resembling eosinophil leukocytes, were detected in
seminiferous tubules (Fig. 4, C and E). In addition, numerous vacuoles
of different sizes were observed within the seminiferous epithelium
(Fig. 4, B and C). The interstitium was enlarged and filled with two
populations of cells, namely hypertrophic Leydig cells and large
multinucleated cells (Fig. 4, C and D). The Leydig cells displayed
swollen cytoplasm and ocular nuclei, and appeared to be larger than
those of the WT males.
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). In line with the
increased corticosterone production, histological examination showed
adrenocortical hyperplasia (Fig. 4, G and I), with pronounced expansion
of the innermost cortical layer, apparently the X-zone, filled with
large centripetal vacuole-filled structures (Fig. 4I).
Histological examination further confirmed the small seminal vesicles
and prostate glands (Fig. 3) with undifferentiated stratified
epithelium and uncanalized bud-like formations surrounded by dense
fibromuscular stroma (Fig. 5B). In the
collecting ducts, squamous epithelial metaplasia (Fig. 5C) was present
in all AROM+ males analyzed, although the extent
varied from animal to animal. In some AROM+ males
a prominent prostatic utricle with keratinized stratified squamous
epithelium was observed (Fig. 5D). In addition, pronounced expansion of
the extracellular spaces in the stromal layer of ejaculatory ducts was
frequently seen bi- or unilaterally (Fig. 5E).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, another transgenic mouse model with P450arom overexpression was developed using mouse mammary tumor virus promoter (29). Mouse mammary tumor virus is active in male reproductive tissues as well as in the mammary gland. About half of these male mice (int-5/aromatase) were infertile and/or had enlarged testes. Histological analysis showed the mice to have Leydig cell hyperplasia and Leydig cell tumors unilaterally or bilaterally (30). However, serum E2 levels measured in the int-5/aromatase mice were much lower than those in the AROM+ mice generated in the present study (5.7 and 150 pg/ml, respectively), and it is likely that this difference is not explained solely by methodological differences. The difference in the peripheral concentrations of E2 between the AROM+ mice and int-5/aromatase mice most probably bears a biological significance as regards the development of the phenotype. In contrast to the severe structural and functional alterations in AROM+ mice, no changes of the male accessory sex glands were reported in int-5/aromatase mice. In addition, in AROM+ mice PRL and corticosterone production is also markedly elevated, whereas these effects were not reported in the int-5/aromatase mice.
The AROM+ males display several of the changes observed in males perinatally exposed to estrogens (19, 20, 21, 22, 23), such as undescended testes, testicular interstitial cell hyperplasia, hypoandrogenism, and growth inhibition of accessory sex glands. The AROM+ mice were generated by using the human ubiquitin C promoter, which is known to be activated at around embryonic day 15 in mice, and which has been shown to drive constitutive expression of transgenes in a wide range of tissues (24). This, together with the phenotypic alterations found in the present study, indicates that the estrogen to androgen ratio in AROM+ mice is also high in the fetal and neonatal periods. In addition, as a sign of long-term excessive estrogen exposure in adulthood, the AROM+ males showed pronounced squamous epithelial metaplasia in periurethral collecting ducts. Interestingly, no hyperplastic or dysplastic lesions were observed in the prostate glands of AROM+ mice, although both perinatal estrogen treatment, as well as chronic combined estrogen-androgen treatment have been shown to induce prostatic neoplasia in rodents (31). This could be a result of the constitutive low T concentration in the AROM+ males.
