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S A Coonrod

Publications and source records attributed to S A Coonrod.

12 recordsLinked to original sources
PubMed2004

Oocyte proteomics: localisation of mouse zona pellucida protein 3 to the plasma membrane of ovulated mouse eggs.

In order to gain a deeper understanding of the molecular underpinnings of sperm-egg interaction and early development, we have used two-dimensional (2D) electrophoresis, avidin blotting and tandem mass spectrometry to identify, clone and characterise abundant molecules from the mouse egg proteome. Two-dimensional avidin blots of biotinylated zona-free eggs revealed an abundant approximately 75-kDa surface-labelled heterogeneous protein possessing a staining pattern similar to that of the zona pellucida glycoprotein, mouse ZP3 (mZP3). In light of this observation, we investigated whether mZP3 specifically localises to the plasma membrane of mature eggs. Zona pellucidae of immature mouse oocytes and mature eggs were removed using acid Tyrode's solution, chymotrypsin or mechanical shearing. Indirect immunofluorescence using the mZP3 monoclonal antibody (mAb) IE-10 demonstrated strong continuous staining over the entire surface of immature oocytes and weak microvillar staining on ovulated eggs, regardless of the method of zona removal. Interestingly, in mature eggs, increased fluorescence intensity was observed following artificial activation and fertilisation, whereas little to no fluorescence was observed in degenerated eggs. The surface localisation of ZP3 on mature eggs was supported by the finding that the IE-10 mAb immunoprecipitated an approximate 75-kDa protein from lysates of biotinylated zona-free eggs. To further investigate the specificity of the localisation of mZP3 to the oolemma, indirect immunofluorescence was performed using the IE-10 mAb on both CV-1 and CHO cells transfected with full-length recombinant mZP3 (re-mZP3). Plasma membrane targeting of the expressed re-mZP3 protein was observed in both cell lines. The membrane association of re-mZP3 was confirmed by the finding that biotinylated re-mZP3 (approximately 75 kDa) is immunoprecipitated from the hydrophobic phase of Triton X-114 extracts of transfected cells following phase partitioning. Immunoprecipitation assays also demonstrated that surface re-mZP3 was released from transfected CV-1 in a time-dependent manner. These results demonstrate that ZP3 is specifically associated with the surface of mature eggs and its subsequent release from the cell surface may represent one mechanism by which ZP3 is secreted. Furthermore, the increase in ZP3 surface expression following fertilisation suggests that ZP3 may have a functional role during sperm-oolemma binding and fusion. These results also validate the usefulness of using the 2D proteomic approach to identify and characterise egg-surface proteins.

Animals↗
PubMed2001

Co-localization of the inositol 1,4,5-trisphosphate receptor and calreticulin in the equatorial segment and in membrane bounded vesicles in the cytoplasmic droplet of human spermatozoa.

Modulation of the intracellular calcium concentration within mammalian spermatozoa is important in several pre-fertilization events including hyperactivated motility and the acrosome reaction. To identify calcium binding proteins (CBP) potentially regulating these processes, a (45)Ca overlay technique was employed on 2-D blots of human sperm extracts. Microsequencing by Edman degradation and CAD mass spectrometry identified a relatively abundant 60.5 kDa CBP with a pI of 4.2 as calreticulin (CRT). Immunofluorescent labelling with anti-CRT antibodies localized CRT to the acrosome, with highest fluorescence in the equatorial segment, and in the cytoplasmic droplets of 94 and 48% of human spermatozoa respectively. Double immunolabelling experiments demonstrated co-localization of CRT and the inositol 1,4,5-trisphosphate receptor (IP(3)R) in the acrosome, in the equatorial segment, and vesicular structures in the cytoplasmic droplets of the neck region. Electron microscopic immunogold labelling localized CRT to the equatorial segment of acrosome-reacted spermatozoa and to membrane-enclosed vesicles within the cytoplasmic droplet of both acrosome-intact and acrosome-reacted spermatozoa. Localization of the IP(3) receptor to the CRT-containing vesicles, in the sperm neck and to the acrosome, suggests that capacitative calcium entry in human spermatozoa may be regulated from these putative calcium storage sites.

Acrosome↗
PubMed2001

Differential nuclear localization of the cancer/testis-associated protein, SPAN-X/CTp11, in transfected cells and in 50% of human spermatozoa.

