bluuebunny/crossref_metadata_2025_split huggingface dataset:
A guanine nucleotide‐binding regulatory protein in human sperm mediates acrosomal exocytosis induced by the human zona pellucida
Authors: Lee Michael A. , Check Jerome H. , Kopf Gregory S. 
Published: February 2005

AbstractGuanine nucleotide‐binding regulatory proteins play key intermediary roles in regulating zona pellucida‐mediated acrosomal exocytosis in mouse and bull sperm. Since human sperm possess a Gi‐like protein and undergo the acrosome reaction in response to the human zona pellucida, we investigated whether this G protein plays a regulatory role in this exocytotic process. Zonae pellucidae isolated from eggs that had been inseminated but had shown no signs of fertilization after retrieval for in vitro fertilization and embryo transfer were pooled into groups of ≥50 in order to reduce variability in biological responses due to the possible presence of ZP that had undergone modifications associated with the polyspermy block. Acid‐solubilized zonae pellucidae were incubated with capacitated sperm, and the sperm then assessed for the acrosome reaction using both the P. sativum agglutinin and chlortetracycline fluorescence assays; both assays gave similar results. Sperm incubated with solubilized zonae pellucidae at a final concentration of 2, 4, or 6 ZP/μl underwent acrosomal exocytosis to a similar extent as compared with A‐23187. Sperm were incubated with 1 μg/ml pertussis toxin during capacitation to functionally inactivate the Gi‐like protein. Pertussis toxin treatment of sperm did not affect sperm motility and the ability of the cells to bind to structurally intact zonae pellucidae. Pertussis toxin, however, completely inhibited the percentage acrosome reactions induced by solubilized zonae pellucidae. By contrast, the A‐23187‐induced acrosome reaction was insensitive to PT treatment. Pertussis toxin inhibition of the zona pellucida‐induced acrosome reaction occurred in a concentration‐dependent manner with maximal effects observed at 100 ng/ml PT. These data suggest that the pertussis toxin‐sensitive Gi‐like protein in human sperm plays an important regulatory role in the acrosome reaction induced by the human zona pellucida.
Cortical granule complements in human oocytes undergoing partial zona dissection
Authors: Lanzendorf Susan E. , Kazer Ralph R. , Patton Phillip E. , Wolf Don P. 
Published: February 2005

AbstractThis study was performed to evaluate the effects of mechanical stimulation and sucrose treatment on the oocyte activation process. Fresh and aged human oocytes were exposed to sucrose and zonae were dissected with microneedles before fixation and quantitative analysis of cortical granules by transmission electron microscopy. Examination of the mean number of cortical granules/analyzed segment revealed no significant differences between control oocytes or oocytes treated with sucrose or sucrose treatment followed by zona dissection. A significant decline in the number of cortical granules/segment was observed for oocytes undergoing prolonged culture after dissection (P < 0.05). Thus, zona dissection and sucrose exposure of freshly aspirated mature human oocytes do not result in classical oocyte activation.
The interaction between exogenous DNA and sperm cells
Authors: Lavitrano M. , French D. , Zani M. , Frati L. , Spadafora C. 
Published: February 2005

AbstractEpididymal sperm cells, incubated with plasmid DNA, showed a spontaneous tendency to interact with the exogenous nucleic acid. We have investigated the molecular basis of such interaction. Exogenous DNA is taken up by sperm cells over a 15‐ to 20‐min period and is specifically localized on the nuclear area of the sperm head. DNA was reversibly bound to spermatozoa since it can be competed out by excess of cold competitor DNA or by other polyanions as heparin and dextran sulphate. By contrast, poly‐L‐lysine, a polycation, favours the uptake. DNA molecules of large size (7 kb) were preferentially taken up as compared to smaller ones (150–750 bp). Acidic proteins were also taken up and concentrated, as for DNA, at the nuclear level. These data strongly suggested that ionic interactions may occur between foreign molecules and a substrate located in the sperm head. On the basis of Southwestern analysis, a sperm head protein(s) of 30–35 KD is identified as potential substrate for exogenous DNA binding. Moreover, we have found that seminal plasma contains factor(s) which abolish sperm permeability, exerting a powerful inhibitor effect on DNA uptake. The presence of a specific binding protein for the DNA and of a factor inhibiting such interaction support the existence of a mechanism controlling, through specific factors, the sperm–DNA interaction.
Evidence for the presence of an integrin cell adhesion receptor on the oolemma of unfertilized human oocytes
Authors: Fusi F. M. , Vignali M. , Busacca M. , Bronson Richard A. 
Published: February 2005

