From the Center for Research on Reproduction and Women's Health and Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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The success of spermatogenesis is dependent upon
closely coordinated interactions between Sertoli cells and germ cells.
To identify specific molecules that mediate interactions between somatic cells and germ cells in the rat testis, Sertoli cell-germ cell
co-cultures and mRNA differential display were used. Two cDNAs,
clone 1 (660 nucleotides) and clone 2 (390 nucleotides) were
up-regulated when Sertoli cells were co-cultured with pachytene spermatocytes or round spermatids. Northern blot analyses confirmed the
differential display expression patterns. Sequence analyses indicated
that clone 1 was similar to a von Ebner's gland protein (87% at the
nucleotide level and 80% at the amino acid level) and clone 2 was
identical to a region of the Huntington disease protein. The von
Ebner's-like protein mRNA was induced after 4 h of
co-culture, while the Huntington disease protein required 18 h of
co-culture for expression. The von Ebner's-like protein was induced in
germ cells by a secreted Sertoli cell factor(s) smaller than 10 kDa
that is sensitive to freezing and thawing or boiling. The Huntington
disease protein was induced in germ cells by a Sertoli cell secreted
factor(s) larger than 10 kDa which survives freezing and thawing, but
is inactivated by boiling.
A prerequisite for normal spermatogeneis is the coordination of
Sertoli cell function with the needs of germ cells in the seminiferous
epithelium. Regulatory interactions between Sertoli cells and germ
cells facilitate the sequential expression of genes needed for male
germ cell differentiation (1-4). Disruption of testicular cell
associations by heat, disease, or cytotoxic agents often affect the
interactions between Sertoli cells and germ cells and lead to
infertility (5-7).
Numerous experiments have utilized co-cultures of Sertoli cells and
germ cells to investigate how Sertoli cell factors influence germ cell
development. Sertoli cells stimulate germ cell RNA and DNA synthesis
(8), induce the appearance of germ cell surface antigens (9), and
maintain spermatogenic cell glutathione synthesis (10). Sertoli cell
secretory products have been proposed to mediate regulatory factors
such as insulin growth factor 1 (11-14) and transforming growth
factors Germ cells can also affect Sertoli cell functions. Germ cells secrete
proteinacious inhibitors or stimulators that modulate Sertoli cell
function (27-29). For instance, germ cell-conditioned medium
stimulates the phosphorylation of proteins in Sertoli cells (30),
stimulates We have previously shown that several genes including an isoform of
casein kinase, an epidermal growth factor, a statin-related protein
and an integral membrane glycoprotein are up-regulated and a basic
fibroblast growth factor, fibronectin, an endoplasmic reticulum stress
protein and a pro- Isolation of Sertoli Cells--
Primary cultures of Sertoli
cells were prepared from 20-day-old Sprague-Dawley rats (Charles River,
Kingston, MA) by sequential enzymatic treatments as described
previously (51-53). The testes were decapsulated and digested with
trypsin (1 mg/ml, Sigma) for 30 min at 37 °C to remove interstitial
cells and then with collagenase (1 mg/ml, Sigma) for 25 min at
37 °C. The cell suspensions were centrifuged at 800 rpm for 2 min.
The supernatants, containing peritubular cells, were discarded. The
pellets were washed twice with phosphate-buffered saline and incubated
in phosphate-buffered saline containing hyaluronidase (1 mg/ml, Sigma)
for 30 min at 37 °C. After incubation, the cell suspensions were
centrifuged at 800 rpm and the pellets were washed twice with
phosphate-buffered saline. The resulting Sertoli cells were plated at a
density of 2 × 106/cm2 in polystyrene
Isolation of Germ Cells--
Germ cells were obtained from the
testes of 60-day-old rats. The testes were decapsulated and incubated
with collagenase (1 mg/ml) for 15 min at 37 °C. After the
seminiferous tubules settled, interstitial cells were removed by
decanting the supernatant. The seminiferous tubules were incubated with
trypsin (1 mg/ml) for 20 min at 37 °C, pipetted up and down several
times to obtain a single cell suspension, and filtered through 20-µm
nylon mesh. Enriched populations of germ cells were obtained from adult
rat testes by Staput sedimentation as described previously (53). Staput
cell separations were performed in bovine serum albumin gradients
(2-4%) in culture medium adjusted to pH 7.4. The cells were allowed
to sediment for 3.5 h, fractions were collected, and cells were
identified by microscopy.
