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Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430
Address all correspondence and requests for reprints to: Gail A. Cornwall, Ph.D., Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, Texas 79430.
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Abstract |
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Introduction |
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The ADAM family (1, 2) or MDC family (3) of proteins are a recently
discovered and rapidly growing gene family encoding mosaic proteins
that possess a disintegrin and metalloprotease domain. These cell
surface proteins have been proposed to play important roles in
cell-cell and cell-matrix adhesion, proteolytic processing, and cell
signaling. Of the many ADAM family members that have been identified,
several of these genes are predominantly expressed in the testis, in
particular in germ cells at varying stages of the spermatogenic cycle
(1, 3, 4, 5), whereas another member is expressed in the epididymis
(6), suggesting that the ADAM proteins may play important roles in male
reproduction. Of the testicular ADAMs, perhaps the most well
characterized proteins are the fertilins 
and ß, which are present
on the sperm surface as a heterodimer and which may be involved in
sperm/egg binding and membrane fusion (7, 8). Other proposed roles for
the ADAM proteins present in the testis include their involvement in
spermatid migration or as mediators of spermatid/Sertoli cell
interactions (1).
During a search to identify genes expressed in the proximal or caput region of the epididymis, which may be important for the initiation of sperm maturation, the mouse homologue of an ADAM family member, which we termed ADAM7, was identified. The epididymal ADAM gene has been shown in the rat to encode an androgen regulated 89 kDa protein (EAP1) (6). In the studies presented here, detailed experiments have been performed examining the tissue-specific expression of the mouse epididymal ADAM7 gene, its regulation by hormones and putative testicular factors, and its chromosomal position. These studies demonstrate that, in addition to its expression in the epididymis, the ADAM7 gene is also expressed in the anterior pituitary, specifically in the gonadotropes. Hormonal, surgical, and genetic approaches demonstrate that ADAM7 gene expression in the epididymis requires, in a region-dependent manner, both the presence of androgens as well as unknown testicular factors. Finally, chromosomal mapping shows that the epididymal ADAM7 gene maps to the central region of mouse chromosome 14, approximately 4–5 cM from the fertilin ß locus. Taken together, these studies provide new information regarding an ADAM family member that may play an important role in reproductive function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation of RNA
Total RNA was isolated from each tissue using a procedure
modified from that of Chomczynski and Sacchi (9). Briefly, the tissues
were pooled from 6–10 mice, placed into RNAzol (guanidinium
thiocyanate, phenol, and ß-mercaptoethanol; Cinna/Biotecx, Houston,
TX) at 4 C and homogenized. The homogenate was extracted with
chloroform followed by isopropanol precipitation. After two 70%
ethanol washes, the RNA pellet was resuspended in diethylpyrocarbonate
(DEPC)-treated water, and the concentration of RNA was determined by
spectrophotometric measurement at A260. Digestive tract
tissues and prostate were quick-frozen in liquid nitrogen before
homogenization in RNAzol. Poly (A)+ RNA was isolated from 500 µg
total RNA using Stratagene (La Jolla, CA) push columns containing
0.25 g oligo dT cellulose. For the isolation of RNA from cultured
cells, media was aspirated from tissue culture plates, the plates were
rinsed quickly with cold sterile PBS, and 5 ml RNAzol (Cinna/Biotecx,
Houston, TX) or 5 ml Trizol (GIBCO-BRL, Grand Island, NY) were added
directly to each plate. The cells were resuspended by scraping the
plate with a sterile spatula followed by repeated pipetting, and RNA
was isolated as described above.
Subtractive hybridization
An oligo dT-primed C57BL6/J mouse epididymal Uni-ZAP phage
complementary DNA (cDNA) library was commercially prepared by
Stratagene (La Jolla, CA) using poly (A)+ RNA prepared as described
above from the mouse epididymis. Approximately 1 x
106 recombinant clones were plated with the
Escherichia coli strain SURE and then transferred to
Nitroplus filters (Micron Separations Inc., Westboro, MA). To identify
cDNAs representing genes expressed in the caput epididymidis, a
caput-specific subtractive probe was prepared and hybridized with the
library filters as previously described (10).
