bluuebunny/crossref_metadata_2025 huggingface dataset:
Phospholipids of rabbit and bull sperm membranes: Structural order parameter and steady‐state fluorescence anistropy of membranes and membrane leaflets
Authors: Hinkovska‐Galcheva Vania , Srivastava Prakash N.
Published: February 2005

The plasma (PM), outer acrosomal (OAM), and inner acrosomal membranes (IAM) were isolated from rabbit and bull spermatozoa and the major phospholipids characterized. Choline‐containing phospholipids, phosphatidylcholine (PC) and sphingomyelin (SM), constituted more than 60% of the total phospholipids (TPL) in all membranes of both species. Approximately more than 50% of PC in membrane preparations contained some form of ether linkage. Compared to OAM and IAM, cholesterol to phospholipid molar ratio was highest in PM of both species. Contrarily, protein to phospholipid ratio for PM was lowest compared to other membranes. The sphingomyelin to phosphatidylcholine ratio increased in the direction from PM to OAM to IAM. The hydrophobic fluorescent probe 1,6‐diphenyl‐1,3,5‐hexatriene (DPH) was used to examine both the steady‐state fluorescence anisotropy parameters and structural order parameter SDPH. The data showed higher rigidity in rabbit spermatozoa compared to bull spermatozoa (SDPH = 0.7582 and SDPH = 0.7326). In both species OAM had higher rigidity compared to the other two membranes (SDPH(OAM) = 0.7809, SDPH(PM) = 0.7308, and SDPH(IAM) = 0.7481 for bull; SDPH(OAM) = 0.8091, SDPH(PM)= 0.7857, and SDPH(IAM) = 0.7663 for rabbit). The inner leaflets of bull and rabbit spermatozoal membranes had significantly higher rigidity than the outer leaflets (for inner leaflet: rabbit‐SDPH(PM) = 0.8391, SDPH(OAM) = 0.8149, and SDPH(IAM) = 0.7675; bull‐SDPH(PM) = 0.8000, SDPH(OAM) = 0.7990, and SDPH(IAM) = 0.7990, and for outer leaflet: rabbit‐SDPH(PM) = 0.7021, SDPH(OAM) = 0.7145, and SDPH(IAM) = 0.6867; bull‐SDPH(PM) = 0.6986, SDPH(OAM) = 0.5980, and SDPH(IAM) = 0.7388). © 1993 Wiley‐Liss, Inc.
Temporal expression of tissue inhibitors of metalloproteinases in mouse reproductive tissues during gestation
Authors: Waterhouse Paul , Denhardt David T. , Khokha Rama
Published: February 2005

Tissue inhibitors of metalloproteinases (TIMPs) appear to play an important regulatory role in tissue remodelling and invasion by malignant cells. Since pregnancy involves morphological changes in existing maternal tissues, as well as a strictly controlled invasion by fetal trophoblasts, we have examined the temporal expression of TIMP‐1, TIMP‐2, and specific metalloproteinases in the mouse uterus, decidua, placenta, amnion, and ovaries throughout gestation by examining mRNA levels on northern and slot blots. Maximal levels of TIMP‐1 mRNA were observed from day 6 to day 10 in the uterus, decidua, and placenta. In clear contrast to the early burst of TIMP‐1 mRNA accumulation, the level of TIMP‐2 mRNA increased steadily throughout gestation in the uterus, decidua, and amnion, while in the placenta it showed a sevenfold increase after day 14. In amnion, TIMP‐1 was induced specifically on day 18. Interestingly, the normally high level of TIMP‐1 mRNA seen in the ovaries of virgin mice was low during gestation, until day 18 and postpartum, when a sixfold increase over the levels in virgin ovary was observed. In contrast, ovarian TIMP‐2 mRNA showed a marginal increase during gestation. The temporal pattern of 72 kDa gelatinase type A followed that of TIMP‐2 in the decidua and ovary. Stromelysin‐2 mRNA was detected at term only in ovary and decidua. Our data show that the temporal accumulation of TIMP‐1 and TIMP‐2 mRNA is precisely coordinated in each of the tissue compartments and is independently regulated during the in vivo remodelling of reproductive tissues in gestation. The peak of TIMP‐1 mRNA levels in the uterus, decidua, and placenta at midgestation is associated with the most invasive period of embryo development. © 1993 Wiley‐Liss, Inc.
Immunoelectron microscope localization of snRNP, hnRNP, and ribosomal proteins in mouse spermatogenesis
Authors: Biggiogera M. , Von Schack M.‐L. , Martin T. E. , Gordon J. , Müller M. , Fakan S.
Published: February 2005

We have studied the ultrastructural distribution of heterogeneous nuclear ribonucleoproteins (hnRNPs), small nuclear ribonucleoproteins (snRNPs), and ribosomal proteins during mouse spermatogenesis and spermiogenesis by means of specific antibodies and immunocytochemistry.

All the above components were detectable from primary spermatocytes until the spermatid elongation phase, when the RNA synthetic activity is known to cease. Ribosomal protein (P1/P2 and L7) labeling disappeared as early as during the acrosome phase, and nucleoli were no longer labeled even during the cap phase. The nucleoplasmic structures labeled with the different anti‐nucleoplasmic RNP immunoprobes corresponded, until the acrosome phase, to those previously observed as targets of the same antibodies in the nucleoplasm of somatic cell nuclei. Clusters of interchromatin granules of spermatocyte and early spermatid nuclei exhibit some labeling for hnRNP when compared with nuclei of Sertoli cells or previously analyzed liver or tissue culture cells, where these structural constituents usually remain weakly labeled or unlabeled.