Co-cultures of Sertoli Cells and Germ Cells--
Sertoli cells
(2 × 106) from 20-day-old rats were co-cultured with
pachytene spermatocytes (8 × 106) or round spermatids
(8 × 106) for 24 h. Germ cells were cultured in
medium supplemented with 2 mM sodium pyruvate and 6 mM DL-lactate at a density of 8 × 106/ml. At the end of culture, cells were scraped from the
plates and RNA was extracted.
To study the time-dependent expression and cellular sites
of the induced genes, Sertoli cells were co-cultured with pachytene spermatocytes or round spermatids for 2, 4, 8, 18, and 24 h. At the termination of each culture, the germ cells were removed from Sertoli cells by aggressive washing. Total RNAs were extracted from
Sertoli cell-germ cell co-cultures and from separated Sertoli cells and
germ cells with the RNAgents Kit (Promega, Madison, WI). Throughout the
cultures, cell viability, monitored by trypan blue exclusion, revealed
greater than 97% viability. To delineate whether secreted factors
induced mRNAs, the germ cells and Sertoli cells were cultured
separately for 24 h and the media were collected. The media
collected from Sertoli cells were added to germ cells and the media
from germ cells were added to Sertoli cells. The Sertoli and germ cells
were then cultured for 24 h and collected for RNA extraction.
To start to characterize the factor(s) up-regulating genes in
co-culture, conditioned media from germ cells or Sertoli cells were
collected after 24 h of culture (35, 44, 45). The media were
boiled for 10 min, frozen and thawed five times, or passed through
Ultrafree-15 columns (Sigma) containing Biomax membranes with a
molecular weight cut-off limit of 10 kDa. Germ cells were cultured with
media prepared from Sertoli cells and Sertoli cells were cultured with
media prepared from germ cells treated as described above. After
24 h, the cells were collected for RNA extraction.
Differential Display and Isolation of Clones--
Preparation of
total RNA from Sertoli cells, from co-cultures of Sertoli cells with
pachytene spermatocytes, or round spermatids, pachytene spermatocytes,
and round spermatids were extracted as described previously (54). The
mRNA differential display was performed as described earlier
(51-53). Aliquots of total RNA (400 ng) were reverse-transcribed with
300 units of reverse transcriptase (Life Technologies, Inc.,
Gaithersburg, MD) in a buffer containing 250 mM Tris-HCl
(pH 8.3), 375 mM KCl, 15 mM MgCl2,
10 µM dithiothreitol, 1 µM anchor primer
(Operon, Alameda, CA), and 5 µM each of dATP, dCTP, dGTP,
and dTTP for 60 min at 42 °C. After inactivating the reverse
transcriptase, 2 µl of reverse-transcribed reaction mixtures were
added to 18 µl of PCR1
buffer containing 100 mM Tris-HCl (pH 8.3), 500 mM KCl, 1.5 mM of MgCl2, 2 µM of each dATP, dCTP, dGTP and dTTP, 1 µl of anchor primer, 10 µCi of [35S]dATP, and 1 unit of
Taq polymerase (Perkin Elmer, Norwalk, CT). The cycling
parameters for PCR were 95 °C for 30 s, 42 °C for 2 min, and
72 °C for 30 s, followed by 72 °C for 5 min. The
radiolabeled cDNAs were electrophoresed in 6% denaturing
polyacrylamide gels and after drying, the gels were exposed to x-ray films.
Subcloning and Sequencing--
cDNA bands that were
reproducibly detected in multiple preparations and in multiple
differential displays were excised from the gels and extracted in a 0.3 M ammonium acetate solution containing 1% SDS and 5 µg
of tRNA. The cDNAs were precipitated by the addition of 3 M sodium acetate (pH 5.2) and 1 ml of 100% ethanol,
resuspended in 10 µl of water and amplified by PCR using the same
reaction conditions as described above. The PCR amplified fragments
were ligated into the PCR-II vector of the TA cloning kit according to
the manufacturer's instructions. Plasmids were prepared by the
QIAprep, Miniprep Kit (Qiagen, Santa Clarita, CA). Both strands of each
cDNA were sequenced using M13 and T7 primers. The sequences of the
isolated clones were compared with the GenBank and EMBL data bases.
