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INSERM, U-407, Communications Cellulaires en Biologie de la Reproduction, Faculté de Médecine Lyon-Sud, F-69921 Oullins, France
Address all correspondence and requests for reprints to: Dr. Mohamed Benahmed, INSERM, U-407, Faculté de Médecine Lyon-Sud, BP 12, F-69921 Oullins Cedex, France. E-mail: benahmed{at}lsgrisn1.univ-lyon1.fr
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Abstract |
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(TNF). Indeed, using a model of purified cultured mouse Sertoli
cells, we demonstrate that FSH inhibits, in a dose
(ED50 = 4 ng/ml)- and time (maximal effect after
24 h)-dependent manner, the levels of OSP mRNA. Such an inhibitory
effect was mimicked by 8-bromo-cAMP, suggesting that FSH may use the
cAMP/protein kinase A pathway to inhibit OSP mRNA levels. TNF was
also shown to inhibit OSP expression in cultured Sertoli cells. The
maximal effect was observed after 48 h of TNF treatment with an
ED50 of 4.5 ng/ml. Together, our results indicate that OSP
expression 1) starts during fetal life at a critical period, probably
under SRY control and during testicular formation; and 2) is regulated
by hormones (FSH) and cytokines (TNF) in the adult testis,
suggesting a critical role for these molecules in the (re)modeling
process of the hematotesticular barrier during spermatogenesis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Spermatogenesis in the adult is still dependent upon Sertoli cells, as it is these cells that are under direct endocrine control (2, 3, 12). Endocrine regulation allows Sertoli cells to develop and reorganize to generate the hematotesticular barrier through their tight junction complexes and to provide nutrients and regulatory factors to the germ cells. This time-modeling process and the production of Sertoli cell factors lead to the constitution of a specific biochemical and cytoarchitectural microenvironment in the adluminal compartment where germ cells will proliferate and differentiate. Among the identified Sertoli cell products are binding transport proteins, proteases and protease inhibitors, energy substrates such as lactate, and local signaling molecules such as growth factors and cytokines (13, 14, 15, 16, 17, 18). Although hormones (LH/testosterone and FSH) exert their control on spermatogenesis via these above-mentioned identified components, there are still other hormonally regulated Sertoli cell factors that remain unknown. To identify these factors that are under FSH control, we have used a method based on messenger RNA (mRNA) differential display in a model of purified mouse Sertoli cells cultured in the absence or presence of FSH. In the present study among the different transcripts differentially regulated, oligodendrocyte-specific protein (OSP) mRNA was identified. OSP was initially shown to be produced in oligodendrocytes, which are supporting cells for the highly specialized neurons (19). Recently, OSP has been identified as a new claudin family member (claudin 11) due to its sequence similarity (30%) and its ability to form tight junction strands in transfected fibroblasts (20). The key role of OSP in central nervous system myelin and Sertoli cell tight junction strands was demonstrated in osp/claudin 11 null mice (21). However, the control of the expression of this potentially crucial factor in the formation of the hematotesticular barrier is yet unknown.
In the present study we investigated the possibility that OSP
expression might be developmentally and hormonally regulated in the
testis. We report here that OSP expression is 1) regulated during
testicular development, as it appears just after Sry
expression and before that of Amh; and 2) targeted by the
actions of FSH and tumor necrosis factor- (TNF) in the postnatal
testis. This last observation clearly supports the concept that the
remodeling process affecting the hematotesticular barrier during
spermatogenesis might be under the control of hormones and local
signaling factors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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was
purchased from TEBU (Le Perray en Yvelines, France). Sigma
(Isle d’Abeau, France) was the source for 8-bromo-cAMP,
transferrin, gentamicin, HEPES, sodium bicarbonate, -tocopherol,
trypsin, and DNase I. RNA image kits were purchased from GenHunter
Corp. (Nashville, TN). Taq polymerase was obtained from
Promega Corp. (Lyon, France). The Amplicycle sequencing
kit was purchased from Perkin-Elmer Corp. (Courtaboeuf,
France). [-33P]dATP (1000–3000 Ci/mmol) and
[-33P]dCTP (3000 Ci/mmol) were purchased
from Amersham Pharmacia Biotech (Aylesbury, UK).
Oligonucleotides were purchased from GENSET (Paris, France). Primary
and secondary antibodies and the chemiluminescence detection kit were
obtained from Covalab (Lyon, France).
Animals
OF1 strain mice were purchased from IFFA CREDDO (Lyon, France).
