bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein databases for compacted eight‐cell and blastocyst‐stage mouse embryos
Authors: Shi C. Z. , Collins H. W. , Garside W. T. , Buettger C. W. , Matschinsky F. M. , Heyner S.
Published: February 2005

High‐resolution two‐dimensional sodium dodecyl sulfate‐polyacrylamide (2D‐SDS) gel electrophoresis combined with computerized analysis of gel images was used to construct and analyze protein databases for two stages of preimplantation mouse embryogenesis, the compacted eight‐cell stage and the fully expanded blastocyst stage. These stages were chosen for their ease in identification of multiple synchronous embryos. Synchronous cohorts of 30–50 embryos were labelled with L‐[35S]methionine for 2 hr. The embryos were then lysed in 30 μl hot SDS sample buffer, and the lysates were stored at −80°C until the gels were run. Five replicates were run for eight‐cell embryos, and four for blastocyst‐stage embryos. The samples were processed for 2D gel electrophoresis and fluorography; multiple exposures were made. Gel images were analyzed using the PDQUEST system, and databases were constructed. Analysis of the databases for both developmental stages showed high reproducibility of protein spots in multiple gel images. Of 1,674 total spots in eight‐cell embryo standards, >79% of spots had a percentage error (S.E.M./average) <50%, and >45% had a percentage error <30%. Similarly, of 1,653 total spots in blastocyst‐stage embryo standards, 74% of spots had a percentage error <50%, and approximately 47% of spots had a percentage error <30%. Forty‐three spots (approximately 3% of the total spots) were found to be detected only in the eight‐cell stage, while 75 spots were detected solely in the blastocyst stage. Sixty‐nine proteins showed a greater than threefold increase in isotope incorporation from the eight‐cell to the blastocyst stage, with a percentage error <50% in both the eight‐cell and the blastocyst stages. In contrast, 41 of the proteins showed a decrease during this period. Analysis of the protein databases described in this study has allowed us to document the overall quantitative changes in proteins from the compacted eight‐cell stage to the blastocyst stage of mouse preimplantation development. These databases provide a valuable tool for further detailed quantitative analysis of specific proteins associated with developmental events. In addition they will permit analysis of the effects of environmental factors, such as growth factors, on early embryo development. © 1994 Wiley‐Liss, Inc.
Relationship between nuclear remodeling and subsequent development of mouse embryonic nuclei transferred to enucleated oocytes
Authors: Cheong H. T. , Takahashi Y. , Kanagawa H.
Published: February 2005

The present study was conducted to examine the relationship between nuclear remodeling and subsequent embryonic development in nuclear transplant mouse embryos. Metaphase II oocytes were enucleated without staining and fused with transferred donor nuclei from two‐, four‐, or eight‐cell embryos. Fusion and oocyte activation were performed by means of electric fields. High rates of enucleation (89.1%), fusion (88.0–91.6%), and activation (95.2–96.9%) were obtained using this system. Nuclear remodeling was characterized by premature chromosome condensation (PCC), followed by various pronuclear‐like formations upon oocyte activation. Development to blastocysts was obtained from both PCC (17.9%) and non‐PCC (NPCC; 52.9%) embryos fused with the two‐cell nuclei. However, development to term was obtained only in PCC embryos with a single pronucleus‐like structure and a polar body (12.5%). In vitro development of nuclear transplant embryos with four‐ and eight‐cell nuclei was limited. All the NPCC embryos examined had tetraploid chromosome constitutions, but chromosome constitutions of PCC embryos varied. Only 37.5% of the PCC embryos had diploid chromosome constitutions. The results indicated that the development of nuclear transplant embryos is affected by the types of nuclear remodeling and that oocyte activation in relation to their chromosome constitutions. The results also indicated that the PCC of the donor nucleus in nonactivated cytoplasm is important for the development of the nuclear transplant embryos. © 1994 Wiley‐Liss, Inc.
Effects of cryopreservation on the intracellular calcium concentration of human spermatozoa and its response to progesterone
Authors: McLaughlin E. A. , Ford W. C. L.
Published: February 2005

The intracellular free calcium concentration [Ca2+]i of sperm from 23 ejaculates was measured before and after cryopreservation using the fluorescent probe Fura‐2. Spermatozoa were treated with 3.18 μM progesterone so that the regulation of [Ca2+]i in a dynamic situation could be studied. [Ca2+]i (nM) was 290 ± 13 in fresh spermatozoa vs. 550 ± 26 in cryopreserved samples (mean ± S.E.M. P < 0.0001 paired t‐test). Progesterone at a dose of 3.18 μM stimulated a large and rapid increase in [Ca2+]i to a peak value > 1 μM after 10–20 seconds. [Ca2+]i then declined to a slightly raised basal level over the next 30–40 seconds. This phenomenon occurred in all the fresh samples, but about half the frozen thawed samples failed to respond. The peak [Ca2+] attained by frozen samples which did respond after the addition of progesterone was similar to that observed with fresh sperm. The calcium channel blocker verapamil (200 μM) completely inhibited the transient rise in [Ca2+]i produced by progesterone, but 100 μM verapamil had only a partial effect. We conclude that (1) cryopreservation causes a substantial elevation of the [Ca2+]i in human spermatozoa and (2) damage to the plasma membrane during cryopreservation may result in the loss of the progesterone receptor. Both factors may contribute to the loss of fertility after cryopreservation. © 1994 Wiley‐Liss, Inc.
Spatially restricted imprinting of mouse chromosome 7
Authors: Villar Angela J. , Pedersen Roger A.
Published: February 2005

