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From the * Department of Neurology and the
Department of Urologic Surgery, Washington
University School of Medicine, St. Louis, Missouri; and the
Department of Veterinary Biosciences,
University of Illinois at Urbana-Champaign, Urbana, Illinois.
| Correspondence to: Dr Cathy K. Naughton, Department of Surgery, Division of Urology, Washington University School of Medicine, 1040 North Mason Road, Suite 122, St. Louis, MO 63141 (e-mail: naughtonc{at}wustl.edu). |
| Received for publication April 27, 2006; accepted for publication June 30, 2006. |
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Abstract |
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Key words: Spermatogenesis, spermiogenesis, testis, Syn-CAM1, IgSF4
TSLC1, like other members of the nectin superfamily, is composed of a large glycosylated extracellular region with 3 immunoglobulin (Ig)-like domains, a small transmembrane region, and a short cytoplasmic tail similar to glycophorin C and neurexin IV (Yageta et al, 2002). By analogy to nectin proteins, the first Ig-like loop is responsible for mediating interactions with nectin proteins on adjacent cells (trans-dimers), while the second Ig-like loop is responsible for nectin protein interactions within the same cell (cis-dimers) (Momose et al, 2002; Yasumi et al, 2003). The cytoplasmic tail of nectin proteins interacts with PDZ domain-containing proteins, like afadin, which may serve to link nectin proteins to the actin cytoskeleton (Miyoshi and Takai, 2005). In addition, nectin proteins have been shown to recruit E-cadherin (Tachibana et al, 2000; Tanaka et al, 2003). As reported for other nectin-like proteins, TSLC1 does not bind afadin or recruit E-cadherin, but it has been shown to interact with several other proteins, including Pals2 (Shingai et al, 2003), Protein 4.1B (Yageta et al, 2002), and MPP3 (Fukuhara et al, 2003), which are hypothesized to link TSLC1 to the actin cytoskeleton.
The role of nectin family proteins in the male gonad has been limited to studies of nectin-2 and nectin-3: nectin-2 is expressed exclusively in Sertoli cells, while nectin-3 expression is limited to spermatids (Ozaki-Kuroda et al, 2002). The heterotypic interaction between these 2 proteins is essential for normal spermatozoa maturation, such that nectin-2-deficient mice display loss of the junctional scaffold between Sertoli cells and spermatids, abnormal sperm morphogenesis, and infertility (Bouchard et al, 2000). Similar to nectin proteins, TSLC1 is robustly expressed in the male testis, where it is localized to germ cells (Wakayama et al, 2003). However, the function of TSLC1 in the male gonad is not known. In an effort to directly examine the role of TSLC1 in male fertility, we generated Tslc1+/– mice and demonstrated that markedly reduced Tslc1 expression results in disrupted Sertoli cell-germ cell junctions, impaired spermatozoa motility, and reduced male fertility.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Reverse Transcriptase-Polymerase Chain Reaction
Total RNA was extracted from Tslc1+/– adult mouse brains
using the Trizol reagent (Invitrogen, Carlsbad, Calif), and first strand cDNA
was synthesized from 5 µg total RNA using a reverse primer in the 5'
end of the targeting vector (Trap-R:
5'-GACAGTATCGGCCTCAGGAAGATCG-3') in the presence of Superscript II
reverse transcriptase (Invitrogen). After heat inactivation, the synthesized
first strand cDNA was used as template in a PCR reaction using the Trap-R
primer with either a forward primer in exon 3 (Ex3-F:
5'-GGGAGATACTTCTGCCAGCTCTACAC-3') or exon 4 (Ex4-F:
5'-GGCAGTTGAAGGGGAGGAGAT-3'). PCR amplification was performed in a
thermocycler at 94°C for 1 minute, 55°C for 1 minute, 72°C for 2
minutes (29 cycles), and 72°C for 10 minutes. Five microliters of the PCR
reaction were electrophoresed on 1% agarose gels, stained with ethidium
bromide, and visualized by UV transillumination.
ß-Galactosidase Staining
Mouse brains were sectioned on a cryostat, and 8 micron sections were
incubated in X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside)
staining solution at 37°C for 24 hours, as previously reported
(Bajenaru et al, 2002).