AbstractThe Arg‐Gly‐Asp peptide (RGD), contained in several extracellular matrix proteins such as fibronectin, laminin, vitronectin, and collagen, is a tripeptide that plays a role as a recognition sequence in many cell‐to‐cell and cell‐to‐matrix adhesion mechanisms, through its interaction with several receptors of the integrin family. We previously described the ability of the oolemma of hamster oocytes to bind GRGDTP coupled to the surface of activated immunobeads and demonstrated that RGD‐containing oligopeptides inhibit the adhesion of human and hamster spermatozoa to zona‐free hamster oocytes and their subsequent penetration. In the present experiments, we show, utilizing immunobeads coated with an RGD‐containing peptide (PepTiteTM 2000), that the oolemma of unfertilized human eggs is also able to recognize this adhesion sequence. The binding of PepTiteTM 2000‐coated immunobeads to the oolemma was inhibited by the oligopeptide GRGDTP as well as by fibronectin and laminin. When immunobeads were prepared with a PepTiteTM concentration of 10 μg/ml, GRGDTP 150 μg/ml, laminin 80 μg/ml, and fibronectin 60 μg/ml inhibited bead rosetting on the egg surface. These data suggest that a specific binding moiety for RGD is present on the human egg surface. The binding of fibronectin to the oolemma was also demonstrated by the rosetting of immunobeads coupled with antifibronectin antibody to human oocytes after their exposure to 1 mg/ml free fibronectin. Such binding of fibronectin to the oolemma could be inhibited by coincubation with a monoclonal antibody directed against the cell adhesion fragment of fibronectin. In addition, oolemmal rosetting of immunobeads coupled with a monoclonal antibody directed against the α5 subunit, usually part of the fibronectin receptor VLA 5 (α5β1), provided additional evidence that a putative fibronectin receptor is present on the oolemma of human eggs.
Subzonal insemination with a single spermatozoon using manipulation assisted sperm adhesion onto the ooplasmic membrane in mouse ova
Authors: Kobayashi Kazuhiko , Okuyama Manabu , Fujimoto Goro , Ogawa Shyoso , Rothman Cappy M. , Hill David L. 
Published: February 2005

AbstractA simple and successful method of microinjection of a single spermatozoon under the zona pellucida of a mouse oocyte has been developed. A characteristic of this method is that the tip of the sperm injection needle pierces the zona pellucida without touching the ooplasmic membrane. All the ova (277) used for this series of experiments had normal morphology after the injection procedure. Spermatozoa preincubated in culture medium for capacitation and those treated with ionophore A23187 for induction of acrosome reaction were used. In combination with some of these injections, a manipulation assisting the adhesion of the sperm head onto the ooplasmic membrane was employed. The fertilization rate (67.3%) of the ova injected with the ionophore‐treated sperm using the sperm‐adhesion treatment was significantly higher (P<0.005) than that obtained by the injection of the preincubated sperm without applying the adhesion treatment (23.6%). All three of the recipients that received the 24 fertilized ova became pregnant and gave birth to 11 off‐spring (45.8%). The inseminations performed with the sperm‐adhesion treatment using the immotile sperm from the preincubated population and/or those from the ionophore‐treated population did not result in fertilization in any case. These results suggest that the fertilization rate of subzonal insemination with motile ionophore‐treated sperm can be improved by applying the sperm‐adhesion treatment and that sperm motility might be involved in the establishment of fertilization, even after the adhesion of the sperm head with the mouse ovum membrane.
Germ cell‐specific expression of a proacrosin‐cat fusion gene in transgenic mouse testis
Authors: Nayernia Karim , Burkhardt Elke , Beimesche Stephan , Keime Sabine , Engel Wolfgang 
Published: February 2005