Virgin female CD-1 mice were individually housed in controlled
conditions of lighting (12 h of light, 12 h of darkness),
temperature (20–24 C), and humidity (40–70%) and were given free
access to water and feed. Females were mated on a one to one basis with
male animals of the same strain and the same supplier. Day 0 of
gestation was when a vaginal plug was observed. At the appropriate
times, mice were killed by asphyxiation in CO2.
All studies were approved by the INSERM animal care committee.
Isolation and culture of Sertoli cells
Sertoli cells were prepared from 16- to 18-day-old mice, as
described by Dorrington et al. (22). First, albuginea was
removed, then testicular tissues were mechanically dispersed with
forceps in DMEM/Ham’s F-12 (1:1) medium (1.2 mg/ml sodium bicarbonate,
15 mM HEPES, and 20 µg/ml gentamicin)
containing DNase I (0.05 mg/ml). Testicular tissues were further
dispersed by collagenase/dispase treatment (0.5 mg/ml, 30 min at 32 C)
in the presence of DNase I (0.05 mg/ml) and FCS (2%) in DMEM/Ham’s
F-12 medium through mild stirring. At the end of the enzymatic
dissociation, testicular cells were submitted three times to gravity
sedimentation (3–5 min), and supernatants were removed. The pellets
containing the sedimented tubules were further dissociated with a
collagenase treatment as described above until small clumps resulted.
The cells where then submitted to a gravity sedimentation (10–15 min).
The supernatant was removed, and the sedimented clumps of Sertoli cells
were further washed by centrifugation (200 x g, 10
min). The Sertoli cell pellets were resuspended in DMEM/Ham’s F-12
medium (supplemented with 5 µg/ml transferrin and 10 µg/ml vitamin
E), and cells were plated on Falcon petri dishes (Becton Dickinson & Co., Franklin Lakes, NJ; 10-mm diameter,
5 x 106 cells/dish) and cultured at 32 C in
a humidified atmosphere of 5% CO2-95% air.
Isolation of RNA
Total RNAs were prepared using TRIzol, a monophasic solution of
phenol and guanidine isothiocyanate. This reagent is an improvement
over the single-step RNA isolation method developed by Chomczynski and
Sacchi (23). The amount of RNA was estimated by spectrophotometry at
260 nm.
mRNA differential display
mRNA differential display was performed with the RNAimage kit
according to the protocol of Liang and Pardee (24). RNA preparations
were submitted to DNase I digestion (1 U DNase I for 1 µg RNA, 15 min
at room temperature) to remove DNA contamination. Complementary DNA
(cDNA) was synthesized from 0.5 µg total RNA using SuperScript
reverse transcriptase (1 U), one of three 1-base anchored
oligo(deoxythymidine) primers (0.2 µM), and 20
µM dNTPs (1 h, 37 C). The cDNAs generated were used for
the PCR display; one of the three reverse primers was used in
conjunction with one of the eight forward arbitrary 13-mer primers
(AAGCT11V). Briefly, 2 µl of 1:4 diluted cDNA
were amplified using Taq polymerase (2 U) in a total volume
of 20 µl containing 10 mM Tris-HCl (pH 9.0), 50
mM KCl, 2.5 mM MgCl2 0.1%
Triton X-100, 0.2 µM dNTPs, 0.2
µM of each primer, and 0.2 µl
[-33P]dATP. After a 94 C, 5-min denaturing
step, PCR cycles consisted of 30 sec at 94 C, 2 min at 40 C, and 30 sec
at 72 C for 40 cycles, followed by a 72 C, 7-min elongating step. The
amplified products were separated on 8 M urea 6%
acrylamide gels, then dried and exposed to Kodak film
(Eastman Kodak Co., Rochester, NY) for 24–48 h. After
development, lanes containing amplified products from FSH-treated cells
or untreated cells were compared. Bands that showed differential
expression were excised from the gel, DNA was extracted by boiling in
100 µl sterile H2O and precipitated with
ethanol 85% (2 vol) and sodium acetate (0.3 M)
in the presence of glycogen (50 µg). DNA was reamplified using the
original set of primers and the same thermal cycling conditions, except
that the deoxynucleoside triphosphate concentration was increased to
250 µM. Reaction products were run on a 1%
agarose gel and stained with ethidum bromide. Bands were excised from
the gel, eluted, and used for sequencing or as a probe for Northern
blot analysis.