In spermatids in step 10, another type of nuclear granule, resembling perichromatin granules, but occurring in aggregates, can be observed. These structural constituents were labeled with antibodies recognizing nucleoplasmic snRNP antigens and therefore suggesting a non‐nucleolar origin of these granules.

Finally, we have observed nucleoplasmic areas of fibrogranular material, occurring only in primary spermatocytes. These components were labeled with anti‐ribosomal protein antibodies but did not contain either hnRNPs or snRNPs. © 1993 Wiley‐Liss, Inc.
Biotinylation of proteins on the surface of zona‐free mouse oocytes
Authors: Flaherty Sean P. , Swann Nicholas J.
Published: February 2005

In this study, we have labelled proteins on the surface of unfertilized, zona‐free mouse oocytes using a nonisotopic biotinylation procedure. The zona pellucida was weakened by brief incubation in chymotrypsin and removed by mechanical pipetting through a narrow‐bore glass pipette. Surface proteins were labelled using sulfo‐NHS‐biotin (sulfosuccinimidobiotin), a water‐soluble, membrane‐impermeable biotinylation reagent. The distribution of biotinylated proteins on the oocyte surface was assessed by fluorescence microscopy using streptavidin–FITC. Bright fluorescence was noted on the surface of the oocyte, except in a circular region overlying the meiotic spindle where the fluorescence was weak or absent. The intensity of fluorescence was markedly reduced by incubation of biotinylated oocytes in trypsin (1 mg/ml) or chymotrypsin (2 mg/ml), and in vitro fertilization experiments showed that biotinylation did not compromise the fertilizability of the oocytes. The biotinylated proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and analyzed by Western blotting using streptavidin–HRP and enhanced chemiluminescence (ECL) detection. The most prominent biotinylated proteins were of Mr 82 and 69 kD, but other major proteins of Mr 93, 78, 61, 52, 49, 40, 28, and 22 kD were detected, as well as 14 minor proteins of Mr 18–100 kD. The major bands could be detected in fewer than 50 oocytes. This biotinylation procedure is fast, versatile and sensitive, and it is therefore an excellent tool for studying proteins exposed on the surface of mammalian oocytes and embryos. © 1993 Wiley‐Liss, Inc.
Multiple effects of seminal plasma on the acrosome reaction of human sperm
Authors: Cross Nicholas L.
Published: February 2005

Mammalian sperm do not respond to inducers of the acrosome reaction immediately after ejaculation. They become responsive after they are removed from seminal plasma and incubated in an appropriate medium. We tested the effects of seminal plasma on the development of acrosomal responsiveness. Washed human sperm incubated 24 hr in vitro with 10% (v/v) seminal plasma did not complete an acrosome reaction when exposed to human follicular fluid, progesterone, or ionomycin. Seminal plasma did not reduce sperm viability or motility. Electron microscopy of sperm incubated 24 hr with 5% seminal plasma and then treated with progesterone revealed no sign of membrane fusion or other changes that are associated with the acrosome reaction. During a 12‐hr incubation, seminal plasma was 50% effective at inhibiting the acrosomal response to progesterone when diluted 821 ± 112 foid (mean ±SD, n = 3). Sperm that were incubated with seminal plasma for 24 hr and then washed free of the seminal plasma became acrosomally responsive over the following 24 hr, at a rate similar to that of sperm not incubated with seminal plasma in vitro. When sperm were incubated 6 hr without seminal plasma and then seminal plasma was added, the sperm population transiently became more responsive to progesterone, and then became unresponsive. During incubation in vitro, the ability of sperm to have an augmented response to a mixture of seminal plasma plus progesterone developed slightly earlier and more rapidly than ability to respond to progesterone alone. When sperm were incubated 24 hr without seminal plasma, a few acrosome reacted in response to the addition of seminal plasma alone. Therefore, depending on how it is applied, seminal plasma can prevent or reverse the development of acrosomal responsiveness, and it can enhance or induce the acrosome reaction. © 1993 Wiley‐Liss, Inc.
Phylogeny of the insulin‐like growth factors (IGFS) and receptors: A molecular approach
Authors: LeRoith Derek , Kavsan Vadim M. , Koval Anatolii P. , Roberts Charles T.
Published: February 2005

The IGFs (IGF‐I and IGF‐II) are essential for normal mammalian growth and development. Their actions are mediated primarily by their interactions with the type I IGF receptor (IGF‐I receptor), a transmembrane tyrosine kinase. The ligands and the IGF‐I receptor are structurally related to insulin and to the insulin receptor, respectively. Analysis of evolutionary conservation has often provided insights into essential regions of molecules such as hormones and their receptors. The genes for insulin and IGFs have been partially characterized in a number of vertebrate species extending evolutionarily from humans as far back as fish. The sequences of the exons encoding the mature insulin and IGF peptides are highly conserved among vertebrate species, and IGF‐I‐Iike molecules are found in species whose origins extend back as much as 550 million years. The insulin receptor is also highly conserved in vertebrate species, and an insulinreceptor‐like molecule has been characterized in Drosophila. In contrast, IGF‐I receptors have only been characterized in mammalian species and partially studied in Xenopus, in which the tyrosine kinase domain is highly conserved. Studies are presently being undertaken to analyze in more detail the regulation of the genes encoding this important family of growth factors and the structure/function relationships in the gene products themselves. © 1993 Wiley‐Liss, Inc.
Functional analysis of the human IGF‐I gene promoters
Authors: Steenbergh P. H. , Jansen E. , Van Schaik F. M. A. , Sussenbach J. S.
Published: February 2005

