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Original Studies |
Departments of Internal Medicine (S.F., P.D., G.P., E.S., G.C., S.N.) and Experimental Medicine (N.R., G.S., S.U.), University of L’Aquila, I-67100 L’Aquila; and Department of Medical Physiopathology, University of Rome "La Sapienza" (L.G., M.A.), I-00100 Rome, Italy
Address all correspondence and requests for reprints to: Sandro Francavilla, M.D., Dipartimento di Medicina Interna, Università dell’Aquila, Via S. Sisto, I-67100 L’Aquila, Italy. E-mail: sandrof{at}univaq.it
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Introduction |
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FasL is a type II membrane-bound protein that belongs to the tumor necrosis factor (TNF) family and is capable of inducing apoptosis in Fas-bearing cells (15, 19). Fas (APO-1, CD95) is a transmembrane receptor protein that shares high homology with members of tumor necrosis factor/nerve growth factor receptor family. It contains an intracellular domain, called the death domain, which is responsible for the activation of multiple intracellular signaling pathways after Fas interaction with FasL or Fas receptor cross-linking (20, 21, 22). The physiological importance of the Fas/FasL system is underscored in lpr (lymphoproliferation) and gld (generalized lymphoproliferative disease) mice, characterized by mutations, respectively, in Fas and FasL genes (23, 24) that determine a dramatic lymphoproliferation and systemic autoimmunity due to altered lymphocyte apoptosis.
Within the mammalian testis, FasL expressed by Sertoli cells (15, 25) has proven to be a major determinant in maintaining the immune privilege of the male gonadal tissue (16, 17). However, a functional role for the Sertoli cell-expressed FasL in inducing apoptosis of Fas-expressing germ cells has been recently demonstrated (26, 27). Indeed, Lee and colleagues demonstrated the involvement of the Fas system in germ cell apoptosis of rat testis occurring either physiologically or after different testicular injury, such as radiation exposure or administration of Sertoli cells toxicants (26, 27). Recent studies have shown that in the human testis also, programmed germ cell death by apoptosis is a conspicuous event during spermatogenesis (28, 29, 30, 31, 32), but the possible involvement of Fas-FasL interaction in the regulation of this event is undefined (33, 34, 35). Recently, Pentikainen and colleagues (35) documented the expression of Fas protein by Western blot in senescent human testis. The same researchers, by means of immunohistochemistry experiments performed on isolated seminiferous tubules, showed a diffuse expression of Fas protein in cells identified as primary spermatocytes and round spermatids after apoptosis induction, whereas no information on Fas expression in normal or control conditions was provided.
In the present study, by means of RT-PCR in solution, in situ RT-PCR, and immunohistochemistry we have investigated the differential expression of Fas and FasL genes in human fetal and normal adult testis as well as in some selected human testicular pathologies. In the adult testis Fas is expressed by scattered degenerating germ cells, and FasL is highly expressed by Sertoli cells, suggesting that in humans also, the Fas-FasL interaction might be involved in the paracrine signaling between Sertoli cells and germ cells. Fas and its natural ligand are not expressed in fetal testes between 20 and 22 weeks gestation or in cases of idiopathic hypogonadotropic hypogonadism, thus suggesting that the expression of the Fas system in the human testis may be developmentally regulated and under the control of gonadotropins.
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Materials and Methods |
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Proteinase K and deoxyribonuclease (DNase), ribonuclease (RNase) free, were purchased from Sigma (Milan, Italy). First strand complementary DNA (cDNA) synthesis kit and 10 mmol/L solutions of deoxy (d)-ATP, dCTP, dGTP, and dTTP were purchased from Amersham Pharmacia Biotech (Milan, Italy). Human FasL, Fas, and ß-actin primers were obtained from Labtek (Milan, Italy). Taq DNA polymerase, silane-coated glass slides, Amplicover discs, and clips were purchased from Perkin-Elmer Corp. (Rome, Italy). The DIG Nucleic Acid Detection Kit and PCR DIG Labeling Mix were purchased from Roche Molecular Biochemicals (Milan, Italy). Two different antibodies to Fas and FasL were used for immunohistochemistry. Polyclonal antibodies to Fas (N-18) and FasL (N-20) and respective inhibitor peptides were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal antibodies to human Fas (lone DX2) and human FasL (clone Nok-1) were purchased from PharMingen (San Diego CA). The ImmunoPure ABC Peroxidase Rabbit IgG and Mouse IgG Staining Kits were obtained from Pierce Chemical Co. (Rockford, IL).