Disruption of spermatogenesis in AROM+ mice could be a consequence of multiple factors, including cryptorchidism, abnormal Leydig cell function, hypoandrogenemia, or hyperestrogenemia. Estrogens are known to inhibit Leydig cell development, growth, and function, resulting in suppression of androgen production (32), and P450arom-deficient mice develop Leydig cell hyperplasia/hypertrophy (12). However, the present findings, together with those from int-5/aromatase mice (30), show that increased E2 to T ratio, including excessive estrogen exposure, disrupt Leydig cell function and can cause hyperplasia, hypertrophy, and Leydig cell tumors. Consistent with these findings, both prenatal exposure to DES and chronic exposure to DES in adulthood have been shown to induce Leydig cell tumors in mice (21, 33). Numerous degenerating germ cells and no spermatids within the seminiferous tubules suggest that germ cell development was arrested at the stage of pachytene in the cryptorchid testes of AROM+ mice. Interestingly, the spermatogenic arrest occurs at a stage where P450arom is typically expressed (34). However, the direct role of P450arom as a local autocrine/paracrine modulator of spermatogenesis remains to be characterized further. The spermatogenic arrest found in the AROM+ mice could be explained, at least partially, by the suppressed FSH action. The morphology of the hypertrophic Leydig cells in AROM+ mice was very different from that in the WT mice. The cryptorchidism alone does not lead to hyperplastic Leydig cells as shown by the relaxin-like factor knockout mice (35, 36). Hence, the structural and functional changes in the Leydig cells of AROM+ males are suggested to be related with the high E2 levels both prenatally and at adulthood. Given that the number of Leydig cells in experimentally cryptorchid testes is increased and T production suppressed (37); however, it is possible that the impaired Leydig cell function is partly associated with the cryptorchidism rather than to a direct effect of E2 on Leydig cells.
The reduced serum FSH levels in AROM+ males are further proof of the role of estrogens in suppressing FSH secretion in males (1). No significant differences in the average LH concentrations were seen between AROM+ and WT male mice. However, there was less variation in serum LH levels in AROM+ mice as compared with the WT males suggesting the possibility of reduced LH amplitude in AROM+ male mice. A similar effect on LH amplitude has been found in men after an exogenous estrogen administration (38). The data suggest that, similar to the situation found in men (39), FSH is more sensitive than LH in regard to the suppressive effect of E2 in AROM+ males. Therefore, the AROM+ mice provide a novel tool to further study the effects of estrogens on the regulation of LH and FSH secretion. Despite the normal LH concentrations, androgen levels were reduced in AROM+ mice, which is in agreement with the idea of testicular failure.
AROM+ males also display pronounced adrenocortical hyperplasia with enlarged cells throughout the cortex, and development of large centripetal vacuole-filled structures in the innermost cortical layer. Morphologically, this resembles the adrenal phenotype we recently observed in female bLHß-CTP mice overexpressing LH. These mice showed high serum corticosterone concentration associated with hyperactive adrenal function due to chronic adrenal gland stimulation (40). In addition to high corticosterone and LH, bLHß-CTP mice present elevated E2, T, and PRL concentrations. Interestingly, in the AROM+ male mice, the adrenocortical stimulation was associated with high circulating levels of E2 and PRL; hence, the role of these hormones in the etiology of adrenocortical hyperplasia in these mouse models remains to be explored.
The high PRL concentration found in the AROM+ males is in line with previous observations showing that estrogen exposure, both at neonatal period and at adult age, may cause hyperprolactinemia in male rats (41, 42). Furthermore, together with estrogens and androgens, increased PRL action has been shown to affect prostate structure and function, and the effect is dependent on the age of the exposure. Neonatal exposure to estrogens induces hyperprolactinemia and decreases prostate weight (43), whereas a chronic (15–20 weeks) estrogen treatment of adult male rats induces persistent hyperprolactinemia, and increased prostate weight (41). The difference is likely to be due to the interaction between sex steroid and PRL as, for example, androgens are necessary for PRL induced prostatic growth (44). Also hyperprolactinemic male mice have enlarged accessory sex glands, including the prostate, due to an increased amount of secretory material and interstitial tissue (45, 46). The fact that the prostate in the AROM+ mice is small and undeveloped is most likely due to the lack of proper amount of androgens at adult age and to the increased estrogen action at the neonatal period.
In conclusion, the AROM+ male mice generated show complex hormonal disturbances with multiple structural and functional abnormalities in the male reproductive system. Several of the abnormalities found resemble those described in mice exposed to exogenous estrogens during perinatal life. Hence, AROM+ mice represent a valuable model for analyzing the direct and indirect effects of unbalanced estrogen and androgen action, especially in the developing males.
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Acknowledgments |
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Footnotes |
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Received October 6, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, oestrogen receptor ß and androgen
receptors in the rat reproductive organs. J Endocrinol 165:359–370[Abstract]
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