Western Blot
Mouse tissues, including brain and testis as well as spermatozoa, were
homogenized in lysis buffer (20 mM Tris pH 7.5, 10 mM EGTA, 40 mM
glycerol-2-phosphate, 1% NP40, 2.5 mM MgCl2, 2 mM sodium
orthovanadate), and total protein concentration was determined using the BCA
kit (Pierce Biotechnology, Inc, Rockford, Ill). Twenty-five micrograms of
total protein were separated by SDS-polyacrylamide gel electrophoresis and
transferred onto Immobilon membranes (Millipore, Billerca, Mass) prior to
blocking in 3% nonfat milk in PBS-Tween. The following primary antibodies were
used: ES1 Tslc1 antibody (1:20 000 dilution;
Surace et al, 2004), actin
A2066 antibody (1:1000 dilution; Sigma Chemical Co, St Louis, Mo), tubulin
T9026 antibody (1:20 000 dilution; Sigma), nectin-2 rat monoclonal antibody
(1:500 dilution; GeneTex, Inc, San Antonio, Tex), and ß-galactosidase
Z3781 antibody (1:500 dilution; Promega, Madison, Wis). Western blots were
developed using horseradish peroxidase-conjugated secondary antibodies (1:20
000) and ECL chemiluminescence (Amersham Biosciences, Fairfield, Conn),
followed by exposure to radiographic film. Quantitation was performed by
scanning densitometry using Gel Pro Analyzer software (Media Cybernetics,
Silver Spring, Md) and normalized to the tubulin expression in each
sample.
Histology and Fluorescence Immunohistochemistry
Mouse testes were fixed in 4% paraformaldehyde or Bouin solution overnight
at 4uC, paraffin-embedded, and serially cut on a microtome at 6 µm. For
gross histological analysis, hematoxylin/eosin (H&E) staining was
performed. For fluorescence immunohistochemistry, sections were
deparaffinized. Antigen retrieval was achieved by incubating the slides in 1
mM EDTA and boiling for 30 minutes in a rice cooker. After blocking
nonspecific antibody binding sites in 5% horse serum/PBS, primary antibody
(ES1, germ-cell nuclear antigen [GCNA], and polyclonal H-112 antibody [GATA4])
was applied to the slides. The ES1 antibody was used at a 1:1000 dilution,
while antibodies against the GCNA (gift from G. Enders, University of Kansas
Medical Center; Enders and May,
1994) and the GATA4 (polyclonal H-112 antibody; Santa Cruz
Biotechnology, Inc, Santa Cruz, Calif) were used at dilutions of 1:100 and
1:200, respectively. Nectin-2 (502–57) and nectin-3 (103-A1) rat
monoclonal antibodies were used at 1:200 dilution (GeneTex, Inc). For GCNA,
nectin-2, nectin-3, and ES1, Alexa 488 or Cy3-conjugated secondary antibodies
(anti-rabbit for ES1 and anti-rat for GCNA, nectin-2, and nectin-3) were used.
For GATA4, a biotinylated anti-goat secondary antibody followed by
streptavidin-Cy3 (Jackson ImmunoResearch Laboratories, Inc, West Grove, Pa)
was used. Images were analyzed using a Nikon Eclipse TE300 fluorescence
microscope equipped with a digital camera. All immunohistochemical studies
were performed alongside negative controls (without primary antibody).
Evaluation of Spermatozoa Motility and Viability
The entire epididymis from wild-type and Tslc1+/– mice was
dissected away from the testis, cut into 3–4 pieces, and incubated in 1
mL M-16 media for 15 minutes at 37uC. Spermatozoa from the entire epididymis
were released by passing the entire milliliter of suspension through a
70-µm mesh. The resulting suspension was collected in a 1.5-mL
microcentrifuge tube, and spermatozoa were collected by centrifugation at 3000
rpm for 5 minutes at 4uC. The supernatant was removed, and the spermatozoa
were resuspended in 1 mL of NIM solution (123 mM KCl, 2.6 mM NaCl, 7.8 mM
NaH2PO4, 1.4 mM KH2PO4, 3 mM EDTA
disodium salt; pH 7.2) and 1% polyvinyl alcohol. Next, 15 µl of a 1:10
dilution of the spermatozoa suspension were counted on a hemacytometer, and
>50 cells per mouse were scored as being motile or immotile. Viability was
assessed by eosinnigrosin staining of released epididymal spermatozoa
(World Health Organization,
1999).