Briefly, 1–1.5 µg poly (A)+ from the caput epididymidis were used to
generate a single-stranded cDNA probe using avian myoblastosis virus
reverse transcriptase (Stratagene, La Jolla, CA) and 1 mCi
[-32P]dATP. After incubation for 3 h at 42 C, the
RNA was removed by hydrolysis. The radiolabeled cDNA was ethanol
precipitated and passed through a G-50 Sephadex column to remove
unincorporated nucleotides and then hybridized overnight in 0.5
M sodium phosphate buffer, pH 6.6, 1.25% SDS, and 0.2
M EDTA at 68 C with 8-fold excess poly (A)+ isolated from
the corpus and cauda epididymidis. To separate the single-stranded
cDNAs from the double-stranded cDNArNA hybrids, the hybridization
mixture was passed through a 60 C water-jacketed column containing
hydroxyapatite (Bio-Gel HTP, Bio-Rad, Richmond, CA) hydrated with 0.12
M phosphate buffer containing 0.1% SDS. The
single-stranded caput cDNAs were eluted off the column with 0.12
M phosphate bufer, 0.1% SDS, whereas the cDNA/RNA hybrids
were eluted off with 0.5 M phosphate buffer. The
single-stranded caput cDNAs were subtracted again with 8-fold excess
poly (A)+ RNA from the corpus/cauda before being used in the
hybridization reaction. Approximately 4 x 107 cpm
caput-specific cDNA probe were used. To optimize hybridization of the
probe, the filters were incubated with the subtractive probe for 4 days
at 42 C in a hybridization buffer containing 5 x SSC, 50
mM sodium phosphate buffer, pH 6.6, 0.1% SDS, 0.1% sodium
pyrophosphate, 5 x Denhardt’s solution, 50 mg/ml salmon sperm
DNA, and 5% dextran sulfate. The filters were washed in 2 x SSC,
0.1% SDS at 42 C followed by washes in 0.1 x SSC, 0.1% SDS at
42 C and 65 C. Putative caput-specific clones were subjected to two
additional rounds of screening with a caput-specific probe to remove
false positive clones. Of the clones that remained positive, the cloned
inserts were in vivo excised and the resulting phagemid
plated with Escherichia coli to prepare plasmid DNA for
subsequent sequencing and expression analysis.
Northern blot analysis
Total RNA from mouse tissues was separated on a 1% agarose gel
containing borate buffer, pH 8.2, and 0.66 M formaldehyde.
The RNA samples were heated at 95 C for 2 min and then loaded onto the
gel and electrophoresed. To verify equal loading of RNA in each lane of
the gel, ethidium bromide was included in the RNA sample. The gels were
washed extensively in water to remove formaldehyde before transferring
to nylon membrane (Nytran, Schleicher and Schuell, Keene, NH). The
blots were prehybridized for 2 h at 42 C in hybridization buffer
containing 50% formaldehyde, 5 x SSC, 0.2 mg/ml salmon sperm
DNA, 0.4 mg/ml yeast RNA, 50 µg/ml BSA, 0.1% SDS, and 12.5
mM sodium phosphate buffer, pH 6.6, followed by
hybridization overnight at 42 C in the presence of cDNA probe at a
concentration of 3 x 105 cpm/ml hybridization buffer.
cDNA probes representing the 600 bp 3'UT region of the ADAM7 cDNA, rat
LHß (a generous gift of P. Mellon, The Salk Institute, La Jolla, CA),
and 18S rRNA were prepared using a random primer labeling method
(Prime-It II, Stratagene, La Jolla, CA). After hybridization, the blots
were washed in 2 x SSC at room temperature for 10 min followed by
washing in 2 x SSC, 1% SDS at 42 C for 30–45 min and then at 65
C before exposure to film. To quantitate the total amount of RNA in
each lane, the autoradiograms were scanned using a computer-assisted
image analysis system (BioImage VISAGE 2000, BioImage, Ann Arbor, MI).
