bluuebunny/crossref_metadata_2025 huggingface dataset:
Protamine–histone replacement and DNA replication in the male mouse pronucleus
Authors: Nonchev Stefan , Tsanev Roumen
Published: February 2005

The protamine to histone replacement in fertilized mouse eggs was studied by using antibodies to these proteins. Its course was followed with respect to DNA replication by autoradiography of 3H‐thymidine‐labeled fertilized eggs. It was found that protamines were replaced by histones before the onset of DNA replication.
Combined effect of glycerol concentration and cooling velocity on motility and acrosomal integrity of boar spermatozoa frozen in 0.5 ml straws
Authors: Fiser P. S. , Fairfull R. W.
Published: February 2005

The interaction of glycerol concentrations of 0–10% and cooling rates from 1 to 1,500°C/min with boar spermatozoa motility and acrosomal integrity (proportion of spermatozoa with normal apical ridge) was studied after thawing 0.5 ml straws at a constant rate. While increasing the glycerol concentration from 0 to 4% progressively improved motility, the percentage of spermatozoa with a normal apical ridge gradually decreased. The magnitudes of the respective changes depended on cooling rate. A peak value of 48.1% and rating 3.8 were obtained in semen protected with 4% glycerol, frozen at 30°C/min. Increasing the glycerol levels above 6% resulted in a gradual decrease in motility. The proportion of spermatozoa with normal apical ridge was highest in semen protected with 0–1% glycerol after cooling at 30°C/min (64.4% and 66.1%, respectively), but at these glycerol concentrations the percentage of motile spermatozoa was low. At the 30°C/min cooling rate, the decline in the proportion of cells with normal apical ridge due to increasing the glycerol levels to 3 and 4% was relatively slow (57.3% and 49.4%, respectively). Cooling at 1°C/min was detrimental to acrosomal integrity, which decreased with increasing glycerol concentration, in contrast to increasing motility, which even at its maximum, remained low. The direct plunging of straws into liquid nitrogen (1,500°C/min) resulted in damaged acrosomes in all spermatozoa with the total loss of motility. Balancing motility and acrosomal integrity, freezing boar semen protected with 3% glycerol by cooling at 30°C/min resulted in optimal survival for boar semen frozen in 0.5 ml French straws.
Ultrastructural study on the spermiogenesis and spermatozoon of the metacercariae of Microphallus primas (digenea), a parasite of Carcinus maenas
Authors: Castilho F. , Barandela T.
Published: February 2005

The thread‐like spermatozoon of the crab parasite Microphallus primas was studied by electron microscopy. A survey of the head region of the spermatozoon reveals three features hitherto unknown in Plathelminthes spermatozoa. The first is the aberrant inclusion of the nucleus within one of the two axonemes, limited to the head end region. The second is the coexistence, in the same axoneme, of two patterns, 9 + 0 (doublets without dynein arms) and 9 + “1”. The third is the presence of a layer of cortical microtubules running longitudinally from the zone where the nucleus goes from axoneme to the tail region (where the two flagella start). The sequence of events in spermatogenesis is similar to that described for most of the Digenea trematodes, and the spermiogenesis process conforms to a common plan in nearly the whole group.
An intercellular adhesion protein of rat testis: Isolation and preliminary characterization
Authors: D'Agostino Angela , Stefanini Mario
Published: February 2005

By using biochemical and immunochemical approaches, we have isolated a rat spermatocyte surface protein of an apparent molecular weight, of 80 kd involved in spermatocyte‐Sertoli cell recognition in culture. Preliminary characterization of such a molecule has demonstrated that (1) it is a nonintegral membrane component; (2) it possesses a small amount of N‐linked oligosac‐charides of the high mannose type; and (c) it is a stage‐specific molecule, being present only in middle‐late pachytene spermatocytes.
Fertilization envelope assembly in sea urchin eggs inseminated in chloride‐deficient sea water: II. Biochemical effects
Authors: Green Jeffrey D. , Glas Patricia S. , Cheng Sou‐De , Lynn John W.
Published: February 2005

Eggs of the sea urchin Strongylocentrotus purpuratus were fertilized in normal and in several chloride‐deficient sea waters ([CI−]: normal > isethionate > methyl sulfonate > bromide). The fertilization envelopes (FE) were thinner and failed to harden, and the characteristic I‐T transition did not occur. The permeability of the experimental FEs, as determined by release of protein from the perivitelline space, increased in the order of decreasing [CI−]. Release of the enzymes β‐1, 3‐glucanase and cortical granule protease were not significantly altered. On the other hand, release of ovoperoxidase was increased three to four times in bromide sea water. Furthermore, a dose‐response was observed in varying concentrations of bromide‐normal sea water. With decreasing chloride (increasing bromide) concentration, more ovoperoxidase activity was observed. Cytochemical localization of ovoperoxidase activity with diaminobenzidine revealed almost a total lack of staining of FEs from bromide‐substituted sea water. The results suggest that in chloride‐deficient sea waters protein incorporation into the nascent FE is impaired. At least in the case of bromide, the incorporation of ovoperoxidase into the nascent FE was also inhibited.
Immunolocalization of hyalin in sea urchin eggs and embryos using an antihyalin‐specific monoclonal antibody
Authors: Vater Carol A. , Jackson Robert C.
Published: February 2005

