bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ram spermatozoa cocultured with epithelial cell monolayers: An in vitro model for the study of capacitation and the acrosome reaction
Authors: Gutiérrez A. , Garde J. , García‐Artiga C. , Vázquez I. 
Published: February 2005

AbstractThe effects of different epithelial cells, namely, hamster oviduct, sheep oviduct, and pig kidney epithelial cells (IBRS‐2), on the viability, percentage of progressive motility (PPM), and acrosome reactions of ejaculated ram spermatozoa were investigated. Sperm aliquots were cultured on cells, cell‐conditioned medium 199, or control medium 199. The PPM of unattached spermatozoa was estimated after 0, 3, 6, 9, 12, and 24 hr of incubation at 37°C under 5% CO2 in air. Viability and the occurrence of true acrosome reactions were assessed using a triple‐stain technique. Spermatozoa started to attach within 1 hr of coculture with the hamster or sheep oviductal epithelial cell (OEC) monolayers, and these spermatozoa showed vigorous tail motion. No spermatozoa were found to attach to the IBRS‐2 monolayer. The PPM of unattached spermatozoa cocultured with the various types of epithelial cell monolayers for 12 hr was significantly higher than that of spermatozoa incubated in conditioned media or medium 199 alone (54% in hamster OEC vs. 40% in conditioned; 68% in sheep OEC vs. 38% in conditioned; 36% in control medium). On the other hand, after 24 hr of incubation, there were no differences in the PPM of spermatozoa cocultured with epithelial cells or incubated in conditioned media. The percentages of cells undergoing a true acrosome reaction reached maximum values (P < 0.05) in spermatozoa incubated for 9 hr in the presence of hamster OEC (22.5%) or for 12 hr on sheep OEC (20.5%) monolayers. IBRS‐2, a commercial nonreproductive cell type, had a positive influence on both PPM and sperm viability but no effect on the occurrence of the acrosome reaction. Interactions leading to the acrosome reaction were thus observed only when spermatozoa were cocultured with OEC monolayers. The values of PPM in unattached sperm cells seen after 12 hr of coculture with OEC or IBRS‐2 were still at a high level (52–67%) for in vitro fertilization. The coculture with OECs provides an “in vitro” model to study the capacitation processes in a situation that may resemble that occurring in vivo. Moreover, the coculture with hamster OECs may provide a convenient and standardized in vitro system to study mechanisms underlying capacitation and the acrosome reaction. © 1993 Wiley‐Liss, Inc.
Relationships between the completion of first cleavage and the chromosomal complement, sex, and developmental rates of bovine embryos generated in vitro
Authors: Yadav B. R. , King W. A. , Betteridge K. J. 
Published: February 2005

AbstractOne thousand eighty‐four two‐cell bovine embryos produced from 1,574 oocytes matured and fertilized in vitro were cultured as groups separated according to the time when they completed their first cleavage (24,30,40,48, or 62 hr postinsemination; hpi). At 5 days after insemination, the proportions of each group that had progressed to the eight‐cell stage or beyond were determined and the 350 that had done so were fixed and examined cytogenetically for cell number, chromosomal abnormalities, and sex. Embryos in the “early” cleaving (24 and 30 hpi) and “late” cleaving (40–62 hpi) groups were compared. Early cleaving embryos were more likely to have developed to the eight‐cell stage or beyond (52.2% vs. 20%), contained more cells (22 vs. 17), and were more likely to be male (3.6:1 vs. 0.93:1). It is suggested that these phenotypic differences between the sexes begin before the embryonic genome is generally thought to become activated and are due either to differential processing of X‐ and Y‐bearing sperm within the zygote or to very early differential expression of genes derived from X‐ and Y‐bearing sperm. © 1993 Wiley‐Liss, Inc.
Video microscopic analysis of ionophore induced acrosome reactions of lobster (Homarus americanus) sperm
Authors: Tsai K. L. , Talbot P. 
Published: February 2005

