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From the Department of Biological Sciences, University of Delaware, Newark, Delaware.
| Correspondence to: Patricia A. Martin-DeLeon, Department of Biological Sciences, University of Delaware, Newark, DE 19716 (e-mail: pdeleon{at}udel.edu). |
| Received for publication April 19, 2006; accepted for publication August 4, 2006. |
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Abstract |
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55–56 kd, contributing modestly to the overall neutral hyase
activity. This activity is associated with in vitro cumulus penetration, since
antibody inhibition of Hyalp1 significantly (P = .034) retarded the
rate of penetration of wild-type (WT) sperm. Antibody-inhibited Spam1
null sperm were more severely retarded (P = 4.2 x
10–19), suggesting an up-regulation of Hyalp1 in
these mice. A functionality test of the hyaluronic acid (HA) receptor domain
identified in the N-terminus by in silico analysis revealed that sperm Hyalp1
is significantly (P = .006) involved in the progesterone-induced
HA-enhanced acrosome reaction. Finally, developmental reverse transcription
polymerase chain reaction (RT-PCR) shows that testicular transcripts of
Hyalp1 are detected as early as 6 days postparturition, similar to
transcripts for Spam1, suggesting that the gene might also play a
role in the developing testes prior to spermiogenesis. Taken together, the
findings reveal that Hyalp1 likely has a unique function in the adult testis,
and redundant overlapping ones with Spam1 and may compensate for it in
Spam1 null mice.
Key words: Sperm antigen, hyaluronidase activity, Spam1 null sperm, acrosome reaction, cumulus penetration, hyaluronic acid receptor
In addition to hyase activity, SPAM1 has been shown to perform other roles in fertilization, including ZP binding and acrosomal exocytosis (Hunnicutt et al, 1996; Sabeur et al, 1998; Cherr et al, 1999; Vines et al, 2001). However, sperm from Spam1 null mice are fertile with a delay in cumulus penetration, indicating functional compensation for a lack of Spam1 by other proteins (Baba et al, 2002). Hyalp1 and Hyal5 are conspicuous candidates for this position because of strong sequence homology and similarity of domains with Spam1 (Zhang et al, 2005). Further, the Rb(6.16) and Rb(6.15) Robertsonian translocations, which were shown to bear point mutations of Spam1 and to be associated with sperm dysfunction in homozygotes, as well as transmission ratio distortions (TRDs) in the heterozygotes (Zheng and Martin-DeLeon, 1997; Zheng et al 2001a,b), were recently shown to also carry mutations in Hyalp1 and Hyal5 (Zhang et al, 2005). Thus, mutations in each of these 3 hyases seemingly contribute to the TRDs. Interestingly, attempts at overexpression of both Spam1 and Hyal5 have met with strong resistance, suggesting stringent regulatory control of the reproductive hyases (Zhang et al, 2006).
Four separate domains for functional activity exist within SPAM1 and account for each of its independent roles in fertilization: neutral and acidic hyaluronidase activity (Cherr et al, 2001), HA-binding (Sabeur et al, 1998; Morales et al, 2004; Zhang et al, 2005), and ZP-binding (Myles and Primakoff, 1997; Cherr et al, 2001). Neutral enzymatic activity, a characteristic unique to the reproductive hyases, dominates the membrane-bound SPAM1 and is responsible for penetration of the cumulus cell-oocyte matrix. Second, soluble SPAM1 cleaved during the acrosome reaction is characterized by acidic hyase activity and is responsible for digestion of hyaluronan or HA in the ZP (Hunnicutt et al, 1996; Cherr et al, 2001). A hyaluronan-binding domain exists in SPAM1 separately from either hyase domain and contributes to the acrosome reaction signaling pathway via a HA-enhanced increase in Ca2+ influx after the acrosome reaction (Sabeur et al, 1998; Cherr et al, 1999; Vines et al, 2001). The signal transduction of the acrosome reaction, required for the release of soluble enzymes that aid in the penetration of the ZP, has been linked to SPAM1 in humans (Cherr et al, 2001) and mice (Morales et al, 2004). Fourth, a ZP binding domain in the carboxy-terminal end of SPAM1 facilitates secondary ZP binding after the acrosome reaction (Hunnicutt et al, 1996; Myles and Primakoff, 1997).