Monoclonal antibodies were raised against purified cortical secretory vesicles (CVs) from the eggs of Stronglocentrotus purpuratus. One of the monoclonal antibodies (MAb 69‐10, an lgA) was shown by immunofluorescence labeling of intact and detergent‐lysed CVs to be directed against a CV content antigen. Immunoblot analysis of CVs revealed that MAb 69‐10 bound to a major CV polypeptide with an Mr similar to that of hyalin (i.e., 300,000). MAb 69‐10 was subsequently shown to bind to purified hyalin prepared from S. purpuratus and to cross react with hyalin prepared from Lytechinus pictus. Immunogold labeling on thin sections of unfertilized S. purpuratus eggs showed that hyalin was localized to the electron‐lucent portion of CVs. This result is in agreement with the labeling pattern obtained by Hylander and Summers (Dev Biol 93:368–380, 1982) using polyclonal antihyalin antibodies. In fertilized eggs and later‐stage embryos, hyalin was observed to be located on the external surface of the embryo. MAb 69‐10 should be useful in studies of the structure of hyalin and its function in morphogenesis.
Effect of antibodies to human seminal plasma inhibin on spermatogenesis and sperm agglutination in adult male rats
Authors: Vanage G. R. , Gopalkrishnan K. , Sheth A. R.
Published: February 2005

In vitro incubation of rat epididymal sperm with antiserum to human seminal plasma inhibin (As hSPl) caused agglutination of the sperm. In vivo administration of As hSPl to adult male rats resulted in a significant decrease in testicular as well as epididymal sperm counts. Furthermore, the majority (almost 90%) of the epididymal sperm were agglutinated. When these animals were mated with normal cycling females, significant reduction in fertility was observed.
Male accessory sex glands produce heparin‐binding proteins that bind to cauda epididymal spermatozoa and are testosterone dependent
Authors: Nass Sharyl J. , Miller David J. , Winer Martin A. , Ax Roy L.
Published: February 2005

Heparin binds to bovine sperm and stimulates capacitation in vitro. Seminal plasma alters the ability of epididymal sperm to bind heparin, and several heparin‐binding proteins (HBPs) have been identified in bull seminal plasma. This study had three objectives: (1) to identify production sites of seminal plasma HBPs, (2) to determine which HBPs bound to cauda epididymal sperm, and (3) to determine whether presence of HBPs was testosterone dependent. Proteins from bull or rat seminal vesicles, prostates, and bulbourethral glands were separated by heparin affinity high‐performance liquid chromatography. HBPs were found in all accessory glands of rats and bulls, but the major source of bovine seminal plasma HBPs appeared to be seminal vesicles. Between 25% and 50% of the protein from each gland bound to the heparin column, and NaCl concentrations required to elute proteins ranged from 0.15 to 1.4 M. One‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) showed that major HBPs were relatively small, with molecular weights between 13 and 31 kDa, but some HBPs also exhibited higher molecular weights, between 40 and 100 kDa. Radioiodinated HBPs from each bovine gland were incubated with epididymal sperm. Labeled HBPs binding to sperm exhibited molecular weights of 14, 16, 24, and 30 kDa as determined by SDS‐PAGE and autoradiography. The HBP content of the accessory sex glands decreased significantly in castrated rats and was restored to levels of sham‐operated controls by testosterone replacement. Heparin‐binding proteins may play a role in fertilization by attaching to sperm surfaces, enabling heparin‐like glycosaminoglycans in the female reproductive tract to induce capacitation.
Activation of mammalian oocytes by a factor obtained from rabbit sperm
Authors: Stice Steven L. , Robl James M.
Published: February 2005

In this study a fraction was prepared from rabbit sperm that activated rabbit and mouse oocytes following injection into the cytoplasm. The sperm factor activated oocytes exhibited cortical granule exocytosis, pronuclear formation, and cleavage. The sperm factor was soluble in aqueous solution and was not active extracellularly. Unlike most artificial activation methods that are only effective with aged oocytes, the sperm factor activated recently ovulated oocytes. The factor appears to be a protein or associated with a protein but not an acrosomal protein. Fractions from both mouse and bull sperm did not activate rabbit or mouse oocytes. Their inactivity may be owing to the techniques used to recover the fractions or differences between species in sperm morphology and fertilization processes. These observations support the hypothesis that oocyte activation is induced by a factor within sperm that is released into the cytoplasm of the oocyte at the time of sperm–oocyte fusion.
Cleavage development of bovine oocytes fertilized by sperm injection
Authors: Keefer Carol L. , Younis Abdelmoneim I. , Brackett Benjamin G.
Published: February 2005

Whole in vitro capacitated bovine spermatozoa were microinjected directly into the ooplasm of in vitro matured bovine oocytes in order to determine whether oocytes fertilized by sperm injection could undergo normal pronuclear formation and cleavage development. Immature oocytes recovered from follicles (2–5 mm) of unstimulated ovaries were cultured for 24–25 h in modified TCM 199 medium supplemented with heat‐treated day 20 cow serum, luteinizing hormone (LH), and estradiol 17‐B. In vitro capacitated, frozen‐thawed spermatozoa were injected into the ooplasm, and the injected oocytes were cultured for an additional 24–28 h. Twenty‐one percent (21/101) of the sperm‐injected oocytes contained a sperm within the ooplasm; however, only 2% (2/101) cleaved. The remaining oocytes either did not contain a sperm or had degenerated. After oocyte activation induced by a 5 min incubation in 1 μM A23187, sperm nuclear decondensation occurred in the A23187‐activated, injected oocytes but not in the unactivated, injected controls (37% vs. 0% after 3 h). Those injected, activated oocytes that contained a male pronucleus also exhibited a female pronucleus and second polar body. Furthermore, a significantly higher number (28%, 6/21) of the injected, activated oocytes cleaved to a two‐ to four‐cell stage after 48 h than did the injected, unactivated oocytes (4%). These results indicate that, unlike hamster and rabbit oocytes, bovine oocytes are not sufficiently stimulated by the injection procedure to complete meiosis, but, upon activation by calcium ionophore, they will undergo normal‐appearing cleavage development following fertilization by sperm injection.
Atypical spermatogenesis in Murex brandaris
Authors: Amor M. J. , Durfort M.
Published: February 2005