Three of the four known imprinted genes (Igf‐2, H19, and Snrpn) map to mouse chromosome 7. We used mRNA phenotyping to examine the tissuespecific transcription of Igf‐1r, H‐ras‐1, and Gabrb3, which map to chromosome 7 between Snrpn and the Igf‐2/H19 domain, and Myod‐1, which maps proximal to Snrpn. We found that all of these genes were expressed by both parental alleles in tissues from day 1 neonates. The fact that imprinted genes can flank or map closely to genes that escape such epigenetic modification suggests that autosomal imprinting is not manifested globally along imprinted chromosomes but rather is spatially restricted, perhaps even defined by specific DNA consensus sequences or an “imprint box” associated with imprintable genes. © 1994 Wiley‐Liss, Inc.
Synthesis and processing of mammalian protamines and transition proteins
Authors: Green G. R. , Balhorn R. , Poccia D. L. , Hecht N. B.
Published: February 2005

Mouse and rat seminiferous tubule fragment cultures were used to examine synthesis and processing of mammalian protamines and transition proteins. The tubule fragments were incubated with [3H]‐arginine, [3H]‐histidine, [35S]‐cysteine, or [32P]‐PO4, and radiolabeled proteins were analyzed by acid/urea polyacrylamide gel electrophoresis and fluorography or autoradiography. Newly synthesized protamines were recovered from sonication‐resistant nuclei (SRN) and could not be detected in cytoplasmic fractions, indicating that protamines are deposited into nuclei immediately after synthesis. Newly synthesized mouse protamine 1 (mP1) and the precursor to mouse protamine 2 (pre‐mP2) migrated more slowly during electrophoresis than their predominant testicular forms, identified by staining with Coomassie blue R‐250. Within 1 hour of synthesis, the electrophoretic mobilities of mP1 and pre‐mP2 increased to match those of their predominant forms. These changes are consistent with initial charge‐neutralizing modifications of the newly synthesized protamines, followed by removal of at least some of the modifying ligands, to unmask protamine basicity. Steady‐state phosphorylation rates were high for rat protamine 1 (rP1) and were independent of phosphate content; both rP1 molecules of low and high phosphate content were rapidly phosphorylated. Pre‐mP2‐3, a major processing intermediate derived by proteolysis of pre‐mP2, was also rapidly phosphorylated. Like the protamines, transition protein 2 (TP2) was rapidly phosphorylated and increased in electrophoretic mobility soon after synthesis. In contrast, transition protein 1 (TP1) was not phosphorylated and did not exhibit multiple electrophoretic forms. © 1994 Wiley‐Liss, Inc.
Effects of freezing of bovine preimplantation embryos derived from oocytes fertilized in vitro on survival of their inner cell mass cells
Authors: Iwasaki S. , Yoshikane Y. , Li X. , Watanabe S. , Nakahara T.
Published: February 2005

The morphology of the inner cell mass (ICM) cells and the proportion of dead ICM cells in frozen‐thawed bovine preimplantation embryos were investigated by differential fluorochrome staining. Embryos at the blastocyst stage of development were frozen and thawed by two different techniques (three‐step and one‐step) in two different basic salt solutions (PBS and TCM 199) containing 1.36M glycerol. After thawing and glycerol removal, embryos were co‐cultured in a cumulus cells monolayer in TCM 199 for 48 hr (morula) or 24 hr (blastocysts). Differential cell counts of the ICM and trophectoderm were then done using differential fluorochrome staining. Overall, there was no significant difference in the viability of embryos frozen in the two basic salt solutions. Low proportions of dead ICM cells were observed in embryos frozen at the morula stage in both PBS (19.1%) or TCM 199 (18.0%). However, blastocyst stage embryos frozen by the three‐step technique had a higher (P < 0.05) proportion of dead ICM cells in TCM 199 (37.7%) than in PBS (18.2%). Blastocysts frozen by the one‐step technique had a higher (P < 0.05) proportion of dead ICM cells (42.2%) than those frozen by the three‐step technique (18.2%), regardless of basic salt solutions. Results indicate that freezing and thawing damages ICM cells in morphologically normal embryos and that the degree of damage depended on the basic salt solution and the freezing method. © 1994 Wiley‐Liss, Inc.
Inhibition of germinal vesicle breakdown in bovine oocytes by 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole (DRB)
Authors: Farin Charlotte E. , Yang Li
Published: February 2005

5,6‐Dichloro‐1‐β‐D‐ribofuranosylbenzimidazole (DRB) is an analog of the nucleoside adenosine that has been used to inhibit transcription in a variety of cell types. The present studies were designed to evaluate the ability of DRB to block germinal vesicle breakdown (GVBD) in bovine oocytes matured in vitro and to characterize culture conditions required for DRB‐mediated arrest of meiosis. Administration of DRB (60–90 μM) at 2–3 h intervals during culture of COC blocked GVBD in approximately 70 percent of oocytes. The inhibitory effect of DRB was reversible and required the presence of cumulus cells. Treatment with DRB was associated with a 57% decrease in 3H‐uridine incorporation into total COC RNA and a 90.8% decrease into mRNA but did not affect the incorporation of 3H‐leucine into COC proteins. The ability of DRB to arrest meiosis was significantly compromised if supplemental gonadotropin preparations were absent from the maturation media. Gonadotropin‐induced GVBD as well as cumulus cell expansion was blocked by treatment with DRB but no with adenosine. GVBD in cultured bovine COC was initially inhibited and then stimulated when supplemental gonadotropin preparations were included in the culture media. DRB treatment in the presence of gonadotropin supplementation blocked the stimulatory effect of gonadotropins on GVBD. In conclusion, DRB can be used to arrest GVBD in bovine COC in a specific and reversible manner. The data support the hypothesis that gene transcription is required for the stimulatory phase of gonadotropin‐mediated GVBD in cultured bovine COC. © 1994 Wiley‐Liss, Inc.
Flow cytometric analysis of mouse sperm using monoclonal anti‐sperm antibody A‐1
Authors: Kawai Yuichi , Ohnishi Shigeru , Miyake Masaharu , Hama Takao , Mayumi Tadanori
Published: February 2005