The integrated areas obtained for the ADAM7 probe were then normalized
to the areas obtained for the 18S ribosomal probe. The Northern blots
were repeated and representative blots are shown.
In situ hybridization
Epididymal tissue sections were hybridized as described
previously (10) with 2 x 104 cpm/µl of the
35S-labeled antisense ADAM7 riboprobe generated from the
600 bp 3'UT region of the ADAM7 cDNA. To determine the specificity of
the labeling, epididymal sections were hybridized under the conditions
described above but with the sense-strand ADAM7 RNA probe. Following
hybridization, the sections were washed in 5 x SSC, 10
mM DTT at 50 C followed by a wash in 50% formamide, 2
x SSC, 100 mM DTT at 65 C. The sections were then washed
in 0.5 M NaCl, 10 mM Tris-Cl, 5 mM
EDTA in the absence and presence of 40 µg/ml RNAse A followed by a
wash in 0.1 x SSC at 65 C and at room temperature. The slides
were dipped Ilford K-5 emulsion, dried, and allowed to expose for
10–14 days. The exposed slides were then developed, fixed, and stained
with toluidine blue, dehydrated with a series of ethanol solutions
(30–100%), and coverslips were applied with Permount.
Chromosomal mapping
Chromosomal mapping was performed with the assistance of The
Jackson Laboratory, Bar Harbor, ME. To identify restriction enzymes
that would produce restriction fragment length polymorphisms, genomic
DNA from C57BL6/J and M. spretus mice was digested with restriction
enzymes BamHI, BglII, EcoRV,
HindIII, MspI, PstI, PvuII,
SstI, TaqI, and XbaI followed by
Southern blot analysis. A 600 bp ADAM7 cDNA probe representing the 3'
untranslated region was prepared by a random primer labeling method as
described above. Restriction enzymes that produced polymorphisms were
selected and then used for Southern blot analysis of DNA from the mice
of 94 backcrosses between (C57BL/6JEi x SPRET/Ei) x SPRET/Ei
mice (11) (BSS panel).
Cell culture
Gonadotrope cell lines T3–1 and LßT2 were a generous gift
of P. Mellon, The Salk Institute (La Jolla, CA). These lines were
derived from targeted oncogenesis in transgenic mice using a hybrid
transgene consisting of the SV40 T-antigen linked to the human
-subunit gene regulatory region (T3–1 cell line) or to the rat
LHß-subunit regulatory region (LßT2 cell line). LßT2 cells and
T3–1 cells were cultured in 100 mm tissue culture dishes and were
maintained in DMEM with 4.5 mg/ml glucose, 5% FCS, 5% calf serum, and
100 U/ml penicillin and 0.1 mg/ml streptomycin at 37 C in an atmosphere
of 5% CO2. The somatotropic/lactotropic GH-3 cells were
maintained in DMEM with 10% FCS, 100 U/ml penicillin, and 0.1 mg/ml
streptomycin. Media and antibiotics were from GIBCO-BRL, and serum was
from Hyclone (Logan, UT).
RT-PCR
RT-PCR was performed to obtain the 5' end of the ADAM7 sequence
not present in the ADAM7 cDNA cloned from the epididymal cDNA library.