Radiolabeling DNA Probes and Northern Blot Analysis--
Plasmid
DNAs (10 µg) were digested with EcoRI and the cDNA
inserts were separated from the vector by electrophoresis on 1% agarose gels. The insert bands were excised from the gels and purified
with the Sephaglas Band Prep Kit (Pharmacia Biotech Inc., Piscataway,
NJ). Approximately 25 ng of cDNAs were radiolabeled with 50 µCi
of [ To define genes whose expression is induced by cell-cell
interactions of specific germ cells and Sertoli cells, we have
co-cultured rat Sertoli cells and pachytene spermatocytes or round
spermatids. Using mRNA differential display, two cDNAs were
detected with RNAs isolated from co-cultures that were not detectable
in equivalent RNA preparations from separate cultures of Sertoli cells,
pachytene spermatocytes, or round spermatids. Both genes, up-regulated
in germ cells, will be discussed in turn.
The mRNA of a von Ebner's-like Gene Is Up-regulated in
Co-cultures--
A cDNA of 660 nucleotides (clone 1 in Fig.
1) was up-regulated in co-cultures of
Sertoli cells with pachytene spermatocytes or round spermatids. It
showed 87% similarity at the nucleotide level (from nucleotides 107 to
767) and a coding region amino acid similarity of 79.5% (from amino
acid 18 to 178) to von Ebner's protein (Fig.
2) (55). The von Ebner's-like gene
encodes a transcript of 1.2 kb in RNA from co-cultures or total testis,
which is not detectable in RNA preparations from brain, lung, or liver
confirming the selective differential display expression of this
cDNA (Fig. 3A). Although
von Ebner's protein is normally expressed in salivary glands, tear
glands, and prostate, we did not detect hybridization with this
660-nucleotide von Ebner's-like protein cDNA to RNA from these
tissues (Fig. 4).
The von Ebner's-like Transcript Is Induced in Germ Cells by a
Sertoli Cell Factor(s) after 4 h of Co-culture--
In order to
study the time dependent induction and cellular site of expression of
the von Ebner's-like gene, Sertoli cells were co-cultured with
pachytene spermatocytes or round spermatids for 2, 4, 8, 18, and
24 h. At the termination of culture, germ cells and Sertoli cells
were separated and RNA was prepared from each. The 1.2-kb transcript
was first detected in RNA from co-cultured germ cells after 4 h
co-culture, but not in individual cultures of Sertoli cells, pachytene
spermatocytes, or round spermatids under identical culture conditions
(Fig. 5A). Surprisingly, no mRNAs were detected in either Sertoli cells or germ cells following their disassociation after co-culture.
To determine whether germ cells or Sertoli cells or both express the
von Ebner's-like protein mRNA, conditioned media were prepared
from germ cells and Sertoli cells. The conditioned medium from Sertoli
cells induced the 1.2-kb von Ebner's-like protein mRNA in germ
cells, while the germ cell-conditioned medium did not induce the
mRNA in Sertoli cells, demonstrating that the germ cells are
transcribing the 1.2-kb mRNA (Fig.
6A). The Sertoli cell
factor(s) that induces the 1.2-kb von Ebner's-like protein mRNA in
germ cells is smaller than 10 kDa and is inactivated by freezing and
thawing or boiling (Fig.
7A).
The Huntington Disease Protein mRNA Is Up-regulated in
Co-cultures--
A cDNA of 390 nucleotides (clone 2 in Fig. 1) was
up-regulated when Sertoli cells were co-cultured with pachytene
spermatocytes or round spermatids. Sequence analysis of the cDNA
revealed 100% identity with nucleotides 5618 to 6008 of the Huntington
disease gene. A transcript of about 10 kb was detected in RNA isolated from co-cultures of Sertoli cells and pachytene spermatocytes, from
co-cultures of Sertoli cells and round spermatids or total testis, but
not in RNA from Sertoli cells cultured alone, germ cells cultured
alone, brain, lung, or liver (Fig. 3B).
The Huntington Disease Protein mRNA Is Induced in Germ Cells by
a Sertoli Cell Factor(s) after 18 h of Co-culture--
When germ
cells are separated from Sertoli cells after co-culture for 18 h
or more, the transcript for the Huntington's disease gene was seen in
RNA isolated from pachytene spermatocytes or round spermatids, but not
from Sertoli cells (Fig. 5B). The 10-kb transcript was also
induced in germ cells with Sertoli cell-conditioned media, but not in
Sertoli cells with germ cell-conditioned media (Fig. 6B).