Northern blot analysis
About 20 µg total RNA were loaded on a 1.2% agarose/2.2
M formaldehyde gel. After 5 h of migration in 0.02
M 3(N-morpholino)propane sulfonic acid
(MOPS) running buffer, RNAs were transferred to nitrocellulose
membrane (Hybond-C Extra, Amersham Pharmacia Biotech) by
capillary transfer with 10 x SSC (standard saline citrate) and
fixed at 80 C for 2 h. The probes used for hybridization were a
339-bp OSP cDNA, and a 1.3-kb PstI rat
glyceraldehyde-3-phosphate dehydrogenase cDNA (GAPDH). Probes were
labeled with 50 µCi [-32P]dCTP using a
random primed DNA labeling kit (SA, 109 dpm/µg
DNA). The labeled probes were separated from free nucleotides by
filtration through a diethylaminoethyl-cellulose column. After 4 h
of prehybridization at 42 C, filters were hybridized with the labeled
probe (1–4 106 cpm/ml) overnight at 42 C in 50%
formamide, 5 x SSPE (0.9 M NaCl, 50
mM sodium phosphate, 5 mM EDTA, pH 7.4), 5
x Denhardt’s solution, 1% SDS, and 100 mg/ml yeast RNA. Then,
membranes were washed four times in 2 x SSC-0.1% SDS (15 min at
room temperature), followed by 30 min at 55 C. Filters were exposed to
Kodak X-OMAT films for 1–2 days at -70 C. The
intensities of the autoradiographic bands were estimated by
densitometric scanning using Intelligent Quantifier software (BioImage,
Cheshire, UK). The data were expressed as the OSP/GAPDH mRNA
ratio.
Semiquantitative RT-PCR
cDNAs were obtained from RT of 3 µg total RNA using random
hexanucleotides as primer (50 µM) in the presence of
dNTPs (250 µM), dithiothreitol (10 µM), and
Moloney murine leukemia virus (10 U/µl) for 1 h at 37 C. cDNAs
(2 µl RT mixture) were amplified by PCR with Taq
polymerase (0.01 U/µl), dNTP (250 µM),
[-33P]dATP (0.75 µCi), and specific
primers (10 µM). PCR amplification was
performed by first heating the mixture at 92 C for 3 min, followed by X
cycles (see Table 1
) at 92 C for 30 sec, Tm C for
30 sec, 70 C for 30 sec, then 70 C for 5 min.
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Sequencing
Samples showing significant changes by Northern blot analysis
were sequenced directly from the PCR product using the Amplicycle
sequencing kit (Perkin-Elmer Corp.) following the
manufacturer’s protocol. Differential display-PCR upstream arbitrary
primers were used to locate the sequence. DNA sequences were analyzed
by the BLAST search program and were screened for homology against the
GenBank/EMBL database sequences.
Western blotting analysis and immunohistochemistry
Two polyclonal antibodies (Covalab, Lyon, France) raised against
synthetic peptides corresponding to the 1) C-amino acid terminal or 2)
the N-amino acid terminal sequence of OSP were used for the
immunological approaches.
Proteins (10 µg) from whole adult testis and brain were analyzed on 15% SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes using 25 mM Tris and 185 mM glycine, pH 8.3, containing 20% methanol. The transfer was performed at a constant voltage of 100 V for 2 h. After transfer, the membrane was incubated in a blocking buffer [Tris-buffered saline (TBS) containing 1% BSA] overnight at 4 C. The membrane was rinsed three times with TBS/0.1% Tween (three times, 10 min each time), then incubated with the primary antibody (1:500 dilution in TBS) for 2 h at room temperature. The membrane was rinsed with TBS/0.1% Tween (three times, 10 min each time) and then incubated with horseradish peroxidase-labeled goat antirabbit IgG (1:1000 dilution in TBS) for 1 h at room temperature. The membrane was thoroughly washed with TBS/Tween (three times, 10 min each time) and then with TBS. Bound antibodies were detected using the chemiluminescence detection kit and Biomax MR films from Eastman Kodak Co. The protein concentration was determined by the Bradford assay.
For the immunohistochemistry analysis, paraffin sections (5 µm) of Bouin-fixed testis were mounted on glass slides. The sections were deparaffinized and rehydrated. The UltraVision Detection System (Lab Vision Corp., Fremont, CA) was used as recommended by the manufacturer. Briefly, endogenous peroxidases were blocked with 3% H2O2 for 15 min. The sections were incubated for 5 min with a protein-blocking solution to minimize nonspecific binding. The rabbit primary antibody was diluted (1:20) in antibody diluent (DAKO Corp., Trappes, France) and incubated with the sections for 2 h at room temperature. After washing and incubation with the biotinylated secondary antibody, a peroxidase-streptavidin complex was applied. Diaminobenzidene was used as the peroxidase chromogen. Sections were briefly counterstained with Harris hematoxylin and mounted in mounting medium. Antibody diluent alone or normal rabbit serum was used as the negative control.