Testicular specimens
The investigation was approved by the local institutional review board for human research.
Fetuses. Testes were collected from three fetuses (20, 21, and 22 weeks gestation), obtained during therapeutic interruption of pregnancy due to maternal illness. Fetuses were carefully removed from the uterine cavity and immediately dissected, and one testis was snap-frozen in liquid nitrogen and stored at -80 C. The other testis of each fetus was in part fixed in Bouin’s solution and in part fixed in 4% paraformaldehyde for 6–8 h and processed for routine paraffin embedding.
Adults. Normal testis tissue was obtained from 2 fertile men, aged 35 and 47 yr, undergoing orchidectomy as treatment for testicular seminoma. A piece of each testis was immediately snap-frozen in liquid nitrogen and stored at -80 C. Adjacent parts of tissue were fixed, respectively, in Bouin’s solutio, and 4% paraformaldehyde for 6–8 h before processing for paraffin embedding. To analyze the FasL/Fas system by immunohistochemistry in cases of deranged spermatogenesis, archived testicular specimens fixed in Bouin’s solution and embedded in paraffin were obtained using an open testicular biopsy technique from men with azoospermia or severe oligozoospermia (mean age ± SD, 33.7 ± 4.5 yr). Informed consent was obtained from all subjects. On the basis of standard qualitative interpretations of hematoxylin- and eosin-stained sections, biopsies were classified as described below: complete Sertoli cell-only syndrome (SCO; n = 10): all seminiferous tubules showed only Sertoli cells; mixed atrophy, including 4 cases of Klinefelter’s syndrome according to results of genetic evaluation (n = 10): the tubules showed a thickened lamina propria associated with a total lack of the seminiferous epithelium (tubule shadow); this coexisted with some tubules with Sertoli cells only, and some tubules with spermatogenesis progressing through rare elongated spermatids; complete meiotic germ cell arrest (n = 10): all tubules showed arrested spermatogenesis at the level of primary spermatocytes; postmeiotic germ cell arrest (n = 10): almost all tubules showed spermatogenesis progressing through elongated spermatids; the latter, however, were greatly reduced to less than 5 in each cross-section of seminiferous tubules; and normal spermatogenesis (n = 10): almost all tubules showed the development of more than 10 elongating spermatids in each tubule cross-section. Testicular specimens of 3 adult men, 28, 32, and 35 yr of age, affected by idiopathic hypogonadotropic hypogonadism were also included. According to the clinical records, the diagnosis was based on the lack of increase in serum gonadotropin levels over the basal levels after iv injection of 100 µg GnRH (LHRH, Serono, Milan, Italy), performed after the age of 18 yr.
RNA isolation and analysis
Total cellular RNA was extracted from fetal and adult human testis using the acid guanidinium thiocyanate-phenol-chloroform method (36). The purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis before carrying out the analytical procedures. Levels of Fas and FasL messenger RNA (mRNA) were determined by the semiquantitative RT-PCR method. Two micrograms of total RNA were reverse transcribed, using the First Strand cDNA synthesis kit from Amersham Pharmacia Biotech. The cDNAs obtained were used as a template for the subsequent PCR coamplification of human (h) FasL (478 bp) and hß-actin (287 bp) as internal control or for coamplification of hFas (427 bp) and hß-actin (287 bp). The following primers were used: hFasL: upstream, 5'-GAAGGAGCTGGCAGAACTCCGAG-3'; and downstream, 5'-GACCAGAGAGAGCTCAGATACGTTGAC-3'; hFas: upstream, 5'-CCAAGTGACTGACATCAACTC-3'; and downstream, 5'-CTCTTTGCACTTGGTGTTGCTGG-3'; and hß-actin: upstream, 5'-AGCGGGAAATCGTGCGTG-3'; and downstream, 5'-CAGGGTACATGGTGGTGCC-3' (37, 38, 39). Conditions for coamplification for hFasL and hß-actin were 94 C for 1 min, 62 C for 1 min, and 72 C for 1 min for 35 cycles for hFasL and 25 cycles for hß-actin. Conditions for coamplification for hFas and hß-actin were 94 C for 1 min, 60 C for 1 min, and 72 C for 1 min for 35 cycles for hFas and 25 cycles for hß-actin. In these conditions preliminary experiments demonstrated that the plateaus for the ß-actin, Fas, and FasL amplification were not reached. Controls for DNA contamination or PCR carryover were performed omitting the reverse transcriptase or the RNA during RT. cDNAs were amplified using a DNA thermal cycler (Perkin-Elmer Corp.-PCR system 9700) and the Taq DNA polymerase (2 U/tube) with 15 pmol of both upstream and downstream primers and 2.2 mmol/L magnesium chloride in a final volume of 50 µL. For each sample 18 µL PCR amplification product were analyzed on 2% agarose gel and stained with ethidium bromide. A standard DNA molecular weight ladder (Ladder VI, Boehringer) was run to provide appropriate size markers. To monitor the specificity of the RT-PCR product, the amplified DNA was recovered from the agarose gel, purified, and subjected to sequencing reactions in the presence of fluorescent-labeled nucleotides. Samples were then analyzed by ABI Prism DNA sequencer (Perkin-Elmer Corp.). The sequences obtained all corresponded to the expected ones.