AbstractSperm from the American lobster (Homarus americanus) are normally nonmotile. However, during fertilization, the sperm undergo a calcium‐dependent acrosome reaction that propels them forward about 18 μMm. The reaction occurs in two phases, eversion and ejection, which take place too quickly to permit analysis by direct observation. The purposes of this study were to examine the structural changes occurring in sperm during the normal acrosome reaction and to determine the rate of the reaction using video microscopy. The reaction was induced in vitro by ionophore A23187 and recorded using a video system attached to a Nikon Nomarski interference microscope. Videotapes were played back frame by frame (30 frames/sec), and images of reactions from 10 sperm were analyzed. The acrosome reaction, including the eversion of the acrosomal vesicle and ejection of the subacrosomal material and nucleus, can be divided into 4 steps: (1) expansion of the apical cap followed by expansion of the remainder of the acrosomal cylinder; expansion of the cylinder begins at its apical end and proceeds toward its base, (2) eversion of the apical half of the acrosomal vesicle and initial contraction of the apical cap, (3) eversion of the basal half of the acrosomal vesicle, continued contraction of the apical cap, and ejection of the subacrosomal material and nucleus, and (4) final contraction of the apical cap and ejection of the acrosomal filament. During steps 2, 3, and 4, the mean forward movement of sperm is 12.7, 3.9, and 1.1 μMm, respectively. Although the time required to complete the reaction ranged from 0.66 to 5.16 s, most sperm reacted in less than 3. s, and these sperm were considered to have typical rates. For sperm that reacted in less than 3 s, both step 1 and step 4 take about 0.2 s and show little variation among sperm. the time required to complete steps 2 and 3 averaged 0.63 and 0.37 s, respectively. Forward movement of the sperm during the acrosome reaction is caused by eversion of the inner and outer acrosomal material and contraction of the apical cap. The protein(s) responsible for this contraction is not yet known. © 1993 Wiley‐Liss, Inc.
Chromatin condensation and acrosome development during spermiogenesis of Ensis ensis (Mollusca, Bivalvia)
Authors: Casas M. T. , Subirana J. A. 
Published: February 2005

AbstractWe describe chromatin condensation and acrosome development during spermiogenesis of Ensis ensis. The overall shape of the mature spermatozoon corresponds to the primitive type. The nucleus is oval and on its superior pole there is an elongated acrosome; the middle piece contains four mitochondria around the centriolar complex. The condensation of the nuclei seems to occur in three steps: first the diameter of chromatin fibers increases slightly from 17 to 20 nm; second, in midspermatids fiber pairs coalesce; and third, the coalescence continues by addition of other fibers until the nuclei become highly compacted. Chromatin changes are related with nuclear protein composition. Small proacrosomal vesicles show two regions of different electron density. At a later stage they fuse to give a single, spherical vesicle in round spermatids, which migrates to the upper pole and transforms into a tapered acrosome (18 μm long) with a central channel filled with finely fibrous material. © 1994 Wiley‐Liss, Inc.
Functions of milk protein gene 5′ flanking regions on human growth hormone gene
Authors: Ninomiya Takashi , Hirabayashi Masumi , Sagara Junko , Yuki Atsushi 
Published: February 2005

AbstractFragments containing 5′ flanking regions of four bovine milk protein genes—alpha lactalbumin (bαLA), alpha S1 casein (bαS1CN), beta casein (bβCN), kappa casein (bkCN)—and mouse whey acidic protein (mWAP) gene were prepared by PCR and ligated to human growth hormone (hGH) gene. These recombinant DNAs were microinjected into rat embryos to produce transgenic rats, and the functions of the 5′ regions to direct secretion of hGH in the milk were tested. Although milk was obtained only in 5 of 19 mWAP/hGH rat lines, more than two‐thirds of the rats carrying the other four DNAs produced milk. More than 80% of the lactated rats carrying bαLA/, bβCN/, and mWAP/hGH, and 33% of the laclated bαS1CN/hGH rats secreted detectable amounts of hGH (> 0.05 μg/ml) in the milk. In some rats, the hGH concentrations in the milk were comparable to or more than that of the corresponding milk protein in bovine milk. The ranges of hGH concentrations in the milk of bαLA/, bβCN/, bαS1CN/, and mWAP/hGH rats were 1.13–4,360 μg/ml, 0.11–10,900 μg/ml, 86.8–6,480 μg/ml, and 6.87–151 μg/ml, respectively. HGH was also detected in the sera of these rats, and some abnormalities of growth and reproduction were observed. All but one virgin mWAP/hGH rat secreted up to 0.0722 μg/ml of hGH in the serum, and more than half of them showed abnormal fat accumulations at their abdomen. None of the bαCN/hGH rats secreted detectable amount of hGH into their milk, whereas 8 of the 11 lines secreted hGH into their sera. For the production of hGH in transgenic rat milk, the 5′ region of bαS1CN was shown most suitable, because the bαS1CN/hGH rat secreted > 6,000 μg/ml of hGH into the milk and could be reproduced. © 1994 Wiley‐Liss, Inc.
Cloning and tissue expression of bovine follistatin cDNA
Authors: Houde A. , Lussier J. G. , Ethier J.‐F. , Gagnon C. , Silversides D. W. 
Published: February 2005