Atypical spermatogenesis in Murex brandaris occurs in specific areas of the testis. In the early stages, a large nucleus is apparent, together with highly electrodense drops of chromatin spread through the caryoplasm. Many mitochondria are observed at one point of the cytoplasm, and numerous centrioles appear. In the following stages, vacuolization and degeneration of the chromatin are apparent, producing half moon‐shaped chromatinic bodies called “caryomerites.” Some degeneration of the chondrioma occurs too. Later, the chromatin degenerates and the centrioles produce cilia, while the Golgi body, highly secretory at this stage, produces electrodense PAS‐ as well as Thiery‐positive granules. Moreover, a vermiform‐shaped anuclear ciliated cell with numerous electrodense cytoplasmic granules is observed in the M. brandaris atypical spermatozoon.
Further characterization of the mouse sperm surface zona‐binding site with galactosyltransferase activity
Authors: Benau Danny A. , McGuire Edward J. , Storey Bayard T.
Published: February 2005

One of the mouse sperm surface binding sites for zona pellucida ligands exhibits galactosyltransferase (GT) enzyme activity. The present study was undertaken to ascertain whether the GT site behaves as a noncatalytic binding site in its physiological capacity, with no glycosylation of zona ligands, or whether glycosylation of zona ligands is an integral part of sperm‐zona binding. The effects of Mn2+, the obligatory cation for GT catalysis, on enzyme activity and sperm‐zona binding were examined. With uridine‐5′‐diphosphogalactose (UDPgal) as galactose donor, and N‐acetylglucosamine (GlcNAc) as galactose acceptor, increasing concentrations of Mn2+ in the range of 0.1–10 mM increased GT enzyme activity, with half‐maximal activation at 0.65 mM Mn2+ (Vmax = 20 pmol/hr/106 cells). In the presence of 0–2 mM Mn2+, sperm‐zona binding was inhibited in a concentration‐dependent manner; 50% inhibition occurred at 1.25 mM Mn2+. At this concentration, GT enzyme activity was at 65% Vmax. To determine the specificity of the GT site for glycoprotein terminal carbohydrate residues, spermatozoa were incubated with, asialo‐ovine submaxillary mucin (N‐acetylgalactosamine residues), asialo‐, ‐α1‐acid glycoprotein (β1–4 galactose residues) ovalbumin (Ov; GlcNAc residues), and asialo‐agalacto‐/α1‐acid glycoprotein (AsAgAGP; GlcNAc residues). Only Ov and AsAgAGP acted as acceptors for galactose in the enzyme assay and inhibitors in the sperm‐zona binding assay. The kinetics of the interaction of AsAgAGP with the GT site were determined: the Km was 3.6 mg/ml, with Vmax of 33 pmol/hr/106 cells. In the absence of Mn2+ and UDPgal, sperm‐zona binding was inhibited in a hyperbolic manner by 0–8 mg/ml AsAgAGP; 50% inhibition was observed with 0.25 mg/ml AsAgAGP. Our model for this site predicts that, in the absence of Mn2+, UDPgal and glycoproteins with GlcNAc terminal residues compete for zona ligands to inhibit sperm‐zona binding; the presence of Mn2+ causes a change in the site from substrate binding conformation to catalytic conformation, which is unfavorable for sperm‐zona interaction.
Evaluation of rabbit sperm acrosomal integrity and fertilizing ability by use of vital stains
Authors: McBride C. E. , Fayrer‐Hosken R. A , Srivastava P. N. , Brackett B. G.
Published: February 2005

Three staining procedures to detect sperm acrosome integrity were compared via electron microscopy. Stains were applied to epididymal, freshly ejaculated, in vivo capacitated, and sonicated sperm cells in addition to spermatozoa displaying sequentially removed plasma and outer and inner acrosomal membranes. Sequential membrane removal procedures resulted in removal of plasma membranes from 73% of all sperm cells, removal of plasma and outer acrosomal membranes from 74% of all sperm cells, and removal of plasma and outer and inner acrosomal membranes from 87% of all sperm cells as determined by electron microscopy. Live/dead staining results were not statistically different from subjective microscopic motility evaluations (P < 0.005) for epiddymal, sonicated, freshly ejaculated, and in vivo capacitated sperm samples. All three stains assessed were similarly capable of detecting the acrosome status of freshly ejaculated and of sonicated spermatozoa compared to data obtained by electron microscopy (P = 0.010). However, only the Bryan‐Akruk stain afforded data that were closely correlated with data obtained via electron microscopy for all sperm types assessed; the latter included in vivo capacitated spermatozoa and sperm cells rendered free of plasma membranes. Results confirmed an earlier report by successfully effecting sequential removal of rabbit acrosomal membrances and documented use of the Bryan‐Akruk acrosomal stain for evaluation of sperm cell popualtions for fertilizing ability. These findings should prove useful in further investigations of mechanisms involved in achievement of fertilizing ability by rabbit spermatozoa.
Origin and early development of female germ cells in Eoleptestheria ticinensis Balsamo‐Crivelli, 1859 (Crustacea, Branchiopoda, Conchostraca)
Authors: Sabelli Franca Scanabissi , Tommasini Stefano
Published: February 2005