For detection of the targeted Tslc1 allele in Tslc1+/– spermatozoa, DNA PCR was performed using primers contained within the ß-geo cassette. Briefly, high molecular weight DNA was extracted from both wild-type and Tslc1+/– epididymal spermatozoa using standard methods. Approximately 100 ng of DNA were used in a PCR reaction containing a forward primer (8117; 5'-GAC ACC AGA CCA ACT GGT AAT GGT AGC GAC-3') and a reverse primer (8118; 5'-GCA TCG AGC TGG GTA ATA AGC GTT GGC AAT-3'). PCR amplification was performed in a thermocycler at 94°C for 3 minutes, 94°C for 1 minute, 62°C for 1 minute, 68°C for 3 minutes (38 cycles), and 68°C for 10 minutes. Five microliters of the PCR reaction were electrophoresed on 1% agarose gels, stained with ethidium bromide, and visualized by UV transillumination.
TUNEL Staining
Cell death was analyzed using the TUNEL assay (Roche Applied Bioscience,
Basel, Switzerland) on 6-µm paraffin-embedded testis sections, representing
50 tubules per mouse. Specific signal was visualized by treatment with sheep
peroxidase-conjugated anti-digoxigenin antibodies (1:1000) followed by
diaminobenzidine (DAB) development.
BrdU Incorporation and Staining
Mice were injected with 5-bromo-2-deoxyuridine (BrdU, Sigma) at a dose of
200 mg/kg 2 hours before euthanasia. Testes were removed and processed for
immunohistochemistry using a mouse anti-BrdU antibody (1:200, Roche Boehringer
Mannheim, Amsterdam, Netherlands). Fifty tubules per mouse were analyzed.
Flow Cytometric Analysis of Testis Cells for DNA Ploidy
Testes were decapsulated and triturated into single cell suspensions in
Hanks balanced salt solution containing 50 µg/mL propidium iodide (Sigma),
1 mg/mL citric acid, and 0.3% NP40 for 30 minutes. Thirty thousand cells were
analyzed for DNA content on a FACScan (Becton Dickinson, San Jose, Calif) with
FlowJo software (Version 4.3, Tree Star, Inc, Ashland, Ore).
Transmission Electron Microscopy
Mice were perfused with PBS, then 4% paraformaldehyde. Testis tissue was
fixed in 2.4% glutaraldehyde, 1% paraformaldehyde, 130 mM cacodylate, and 1 mM
calcium chloride (CaCl2) solution. Following fixation, the tissues
were cut into approximately 1 mm x 1 mm pieces, which were rinsed in 0.1
M cacodylate buffer and postfixed in 1% osmium tetroxide prior to embedding in
Epon media. Ultrathin sections were visualized under a Morgagni transmission
electron microscope as previously described
(Nakai et al, 2002).
In Vitro Fertilization
In vitro fertilization (IVF) was performed by the Mouse Genetics Core at
the Washington University School of Medicine. Briefly, fresh epididymal
spermatozoa were obtained as described above from Tslc1+/– mice
and incubated in the presence of ova obtained from C57/Bl6 female mice. Four
to 6 hours after incubation, ova were washed to remove excess spermatozoa and
incubated overnight in HTF media (Quinn et
al, 1985) and embryos were transferred to pseudopregnant female
mice.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Tslc1+/– Male Mice Exhibit Reduced Fertility
While the Tslc1+/– mice did not have any obvious abnormal
phenotype and did not exhibit reduced survival, we noticed that male
Tslc1+/– mice consistently demonstrated reduced breeding. While
some matings were able to produce a very small number of pups harboring the
targeted Tslc1 gene, 8 of 14 matings of proven fertile female mice
with Tslc1+/– male mice did not result in the birth of any
Tslc1+/– pups. Pregnancies were routinely verified by plugs. In
contrast, all female Tslc1+/– mice were able to breed normally
and gave birth to pups harboring the mutant Tslc1 gene in an expected
Mendelian fashion (n > 15 matings; 46 Tslc1+/– pups out of
71 total pups).