The Sertoli cell factor(s) appears to be larger than 10 kDa and
survives freezing and thawing, but is inactivated by boiling (Fig.
7B).
Local secretory factors and cell to cell communication within the
seminiferous tubule are essential to create and maintain microenvironments that allow the normal progression of spermatogenic events. Inappropriate or absent intratesticular signals from either germ cells or Sertoli cells may cause abnormal spermatogenesis leading
to infertility. Many paracrine and autocrine factors mediate interaction between different testicular cell types (3, 4, 56, 57). The
expression of inhibin, transferrin, testin, and prepro-enkephalin in
Sertoli cells is under the influence of paracrine factors potentially
of germ cell origin (37, 44, 45, 58, 59). Here we report that Sertoli
cells in culture can induce meiotic or post-meiotic male germ cells to
up-regulate a von Ebner's-like protein and the Huntington disease protein.
von Ebner's gland protein belongs to the lipocalin protein family, a
large group of small proteins including bilin-binding protein,
retinol-binding protein, retinoic acid-binding protein, Several lipocalin proteins including apolipoprotein D, murine Our finding that induction of a von Ebner's-like protein in meiotic
and post-meiotic germ cells suggests the presence of an additional
lipocalin in the testis. A number of different transport proteins (73,
74), including ceruloplasmin (75, 76), retinoid-binding protein (77,
78), and clusterin (79, 80) have been detected in Sertoli cells. Many
of these proteins have been implicated in the transport of nutritional
components to germ cells. Our detection of a von Ebner's-like protein
induced in germ cells suggests that a similar transport molecule could
carry small molecules in germ cells or from germ cells to Sertoli
cells. Although the von Ebner's-like protein induced in germ cells
shows homology (Fig. 2) to a gene expressed in salivary glands (57),
tear glands (68), and the prostate (81), under the stringent
hybridization conditions we have used our rat testicular cDNA does
not hybridize to RNAs from these tissues, suggesting it encodes a
distinct isoform of this family of proteins.
Our time course co-culture experiment (Fig. 5) reveals that the
mRNA of the von Ebner's-like protein is induced after 4 h of
co-culture and upon cell separation the turnover of its mRNA is
very rapid as no RNA is detected in separated Sertoli cells or germ
cells following co-culture (Fig. 6). Continued cellular contact does
not seem to be essential for the stability of the von Ebner's-like
protein mRNA, since a soluble factor(s) secreted by Sertoli cells
induces the 1.2-kb mRNA in germ cells (Fig. 6A). More
likely, a continued presence of the inducing factor is needed. Preliminary analyses indicate the factor(s) is less than 10 kDa, does
not survive freezing and thawing, and is inactivated by boiling.
The Huntington disease protein gene is also up-regulated in pachytene
spermatocytes or round spermatids co-cultured with Sertoli cells.
Huntington disease is a late onset progressive lethal neurogenerative disorder of autosomal dominant inheritance. The Huntington disease gene
(82, 83) contains repeating CAG triplets which are translated as a
polyglutamine stretch near the amino terminus of the protein (84-87).
In rats and humans, Huntington disease is transcribed as two
transcripts of 10 and 13.7 kb (88). Using Northern blot hybridization we have detected the 10-kb mRNA in co-cultures and in total testis (Fig. 3), but not in RNA preparations from brain, lungs, or liver. Our
results are in agreement with previous studies (88, 89) that have
detected the shorter transcript of the Huntington gene in testis, but
not in non-neuronal tissues such as liver, lung, and kidney. It has
recently been shown that the 10-kb transcript is due to a different
polyadenylation signal (90). Although a 13-kb transcript has been
reported in the rat brain (88), our inability to detect this brain
transcript is likely due to differences in hybridization conditions.
Our detection of Huntington disease protein mRNA in pachytene
spermatocytes and round spermatids is in agreement with the finding of
Schmitt et al. (88) and Lin et al. (90) who have shown by in situ hybridization a stage-dependent
expression in spermatocytes and spermatids. The presence of Huntington
disease protein mRNA in total testis and separated germ cells
following co-culture indicates it is induced in germ cells by Sertoli
cells. Unlike the apparently unstable von Ebner's-like mRNA, both
pachytene spermatocytes and round spermatids contain the 10-kb mRNA
after the germ cells are separated from the inducing Sertoli cells
(Fig. 5B). The Sertoli cell factor(s) inducing the
Huntington disease gene appears larger than 10 kDa, survives freezing
and thawing, but is inactivated by boiling, suggesting that it is a
protein. The Huntington disease gene is localized in the cytoplasm of
most cells including testicular germ cells. However, in some
spermatocytes the Huntington disease protein has been detected in the
nucleus, suggesting an additional but unknown function during
spermatogenesis (91). The induction of Huntington disease protein
mRNA in germ cells by a soluble factor from Sertoli cells suggests
its regulation is under paracrine control in the testis.
