bluuebunny/crossref_metadata_2025 huggingface dataset:
Morphological development and sex of bovine in vitro‐fertilized embryos
Authors: Avery Birthe , Jørgensen Claus B. , Madison Valerie , Greve Torben
Published: February 2005

Bovine in vitro‐fertilized embryos at the blastocyst stage were collected at days 7, 8, and 10 postinsemination and sex was determined via the polymerase chain reaction (PCR) to compare the embryonic development with the sex of the embryos. The percentages of males (sex ratio) after division of the embryos into three developmental groups were 68%, 48%, and 35% in the fast, intermediate, and slow groups, respectively (P = 0.014). The percentages of males on days 7, 8, and 10 were 60%, 40% and 33%, respectively (P = 0.043). The average sex ratio for the whole material was 50%. It is thus concluded that male bovine preimplantation embryos develop faster than female embryos.
Synthesis and modification of D7 protein during Xenopusoocyte maturation
Authors: Smith Rosamund C. , Dworkin Mark B. , Dworkin‐Rastl Eva
Published: February 2005

The Xenopus maternal mRNA D7 is translationally repressed during oogenesis, only becoming recruited into polysomes during oocyte maturation, with D7 protein being detectable for the first time prior to germinal vesicle breakdown (GVBD). The synthesis of D7 protein was found to be induced by a variety of maturation‐promoting agents including cyclin, c‐mos and crude preparations of MPF. D7 protein induced by all these agents is post‐translationally modified and exists as a number of variants of differing molecular weight. In contrast to endogenous D7 mRNA, D7 RNA injected into the stage VI oocyte is efficiently translated, resulting in the accumulation of predominantly unmodified D7 polypeptides, which become increasingly modified during oocyte maturation to produce a pattern of polypeptides similar to those derived from endogenous D7 mRNA. Thus, the system that results in the post‐translational modification of the D7 protein is itself activated during oocyte maturation. The nature of the protein modification is not known but does not appear to be phosphorylation. The translation of exogenous D7 RNA in the stage VI oocyte does not lead to translational derepression of endogenous D7 mRNA.
Glycogen breakdown in cleaving xenopus embryos is limited by ADP
Authors: Dworkin Mark B. , Dworkin‐Rastl Eva
Published: February 2005

Xenopus eggs contain large stores of glycogen, but this glycogen is not glycolytically processed during cleavage. The Embden‐Meyerhof pathway is inhibited by the absence of pyruvate kinase activity in vivo, and lactate and pyruvate are present at relatively low levels. In the late blastula, just preceding gastrulation, lactate levels increase, indicating the onset of glycogen breakdown and glycolytic flux. Glycolysis from microinjected [14C]glucose‐6‐phosphate could be transiently activated, however, by the coinjection of ADP into fertilized eggs, and constitutively activated by the injection of the ATPase potato apyrase, indicating the presence of all enzymes necessary for glycolytic activity. The isozyme profiles of pyruvate kinase and malic enzyme, two enzymes involved in carbon metabolism during cleavage or in the subsequent activation of glycogen breakdown, do not change between the egg and gastrula stages. These data suggest that the activation of glycogen breakdown and glycolysis in the late blastula is probably not a result of new gene activity but may be the metabolic consequence of increased free ADP that is then able to support the pyruvate kinase reaction.
Spatial distribution of active genes implicated in the regulation of insulin‐like growth factor stimulatory loops in human decidual and placental tissue of first‐trimester pregnancy
Authors: Glaser Anna , Luthman Holger , Stern Ingrid , Ohlsson Rolf
Published: February 2005

We have previously shown that the insulin‐like growth factor‐2 (IGF‐2) gene is partially coexpressed with the IGF‐1 and ‐2 receptor genes in proliferative cytotrophoblasts of the human extraembryonic tissue. Here we show that high levels of IGF‐2 gene expression are not restricted to the embryonic tissue but can also be found in the decidua compacta. The IGF‐2 gene is thus expressed at high levels in the mesenchymal stroma of the decidua to establish potentially short‐range communication with primarily IGF‐1 receptor‐positive mesenchymal stroma cells. Conversely, the glandular and surface epithelia coexpress the IGF‐1 receptor and IGF‐1 genes, while the IGF‐2 gene is not detected above background levels. The potential control machanisms of these cell‐cell signalling pathways were investigated by the analysis of the spatial distribution of active IGF binding proteins (IGFBP) genes. The IGFBP‐3 gene is coexpressed with the IGF‐2 gene in proliferative cytotrophoblasts of the embryonic placenta. While active IGFBP‐1 and ‐2 genes in our hands cannot be detected in the embryonic placenta, all three IGFBP genes are expressed in complex and overlapping patterns in the decidua compacta. The results are discussed in terms of how the various IGFBP genes may operate in different cell types to restrict IGF local stimulatory pathways. © 1992 Wiley‐Liss, Inc.
Functional properties of caltrin proteins from seminal vesicle of the guinea pig
Authors: Coronel Carlos E. , Lardy Henry A.
Published: February 2005

