Biomedical subjects

Patricia A Martin-DeLeon

Publications and source records attributed to Patricia A Martin-DeLeon.

9 recordsLinked to original sources

PubMed2006

Hyalp1 in murine sperm function: evidence for unique and overlapping functions with other reproductive hyaluronidases.

While Sperm adhesion molecule 1 (SPAM1) is the highly conserved mammalian sperm hyaluronidase (hyase), multiple hyases are present in the mouse testis. In this study we show that one of the murine hyases, Hyalp1, which is predominantly expressed in the testis in a 24-kd isoform has neutral enzymatic activity. On sperm, Hyalp1 is localized on the plasma membrane of the anterior head and was shown to have neutral hyase activity for an isoform of approximately 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.

Amino Acid Sequence↗

PubMed2006

Epididymal SPAM1 is a marker for sperm maturation in the mouse.

Sperm adhesion molecule 1 (SPAM1), is a glycosyl phoshatidylinositol-linked sperm membrane protein that is dually expressed in testis and epididymis. Epididymal SPAM1 is secreted in all three regions of the epididymis in all mammalian species studied, including humans. It shares the same molecular mass and neutral hyaluronidase activity as the testicular and sperm isoforms that are responsible for the penetration of the cumulus during fertilization. Using wild-type (W/T) sperm and those from mice homozygous for either a null (Spam1-/-) or mutant Spam1 allele, which results in decreased mRNA and protein, we demonstrate that sperm binding of epididymal SPAM1 occurs in vitro after exposure to W/T sperm-free epididymal luminal fluid (ELF). Binding or adsorption that occurred after incubation at room temperature or 32 degrees C was detected immunocytochemically and confirmed quantitatively using flow cytometry. The localization of SPAM1 on the plasma membrane of Spam1-null sperm mimicked that seen in the W/T. The remarkable increase in binding on W/T caudal sperm indicates that they are not fully saturated with SPAM1 during storage, and suggests that uptake of epididymal SPAM1 in vivo augments testicular SPAM1. Spam1-null sperm exposed to W/T ELF for 45-60 min during in vitro capacitation to allow epididymal SPAM1 binding showed a highly significant (P < 0.001) increase in cumulus penetration after 6-7 h compared to those incubated in ELF from null males. Similarly, the number of cumulus-free oocytes was also highly significantly greater (P < 0.001) than that for sperm capacitated in W/T SPAM1-antibody-inhibited ELF. Because epididymal SPAM1 uptake significantly increases cumulus penetration, we conclude that it is a marker of sperm maturation.

Animals↗

PubMed2006

Epididymal SPAM1 and its impact on sperm function.

The most widely conserved mammalian sperm antigen is sperm adhesion molecule 1, SPAM1/PH-20, which is also the major testicular hyaluronidase. This multifunctional glycosyl phosphatidylinositol (GPI)-linked protein plays several roles in fertilization and is encoded by a gene that resides among hyaluronidase family members in a cluster on human 7q31/mouse 6A2. In the human cluster, SPAM1 is the only functional hyaluronidase and of all six hyaluronidases in the genome it is the best characterized, both structurally and functionally. While SPAM1 transcripts are abundantly expressed only in the testis, specifically in spermatids, the RNA and protein are present in the male reproductive tract and accessory organs and in the female tract of mice. Our investigation of the post-testicular expression of SPAM1 shows that the protein is widely expressed in the epididymis. Like testicular SPAM1, epididymal SPAM1 (ES) has hyaluronidase activity and is conserved in at least five species (mouse, rat, bull, macaque, and human) all of which have putative androgen response elements in the gene promoters, consistent with androgen regulation. Testicular lumicrine factors have also been implicated in ES regulation. Based on regional expression, the protein is likely to play a role in both sperm maturation and storage. A minor secretory glycoprotein, ES is present in the epididymal luminal fluid in both a soluble and insoluble form (epididymosomes), with the latter having an intact lipid anchor. Genetic approaches have provided evidence for sperm uptake of ES in vivo, and in vitro uptake has been demonstrated with the use of Spam1 null mice. In vitro acquisition of ES on the sperm surface results in a pattern that mimics the wild-type distribution. More importantly it significantly increases the ability of null sperm to penetrate the cumulus of oocytes via hyaluronidase activity, directly relating ES uptake with fertilizing ability and indicating that ES is a marker of sperm maturation.

Animals↗

PubMed2005

Spam1-associated transmission ratio distortion in mice: elucidating the mechanism.

