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Center for Experimental Therapeutics, University of Pennsylvania School of Medicine, 905 Stellar-Chance Laboratories (W.-C.S., Y.Q., X.S.), Philadelphia, Pennsylvania 19104; and Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences (M.N.), Research Triangle Park, North Carolina 27709
Address all correspondence and requests for reprints to: Dr. Wen-Chao Song, Center for Experimental Therapeutics, University of Pennsylvania School of Medicine, 905 Stellar-Chance Laboratories, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104. E-mail: song{at}spirit.gcrc.upenn.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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With the availability of EST complementary DNAs (cDNAs) and antibodies, the regulation and tissue expression pattern of EST have also become amenable for further analysis. An emerging picture from these investigations is that EST may play equally, if not more important, roles in extrahepatic, estrogen-responsive tissues by controlling the activity of estrogen in the local microenvironment (10, 13, 14). For example, the recent demonstration that EST was expressed in normal human mammary epithelial cells, but not in breast cancer cell lines, has raised a question regarding the roles of EST in normal mammary gland physiology (14). Previously, we cloned mouse EST cDNA and found, somewhat surprisingly, that the enzyme was expressed abundantly in the testis, but was not detected in the liver of either sex by Northern blot analysis of total RNA (10). This result suggested that there might be a species variation in the hepatic expression of EST (8, 10) and also raised the possibility that EST may play a role in testicular biology. The testis as a source of estrogen production in both animals and man has been well established (15, 16). For example, it has been estimated that in man up to 25% of the circulating estradiol is derived from the testis (16, 17). Expression of the estrogen biosynthetic enzyme P450 aromatase has also been detected in the Leydig cells and germ cells of a number of species, including rodents (18, 19) and man (20). It is equally well documented, both biochemically and pharmacologically, that estrogen receptor is expressed and functional in testicular cells (21, 22, 23). The recent finding in estrogen receptor knock-out mice that spermatogenesis and male fertility were severely impaired (24) further established the testis as a major estrogen target site in the male. Thus, it is conceivable that expression of EST in the testis may offer a regulatory mechanism, preventing testicular cells from excessive stimulation by the locally synthesized estrogen.
In exploring the physiological roles of EST in the testis, several important questions stood out. First, is the testicular expression of EST specific to the mouse or is it a common feature among different species? Secondly, what is the cellular distribution of EST within the testis? Thirdly, how is the expression of EST regulated in the testis and correlated with Leydig cell function and the reproductive process of the animal? This report describes the results of our study, which was directed at addressing these specific questions. In addition, the hepatic expression of EST in the mouse liver was reinvestigated and compared with that in the rat and human using the more sensitive reverse transcription-PCR (RT-PCR) method to ascertain whether the mouse is indeed different from other species in the sexually dimorphic expression of EST in the liver.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RT-PCR and ribonuclease (RNase) protection assays
First strand cDNA was synthesized from 20 µg total RNA using
400 ng oligo(deoxythymidine)18 and 400 U Moloney murine
leukemia virus reverse transcriptase (Life Technologies, Gaithersburg,
MD). The cDNA was ethanol precipitated and dissolved in 100 µl water.
Two microliters of this cDNA were used in a 50-µl PCR reaction. The
following pair of primers was used in PCR reactions to amplify a 369-bp
EST cDNA fragment: 5'-CTT-CCA-GT(C)A(C)-TCA-TTT-TGG-GAA-AAG-3'
(upstream) and 5'-TGG-ATT-GTT-CTT-CAT-CTC-3' (downstream). They
correspond to the amino acid sequences LPASFWEK and EMKNNP,
respectively. These two amino acid sequence motifs are conserved and
are specific to EST proteins from various species, including the mouse,
rat, and human. The PCR reaction mixture consisted of 10 mM
Tris-HCl (pH 8.3), 50 mM KCl, 3 mM
MgCl2, 0.2 mM of each deoxy-NTP, 20 pmol of
each primer, and 1.25 U Taq DNA polymerase (Perkin-Elmer,
Norwalk, CT). The temperature program used was 94 C for 1 min, 50 C for
1 min, and 72 C for 1 min for 35 cycles.