Caltrin proteins from seminal vesicle content of the guinea pig bind with great specificity to different regions of the spermatozoa. Indirect immunofluorescence studies with polyclonal antibodies showed that caltrin I binds to the head, on the acrosomal cup, while caltrin II binds on the principal tail and the neck. No fluorescence was detected either in the midpiece or in the postacrosomal area of the head when sperm were exposed to either of the caltrins. Calcium‐induced hyaluronidase release, which occurs during the acrosomal reaction, was dramatically inhibited by caltrin I (∼85% inhibition). Caltrin II was less effective in preventing the enzyme release (∼50% inhibition). Chemical modification of the structure modified the biological activity of the two caltrins. Reduction and carboxymethylation of the cysteine residues diminished the inhibitory activity on 45Ca2+ uptake and reduced the ability of the proteins to react with their antibodies. Removal of the carbohydrate portion by chemical deglycosylation transformed the inhibitor proteins into enhancers of calcium uptake into the spermatozoa. Caltrin proteins from the guinea pig appear to play the same physiological role as bovine caltrin, regulating specifically calcium transport across the spermatozoal membranes related with the acrosome reaction and hyperactivation process. The dual behavior of caltrins to inhibit or enhance Ca2+ uptake enables them to fulfill this function. Nevertheless, molecular mechanisms different from those described for bovine caltrin seem to be involved in the control of the functional activity of the guinea pig caltrins. © 1992 Wiley‐Liss, Inc.
Structural organization of surface domains of sperm mitochondria
Authors: Olson Gary E. , Winfrey Virginia P.
Published: February 2005

Sperm mitochondria are assembled into an organized sheath surrounding the outer dense fibers and axoneme of the flagellar midpiece. Each mitochondrion is arranged so that its surface faces three different cellular organelles including the plasma membrane, neighboring mitochondria and the outer dense fiber–axoneme complex. In this manuscript we present data on structural differentiations of these three different surface domains of the outer mitochondrial membrane. We demonstrate that the apposed surfaces of adjacent mitochondria are joined by a two dimensional network of studs unique to this domain. By contrast, the surface domain facing the outer dense fiber–axoneme complex exhibits a different, but highly ordered structural organization, evident as a parcrystalline network of parallel stripes; this domain is further distinguished by its exclusive association with a midpiece‐specific cytoskeletal complex. These differentiations are not seen on the surface domain of mitochondria which faces the plasma membrane. The implications of the mosaic composition of the outer mitochondrial membrane in the assembly and function of the mitochondrial sheath are discussed. © 1992 Wiley‐Liss, Inc.
Cyclin B in fish oocytes: Its cDNA and amino acid sequences, appearance during maturation, and induction of p34cdc2 activation
Authors: Hirai T. , Yamashita M. , Yoshikuni M. , Lou Y.‐H. , Nagahama Y.
Published: February 2005

Under the influence of maturation‐inducing hormone (MIH) secreted from follicle cells, oocyte maturation is finally triggered by maturation‐promoting factor (MPF), which consists of a homolog of the cdc2+ gene product of fission yeast (p34cdc2) and cyclin B. Two species of cyclin B clones were isolated from a cDNA library constructed from mature goldfish oocytes. Sequence comparisons revealed that these two clones are highly homologous (95%) and were found to be similar to Xenopus cyclin B1. Using monocional antibodies against Escherichia coli produced goldfish cyclin B and the PSTAIR sequence of p34cdc2, we examined the levels of cyclin B and p34cdc2 proteins during goldfish oocyte maturation induced in vitro by 17α,20β‐dihydroxy‐4‐pregnen‐3‐one (17α,20β‐DP), a natural MIH in fish. Protein p34cdc2 was found in immature oocyte extracts and did not remarkably change during oocyte maturation. Cyclin B was not detected in immature oocyte extracts and appeared when oocytes underwent germinal vesicle breakdown. Cyclin B that appeared during oocyte maturation was labelled with [35S]methionine, indicating its de novo synthesis. Introduction of E. coli‐produced cyclin B into immature oocyte extracts induced p34cdc2 (MPF) activation. Although the possibility that immature goldfish oocytes contain an insoluble cyclin B is not completely excluded, these results strongly suggest that 17α, 20β‐DP induced oocytes to synthesize cyclin B, which in turn activates preexisting p34cdc2, forming active MPF. © 1992 Wiley‐Liss, Inc.
Vitrification of mouse oocytes using short cryoprotectant exposure: Effects of varying exposure times on survival
Authors: Shaw P. W. , Bernard A. G. , Fuller B. J. , Hunter J. H. , Shaw R. W.
Published: February 2005

The effects on oocyte viability of varying the duration of exposure to cryoprotectants before rapid cooling to – 196°C were examined, using the vitrification protocol of Nakagata. A very short exposure (15 sec) was found to be optimal, resulting in an overall rate of development from vitrified oocytes to hatching blastocysts of 31.8%. Very high rates of survival (77–89%) of oocytes exposed to the cryoprotectant media, but without the vitrification, together with extreme variability in results between straws in the vitrified groups, suggest that losses in viability during vitrification may result from ice damage during devitrification of the medium. (c) 1992 Wiley‐Liss, Inc.
Demonstration of a stage‐specific expression of the zfy protein in fetal mouse testis using anti‐peptide antibodies
Authors: Su Hua , Lau Yun‐Fai Chris
Published: February 2005