In summary, we have detected two genes that are up-regulated in meiotic
or post-meiotic germ cells by Sertoli cell factors. We believe these
inductions are cell- and factor-specific, since no inductions are seen
when Sertoli cells are co-cultured with a rat kidney cell line
(NRK-5ZE) (data not shown). These studies demonstrate that information
transfer between Sertoli cells and germ cells can be maintained through
soluble factors and the von Ebner's-like gene and the Huntington
disease gene provide specific markers for physiological studies of germ
cells and Sertoli cells in culture.
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and
(15-17). Sertoli cells secrete transferrin (18,
19) and an acute-phase protein identical to rat haptoglobin (20),
matrix metalloproteases, plasminogen activators, cyclic protein-2, and
protease inhibitors such as tissue inhibitor of metalloproteases-2 and
2-macroglobulin (21-24). The activities of Sertoli cell
proteases and protease inhibitors display pronounced cyclic changes
(25). The highest activities of plasminogen activator and cyclic
protein-2 are observed at stages where germ cells translocate to the
adluminal compartment, stages VII-VIII and VI-VII, respectively. At the
same time, protease inhibitors such as cystatin (stages VII-VIII) and
2-macroglobulin (stages VII-X) show low levels of
activity (25). The number of germ cells in the testis is also regulated
by Fas ligand from Sertoli cells, a molecule initiating apoptosis in
germ cells expressing Fas (26).
-glutamyl transpeptidase activity of Sertoli cells (31),
and decreases RNA synthesis in Sertoli cells (32). Germ cells in
co-cultures can influence Sertoli cell protein glycosylation (33),
increase basal and follicle-stimulating hormone-induced androgen-binding protein production (34, 35), inhibit CP-2/cathepsin L
mRNA expression (36), enhance inhibin and transferrin secretion and
transferrin gene expression (37-39), inhibit Sertoli cell estradiol production (38), stimulate fibroblast growth factor receptor type-1
expression (40), stimulate
2-macroglobulin expression (41), and N-cadherin expression (42). Testin, a glycoprotein secreted
by Sertoli cells, shows a sharp increase when Sertoli-germ cell
junctions are disturbed suggesting that germ cells may down-regulate testin gene expression (43-45). Nerve growth factor expressed in spermatocytes and early spermatids (46, 47) has also been proposed to
be a germ cell paracrine factor for the nerve growth factor receptor in
Sertoli cells (48). Differential regulation of Sertoli cells by germ
cells has been studied by examining the effects pachytene spermatocytes
or round spermatids have on Sertoli cells. A 24.5-kDa protein,
homologous to phosphatidylethanolamine-binding protein, produced by
cultured round spermatids stimulates the secretion of proteins from
Sertoli cells, suggesting that the phosphatidylethanolamine-binding
protein participates in the negative regulation of Sertoli cell
secretory function during spermatogenesis (49). Soluble protein(s) from
pachytene spermatocytes stimulate ceruloplasmin, transferrin, and
sulfated glycoprotein 1 and 2 in Sertoli cells (50). Clearly,
interactions between somatic and germ cells lead to the induction of
factors that selectively up- or down-regulate gene expression in
recipient cells.
2(XI) collagen are down-regulated when Sertoli
cells are co-cultured with a mixed population of germ cells (51). Here
we begin to define the molecular mechanisms regulating cellular
interactions between Sertoli cells and specific populations of meiotic
or post-meiotic male germ cells. We demonstrate that a von
Ebner's-like protein and the Huntington disease protein are
up-regulated in male germ cells by soluble factors secreted by Sertoli cells.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-irradiated Petri dishes (Falcon, Oxnard, CA) in serum-free Ham's
F-12 and Dulbecco's modified Eagle's medium (Life Technologies, Inc.,
Gaithersburg, MD) supplemented with 10 µg/ml insulin (Sigma), 5 µg/ml transferrin (Sigma), and 4 µg/ml gentamycin (Life
Technologies, Inc.). Cells were maintained in a humidified atmosphere
of 95% air and 5% CO2 at 32 °C. To obtain Sertoli
cells with a purity greater than 97%, cultures were hypotonically shocked with 20 mM Tris (pH 7.4) 48 h after plating to
lyse contaminating germ cells, and then washed twice with culture
medium. Twenty-four hours later, germ cells were added to the cultured
Sertoli cells for different time periods as described.