The two putative promoter regions of the human insulin‐like growth factor‐I (IGF‐I) gene, located upstream of the two alternatively used leader exons 1 and 2, were tested for promoter activity in transient transfection assays. Both regions were shown to possess promoter activity. The results further indicate cell‐type‐specific regulation of the activities of the two promoters in endogenously IGF‐I producing human cell lines of different origin. © 1993 Wiley‐Liss, Inc.
Identification of an antisense transcript from the IGF‐II locus in mouse
Authors: Rivkin Michael , Rosen Kenneth M. , Villa‐Komaroff Lydia
Published: February 2005

We have identified a novel antisense RNA transcribed from the insulin‐like growth factor‐II (IGF‐II) locus in mouse. This transcript was identified using probes derived from cloned genomic DNA lying between the insulin II locus and the IGF‐II locus. S1 protection assays confirmed that this transcript is transcribed from the strand complementary to the stand encoding IGF‐II. A 3.75 kb RNA was consistently detected on northern analysis of mouse tissue using different randomly primed DNA probes generated from this region. S1 nuclease protection analysis identified three exons contained in the transcript. Developmental northern analysis was performed using RNA from embryonic (E) and postnatal (P) tissues of E10, E13, E18, P1, P4, P10, and adult mice. The antisense RNA was most abundant in E13 and E18 mouse and was present in greatest amounts in skull, skeletal muscle, cardiac muscle, and placenta. No signal for this RNA was detected after the fourth day of life in any tissue studied. © 1993 Wiley‐Liss, Inc.
Expression of IGF ligand and receptor genes during preimplantation mammalian development
Authors: Schultz Gilbert A. , Hahnel Ann , Arcellana‐Panlilio Mayi , Wang Liangsu , Goubau Sabine , Watson Andrew , Harvey Mark
Published: February 2005

The temporal patterns of expression of genes encoding insulin‐like growth factor (IGF) ligands and receptors during very early development have been investigated in several laboratories in several different mammalian species. Both reverse transcription‐polymerase chain reaction (RT‐PCR) and immunocytochemical techniques have been used to identify the time of appearance of gene transcripts or end‐products. In preimplantation mouse embryos, IGF‐II ligand and receptor gene activity is detectable as early as at the two‐cell stage, the time when transcription from the embryonic genome is activated, but receptors for insulin and IGF‐I are not detectable until the compacted eight‐cell stage. Transcripts for insulin or IGF‐I are not detectable in preimplantation mouse embryos, although the ligands are present in the reproductive tract. The pattern of IGF gene expression is not, however, identical in all mammalian species. In cow embryos, for example, transcripts for IGF‐I and IGF‐II ligands and receptors and insulin receptors have been detected at all stages of preimplantation development from mature oocyte to blastocyst (Watson et al., 1992). Attempts to quantitate transcript abundance in these early embryos are in progress in our laboratory. In the preimplantation mouse embryo, transcripts for several different IGF‐binding proteins (IGFBP‐2, ‐3, ‐4, and ‐6) have been detected by RTPCR procedures. In addition, transcripts for IGFBPs have been identified in RNA derived from cumulus cells, the ovary, the oviduct, the uterus, and the decidua. These findings suggest that the interactions of IGF ligands and receptors in preimplantation development might, indeed, be modulated by IGFBPs. Approaches to function of IGFs in preimplantation embryos have involved analysis of the stimulatory effects on metabolism and cell division when IGFs are added exogenously to embryos in culture in simple defined medium (for example, see Harvey and Kaye, 1991) or observations in the reduction in rate of development following interference with IGF expression (see Rappolee et al., 1992). In general, members of the insulin and IGF gene family of polypeptides have been shown to lead to an enhahancement of development in vitro in both laboratory and domestic mammalian species. © 1993 Wiley‐Liss, Inc.
Functions of the IGFs in early mammalian development
Authors: Heyner Susan , Shi Cong‐Zhu , Garside William T. , Smith Robert M.
Published: February 2005

The identification of growth factors and/or receptors produced by mammalian embryos or present in the maternal reproductive tract is of basic interest, as well as having practical application. Early studies established that receptors binding insulin and the insulin‐like growth factors (IGFs) are expressed by preimplantation mouse embryos. These studies have been confirmed at the molecular level using RT‐PCR techniques. In addition, high resolution electron microscopy has shown that insulin is internalized by the cells of the blastocyst stage mouse embryo, and that immunologically intact insulin is detectable in the cells of the trophectoderm and inner cell mass. Similar studies with gold labelled IGF‐I have shown that this ligand is also bound and internalized by mouse blastocysts. However, although all blastocysts express receptors that bind IGF‐I on the basolateral cell surface of the trophectoderm, only 30% exhibit apically located receptors. In order to elucidate the functions of IGFs in early mouse development, we are in the process of constructing protein databases for embryos at the eight‐cell and blastocyst stage. By the use of the database, it should prove possible to elucidate targets of growth factor action. © 1993 Wiley‐Liss, Inc.
Acrosome reaction of boar spermatozoa in homologous in vitro fertilization
Authors: Vázquez J. M. , Martínez E. , Roca J. , Coy P. , Pastor L. M.
Published: February 2005

Ejaculated spermatozoa from four different boars were used to evaluate the acrosome reaction during in vitro fertilization with homologous ovulated oocytes. The acrosome reaction was assessed according to a peroxidase‐labeling peanut agglutinin method and a triple‐stain technique. An increase in the proportion of living sperm with reacted acrosomes was observed after preincubation and 2 hr of coincubation (P < 0.05). The percentage of true acrosome‐reacted sperm remained reasonably constant throughout coincubation. In vitro penetration rates of oocytes varied among boars, but no relationship was found between fertilization rates of oocytes and maximum percentages of acrosome reacted living sperm. © 1993 Wiley‐Liss, Inc.
The abundant 19‐kilodalton protein associated with human sperm nuclei that is related to seminal plasma α‐inhibins
Authors: Zalensky Andrei O. , Yau Peter , Breneman John W. , Bradbury E. Morton
Published: February 2005