In situ RT-PCR
Fas and FasL distribution within the human fetal and adult testis was investigated by in situ RT-PCR (40, 41). Sections of 5 µm from paraformaldehyde-fixed paraffin-embedded fetal and normal adult human testis were placed on silane-coated in situ PCR glass slides (Perkin-Elmer Corp.) and incubated at 50–60 C for 36–48 h to ensure maximum adhesion to the slides. After heating, sections were dewaxed in xylene for 10 min three times, then immersed in fresh 100% ethanol, rehydrated in 70% ethanol and dH2O (RNase free), washed in phosphate-buffered saline (PBS), and permeabilized with 1 µg/mL proteinase K for 10 min at room temperature. After dehydration in ethanol 70–100% and air drying, sections were incubated in 1 IU/mL DNase (RNase free) in buffer B1 [40 mmol/L Tris-HCl (pH 7.4), 6 mmol/L MgCl2, and 2 mmol/L CaCl2], for 18 h in a moist chamber at 37 C. The next day, sections were dehydrated in 70–100% ethanol, air-dried, and incubated for 1 h at 42 C in moist chamber for RT. In the positive control section, treatment with DNase was omitted, whereas in both positive and negative control sections, the reverse transcriptase was omitted. The cDNA obtained was amplified in a thermal cycler in the presence of hFas, hFasL, or hß-actin downstream and upstream primers using digoxigenin-labeled dNTPs. Conditions for amplifications were 95 C for 1 min, 60 C for 1 min, and 72 C for 1 min for 30 cycles. After PCR, sections were washed twice in PBS, left in 1% BSA in PBS for 15 min, and incubated with anti-digoxigenin alkaline phosphatase conjugate (1:250) for 1 h at room temperature. After two washings in PBS for 10 min each time, sections were incubated for 5 min in the dark in the presence of nitro blue tetrazolium salt/5-bromo-4-chloro-3-indolyl phosphate, which produces, through the enzyme-catalyzed reaction, an insoluble blue precipitate. Slides were mounted with a mounting medium and observed under the microscope.
Immunohistochemistry
Five-micron-thick tissue sections were deparaffinized, and then subjected to immunohistochemical labeling according to the following protocol. 1) Endogenous peroxidase activity was eliminated by treating sections with 0.3% H202 in PBS for 30 min at room temperature. 2) Nonspecific binding sites were blocked by incubation for 1 h in 5% normal goat serum in PBS. 3) Sections were incubated overnight at 4 C in PBS containing the anti-FasL and Fas antibodies at a concentration of 1 µg/mL. 4) After repeated washing in PBS, the immunoreaction was detected by an avidin-biotin peroxidase complex method using a biotinylated goat antirabbit or antimouse IgG (ImmunoPure ABC Peroxidase Rabbit or Mouse IgG Staining Kit). Sections were left unstained or were counterstained in hematoxylin, mounted with a mounting medium (Histovitrex, Carlo Erba, Milan, Italy), and visualized in a Leitz photomicroscope (Rockleigh, NJ). Controls were performed by omission of the primary antibody or by incubating slides with the first antibody in the presence of inhibitor peptides (2 µg/mL). In a preliminary study, the different antibodies were tested with paraffin-embedded testicular tissue fixed with different fixatives and also with frozen tissue sections fixed in acetone before immunostaining. Bouin’s fixative and paraffin embedding gave results comparable to those obtained with frozen, acetone-fixed sections, and it was therefore selected for the study, because it allowed a large number of archived paraffin-embedded specimens to be evaluated.