We have studied the early stages of the development of the female germ cells in the Conchostraca Eoleptestheria ticinensis Balsamo‐Crivelli, 1859. The gametes originate in a scattered way throughout the tubular units of the gonad, with no development gradient recognizable. The female germ cells arise from successive karyokineses not followed by cytokinesis, within an unorganised area in which a sort of plasmodium is formed. Each primordial nucleus of this germarium develops and then forms an individual plasmic membrane. Subsequently, the usual organules differentiate in the cytoplasm. The presence of synaptinemal complexes and the beginning of the endogenous vitellogenesis by the rough endoplasmic reticulum and the Golgi apparatus qualify the previtellogenesis. The general characteristics of this early development phase of the gametes, as well as several substantial differences in the gametogenesis with respect to the other Phyllopoda studied, lead us to suggest the systematic positioning of the Leptestheriidae.
Effect of culture conditions and media on the frequency of chromosomal abnormalities in human sperm chromosome complements
Authors: Martin Renee H. , Templado Cristina , Ko Evelyn , Rademaker Alfred
Published: February 2005

Human sperm chromosomes can be visualized after fusion with hamster eggs. Most laboratories use one of two methods of sperm treatment for capacitation: incubation in a modified Krebs‐Ringer medium (BWW) for 5–7 h at 37°C or storage in a TES‐Tris yolk buffer (TYB) for 24–72 h at 4°C. To determine whether data from the two methods were comparable, we performed a series of controlled experiments on one normal donor in which ejaculates were split and one aliquot of sperm was capacitated in BWW for 5–7 h at 37°C (fresh) and the second aliquot was capacitated in TYB for 48 h at 4°C (TYB). After capacitation, the technique used to obtain human sperm chromosome complements was identical for both aliquots. Both fresh and TYB sperm were further subdivided into two groups, which were subjected to either a short (1 h) or a long (3 h) gamete coincubation in BWW. This experiment was performed to determine if the longer incubation in BWW might induce chromosomal fragile sites and breaks because of nutritional depletion of the medium. A total of 458 human sperm chromosome complements was analysed. There was no significant difference in the frequency of sperm chromosomal abnormalities or in the sex ratio in the sperm coincubated with eggs for a short (1 h) or long (3 h) time in BWW. When sperm pretreatments were compared, there was a significant increase in the frequency of total sperm chromosomal abnormalities after TYB storage compared to fresh treatment. This difference was due to an increased frequency of structural chromosomal abnormalities in sperm stored in TYB, not in hyperhaploid or hypohaploid sperm. There was no difference in the sex ratio of fresh (47.2% X) or TYB sperm (47.2% X). Thus our results suggest that storing sperm at 4°C for 48 h in TYB does not alter the frequency of aneuploidy but significantly increases the frequency of structural chromosomal abnormalities.
Quantitative changes in cytoskeletal β‐ and γ‐actin mRNAS and apparent absence of sarcomeric actin gene transcripts in early mouse embryos
Authors: Taylor Kent D. , Pikó Lajos
Published: February 2005

Actin is known to be synthesized both during oogenesis and in cleavage‐stage embryos in mice. Cytoskeletal β‐actin appears to be the major component, followed by γ‐actin, but the synthesis of α‐actin has also been inferred from protein electrophoretic patterns. We have studied the expression of cytoskeletal (β‐ and γ‐) and sarcomeric (α‐cardiac and α‐skeletal) actin genes at the level of the individual mRNAs in blot hybridization experiments using isoform‐specific RNA probes. The results show that there are about 2 × 104 β‐actin mRNA molecules in the fully grown oocyte; this number drops to about one‐half in the egg and less than one‐tenth in the late two‐cell embryo but increases rapidly during cleavage to about 3 × 105 molecules in the late blastocyst. The amount of γ‐actin mRNA is similar to that of β‐actin in oocytes and eggs but only about 40% as much in late blastocysts, indicating a differential accumulation of these mRNAs during cleavage. The developmental pattern of β‐ and γ‐actin mRNA provides a striking example of the transition from maternal to embryonic control that occurs at the two‐cell stage and involves the elimination of most or all of the maternal actin mRNA. There was no detectable α‐cardiac or α‐skeletal mRNA (i.e., < 1,000 molecules per embryo) at any stage from oocyte to late blastocyst, suggesting that the sarcomeric actin genes are silent during preimplantation development.
Immunoelectron microscopical visualization of ribonucleoproteins in the chromatoid body of mouse spermatids
Authors: Biggiogera M. , Fakan S. , Leser G. , Martin T. E. , Gordon J.
Published: February 2005

The chromatoid body (CB), a cytoplasmic organelle present only in germ cell line, was studied at the electron microscopic level in mouse spermatids using cytochemical techniques and specific antibodies directed against sn‐RNPs, hnRNPs, and ribosomal proteins. We found that specific staining for DNA as well as the use of monoclonal anti‐DNA antibodies show a complete absence of DNA in the CB. The CB remains stained, however, after the application of the ethidium bromide‐PTA technique, suggesting the presence of RNA within this organelle. snRNP as well as hnRNP proteins are demonstrated within the CB by means of specific monoclonal or polyclonal antibodies, especially during earlier spermiogenic stages. Monoclonal antibodies directed against the large ribosomal subunit proteins P1/P2 detect these antigens on the CB essentially along the internal threads of dense fibrillar material. Our findings suggest that the CB may function as a source of mRNA and/or of its partially processed precursors during the late stages of spermiogenesis, when the spermatid nucleus becomes gradually inactive.
Dimethylsulphoxide affects the organisation of microfilaments in the mouse oocyte
Authors: Vincent C. , Pickering S. J. , Johnson M. H. , Quick S. J.
Published: February 2005