The zinc finger Y (Zfy) gene is located on the Y chromosome of all placental mammals. Although it is phylogenetically conserved and is expressed in mouse fetal testis, it is not the sex determining Y (Tdy) gene. To address the possible function of the Zfy gene in mice, the distribution of Zfy protein in fetal mice was investigated by immunocytochemical staining using several specific antisera against synthetic peptides of the mouse Zfy protein. Analysis of various fetal tissues at different embryonic stages demonstrated a specific staining only in fetal testis. In particular, reactive protein was initially observed in male fetal gonads at day 11.5 postcoitum (p.c.). The immuno‐staining intensified in fetal testes at day 12 and 12.5 p.c., decreased drastically in those at day 13 and 14 p.c. and became undetectable in those at day 15 p.c. and beyond. The reactive molecules were distributed mostly within the seminiferous tubules of the embryonic testis. The present observations confirm the previous findings with RT‐PCR analysis and indicate that Zfy or Zfy‐like protein is expressed in stage‐specific manner during early testis differentiation. Its location in the seminiferous tubules suggests a possible role in early germ cell development. © 1992 Wiley‐Liss, Inc.
Characterization of male meiotic germ cell‐specific antigen (Meg 1) by monoclonal antibody TRA 369 in mice
Authors: Watanabe Daisuke , Sawada Ken , Koshimizu Uichi , Kagawa Tsutomu , Nishimune Yoshitake
Published: February 2005

We have identified a male meiotic germ cell‐specific antigen (Meg 1) with monoclonal antibody (mAb) TRA 369 in mice. The Meg 1 antigen was strongly expressed in specific steps of meiotic germ cells from pachytene spermatocyte to early spermatid, and not in other germ cells or somatic cells. Immunohistochemical examination revealed that the antigen was localized to the cytoplasm and was not distributed in the nucleus or on the cell surface. This antigen was demonstrated to have a molecular weight of 93 kDa and an isoelectric point of 5.2 by Western blotting. This molecule was first detected in the testis of 13‐day‐old mouse when pachytene spermatocytes first appeared. Thus this is a differentiation‐specific antigen in male meiotic germ cells, and mAb TRA 369 is a useful tool to study the regulation of germ cell differentiation and to define germ cell development in a molecular level. © 1992 Wiley‐Liss, Inc.
Protein synthesis requirements during resumption of meiosis in the hamster oocyte: Early nuclear and microtubule configurations
Authors: Plancha Carlos E. , Albertini David F.
Published: February 2005

The organization of chromatin and cytoplasmic microtubules changes abruptly at M‐phase entry in both mitotic and meiotic cell cycles. To determine whether the early nuclear and cytoplasmic events associated with meiotic resumption are dependent on protein synthesis, cumulus‐enclosed hamster oocytes were cultured in the presence of 100 μg/ml puromycin or cycloheximide for 5 hr. Both control (untreated) and treated oocytes were analyzed by fluorescence microscopy after staining with Hoechst 33258 and tubulin antibodies. Freshly isolated oocytes exhibit prominent nucleoli and diffuse chromatin within the germinal vesicle as well as an interphase network of cytoplasmic microtubules. After 4–4.5 hr in culture, most oocytes were in prometaphase I of meiosis as characterized by a prominent spindle with fully condensed chromosomes and numerous cytoplasmic asters. After 5–5.5 hr in culture, microtubule asters are no longer detected in most cells and the spindle is the only tubulin‐positive structure. Incubation for 5 hr in the presence of inhibitors does not impair germinal vesicle breakdown, chromatin condensation, kinetochore microtubule assembly, or cytoplasmic aster formation in the majority of oocytes examined; however, under these conditions, a population of oocytes retains a germinal vesicle, exhibiting variable degrees of chromatin condensation and cytoplasmic aster formation. Meiotic spindle formation in inhibited in all oocytes. These effects are fully reversible upon culture of treated oocytes in drug‐free medium for 5 hr. The data indicate that meiotic spindle assembly is dependent on ongoing protein synthesis in the cumulus‐enclosed hamster oocyte; in contrast, chromatin condensation and aster formation are not as sensitive to protein synthesis inhibitors during meiotic resumption. © 1992 Wiley‐Liss, Inc.
Parthenogenetic development of bovine oocytes treated with ethanol and cytochalasin b after in vitro maturation
Authors: Fukui Yutaka , Sawai Ken , Furudate Makoto , Sato Noriyuki , Iwazumi Yasuaki , Ohsaki Kazue
Published: February 2005