Briefly, 1 µg of total RNA from the mouse caput epididymidis was
incubated at 42 C for 20 min in the presence of 5 mM
MgCl2, 50 mM KCl, 10 mM Tris, pH
8.3, 1 mM dNTPs, 20 U RNAsin, 2.5U MuLV reverse
transcriptase (Perkin-Elmer, Foster City, CA) and 0.75 µM
ADAM7 antisense primer (5'GGG TAA GCA ATT CCT TGC ATA 3'). The reaction
was then incubated at 99 C for 5 min to inactivate the reverse
transcriptase. The entire reverse transcriptase reaction was used for
PCR in the presence of 2 mM MgCl2, 50
mM KCl, 10 mM Tris, pH 8.3, 0.4 mM
dNTPs, 2.5U Amplitaq DNA polymerase (Perkin-Elmer, Foster City, CA) and
0.2 µM ADAM7 sense primer (5' ATG TTT CCC ACA GGT ATA TTT
TTG 3'). The ADAM7 sense primer was designed from the rat EAP1 sequence
(6). Following an initial denaturation step at 94 C for 1 min, the
reaction was incubated at 94 C, 30 sec; 50 C, 30 sec; 60 C, 60 sec for
36 cycles followed by 72 C, 15 min using a minicycler (MJ Research,
Watertown, MA). The single resulting PCR product was of the
expected size (890 bp) and following selective precipitation in the
presence of 0.1 M NaCl, 20 mM Tris, pH 7.5, 10
mM EDTA, 0.4 M ammonium acetate and 100% EtOH
to remove excess primers, the PCR product was cloned into the pGEM-T
vector (Promega, Madison, WI) and sequenced using SP6 and T7
primers.
Sequence analysis
Double-stranded sequence analysis was performed using
[35S] dATP and the Sequenase 2 sequencing kit (Amersham,
Cleveland, OH). Automated sequencing was performed through the Texas
Tech University Biotechnology Core facility under the direction of
Susan San Francisco, Ph.D.
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Results |
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, B). However, the mouse ADAM protein,
like the rat, does not possess a conserved metalloprotease active site,
the EGF-like repeat sequence found in most ADAM proteins nor the
putative fusion peptide present in a limited number of ADAM proteins
such as fertilin .
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). According to
the composite linkage map provided by the Mouse Genome Database (MGD,
The Jackson Laboratory), epididymal ADAM is located 4–5 cM distal to
the fertilin ß (Ftnb) locus and has been assigned the
locus symbol Adam7 (MGD Data Accession number
MGD-JNUM-37805). Due to this locus assignation, we have termed the
epididymal ADAM gene, Adam7.
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) the ADAM7
cDNA recognized a 4-kb transcript that was most abundant in the
proximal caput epididymidis with less expression in the distal
epididymal regions.
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),
silver grains representing ADAM7 mRNA were predominantly located in the
proximal caput epididymidis with decreasing levels of ADAM7 mRNA
present in the distal epididymal regions. No detectable expression of
the ADAM7 gene was observed in the efferent ducts. Higher magnification
of the caput epididymidis indicated that the ADAM7 mRNA was expressed
by the principal cells; however, expression by other minor cell types
cannot be ruled out (Fig. 4, B and C). Interestingly, in the proximal
caput epididymidis, the ADAM7 mRNA was concentrated at the apical cell
surface in addition to being distributed throughout the cytosol (Fig. 4, B and C). However, at the approximate junction between the proximal
caput and the midcaput epididymidis, this apical localization of the
ADAM7 mRNA disappeared, whereas the dispersed cytosolic localization of
ADAM7 mRNA continued, although with decreasing levels, throughout the
remainder of the epididymis (Fig. 4D). Hybridization of similar mouse
epididymal tissue sections with the control sense riboprobe showed only
a background hybridization (Fig. 4E). These observations support the
Northern blot studies and demonstrate that the ADAM7 mRNA is primarily
expressed in the proximal caput epididymal epithelium with less
expression in the distal epididymis.
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), the 4 kb ADAM7 mRNA was detected in
the mouse epididymis with no detectable expression in the other
twenty-five tissues examined including testis, vas deferens, seminal
vesicle, adrenal, submaxillary gland, kidney, heart, spleen, skeletal
muscle, lung, brain, thymus, bladder, prostate, small intestine, large
intestine, pancreas, stomach, gall bladder, lacrimal gland, and thyroid
of the male and in the ovary, oviduct, uterus, and liver of the female.