A basic protein with a relative molecular mass of 19 kDa has been identified and isolated to purity from sonication‐resistant, partially demembranized human sperm nuclei. Several criteria prove that this is the unique sperm‐specific protein, which was previously thought to be a sperm/testis histone. Partial primary structure sequencing demonstrates homologies with human seminal α‐inhibins and semenogelin. From the sequence and Western‐blotting data with antibodies against basic seminal inhibin‐like peptide, we propose that this 19‐kD protein is a product of 52‐kDa semenogelin processing. The 19‐kDa protein was not found among seminal plasma proteins and may be protected from further cleavage into inhibin‐like peptides by its association with the sperm head Immunofluorescence data indicate its localization in the nuclear periphery, with preferential concentration at the acrosome calyx boundary. © 1993 Wiley‐Liss, Inc.
Actin polymerization in boar spermatozoa: Fertilization is reduced with use of cytochalasin D
Authors: Castellani‐Ceresa L. , Mattioli M. , Radaelli G. , Barboni B. , Brivio M. F.
Published: February 2005

The aggregational state of actin in boar spermatozoa after capacitation and the acrosome reaction has been examined by several methods. In vitro fertilization (IVF) experiments were conducted in the presence and absence of cytochalasin D (CD) to evaluate the role of actin polymerization in the events of fertilization. The fertilizing capacity was very high in controls, but, when CD (an inhibitor of the polymerization of actin) was added to the capacitation medium, there was a marked decrease in the fertilizing capacity of the boar spermatozoa. There was a further decrease when CD was present during both capacitation and fertilization processes. In addition to the IVF tests, biochemical and immunoelectron microscopic methods were used to analyze the state of aggregation of actin in boar spermatozoa after capacitation, and the acrosome reaction. By immunoelectron microscopy with a phalloidin probe, there were no gold particles, indicating the presence of F‐actin on boar sperm heads capacitated and acrosome‐reacted in media containing CD. By sodium dodecyl sulfate‐polyacrylamide gel electrophoresis there were differences in NP‐40 solubility, reflecting actin polymerization, between CD‐treated and untreated sperm. These results suggest that actin polymerizes during capacitation and the acrosome reaction and that this polymerization is essential to the fertilization process. © 1993 Wiley‐Liss, Inc.
Mammalian oocytes exhibit specific recognition of the RGD (ARG‐Gly‐Asp) tripeptide and express oolemmal integrins
Authors: Fusi F. M. , Vignali M. , Gailit J. , Bronson R. A.
Published: February 2005

Integrins are a family of cell adhesion receptors involved in many cell–cell and cell–matrix interactions. Some of these heterodimeric receptors, such as α5b̃1 and αvb̃1, specifically recognize the amino acid sequence Arg‐Gly‐Asp (RGD) within their ligands. The RGD sequence is found in fibronectin, vitronectin, and other extracellular matrix proteins. Our results demonstrate that the oolemmas of eggs from human and several other mammalian species contain receptors capable of binding to RGD ligands, and that integrin subunits are expressed by oocytes. Four distinct techniques were utilized to identify the presence of functional integrins on mammalian eggs. RGD‐binding receptors were detected on the surfaces of zona‐free eggs from all species tested. Covaspheres coated with PepTite‐2000, which contains RGD, bound to the eggs and formed rosettes. Rosetting was competitively inhibited by PepTite‐2000 and by GRGDTP, a soluble RGD peptide, but not by RGES. An ELISA using polyclonal antibodies directed against the cytoplasmic tails of the integrin subunits identified the integrin subunits α5, b̃1, and αv, but not b̃3, in detergent extracts of Syrian hamster eggs. A dot blot confirmed the presence of αv in hamster egg lysates. Finally, the integrin subunits α2, α5, α6, but not α4, were detected on the surfaces of zona‐free eggs from human and Syrian hamster. Immunobeads coated with monoclonal antibodies specific for α2, α5, and α6 bound to the eggs and formed rosettes. A similar rosetting was obtained from beads coated with polyclonal antibody against the vitronectin receptor (αvb̃3/b̃5). These observations suggest that the integrins α2b̃1, α5b̃1, α6b̃1, and one or more integrins containing αv are expressed by oocytes. We propose that these integrins may play important roles in fertilization and implantation. © 1993 Wiley‐Liss, Inc.
Modulation of embryonic NA+‐K+‐ATPase activity and mouse preimplantation development in vitro in media containing high concentrations of potassium
Authors: Dumoulin John C. M. , Evers Johannes L. H. , Michiels Anton H. J. C. , Pieters Math H. E. C. , Bras Marijke , Land Jolande A. , Geraedts Joep P. M.
Published: February 2005

The effect of various potassium concentrations (ranging from 1.4 mM to 30 mM K+) in modified Tyrode's medium on the culture of mouse zygotes obtained after in vitro fertilization to the blastocyst stage was examined. A clear dose‐dependent negative effect of increasing K+ concentrations on the preimplantation embryonic development in vitro was found. We have previously shown that significantly more two‐cell embryos reach the blastocyst stage when cultured during the second day postinsemination in medium supplemented with taurine. Because taurine, an amino acid that abounds in the reproductive tract, has been reported to inhibit the enzyme Na+‐K+‐adenosine triphosphatase (Na+‐K+‐AT‐Pase), we used two other conditions known to inhibit the Na+‐K+‐ATPase to study their effect on mouse embryo development. Culturing embryos during a short period (the second day postinsemination) in low extracellular K+ concentrations (1.4 mM) or in medium supplemented with ouabain (50 μM) showed positive effects similar to those of culturing in medium with taurine (10 mM). This beneficial effect of ouabain was found in various K+ concentrations tested, including the high concentrations present in the oviduct. Although the effects of low K+ and taurine can possibly be ascribed to their other cellular effects, the effect of ouabain shows that inhibition of the Na+‐K+‐ATPase during the two‐cell stage in the mouse is beneficial for further embryonic development to the blastocyst stage. © 1993 Wiley‐Liss, Inc.
Sperm penetration through the zona pellucida of salt‐stored hamster eggs is delayed
Authors: Stewart‐Savage J.
Published: February 2005