The present study was conducted to investigate the effects of different culture durations (24–36 hr) on bovine oocyte maturation in vitro and the effect of the presence or absence of cumulus cells at the time of treatment to induce parthenogenetic activation (exposure to ethanol and cytochalasin B; CB) (experiment I). The effects of dosage (2.5 or 5.0 μg/ml) and incubation time (2.5, 5, or 10 hr) in CB (experiment II) on the subsequent development to the blastocyst stage in vitro was also investigated. In experiment I, cleavage and development to the blastocyst stage were not affected by the presence or absence of cumulus cells at the time of parthenogenetic activation. However, the 24‐hr culture duration for in vitro maturation had a significantly lower rate of development to the blastocyst stage than the longer culture durations (27‐36 hr). In experiment II, treatment with 5 μg/ml CB for 5 hr showed the highest percentage of development to blastocyst in the oocytes matured for both 27 and 30 hr. To determine the viability of the parthenogenetic embryos (morulae and blastocysts), four recipient heifers received two embryos each, and one heifer was found to be pregnant on day 35 following transfer. Although fetal heartbeat was not observed, the subsequent estrus was prolonged in all heifers. The present results demonstrate development of in vitro‐matured, parthenogenetically activated bovine embryos up to the preimplantation stage. © 1992 Wiley‐Liss, Inc.
Stage‐Specific expression of rat transition protein 2 mrna and possible localization to the chromatoid body of step 7 spermatids by in situ hybridization using a nonradioactive riboprobe
Authors: Saunders P. T. K. , Millar M. R. , Maguire S. M. , Sharpe R. M.
Published: February 2005

The present study has used methoxyacetic acid (MAA)‐induced depletion of specific germ cell types in the rat and in situ hybridization with nonradioactive riboprobes to determine the stages of the spermatogenic cycle at which there is expression of the mRNA for the basic chromosomal protein transition protein 2 (TP2). On Northern blots, an abundant mRNA was detectable in samples from control adult rats, but the amount of message was markedly reduced when RNA was extracted from the testes of rats treated 14 and 21 days previously with methoxyacetic acid. These testes were depleted specifically of step 7–12 spermatids, suggesting that these cells contain TP2 mRNA. When tissue sections were subjected to in situ hybridization, the TP2 mRNA was localized at the cellular and subcellular levels. Messenger RNA for TP2 was first detectable in spermatids at step 7. In these spermatids, at high magnification, in addition to some positive reaction in the cytoplasm, intense staining was located to a perinuclear structure consistent with localization of mRNA within the chromatoid body. The amount of TP2 mRNA in the cytoplasm increased as remodelling of the early spermatid nucleus progressed and was highest in step 10 and 11 spermatids at stages X and XI. Thereafter, the mRNA decreased until it was undetectable in step 14 spermatids at stage XIV. The localization of TP2 mRNA to the chromatoid body of step 7 spermatids would be consistent with this organelle being a storage site for long‐lived mRNAs utilized later in spermiogenesis. © 1992 Wiley‐Liss, Inc.
The third IGF‐II promoter specifies transcription of three transcripts out of five in human placenta
Authors: Daimon Makoto , Johnson Thomas R. , Ilan Joseph , Ilan Judith
Published: February 2005

Insulin‐like growth factor‐II (IGF‐II) mRNA exists as multiple transcript size classes, such as 6.0, 5.3, 4.9, 3.2, and 2.2 kb mRNAs in various human tissues. Three different promoters, 2 different polyadenylation sites, and alternative splicing are involved in producing these multiple transcripts. Initiation of transcription at the 3 different promoters results in multiple mRNAs which contain identical coding regions but different 5′‐untranslated regions (5′‐UTRs). The first promoter is thought to direct expression of 5.3 kb mRNA in adult human liver. The second promoter region directs expression of 6.0, 3.2, and 2.2 kb mRNAs in human fetal tissues and several adult nonliver tissues. The third promoter specifies transcription of a 4.9 kb mRNA in various tissues. We isolated and sequenced a cDNA clone (pIGF‐II‐1‐70) from a human plcental cDNA library, which contains the IGF‐II coding region and the 5′‐UTR associated with the third promoter. By using a 5′‐UTR‐specific probe from the clone, we found that this third 5′‐UTR is contained in the IGF‐II mRNA of 2.2 kb and is absent in the 3.2 kb IGF‐II mRNA. We also found an 0.9 kb transcript expressed in placenta, which hybridized strongly to the third 5′‐UTR specific probe but not to IGF‐II coding region probes. This finding might indicate the existence of an mRNA encoding an IGF‐II‐associated peptide. © 1992 Wiley‐Liss, Inc.
Transforming growth factor β and basic fibroblast growth factor synergistically promote early bovine embryo development during the fourth cell cycle
Authors: Larson R. C. , Ignotz G. G. , Currie W. B.
Published: February 2005

Developmentally competent bovine blastocysts were produced by adding transforming growth factor β(TGFβ) and basic fibroblast growth factor (bFGF) to serum‐free cultures of in vitro produced, 2‐cell bovine embryos. The effects of TGFβ were evaluated because this growth factor signals synthesis and secretion of the extracellular matrix component fibronectin and its receptor. Previous investigations have demonstrated that fibronectin promotes early bovine embryo development in vitro. The effects of TGFβ can be potentiated by bFGF; bFGF itself is an effector of protein synthesis and a potent mitogen.

A positive interaction between the 2 growth factors resulted in 38.8% of fertilized oocytes maturing beyond the 16‐cell stage; of these, 24.6% formed blastocysts. Transfer of early blastocysts produced using serum‐free medium supplemented with growth factors resulted in pregnancy in 3 of 9 recipients.