-32P]dCTP (NEN Life Science Products Inc.) and the
random primer labeling system (Life Technologies Inc.). Unincorporated
dNTPs were removed by centrifugation through Chroma-Spin columns
(CLONTECH, Palo Alto, CA) and the probes were
denatured at 100 °C for 5 min immediately before hybridization.
Northern blot analysis was performed with GeneScreen membranes (NEN
Life Science Products Inc.) as described earlier (51-54). Equal
amounts of RNA (10 µg) were hybridized and after hybridization, the
blots were stripped, and rehybridized with an actin cDNA to monitor
for equal RNA loading and transfer.
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
mRNA differential display profile of
cDNAs expressed in co-cultures of Sertoli cells and germ
cells. RNAs were isolated from 24-h cultures of Sertoli cells
(SC), Sertoli cells co-cultured with pachytene spermatocytes
(SC+P), pachytene spermatocytes (P), Sertoli
cells co-cultured with round spermatids (SC+RS), and round
spermatids (RS) and analyzed by reverse transcriptase-PCR.
The primers used were OPA-12 (TCGGCGATAG) and T11GT. The
arrowheads show two cDNAs (1 and
2) that are differentially expressed. Size markers are shown
on the right. bp, base pair.
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Fig. 2.
Amino acid sequence of clone 1. Amino
acid sequence comparison of clone 1 with rat von Ebner's gland protein
(GenBank accession number P20289). 159 amino acids of open reading
frame of clone 1 (lower sequence) share high extent of
similarity (79.5%) with amino acids 18 to 178 of the von Ebner's
protein (upper sequence). The shaded area
indicates identity.
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Fig. 3.
Northern blot of RNAs differentially
expressed in Sertoli cell-germ cell co-cultures. Total RNAs (10 µg) were extracted from 24-h cultures of Sertoli cells alone
(SC), Sertoli cells co-cultured with pachytene spermatocytes
(SC+P), Sertoli cells co-cultured with round spermatids
(SC+RS), pachytene spermatocytes alone (P), round
spermatids alone (RS) and from testis, brain, lung, and
liver. RNAs were electrophoresed and hybridized with the cDNAs
encoding the von Ebner's-like protein (A) and Huntington
disease protein (B). The blots were rehybridized with an
actin coding region cDNA.
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Fig. 4.
Northern blot showing selective expression of
the von Ebner's-like gene. Total RNAs (10 µg) were extracted
from testis, brain, prostate, lacrymal gland, salivary gland, and
interstitial cells. RNAs were electrophoresed and hybridized with the
cDNA encoding the von Ebner's-like protein. The blots were
rehybridized with an actin coding region cDNA.
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Fig. 5.
Time-dependent expression and
cellular localization of cDNAs that are up-regulated in co-cultures
of Sertoli cells with pachytene spermatocytes or round spermatids.
Total RNAs (10 µg) were isolated from 2-, 4-, 8-, 18-, and 24-h
cultures of Sertoli cells with pachytene spermatocytes
(SC+P), Sertoli cells co-cultured with either pachytene
spermatocytes or round spermatids and then separated (SC),
pachytene spermatocytes that had been co-cultured with Sertoli cells
and then separated (P), Sertoli cells co-cultured with round
spermatids and then separated (SC+RS), and round spermatids
that had been co-cultured with Sertoli cells and then separated
(RS). The three lanes underlined with
C represent Sertoli cells (SC), pachytene
spermatocytes (P), or round spermatids (RS)
cultured alone for 24 h as controls. The blot was hybridized with
cDNAs encoding the von Ebner's-like protein (A) and the
Huntington disease gene (B). The blot was rehybridized with
an actin coding region probe.
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Fig. 6.