Statistical analysis
Experiments were repeated at least three times with independent
cell preparations. The statistical significance of the results was
determined by Student’s t test when comparing data from
three experiments. Data are presented as the mean ±
SD.
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Results |
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Total RNA from a large variety of mouse tissues (lung, heart, brain,
kidney, skeletal muscle, ovary, and testis) were hybridized with
32P-labeled OSP probe to identify the specific
expression of OSP. The results obtained show specific expression of OSP
in the testis and, as expected, in the brain (Fig. 1A). It is interesting to note that OSP
was expressed in the testis but not in the ovary, whereas it was
expressed in both male and female brains (Fig. 1B). Finally, the data
in Fig. 1C show that testicular OSP mRNA is translated into protein.
Indeed, by using polyclonal antibodies raised against the N-and
C-terminal parts of OSP, we identified through Western blotting
analysis a protein with a molecular mass of 21 kDa and showed through
an immunohistochemical approach the specific expression of OSP in
Sertoli cells, but not in the other cell types (Fig. 2).
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show that SRY expression was
detected at 11 dpc and then was almost absent at 12 dpc. AMH was
expressed soon after OSP at 13 dpc. Thus, OSP expression appears and is
up-regulated just before the first sign of testis development;
i.e. the formation of the testis cords that is observable at
12 dpc. In the female (XX), no OSP expression was detected during fetal
life (data not shown).
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). From day 16, there was an apparent
decline in OSP mRNA. Such a decrease in OSP mRNA levels might be due,
at this age, to the increasing number of germ cells that do not express
OSP.
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in cultured
Sertoli cells. In Fig. 5, Sertoli cells were exposed to various
FSH concentrations (0.5–50 ng/ml) for different times (0–48 h), and
total RNA was extracted and analyzed through Northern blotting
analysis. The data obtained show that FSH decreased OSP mRNA levels.
The inhibitory effect of FSH on OSP mRNA levels was dose dependent, as
it was detectable at a concentration of 0.5 ng/ml and was maximal at 20
ng/ml (P < 0.001; Fig. 5A). This inhibitory effect on
OSP mRNA was also time dependent, as FSH decreased OSP mRNA levels
after 6 h of exposure, with a maximal inhibition observed at
24 h (P < 0.001) but not at 48 h (Fig. 5B).
To determine whether the effect of FSH is mediated by the cAMP/protein
kinase A (PKA) pathway, cultured Sertoli cells were exposed to 0.5
mM 8-bromo-cAMP for 1, 6, 12, 24, and 48 h.
The data in Fig. 5C show an inhibition of OSP mRNA levels in
8-bromo-cAMP-treated Sertoli cells close, if not similar
(P < 0.001), to that observed in FSH-treated
cells.
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, OSP mRNA levels were shown to
be inhibited by TNF in a dose- and time-dependent manner. The
inhibitory effect of TNF on OSP mRNA levels was detectable at a
concentration of 2.5 ng/ml and was maximal at 20 ng/ml
(P < 0.001; Fig. 6A). The TNF inhibitory action was
also evidenced after 12 h of exposure and was maximal at 48 h
(P < 0.001; Fig. 6B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Concerning OSP expression in the course of testis development, it was found to be present in both the early fetal testis as well as the postnatal testis, but with a different pattern of expression. The testis exhibits two main and distinct activities in the course of its development. The first occurs during fetal life and is mainly related to the process of organogenesis, whereas the second major activity, which occurs throughout adult life, is mainly related to spermatogenesis. During fetal life, the indifferent gonad has the ability to develop into an ovary or a testis depending on several genes, including Sry (sex-determining region 4 chromosome) as well as Sox-9 (an Sry-related gene), WT-1 (Wilm’s tumor gene), and the nuclear receptors DAX-1 and SF-1. Genetic analysis of XX-XY chimeras indicates that SRY functions within the cell lineage that will differentiate into Sertoli cells (9). OSP is expressed in the same lineage and immediately after the peak of SRY, suggesting that SRY could, theoretically, induce OSP expression. As SRY expression is turned off at 12 dpc, whereas OSP continues to be expressed, it is possible that SRY is not required for its maintenance. It is also possible that Sox-9 controls OSP, as Sox-9, which is already expressed in undifferentiated gonad, becomes abundant after 10.5 dpc in XY embryos and is absent in XX embryos (9). Furthermore, AMH, which is also a key gene in Sertoli cells, is expressed on 13 dpc, immediately after the expression of OSP. AMH, which belongs to the transforming growth factor-ß peptide family and is classically known to be responsible for Mullerian duct regression, may also play a role in the development of fetal testicular cells, such as Leydig cells and germ cells (11, 26, 27). Together, these observations indicate that OSP is expressed at a critical period in the cascade of gene expression, between SRY and AMH, during Sertoli cell differentiation. In addition, the absence of OSP expression in the ovary, but not in the female brain, clearly indicates that OSP expression is tissue specific. The promoter of the testicular OSP gene is potentially responsive to the genes involved in the testis (Sertoli cell) differentiation, indicating that in the gonad, but not in the brain, SRY expression is probably required for that of OSP.