We have previously prepared an anti‐mouse sperm monoclonal antibody (A‐1) which inhibited sperm penetration into the egg zona pellucida. By indirect immunofluorescence (IIF), the A‐1 antibody was shown to recognize an antigen localized in the acrosomal area of sperm. This antibody bound negligibly to fresh sperm, while binding to methanol‐fixed sperm was almost complete. After methanol fixation, no sperm that penetrated into the zona were immunoreactive for this antibody. In the present study we examined the localization and fate of A‐1 antigen during the acrosome reaction by IIF and flow cytometry (FCM). Cauda epididymal sperm were treated with either calcium ionophore A23187 or zona solution, immunostained indirectly, and subjected to FCM. Treatment with A23187 reduced the percentage of immunoreactive sperm to 59% from the 80% obtained in the untreated sperm. The treatment also reduced the average fluorescence intensity per fluorescence‐positive spermatocyte to 65 channels, while this intensity was 89 channels in the untreated sperm. A similar result was obtained from treatment with zona solution. The proportion of sperm that was immunoreactive with A‐1 antibody was reduced to 55% by incubation in zona‐containing media from the 80% obtained in zona‐free media. On the other hand, neither A23187 nor the zona solution affected the immunoreactivity or the fluorescence intensity of caput epididymal sperm, while the A‐1 antigen was present in both the immature sperm from the caput epididymis of adult mice and in the mature sperm from the cauda epididymis of the same mice. These findings suggest that the intramembrane antigen recognized by the A‐1 monoclonal antibody is released from sperm as a result of the acrosome reaction. © 1994 Wiley‐Liss, Inc.
Production of motile acrosome‐reacted mouse sperm with nanomolar concentration of calcium ionophore A23187
Authors: Tanphaichitr N. , Hansen C.
Published: February 2005

A method to generate a population of motile, acrosome‐reacted mouse sperm is described. Sperm retrieved from the cauda epididymis and vas deferens were first capacited in a 3% bovine serum albumin (BSA) containing medium. Sperm were then resuspended in medium with low BSA content (0.01%) and treated with 30 nM of the calcium ionophore, A23187, which was added as a singel dose of 30 nM for 15 min at 37°C; or three sequential 10 nM doses over three 5 min intervals. Approximately 55–60% of the treated sperm population became acrosome reacted. The motility of the treated sperm sample was 40–65%, slightly lower than that of the control sperm, following addition of medium containing 3% BSA, This is in contrast to the <10% motility observed for capacitated mouse sperm treated with 10 μM A23187, a concentration that had been used by other investigators to induce the acrosome reaction. The ultrastructure of the 30 nM A23187‐induced acrosome‐reacted sperm ws similar to that of the acrosome‐reacted sperm induced by solubilized zonae pellucidae. These motile, acrosome‐reacted sperm were able to penetrate zone‐free mouse eggs at a higher rate than the control sperm. Thus this method of treatment will be useful for further physiological experimentation with acrosome‐reacted sperm. © 1994 Wiley‐Liss, Inc.
Expression of insulin‐like growth factors I and II in conceptuses from normal and diabetic mice
Authors: Chernicky C. L. , Redline R. W. , Tan H. Q. , Gwatkin R. B. L. , Johnson T. R. , Ilan Joseph , Ilan Judith
Published: February 2005

Insulin‐like growth factors (IGF‐I and IGF‐II) play an important regulatory role in fetal growth and development. Alterations in expression of these growth factors may result in developmental abnormalities, macrosomia, and intrauterine growth retardation, which occur with a higher incidence in diabetic pregnancies. In situ hybridization histochemistry was employed to investigate the distribution and abundance of IGF‐I and IGF‐II in peri‐implantation and postimplantation conceptuses from normal and streptozotocin‐treated diabetic mice. Animals were sacrificed on gestational days 5, 6, 7, 8, and 9. The entire uterine horn was prepared for hybridization with antisense and sense α‐35S‐dATP labeled oligonucleotide probes for IGF‐I, IGF‐II, and mouse β‐actin. IGF‐I transcript was apparent only in myometrium at 6 days of gestation in normal and diabetic mice. IGF‐II transcripts were restricted to trophoectoderm cells within the implantation chamber on day 5. Following implantation, IGF‐II transcripts were found in trophoectodermal derivatives, primitive endoderm, mesoderm, heart, walls of the foregut, and mesenchyme in normal and diabetic postimplantation conceptuses. There were no apparent differences between normal and diabetic samples in the distribution and abundance of the IGF‐II transcript from gestational days 7, 8, and 9. The embryos from the diabetic mother at day 6 were growth retarded and had a significant decrease in the expression of IGF‐II. These results suggest that maternal hyperglycemia may retard development of the early implanting conceptus in a narrow window around day 6 through a mechanism involving decreased IGF‐II expression. Fetuses from diabetic pregnancies that escape this critical period appear to develop and express IGF‐II in an equivalent manner to those of the control group. © 1994 Wiley‐Liss, Inc.
Characterization of the major proteins of bovine seminal fluid by two‐dimensional polyacrylamide gel electrophoresis
Authors: Desnoyers L. , Théarien I. , Manjunath P.
Published: February 2005