One of the problems in assaying sperm capacitation is that live eggs mount a block to polyspermy at the zona pellucida. To circumvent this problem, researchers have started to use salt‐stored eggs to assay sperm capacitation (Boatman et al., Gamete Res 19:19–29, 1988; Yoshimatsu et al., Gamete Res 21:115–126, 1988). These researchers have demonstrated that the zonae of salt‐stored eggs were penetrable by capacitated sperm, but they did not examine whether the timing of penetration of fully capacitated sperm was altered. This report shows that zona penetration by fully capacitated sperm is delayed in both short‐term salt‐treated eggs (2 hr) and in eggs stored in salt for >2 days. These results indicate that the zona is modified when eggs are treated with a high‐salt solution. © 1993 Wiley‐Liss, Inc.
Binding of a murine proteinase inhibitor to the acrosome region of the human sperm head
Authors: Boettger‐Tong Holly L. , Aarons David J. , Biegler Beth E. , George Bobby , Poirier Gary R.
Published: February 2005

Proteinase inhibitors are present in the various glands, tissues, and secretions of the male reproductive tract. Some of these inhibitors bind to the acrosomal region of the sperm, and their release during in vitro or in utero incubation suggests that they may play a role in capacitation. In the mouse, the binding site for a trypsin‐acrosin inhibitor, the acceptor, has been implicated in capacitation, zona binding, and the acrosome reaction. This presentation demonstrates that a component, molecular weight ˜T20,000, on the human sperm head may recognize the murine inhibitor. Furthermore, the acrosome reaction can be induced in capacitated human sperm by immunoaggregation of bound murine inhibitor. The data indicate that the proteinase inhibitor binding site on the human sperm head may, as with a similar site on murine sperm, play a role in the early events of fertilization. © 1993 Wiley‐Liss, Inc.
Effects of electrical stimulation before or after in vitro fertilization on sperm penetration and pronuclear formation of pig oocytes
Authors: Funahashi Hiroaki , Stumpf Todd T. , Terlouw Steve L. , Day Billy N.
Published: February 2005

The effects of exposure of pig oocytes to an electrical pulse on sperm penetration and pronuclear formation were determined before or after in vitro fertilization (IVF). After in vitro maturation (IVM) or after collection from oviducts of unmated gilts, pig oocytes either were not exposed or were exposed to an electrical pulse (a 10 sec pulse at 4.0 V mm−1 AC followed by a 30 μsec pulse at 120 V mm−1 DC), followed 30 min later by IVF. The incidence of male pronuclear formation of both IVM and in vivo‐matured oocytes at 12 hr after insemination was decreased from 59% and 100%, respectively, to 2% and 36%, respectively, by the electrical pulse, but the penetration rates (88–100%) and polyspermic rates (79–100%) were not affected by exposure to an electrical pulse. Similarly, when pig IVM oocytes were exposed to an electrical pulse at 6 hr after insemination, electrical activation did not decrease penetration rates (93% vs. 90%), polyspermic rates (83% vs. 91%), or number of spermatozoa in penetrated oocytes (4.0 ± 0.5 vs. 4.6 ± 0.5) but did decrease the rate of male pronuclear formation from 58% to 18%. When oocytes were examined at 6 hr after insemination, 75% of them had been penetrated and resumed meiotic progression, but all sperm heads in penetrated oocytes were fully condensed or only partially decondensed. The percentage of penetrated eggs with multiple female pronuclei was increased when oocytes were exposed to an electrical pulse in all experimental series. In summary, electrical activation of pig oocytes before or just after IVF does not prevent sperm penetration but does inhibit male pronuclear formation and increases the formation of multiple female pronuclei. © 1993 Wiley‐Liss, Inc.
Does the zona pellucida select spermatozoa from the medium with higher fertilizing potential?
Authors: Tarín J. J. , Trounson A. O.
Published: February 2005

The present study was carried out to test whether the zona pellucida selects spermatozoa with higher fertilization potential. Fertilization rates of mouse oocytes after sperm microinjection under the zona pellucida (SMUZ) of zona‐bound spermatozoa and of spermatozoa incubated in the absence of oocytes and treated (acid‐treated group) or not (control group) with acid Tyrode's solution were compared. SMUZ was performed at 15, 30, 60, and 90 min after the insemination of fresh oocytes required for selecting spermatozoa bound to the zona pellucida. At these times, the percentages of acrosome‐reacted spermatozoa (PARS) were evaluated using the chlortetracycline fluorescence method. Fertilization rate in the three groups analysed increased from 25.9% to 47.3% in the control group, from 29.3% to 44.0% in the acid‐treated group, and from 19.5% to 40.0% in the zona‐bound group from 15 to 90 min after insemination, respectively. The global fertilization potential was significantly lower in the zona‐bound group compared to the other two groups. The PARS in the zona‐bound group at 15 (11.48 ± 3.02); 30 (16.74 ± 3.71), and 90 (19.68 ± 3.68) min after insemination were significantly (P < 0.05) lower than those found in the acid‐treated group (39.26 ± 6.69, 38.20 ± 6.24, and 42.83 ± 5.39, respectively). At 90 min after insemination, the PARS in the zona‐bound group was also significantly (P 0.05) lower than the control group (36.72 ± 4.51). No significant correlation between either time and PARS or PARS and fertilization rate was observed. It appears that the zona pellucida does not select from the medium spermatozoa with higher fertilization potential. © 1993 Wiley‐Liss, Inc.
Confocal and fluorescence microscopic study using lectins of the distribution of cortical granules during the maturation and fertilization of pig oocytes
Authors: Yoshida Mitsutoshi , Cran David G. , Pursel Vernon G.
Published: February 2005