AbstractBovine follistatin cDNA sequences were derived using a cloning strategy based entirely on reverse transcription and polymerase chain reaction amplification of RNA derived from bovine ovarian and testicular tissues. Complete bovine follistatin cDNA coding sequences are presented including 1,029 bases of open reading frame, the 5′ translational start codon, and the 3′ translational stop codon. Homologies of bovine follistatin cDNA with pig, human, rat, and partial sheep sequences are 94.3%, 92.4%, 89.9%, and 98.4% at the nucleic acid level and 98.3%, 97.1%, 95.6%, and 100% at the deduced amino acid level, respectively. Northern blot analysis on a survey of bovine reproductive tissues showed strongest expression in ovaries collected from superovulated cows and major RNA species at 2.8 Kb and 1.75 Kb. © 1994 Wiley‐Liss, Inc.
Identification of anti‐agglutinin for spermatozoa in epididymal boar plasma
Authors: Harayama H. , Miyano T. , Miyake M. , Kusunoki H. , Kato S. 
Published: February 2005

AbstractThe present report identifies epididymal boar anti‐agglutinin and examines its effect on sperm motility. Boar spermatozoa from the cauda epididymidis were washed and incubated in modified Krebs–Ringer bicarbonate at 37°C (5% CO2 in air). In the samples washed three or five times and then incubated for 3–5 h, higher rates (72–79%) of spermatozoa were associated with one another at the acrosomal region, mainly in groups of 2–5 cells (head‐to‐head agglutination), and many cells exhibited intensively flagellant and/or circular types of movement but rarely progressive motility. The addition of epididymal plasma or 25 kDa protein purified from it markedly inhibited the occurrence of head‐to‐head agglutination in washed spermatozoa, whereas heat treatment and subsequent removal of insoluble materials reduced the anti‐agglutination activity of epididymal plasma. The percentages of progressively motile cells in the samples incubated with epididymal plasma or 25 kDa epididymal protein rose coincident with the reduction of sperm agglutination. These findings demonstrate that the 25 kDa epididymal protein is an anti‐agglutinin for the cauda spermatozoa and that it effectively functions to maintain progressive motility of the cells in vitro. © 1994 Wiley‐Liss, Inc.
Characterization of mouse‐hamster‐human cross‐reacting antioocyte monoclonal antibodies produced by intrasplenic immunization of mice with 12 zona‐free mouse oocytes
Authors: Jin Meishan , Johansson Lars , Sundstrōm Per , Nilsson B. Ove 
Published: February 2005

AbstractSeveral intrasplenic immunizations with batches of ∼15 or ∼30 zona‐free, unfertilized mouse oocytes resulted in 200–300 hybrids, respectively, among which about 20 positive clones were selected from each fusion between splenic plasma cells and SP2/0 myeloma cells. When nonimmunized splenic plasma cells were used, only one antibody, showing weak immunoreaction, was obtained from ∼370 hybrids collected from 2 fusions. From one immunization with a total of 12 zona‐free, unfertilized mouse oocytes, 15 positive clones were selected for further study. Eleven of these 15 antibodies reacted with antigens only in unfertilized oocytes but not in fertilized, pronuclear stage oocytes. Three antibodies, which recognized antigens in paraffin‐embedded oocyte sections, did not label growing ovarian oocytes, indicating that the antibodies were specific to ovulated, unfertilized oocytes. These antibodies did not detect any antigen epitopes in the panel of tissues examined. The molecular weight of one antigen, corresponding to a IgM antibody that is present both in ooplasma and zona pellucida, was ∼116 kDa. Cross‐reactivity to blots of unfertilized zona‐free hamster oocytes was demonstrated by 6 antibodies and to unfertilized human oocytes by 7 antibodies. Three antibodies cross‐reacted with both hamster and human oocytes. The study indicates that the intrasplenic immunization is an appropriate means of raising antibodies against unfertilized, zona‐free mouse oocytes and that the method applied offers an easy way to select antibodies against human oocytes for functional studies. © 1994 Wiley‐Liss, Inc.
Calmodulin during human sperm incorporation into hamster oocyte: An immunogold electron microscope study
Authors: Courtot Anne‐Marie J. , Feinberg Jacqueline M. , Schoevaert Damien A. , Rainteau Dominique P. , Weinman Serge J. 
Published: February 2005