AbstractAcrosin is a serine proteinase located in a zymogen form, proacrosin in the acrosome of the sperm. It is released as a consequence of the acrosome reaction and is believed to be the most important enzyme in the fertilization process. In the mouse, the proacrosin gene is transcribed premeiotically in spermatocytes, but protein biosynthesis starts in haploid spermatids and is restricted to the emerging acrosome. Four lines of transgenic mice harboring 2.3 kb of 5′ untranslated region of the rat proacrosin gene fused to the CAT‐reporter gene were generated by microinjection of fertilized eggs. The chimeric gene was found to be present in 10–100 copies per genome in the different strains. The 5′ untranslated region of rat proacrosin gene could properly direct CAT‐gene expression to spermatocytes and CAT‐mRNA translation to round spermatids as it is known for mouse proacrosin gene. However, CAT protein is not restricted to the acrosome; rather, it is distributed in the spermatid cytoplasm. This could be due to the lack of DNA sequences for a hydrophobic leader peptide that have been found in all mammalian proacrosins studied until now but that was not present in transgene. It can be concluded from our results that cis‐acting sequences required for tissue specific proacrosin expression reside on a 2.3‐kb restriction fragment and are conserved in the proacrosin genes of mouse and rat.
Localization of DNase I‐hypersensitive regions during rat spermatogenesis: Stage‐dependent patterns and unique sensitivity of elongating spermatids
Authors: McPherson S. M. , Longo F. J. 
Published: February 2005

AbstractDNase I‐hypersensitivity of rat spermatogenic cells was analyzed (1) to establish overall patterns of hypersensitivity in individual cell types, (2) to correlate these patterns with known changes in chromatin organization and function, and (3) to provide a foundation for further analyses examining DNase I‐hypersensitivity and the localization of specific genes during spermatogenesis. Parameters for in situ nick translation, using radioactive and fluorescent probes to visualize DNase I‐hypersensitive regions (DHR), were established for fixed and sectioned testicular preparations, permeabilized cells, and isolated germ cell nuclei. As anticipated, the pattern of DHR changed in a cell‐type specific manner during the course of spermatogenesis, reflective of known stage‐dependent alterations in the composition and structure of both the chromatin and the nuclear lamina/matrix as well as changes in gene expression. DHR in preleptotene spermatocytes were primarily peripheral, while in pachytene spermatocytes they were localized along the condensed chromosomes. The pattern of DHR changed from “checkerboard” in steps 7–8 round spermatid nuclei to “lamellar” in steps 10–11 elongating spermatids. In steps 12–13 elongating spermatids, DHR were localized throughout the nuclei or in a graded manner—increasing from anterior to posterior and mirroring the pattern of chromatin condensation. However, unlike the case in other stages, DNA of steps 12–13 elongating spermatids was exquisitely sensitive to nick translation even in the absence of exogenous DNase I. In contrast to the labeling of earlier stages, steps 16–19 spermatids and mature spermatozoa did not demonstrate DNase I‐hypersensitivity under any conditions employed. A variety of agents that interact with topoisomerase II and DNA (teniposide, novobiocin, ethidium bromide, and adenosine triphosphate) were tested to determine the basis for the unique sensitivity to nick translation of steps 12–13 elongating spermatids. None of the agents tested, however, affected this unique labeling. The sensitivity of steps 12–13 elongating spermatids to nick translation in the absence of exogenous nuclease indicates the presence of endogenous nicks, which may relieve torsional stress and aid rearrangement as the chromatin is packaged into a form characteristic of the mature spermatozoon.
Activation of a G protein in mouse sperm by the zona pellucida, an egg‐associated extracellular matrix
Authors: Wilde Mary W. , Ward Cynthia R. , Kopf Gregory S. 
Published: February 2005

AbstractMammalian sperm possess a guanine nucleotide‐binding regulatory protein (G protein), with properties similar to Gi, that appears to be involved in the signal transduction pathway required for zona pellucida (ZP)‐mediated acrosomal exocytosis. Mouse sperm treated with pertussis toxin (PT), a toxin that functionally inactivates Gi proteins, bind to the ZP of mouse eggs but are inhibited from undergoing acrosomal exocytosis. We have measured high‐affinity GTPase activity and GTPγ[35S] binding in mouse sperm homogenates incubated in the absence and presence of ZP glycoproteins isolated from either ovulated eggs or from ovarian homogenates to determine whether this extracellular matrix can activate the sperm‐associated Gi protein. An increase in GTP hydrolysis (∼50% over basal activity) and GTPγ[35S] binding (∼25–60% over basal activity) is observed when sperm homogenates are incubated in the presence of solubilized ZP glycoproteins, and the increase in GTPase activity is dependent on the concentration of ZP added to the homogenates. Accompanying this increase is a reduction in the ability of PT to catalyze in vitro [32P]ADP‐ribosylation of a Mr = 41,000 sperm Gi protein, suggesting that the increase in GTPase activity and GTPγ[35S] binding is associated with the activation of a PT‐sensitive sperm G protein(s). The ability of the ZP to stimulate high‐affinity GTPase activity in these homogenates appears to be dependent on the capacitation state of the sperm from which the homogenates are prepared. These data suggest that a component(s) of the ZP may function in a manner similar to that of other ligands by binding to a sperm surface‐associated receptor and subsequently activating a G protein coupled to an intracellular signal transduction cascade(s) required for induction of acrosomal exocytosis.
Intracellular CA2+ response of rabbit oocytes to electrical stimulation
Authors: Fissore Rafael A. , Robl James M. 
Published: February 2005