The effect of dimethylsulphoxide (DMSO) on microfilament organisation has been studied in the mouse oocyte after staining with (NBD)‐phallacidin. The cortical actin meshwork was disrupted by exposure of oocytes to 1.5 M DMSO at 37°C, and this disruption was associated with changes in the cell surface, especially microvilli length and distribution, as observed by scanning electron microscopy. The irregular distribution of actin filaments observed also appears to lead to an irregular expansion of the cell after DMSO removal. However, when exposure to DMSO was combined with cooling, the effects on the microfilament system were much reduced. The reversibility of DMSO action is considered and the potential implications of microfilament disruption on the viability and functions of the oocyte discussed.
Chymotrypsin‐like enzymes are involved in sperm penetration through the vitelline coat of Ciona intestinalis egg
Authors: Pinto Maria Rosaria , Hoshi Motonori , Marino Rita , Amoroso Alessandro , De Santis Rosaria
Published: February 2005

In Ciona intestinalis, sperm penetration through the egg vitelline coat is an essential event of fertilization. We investigated whether trypsin‐ and chymotrypsin‐like enzymes are involved in this event. Inhibitors and peptide substrates for chymotrypsin‐like enzymes blocked the overall process of fertilization in a concentration‐dependent manner. The inhibitory activity was specifically exerted on the step of sperm penetration. Chymotrypsin‐like protease activity was identified in spermatozoa with the fluorogenic synthetic substrate Suc‐Ala‐Ala‐Phe‐AMC, which was the most effective substrate in blocking sperm penetration. These data indicate that a chymotrypsin‐like protease activity is a sperm lysin of Ciona intestinalis.
Stage‐specific expression of phosphoprotein p19 during spermatogenesis in the rat
Authors: Amat Jose A. , Fields Kay L. , Schubart Ulrich K.
Published: February 2005

The expression of phosphoprotein p19, a 19‐kDa cytosolic substrate for cyclic adenosine monophosphate (cAMP)‐dependent protein kinase, occurs abundantly in brain and testis and is developmentally regulated. In the present study we have identified the cell types of adult rat testis that contain p19. Using cryostat sections, which were first incubated with rabbit anti‐p19 for immunohistochemistry followed by counterstaining with periodic acid‐Schiff (PAS)‐hematoxylin to reveal nuclear morphology, we demonstrate that immunoreactive p19 is detectable only in germ cells and is restricted to a limited stage of spermatogenesis. Expression first appears after the differentiating gametes have entered the prophase of meiosis, is abundant in spermatocytes until meiosis is completed, and declines to undetectable levels in maturing spermatids. We have ruled out immunocross‐reactivity with SCG10, a 22‐kDa protein that is closely related in structure to p19, by demonstrating, using Northern blot analysis, that RNA transcripts encoding SCG10 are not detectable in adult rat testis, whereas p19 is abundantly expressed. The transient expression of p19 during spermatogenesis suggests that the protein plays a role during male gamete differentiation.
Ectopic expression of chloramphenicol acetyltransferase (CAT) in the cerebellum in mice transgenic for a carbonic anhydrase II promoter‐CAT construct that is without apparent phenotypic effect
Authors: Erickson Robert P. , Bevilacqua Arturo , Venta Patrick J. , Karolyi Jill , Tashian Richard E.
Published: February 2005

We have developed six transgenic lines of mice with constructs containing presumptive 5′ regulatory regions of carbonic anhydrase II (CA II). Four of the lines contained 1,100 bases of the 5′ flanking region of the human CA II gene, and two transgenic lines resulted from a construct containing 500 bases of the 5′ flanking region of the mouse CA II gene. Tissue‐specific expression of the chloramphenicol acetyltransferase (CAT) gene was not obtained in any of the transgenic lines. One of the transgenic lines was found to have high levels of expression of CAT in cerebellum. This expression persisted through multiple generations and was independent of the parental origin of the transgene. On the assumption that the expression was due to the insertion of the transgene in or near a gene expressed normally in cerebellum, homozygous mice were bred for the transgenic insert to see if a mutation might have been induced. Homozygous mice were found and seemed to be normal in all aspects of their phenotype studied. Thus, in this case, neither the insertion of the gene nor the ectopic expression of CAT seemed to be harmful to the animals.
Potential of hypertonic medium treatment for embryo micromanipulation: I. Survival of rabbit embryos in vitro and in vivo following sucrose treatment
Authors: Yang X. , Chen Y. , Chen J. , Foote R. H.
Published: February 2005