Recently, we demonstrated that the major proteins from bovine seminal plasma BSP‐A1, ‐A2, ‐A3 and ‐30‐kDa (collectively called BSP proteins) specifically interact with choline phospholipids. These proteins coat the surface of the spermatozoa after ejaculation and are believed to play an important role in membrane modifications occurring during capacitation. In this study we determined the isoelectric point (pl) and analysed the molecular heterogeneity of BSP proteins. Total protein from bovine seminal plasma (CBSP) and purified BSP proteins were iodinated using chloramine T. Samples were reduced, denatured, separated by two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE), and visualized by autoradiography. Analysis of CBSP proteins demonstrated the presence of polypeptides migrating in the pH range of 3.5–7.8 and at molecular weights (Mr) between 6 and 100 kDa. Many isoforms of each BSP protein were found when purified iodinated proteins were analysed by 2D‐PAGE. BSP‐A1 was found at a Mr of 16.5 kDa and in the range of pl of 4.7–5.0; BSP‐A2 at 16 kDa and at a pl of 4.9–5.2; BSP‐A3 at 15 kDa and at a pl of 4.8–5.2, and BSP‐30‐kDa at 28 kDa and at a pl of 3.9–4.6. Similar results were obtained with immunolocalization of BSP proteins after Western blot using specific antibodies. The treatment of purified iodinated BSP proteins with neuraminidase increased the pl of BSP‐30‐kDa to 4.8–5.0 and decreased its Mr to 25 kDa, but no change was observed for BSP‐A1, ‐A2 and ‐A3. The treatment of BSP proteins with sulfatase or acid phosphatase modified neither their Mr nor their pl. Furthermore, when CBSP proteins were separated in 2D‐PAGE and the gels stained for glycoproteins with dansyl hydrazine, BSP proteins were among the major glycoproteins found in the bovine seminal plasma. In conclusion, BSP proteins are acidic and have several isoforms. Furthermore, the heterogeneity of BSP‐30‐kDa is mainly due to its sialic acid content. © 1994 Wiley‐Liss, Inc.
Spermophagy in semen in the red wolf, Canis rufus
Authors: Koehler James K. , Platz Carrol C. , Waddell Will , Jones Michael H. , Smith Roland , Behrns Susan
Published: February 2005

The red wolf (Canis rufus) is an endangered species with 194 individuals remaining in the wild and in various captive facilities. Breeding efforts at the Graham, WA site (Point Defiance Zoo and Aquarium) have involved artificial insemination with fresh or frozen semen in an effort to increase population and maximize the genetic potential of the stock. Electron microscopic observations were made in semen specimens obtained by electroejaculation from mature males prior to their use in an effort to determine semen parameters that might be useful in guiding breeding procedures. Sperm samples were either fixed immediately or treated with capacitating media and fixed after 4 to 7 hr of incubation. Many of the specimens examined were pyospermic (white cell in semen) and showed evidence of spermophagy, primarily by neutrophils. Of the six animals surveyed, only one showed little evidence of spermophagy, and three had extensive pyospermia and spermophagy but this finding was not correlated with fertility. Samples fixed immediately as well as those incubated for several hours showed evidence of spermophagy, indicating that the phagocytosis was not the result of culture. Gene pool restriction and/or captive stress may be contributing factors of reduced semen quality. © 1994 Wiley‐Liss, Inc.
Effects of gonadotropins upon the incidence and kinetics of meiotic maturation of macaque oocytes in vitro
Authors: Schramm Ralph D. , Tennier Michael T. , Boatman Dorothy E. , Bavister Barry D.
Published: February 2005

The specific aim of this study was to determine the effects of gonadotropins in vitro upon the incidence of and precise time interval to germinal vesicle breakdown (GVB) and extrusion of the first polar body (PB1) in oocytes from nonstimulated rhesus monkeys. Cumulus‐enclod germinal vesicle (GV) stage oocytes from 10 normal, cycling rhesus monkeys in the follicular phase of the menstrual cycle were cultured with either: (1) 1.0 μg/ml human follicle‐stimulating hormone (hFSH), (2) 10 μg/ml human luteinizing hormone (hLH), (3) 1.0 μg/ml hFSH and 10 μg/ml hLH, or (4) no gonadotropins (controls). Oocytes (n = 234) were examined at 3‐hr intervals from 0 to 21 hr and at 4‐hr intervals from 24 to 52 hr for GVB and PB1. Neither the incidence of GVB (hFSH: 63.5%; hLH: 56.1%; both gonadotropins: 63.1%; no gonadotropins: 53.6%) nor extrusion of PB1 (hFSH: 41.3%; hLH: 36.4%; both gonadotropins: 36.9%; no gonadotropins; 31.9%) differed (P > 0.05) among treatments. The time to GVB was accelerated (P < 0.05) by gonadotropins (hFSH: 10.8 ± 1.7 hr; hLH: 10.1 ± 1.8 hr; both gonadotropins: 8.8 ± 1.1 hr) when compared to controls (17.4 ± 2.0 hr). However, the time interval to extrusion of PB1 did not differ (P > 0.05) among treatments (hFSH: 32.3 ± 1.2 hr; hLH: 35.1 ± 1.4 hr; both gonadotropins: 35.2 ± 1.3 hr; no gonadotropins: 34.1 ± 1.2 hr). The mean interval to extrusion of PB1 was 34.1 ± 0.6 hr. In conclusion, GVB and PB1 extrusions appear to be, in part, independently regulated events in macaque oocytes matured in vitro since the timing of PB1 extrusion is not tightly coupled with the onset of GVB. Although the developmental potential of oocytes may be enhanced by gonadotropins, alternative approaches must be developed to improve the poor competence of oocytes from nonstimulated monkeys to mature in vitro. © 1994 Wiley‐Liss, Inc.
Developmentally regulated expression during gametogenesis of the murine gene meg1 suggests a role in meiosis
Authors: Don Jeremy , Winer Martin A. , Wolgemuth Debra J.
Published: February 2005