Quantitative evaluation of Fas-positive germ cells
Counts of Fas-positive germ cells were performed on slides
lightly counterstained in hematoxylin and examined with a x63 oil
immersion objective lens and a x12.5 eyepiece. S.F. performed counting
on coded slides. The counting method was similar to that proposed by
Rowley and Heller (42). Longitudinal sections and cross-sections of
tubules with clear lumen were used for scoring. Approximately 30
seminiferous tubules were scored in each biopsy; the total number of
Fas-positive germ cells was divided by the total number of Sertoli cell
nuclei, and the rate of positive cells was expressed as the number of
Fas-positive germ cells per 100 Sertoli cell nuclei. Two hundred
Sertoli cell nuclei with clear nucleolus were counted in each biopsy.
Kruskal-Wallis one-way ANOVA by ranks was used to compare the number of
Fas-positive germ cells per 100 Sertoli cell nuclei in 3 groups of
biopsies with normal spermatogenesis (n = 10), postmeiotic arrest
(n = 10), and complete meiotic arrest (n = 10). The small
sample size of the three groups suggested using nonparametric tests for
data analysis. Post-hoc comparison between pairs of groups
was assessed by Wilcoxon’s rank sum test, with a downward adjustment
of the 
level to compensate for multiple comparisons. To maintain
the overall probability at a level of 0.05 in the 3 independent
comparisons, the value was divided by 3 to obtain a comparisonwise
= 0.017 (0.05/3). Thus, each comparison was significant at the
0.017 level. Data analysis was performed with SAS, version 6.12, 1996
(SAS Institute, Inc., Cary, NC).
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Results |
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The expression of Fas and FasL in fetal and normal adult human
testis was first evaluated at the mRNA level by semiquantitative
RT-PCR. Figure 1
shows that Fas and FasL
mRNAs are present in adult, but not fetal, human testis. The same
set of primers used in the above experiments was also used for in
situ RT-PCR to evaluate the distribution of both Fas and FasL mRNA
expression within fetal and adult human testicular tissues. In these
experiments, the fidelity of in situ mRNA amplification
(Fig. 2) was monitored on one slide for
each testicular specimen from the two fertile men (see Materials
and Methods). Each slide included, on three serial tissue
sections, a positive control (no DNase treatment, no reverse
transcription; Fig. 2A) and a negative control (DNase treatment, no
reverse transcription; Fig. 2B). The results obtained clearly
demonstrated the presence in the adult human testis of FasL mRNA
expression in both the seminiferous epithelium (Fig. 2, C and D) and
the interstitial cells (Fig. 2E). The irregular profiles of stained
nuclei and their regular localization in the basal compartment of the
seminiferous tubule (Fig. 2D) suggest that the cells expressing FasL
mRNA in the human seminiferous epithelium are Sertoli cells. FasL mRNA
was also expressed by interstitial Leydig cells, as judged by the
intense staining of the nucleus of cells with a large cytoplasm and
grouped in small perivascular clusters (Fig. 2E). On the other hand,
Fas mRNA expression was confined to scattered cells of the seminiferous
epithelium (Fig. 2F). The large nuclear size of most of the stained
cells and the localization in the basal compartment of the seminiferous
tubule suggest that Fas mRNA is expressed by primary spermatocytes
(Fig. 2F). Perinuclear staining was seldom found in cells identified as
round spermatids, as judged by the small nuclear size and the
localization very far from the basal lamina (Fig. 2G). A perinuclear
stain was also present in clusters of interstitial Leydig cells (Fig. 2H). When the same experiments were repeated on fetal testis sections
no expression of either Fas or FasL mRNA could be observed (data not
shown). However, when in situ RT-PCR on fetal testis
sections was performed using hß-actin primers, a strong positivity
was observed in nearly all testicular cell types (data not shown).