Rabbit zygotes and embryos were exposed to hypertonic sucrose in phosphate‐buffered saline (SPBS). In experiment one, 144 zygotes shrank to 32–36% of their initial volume in 1.0 M SPBS within 30 min. Neither hypertonic treatment with 0.5 M or 1.0 M SPBS nor micropuncture of the zona pellucida after shrinkage affected embryo development into blastocysts in vitro (88%, 83%, and 82%, respectively), compared to that of the controls (93%, P > .05). In experiment two, 252 two‐ to four‐cell‐ and 177 morula‐stage embryos were exposed to isotonic PBS control or 0.5 M, 1.0 M, or 1.5 M SPBS for 30, 60, 90, 120, and 150 min before transfer to PBS (290 mOsm). Embryo development was significantly reduced (P < .05) when embryos were exposed in 0.5 M and 1.0 M SPBS for more than 60 min or in 1.5 M SPBS for more than 30 min. In experiment 3, morulae exposed for 60 min to 0.5 M or 1.0 M SPBS shrank to 37–39% or 32–35% of their initial volume and then expanded to 87–94% or 81–90% of their initial volume, respectively, after being returned to isotonic PBS for 60 min, but embryos in 1.5 M SPBS had erratic osmotic behavior. In experiment four, 192 two‐ to four‐cell embryos exposed to 0.5 M SPBS for 0, 30, and 60 min before transfer to oviducts of recipients resulted in the production of 39%, 42% and 31% young, respectively (P > .05). Exposure of embryos to 0.5 M sucrose for 60 min clearly does not compromise developmental potential and can simplify and speed up micromanipulation procedures.
Ultrastructure of porcine embryos following development in vitro versus in vivo
Authors: Hyttel P. , Niemann H.
Published: February 2005

The present study examined the ultrastructural appearance of porcine embryos from the four‐cell stage to the blastocyst grown either in vivo or in vitro. Embryos were collected at slaughter from superovulated gilts and were fixed for transmission electron microscopy either immediately or after various periods of in vitro culture. In general, the morphology of in vivo and in vitro grown embryos was similar. In vivo grown four‐cell stages contained dense fibrillar nucleoli. At the eight‐cell stage the nucleoli possessed increasing amounts of chromatin and granules. In both stages the mitochondria were spherical or ovoid in shape and had only few cristae. In morulae and blastocysts the nucleoli were mainly of the fibrillogranular type, and the mitochondria were filamentous and possessed more cristae, of which many were tubular. Two major ultrastructural deviations were observed in about half of the in vitro cultured embryos. First, nucleolus‐like structures were found outside the nuclei in the cytoplasm of blastomeres. These structures were spherical and composed of chromatin‐like material containing characteristically a single large and several small vacuoles. The structures were frequently associated with profiles of smooth endoplasmic reticulum (SER). A second type of deviation was aggregates of SER appearing as spiral coils or multiangular complexes. Some embryos displayed both types of deviations. The physiological significance of these deviations remains speculative. They may be involved in the considerably reduced capability of porcine embryos to develop to piglets following in vitro culture.
Sperm DNA and sex chromosome differences between two geographical populations of the creeping vole, Microtus oregoni
Authors: Johnson Lawrence A. , Clarke Robert N.
Published: February 2005

The relative DNA content of the “O” and Y chromosome‐bearing sperm is presented for the creeping vole, Microtus oregoni. The animals had been trapped in Oregon and in Washington State. The two populations had very similar autosomal chromosome relationships but differed greatly in the size of their X chromosome (which is not carried by vole sperm) and in their Y chromosome. The greater size and banding differences of the Y chromosome of the Washington State vole compared to the Oregon vole paralleled the greater differences in sperm DNA between the Y‐bearing sperm and the sperm carrying no sex chromosome (O). The actual DNA differences between O and Y sperm was 12.5% for the sperm from the Washington State voles and 9.1% for sperm from the Oregon voles. The difference in sperm DNA content (12.5%) for Washington State voles was far greater than the difference shown for other voles or other mammals.
Chromosome constitution of highly motile mouse sperm
Authors: Estop A. , Catala V. , Santalo J.
Published: February 2005

In this study, we address the relationship between motility and genetic content of mouse sperm. The chromosome complements of highly motile mouse sperm, selected using the swim‐up technique, were analyzed after in vitro fertilization, at the first cleavage state. They were compared to those of unselected sperm. Identification of male and female chromosome sets was possible because of their differential condensation at the first mitotic division. In vitro fertilization, swim‐up separation, chromosome preparation, and staining were carried out using standard techniques. The results indicate that highly motile mouse sperm did not differ in types and frequencies of chromosomal abnormalities from those not selected for motility. Moreover, separation of motile sperm does not deviate the sex ratio from the theoretical 1:1.
Changes in exposed membrane proteins during in vitro capacitation of boar sperm
Authors: Berger Trish
Published: February 2005

Exposed plasma membrane proteins were labeled with 125I before and after incubation of boar sperm under capacitating conditions. Labeled protein profiles were compared to the ability of the sperm to penetrate zona‐free hamster ova. Quantitatively, the labeled sperm membrane proteins were primarily low Mr prior to capacitation. The majority of the labeled seminal plasma protein was also low Mr. After capacitation, two new proteins (64,000 Mr and 78,000 Mr) were labeled. Sperm did not exhibit these exposed membrane proteins when incubated under noncapacitating conditions. Appearance of these proteins was not correlated to the percentage of acrosome‐reacted sperm. Although the 64,000 Mr protein was not consistently observed, the relative labeling of the 78,000 Mr protein was highly correlated with the ability of sperm to fuse with zonafree hamster ova. The 78,000 Mr protein may be a sperm protein involved in fusion with the egg plasma membrane.
Effects of follicle stimulating hormone and purines on rat oocyte maturation
Authors: Törnell Jan , Brännström Mats , Magnusson Claes , Billig Håkan
Published: February 2005