Northern blot of RNAs from Sertoli cells
cultured with germ cell conditioned medium or germ cells cultured with
conditioned medium from Sertoli cells. RNAs (10 µg) were
isolated from 24-h cultures of Sertoli cells and germ cells
(SC+GC), Sertoli cells cultured with germ cell conditioned
media (SC+GCM), or germ cells cultured with Sertoli cell
conditioned media (GC+SCM). RNAs were electrophoresed and
hybridized with cDNAs encoding the von Ebner's-like protein
(A) and the Huntington disease gene (B). Blots
were rehybridized with an actin coding region cDNA.
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Fig. 7.
Partial characterization of Sertoli cell and
germ cell factor(s). Total RNAs (10 µg) were extracted from
Sertoli cells co-cultured with germ cells for 24 h
(SC+GC), Sertoli cells cultured with germ cell conditioned
medium (SC+GCM), Sertoli cells cultured with germ cell
conditioned medium that had been passed through a Millipore
Ultrafree-15 column with a 10-kDa molecular mass cut-off limit
(SC+GCM >10 kd), Sertoli cells cultured with
germ cell medium that had been frozen and thawed 5 times (SC+GCM
F & T), Sertoli cells cultured with germ cell medium previously
boiled for 10 min (SC+GCM 100 C), germ cells cultured with
Sertoli cell conditioned medium (GC+SCM) germ cells cultured
with Sertoli cell medium that had been passed through a Millipore
Ultrafree-15 column of 10-kDa molecular mass cut-off limit
(GC+SCM >10 kd), germ cells cultured with
Sertoli cell medium that had been frozen and thawed 5 times
(GC+SCM F & T), and germ cells cultured with Sertoli cell
medium previously boiled for 10 min (GC+SCM 100 C). RNAs
were electrophoresed and hybridized individually with cDNAs
encoding the von Ebner's-like protein (A) and the
Huntington disease gene (B). The blots were rehybridized
with an actin coding region cDNA.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-lactoglobulin, odorant-binding protein, apolipoprotein D, and murine
-trace (60). Members of this family bind and transport small
hydrophobic molecules such as retinoids, steroids, bilins, and lipids
(61). Some members of this family such as the odorant-binding protein
show little ligand specificity, while others, such as the
retinol-binding protein, are highly specific. The lipocalins have been
implicated in the modulation of immune responses and serve as carrier
proteins helping to clear both endogenous and exogeneous compounds
(60). In the tongue, von Ebner's protein is secreted from the lingual
gland where it is believed to help clear bitter tasting compounds (55).
Lipocalin from human von Ebner's gland contains three sequence motifs
corresponding to the papain-binding domains of cysteine proteinase
inhibitors. Since von Ebner's gland protein is able to inhibit papain
activity to a similar extent as salivary cystatin, it may also function as an inhibitor of cysteine proteinases and have a role in the control
of inflammatory processes in oral tissues (62).
trace, lipocalin-type prostaglandin D synthase, and odorant receptors
are expressed in the testis (63-65). Substantial levels of
apolipoprotein D have been detected in rat and rabbit Sertoli cells
(63, 64). Apolipoprotein D may serve as a transport protein carrying
small hydrophobic molecules such as unesterified and esterified
cholesterol and lecithin (66). Lipid metabolism is essential for
steroid hormone biosynthesis in the testis and a role for
apolipoprotein D in steroid hormone-binding and transport in the testis
could be envisaged. Murine
trace has been localized to Leydig cells
of postpubertal animals and has been proposed to function in transport
of small hydrophobic molecules across the blood testis barrier (65).
Lipocalin-type prostaglandin D synthase also is expressed in Leydig
cells (67). The receptors of a subfamily of lipocalin proteins,
odorant-binding proteins (68), have been detected in rat spermatids
(69-72).
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ACKNOWLEDGEMENT |
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We are indebted to Dr. Vargheese Chennathukuzhi for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant HD-11878.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF 123454.
Supported by Grant T32HD07305 from NICHD, National Institutes of Health.
§ Present address: Dept. of Anatomy and Physiology, University of Dundee, Dundee, United Kingdom.
¶ To whom correspondence should be addressed: Center for Research on Reproduction and Women's Health, 415 Curie Blvd., 752b CRB/6142 University of Pennsylvania, Philadelphia, PA 19104. Tel.: 215-898-0144; Fax: 215-573-5408; E-mail: nhecht{at}mail.med.upenn.edu.
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ABBREVIATIONS |
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The abbreviations used are: PCR, polymerase chain reaction; kb, kilobase(s).
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REFERENCES |
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