Previous studies have shown that in adult male mice, expression of the meg1 gene is restricted to meiotic and early postmeiotic testicular germ cells. We have now analyzed the expression of meg1 during postnatal testicular development and the comparable meiotic stages in the female. The 0.75 kb transcript for meg1 begins to accumulate in testes at d8‐9 of postnatal (pn) development, coincident with the entry of germ cells into meiosis, and is expressed most abundantly at pn d14 and subsequent stages, when the spermatocytes have entered pachytene. In situ hybridization analysis shows that meg1 is expressed at very low levels in leptotene cells and increases as the cells progress through zygotene and pachytene stages. In the embryonic ovary, meg1 is not detected until after day 15 of gestation when the cells have entered the pachytene stage of meiosis I. In situ hybridization analysis suggests that meg1 transcripts are expressed at higher levels in degenerating rather than in healthy pachytene stage oocytes; meg1 is not expressed in any cells of the adult ovary, regardless of the stage of follicular development. These results suggest that meg1 is indeed a meiosis‐associated gene in both male and female germ cells through the pachytene stage of meiosis I and appears to exhibit sex‐specific differences in its expression thereafter. © 1994 Wiley‐Liss, Inc.
Early gestational expression of retinol‐binding protein mRNA by the ovine conceptus and endometrium
Authors: Doré Jules J. E. , Roberts Mary P. , Godkin James D.
Published: February 2005

Communication between the mother and the early developing embryo is mediated by a variety of signals secreted by either the uterus or the embryo to elicit a response from the other. These signals include prostaglandins, proteins, and steroids. Recently, retinol‐binding protein (RBP) has been described as a product of both the conceptus and endometrium in several species. Utilizing a cDNA clone to bovine RBP, we have described RBP mRNA expression in the endometrium, early conceptus, and extraembryonic membranes of sheep. Endometrial RBP mRNA expression did not differ between samples collected on day 13 of the estrous cycle and early pregnancy. In cyclic animals, RBP mRNA expression decreased two‐fold between days 13 and 16, presumably a result of luteal regression and the consequential withdrawal of progesterone. In pregnant animals, endometrial RBP mRNA expression likewise decreased between days 13 and 16 and remained at this reduced level through day 30, despite the presence of a functional corpus luteum. Initiation of embryonic RBP expression appeared to coincide with early stages of blastocyst elongation at day 13. Levels of expression increased dramatically with conceptus development, peaked on day 23, and declined afterwards. Results from restriction enzyme analysis of genomic DNA indicated that RBP was encoded by a single gene per haploid genome. Differences in the temporal and tissue‐specific expression of the protein, despite the apparent utilization of a single gene, suggest complex regulation of RBP gene expression. © 1994 Wiley‐Liss, Inc.
Calcium concentration and fertilization by subzonal insemination of a single spermatozoon in mouse oocytes
Authors: Fujimoto Goro , Tsuchiya Hideki , Kobayashi Kazuhiko , Saga Masahiko , Rothman Cappy M. , Ogawa Shyoso
Published: February 2005

The effect of calcium concentration in culture medium on the fertilization of subzonally microinseminated mouse oocytes was examined. Oocytes were injected with a single spermatozoon so that the sperm head was forced to adhere onto the ooplasmic membrane with a micromanipulation technique. For the inseminations, epididymal spermatozoa preincubated in culture medium and those treated with ionophore A23187 were used. Inseminated oocytes were cultured using media with three different calcium concentrations of 1.71, 3.42, and 5.13 mM; 40.0%, 71.6%, and 47.9% of oocytes microinjected with preincubated sperm were fertilized after incubation with those media, respectively. When the oocytes inseminated with ionophore‐treated sperm were incubated in media containing 1.71 and 3.42 mM calcium, their fertilization rates were 58.2% and 87.5%. Thus fertility of subzonally microinseminated oocytes was obviously enhanced when cultured in medium with 3.42 mM of calcium, irrespective of being inseminated with preincubated sperm (P < 0.01) or with ionophore‐treated sperm (P < 0.005). Some of the microinseminations with preincubated sperm were performed without sperm adhered to the oolemma. In these cases, the incidence of fertilization was not improved by incubating the inseminated oocytes in medium containing 3.42 mM calcium (32.6%) as compared to those incubated in medium with 1.71 mM calcium (28.3%). These results suggest that the concentration of extracellular calcium exerts an important effect on the progress of fertilization events subsequent to sperm adherence onto the ooplasmic membrane. Almost 80% of the zygotes fertilized via incubation in medium with 3.42 mM of calcium developed into blastocysts after culturing in vitro. © 1994 Wiley‐Liss, Inc.
Bovine nuclear transfer embryos: Oocyte activation prior to blastomere fusion
Authors: Stice Steven L. , Keefer Carol L. , Matthews Lee
Published: February 2005