AbstractIn the present study, immunogold labeling of ultrathin sections of human sperm, before and after incorporation into hamster oocyte, was used to obtain insight into the ultrastructural localization and possible function of calmodulin during fertilization. In heads of ejaculated, capacitated, and acrosome‐reacted fixed human sperm, calmodulin was mainly found in two compartments, the subacrosomal layer and the postacrosome. After sperm‐egg fusion, the subacrosomal calmodulin was unaltered and surrounded by the fertilization cone in which actin was abundant. There was no co‐localization of calmodulin and actin. In contrast, postacrosomal calmodulin disappeared as soon as the sperm head was incorporated into egg cytoplasm. These unique localizations and redistributions are in agreement with the concept of a calmodulin targeting from acrosome toward postacrosome through the subacrosomal layer during spermatogenesis (Weinman et al., 1986b: J Histochem Cytochem 34:118). Moreover, they strongly suggest a role for calmodulin both in sperm‐egg fusion and in the initial pulse of Ca2+ occurring during fertilization. © 1994 Wiley‐Liss, Inc.
Stimulating effect of pyroglutamylglutamylprolineamide, a prostatic, TRH‐related tripeptide, on mouse sperm capacitation and fertilizing ability in vitro
Authors: Green Catherine M. , Cockle Sheena M. , Watson Paul F. , Fraser Lynn R. 
Published: February 2005

AbstractPyroglutamylglutamylprolineamide, a prostatic tripeptide with structural similarities to thyrotrophin‐releasing hormone (TRH), has been found in the seminal plasma of several mammalian species, suggestive of a biological function relating to spermatozoa. Using chlortetracycline (CTC) fluorescence analysis and in vitro fertilization, we have obtained evidence that the tripeptide stimulates mouse sperm capacitation and fertilizing ability in vitro. The tripeptide at concentrations from 5–500 nM was added to sperm suspensions and cells were assessed with CTC after 40 min, insufficient time for complete capacitation by a majority of spermatozoa under standard conditions of incubation. Concentrations of 25 nM and higher significantly promoted capacitation, as evidenced by a decrease in the proportion of acrosome‐intact F pattern spermatozoa, characteristic of uncapacitated cells, and an increase in the proportion of acrosome‐intact B pattern spermatozoa, characteristic of capacitated cells. However, there was no significant stimulation of acrosomal exocytosis. These results suggested that peptide‐treated cells would be more fertile than their untreated counterparts. This was confirmed using in vitro fertilization, where the presence of 100 nM peptide during sperm preincubation and gamete coincubation significantly stimulated fertilizing ability (peptide, 56.5% of oocytes fertilized; controls, 26.5%). Comparison of the prostatic tripeptide and TRH effects on capacitation revealed that TRH at a concentration of 250 nM was as effective as the prostatic tripeptide in promoting the F & B transition but was less effective or ineffective at lower concentrations. In vitro fertilization assessment of the two peptides, at 100 nM, revealed that only the prostatic tripeptide significantly stimulated fertility. Again, this was consistent with the CTC analyses. Because the prostatic tripeptide can stimulate sperm function in vitro, it is possible that it plays a similar role in vivo and promotes fertilizing ability of ejaculated spermatozoa. We therefore propose that this tripeptide be referred to as fertilization promoting peptide (FPP). © 1994 Wiley‐Liss, Inc.
Evidences for the presence of chymotrypsin‐like activity in human spermatozoa with a role in the acrosome reaction
Authors: Morales Patricio , Socias Teresa , Cortez Jacqueline , Llanos Miguel N. 
Published: February 2005