The 369-bp PCR fragment amplified from the reverse transcribed first
strand mouse EST cDNA was cloned into pCRII vector (TA cloning kit,
Invitrogen, Richmond, CA) and used to generate a radioactive antisense
RNA probe for RNase protection assays of the mouse EST messenger RNA
(mRNA) transcript. [
-32P]-CTP-labeled (800 Ci/mmol;
Amersham, Arlington Heights, IL) RNA probe was synthesized from the DNA
template with the Maxiscript in vitro RNA synthesis kit
(Ambion, Austin, TX). RNase protection assays were carried out with 30
µg total RNA using the RPAII kit (Ambion) and analyzed on 8% DNA
sequencing gels. Before the experiments were performed, the methodology
of RNase protection assay was validated by appropriate control
experiments, as suggested by the RPAII kit manual. These included a
control RNA (yeast RNA) and reactions with or without RNase
treatment.
Northern and Western blot analysis
Total RNA samples (20 µg each lane) were separated on a 1.2%
formaldehyde-agarose gel and transferred onto a nylon membrane
(Hybond-N, Amersham) via capillary action overnight in 6 x SSC
(standard saline citrate). The membrane was cross-linked under UV and
hybridized with a 32P-labeled cDNA probe synthesized with
random primers from the full-length mouse EST cDNA. Hybridization was
carried out in QuikHyb solution (Stratagene, La Jolla, CA) at 68 C for
1 h. The membrane was washed first in 2 x SSC-0.1% SDS at
65 C for 15 min and then in 0.1 x SSC-0.1% SDS at 50 C and
exposed to x-ray film. Polyclonal antiserum for mouse EST was generated
in rabbits using the bacterially expressed protein as described
previously (10). The antiserum was affinity purified on Affigel-15
beads (Sigma) to which pure mouse EST protein had been coupled. Testes
were homogenized at 4 C in 10 vol Tris-EDTA buffer (10 mM
Tris-HCl, pH 7.5, and 1.5 mM EDTA). The homogenate was
centrifuged at 100,000 x g for 1 h, and the
resulting supernatant was used for Western blot analysis. The protein
concentration was determined by the Bradford method with a colorimetric
assay kit from Bio-Rad (Richmond, CA). Samples were electrophoresed on
10% SDS-polyacrylamide gels, transferred onto nitrocellulose membranes
(Schleicher and Schuell, Keene, NH; BA85, 0.45 µm) and probed with
purified anti-EST. Immunodetections were performed with the enhanced
chemiluminescence Western blotting detection system from Amersham.
Immunohistochemical studies
For immunohistochemistry, testes were isolated from mature CD-1
mice and whole embryos at 13.5 and 15.5 days gestation (day of plug
detection = day 0.5) were dissected out from pregnant C57BL/6
mice. They were fixed in cold (4 C) Bouin’s solution overnight,
dehydrated, and paraffin embedded. Serial sections of whole embryos
were prepared at 8 µm, and sections of isolated testes were prepared
at 5 µm. The slides were deparaffinized in xylene and graded ethanol,
washed in distilled water, and then treated with 0.3%
H2O2 in methanol for 30 min to quench
endogenous peroxidase activity. After rinsing twice with PBS, diluted
normal goat serum (1.5%) was added to the slides to reduce nonspecific
binding. The slides were incubated for 20 min and rinsed with PBS, and
purified EST antiserum or control nonimmunized rabbit serum was added.
Formation of antigen-antibody complexes was allowed to take place for
60 min at room temperature. EST antigen was localized with a secondary
antibody coupled to a peroxidase-biotin-streptavidin detection system
(Vectastain ABC Elite kit, Vector Laboratories, Burlingame, CA) and
with diaminobenzidine tetrahydrochloride as the peroxidase substrate
for color detection.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, using a pair of oligonucleotide
primers corresponding to conserved amino acid sequences in EST
proteins, we could detect EST mRNA expression in testis and male liver
of all three species. In contrast, no signal was detected in female
mouse liver. Thus, it appears that the testicular and male-specific
hepatic expression of EST is a common phenomenon. It is evident,
however, that the relative levels of EST expression in testis and male
liver differ considerably in the three species. It is more prominent in
liver than in testis in rats and humans, whereas the opposite is true
in the mouse (Fig. 1). This result is consistent with the Northern blot
data from our previous study (10).