Successful bovine nuclear transfer (NT) embyro production requires proper oocyte activation and transfer of a nucleus into this oocyte. However, the temporal relationship between these two events is unclear. The current study examined whether activation of the oocyte prior to fusion would induce nuclear swelling while also affecting development to morula and blastocyst stage and finally development to offspring. Aged oocytes can be activated by a number of techniques including exposure to room temperature. In this study oocyte activation was induced through three different means: reduced temperature culture alone, reduced temperature culture and calcium ionophore, and naturally, through the fertilization process. Electrofusion was carried out after the activation stimulus. When used in the NT procedure, activation of oocytes prior to fusion resulted in NT embryos that under went nuclear swelling and had a high developmental rate to morula and blastocyst stages. Also, these NT embryos developed to normal offspring when transferred to recipient animals. The addition of a calcium ionophore treatment to the reduced temperature culture was not beneficial and resulted in less nuclear swelling. The use of enucleated fertilized oocytes as recipient cytoplasm for the new nucleus resulted in NT embryos developing to morula and blastocyst stages at the same rate as room temperature activated NT embryos. Therefore, improved embryo development can be obtained from NT embryos if the aged recipient oocyte is activated prior to the time of fusion. Also, offspring were obtained from these pre‐activated NT embryos. © 1994 Wiley‐Liss, Inc.
Evidence for presence of hyaluronan binding protein on spermatozoa and its possible involvement in sperm function
Authors: Ranganathan Sripriya , Ganguly Amit Kumar , Datta Kasturi
Published: February 2005

Hyaluronic acid, a major component of the extracellular matrix, plays an important role in the regulation of different cellular processes, e.g., locomotion, cell‐cell interaction during morphogenesis, and differentiation. Distribution of hyaluronic acid with respect to the role of sperm hyaluronidase in sperm penetration and gamete interaction is well established. In order to elucidate this mechanism, in our current study we have identified and demonstrated, for the first time, the presence of a 68‐kDa cell surface hyaluronic acid binding glycoprotein (HABP) in spermatozoa of different species (rat, mice, bull, and human) by immunoblot analysis and indirect immunofluorescence using the polyclonal antibodies raised against purified HABP. Furthermore, we were able to demonstrate a differential distribution of 68‐kDa HA binding protein on the sperm head, midpiece, and tail of different species. To identify its role in sperm function, we observed its declining pattern during epididymal maturation and also the inhibition of sperm‐oolemmal adherence by pretreatment of the sperms with anti‐HABP antibodies. We have further observed its in vivo phosphorylation in motile spermatozoa. All our data clearly indicate that sperm hyaluronan binding protein may have a specific role in sperm maturation, motility, and fertilization processes. © 1994 Wiley‐Liss, Inc.
MPF components and meiotic competence in growing pig oocytes
Authors: Christmann Leandro , Jung Thomas , Moor Robert M.
Published: February 2005

Growing pig oocytes (≤90 μm in diameter) are unable to resume meiosis in vitro. The objective of our present experiments has been to identify the reasons for meiotic competence in these cells. By comparing histone H1 kinase activity in growing and fully grown oocytes we demonstrate that incompetence is associated with an inability to activate H1 kinase in growing oocytes. Immunoblotting was used to determine whether this kinase inactivity resulted from a lack of either p34cdc2 protein or B‐type cyclin. The results established that each of these cell cycle molecules are present in comparable amounts in both growing and fully grown oocytes. In the third series of studies experiments were carried out in an attempt to induce p34cdc2 activation during growth. Treatment with okadaic acid, an inhibitor of phosphatase 1 and 2A known to stimulate and accelerate the transition into M‐phase of the meiotic cycle in a number of different species, was able to induce p34cdc2 kinase activity and facilitated the G2‐ to M‐phase in growing oocytes.

We conclude that although growing oocytes in pigs have sufficient key cell cycle components for the G2 to M transition, they remain incapable of converting these components to active maturation‐promoting factor (MPF) until growth is virtually completed. Inhibition of phosphatase 1 or 2A induces the formation of active MPF during growth by an as yet unidentified pathway. © 1994 Wiley‐Liss, Inc.
Computerized analysis of the motility parameters of hamster spermatozoa during maturation
Authors: Devi L. Girija , Shivaji S.
Published: February 2005

A computer‐aided semen analysis system was used for the objective assessment of hamster spermatozoa during epididymal maturation. The caput epididymal spermatozoa were extremely sluggish, achieved very little progression, and the three velocity parameters, namely curvilinear velocity (VCL), progressive velocity (VSL), and path velocity (VAP), were low. These spermatozoa during progressive movement alternated between the linear shape and “U” shape or attained an “S” shape prior to changing to the “U”; shape. The corpus epididymal spermatozoa were faster, displayed greater VSL, VAP, and VCL compared to caput epididymal spermatozoa, and, during forward motility, attained “U,” “C,”; and (or) “?” shape as in the wriggling motility pattern. The proximal cauda epididymal spermatozoa were actively motile and VSL, VAP, and VCL in these spermatozoa were more than 10 times greater compared to the caput epididymal spermatozoa. The proximal cauda epididymal spermatozoa predominantly moved in circles and with time became slower and more circular in their trajectories and exhibited a reduction in LIN (linearity). The distal cauda epididymal spermatozoa were very similar to the proximal cauda epididymal spermatozoa with respect to their fast motility (VSL, VAP, and VCL are similar) and beat cross frequency (BCF), but showed larger values for STR (straightness) and LIN and moved along curved trajectories. The amplitude of lateral head displacement (ALH) was also considerably lower in the distal cauda epididymal spermatozoa compared to the proximal cauda epididymal spermatozoa. Thus, this study provides for the first time data related to seven motility parameters for caput and corpus epididymal spermatozoa of hamster. It also provides additional data with respect to VCL, LIN, BCF, and ALH for proximal and distal cauda epididymal spermatozoa of hamster. © 1994 Wiley‐Liss, Inc.
Effects of transforming growth factors and activin‐A on in vitro porcine oocyte maturation
Authors: Coskun Serdar , Lin Young C.
Published: February 2005