Tslc1 Is Normally Expressed in Testicular Germ Cells But Is Nearly Absent in Tslc1+/– Testis
Tslc1 expression has been previously demonstrated in male germ cells of the
mouse testis using both histological and electron microscopic methods
(Wakayama et al, 2003). In
contrast, other members of the nectin family (e.g., nectin-2 and nectin-3)
show expression in Sertoli cells and not germ cells, or expression at
different stages of germ cell maturation, suggesting that each of these
molecules may be critical in certain gonadal cell types at specific times
during germ cell development (Ozaki-Kuroda
et al, 2002). To define the cell types expressing Tslc1 in the
normal mouse testes, we performed colocalization studies with known cell-type
specific markers (GCNA for germ cells and GATA4 for Sertoli cells) by
fluorescence immunohistochemistry using a previously generated anti-Tslc1
antibody (Surace et al, 2004).
We found that Tslc1 was expressed at the cytoplasmic membrane exclusively in
germ cells (nuclear GCNA-immunoreactive spermatogonia and primary
spermatocytes), but not in Sertoli cells with nuclear GATA4 immunostaining
(Figure 2A). Our results are
similar to those obtained using a different Tslc1 antibody, in which cell
surface expression of Tslc1 was found in intermediate/type B spermatogonia and
spermatocytes (Wakayama et al,
2003). In addition, Tslc1 expression was also detected in
elongating spermatids.
As observed in brains from Tslc1+/– mice, Tslc1 protein expression in the testes of heterozygous Tslc1 males (n = 9) also showed greater than 90% reduction compared to wild-type littermates (Figure 2B). To exclude the possibility that markedly reduced Tslc1 expression resulted in alterations in nectin-2 and nectin-3 expression in the testis, we performed immunohistochemistry and Western blotting. We observed no changes in nectin-2 expression by Western blot in whole testis lysates from Tslc1+/– mice compared to wild-type controls (Figure 2C). Similarly, no changes in the subcellular localization or expression of nectin-2 and nectin-3 by immunohistochemistry were seen in the testis from Tslc1+/– mice compared to controls (data not shown).
Tslc1+/– Testis Exhibits Disrupted Germ Cell/Sertoli Cell Association
In order to assess the effect of reduced Tslc1 expression on cellular
organization in the testis, we performed histological analysis using H&E
staining and transmission electron microscopy. H&E staining revealed
disruption of the basal compartment at the junction between germ cells and
Sertoli cells in the Tslc1+/– mouse testis at various ages (3
weeks to 7 months; n = 15 mice; Figure
2D). Even in histologically normal appearing tubules, we detected
disrupted architectural associations between germ cells and Sertoli cells by
transmission electron microscopy (Figure
2E). In contrast to wild-type testes, Tslc1+/– mice
exhibit a disorganization of the leptotene/pachytene spermatocyte region of
the seminiferous epithelium. The junctions between Sertoli cells and these
spermatocytes also appear to be disrupted, with the formation of extensive
amounts of vacuolation and spaces in the base of the seminiferous tubules
(denoted by asterisks).
Tslc1+/– Spermatozoa Exhibit Reduced Cell Motility
Given the gonadal histologic abnormalities observed in
Tslc1+/– males, we sought to determine whether the disrupted
Sertoli cell-germ cell associations result in abnormal spermatozoa maturation.
We first examined Tslc1+/– mice for changes in gonadal cell
proliferation or apoptosis. No differences in cell proliferation or apoptosis
as assessed by BrdU incorporation and TUNEL staining, respectively, were
observed between wild-type and Tslc1+/– testes (data not
shown). Next, we performed flow cytometric analyses of testis cells to detect
defects in cell ploidy, and found no statistically significant differences in
haploid (1N), diploid (2N), or tetraploid (4N) cell number between wild-type
and Tslc1+/– mice (Figure
3A). Moreover, light (n = 15 mice) and electron (n = 3 mice)
microscopic analysis of spermatozoa collected from Tslc1+/–
epididymii did not reveal any structural alterations (data not shown).
Collectively, these results suggest that cell maturation, as measured by DNA
content and morphology occurs normally in Tslc1+/– mice.