BACKGROUND: While transmission ratio distortion, TRD, (a deviation from Mendelian ratio) is extensive in humans and well-documented in mice, the underlying mechanisms are unknown. Our earlier studies on carriers of spontaneous mutations of mouse Sperm Adhesion Molecule 1 (Spam1) suggested that TRD results from biochemically different sperm, due to a lack of transcript sharing through the intercellular cytoplasmic bridges of spermatids. These bridges usually allow transcript sharing among genetically different spermatids which develop into biochemically and functionally equivalent sperm. OBJECTIVES: The goals of the study were to provide support for the lack of sharing (LOS) hypothesis, using transgene and null carriers of Spam1, and to determine the mechanism of Spam1-associated TRD. METHODS: Carriers of Spam1-Hyal5 BAC transgenes were mated with wild-type female mice and the progeny analyzed for TRD by PCR genotyping. Sperm from transgene and Spam1 null carriers were analyzed using flow cytometry and immunocytochemistry to detect quantities of Spam1 and/or Hyal5. Transgene-bearing sperm with Spam1 overexpression were detected by fluorescence in situ hybridization. In wild-type animals, EM studies of in situ transcript hybridization of testis sections and Northern analysis of biochemically fractionated testicular RNA were performed to localize Spam1 transcript. Finally, AU-rich motifs identified in the 3' UTR of Spam1 RNA were assayed by UV cross-linking to determine their ability to interact with testicular RNA binding proteins. RESULTS: The Tg8 line of transgene carriers had a significant (P < 0.001) TRD, due to reduced fertilizing ability of transgene-bearing sperm. These sperm retained large cytoplasmic droplets engorged with overexpressed Spam1 or Hyal5 protein. Caudal sperm from transgene carriers and caput sperm of null carriers showed a bimodal distribution of Spam1, indicating that the sperm in a male were biochemically different with respect to Spam1 quantities. Spam1 RNA was absent from the bridges, associated exclusively with the ER, and was shown to be anchored to the cytoskeleton. This compartmentalization of the transcript, mediated by cytoskeletal binding, occurs via protein interactions with 3' UTR AU-rich sequences that are likely involved in its stabilization. CONCLUSION: We provide strong support for the LOS hypothesis, and have elucidated the mechanism of Spam1-associated TRD.

Alleles↗

PubMed2004

Spam1 (PH-20) expression in the extratesticular duct and accessory organs of the mouse: a possible role in sperm fluid reabsorption.

A widely conserved sperm antigen, the sperm adhesion molecule 1 (SPAM1 or PH-20) is a glycosylphosphatidyl inositol-linked protein with multiple roles in mammalian fertilization. It has been shown to be dually expressed in testis and epididymis and this is conserved in the four species (mouse, rat, macaques, humans) that have been studied to date. Here, we report Spam1 RNA and protein expression in the murine vas deferens and efferent ducts. In situ hybridization and immunohistochemistry indicate that transcript and protein are distributed in the nonciliated epithelial cells and that the efferent ducts have the most intense staining of all three regions of the excurrent ducts. Spam1 products were also present in the accessory organs, the prostate, and seminal vesicles and its fluid. Using hyaluronic acid substrate gel electrophoresis, hyaluronidase activity at pH 7.0 was detected in the vas deferens but was absent from the efferent ducts, the prostate, and the seminal vesicles/fluid. This suggests that Spam1 may play a nonenzymatic role in these organs. In the efferent ducts, where Spam1 is enriched in the apical (but not basolateral) membrane of nonciliated cells, it is likely to play a role in sperm concentration, which is the established function of that organ.

Animals↗

PubMed2004

Cytoplasmic localization during testicular biogenesis of the murine mRNA for Spam1 (PH-20), a protein involved in acrosomal exocytosis.

The Sperm Adhesion Molecule1 (SPAM1) is the most widely conserved sperm antigen with important roles in mammalian fertilization. Light and electron microscopy were used to localize, by in situ hybridization, the cellular and subcellular sites of Spam1 mRNA in the murine testis. Transcripts were first detected in step 3 round spermatids, gradually increased until step 8 and abruptly decreased between steps 9-11. They were predominantly localized near the ER and were not dispersed throughout the cytoplasm. Immunohistochemistry revealed that Spam1 is present on both the head and tail of sperm in the seminiferous tubules, and provided support for transcriptional regulation of its transcript. Immunocytochemistry confirmed the location of Spam1 on the tail of testicular sperm and demonstrated that it is localized to both the principal piece and the midpiece. Spam1 on epididymal sperm is localized to the midpiece of the tail and changes from a uniform distribution on the head in the caput to a regionalized pattern, first on the posterior and then on the anterior head, in caudal sperm. Spam1 on the surface of caudal sperm was shown to mediate the increase in acrosome reactions induced by the synergistic effects of HA and progesterone, as confirmed in sperm from the Rb(6.16) translocation-bearing mice which are Spam1 mutants. The similar response of human and mouse sperm to these agonists of the acrosome reaction, underscores the usefulness of the mouse as a model to study physiological aspects of SPAM1 in humans where, unlike the mouse, it is the only sperm hyaluronidase.

Acrosome↗

PubMed2004

Expression and secretion of rat SPAM1(2B1 or PH-20) in the epididymis: role of testicular lumicrine factors.