These results support the hypothesis that TGFβ and bFGF act synergistically to promote development of bovine embryos beyond the “8‐cell block” observed in vitro. (c) 1992 Wiley‐Liss, Inc.
Fate of microinjected genes in preimplantation mouse embryos
Authors: Burdon Thomas G. , Wall Robert J.
Published: February 2005

The state of genes microinjected into mouse embryos was followed from the one‐cell to the blastocyst stage using the polymerase chain reaction (PCR). Microinjected DNA was detected in all one‐, two‐, and four‐cell injected embryos and in 44% of morula and 26% of blastocysts. Head‐to‐tail ligation of microinjected genes, a common feature of stably integrated transgene arrays, was detected in all embryos after injection of microinjected genes and occurred irrespective of the structure at the ends of the injected genes. Sensitivity of microinjected DNA to a methylation‐dependent restriction endonuclease Dpn I was lost in all embryos by the two‐cell stage (24 hr), indicating a change in DNA methylation, independent of transgene integration. Dissociation of blastomeres prior to compaction revealed a mosaic distribution of the microinjected DNA within the embryo and supports the notion that injected genes form a limited number of arrays, which segregate independently until they integrate into the genome or are degraded. Published 1992 Wiley‐Liss, Inc.
Factor in urinary extracts from pregnant women that inhibit mouse oocyte maturation in vitro
Authors: Sakakibara Ryuzo , Sakai Kazuya , Sakurai Yuko , Kohnoura Tae , Ishiguro Masatsune
Published: February 2005

Mouse oocyte maturation inhibitory factors, on the basis of inhibitory activity of spontaneous germinal vesicle breakdown (GVBD) of denuded mouse oocytes in culture, were extracted and partially purified by reversed‐phase resin adsorption and Sephadex G‐100 and G‐50 column chromatographies from the urine of pregnant women. Denuded oocytes obtained from ovaries of ICR mice underwent spontaneous GVBD by cultivation for 3 h in modified Krebs–Ringer's buffered solution, while this spontaneous GVBD was found to be inhibited by adding the final preparation (U‐D‐4) of urine. The inhibition was dose dependent, ranging from 0.6 to 10 μg protein/ml medium. Oocytes treated with U‐D‐4 and resuspended in control medium resumed GVBD. The molecular mass of U‐D‐4 was estimated to be less than 2,000 Da with gel filtration. Ether treatment failed to extract inhibitory factor(s) from U‐D‐4 and pepsin treatment inactivated U‐D‐4, indicating that inhibitory factor(s) in U‐D‐4 are peptide‐like substances. The inhibitory effect of U‐D‐4 on spontaneous GVBD was partially reversed in the presence of naloxone, a potent opioid antagonist. U‐D‐4s obtained from urine samples of pregnant women, nonpregnant women, and men showed the inhibitory effect on spontaneous GVBD; however, the activity of U‐D‐4 obtained from pregnancy urine was significantly more potent than those of the other urine samples. © 1993 Wiley‐Liss, Inc.
Isolation of sperm bindin from the oyster (Crassostrea gigas)
Authors: Brandriff Brigitte , Moy Gary W. , Vacquier Victor D.
Published: February 2005

A method was devised for isolating the insoluble content of the acrosome granule of sperm of the oyster Crassostrea gigas. The method involves the dissolution of the entire cell, except for the acrosome granule, in the detergent sodium lauroyl sarcosinate (sarcosy I). The isolated acrosome granule content is ring‐shaped and is 84% protein by weight. SDS‐polyacrylamide gel electro‐phoresis of this material yields from 1 to 4 bands of 65,000; 53,000; 43,000 and 34,000 apparent molecular weight, all of which stain with the PAS reaction indicating the material is a glycoprotein. The 65,000 molecular weight component is always present, but the presence of the other three bands varies with each preparation. The isolated acrosome granules agglutinate formaldehyde‐fixed oyster eggs. A trypsin‐generated glycopeptide digest of oyster egg surfaces inhibits the agglutinin activity of the isolated acrosome granules. We propose that the acrosomal glyco‐protein material is oyster sperm bindin which functions as the adhesive substance bonding the sperm to the egg during fertilizaion.
Two pathways from glycogen to glucose‐6‐phosphate in sea urchin eggs with special reference to the difference between the species in the contribution ratio of each pathway
Authors: Hino Akiya , Tazawa Eigoro , Yasumasu Ikuo
Published: February 2005