While HYALP1 is a pseudogene in humans
(Csoka et al, 1999), its RNA
and protein are abundantly expressed in the testis of the mouse
(Zhang et al, 2005), where it
is located 27.2 kb upstream of Spam1. However, apart from a
knowledge of the location of the murine gene (Hyalp1) and the size of
the mRNA and protein (Zhang et al,
2005), nothing has been published on its functional role. This
investigation thus focuses on the expression and physiological role of
Hyalp1 in murine reproduction and its potential to compensate for a
lack of functional Spam1. Our results reveal a possible unique role
for Hyalp1 in the testis as well as in sperm, where it has a functional
overlap with the well-characterized Spam1. Importantly, the data suggest a
cross-talk between the encoding genes.
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Materials and Methods |
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Animals and Reagents
Sexually mature ICR or C57BL/6 (Harlan Sprague-Dawley, Indianapolis, Ind)
mice were used in all studies except developmental reverse transcription
polymerase chain reaction (RT-PCR), where ages are indicated. Studies were
approved by the Institutional Animal Care and Use Committee at the University
of Delaware and conform to the Guide for the Care and Use of Laboratory
Animals published by the National Institutes of Health (publication
85–23, revised 1985). Spam1 null mice on the ICR background
were obtained from the laboratory of Tadashi Baba in Japan. All chemicals were
purchased from Sigma Chemical Company (St Louis, Mo) or Fisher Scientific
Company (Malvern, Pa), unless otherwise specified.
Development of Hyalp1 Antibody
A polyclonal anti-peptide (Zymed Laboratories, South San Francisco, Calif)
was created for a unique C-terminus sequence for Hyalp1 (GenBank accession
number AK106575) by way of a PolyQuik rabbit antiserum. C-terminus specificity
was assessed by competing with the peptide (amino acid residues 477–494:
C YNGNFSLKPLKRREIIFL) used in development of anti-serum, which has been
previously characterized (Zhang et al,
2005).
Protein Extraction From Whole Testicular Tissue and Sperm
This was performed as described in Zhang et al
(2004). Briefly, whole
testicular tissue was manually homogenized (using a mortar and pestle) with a
solubilization buffer (62.5 mM Tris-HCl, 10% glycerol, 1% SDS, pH 6.8)
containing a protease inhibitor, 1% phenylmethylsulfonyl fluoride (PMSF) at
4°C. The suspension was then centrifuged at 12 000 x g at
4°C for 15 minutes and the supernatant collected. Cauda sperm were
collected from minced epididymides and incubated in 5 mL sperm suspension
buffer (50 mM Tris, 20 mM EDTA containing 1% PMSF at 37°C) to disperse
sperm and allow them to swim out. Tissues were pelleted at 500 x
g for 2 minutes and sperm collected from the suspension by
centrifugation at 1000 x g for 10 minutes at 4°C. Protein
extracts were prepared by lysing sperm with solubilization buffer as above and
the suspension vigorously vortexed for 4 minutes prior to centrifugation. The
supernatants from testis and sperm were collected and protein concentrations
measured using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford,
Ill), using different concentrations of BSA as standards.
HA Substrate Gel Electrophoresis
Hyase activity in testicular and sperm protein extracts was measured using
HA substrate gel electrophoresis (HASGE), as described by Guntenhoner et al
(1992) and Deng et al
(2000). HA from bovine vitreous
humor or human umbilical cord was added to a 15% (10% in the case of sperm
protein) SDS-polyacrylamide gel (final concentration 0.15–0.3 µg/mL).
Approximately 30 minutes before the gels were loaded, the protein samples were
treated with either 1:10 dilution of preimmune serum or Hyalp1 antiserum. Gels
were loaded with 60 µg protein for the testis or 6 µg for sperm and run
at 15 mA. BSA negative controls were also loaded. After completion of
electrophoresis, gels were incubated in 3% TritonX-100 in PBS for 2 hours at
RT, then at 37°C for 24 to 36 hours in 100 mM sodium acetate (pH 7.0). To
visualize digestion of HA, gels were stained with 0.5% alcian blue in 3%
acetic acid for 2 hours, and then destained in 7% acetic acid until digestion
was visible. Gels were counterstained with Coomassie Brilliant Blue G-250 and
destained with methanolacetic acid and scanned to image the bands.