Rat sperm surface antigen Sperm Adhesion Molecule1, SPAM1 (a.k.a. 2B1 or PH-20) is a plasma membrane-bound glycoprotein with hyaluronidase activity and putative roles during fertilization. Previously the antigen was thought to be testis-specific but recently it has been shown to be synthesized in the epididymis (mouse, macaque and human). Using the efferent ductule ligated (EDL) rat as a model to produce a sperm-free androgen-maintained epididymis, we have examined the factors regulating the expression of epididymal 2B1. RT-PCR and in situ transcript hybridization (ISH) studies showed that 2B1 mRNA is transcribed in the principal cells in all three regions of the epididymis. Its cognate protein was also detected by Western blot analysis in sperm-free cytosols from normal epididymis and found to undergo endoproteolytic cleavage into 2 subunits of similar size to the sperm-bound form. Immunohistochemistry with a monoclonal antibody to 2B1 confirmed that the protein is present in the epididymal epithelium and luminal secretions. The intensity of staining was much stronger in the sperm-free EDL epididymis than that in the normal (sperm-present) epididymis. The protein was shown to have hyaluronidase activity at neutral pH and both its quantity and activity appeared to be greater in the EDL epididymis. It is suggested that a soluble form of SPAM1 glycoprotein is synthesized and released in the epididymis and that in addition to androgens, its regulation may involve a cross-talk between the tubule epithelium and lumicrine factors, the latter possibly of testicular origin.

Animals↗

PubMed2003

SPAM1 (PH-20) protein and mRNA expression in the epididymides of humans and macaques: utilizing laser microdissection/RT-PCR.

BACKGROUND: The Sperm Adhesion Molecule 1 (SPAM1) is an important sperm surface hyaluronidase with at least three functions in mammalian fertilization. Previously our laboratory reported that in the mouse, in addition to its expression in the testis, Spam1 is synthesized in the epididymis where it is found in membranous vesicles in the principal cells of the epithelium in all three regions. Since SPAM1 is widely conserved among mammals the aim of the study was to determine if its expression pattern in the epididymis is conserved in rodents and primates. METHODS: We used laser microdissection (LM)/RT-PCR on frozen and paraffin-embedded epididymal sections of humans (n = 3) and macaques (n = 2) as well as in situ transcript hybridization to determine if transcripts are present in the epididymal epithelium. Western analysis and immunohistochemistry were used to detect and confirm the protein expression, and hyaluronic acid substrate gel electrophoresis analyzed its hyaluronidase activity. An in silico analysis of the proximal promoter of SPAM1 was also performed to identify relevant putative transcription binding sites for the androgen receptor. RESULTS: We demonstrate that mRNA unique to SPAM1 is present in the principal cells of the epididymal epithelium in all individuals of both species studied. SPAM1 protein is present in all three regions of the epididymis, as well as the vas deferens, and is localized similarly to the transcripts. SPAM1 was shown to have hyaluronidase activity at pH 7.0. In the proximal promoter of SPAM1 were uncovered putative epididymal transcription factor binding sites including androgen receptor elements (AREs), consistent with epididymal expression. CONCLUSIONS: These findings allow us to conclude that epididymal SPAM1 is conserved in at least two mammalian classes, rodents and primates. This conservation of expression suggests that the protein is likely to play an important function, possibly in sperm maturation.

Adult↗

PubMed2003

Mouse Spam1 (PH-20) is a multifunctional protein: evidence for its expression in the female reproductive tract.

Sperm adhesion molecule 1 (Spam1) is a widely conserved sperm surface protein with multiple roles in mammalian fertilization. Although the gene for this protein has been thought to be testis specific based on Northern blot analysis, there is evidence for nontesticular expression when transcripts are analyzed by more sensitive techniques. In the present investigation, results of a reverse transcription polymerase chain reaction assay, an RNase-protection assay (RPA), and an in situ transcript hybridization assay revealed that the murine Spam1 gene is transcribed in the female genital tract. RPA revealed that Spam1 transcripts are synthesized in a region-dependent manner, with the oviduct having lower transcript levels than the uterus and vagina. The transcripts levels were 3- to 10-fold lower in the female genital tract than in the testis. In situ transcript hybridization assay revealed RNA in the luminal epithelium in all three regions of the genital tract and in the uterine myometrium and the oviductal mesothelium. Western blot analysis and immunohistochemistry demonstrated that the protein concentration is 1.5- to 3-fold lower in female tissues than in sperm, and localization is similar to that of the transcripts. The protein has hyaluronidase activity at neutral pH, which is unique for sperm hyaluronidase, but not at acidic pH. In the uterus, Spam1 expression fluctuated during the estrous cycle. Its localization suggests that in addition to functioning as a secretory protein, it may be involved in hyaluronic acid metabolism or turnover in the female genital tract. Our results provide further evidence that Spam1 is a multifunctional protein and that it is less restricted in its expression than previously reported.

Animals↗