A study was carried out to determine whether pig cortical granules (CGs) could be visualized using fluorescein isothiocyanate (FITC)‐labelled lec‐tins. Following labelling with FITC‐labelled peanut agglutinin (FITC‐PNA), fluorescent spots were observed that had a distribution during maturation and fertilization entirely consistent with that observed by electron microscopy. For the first 18 h of in vitro maturation, most of the fluorescent spots of FITC‐PNA were distributed throughout the cortical cytoplasm. Thereafter, the CGs underwent centrifugal migration to form a monolayer next to the plasma membrane. Following penetration by sperm, fluorescent spots were extruded into the perivitelline space, where they aggregated forming fluorescent clumps, which subsequently formed a reticulate structure surrounding the egg. Fluorescence was gradually lost such that by 18 h after insemination none could be detected in 70% of the eggs. The results indicate that CGs in pig oocytes contain galactosyl‐rich glycoconjugates and that FITC‐PNA is a useful probe for their rapid visualization and examination. © 1993 Wiley‐Liss, Inc.
Effect of follicle‐stimulating hormone and luteinizing hormone during bovine in vitro maturation on development following in vitro fertilization and nuclear transfer
Authors: Keefer C. L. , Stice S. L. , Dobrinsky J.
Published: February 2005

The effects of luteinizing hormone (LH) (0, 100, 10,000 lU/ml) and follicle‐stimulating hormone (FSH) (20 μg/ml) supplementation during in vitro maturation of slaughterhouse‐derived oocytes on polar body formation and embryo development subsequent to in vitro fertilization and nuclear transfer were evaluated. Go‐nadotropin supplementation of maturation medium in the presence of serum neither enhanced the proportion of oocytes forming a polar body nor significantly affected development following in vitro fertilization or nuclear transfer, except at the highest LH concentration. A very high concentration of LH (10,000 lU/ml) significantly decreased polar body formation, initial cleavage, and blastocyst development (P < 0.05). © 1993 Wiley‐Liss, Inc.
Origin of a sperm motility inhibitor from boar seminal plasma
Authors: Iwamoto Teruaki , Tanaka Hiroki , Osada Takao , Shinagawa Toshihito , Osamura Yoshiyuki , Gagnon Claude
Published: February 2005

We have investigated the origin of the sperm motility inhibitor (SPMI) from boar seminal plasma. SPMI was measured by its capacity to inhibit the motility of demembranated spermatozoa and by an enzyme‐linked immunosorbant assay (ELISA). Among the various reproductive and now reproductive tissues and fluids tested, only the seminal vesicle fluid and seminal plasma contained significant amounts of SPMI biological activity and SPMI antigen. Like other seminal vesicle fluid proteins, SPMI is diluted 6‐ to 8‐fold upon ejaculation. By immunohistochemical detection at the light microscope with antibodies obtained from rabbits immunized with SPMI purified from boar seminal plasma, SPMI was found in the cytosol and/or on the plasma membrane bordering the lumen of the seminal vesicles. At the electron microscope level, SPMI appeared to be present only on the surface of the secretory cells. The data indicate that SPMI originates from a single tissue, the seminal vesicle, and suggest that only the mature form present on the luminal surface of the gland can react with the antibody generated from rabbits immunized with the secreted form of SPMI. © 1993 Wiley‐Liss, Inc.
Cryogenic protection of oocytes with antifreeze proteins
Authors: Arav A. , Rubinsky B. , Fletcher G. , Seren E.
Published: February 2005

Proteins belonging to a family of compounds known as “antifreeze proteins” interact with occytes and protect the oolemma from damage at cryogenic temperatures. Experiments were performed with pig oocytes rapidly cooled to cryogenic temperatures in vitrifying solutions with and without antifreeze proteins. Four different types of antifreeze polypeptides and glycoproteins were tested. The integrity of the oolemma was examined with Fluoroscein Diacetate (FDA) staining and morphological examinations. Results show that the pig oocyte oolemma is a primary site of injury during exposure to low temperatures and that all the different proteins have a similar ability to interact with and protect the oolemma. Our results may be important in developing solutions for long‐term preservation of oocytes at cryogenic temperatures (cryopreservation). © 1993 Wiley‐Liss, Inc.
Protein phosphorylation patterns during in vitro maturation of the goat oocyte
Authors: Gall L. , Le Gal F , De Smedt V.
Published: February 2005