Cancer-testis antigens (CTAs) represent potential targets for cancer immunotherapy because these proteins are widely distributed in tumors but not in normal tissues, except testes. In this paper, we identify homology of the CTA CTp11 with SPAN-X (sperm protein associated with the nucleus mapped to the X chromosome). On two-dimensional Western blots of human sperm extracts, SPAN-X antibodies recognized 19 spots ranging from 20 to 23 kDa with isoelectric points from 5.0 to 5.5. Differential extraction of spermatozoa demonstrated that the SPAN-X protein is highly insoluble. Only 50% of ejaculated spermatozoa exhibited SPAN-X immunofluorescent staining. Dual localization of the sex chromosomes and the SPAN-X protein demonstrated that an equal number of X- and Y-bearing spermatozoa exhibited SPAN-X staining. In transfected mammalian CV1 cells, the SPAN-Xa and SPAN-Xb proteins were localized to the nucleus and cytoplasm, respectively, by indirect immunofluorescence. On immunoblots of CV1 cells, the SPAN-Xa protein migrated at 15-20 kDa, whereas the SPAN-Xb protein migrated at a higher molecular weight of 21-22 kDa. The SPAN-X protein was ultrastructurally associated with nuclear vacuoles and the redundant nuclear envelope. SPAN-X is the first protein specifically localized to these poorly characterized structures of the mammalian sperm nucleus and provides a unique biochemical marker for investigation of their function in spermatozoa as well as the role of SPAN-X/CTp11 in human tumors.

Amino Acid Sequence↗
PubMed1999

Role of the integrin-associated protein CD9 in binding between sperm ADAM 2 and the egg integrin alpha6beta1: implications for murine fertilization.

CD9 is a tetraspan protein that associates with several beta1 integrins, including alpha6beta1. Because alpha6beta1 is present on murine eggs and interacts with the sperm-surface glycoprotein ADAM 2 (fertilin beta), we first asked whether CD9 is present on murine eggs and whether it functions in sperm-egg binding and fusion. CD9 is present on the plasma membrane of oocytes in the ovary as well as on eggs isolated from the oviduct. The anti-CD9 mAb, JF9, potently inhibits sperm-egg binding and fusion in vitro in a dose-dependent manner. JF9 also disrupts binding of fluorescent beads coated with native fertilin or a recombinant fertilin beta disintegrin domain. (Both ligands bind to the egg via alpha6beta1.) Immunohistochemistry showed that CD9 is undetectable in the uterine epithelium, appears basolaterally and as prominent apical patches on the epithelium in the region between the uterus and the oviduct, and then persists apically in the oviduct. The integrin alpha6A subunit is found in similar apical patches in the region between the uterus and oviduct, but is confined to the basal aspect of the epithelium in the uterus and oviduct. Hence, alpha6A and CD9 both are expressed on the apical epithelial surface at the uterine-oviduct junction. These findings correlate with the observation that fertilin beta "knockout" sperm traverse the uterus but do not progress into the oviduct, contributing to the infertility of fertilin beta(-/-) male mice. Our results suggest that high-avidity binding between fertilin beta (ADAM 2) and alpha6beta1 requires cooperation between alpha6beta1 and CD9. Such cooperation may assist sperm passage into the oviduct as well as sperm-egg interactions.

ADAM Proteins↗
PubMed1999

Treatment of mouse oocytes with PI-PLC releases 70-kDa (pI 5) and 35- to 45-kDa (pI 5.5) protein clusters from the egg surface and inhibits sperm-oolemma binding and fusion.

The effect of phosphatidyinositol-specific phospholipase C (PI-PLC) on mouse sperm-egg interaction was investigated in this study to determine if glycosyl-phosphatidylinositol (GPI)-anchored proteins are involved in mammalian fertilization. When both sperm and zona-intact oocytes were pretreated with a highly purified preparation of PI-PLC and coincubated, there was no significant effect on sperm-zona pellucida binding; however, fertilization was reduced from 59.6% (control group) to 2.8% (treatment group). A similar reduction in fertilization rates was found when zona-intact oocytes were treated with PI-PLC and washed prior to incubation with untreated sperm. The effect of PI-PLC on sperm binding and fusion with zona-free oocytes was then investigated. Treatment of sperm with PI-PLC had no significant effect on sperm-egg binding or fusion. However, treatment of eggs with PI-PLC significantly reduced sperm-egg binding and fusion from 6.2 bound and 2.1 fused sperm per egg in the control group to 2.1 bound and 0.02 fused sperm per egg in the treatment group. This decrease in sperm-egg binding and fusion depended on the dose of PI-PLC employed, with a maximal inhibitory effect on binding and fusion at 5 and 1 U/ml, respectively. PI-PLC-treated oocytes could be artificially activated by calcium ionophore, demonstrating that the oocytes were functionally viable following treatment. Furthermore, treatment of oocytes with PI-PLC did not reduce the immunoreactivity of the non-GPI-anchored egg surface integrin, alpha6beta1. Taken together, these observations support the hypothesis that PI-PLC affects fertilization by specifically releasing GPI-anchored proteins from the oolemma. In order to identify the oolemmal GPI-anchored proteins involved in fertilization, egg surface proteins were labeled with sulfo-NHS biotin, treated with PI-PLC, and analyzed by two-dimensional gel electrophoresis followed by avidin blotting. A prominent high-molecular-weight protein cluster (approximately 70 kDa, pI 5) and a lower molecular weight (approximately 35-45 kDa, pI 5.5) protein cluster were released from the oolemmal surface as a result of PI-PLC treatment. It is likely that these GPI-anchored egg surface proteins are required for sperm-egg binding and fusion.