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, which revealed
that EST is localized selectively to the androgen-producing Leydig
cells. The staining of Leydig cells was uniform and, to the extent that
could be determined by light microscopy, appeared to be limited to the
cytoplasm, with little staining of the nucleus. As Leydig cells are the
site where testicular androgen biosynthesis takes place, the intense
and restrictive expression of EST in these cells raised the question of
whether there is a correlation between EST expression and androgen
production. To address this issue, we investigated and compared the
expression of EST in the testes of immature and adult mice by RNase
protection assay and Western blot analysis. Figure 3 shows clearly that the expression of
EST is regulated developmentally. Although highly expressed in the
adult testis, EST mRNA or protein in the immature prepubertal day 5 and
day 17 mouse testis was completely absent. Immunohistochemical analysis
of day 5 and day 17 mouse testis sections also confirmed that although
numerous interstitial cells are present at these developmental stages,
none of them expressed EST protein (data not shown).
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suggested that expression of EST may be a feature unique to the adult
type of Leydig cell. To determine whether EST is expressed prenatally
during embryogenesis when fetal Leydig cells are most prominent and
active, we performed additional immunohistochemical studies on
paraffin-embedded sections of whole embryos on days 13.5 and 15.5 post
coitum (pc). In the mouse, gonadal differentiation can be recognized
histologically on day 13 pc, and by day 15 pc, interstitial Leydig
cells and seminiferous cords are well developed in the fetal testes.
However, we found no EST expression in the testes of either day 13.5 pc
or day 15.5 pc fetuses (Fig. 4). These
results established that EST expression is a feature associated only
with the adult type of Leydig cell.
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) suggests that expression of EST is not an inherent
property of adult Leydig cells, but is probably regulated hormonally
during sexual maturation. In the mature testis, many of the functions
of Leydig cells are dependent on pituitary gonadotropins, most
critically on LH. To begin to understand the hormonal mechanisms of
testicular EST regulation, we investigated whether its expression is
similarly under the control of pituitary hormones. As shown in Fig. 5, we found that hypophysectomy
effectively abolished EST expression in the adult mouse testis.
Furthermore, daily administration of hCG to hypophysectomized mice for
4 consecutive days restored EST expression in the testis (Fig. 6). Thus, it can be concluded that the
Leydig cell expression of EST in the mouse testis is controlled by
pituitary LH.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our demonstration by RT-PCR in this study that EST was expressed in the rat and human testes has extended our earlier observation of prominent EST expression in the mouse testis (10). We have also established by immunohistochemistry that within the mouse testis, EST was localized selectively to the androgen-producing Leydig cells. A previous study by Payne (28) localized a steroid sulfotransferase activity in the rat testis to seminiferous tubules. As dehydroepiandrosterone was used as a substrate in the assay, and the activity was markedly inhibited by pregnenolone but marginally by estradiol (28), it is quite possible that the steroid sulfotransferase activity in rat seminiferous tubules demonstrated by Payne (28) reflected the presence of hydroxysteroid sulfotransferase, which, as we now know, is a different enzyme from EST (28). Our immunohistochemical data clearly showed no EST expression in mouse seminiferous tubules. It is doubtful that the rat and mouse express EST in different compartments of the testis. The limited EST activity demonstrated by Payne (28) in rat seminiferous tubules could be due to cross-reactivity by hydroxysteroid sulfotransferase or the presence of Leydig cells in the tubule fraction. Both the cell type specificity and the fact that EST expression was correlated with male maturity and dependent on LH indicated that EST may play a specific physiological role in testicular function. Previously, a guinea pig adrenal cortical EST was purified and cloned as a pregnenolone-binding protein (7, 29). One intriguing implication was that EST might participate in steroidogenesis by acting as a pregnenolone-binding carrier protein in adrenal gland and testis. However, the lack of EST expression in fetal Leydig cells, where steroidogenesis also takes place, does not support this hypothesis.