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Immunohistochemistry demonstrated the expression of FasL
and Fas in adult, but not fetal, testes of 20–22 weeks gestation (Fig. 3, A–C). Intense
and consistent FasL staining was found in the basal compartment of the
seminiferous tubules and in interstitial Leydig cells of testes with
normal spermatogenesis (Fig. 3, D and E). The positive staining in the
seminiferous tubule was confined to Sertoli cells and was intense along
the plasma membrane at the level of inter-Sertoli cell junctions as
well as in the basal region surrounding spermatogonia (Fig. 3F). Fas
expression was detected on occasional seminiferous tubules, where it
was confined to isolated germ cells that, according to cell size and
cell localization in the seminiferous epithelium, were primary
spermatocytes (Fig. 3H). In most cases Fas staining was confined to
apoptotic primary spermatocytes, as judged by the typical cytoplasm and
nuclear changes (43, 44) (Fig. 3, I and J) and to rare round spermatids
(not shown). The mean number of immunostained germ cells per 100
Sertoli cells recorded in 10 biopsies with normal spermatogenesis was
2.02 ± 0.29 (Table 1). An intense Fas staining was finally
detected in Leydig cells. (Fig. 3K). Staining was abolished by omitting
the antibody against FasL (Fig. 3G) or Fas (not shown) or by
preincubating the antibody with the inhibitory peptides (not
shown).
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FasL and Fas were not expressed in testes of men with idiopathic
hypogonadotropic hypogonadism (Fig. 4, A–C), whereas an intense immunostaining for both proteins was observed
in primary hypogonadism (Fig. 4, D–H). A diffuse immunostaining for
FasL was detected in the Sertoli cells and Leydig cells of testes with
complete SCO syndrome (Fig. 4D). Cases of mixed atrophy showed intense
immunostaining for FasL in tubules that contained only Sertoli cells,
whereas the immunoreaction disappeared in tubule shadows (Fig. 4E).
FasL was diffusely expressed in hyperplastic Leydig cells of men with
Klinefelter’s syndrome (Fig. 4F). Fas protein was never detected in
SCO tubules (Fig. 4G), whereas intense immunostaining was found in
Leydig cells of all cases of primary hypogonadism (Fig. 4H). The
intense coexpression of FasL and Fas in Leydig cells of normal testes
as well as in cases of primary hypogonadism was never associated with
the presence of apoptotic interstitial cells. This was confirmed by
microscopic analysis of epon-embedded 1-µm-thick sections, which
allows accurate identification of cellular changes associated with
apoptosis (45). Fas protein was expressed by primary degenerating
spermatocytes and rare round spermatids. The variation of Fas
expression in germ cells of testes with deranged spermatogenesis was
quantitatively assessed in biopsies with complete meiotic arrest or
postmeiotic arrest and compared to that in biopsies with normal
spermatogenesis (Table 1
). A
significantly increased number of Fas-positive germ cells was observed
in cases of postmeiotic arrest compared with normal testis
(P = 0.0001, by Wilcoxon’s rank sum test) and meiotic
arrest of spermatogenesis (P = 0.002). It is worth
mentioning that there was a huge variation of rate of Fas-positive germ
cells in cases of meiotic arrest (ranging from 0.42/100 Sertoli cells
to 4.28/100 Sertoli cells; Table 1).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A major unresolved question raised in studies on the Fas-FasL system in the gonadal tissue is the identification of factors that regulate the expression of FasL in the Sertoli cell. Here we report that the Fas-FasL system is not expressed at both mRNA and protein levels in fetal testis between 20 and 22 weeks gestation. At this age serum levels of fetal LH and FSH are still close to the detection limit of the assay, and the serum level of CG has already declined (48). Moreover, a negative immunostaining for both Fas and FasL was observed in cases of idiopathic hypogonadotropic hypogonadism. Both observations suggest that the expression of Fas/FasL in human testis is developmentally regulated, and it is probably activated during puberty. This hypothesis fits and enlarges available findings on the variation in apoptosis of germ cells postnatally in male rodents and also in man. In the human testis, apoptosis of germ cells is a rare and occasional event before puberty (28). In rodents, the initial apoptosis of germ cells occurs concurrently with maturation of Sertoli cells (10–15 days of age) and with the first wave of spermatogenesis, whereas it declines to occasional findings in adult normal testis (1, 49, 50, 51). The temporary and massive burst of germ cell apoptosis restricted to the pubertal activation of spermatogenesis and the subsequent decline concurrent with an increase in efficiency of spermatogenesis have been suggested to serve to adjust the number of maturing germ cells to the supporting capacity of Sertoli cells (1, 51). FasL expression might represent the molecular mechanism by which the Sertoli cell at puberty tunes the number of germ cells committed to give rise to mature spermatids. A potential role for FSH as a modulator of the expression of FasL by Sertoli cells is suggested by the lack of this factor in physiological and in pathologicsl conditions associated with absent gonadotropin stimulation. The induction of FasL expression might be viewed as an aspect of the maturation of Sertoli cells that is promoted by FSH (52), although a role for LH, through testosterone stimulation, should also be considered.