Further analysis revealed that the ADAM7 gene was also expressed in the
male and female anterior pituitary (Fig. 5B). In addition to the
predominant 4 kb ADAM7 mRNA, a 2 kb mRNA was also detected in both the
male and female pituitary. The Northern blots of pituitary RNA were
then stripped and reprobed with an LHß cDNA as a marker for anterior
pituitary tissue. As shown in (Fig. 5B), LHß mRNA is expressed in
both the male and female anterior pituitary RNA preparations.
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T3 and LßT2 cell
lines and was examined for ADAM7 gene expression in Northern blot
analysis. As shown in (Fig. 5B
), the ADAM7 4 kb mRNA was detected in
the LßT2 cells with less expression in T3 cells and no detectable
expression in the GH-3 cells. The absence of ADAM7 expression in the
GH-3 cells was not due to an inability of the mouse ADAM7 cDNA to
recognize the rat ADAM7 mRNA since Northern blot analysis of rat
epididymal RNA with the mouse ADAM7 cDNA detected the 4 kb ADAM7 mRNA
(data not shown). In addition to the predominant 4 kb ADAM7 mRNA, two
additional transcripts of 3.5 kb and 2 kb were also detected in the
LßT2 gonadotrope cells. When the Northern blots were stripped and
reprobed with an LHß cDNA the LßT2 cells, which represent a
differentiated gonadotrope cell line, expressed the LHß mRNA. In
contrast, the T3 cells which represent an undifferentiated
gonadotrope line, did not express the LHß mRNA. Similarly, the
somatotropic/lactotropic GH-3 cells did not express LHß mRNA.
Hormonal regulation
Because several genes expressed in the epididymis have been found
to be regulated by androgens, studies were undertaken to examine the
influence of androgens on ADAM7 gene expression in the epididymis.
Northern blot analysis was performed on total RNA isolated from the
epididymides of intact mice, mice that had been bilaterally castrated
12 h, 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, and 4 weeks
previously, and mice which had been bilaterally castrated for 4 weeks
followed by 4 weeks of testosterone administration. Within 12 h
following castration, the ADAM7 mRNA levels had dramatically decreased
with no ADAM7 mRNA detected after 24 h, suggesting that androgens
were necessary for ADAM7 gene expression in the epididymis (Fig. 6A). The administration of testosterone
to castrate mice however, resulted in only a partial recovery of ADAM7
mRNA levels to precastrate mRNA levels (Fig. 6A).
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legend). Northern blot
analysis demonstrated that the administration of testosterone at the
time of castration also resulted in only a partial recovery of ADAM7
gene expression (Fig. 6A). Similarly, the administration of
dihydrotestosterone (DHT) rather than testosterone to castrate mice,
also resulted in an incomplete recovery of ADAM7 mRNA levels (Fig. 6A).
RIA analyses showed that the circulating levels of DHT in the serum of
these mice were approximately 6-fold higher that in the control mice,
suggesting that the incomplete recovery of ADAM7 gene expression was
not due to the absence of DHT (Fig. 6 legend). Because estrogen
receptors have been identified in the efferent ducts and epididymis
(12, 13), estradiol was also administered to castrate mice to determine
its effect on ADAM7 gene expression. As shown in (Fig. 6B), the
administration of estradiol (E2) either in a replacement or
maintenance strategy to bilaterally castrate mice did not induce ADAM7
mRNA expression, even in part, indicating that androgens rather than
estrogens are important for ADAM7 gene expression in the
epididymis.
To date, the expression of several genes in the epididymis have been
shown to be regulated by putative factors from the testis other than,
or in addition to, androgens (14). Because ADAM7 gene expression only
exhibited a partial recovery to precastrate levels following castration
and testosterone administration, the role of testicular factors in the
regulation of ADAM7 gene expression in the epididymis was examined.