AbstractThe effect of chymotrypsin inhibitors and substrates on the human sperm acrosome reaction stimulated by the human zonae pellucidae or follicular fluid were evaluated. Motile spermatozoa, selected by a Percoll gradient, were incubated at 1 × 107 cells/ml, 37°C, and 5% CO2, After 4.5 hr, the chymotrypsin inhibitor TPCK (N‐Tosyl‐L‐Phenylalanine‐Chloromethyl Ketone) or the substrate ATEE (N‐Acetyl‐L‐Tyrosine Ethyl Ester) were added for 30 min. Then, four oocytes were added and the percentage of acrosome‐reacted spermatozoa on the zona was determined. TPCK and ATEE inhibited the zona pellucida‐induced acrosome reaction. The chymotrypsin inhibitors TPCK and chymostatin and the chymotrypsin substrates ATEE, BTEE (N‐Benzoyl‐L‐Tyrosine Ethyl Ester), Succinyl‐Ala‐Ala‐Phe‐7‐Amido‐4‐Methyl‐Coumarin (Suc‐Ala‐Ala‐Phe‐AMC), and Succinyl‐Leu‐Leu‐Val‐Tyr‐7‐Amido‐4‐Methyl‐Coumarin (Suc‐Leu‐Leu‐Val‐Tyr‐AMC) inhibited the human follicular fluid‐induced acrosome reaction. Sperm extracts exhibited hydrolytic activity toward Suc‐Ala‐Ala‐Phe‐AMC and Suc‐Leu‐Leu‐Val‐Tyr‐AMC. This enzyme activity was abolished by TPCK and chymostatin, was independent of Ca2+, and was not modified by 1,10 phenanthroline. In addition, the activity was present in the supernatant after the acrosome reaction was induced with calcium ionophore and in epididymal spermatozoa recovered from the cauda region. Electron microscopic observations indicated that the inhibitors prevented the membrane events of the acrosome reaction. These data suggest an association between human spermatozoa and chymotrypsin‐like activity with a possible role in the acrosome reaction. © 1994 Wiley‐Liss, Inc.
Analysis of mRNA for class I HLA on human gametogenic cells
Authors: Janitz Michal , Fiszer Dorota , Michalczak‐Janitz Karolina , Lukaszyk Andrzej , Fernandez Nelson , Skorupski Witold , Kurpisz Maciej 
Published: February 2005

AbstractWe have studied mRNA expression for Class I HLA (human leukocyte antigen) on male germ cells by amplification of gene fragments in PCR techique and by Northern hybridization. RNA was extracted from fractionated gametogenic cells (isolated from testis) and reversely transcribed. Then, cDNA was amplified for specific HLA sequence (1151 bp) representing whole‐length coding sequence (HLA, ‐A, ‐B, ‐C). The specificity of this product was confirmed in “nested” PCR of 400 bp gene fragment coding for alpha 2 domain, alpha 3 domain, and the transmembrane portion of Class I HLA. The results indicate minimal expression of classical Class I HLA on gametogenic cells. Northern hybridization with 669 bp cDNA fragment (spanning for alpha 3 domain, transmembrane, cytoplasmic, and 3′ untraslated region) resulted in a low intensity signal from gametogenic cell fractions and confirmed our findings obtained by PCR. The minimal expression of classical HLA antigens may create a neutral cover for the male reproductive system, thereby preventing an immunological response during germ cell differentiation. © 1994 Wiley‐Liss, Inc.
Expression of the imprinted gene H19 in the human fetus
Authors: Lustig O. , Ariel I. , Ilan J. , Lev‐Lehman E. , De‐Groot N. , Hochberg A. 
Published: February 2005

AbstractThe H19 gene is a parenterally imprinted maternally expressed gene which has a pivotal role in embryogenesis and fetal development. It is tightly linked to the IGF‐II gene on chromosome 11p15.5 which is reciprocally imprinted. We studied the expression of the human H19 by in situ hybridization in an embryo 35 days post coitus (dpc) and in a fetus from the second trimester of pregnancy. The expression pattern of H19 in the human fetal tissues was similar to its expression in the mouse, and paralleled, with some exceptions, the expression of IGF‐II in human fetuses. Abundant expression was found in organs comprising the fetoplacental unit: the placenta, the fetal adrenal, and liver. The expression in the fetal adrenal cortex was most prominent in the definitive cortex and somewhat weaker in the fetal zone. Considerable expression of H19 was found in the fetal liver as early as 35 dpc and in the second trimester. Hematopoietic cells in fetal liver did not express the gene. Moderate expression of H19 was detected in the epithelium of the small intestines, in endometrial stroma and Fallopian tube. In the kidney conspicuous labeling of the metanephric blastema was noted, which was markedly reduced with differentiation to tubules. This pattern of expression is identical to that of IGF‐II in the fetal kidney and is relevant to the evolution of Wilms' tumor. No expression of H19 was found in the neural tube of the first trimester embryo or in the developing fetal brain in the second trimester, nor were transcripts detected in the choroid plexus. The localization of H19 expression within human fetal tissues may help to elucidate its role in human embryogenesis and fetal development. © 1994 Wiley‐Liss, Inc.