Animals↗
PubMed1999

FSP95, a testis-specific 95-kilodalton fibrous sheath antigen that undergoes tyrosine phosphorylation in capacitated human spermatozoa.

Protein tyrosine phosphorylation has been associated with both capacitation and motility of mammalian sperm. During capacitation, human spermatozoa undergo tyrosine phosphorylation of a characteristic set of proteins, only one of which has thus far been cloned and localized. We report here the sequence of a fibrous sheath protein of 95 kDa (FSP95) that undergoes tyrosine phosphorylation during capacitation of human spermatozoa and has similarity to sperm A-kinase anchor proteins (AKAPs). FSP95 is both auto- and iso-antigenic in humans as it is recognized by sera containing antisperm antibodies from infertile men and women. The 853-residue protein has a calculated molecular weight of 94.6 kDa and an isoelectric point (pI) of 6.0, and it contains multiple potential phosphorylation sites for protein kinase C and casein kinase II as well as one potential tyrosine kinase phosphorylation site at amino acid 435. The sequence has amino acid homology to mouse sperm fibrous sheath AKAP82 (pro-mAKAP82, 34% identity) and to human sperm fibrous sheath AKAP82 (pro-hAKAP82, 32% identity). The gene encoding FSP95 has 5 exons separated by 4 introns and is located on chromosome 12 at locus p13.3. Northern analysis detected a single transcript of approximately 3.0 kilobases, and Northern dot blot analysis of 50 human tissues revealed FSP95 mRNA expression only in testis. By employing sperm immobilization, indirect immunofluorescence, and immunoelectron microscopy with antisera to purified recombinant FSP95, the protein was localized to the ribs of the fibrous sheath in the principal piece of the sperm tail. FSP95 is the second fibrous sheath protein to be cloned, sequenced and localized in human spermatozoa.

Amino Acid Sequence↗
PubMed1996

Refinement of the differentiated phenotype of the spermatogenic cell line GC-2spd(ts)

A transformed spermatogenic cell line GC-2spd(ts), recently reported to express a protein marker of spermiogenesis, was tested for the presence of several mRNAs encoded by genes transcribed specifically in the testis and at precise stages of spermatogenesis. Northern blotting and reverse-transcriptase polymerase chain reaction techniques showed that mRNAs for the stage-specific marker proteins LDH-C4 (preleptotene), acrosin (premeiotic), protamine-2 (postmeiotic), and SP-10 (postmeiotic round spermatid stage) were not detected in GC-2spd(ts) cells. Flow cytometric analysis of GC-2spd(ts) failed to detect a peak indicative of the presence of haploid chromosomes. Furthermore, the HS-63 monoclonal antibody, employed in an earlier report to demonstrate putative proacrosomal granules, failed to recognize the SP-10 protein in extracts of human or mouse sperm or in GC-2spd(ts) cells and instead recognized proteins of different masses. In view of interest in this line as a model for analyzing molecular events of spermatogenesis, this refinement of the GC-2spd(ts) phenotype may aid others considering these cells for studies of terminal stages of sperm differentiation.

Acrosin↗
PubMed1996

Inhibition of bovine fertilization in vitro by antibodies to SP-10.

The inhibiting effect on fertilization of antibodies to the intra-acrosomal protein SP-10 was studied in a bovine in vitro fertilization model. Indirect immunofluorescence showed that antibodies to human SP-10 localized to the acrosomal region of methanol-fixed, but not live, bovine spermatozoa, confirming the intra-acrosomal localization of bovine SP-10. The presence of the SP-10 protein in bovine spermatozoa was further established by immunoblotting of extracts of bovine spermatozoa with monoclonal antibodies and polyclonal antibodies to human SP-10. The antibodies identified a pattern of immunoreactive peptides similar to human, baboon, monkey and pig SP-10. Fertilization experiments in vitro demonstrated that monoclonal antibodies, as well as polyclonal antibodies, to human SP-10 significantly reduced fertilization rates of bovine oocytes by bovine spermatozoa. SP-10 antibodies exerted their anti-fertilization effect by reducing sperm-zona secondary binding. Consistent with the accessibility of the SP-10 antigen following capacitation, SP-10 antibodies also reduced the ability of capacitated spermatozoa to complete the acrosome reaction. Furthermore, SP-10 antibodies affected the motility of capacitated spermatozoa, while not affecting the motility of noncapacitated spermatozoa.