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Lastly, to determine whether Tslc1 is expressed in mature epididymal spermatozoa, Western blot analysis of wild-type mouse spermatozoa and whole testis was performed (Figure 3C). We found that Tslc1 was not expressed in mature epididymal spermatozoa, suggesting that it most likely functions as an adhesion molecule important for defining the germ cell-Sertoli cell niche required for normal germ cell maturation.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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It is not known what specific molecular cues emanate from Sertoli cells to dictate normal germ cell maturation. Recent studies have implicated nectin molecules as important proteins that mediate cell-cell interactions which result in the generation of specific intracellular signals that drive normal male gametogenesis. Examination of nectin-2 and nectin-3 expression and function in the male gonad demonstrate that these molecules are critical not only for the proper maintenance of normal cell architecture within the seminiferous tubule but also for the formation of mature spermatozoa (Bouchard et al, 2000; Ozaki-Kuroda et al, 2002; Mueller et al, 2003). In this regard, male mice lacking either nectin-2 or nectin-3 are infertile (Bouchard et al, 2000; Ozaki-Kuroda et al, 2002; Mueller et al, 2003; Inagaki et al, 2005; Miyoshi and Takai, 2005).
Immunohistochemical analyses have shown that each nectin protein has a distinct pattern of expression: Sertoli cells express nectin-2, but lack nectin-3 and Tslc1 expression (Ozaki-Kuroda et al, 2002; Wakayama et al, 2003). Whereas Tslc1 is expressed in spermatogonia, spermatocytes, and spermatids, nectin-3 expression is found only in spermatids (Guttman et al, 2004). This temporal pattern of Tslc1 and nectin-3 expression suggests that their functions are nonredundant and that nectin-3 cannot compensate for Tslc1 loss during early spermatogenesis. Moreover, we performed immunostaining and Western blot analysis of nectin-2 and -3 in Tslc1+/– testis and found no differences in their overall expression patterns or cellular localization compared to wild-type controls. This result further supports the notion that other nectins expressed in the male gonad do not compensate for Tslc1 loss.
While no detailed reports exist describing the structural or functional abnormalities in nectin-3 knockout mice, nectin-2-deficient mice produce severely deformed spermatozoa with malformations of the head and midpiece but no defects in either sperm viability or motility (Mueller et al, 2003). The infertility phenotype of mice lacking nectin-2 likely reflects defects in spermatozoa-zona binding and sperm-oocyte fusion. Moreover, the Sertoli-spermatid junctions in these nectin-2-deficient mice are virtually devoid of the actin-binding protein, espin. The defect in spermatogenesis in Tslc1+/– mice is distinct from that observed in nectin-2-deficient mice, suggesting a unique function for Tslc1. In contrast to nectin-2-deficient mice, Tslc1+/– spermatozoa were morphologically normal and demonstrated disrupted interactions with Sertoli cells at an early stage in sperm maturation. Since Tslc1 is only expressed in germ cells and not in mature epididymal spermatozoa, binding between Tslc1 on germ cells and cell surface proteins expressed on Sertoli cells imparts a novel signal important for male germ cell maturation. We hypothesize that Tslc1 is critical for specifying interactions between germ cells and Sertoli cells during early testicular germ cell maturation. Impairment of this interaction in Tslc1+/– mice results in incomplete spermatogonia maturation, abnormal spermatozoa motility, and reduced male fertility.
In this report, we show that Tslc1 (nectin-like 2 molecule) has a unique role in the maintenance of the Sertoli cell/germ cell association, which is critical for the acquisition of the motile sperm phenotype. These results extend our understanding of the role of the nectin family of proteins in specifying male germ cell maturation and demonstrate that each nectin protein functions in a nonredundant fashion at a temporally distinct phase of germ cell development. Additional studies on the role of TSLC1 in gonadal maturation relevant to the acquisition of a motile sperm phenotype may provide important insights into the molecular pathogenesis of male-factor fertility in men with isolated asthenospermia.
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Acknowledgments |
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 C. Q.F. Wang and C. Y. Cheng A seamless trespass: germ cell migration across the seminiferous epithelium during spermatogenesis J. Cell Biol., August 9, 2007; 178(4): 549 - 556. [Abstract] [Full Text] [PDF]
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 T. Wakayama, Y. Sai, A. Ito, Y. Kato, M. Kurobo, Y. Murakami, E. Nakashima, A. Tsuji, Y. Kitamura, and S. Iseki Heterophilic Binding of the Adhesion Molecules Poliovirus Receptor and Immunoglobulin Superfamily 4A in the Interaction Between Mouse Spermatogenic and Sertoli Cells Biol Reprod, June 1, 2007; 76(6): 1081 - 1090. [Abstract] [Full Text] [PDF]
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