AbstractElectrical stimulation is known to cause activation in mammalian oocytes, possibly by eliciting an elevation in intracellular calcium (Ca2+). This study reports intracellular Ca2+ concentrations in mature rabbit oocytes using the Ca2+ indicator fura‐2. Calcium levels were determined prior to, during, and after the administration of an electrical pulse (3.6 kV/cm for 60 μsec). Baseline Ca2+ levels ranged from 30 to 90 nM. The intracellular Ca2+ transient evoked by a pulse, peaked at 11 sec, was highly variable in amplitude (40–300 nM) and returned to prepulse levels within 300 sec. Electrically stimulated oocytes did not exhibit repetitive Ca2+ transients. The size of the cytoplasmic Ca2+ rise was influenced by the duration of the pulse, the field strength and the concentrations of external Ca2+ (P < 0.05). Oocytes electrically stimulated in the presence of 100 μM CaCl2, which evoked Ca2+ transients with a mean magnitude of 120 nM, activated at a higher rate (P < 0.05) than oocytes stimulated in the presence of either higher or lower levels of external Ca2+. Although oocytes electrically shocked at 16–18 hr after administration of human chorionic gonadotropin (hphCG) activated at a lower rate than oocytes stimulated at 22–24 hphCG (P < 0.05), their intracellular Ca2+ response to the pulse was similar (P < 0.05). These results indicate that electrical pulse parameters and extracellular Ca2+ concentrations can be used to modulate intracellular Ca2+ levels and optimize oocyte activation rates. Furthermore, the data suggest that as the oocyte ages it becomes more responsive to a given intracellular Ca2+ elevation. © 1992 Wiley‐Liss, Inc.
Role of a germ cell‐specific sulfolipid‐immobilizing protein (SLIP1) in mouse in vivo fertilization
Authors: Tanphaichitr Nongnuj , Tayabali A. , Gradil C. , Juneja S. , Léveillé M. C. , Lingwood C. A. 
Published: February 2005

AbstractSulfolipid‐immobilizing protein 1 (SLIP1) is a germ cell plasma membrane protein that binds specifically to sulfogalactosylglycerolipid, a sulfoglycolipid found preferentially in mammalian male germ cells (Lingwood, Can. J. Biochem. Cell. Biol. 63:1077–1085, 1985b). SLIP1 in mouse and rat sperm exists on the periacrosomal membrane, where sperm initially bind to eggs. Using the in vitro mouse sperm‐egg binding assay with in vitro‐capacitated sperm, we obtained results previously suggesting that sperm SLIP1 is involved in mouse sperm‐zona pellucida interaction. In this study, using the in vitro sperm‐egg binding assay, we showed that SLIP1 in uterine sperm was similarly engaged in this process. Involvement of mouse sperm SLIP1 was also shown to be important in the vivo fertilization process. Superovulated females inseminated with caudal epidididymal and vas deferens sperm preexposed to anti‐SLIP1 lgG yielded only 20% fertilized zygotes, while 80% fertilization was observed in females inseminated with sperm preincubated with preimmune serum lgG. The lower fertilization rate was not due to changes in the sperm capacitation rate as assessed by chlortetracycline staining. © 1992 Wiley‐Liss, Inc.
Comparison of embryonic developmental competence of mouse oocytes grown with and without serum
Authors: Eppig John J. , Wigglesworth Karen , O'Brien Marilyn J. 
Published: February 2005