After birth, OSP mRNA starts to increase from postnatal day 3 and reaches a maximal level between days 6 and 16 postnatally. At this period, during which OSP mRNA levels are at their highest, a crucial morphogenetic event occurs for the development of the adult testis: the formation of the hematotesticular barrier. Based on this timing of OSP expression in postnatal life, the hypothesis was that OSP may play a key role in the hematotesticular barrier. Such a possibility was demonstrated by two different groups during the course of our present study. Firstly, Morita et al. (20) identified OSP as a new claudin family member due to its sequence similarity (30%) and its ability to form tight junction strands in transfected fibroblasts. Secondly, the absence of tight junction intramembranous strands in central nervous system myelin and between Sertoli cells of osp/claudin 11 null mice (21) reinforced the concept for a key role for OSP in the hematotesticular barrier formation.
One of the major contributions of our present work is the first
demonstration that such a key factor involved in the formation of the
hematotesticular barrier is targeted by gonadotropin and local
signaling factor action, specifically by FSH and TNF action. The
actions of both molecules were identified using a model of cultured
Sertoli cells. The action of FSH was mimicked by 8-bromo-cAMP,
indicating that the hormone may use the cAMP/PKA/cAMP response
element-binding protein transducing pathway to reduce OSP mRNA levels.
Interestingly, we confirmed such an inhibitory effect of FSH on
testicular OSP expression in an in vivo model. Indeed, the
daily sc administration of 25 IU FSH (for 2 days) to 32-day-old
hypophysectomized rats induced a decline of about 50% in OSP mRNA
levels in the testis (our unpublished data). TNF also inhibited OSP
expression at a concentration compatible with the
Kd of its receptors, particularly the P55 form,
which has been reported to be present in Sertoli cells (28, 29). The
inhibitory effect of TNF on OSP mRNA levels in primary cultures of
Sertoli cells was exerted in the nanomolar concentration range. Such a
concentration is consistent with the amounts of TNF reported to be
present in the Sertoli cell environment (28). As this testicular TNF
originates from the postmeiotic germ cells (28), it may be that in the
adult testis, Sertoli cell OSP is also under the control of germ cells
in the context of Sertoli cell-germ cell cooperation during
spermatogenesis. Such a possibility is reinforced by the fact that
higher levels of OSP mRNA were observed in genetically modified mice
(white-Spotting, W/Wv) with testes devoid of germ cells (our
unpublished data). Finally, for both FSH and TNF, it remains to be
established whether these factors reduce OSP mRNA levels by acting at a
transcriptional level and/or mRNA stabilization levels.
The effects of FSH and TNF on OSP expression we report here might be
of particular interest concerning the regulatory mechanisms and factors
entering the process of the hematotesticular barrier complex
dissolution and reformation that occur during germ cell passage into
the adluminal compartment. Indeed, as we previously mentioned, the
intratesticular factors involved in such a process remain to be
identified. We hypothesize that the modulatory actions of FSH and
TNF on OSP levels may participate as a key event in the dissolution
and reformation of the testicular barrier, allowing germ cell
translocation in the adluminal compartment. The inhibitory effects of
the two signaling molecules, FSH and TNF, on OSP levels may favor
opening of the junctions between Sertoli cells and therefore allow germ
cell translocation into the adluminal compartment. Concerning FSH
action, its inhibitory effect on OSP testicular expression may appear
as a contradictory observation, assuming its well known positive action
on the spermatogenesis process. In fact, by inhibiting OSP levels, FSH
may favor the remodeling process (opening?) of the hematotesticular
barrier and translocation of germ cells into the adluminal compartment.