Immunocytochemistry of Epididymal Sperm
To visualize the localization of Hyalp1 relevant to cumulus penetration
(ie, protein available for activity on the sperm surface), immature and mature
sperm were collected in PBS by mincing caput and cauda epididymides and
allowing sperm to float or swim out, respectively, after slight agitation of
the dish. For caput epididymides, mincing tissues allows sperm to disperse in
the PBS, albeit at a lower concentration than that for motile sperm, which are
able to swim. However, adequate numbers of caput sperm were obtained for
immunocytochemistry in each trial using this method. Briefly, sperm were
washed and blocked for 30 minutes in 3% bovine serum albumin (BSA) and 1%
normal donkey serum in PBS. Sperm were incubated overnight at 4°C in
rabbit polyclonal antibody against mouse Hyalp1, then for 30 minutes at RT in
FITC-labeled donkey anti-rabbit IgG. Preimmune rabbit serum was used in place
of Hyalp1 antibody as a control. Sperm were then mounted in p-phenylenediamine
antifade with 1.5 µg/mL of 4' 6-diamidino-2-phenylinodole (DAPI) and
viewed on a Zeiss Axiophot fluorescence microscope (Thornwood, NY) using the
appropriate FITC or DAPI filter set. Imaging was done using a CCD-cooled
camera.
In Vitro Fertilization Vestment Penetration
Female ICR mice 8–10 weeks old were superovulated using successive
intraperitoneal injections of pregnant mare's serum gonadotropin (PMSG) and
human chorionic gonadotropin (HCG) 48 hours apart (7.5 IU each). Eggs were
collected from oviductal ampulla 13.5–14 hours after HCG injection and
placed in 1 mL human tubal fluid (HTF), covered with mineral oil, and
equilibrated at 37°C under 5% CO2, 5% O2, 90%
N2. Fresh caudal epididymal sperm from both WT (Spam1 +/+)
sperm and sperm lacking functional Spam1 (Spam1 –/–) were
capacitated for 30–45 minutes in 1 mL HTF under the same conditions, as
described by Chen et al (2006).
Capacitated sperm (7.5 x 104) were added to treatment dishes
containing 0.5 mL HTF and either preimmune serum or Hyalp1 antiserum (1:5) for
30 minutes. Eggs were then incubated with sperm, and individual eggs were
scored at 20-minute intervals (total 6 hours) for the degree of cumulus
penetration.
Assessment of penetration through the cumulus of oocytes was similar to that of Baba et al (2002). Briefly, the progress of cumulus cell-removal was categorized in 4 stages (1–4), based on the appearance of the cumulus masses after microscopic observation. Stage 1 had eggs with tightly packed cumulus cells (such that the eggs were fully occluded); Stage 2 had cumulus masses in which the eggs could be visualized with loosely associated cumulus cells; Stage 3 had eggs in which most of the cumulus cells had been dispersed from the masses; and Stage 4 had eggs with complete dispersal of the cumulus cells. The data were subjected to statistical analysis, using Fisher's exact test, to determine significant differences in the number of eggs penetrated for the different treatments.
Progesterone Enhanced Acrosome Reaction Assay
Induction of acrosome reactions was similar to that of Sabeur et al
(1998) and Morales et al
(2004). Briefly, fresh cauda
epididymal sperm (2.5 x 105) from age-matched sexually mature
WT ICR males were suspended in Biggers Whitten Whittingham (BWW) medium and
then treated under several conditions. Controls were treated with 0.1% DMSO in
BWW-Hepes for 5 minutes. Preimmune serum or Hyalp1 antiserum (1:50) was
incubated for 10 minutes at RT. Samples treated with HA (100 µg/mL) for 30
minutes were successively treated with progesterone (PG; 3.18 µM) for 5
minutes before fixation with 4% paraformaldehyde. Slides were prepared,
stained with 0.44% Coomassie Brilliant Blue G-250 in 60% methanol-acetic acid,
rinsed in ddH2O, and sealed with Permount. Slides were
microscopically analyzed, and at least 200 sperm were scored for the presence
(+) or absence (–) of the acrosome cap, as previously described
(Morales et al, 2004). The
results were subjected to a Kruskal-Wallis one-way ANOVA on ranks for
statistical analysis.