Growth factors are known to regulate ovarian function. In the present study, effects of these growth factors, TGF‐α, TGF‐β, and activin‐A were tested on spontaneous porcine oocyte maturation. Cumulus‐oocyte complexes (COC) were cultured in the presence of TGF‐α, TGF‐β, and activin‐A for 48 hr. Stages of meiotic maturation were assessed by staining with acetic orcein. Among these factors, only TGF‐α significantly enhanced the maturation rate, whereas TGF‐β suppressed the spontaneous maturation rate. The site of action of TGF‐α on COC and the interaction between TGF‐α and EGF receptor was also examined. Denuded oocytes, alone or in coculture with cumulus cells, were cultured in the presence of TGF‐α for 48 hr. TGF‐α did not have any significant effect on denuded oocyte maturation. Heptanol was employed to investigate the role of gap junctions on TGF‐α‐induced oocyte maturation in COC. Although heptanol did not have any significant effect in the control medium, heptanol reversed the stimulatory effect of TGF‐α on porcine oocyte maturation. TGF‐α was able to displace 125I‐EGF binding on COC. In conclusion, TGF‐α enhances the spontaneous maturation of porcine oocytes by generating positive signal(s) in cumulus cells that are transferred to the oocyte via gap junctions. TGF‐α shares the same receptor with EGF on porcine COC. TGF‐β, in contrast, inhibits porcine oocyte maturation. © 1994 Wiley‐Liss, Inc.
Actin in the acrosome of the spermatozoa of the crab, Cancer pagurus L. (decapoda, crustacea)
Authors: Tudge C. C. , Grellier P. , Justine J.‐L.
Published: February 2005

The cytoskeletal protein actin was identified in the mature spermatozoon of the European edible crab, Cancer pagurus Linnaeus, by indirect immunofluoresce with monoclonal and polyclonal anti‐actin antibodies, fluorescent phalloidin, and DNAase I. The actin was localized in two distinct concentric rings within the acrosome vesicle of the spermatozoon and appeared to correlate with the internal zonation of the vehicle. Modifications of the fluorescent pattern for actin were observed in sperm cells which were undergoing changes associated with the acrosome reaction. In these cases, fluorescent staining was observed in the nucleocytoplasm immediately subjacent to the perforatorial column and sometimes in the perforatorial column within the acrosome vesicle. Equally intense fluorescence was observed in an apical perforatorial projection. SDS‐PAGE of C. pagurus sperm confirmed the presence of actin in the cells. A single band of actin (approximately 43 kDa) comigrated with rabbit muscle actin when immunoblotted onto nitrocellulose with mouse monoclonal anti‐actin. The actin‐associated cytoskeletal proteins α‐actinin, tropomyosin, and spectrin were also identified within the spermatozoon of C. pagurus using specific polyclonal antibodies, but their presence was not confirmed by SDS‐PAGE and immunoblotting. © 1994 Wiley‐Liss, Inc.
Development of IVM‐IVF produced 8‐cell bovine embryos in simple, serum‐free media after conditioning or Co‐culture with buffalo rat liver cells
Authors: Rehman N. , Collins A. R. , Suh T. K. , Wright R. W.
Published: February 2005

Development of 8‐cell bovine embryos derived from in vitro matured/in vitro fertilized (IVM/IVF) oocytes was evaluated in two simple, serum‐free media (CZB and SOM) with buffalo rat liver cells co‐culture (BRLC) or after conditioning compared to a commonly used, serum‐supplemented complex medium TCM‐199. In a 3 x 4 factorial design, 578 eight‐cell embryos were randomly assigned to 12 treatment groups. The factors were: first, type of culture medium (M199/FBS, CZBg and SOM), and second, the use of BRLC (as co‐culture or to condition media for 24 hr and 48 hr) and unconditioned media. Development to morula was not affected by the type of medium, but co‐culture and 48 hr conditioning within media type resulted in better development when compared to the 24‐hr conditioned or unconditioned groups. Blastocyst development in SOM (38.9%) was different (P < 0.05) than in CZBg (46.6%) and M199/FBS (48.7%) and was lowest in the unconditioned group (27.8%) followed by 24 hr conditioned (33.3%), 48 hr (56.3%), and co‐culture (59.6%). No blastocyst expansion was observed with unconditioned media and 24 hr conditioned SOM. Significant differences (P < 0.05) were found among all treatment groups except the co‐culture and 48‐hr conditioned groups. Hatching occurred only with co‐culture and 48‐hr conditioned groups of M199/FBS and CZBg media. These data show that CZB with glucose conditioned by BRLC monolayers for 48 hr can support the development of IVM/IVF produced bovine embryos to blastocyst compared to culture in TCM‐199 with serum. © 1994 Wiley‐Liss, Inc.
Induction of meiotic maturation in vivo in the mouse by IMP dehydrogenase inhibitors: Effects on the developmental capacity of ova
Authors: Downs Stephen M.
Published: February 2005