An unexpected finding of our study was the coexpression of Fas and FasL in Leydig cells of adult testis. Immunostaining for Fas was reported in Leydig cells of human testis (33), but for the first time, coexpression of Fas and FasL at both mRNA and protein levels in the Leydig cell, was documented. This was not associated with the evidence of apoptotic degeneration of Leydig cell, suggesting that the coupling of Fas by its ligand does not transduce a death signal in this cell type. Coexpression of Fas and FasL has been described in different human cells. In endometrial glandular cells, the coexpression of both molecules does not trigger apoptosis that is indeed inhibited by the antiapoptotic factor Bcl-2 (53). Normal thyreocytes constitutively express FasL (54) and Fas receptor (55), but the induction of apoptosis through the Fas pathway is blocked by a labile protein inhibitor (55). Therefore, the coexpression of Fas receptor and its ligand is not a rare occurrence, and it is not necessarily associated with an activation of the intracellular death signal. It is interesting that in vivo treatment of rats with a Leydig cell toxicant, ethane 1,2-dimethanesulfonate, is followed by a quick, massive death of Leydig cells by apoptosis; this is not associated with a concurrent increase in the expression of Fas protein, as detected by Western blot (56). It is possible that under normal conditions apoptosis of Leydig cells is inhibited by an antiapoptotic factor downstream from the engagement of Fas by FasL, which is quickly released by a toxic insult.
In conclusion, this study showed that Fas/FasL is expressed in adult human testes at both mRNA and protein levels. The expression seems to be developmentally regulated and gonadotropin dependent, as it is not present in fetal testes between 20 and 22 weeks gestation or in testes of individuals affected by hypogonadotropic hypogonadism. In the seminiferous tubule, FasL is expressed by Sertoli cells, whereas Fas is expressed only by degenerating spermatocytes, and the number of Fas-positive germ cells is increased in conditions of reduced efficiency of spermatogenesis. This suggests that the Fas/FasL interaction is a component of the paracrine signaling between Sertoli cells and germ cells, which contributes to promptly eliminate abnormal germ cells already committed to accomplish differentiation to gametes.
Received November 11, 1999.
Revised April 6, 2000.
Accepted April 18, 2000.
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E. Yakirevich, O. Yanai, Y. Sova, E. Sabo, A. Stein, J. Hiss, and M.B. Resnick Cytotoxic phenotype of intra-epithelial lymphocytes in normal and cryptorchid human testicular excurrent ducts Hum. Reprod., February 1, 2002; 17(2): 275 - 283. [Abstract] [Full Text] [PDF]
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S. Francavilla, M. A. Bianco, G. Cordeschi, P. D'Abrizio, C. De Stefano, G. Properzi, and F. Francavilla Ultrastructural analysis of chromatin defects in testicular spermatids in azoospermic men submitted to TESE-ICSI Hum. Reprod., July 1, 2001; 16(7): 1440 - 1448. [Abstract] [Full Text] [PDF]
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 A. D'Alessio, A. Riccioli, P. Lauretti, F. Padula, B. Muciaccia, P. De Cesaris, A. Filippini, S. Nagata, and E. Ziparo Testicular FasL is expressed by sperm cells PNAS, February 22, 2001; (2001) 51566098. [Abstract] [Full Text]
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A. D'Alessio, A. Riccioli, P. Lauretti, F. Padula, B. Muciaccia, P. De Cesaris, A. Filippini, S. Nagata, and E. Ziparo Testicular FasL is expressed by sperm cells PNAS, March 13, 2001; 98(6): 3316 - 3321. [Abstract] [Full Text] [PDF]
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