In the unfertilized eggs of the sea urchins, Anthocidaris crassispina, Pseudocentrotus depressus, Hemicentrotus pulcherrimus, Mespilia globulus, Temnopleurus toreumaticus, Toxopeneustes pileolus, and Clypeaster japonicus, the activities of phosphorylase [EC 2.4.1.1], phosphoglucomutase [EC 2.7.5.1], exo‐l,4‐α‐glucosidase [EC 3.2.1.3], and hexokinase [EC 2.7.1.1] are very similar. In all species, only phosphorylase activity is higher in fertilized eggs than in unfertilized eggs. The concentrations of glycogen, glucose, GIP, G6P, ATP, ADP, and Pi; the products and substrates in reactions catalyzed by these enzymes, were measured in these eggs. Based on the concentrations of these compounds in the eggs, it is assumed that G6P is produced by the combined action of glucosidase and hexokinase in all species examined, and that it is also produced in the reaction catalyzed by phosphorylase and phosphoglucomutase in all species except A crassispina and P depressus. Glycogen was found both in supernatant and in precipitate fractions, which were obtained by adding perchloric acid. Glycogen in the precipitate seems to be protein‐bound. Whole glycogen level in the eggs is almost the same in all species examined, but the level of acid‐soluble glycogen, as well as GIP, is markedly lower in the eggs of A crassispina and P depressus than in the eggs of other species examined. Protein‐bound glycogen is utilized by glucosidase activity but not by phosphorylase activity, in contrast to acid‐soluble glycogen, which is utilized by both enzyme activities. Hence, it is assumed that the failure of G6P production by phosphorylase and phosphoglucomutase‐in A crassispina and P depressus eggs is due to a low level of acid‐soluble glycogen in these eggs.
Possible causal relation between the acrosome reaction and cross‐fertilization in the sea urchins hemicentrotus pulcherrimus and pseudocentrotus depressus
Authors: Aketa Kenji , Ohta Tadayuki
Published: February 2005

Sperm of Hemicentrotus pulcherrimus undergo the acrosome reaction in the jelly coat or on the surface of the vitelline layer of Pseudocentrotus depressus eggs and can fertilize the latter, whereas those of P depressus do not undergo the acrosome reaction even after they strike the vitelline layer of H pulcherrimus eggs and cannot cross‐fertilize.

In the latter combination, however, if cross‐insemination is performed in the presence of homologous egg water, cross‐fertilization becomes less difficult than in normal seawater, and percentage cross‐fertilization rises as does percentage acrosome reaction. It is suggested that the cross‐fertilizability of this combination depends highly on the inductivity of the acrosome reaction. The reason why such a causal relation is observed is discussed.
Transfer of cultured rabbit embryos
Authors: Binkerd Pamela E. , Anderson Gary B.
Published: February 2005

Embryo transfer experiments were carried out to study the developmental capacity of cultured rabbit embryos when transferred to recipients of variable postovulatory maturity. Rabbit embryos were flushed from the oviduct at 26 hours postcoitum (pc) and cultured in a modified Ham's F‐10 medium supplemented with bovine serum albumin (BSA) for a period of 70 hours. At 96 hours pc the cultured embryos, which ranged from the early morula to the expanding blastocyst stage, were transferred to pseudopregnant recipients mated to vasectomized males 36 to 96 hours prior to the transfer procedure. Greatest embryo survival occurred when transfers were made to either the oviducts or uterine horns of recipients at 48 hours pc. Intermediate results for both implantation rates and number of young born were obtained with recipients at 36, 60, 72, and 84 hours pc. Transferred embryos consistently failed to survive the uterine environment of recipients 96 hours pc at transfer although this group was synchronous with embryonic chronological age. Oviductal transfers were generally more successful than uterine transfers. Markedly higher rates of embryo survival resulted from embryos that were collected 60 and 72 hours pc and transferred directly to synchronous recipients without an interim period of culture. Dissimilarity of development for in vivo grown rabbit embryos and those cultured in synthetic medium is demonstrated.
Effects of hydrolase inhibitors on fertilization of sea urchins: I. Protease inhibitors
Authors: Hoshi Motonori , Moriya Tsuneo , Aoyagi Takaaki , Umezawa Hamao , Mohri Hideo , Nagai Yoshitaka
Published: February 2005

To search the spermatozoa of sea urchins for their lysins, the eggs were inseminated in the presence of various protease inhibitors. Among them, two chymotrypsin‐specific inhibitors, chymostatin and N‐tosyl‐L‐phenylalanyl‐chloro‐methane, as well as p‐nitrophenyl p′‐guanidinobenzoate, inhibit fertilization of the sea urchins, Hemicentrotus pulcherrimus and Strongylocentrotus intermedius.

A chymotrypsin‐like protease is presumed to be a lysin of the sea urchins, since the inhibition of fertilization by chymostatin is remarkably diminished if the eggs are pretreated with trypsin or chymotrypsin to break the vitelline coat before insemination, and since N‐tosyl‐L‐phenylalanyl‐chloromethane, and p‐nitrophenyl p′‐guanidinobenzoate, as well as chymostatin, inhibit the fertilization.

In all the sea urchins so far studied, elevation of fertilization envelopes is inhibited by leupeptin, antipain, soybean trypsin inhibitor, and p‐nitrophenyl p′‐guanidinobenzoate, all of which are potent trypsin inhibitors.