Reverse Transcriptase-PCR
Total testicular RNA was extracted from whole testis using Trizol reagent
according to manufacturers protocol, and samples were treated with RNase-free
DNase (final concentration 5–10 U/mL; Boehringer Mannheim, Basel,
Switzerland) followed by phenol/chloroform extraction. Reverse transcriptase
control (–) and test (+) samples were prepared using 2 µL of RNA
extract with a SuperScript Preamplification system. Amplification was carried
out using a forward primer (nts 1976–1999) 5' agg cat tag gaa gta
cta gag agc 3' and reverse primer (nts 2288–2305) 5'tct tct
ggg ttt gct cct 3'. PCR was performed under the following conditions: 35
cycles of 94°C for 3 minutes, 54°C for 2 minutes, 72°C for 12
minutes. PCR products were resolved on a 1% agarose gel stained with ethidium
bromide.
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Results |
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41% homologous along the length of the
proteins, homology and identity in the putative regions for enzymatic activity
are considerably greater than that along the entirety of the proteins. Further
examination of ClustalW alignment of the reproductive hyases
(Zhang et al, 2005) revealed
that Spam1 and Hyalp1 are 29% identical along the length of the proteins, but
within the neutral hyase domain of Spam1, this identity increases to 49%. The
2 proteins have the highest identity, 59%, in the region for HA-binding
receptor. There is 41% identity in the acidic hyase domain and 54% in the ZP
binding domain. These high levels of identity within the functional domains
suggest important physiological roles for Hyalp1 in fertilization. Immunocytochemical analysis reveals the presence of Hyalp1 on the surface of both caput (immature) and caudal (mature) epididymal sperm (Figure 1). On caput sperm the protein is dispersed on the head, with heavier deposits near the posterior. However, it is very distinctly localized to the acrosome cap of caudal sperm. This localization is similar to that of membrane-bound Spam1 (Deng et al, 1999) and implies functionality on the sperm surface before the acrosome reaction, that is, in cumulus penetration. Both preimmune serum and Hyalp1 peptide-inhibited antiserum served as controls and assured antibody specificity, as treatment of sperm under these conditions did not produce specific staining.
For sperm where Western analysis showed 2 Hyalp1 isoforms of 40 and 66
kd (Zhang et al, 2005), HASGE
assays revealed that the bands for all the murine hyases comigrate
(Figure 2A), as previously
shown (Deng et al, 2000). In
Figure 2A, the addition of
Hyalp1-specific antiserum slightly diminished the overall hyase activity in
the comigrated bands compared to those samples that were treated with
preimmune serum. The missing region of the comigrated bands was in the range
55–56 kd, suggesting that the neutral hyase activity of Hyalp1
results from an isoform of this MW. Based on the size of the missing region,
it appears that Hyalp1 contributes only modestly to the overall neutral hyase
activity.
Previous studies have shown via Western blots
(Zhang et al, 2005) that the
predominant isoform of Hyalp1 in the testis is a 24-kd protein, and HASGE
analysis in this study indicates that this isoform is enzymatically active.
HASGE assays showed the disappearance of a single distinct clear band of
24 kd at pH 7.0 when testicular proteins were treated with Hyalp1
antiserum (Figure 2B). The
clear band seen in the protein sample treated with preimmune serum
demonstrates hyase activity, and the identity of the contributing protein is
confirmed by its disappearance with the addition of Hyalp1-specific antiserum.
Other clear digestion bands with higher MWs were not noticeably affected by
the addition of Hyalp1 antiserum. BSA controls revealed no hyase activity
(data not shown).
A total of 389 cumulus oocyte complexes were examined in 4 groups for sperm penetration. Groups 1 and 2 were incubated with WT sperm treated or untreated with Hyalp1 antiserum, while groups 3 and 4 were Spam1 null sperm similarly treated and incubated. No significant difference was observed between sperm incubated with and without preimmune serum in HTF (data not shown). However, antibody inhibition of Hyalp1 on WT (Spam1 +/+) sperm was shown to retard cumulus penetration through all 4 stages (Figure 3Aa and b). A Fisher's exact test indicates statistical differences (P = .034) between the cumulus penetration of antibody inhibited and noninhibited WT sperm after 6 hours of incubation (Figure 3B).