Several strategies were used including a surgical approach in
which the testicular fluid was prevented from entering the epididymis,
and a genetic strategy in which mice having altered testicular function
due to a genetic defect were examined. In the first approach to prevent
testicular fluid from entering the epididymis, unilateral castration
experiments were performed. Upon removal of one testis, the
contralateral testis responds by increasing its secretion of
testosterone so the circulating levels of testosterone in unilateral
castrate mice are normal. RIA analyses of the serum from unilateral
castrate mice confirmed that the levels of testosterone and
dihydrotestosterone were similar to that in normal, intact mice (Fig. 6
legend). In this paradigm, the effect of removing testicular input can
be assessed independent of the effects of circulating androgens. ADAM7
mRNA levels in the epididymis in which testicular input was removed by
castration was less than mRNA levels in the contralateral intact
epididymis suggesting that the ADAM7 gene required, in addition to
androgens, the presence of other testis factors for normal levels of
expression (Fig. 6B). In a second series of experiments, rather than
removing the entire testis and disrupting the blood flow to the
epididymis as in a castration experiment, the efferent ducts that
connect the testis to the epididymis were carefully ligated
bilaterally. As shown in (Fig. 6B), a decrease in ADAM7 expression was
observed in epididymides that did not receive input from the testis due
to efferent duct ligation (edl). Furthermore, the decrease in ADAM7
mRNA in the efferent duct ligated mice was similar to that observed in
the epididymides of unilaterally castrate mice.
A second approach to examine the effect of testicular input on
ADAM7 gene expression in the epididymis was to use mice which have
impaired testicular function due to a genetic mutation. Specifically,
mice that were homozygous for the white spotting (W) locus mutation
(c-kit) were used. The c-kit gene encodes a
tyrosine kinase receptor and one of the traits of mice lacking a
functional c-kit gene is that they are germ cell-deficient.
The c-kit mutant mice appear to have normal Sertoli, Leydig,
and peritubular cells (15, 16). In fact, circulating and tissue
testosterone levels are not significantly different between wild-type
and mutant mice (17). ADAM7 mRNA levels in the epididymides of
homozygous germ cell-deficient mutant mice were less than that of
homozygous wild-type and heterozygous mice (Fig. 6B). Taken together,
these data suggest that expression of the ADAM7 gene in the epididymis
requires not only the presence of androgens but also the input from a
testis actively carrying out spermatogenesis.
A detailed analysis of ADAM7 mRNA in the epididymis following
castration and testosterone administration was performed by carrying
out in situ hybridization experiments. Epididymal tissue
sections from intact, 2-week castrate, and 2-week castrate and
testosterone maintained mice were hybridized with the ADAM7 antisense
riboprobe as described. As shown in (Fig. 7, A, D, G, and J) in the proximal caput
region of an intact epididymis ADAM7 mRNA was concentrated at the
apical cell surface in addition to being distributed throughout the
cytosol. Following castration and as expected from the Northern
analyses, the ADAM7 mRNA disappeared and only background hybridization
was detected (Fig. 7, B, E, H, and K). When mice were given
testosterone at the time of castration, ADAM7 mRNA was detected in all
regions of the epididymis (data not shown). A striking difference in
the ADAM7 mRNA localization was observed, however, between the proximal
caput epididymidis from intact and testosterone maintained mice. In
contrast to the apical localization of ADAM7 mRNA observed in the
intact epididymis, ADAM7 mRNA in the proximal caput epididymides from
testosterone maintained mice only appeared throughout the cytosol; no
apical localization of ADAM7 mRNA was detected (Fig. 7, C, F, I, and
L).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, contain hydrophobic regions that may promote
membrane fusion while other ADAM proteins lack this sequence (8). All
ADAM genes sequenced to date possess disintegrin-like domains and may
be potential integrin ligands; however, they do not possess the RGD
binding sequence typical of many integrin ligands. The mouse epididymal
cDNA, which we identify as ADAM7, does not contain the consensus
metalloprotease active site nor a potential fusion peptide in its
sequence suggesting that it may primarily function via its disintegrin
domain.