A more likely role for EST in the testis is to protect testicular cells from excessive stimulation by the locally synthesized estrogen. Sulfated estrogens do not bind to the estrogen receptor and are, therefore, devoid of biological activity. As has been alluded to previously, both Leydig cells and germ cells express cytochrome P450 aromatase (18, 19, 20), the enzyme responsible for converting testosterone to estradiol. Previous biochemical data and recent results from estrogen receptor knock-out mice have established that estrogen receptor is also present and functional in the testis (21, 22, 23, 24). Unregulated estrogen activity will certainly be detrimental to both spermatogenesis and androgen biosynthesis. Spermatogenesis is a process controlled by a complex interaction of endocrine and paracrine factors (30). A properly calibrated action of estrogen is likely to contribute to the maintenance of an optimum intratesticular milieu for this process to take place. The fact that sperm count in estrogen receptor knock-out male mice was only about 10% of that in wild-type animals (24) has underscored the importance of estrogen in this process. At the opposite end of the spectrum, it is well known that exogenous estrogen is a rather effective male contraceptive. Administration of estrogen to mice and men could effectively reduce sperm number and motility (31, 32). Although undoubtedly estrogen achieved this effect at least in part by inhibiting androgen biosynthesis through the hypothalamus-pituitary-testis axis, a direct toxic effect of estrogen on spermatogenesis is also a distinct possibility.
The inhibitory effect of estrogen on testosterone biosynthesis both
in vivo and in vitro has been shown in numerous
studies (33, 34, 35). Although such an effect in intact animals again could
be due to the suppression of pituitary hormone secretion, studies in
hypophysectomized rats have established that estrogen can exert a
direct, pituitary-independent effect on testosterone production (36, 37). There is now good evidence to suggest that estrogen causes a
steroidogenic lesion at the step of 17-hydroxylase/17,20-lyase,
through both a reduction in the level of the enzyme and inhibition of
its activity (38, 39, 40). Work in cultured Leydig tumor cells has also
identified an inhibitory effect of estrogen at an earlier site before
17-hydroxylase/17,20-lyase action in the steroidogenic pathway (41).
Significantly, Leydig cell expression of EST under these experimental
conditions is likely to be impaired. For example, we have shown in this
study that hypophysectomy abolished EST expression in the Leydig cells
of the mouse. Additionally, the mouse Leydig tumor cell line used in
some of the in vitro studies (41) does not express EST (our
unpublished observation). Thus, results that demonstrated estrogen
toxicity in Leydig cell lines or Leydig cells of hypophysectomized
animals may, in fact, have reflected the important protective role of
EST under normal physiological conditions.
A role for EST in modulating estrogen activity in the testis is also
supported by several other lines of circumstantial evidence. First, the
lack of EST expression in fetal type Leydig cells is consistent with
previous findings that less estrogen was synthesized in the fetal
testis due to low aromatase expression (19, 42). It also agrees with
the recent observation that expression of 17-hydroxylase/17,20-lyase
in the fetal rat testis was particularly sensitive to estrogen
inhibition and was reduced markedly by maternal exposure of this
hormone (39). Secondly, ex vivo studies using decapsulated
testes isolated from normal mice and rats showed that relatively high
doses (>5 µg) of estradiol were required to achieve an inhibition on
testosterone production (33, 34). Furthermore,
3-mono-benzoate-estradiol, a derivative of estradiol that can be
hydrolyzed to active estrogen and an unlikely substrate for EST, was
shown to be more potent than estradiol in the inhibition (34). These
results suggested the presence of a rapid and efficient inactivation
mechanism for free estrogens in the testis of a normal animal.
The results of the present study provide a new perspective for discussion of the connection between estrogenic chemicals in the environment and the possible decline in male reproductive function in animals and man (43, 44). This is a subject that has attracted considerable interest and debate in recent years. One fact that has been difficult to rationalize in considering such a theory is the comparatively low activity of xenobiotic chemicals, which are often orders of magnitude less potent as estrogen receptor ligands than the endogenous hormone. Given the high substrate specificity of EST, however, one might expect that many of these environmental chemicals are also poor substrates for the testicular EST. Thus, the relative activity displayed by these compounds in vivo as estrogen mimics may be substantially higher than that in receptor activation assays in vitro. On the other hand, it is also possible that some of the environmental estrogens may act as effective inhibitors of EST and thus potentiate the activity of endogenous estrogen in the testis. Whichever mechanism is true for a given estrogenic chemical, the differential expression of EST in fetal and adult testis lends further support to the concept that if estrogenic chemicals in the environment have a negative impact on male reproduction, the developing fetal testis may be a target site particularly sensitive to this detrimental effect (39, 45).
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Acknowledgments |
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Received May 13, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase/C17–20
lyase in the fetal rat testis is reduced by maternal exposure to
exogenous estrogens. Endocrinology 137:1063–1070[Abstract]
-hydroxylase and 17-desmolase
activities in BALB/c mice. Acta Endocrinol (Copenh) 45:487–491
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