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To test the functionality of the HA-binding domain on Hyalp1, mature WT sperm were subjected to a progesterone-induced HA-enhanced acrosome reaction assay. Progesterone increased the rate of acrosome reaction in the presence of HA (P = .005); however, the addition of Hyalp1 antiserum significantly reduced the rate of acrosome reaction despite treatment with HA and PG (P = .006), as seen in Figure 4. Thus the HA receptor on Hyalp1 is functional and contributes to the HA-binding activity that induces acrosomal exocytosis.
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Testicular Hyalp1 transcripts were detected as early as 6 days postparturition, but not in the testis of 3-day neonates (Figure 5). The negative PCR result at 3 days was not due to the quality of the RNA or PCR failure, as PCR on the identical cDNA samples revealed the presence of transcripts for Hyal3 (K. Reese, personal communication), a somatic hyase on chromosome 9 (Csoka et al, 1999). Developmental RT-PCR reveals that expression of Hyalp1 RNA continues through adulthood. Though seeming to be expressed earlier than Spam1 and Hyal5, which by Northern analysis were previously reported to first appear at 21 days (Zheng et al, 2001b; Kim et al, 2005), re-evaluation of the expression of these hyases revealed that both their RNA and protein can be detected in the testis of 6-day neonates by RT-PCR and Western blot, respectively (data unpublished). Thus the 3 reproductive hyases are expressed in early testis development.
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Discussion |
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Immunocytochemical analysis in this study confirms the finding from Western analysis that the protein is present on sperm (Zheng et al, 2005), and localizes it to the acrosome cap of mature cells. The presence of Hyalp1 in a more uniform distribution on immature caput sperm and its localization on caudal sperm is similar to Spam1 (Deng et al, 1999) and suggests that it undergoes reorganization on the sperm surface during epididymal maturation, similar to Spam1. In this regard it should be mentioned that, similar to Spam1 (Martin-DeLeon, 2006), Hyalp1 has been detected in sperm-free epididymal tissues and epididymal luminal fluid (unpublished data).
At pH 7.0, hyase activity of Hyalp1 was shown for an 55–56-kd
isoform in sperm, as there was a detectable reduction of the comigrated HASGE
bands in this region in the presence of Hyalp1 antiserum. This isoform is
smaller than the 66-kd protein found in mature sperm
(Figure 2A;
Zhang et al, 2005), so it is
likely that hyase activity of Hyalp1 results from processing of a larger
isoform either by deglycosylation or endoproteolytic cleavage, as seen for
Spam1 (Deng et al, 1999).
Neutral hyase activity on mouse sperm has been shown to occur for a 52-kd
Spam1 isoform (Baba et al,
2002) and a 55-kd Hyal5 isoform
(Kim et al, 2005), and we have
reported these sperm proteins to be 67 and 66 kd, respectively
(Deng et al, 1999,
Zhang et al, 2005). Since
neutral hyase activity is unique to the reproductive hyases
(Csoka et al 1999), the finding
of an 55–56-kd Hyalp1 isoform with neutral hyase activity supports
the inclusion of Hyalp1 in this category of sperm proteins. It also indicates
that sperm hyase activity in the mouse is controlled by at least 3 genes. In
testicular proteins where the 24-kd isoform of Hyalp1 was the only detectable
digestion band to disappear with Hyalp1 antiserum inhibition, there was no
other hyase activity from proteins of a similar MW. This suggests that hyase
activity of this Hyalp1 isoform may be unique in the testis.
Vestment penetration assays conducted in vitro illustrate that the neutral
hyase activity detected for Hyalp1 plays a role in the penetration of the
cumulus oocyte complex. Antibody-inhibition of Hyalp1 on mature sperm
significantly retarded cumulus penetration on WT sperm, which is consistent
with its hyase activity. While the difference between penetration by WT and
Spam1 null sperm (60%; Figure
3B) was similar to that reported by Baba et al
(2002), penetration for null
sperm was more significantly retarded by Hyalp1 antibody-inhibition than in WT
sperm. The data therefore suggest an up-regulation of Hyalp1 in
Spam1 null sperm, which were more severely inhibited by Hyalp1
antiserum.