Protein phosphorylation patterns were studied by radiolabelling goat cumulus oocyte complexes with [32P]orthophosphate for various periods of time. The radiolabelled denuded oocytes were assessed for nuclear status and were used individually for gel electrophoresis. This study demonstrated that specific changes in protein phosphorylations were programmed during goat oocyte maturation. One of the most prominent changes was a general increase in the phosphorylation rate at germinal vesicle breakdown (GVBD). From 8 hr of culture, dominant phosphoprotein bands with apparent molecular weights of 27, 31, 40, and 50 kD were observed; they remained at this level until the metaphase II stage. In the molecular weight range of 65–80 kD, the protein phosphorylation pattern exhibited characteristic differences, with a complex series of phosphoproteins appearing and disappearing, during maturation. Addition of 6‐dimethylaminopurine (6‐DMAP) at the onset of culture blocked the maturation process after GVBD and induced a dramatic condensation of chromatin. When added at different times after GVBD, 6‐DMAP invariably induced chromosome condensation. This inhibition was partly reversible; i.e., after removal of the drug, oocytes were able to progress only until metaphase l. © 1993 Wiley‐Liss, Inc.
In vitro‐cultured bovine granulosa and oviductal cells secrete sperm motility‐maintaining factor(s)
Authors: Ijaz Ahmad , Lambert Raymond D. , Sirard Marc‐Andre
Published: February 2005

Since bovine cumulus oophorous and oviductual cell cultures are known to support and maintain frozen‐thawed bovine sperm viability and motility for extended time periods, we investigated whether granulosa cell (GC)‐ and oviductual cell (OC)‐conditioned media have similar effects. GC and OC were cultured for 3 days in TCM‐199 medium supplemented with 10% fetal calf serum. At that time, the supernatant was discarded from GC and the monolayers were covered with Sp‐TALP medium containing 6 mg/ml bovine serum albumin, while the OC were recovered by centrifugation and transferred to culture bottles containing Sp‐TALP. Two days later, GC‐conditioned and OC‐conditioned Sp‐TALP were recovered and dialyzed, and their retentates were lyophilized. Bovine follicular fluid (BFF) was also dialyzed, and its retentate was lyophilized. When sperm were incubated in GC‐ or OC‐conditioned media, motility remained above 62% and 42% at 6 hr and 30 hr, respectively, and motility was higher than that of the control both at 6 hr (39%; P < 0.001) and at 30 hr (9%; P < 0.0001). Similarly, when sperm were incubated in the lyophilized retentates of GC‐ and OC‐conditioned media and in BFF at a dose of 0.1, 0.5, or 1.0 mg/ml, the motility rates were higher both at 6 hr (P < 0.05) and at 30 hr (P < 0.01) compared to the control. The increase in motility was dose dependent; a 1.0 mg/ml dose improved (P < 0.05) motility compared to a 0.1 mg mg/ml dose. Heat treatment of the retentates of GC, OC, and BFF at 55°C for 30 min did not destroy their ability to support and maintain motility. However, heating at 100°C for 5 min destroyed their ability to support motility. Molecular sieving of retentates on Sephancryl S‐300 yielded fractions that were highly effective (P < 0.01) in enhancing and maintaining motility compared to the other fractions. In conclusion, GC and OC secrete nondialyzable, heat‐labile factor(s), which support and maintain sperm viability and motility for up to 30 hr. © 1994 Wiley‐Liss, Inc.
Relationship between sperm nucleus remodelling and cell cycle progression of fragments of mouse parthenogenotes
Authors: Szöllösi Maria S. , Borsuk Ewa , Szöllösi Daniel
Published: February 2005

Nucleate and anucleate fragments of parthenogenetically activated mouse oocytes, as well as cybrids obtained by fusion of anucleate fragments (cytoplasts) of maturing and activated matured oocytes were fertilized at different time after activation. Remodelling of the sperm nucleus was studied by electron microscopy at 1.5 and 3 h after fertilization and, in addition, at 14 h in cybrids. Results show that (1) the nuclear envelope of the sperm nucleus can break down when the insemination takes place after the end of M‐phase, but the capacity of the parthenote cytoplasm to remodel the sperm nucleus is restricted in time. (2) Male chromatin can decondense within the old, unbroken nuclear envelope, but in such cases formation of a male pronucleus, one of the two nuclei of zygote possessing inactive nucleoli, is never observed. © 1994 Wiley‐Liss, Inc.
Introduction of exogenous DNA into somatic and germ cells of chickens by microinjection into the germinal disc of fertilized ova
Authors: Naito Mitsuru , Sasaki Erika , Ohtaki Megumi , Sakurai Michiharu
Published: February 2005

The plasmid DNA, pAcZ, containing Escherichia coli β‐galactosidase (lacZ) gene under the control of chicken β‐actin gene promoter was injected in a linearized form into the germinal disc of fertilized chick ova at the single‐cell stage. The manipulated embryos were cultured by the method of Naito et al. (1990) until hatching. The rate of hatching was 11.8% (31/263), and 19 males and 6 females were matured. DNA from blood and semen samples of the 25 matured chickens was analyzed for the presence of the injected DNA by Southern blot hybridization. The injected DNA was detected in the blood DNA of one male and in the sperm DNA of another male up to 13 months after hatching, indicating that the injected DNA was stably maintained in these chickens. Restriction digestion analysis of the injected DNA suggested that it was not rearranged and was organized as head‐to‐tail multimers. The copy numbers of the DNA were 0.07–0.02 in the blood DNA of one male per diploid genome, and 0.02–0.015 in the sperm DNA of another male, indicating that the exogenous DNA was present in limited populations of blood and sperm cells. © 1994 Wiley‐Liss, Inc.
Peroxidase activities in bull spermatozoa
Authors: Pavlova S. K. , Kanchev L. N. , Alexandrov M. Ts.
Published: February 2005