Synthetic inhibitors have cytotoxic side effects on the eggs, but the microbial and plant inhibitors have no such effects.
Isolation of bovine zonae pellucidae from ovaries with collagenase: Antigenic and sperm receptor properties
Authors: Gwatkin R. B. L. , Williams D. T. , Meyenhofer M.
Published: February 2005

Zonae pellucidae were collected from bovine ovaries by chopping, dispersing the chopped tissue with collagenase, sieving through nylon mesh screens, and pipetting. The zonae were free of corona cell processes when examined under the scanning electron microscope. Solutions of zonae obtained with collagenase exhibited the same antigenic and sperm receptor properties as those obtained without enzyme treatment.
Effect of the inhibitors of ion movements, verapamil and tetraethylammonium, on fertilization of mouse eggs in vitro
Authors: Shellenbarger David L. , Shapiro Bennett M.
Published: February 2005

Better than 75% fertilization of C57BL/6 mouse eggs with C57BL/6 sperm is obtained in vitro in a modified Kreb's‐Ringer‐bicarbonate medium containing 8 mM HEPES. No fertilization of obtained when Ca2+ is omitted from this medium. The drug verapamil, which interferes with Ca2+ channels and blocks the acrosome reaction [Schackmann et al, 1978] and fertilization in the sea urchin, also blocks fertilization of mouse eggs in vitro when included in complete medium at a concentration of 80 μg/ml. Tetraethylammonium, which inhibits delayed axonal potassium currents and prevents the acrosome reaction in sea urchin sperm, also completely inhibits fertilization of mouse eggs in vitro at a concentration of 5 mM. Tetramethylammonium, which does not inhibit potassium movements at the same concentration reduces fertilization by about 50%. The data are consistent with the hypothesis that ion movements are necessary for activation of the sperm and/or egg in mouse fertilization.
Induction of meiosis of Xenopus laevis oocytes by mianserine
Authors: Hanocq‐Quertier J. , Baltus E.
Published: February 2005

Maturation of Xenopus laevis oocytes can be induced by mianserine, a tricyclic antidepressant. K+‐free medium facilitates this maturation process. Mianserine must be kept in contact with the oocytes during the whole process of maturation for maximal efficiency. It is inactive after injection into the oocytes. Mianserine induces the formation of maturation‐promoting factor (MPF) in the treated oocytes. Mianserine‐induced maturation is strongly inhibited by theophylline, even in K+‐free medium. Progesterone displays synergistic effects with mianserine for the induction of maturation. Likewise, oestradiol shows cooperative maturing effects with progesterone as well as with mianserine. It is suggested that mianserine exerts its primary effects on oocyte maturation by inhibiting a membrane adenylate cyclase.
Lectin‐mediated sperm agglutination of bufo arenarum and leptodactylus chaquensis spermatozoa
Authors: del Pino Enrique J. , Cabada Marcelo O. , Fonio María C.
Published: February 2005

Various plant lecins were employed in cell agglutination experiments to ascertain the presence of specific saccharides in the surface of B arenarum and L chaquensis spermatozoa. B arenarum spermatozoa were specifically agglutinated with Concanavalin A (Con A), phytohemagglutinin P (PHA‐P), and wheat germ agglutinin (WGA), but not with soybean agglutinin (SBA). In contrast, L chaquensis spermatozoa were strongly agglutinated by SBA, WGA, and PHA‐P. L chaquensis spermatozoa did not agglutinate with Con A even at high concentrations. Lectinmediated sperm agglutination was inhibited in the presence of specific lectinbinding sugars. Spermatozoa from both species were agglutinated randomly with all lectins suggesting a uniform distribution in the sperm surface of the lectinbinding saccharide ligands.

B arenarum sperm agglutination induced by Con A is sensitive to temperature. B arenarum spermatozoa are more agglutinable at 24°C than at 4°C. These results suggest that lectin‐binding site mobility is necessary for sperm agglutination.
Differences in the rate of redistribution of receptors for concanavalin A in vivo and in vitro on spermatozoa from normal mice and from sterile mice carrying different T/t locus haplotypes
Authors: Dooher Gerald B.
Published: February 2005

The redistribution of receptors for fluorescein isothiocyanate‐conjugated concanavalin A (FITC‐con A) on mouse spermatozoa during maturation has been studied in vivo and in vitro using normal mice and sterile mice carrying two different lethal T‐locus haplotypes (tx/ty). Receptors for FITC‐con A, uniformly distributed on the head of functionally immature sperm within the proximal epididymis, become localized as the cells acquire functional maturity within the distal portion of the epididymis. Examining samples of epididymal sperm incubated for short periods of time in vitro, a similar increase in the proportion of discretely labeled cells is observed with time. Sperm from sterile tx/ty males also show redistribution of receptors for FITC‐con A in vivo and in vitro; however, a significantly smaller proportion of cells of the population achieve a discrete distribution of receptors comparable to that displayed on sperm from fertile mice. It is concluded that mouse sperm undergo redistribution of receptors for con A normally in acquiring functional maturity within the epididymis and upon liberation from the male urogenital tract; the sterility of tx/ty mice may be due to a genetically based impairment of plasma membrane reorganization reflected in sluggish redistribution of receptors for con A shown by these cells in vivo and in vitro.
Ultrastructural changes in the spermatid nucleolus during spermiogenesis in the guinea pig
Authors: Ohtomo Kazuo
Published: February 2005