In Spam1 null mice, up-regulation of Hyalp1 would increase the contribution of Hyalp1, which would thus constitute proportionately more of the total sperm hyases than present in WT sperm. When Hyalp1 is antibody-inhibited, effectively creating "double null" sperm, cumulus penetration is severely impeded. This could result if Hyalp1 is a major contributor in the absence of Spam1, and strongly suggests compensatory activity of this hyase. The failure of complete blockage of cumulus penetration in the "double null" sperm argues for the presence of other functional sperm hyase(s). Recently, Hyal5 was shown to be involved in murine sperm penetration through the cumulus (Kim et al, 2005). However, Kim et al (2005) concluded that mouse epididymal sperm contain only 2 hyaluronan-hydrolyzing proteins, Hyal5 and Spam1 (PH-20). Our previously published work (Zhang et al, 2005) and the results in the present study argue cogently against this and show that Hyalp1 contributes to hyaluronan-hydrolyzing activity on sperm. Importantly, our findings make a strong case that hyaluronan-hydrolyzing activity of the extracellular matrix of the cumulus is a polygenic trait in the mouse.
It is important to consider the role that may be played by the predominant
40-kd Hyalp1 sperm isoform that is not involved in hyaluronan-hydrolyzing
activity. Kim et al (2005),
using biotinylated ZP, showed that the ZP-binding domain of Spam1 and Hyal5
functions very little in sperm-egg interaction. We speculate that the
predominant 40-kd Hyalp1 isoform on sperm may function in this capacity
and may be the major ZP-binding hyase.
Hyalp1 could also have compensatory effects with respect to the induction of the acrosome reaction. Binding of SPAM1/Spam1 to HA, via its receptor, in the presence of progesterone in the uterine environment induces acrosomal exocytosis and has been demonstrated in vitro (Sabeur et al, 1998; Morales et al, 2004). In silico analysis indicates that Hyalp1 retains 59% identity with Spam1 in this HA-binding domain, the highest identity for the functional domains. The significant reduction in the rate of acrosome reaction for WT sperm after incubation with Hyalp1 antiserum indicates the involvement of Hyalp1 in the acrosome reaction signaling pathway in WT mice, which likely acts by the same mechanism as does that for Spam1. However, further investigation is required to implicate the involvement of Hyalp1 in compensation for Spam1 with respect to HA-binding.
Taken together, the data reveal that Spam1 and Hyalp1 have redundant, overlapping functions. Hyalp1 should therefore be considered a likely candidate in the compensation for Spam1 in Spam1 null sperm. The discovery of Hyalp1 functionality does not detract from the potential for additional compensatory or perhaps even cooperative activity of other proteins in Spam1 null mutants. With the knowledge that Hyalp1 can compensate for Spam1 physiologically, it is important to investigate the mechanism of gene control and cross-talk between the reproductive hyases.
Finally, early detection of Hyalp1 RNA in the testis of 6-day postnatal mice indicates for the first time a role for the reproductive hyases in the premeiotic stage of spermatogenesis. Further evaluation of Spam1 and Hyal5, which had previously been studied only with Northern analysis (Zheng and Martin-DeLeon, 1997; Kim et al, 2005), revealed a similar early expression of transcripts using RT-PCR, as well as the proteins (our unpublished data). This finding suggests a potentially novel role for the proteins of this gene family during spermatogenesis. Germ cells begin migration through the testis at birth; at 4–6 days postparturition, these germ cells implant into the basement membrane of the seminiferous tubules and are capable of differentiation into type A spermatogonial stem cells (McLean et al, 2003). While Hyalp1 RNA is undetected in 3-day testes, suggesting that the protein might not be involved in the migration of the gonocytes from the center of the seminiferous tubule to the basement membrane, its early expression at 6 days suggests that it might be present in type A spermatogonia and may play a possible role in the initiation or progression of spermatogenesis. Further studies are underway to investigate the role of this protein in the testis during spermatogenesis.
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Acknowledgments |
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Footnotes |
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P = .007), but
antibody-inhibition of Hyalp1 significantly reduces the rate of acrosome
reaction in the presence of HA and PG (
P = .006)
(Kruskal-Wallis one-way ANOVA on ranks).