To date, several of the mouse ADAM family members have been mapped and
have been shown to be dispersed throughout the mouse genome. The
testis-expressed ADAM genes including fertilin , fertilin ß,
ADAM4, and ADAM5 are localized to mouse chromosomes 5, 14, 9, and 8,
respectively (18). The testis-specific ADAM gene, cyritestin, also maps
to chromosome 8; however, it is located about 8 cM distal to the ADAM5
locus (19). Our studies map the epididymal ADAM7 gene to mouse
chromosome 14, approximately 4–5 cM distal to the fertilin ß
(Ftnb) locus.
The expression of several unique ADAM genes in the testis and epididymis suggests that they may play important roles in male reproduction. While several of these ADAM genes are also expressed in other nonreproductive tissues, cyritestin, fertilin ß, and ADAM6 appear to be exclusively expressed in the germ cells of the testis (1). Our studies presented here show that the mouse ADAM7 gene is also restricted in its expression. Northern blot analysis and in situ hybridization demonstrated that the ADAM7 mRNA was predominantly expressed in the proximal caput epididymidis with decreasing levels of expression in the distal epididymal regions. ADAM7 mRNA expression was not detected in the efferent ducts. Furthermore, in situ hybridization experiments showed an unusual localization of the ADAM7 mRNA in the epididymal epithelium. Within the proximal caput epididymidis, there was a strong localization of ADAM7 mRNA near the apical cell surface. However, at the approximate junction between the proximal caput and the mid-caput epididymidis, this apical localization of ADAM7 mRNA disappeared. Histological studies at the EM level have established that within the most proximal region of the epididymidis, in particular within the initial segment, a unique body of sparsely granulated endoplasmic reticulum is present immediately adjacent to the apical cell surface (20, 21). Further studies of ADAM7 mRNA localization at the EM level will establish whether the ADAM7 mRNA is associated with this unique endoplasmic reticulum. It is of interest that immunohistochemical studies of the rat epididymal ADAM protein (EAPI) have shown localization of the protein near the apical cell surface (6). It is therefore tempting to postulate that ADAM7 mRNA is preferentially translated off the apical endoplasmic reticulum which may allow for immediate transport to the adjacent cell surface.
In addition to exhibiting regional-specific expression in the epididymidis, the ADAM7 gene is also highly tissue specific. An examination by Northern blot analysis of 28 mouse tissues including the reproductive tract of the female mouse indicated that the ADAM7 gene was predominantly expressed in the epididymis and in the male and female anterior pituitary. An examination of several pituitary cell lines revealed that the ADAM7 gene was expressed by gonadotrope cell lines but not a somatotropic/lactotropic cell line. That the ADAM7 mRNA was expressed by the gonadotropes was not surprising considering the well established relationship that exists between the anterior pituitary gonadotropes and the testis. Further studies of ADAM7 gene expression in the anterior pituitary will determine if it is regulated by hormones and/or testicular factors similar to that observed in the epididymis.
A comparison in the ADAM7 mRNA expression between the two gonadotrope
cell lines indicated that the ADAM7 gene was predominantly expressed in
the LßT2 cells rather than the T3 cells. The significance of this
observation is that the ADAM7 gene expression may be associated
primarily with a differentiated gonadotrope cell, LßT2, rather than
with an undifferentiated gonadotrope cell, T3. Indeed, the LßT2
cell line is characterized by its ability to express the ß subunit,
which determines hormone specificity, as well as the subunit of
luteinizing hormone (22). The T3 cells, however, only express the
subunit which is present in several peptide hormones including LH,
FSH, TSH, and hCG (23).