Acrosome↗
PubMed1995

Interspecific transfer of IVM IVF-derived red sheep (Ovis orientalis gmelini ) embryos to domestic sheep (Ovis aries ).

Two embryo production methodologies were investigated to generate Red sheep embryos for use in an interspecific embryo transfer program. In Experiment 1, 4 multiparous female Red sheep (Ovis orientalis gmelini ) were implanted with CIDR type G devices for 11 d. Forty-eight hours prior to CIDR removal, a total of 22.5 mg bid of FSH-P was administered over a 3-d period. Laparoscopic embryo collection was performed 5 d post breeding, and embryos were transferred to domestic recipient ewes (Ovis aries and Ovis orientalis musimon ). In Experiment 2, 7 nulliparous female Red sheep were implanted with CIDR devices and injected with 200 IU of PMSG and 25 mg of FSH-P on the 8th day of implant insertion. At 60 to 70 h post PMSG/FSH-P treatment, follicular oocytes were aspirated laparoscopically. The recovered oocytes were matured in M199 (with fetal calf serum, FSH, LH, penicillin and streptomycin) at 39 degrees C in a humidified atmosphere containing 5% CO(2). At 24 h oocytes were fertilized with frozen-thawed semen at a concentration of 1.6 x 10(6) sperm/ml. The ova/embryos were placed in CR2 or BOEC culture medium at 20-22 h post IVF. Following 3 to 4 d in culture, embryos were transferred laparoscopically to the uterine horn of synchronized recipients. In Experiment 1, 4 embryos and 6 UFO were collected from 2 embryo donors, respectively. Two embryos were transferred with the aid of a laparoscope to each of 2 Rambouillet recipients, one of which gave birth to a healthy Red sheep lamb at 158 d of gestation. In Experiment2, a total of 62 oocytes was collected from 7 oocyte donors; 16 developed to the 16- to 32-cell stage and were transferred to 8 recipients. Three of these IVM-IVF embryos were transferred laparoscopically to 2 Mouflon recipients, resulting in no pregnancies. Thirteen IVM-IVF embryos were transferred to 6 Rambouillet recipients. Each of these gave birth to a single healthy Red sheep lamb. Gestation lengths of the 3 IVM-IVF lambs ranged from 152 to 162 d. This research demonstrates that when using compatible species IVM-IVF technology in conjunction with interspecific ET can lead to the production of live offspring and can be used to propagate exotic ovine species.

Journal Article↗
PubMed1994

Monoclonal antibody to human fertilization antigen-1 (FA-1) inhibits bovine fertilization in vitro: application in immunocontraception.

A monoclonal antibody (mAb) to the human sperm plasma membrane protein, fertilization antigen-1 (FA-1), was tested for its reactivity with bovine spermatozoa and its effects on bovine fertilization in vitro. Western blot analysis revealed that the FA-1 mAb reacted with proteins of similar molecular mass (53 +/- 2 kDa) in human and bovine sodium deoxycholate (DOC)-solubilized sperm extracts. Indirect immunofluorescence, using epifluorescence microscopy and laser scanning confocal microscopy, revealed that the FA-1 antigen is present in the post-acrosomal region of bovine spermatozoa, which is similar to human FA-1 localization. In bovine in vitro fertilization (IVF) trials, using oocytes obtained from slaughterhouse ovaries, addition of 20, 40, or 80 micrograms/ml of FA-1 mAb to the IVF medium resulted in a linear decrease in the fertilization rate from 86.3% in the controls to 54.6%, 21.6%, and 1.8% in the respective experimental groups (p < 0.01). There was no inhibitory effect (p > 0.10) of the FA-1 mAb on percent sperm motility or other motility characteristics tested, suggesting that human FA-1 mAb inhibits bovine sperm cell function at some point after capacitation. In conclusion, the evolutionarily conserved antigen FA-1 has a molecular identity in bovine sperm similar to that in human sperm, and mAb to human sperm FA-1 inhibits fertilization of bovine oocytes. These results indicate that FA-1 is a promising candidate for the development of a contraceptive vaccine. The research also suggests that bovine species could be used as a model for investigating the use of FA-1 as an immunovaccine in ruminants.

Animals↗