AbstractThe first objective of this study was to determine whether oocyte growth in serum‐free medium affects the solubility of the zona pellucida to α‐chymotrypsin digestion, which is an index of zona pellucida “hardening” and reflects the potential penetrability of the zona pellucida by sperm. Oocyte‐granulosa cell complexes were isolated from the preantral follicles of 12‐day‐old mice and cultured for 10 days in medium containing 5% fetal bovine serum (FBS) or in serum‐free medium. The zonae pellucidae of oocytes grown in serum‐free medium were four times as hard as freshly isolated germinal vesicle (GV)‐stage oocytes grown in vivo or oocytes grown in vitro in FBS‐containing medium. The hardening of the zonae pellucidae of oocytes grown in serum‐free medium was prevented by addition of fetuin. The second objective was to compare the competence to undergo embryogenesis of oocytes that grew in serum‐free vs. FBS‐containing medium. Approximately 70% of the oocytes underwent maturation regardless of whether the medium was serum‐free or contained FBS. Of the mature ova grown in medium containing FBS, 53% cleaved to the two‐cell stage after insemination compared with only 6% of the ova grown in serum‐free medium. Addition of fetuin to the serum‐free medium used for oocyte growth increased the frequency of cleavage to the two‐cell stage. Of the embryos derived from oocytes that grew in FBS‐containing medium, 70% completed the two‐cell stage to blastocyst transition. In contrast, only about 40% (P < 0.01) of the embryos derived from oocytes that grew in serum‐free medium completed this transition regardless of whether the medium for oocyte growth was supplemented with fetuin. Nevertheless, 12% of the two‐ to four‐cell‐stage embryos derived from oocytes that grew and matured in serum‐free medium containing fetuin developed to live young after transfer to the oviducts of foster mothers. These results suggest that serum‐borne growth factors may function during oocyte growth and maturation to promote events leading to successful fertilization and embryogenesis.
A transient rise in intracellular Ca2+ is a precursor reaction to the zona pellucida‐induced acrosome reaction in mouse sperm and is blocked by the induced acrosome reaction inhibitor 3‐quinuclidinyl benzilate
Authors: Storey Bayard T. , Hourani Catherine L. , Kim J. B. 
Published: February 2005

AbstractThe acrosome reaction induced by the zona pellucida in mouse sperm has been shown to proceed in two stages experimentally distinguishable by the fluorescent probe chlortetracycline. Entry into the first stage of sperm bound to isolated, structurally intact zonae pellucidae is blocked by the compound 3‐quinuclidinyl benzilate. In this study, we show, utilizing the fluorescent Ca2+ indicator fluo‐3, that the first stage of the zona‐induced acrosome reaction is characterized by an increase in intracellular Ca2+, followed by a decrease as the acrosome reaction proceeds. This calcium transient is completely suppressed by 3‐quinuclidinyl benzilate. We conclude that the Ca2+ transient is induced by the zona pellucida and is required for the zona‐induced acrosome reaction. Blockage of this sperm intracellular Ca2+ transient provides a mechanism for the inhibitory action of 3‐quinuclidinyl benzilate on the zona‐induced acrosome reaction in mouse sperm. © 1992 Wiley‐Liss, Inc.
Specific distribution of an epididymal 50 KDa protein revealed by an anti‐mos protein monoclonal antibody
Authors: Koyama Yoh‐Ichi , Ishikawa K. , Marunouchi T. 
Published: February 2005

AbstractAn anti‐Mos protein monoclonal antibody, 4A6, was used to investigate the distribution of the antigen in the epididymis, in which the c‐mos gene is reportedly expressed. The 4A6‐reactive antigen was found on the basement membrane and luminal surface of the epithelial cells in the caput epididymis of BALB/c male mice as well as in the proximal corpus epididymis, the cauda epididymis, and the vas deferens. The 4A6 antigen was also found on the luminal surface of the epithelial cells in the epididymis of male germ cell‐deficient C57BL/6J‐Wv/Wv mice. This confirmed that the 4A6 antigen does not derive entirely from the testicular c‐Mos protein but is synthesized in the epididymis. Western blot analysis revealed that the molecular weight of the epididymal 4A6 antigen was 50 kDa, which is unusually high for the c‐Mos protein. With its specific distribution in the epididymis, the protein should play a specific role in functions of the epididymis. © 1992 Wiley‐Liss, Inc.