A distinctive characteristic of ADAM7 gene expression in the epididymis is its regulation by androgens (24). We used three approaches including hormonal, surgical, and genetic strategies to examine the regulation of ADAM7 gene expression in the epididymis. Northern blot analyses demonstrated that the mouse ADAM7 mRNA levels were drastically reduced to approximately 4% its normal intact levels within 24 h of bilateral castration. Following the administration of testosterone or dihydrotestosterone, the active androgen in the epididymis, ADAM7 mRNA levels recovered to approximately 4–15% of the mRNA levels present in an intact, noncastrated mouse. The incomplete recovery in ADAM7 mRNA most likely was not due to inadequate levels of hormone because RIA analysis of serum from testosterone maintained and DHT replaced mice were 16- and 6-fold greater, respectively, than the circulating levels in control, intact mice. A possibility, however, is that the circulating levels of androgen may not reflect the local epididymal levels of androgen. We think this is unlikely because we have examined the expression of other androgen-regulated genes in the caput epididymidis following castration and hormone replacement and a complete restoration of the mRNA levels to precastrate levels were observed for several of these genes after testosterone administration (25). Therefore, while we cannot rule out that the local epididymal androgen levels were not sufficient to induce ADAM7 gene expression, our studies suggest that testicular factors in addition to androgens are necessary for normal ADAM7 gene expression. In further support of this conclusion are the observations from the surgical studies in which unilateral castrations and efferent duct ligations were performed. In both cases, when the connection between the testis and the epididymis was not maintained, either due to the removal of the testis in the unilateral castrations or the suturing of the efferent ducts in the ligation experiments, ADAM7 mRNA levels were reduced. Finally, when ADAM7 gene expression was examined in mice which are germ cell deficient due to a genetic mutation in the white spotting (W) locus, ADAM7 mRNA levels were reduced. The similarity in the response, or lack thereof, of the ADAM7 mRNA to regain precastrate levels following the various hormonal, surgical, and genetic manipulations suggests a common factor is affected or is absent in all three experiments. Taken together, these studies suggest that either spermatozoa themselves or factors associated with a normal, sperm-producing testis are critical for normal ADAM7 gene expression in the epididymis.
In addition to our observations, other studies have suggested that
testicular factors other than androgens regulate the epididymis and
that the effect of these testicular factors may impact particularly in
the proximal caput epididymal region. Histological studies have
demonstrated that the most proximal caput region does not regain its
precastrate state even after prolonged androgen replacement (26).
Furthermore, several genes expressed in the proximal caput epididymal
region including proenkephalin (27), CRES (10), gamma-glutamyl
transpeptidase (28), and 5 reductase (29) require testicular factors
for mRNA expression. Our in situ hybridization studies of
the ADAM7 mRNA levels in the mouse epididymis before and after
castration and testosterone maintenance suggest that ADAM7 mRNA levels
do not fully recover to the precastrate state particularly in the
proximal caput epididymidis. Interestingly, the intense localization of
ADAM7 mRNA present in the apical region of the proximal caput
epididymal epithelium of an intact mouse does not appear to be present
in the epithelium of a castrate and hormone replaced mouse. It is not
known, however, if the unique sparsely granulated endoplasmic reticulum
present near the apical cell surface in this epididymal region is
affected by castration.
The studies presented herein suggest that the ADAM7 gene may play an important role in several aspects of male reproduction including sperm maturation and gonadotrope function. The putative protease and adhesion domains of the ADAM7 protein imply roles in cell-cell interactions, protein processing, or cell signaling. Further studies will be aimed at identifying the functional domain(s) of the ADAM7 protein to ultimately determine its role in reproductive function.
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Acknowledgments |
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Footnotes |
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Received April 3, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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6ß1 functions as a sperm
receptor. Cell 81:1095–1104[CrossRef][Medline]
reductase messenger ribonucleic acid
levels along the rat epididymis. Endocrinology 128:2407–2414This article has been cited by other articles:
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