The presented data demonstrate a dose‐dependent inhibition of spontaneous meiosis of cumulus‐enclosed rat oocytes by guanosine, hypoxanthine, and adenosine. The inhibition by adenosine was transient whereas guanosine and hypoxanthine exerted a persistent effect over 24 h of incubation. The order of potency of the substances was guanosine>hypoxanthine>adenosine and the inhibition was reversible. The inhibitory effect was reduced when the cumulus cells around the oocyte were removed. The inhibition during the first 12 h of incubation was potentiated by FSH. However, at 24 h of incubation FSH partially overcame the inhibitory effect by hypoxanthine but did not influence the inhibitory effect by guanosine. Also 8BrcAMP potentiated the inhibitory effect observed by guanosine, hypoxanthine, and adenosine, suggesting that the potentiating effect of FSH was mediated via cAMP. Our data demonstrate that adenosine, hypoxanthine, and guanosine synergized with FSH in inhibiting spontaneous rat meiosis, as previously shown in mouse. FSH could partially overcome the inhibitory effect exerted by hypoxanthine but did not counteract the inhibitory effect of guanosine.
Selective binding of mouse and human spermatozoa to beads coated with extracellular matrix components
Authors: De Felici Massimo , Camaioni Antonella , Russo Mario A. , Siracusa Gregorio
Published: February 2005

The ability of mouse epididymal and human ejaculated spermatozoa to bind to beads coated with various extracellular matrix components was examined. Mouse spermatozoa preferentially bound to beads coated with heparin (average values ranging between 6.2 and 8.8 sperm per bead were obtained in different experiments) and with chondroitinsulfate (6.2–7.0), and also, although with significant differences across replicate experiments, to beads coated with laminin (7.9–15.6 sperm per bead) and with collagen type I (6.1–18.5). Human spermatozoa bound to collagen‐coated beads (15.4–22.6 sperm per bead) and, to a much lower extent, to chondroitinsulfate‐coated beads (3.2–4.7); they were also able to bind heparin‐coated beads, although with ample differences between individual sperm donors (ranging between 0.8 and 18.7 sperm per bead). Very few human and mouse sperm bound fibronectin‐coated beads; beads coated with albumin, hyaluronic acid, and chondronectin were always totally free of adhering sperm. The possible physiological role of the interactions between spermatozoa and extracellular matrix components are discussed.
Asynchrony between pronuclear development and protein synthetic changes in zygotes of the mouse derived from oocytes aged postovulation in vivo and fertilized in vivo
Authors: Boerjan M. L. , Op Het Veld C. W. , Koopman M. B. H.
Published: February 2005

The pattern of proteins synthesized by one‐cell embryos derived from unaged oocytes and oocytes aged postovulation in vivo was analyzed by means of 35S‐methionine labeling and gel electrophoresis. The oocytes were obtained after ovulation induction by an injection of luteinizing hormone‐releasing hormone (LHRH) at proestrus or after a superovulation procedure. The analysis was performed in unfertilized aged and unaged secondary oocytes and in zygotes derived from them. The patterns of proteins synthesized by secondary oocytes from all experimental groups were very similar: The oocytes showed a predominant synthesis of 35 kDa proteins. Zygotes from aged as well as unaged LHRH‐induced oocytes also showed a predominant synthesis of one group of polypeptides with a relative molecular weight of about 35 kDa. The proteins of the 35 kDa protein complex migrated in an upper (u), middle (m), or lower (l) band in 10% polyacrylamide sodium dodecyl sulfate (SDS) gels. The u‐ and m‐band 35 kDa proteins were shown to be synthesized by secondary oocytes. Early pronuclear zygotes from unaged LHRH‐induced oocytes synthesized u‐ and m‐ but no l‐band 35 kDa proteins. In contrast, part (38%, n = 47) of the early pronuclear zygotes from aged LHRH‐induced oocytes did synthesize the l‐band 35 kDa proteins. Late pronuclear zygotes (LPZ) from aged as well as unaged oocytes synthesized predominantly the l‐band 35 kDa proteins. However, although only 5.8% (n = 51) of LPZ from unaged oocytes synthesize m‐ and l‐band 35 kDa proteins, these bands of proteins are present in 25% (n = 24) of the LPZ from aged oocytes. Thus, in zygotes from aged oocytes, at least part of the fertilization‐dependent changes in the 35 kDa protein synthetic pattern are advanced with reference to the morphological stage of pronuclear progression. Most (80.6%, n = 36) of the early pronuclear zygotes from unaged superovulated oocytes synthesized u‐ and m‐band 35 kDa proteins, like early pronuclear zygotes from unaged LHRH‐induced oocytes. However, 16.7% did not synthesize any of the 35 kDa proteins at all. Thus with respect to the protein synthetic pattern of 35 kDa proteins, zygotes from unaged superovulated oocytes are different from zygotes derived from unaged LHRH‐induced oocytes.
Subzonal microinjection of mouse spermatozoa: Insufficient sperm motility might induce phagocytosis
Authors: Klemm Martina , Engel Wolfgang
Published: February 2005

Acrosome‐reacted CB6F1 mouse spermatozoa with slight flagellar motility were microinjected under the zona pellucida of CB6F1 mouse oocytes. Electron microscopy revealed the presence of swollen and decondensed sperm heads in the oocyte cytoplasm. Sixty‐one percent of the microinjected oocytes reached a morphologically apparent two‐cell stage, but chromosomal analysis demonstrated only haploid chromosomal complements in all cases. The exposure of microinjected oocytes to suspensions of spermatozoa of mice homozygous for a 2,4 reciprocal translocation resulted in normal fertilization and embryonic development with a maternally as well as a paternally derived haploid genome. Identical results were obtained with oocytes microinjected with medium and subjected to in vitro fertilization thereafter. Thus it can be suggested that the microinjected spermatozoa with insufficient flagellar motility are incorporated into the oocyte cytoplasm by phagocytosis. These spermatozoa do not induce a polyspermy block but induce the oocyte to parthenogenetic development.
Further observations on the phagocytosis of Candida albicans by Hamster and Human Oocytes
Authors: Koeheler James K. , Clark Judy M. , Dianne Smith W.
Published: February 2005