The present study was conducted to examine the effects of priming duration and inosine monophosphate (IMP) dehydrogenase inhibitors on the fertilization and pre‐ and postimplantation development of ova from immature mice. Mice were primed with equine chorionic gonadotropin (eCG) and 1 d later were treated with mycophenolic acid (MA) or mizoribine (Mz; also known as bredinin) or an equal volume of the vehicle. Two days after priming, mice received human chorionic gonadotropin (hCG) and were mated with fertile males. Some mice received hormone but were not mated. Ova were isolated from the oviducts 23–24 hr post‐hCG and cultured in Whitten's medium. Some mice were not killed until 7 or 19 d postvaginal plug to determine the extent of implantation or development to term, respectively. In superovulated control mice, 94% of ova developed to two‐cells and 82% of these progressed to the blastocyst stage. Nineteen of 23 mice (83%) had implantations (24/mouse) and 20 of 24 mice (83%) had term embryos (11/mouse). Induction of meiotic maturation with Mz or MA after 1 d of priming, followed 1 d later by hCG injection and mating, resulted in a significant loss of preimplantation developmental capacity (20–22% two‐cells; 29–50% blastocysts). These numbers were similar whether or not the mice were mated, indicating that the development was parthenogenetic. In addition, Mz treatment reduced the number of mice with implantion sites (13 of 19, 68%), the number of implantations per mouse (10), the viability of the implantations, and development to term (four of 24, mice, 17%; one embryo/mouse). Thus, most of the implanted embryos were resorbed after imlantation. This effect could be attributed, in part, to each of the following: (1) parthenogenetic activation; (2) aging of the metaphase II oocyte before insemination; and (3) resumption of meiotic maturation before the oocyte had reached its full developmental potential. Coincident gonadotropin injection reversed the loss of development brought about by IMP dehydrogenase inhibitors. These data show that induction of premature meiotic maturation by IMP dehydrogenase inhibitors results in a significant loss of developmental capacity and indicates that active purine metabolic pathways in situ maintain meiotic arrest and prevent premature meiotic maturation that would result in compromised development. © 1994 Wiley‐Liss, Inc.
Evidence against a functional ATP‐dependent calcium extrusion mechanism in bovine epididymal sperm
Authors: Vijayaraghavan Srinivasan , Trautman Kevin , Mishra Santosh K. , Herrmsmeyer Kent
Published: February 2005

Bovine epididymal sperm resuspended in ionic buffers take up relatively large amounts of calcium. This uptake, which is almost entirely mitochondrial, apparently bypasses the sperm cytosol. The direct mitochondrial loading is an unusual aspect of sperm calcium uptake, which suggests that the plasma membrane region surrounding the mitochondria should be highly permeable to calcium, whereas the membrane domains surrounding the head and tail regions of sperm should be impermeable. This study was undertaken to determine the role of a plasma membrane calcium ATPase in sperm calcium homeostasis. Kinetics of calcium (45Ca2+) uptake into intact and permeabilized caudal epididymal sperm confirmed that mitochondrial calcium uptake occurs with virtually no resistance from the surrounding plasma membrane. Cytoplasmic calcium accumulation by sperm depleted to intracellular ATP, measured in the presence of mitochondrial calcium uptake inhibitors, showed no increase upon energy depletion as would be expected if an ATP‐dependent calcium extrusion mechanism were present. Furthermore, lowering the incubation temperature to further reduce the activity of the calcium ATPase in these energy‐depleted sperm was also without effect on calcium accumulation. The calcium ATPase inhibitor vanadate, even at high concentrations, failed to increase intracellular 45Ca2+ accumulation. However, vanadate was effective in inhibiting motilty showing that the compound was accumulated into sperm to inhibit flagellar dyenin ATPase. Therefore, the lack of effect of vanadate on 45Ca2+ accumulation was not due to its inability to enter sperm. Other calcium ATPase inhibitors such as quercetin, thapsigargin, and cyclopiazonic acid, which readily demonstrate ATP‐dependent calcium extrusion in other somatic cells, were also without effect on sperm caclium accumulation. These results with 45Ca2+ were also confirmed using single‐cell calcium distribution measurements with a fluorescent indicator. Our data strongly suggest that sperm, contrary to previous assumptions, do not possess a functional plasma membrane, ATP‐dependent, calcium extrusion mechanism. © 1994 Wiley‐Liss, Inc.
Expression of scatter factor/hepatocyte growth factor is regionally correlated with the initiation of sperm motility in murine male genital tract: Is scatter factor/hepatocyte growth factor involved in initiation of sperm motility?
Authors: Naz Rajesh K. , Joseph Ansamma , Lee Yuan , Ahmad Khaliq , Bhargava Madhu M.
Published: February 2005

Based upon findings that the scatter factor/hepatocyte growth factor (SF/HGF) has strong mitogenic and motogenic properties, and that the sperm cell acquires its fertilizing capacity and motility in the distal parts of mammalian epididymis, the present study was conducted to investigate the role of SF/HGF in initiation of sperm cell motility. This was investigated by determining the expression of SF/HGF in various regions of the murine male genital tract by scatter and cell tracking assays using MDCK epithelial cells, Western blot procedure, and the immunohistochemical procedure using paraffin sections of various regions of the male genital tract. The findings from all these assays indicate that SF/HGF is differentially expressed in various parts of the male genital tract with slight or no expression in the testes, caput epididymis, and vas deferens, and with the highest expression in cauda and corpus (distal) epididymis followed by expression in the corpus (proximal) epididymis. This region‐specific SF/HGF expression pattern coincides with the pattern of acquiring the fertilizing capacity and motility by the sperm cell during its transit through the male genital tract. However, wherever SF/HGF was expressed in the male genital tract, its molecular weight was slightly higher (Mr, 82 kD), compared to the SF/HGF expressed in various other somatic tissues (Mr, 78 kD), indicating that the genital tract SF/HGF may be a different molecular species that shares some immunoreactive epitopes with the somatic cell SF/HGF. Incubation of immotile sperm from caput epididymis with the purified human placental SF/HGF of 78 kD initiated motility in 5–15% of sperm population. These results strongly suggest that the SF/HGF‐like activity is expressed in the male genital tract in a region‐specific manner, and this activity may have a role in initiation of sperm motility acquired during its transit through the epididymis in mammals. © 1994 Wiley‐Liss, Inc.