As such, FSH still positively regulates the spermatogenic process.
Although we identified here two inhibitory signaling molecules of OSP
expression, FSH and TNF, it is reasonable to suggest that other
signaling molecules also exist that enhance OSP expression and will
therefore contribute to a coordinated hematotesticular reformation once
the translocated germ cells are present in the adluminal compartment.
The identification of such factors stimulating OSP expression is
currently under investigation in our laboratory.
Finally, whether alterations in OSP expression might be involved in male infertility is as yet unknown. However, such a possibility exists in view of the localization of OSP on chromosome 3 (19). Indeed, this chromosome is of interest in relation to male fertility because 1) several genes, such as DAZH, DAZLA, and TAK1, suspected to be involved in the regulation of spermatogenesis (30, 31) are localized on chromosome 3; and 2) several cases of infertility have been observed in patients with genetic disorders involving this chromosome either in the context of translocations occurring on somatic (autosomal) chromosomes or of translocations between somatic and sex chromosomes (32, 33, 34).
In summary, by using an approach initially based on DD-PCR, we have identified the developmentally and hormonally dependent expression of OSP, a gene expressed in testicular Sertoli cells that has a potential key role in hematotesticular barrier formation. We reported here on 1) its tissue-specific expression, which appears dependent upon SRY in the testis; indeed, OSP is not present before SRY expression, and is absent in the ovary; and 2) its regulation by hormones and cytokines, supporting the concept that the remodeling process affecting the hematotesticular barrier during spermatogenesis is targeted by both the endocrine system and local regulatory factors.
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Acknowledgments |
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Footnotes |
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Received February 22, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in mouse
spermatogenic cells. Endocrinology 133:389–396[Abstract]
antagonizes follicle-stimulating hormone action in cultured Sertoli
cells. Endocrinology 133:69–76[Abstract]
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P.J. O'Shaughnessy, L. Fleming, P.J. Baker, G. Jackson, and H. Johnston Identification of Developmentally Regulated Genes in the Somatic Cells of the Mouse Testis Using Serial Analysis of Gene Expression Biol Reprod, September 1, 2003; 69(3): 797 - 808. [Abstract] [Full Text] [PDF]
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M. O. Suescun, C. Rival, M. S. Theas, R. S. Calandra, and L. Lustig Involvement of Tumor Necrosis Factor-{alpha} in the Pathogenesis of Autoimmune Orchitis in Rats Biol Reprod, June 1, 2003; 68(6): 2114 - 2121. [Abstract] [Full Text] [PDF]
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 F. Hollande, D. J. Lee, A. Choquet, S. Roche, and G. S. Baldwin Adherens junctions and tight junctions are regulated via different pathways by progastrin in epithelial cells J. Cell Sci., April 1, 2003; 116(7): 1187 - 1197. [Abstract] [Full Text] [PDF]
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M. K. Y. Siu, W. M. Lee, and C. Y. Cheng The Interplay of Collagen IV, Tumor Necrosis Factor-{alpha}, Gelatinase B (Matrix Metalloprotease-9), and Tissue Inhibitor of Metalloproteases-1 in the Basal Lamina Regulates Sertoli Cell-Tight Junction Dynamics in the Rat Testis Endocrinology, January 1, 2003; 144(1): 371 - 387. [Abstract] [Full Text] [PDF]
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 C. Y. Cheng and D. D. Mruk Cell Junction Dynamics in the Testis: Sertoli-Germ Cell Interactions and Male Contraceptive Development Physiol Rev, October 1, 2002; 82(4): 825 - 874. [Abstract] [Full Text] [PDF]
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L. Benbrahim-Tallaa, E. Tabone, G. Tosser-Klopp, F. Hatey, and M. Benahmed Glutathione S-Transferase Alpha Expressed in Porcine Sertoli Cells Is under the Control of Follicle-Stimulating Hormone and Testosterone Biol Reprod, June 1, 2002; 66(6): 1734 - 1742. [Abstract] [Full Text] [PDF]
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N. P. Y. Chung and C. Y. Cheng Is Cadmium Chloride-Induced Inter-Sertoli Tight Junction Permeability Barrier Disruption a Suitable in Vitro Model to Study the Events of Junction Disassembly during Spermatogenesis in the Rat Testis? Endocrinology, May 1, 2001; 142(5): 1878 - 1888. [Abstract] [Full Text]
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