Pathogenic yeast, Candida albicans, were incubated with hamster and human oocytes for up to 21 hours in order to determine the nature and time course of phagocytosis of these organisms. Aliquotes of the interacting cells were taken at various time intervals for electron microscopic examination. Some specimens had their zona pellucidae enzymatically removed prior to incubation with yeast, and these specimens showed the most extensive interaction and phagocytosis of Candida. The zona pellucida appears to be an effective barrier to yeast, at least over the time span studied. The observations are consistent with the hypothesis of an initial attachment of yeast via a surface component to oocyte microvilli followed by phagocytic uptake into an endosome. There is no compelling evidence of lysosomal degradation of the yeast over the time course of this study; however, the oocytes appear to undergo some degenerative changes at long incubation times.
Isolation, characterization, and localization of the zona pellucida binding proteins of boar sperm
Authors: Hanqing Mo , Tai‐Ying Yan , Zhao‐Wen Shen
Published: February 2005

In order to isolate the putative zona pellucida‐binding proteins (ZPBPs) from boar spermatozoa, a new, simple method has been developed. The new isolation strategy made the most of the highly specific interactions between the components of the gametes. Detergent‐extracted boar sperm proteins were submitted to affinity chromatography on a ZP‐Sepharose column. SDS‐PAGE analyses of the retained fraction under reducing conditions revealed that in addition to a component of Mr 38,000, the predominant ZPBPs contained at least three low molecular weight proteins (Mr <20 kDa). The isolated ZPBPs were effective in blocking sperm‐oocyte binding in vitro. Using immunofluorescence microscopy, the ZPBPs were shown to be localized primarily in the sperm head, especially in the acrosomal cap region.
Distribution of filipin‐sterol complexes in the plasma membrane of stallion spermatozoa during the epididymal maturation process
Authors: López A. M. L. , De Souza W.
Published: February 2005

The presence and distribution of cholesterol in mature and immature epididymal spermatozoa was analyzed using filipin as a cytochemical tool in freeze‐fracture replicas and thin section preparations. The polyenic‐antibiotic filipin formed complexes with 3,β‐OH sterols, producing characteristic protrusions, or pits, that were heterogeneously distributed in the plasma membrane of stallion spermatozoa, revealing a specific organization in a functionally specialized area of the gamete. The acrosomal region of the sperm head presented a significantly higher density of filipin sterol complexes than the postacrosomal region, which was usually free of these complexes. The plasma membrane of the flagellum also showed filipin sterol complexes randomly distributed in freeze‐fracture replicas. The strong filipin labeling observed in the membrane of spermatozoa obtained from the caput region of the epididymis decreased significantly during epididymal passage. The significance of these changes is not completely understood, but they might contribute to establishing the molecular organization necessary for sperm transit and storage in the epididymis as well as to development of motile spermatozoa that are able to fertilize the oocyte and induce normal embryonic development.
A preferential site for sperm‐egg fusion in mammals
Authors: Talansky Beth E. , Malter Henry E. , Cohen Jacques
Published: February 2005

The tendency of mammalian sperm‐egg fusion to occur at a site away from the first polar body was investigated in a homologous (mouse oocytes and mouse spermatozoa) and in a heterologous model (hamster oocytes and mouse spermatozoa). Following micromanipulation of the zona pellucida either in proximity to or opposite the first polar body, in vitro fertilization was performed and subsequent differences in sperm‐egg interaction were evaluated. Since spermatozoa from random‐bred mice do not readily penetrate intact zonae pellucidae in vitro, it is likely that zona penetration occurred through the artificial holes in both models. The creation of a gap in the zona pellucida opposite the first polar body was associated with levels of sperm fusion that were significantly higher than those resulting from manipulation near the first polar body. Spermatozoa were rarely found to penetrate the hole completely, and in general few spermatozoa were observed in the perivitelline space. The proximity between pronuclei following sperm penetration was correlated with the position of the incision with respect to the polar body. The findings suggest that breaching the zona pellucida for microsurgical fertilization should be performed away from the microvillus‐free area of the oocyte.
Studies on zona hardening in rat oocytes that are matured in vitro in a serum‐free medium
Authors: Zhang X. , Rutledge J. , Armstrong D. T.
Published: February 2005

The present study confirms that zonae pellucidae of rat oocytes became resistant to chymotrypsin digestion (zona hardening) after undergoing in vitro maturation (IVM) in a serum‐free medium. However, zona hardening and did not occur when empty zonae without oocytes were cultured in the same IVM conditions, suggesting that oocyte‐derived factor(s) is responsible for zona hardening in oocytes matured in the serum‐free medium. Zona hardening occurred primarily after dictyate oocytes were cultured for 6–8 hours in the IVM medium without serum. Zona hardening could be prevented or alleviated if oocytes were cultured in the IVM medium containing bovine foetal calf serum, a soybean trypsin inhibitor, or β‐mercaptoethanol, and in vitro fertilization rates for such oocytes were normal. Possible mechanisms of the phenomenon of zona hardening in oocytes matured in serum‐free conditions are discussed in relation to its possible relevance to the cortical reaction and the physiological block to polyspermy.