The present study was carried out to determine the localization of peroxidase activity in bull spermatozoa. 3,3′‐Diaminobenzidine (DAB) was used as a substrate for revealing peroxidase activity, and light and electron microscopic analysis of the results obtained was performed. Peroxidase activity was detected in the mitochondria of the middle piece and the outer acrosomal membrane. Catalase was excluded as an enzyme, catalyzing the detected peroxidase activity. Concerning the biochemical properties of bull sperm peroxidases, peroxidase activity was found to be manifested in a large pH range, 4–10.5. Bull sperm peroxidase activity appeared to be temperature sensitive and azide sensitive and could be readily inhibited by phenylhydrazine. Electrophoretic analysis of the proteins from bull sperm extracts separated in a Davis‐Ornstein system of 7% polyacrylamide gel, followed by the determination of peroxidase activity on the polyacrylamide gels, revealed that all 14 sperm protein fractions available on the gel possessed peroxidase when benzidine was used as a substrate. The possible reasons for the electrophoretic heterogeneity of bull sperm peroxidases are discussed. © 1994 Wiley‐Liss, Inc.
Chromatin condensation and histone H1 kinase activity during growth and maturation of rabbit oocytes
Authors: Jelíanková Ladislava , Kubelka Michal , Motlík Jan , Guerrier Pierre
Published: February 2005

Fully grown rabbit oocytes, isolated from preovulatory follicles, exhibit highly condensed bivalents within an intact germinal vesicle while a very low level of histone H1 kinase activity could be detected in their extracts. Chromatin condensation started in growing oocytes isolated from antral follicles presenting a diameter of 0.5 mm. This event was accompanied by a transient rise in histone H1 kinase activity which culminated in large antral follicles measuring 0.75 to 1 mm in diameter.

However, the extent of histone H1 kinase activity observed in these growing oocytes remained far less important than that recorded in extracts prepared from in vitro cultured metaphase I and metaphase II oocytes. Moreover, this activity was insufficient to induce germinal vesicle breakdown which will only occur with an increasing efficiency, following in vitro culture of medium, large, and fully grown antral follicles. © 1994 Wiley‐Liss, Inc.
Fate of sperm organelles during early embryogenesis in the rat
Authors: Shalgi Ruth , Magnus Amnon , Jones Roy , Phillips David M.
Published: February 2005

The report is part of a continuing study in which we employ monoclonal antibodies to membrane domains and internal organelles of rat spermatozoa in order to trace events during maturation, capacitation, fertilization, and early development. In the present study, we have used immunocytochemistry at the light and EM levels to localize one antibody, 5A5, to the fibrous sheath and a second, 3D5, to the outer mitochondrial membrane. Antibody 5A5 does not stain the fibrous sheath of spermatozoa of rodents other than the rat, while 3D5 can be localized to the outer mitochondrial membrane of rat, hamster, and mouse spermatozoa. In order to follow these antibodies during fertilization and early embryogenesis, we developed a method to stain internal components of zygotes and early embryos. Our findings suggests that the fibrous sheath disappears prior to the first cleavage and that mitochondria can be detected up to the 2‐cell stage in mouse and the 4‐cell stage in rat. © 1994 Wiley‐Liss, Inc.
Separate effects of caffeine and dbcAMP on macaque sperm motility and interaction with the zona pellucida
Authors: Vandevoort Catherine A. , Tollner Theodore L. , Overstreet James W.
Published: February 2005

Capacitation of macaque sperm with caffeine and dbcAMP is required for fertilization in vitro. This study determined the separate effects of caffeine and dbcAMP on sperm‐zona pellucida binding and the acrosome reaction of zona bound sperm. Semen from 6 cynomolgus macaques was washed through 60% Percoll, resuspended, and washed with BWW media and incubated for 2.5 hr. Caffeine, dbcAMP (2 mM each), or both (1 mM each) were added to aliquots of the sperm suspensions. Immature macaque oocytes were placed into drops of sperm suspensions, coincubated with sperm for 30 sec, and either fixed immediately or removed to sperm‐free media and incubated 1 hr before fixation. There were no significant diffences between groups in the percentage of live, acrosome‐reacted sperm in suspension. Treatment with caffeine and dbcAMP or with caffeine alone, significantly increased the number of sperm bound to each zona pellucida (96 ± 16 and 81 ± 17, respectively) compared to control and dbcAMP treatment (15 ± 4 and 28 ± 13). However, treatment with dbcAMP, alone and with caffeine, resulted in a higher percentage of acrosome‐reacted sperm on the zona (15.2 ± 2.1 and 9.0 ± 0.6) than control or caffeine treatment (3.0 ± 1.4 and 2.4 ± 0.5). Effects on sperm motility consistent with hyperactivation were detected only when both caffeine and dbcAMP were present. Although both caffeine and dbcAMP are presumed to increase or to produce the same effects as increased intracellular cAMP levels, these compounds have different effects on the ability of sperm to bind to the zona and to undergo the acrosome reaction. © 1994 Wiley‐Liss, Inc.
Sperm nuclear chromatin transformations in somatic cell‐free extracts
Authors: Banerjee Subhasis , Hulten Maj
Published: February 2005

HeLa cell extracts induced decondensation of lysolecithin permeabilized Xenopus, pig, and human sperm chromatin; decondensation began almost immediately on incubation in the extract and was completed within 10–20 min. The average enlargements of human and pig sperm nuclei were 15‐fold and 3‐fold, respectively. The structural organization of pig and human sperm chromatin was significantly differnt. Decondensation was differentially inhibited by Mg++ and polyamines; inhibition was least for Xenopus and most for pig sperm nuclei. The nuclear membrane was disintegrated on chromatin dispersion, whereas the nuclei which failed to decondense exhibited distinct nuclear envelopes. The decondensing factors were stable at 65°C for 15 min. The dispersed chromatin was remodelled to somatic nucleosomal structures within 60 min. The remodelled chromatin could be recondensed to chromosome‐like structures, when incubated further in extracts from mitosis arrested HeLa cells. © 1994 Wiley‐Liss, Inc.