Morphological changes in the ring‐shaped nucleoli of spermatids were observed by electron microscopy during spermiogenesis in the guinea pig. In the early acrosomal phase each nucelolus was composed of a bundule of fibers about 25 nm in diameter and 250 nm in length. In cross section the fibers were seen to be closely packed in hexagonal fashion. From the early maturation phase through maturation of the spermatids the fibers of the nuceloli appeared to be replaced by a fine filamentous structure. As the condensation of chromatin proceeded, the filamentous structure decreased in density, and finally the space of the nucleolus was replaced by a nucleolar vacuole.
The effects of aphidicolin and α‐amanitin on DNA synthesis in preimplantation mouse embryos
Authors: Cozad Karen M. , Warner Carol M.
Published: February 2005

The effects of aphidicolin and α‐amanitin on DNA synthesis by preimplantation mouse embryos were studied. It was found that both blastocyst and 8‐cell embryos showed marked inhibition of 3H‐thymidine incorporation into DNA by aphidicolin at concentrations of 20–50 μg/ml. However, aphidicolin did not inhibit the conversion of morula embryos to blastocyst embryos, although aphidicolin‐treated blastocysts lost their blastocoel and collapsed into a compact form after prolonged exposure to the drug. Both 8‐cell and blastocyst embryos were found to be susceptible to inhibition of DNA synthesis by α‐amanitin.
A strategy for an improved separation of mammalian spermatids
Authors: Loir Maurice , Lanneau Maryvonne
Published: February 2005

A collection procedure has been developed to improve the homogeneity of mammalian spermatid populations separated by elutriation. Trypsinizied ram testis cells were elutriated at 18C. Every cell population was eluted by progressive changes in the flow rate and/or rotor speed, instead of by abrupt changes, to reduce the contamination by cells from the next population. Pure populations were collected alternating with mixed populations corresponding to the overlap between two adjacent pure populations. Furthermore, each pure population was collected into two subfractions, the second of which, contamined by cells from the following population, was pooled with the following fraction. In less than 2 hr after castration, three populations of at least 1 × 108 viable round or elongated or elongating spermatids were obtained with respective purities of 95%, 82%, and 99% of the nucleated cells. In addition, two mixed populations containing only two adjacent spermatid types (round plus elongating spermatids: 98%; elongated plus elongating spermatids: 98%) were obtained, as well as a population containing around 60% pachytene spermatocytes.
Sperm motility in the horseshoe crab. IV. Extracellular ions and intracellular pH are not mediators of motility initiation
Authors: Clapper David L. , Epel David
Published: February 2005

Upon dilution into sea water, Limulus spermatozoa undergo a brief flurry of motility (duration < 60 sec), after which they are nonmotile until encountering a sperm motility initiating peptide (SMI) that emanates from eggs. Utilizing highly purified SMI extracts and simplified seawater formulations (from which individual ions have been deleted), we found that no specific extracellular ion is required for either dilution‐initiated or SMI‐initiated motility. Indeed, deletion of one ion (Na+) produced dilution‐initiated motility of very long duration (several hours). When motility is initiated by SMI (in normal seawater) there is an increase in intracellular pH (pHi), as indicated by the fluorescent probe, 9‐amino acridine; however, this pH, change is not a trigger for motility. As a more general method examining ion movements, the fluorescent probe diS‐C3‐(5) was used to qualitatively measure changes in the membrane potential of spermatozoa. Although crude SMI extracts caused membrane depolarization, further purification resulted in an almost complete separation of this activity from SMI, thus showing that SMI activation is apparently an electroneutral event. (The membrane‐depolarizing factor has a molecular weight > 30,000 and does not initiate acrosome reactions.) Experiments utilizing the ionophore A23187 and Ca+2‐blocking agents (verapamil and TMB‐8) provided tentative evidence that mobilization of intracellular Ca+2 may be required for motility initiation. These results show that neither changes in pHi nor the influx of specific extracellular ions are direct mediators of SMI‐initiated motility; however, experiments with pharmacologic agents indicate a possible role for intracellular Ca+2.
Mitochondrial movement during the ascidian sperm reaction
Authors: Lambert Charles C. , Lambert Gretchen
Published: February 2005

The ascidian sperm reaction, Which involves swelling, migration, and loss of the single large mitochondrion, can be triggered in vitro by raising the seawater pH to 9.3 or lowering Na+ to 20 mM, but only if the sperm are allowed to attach to a suitable Substate. Mitochondrial translocation does not usually occur in the absence of sperm attachment. Extracellular Ca2+ is necessary for triggering the reaction with low Na+ but not high pH; however, the intrecellular Ca2+ blocker, TMB‐8, inhibits high pH‐induced mitochondrial movement in the absence of extracellular Ca2+. After swelling, the mitochondrion fluoresces in the presence of chlortetracycline, suggesting that Ca2+ becomes membranebound after activation. Elevated cAMP and theophylline both inhibit mitochondrial move ment but not sperm motility. The antiactin drug cytochalasin B(10μM) and the calmodulinblocking drugs TFP (1 μM) and W‐13 (10 μM) block mitochondrial movement, suggesting roles for actin and calmodulin in mitochondrial movement. A model is proposed relating intracellular alkalinization, Ca2+ influx, actin, myosin, and calmodulin in mitochondrial translocation.
N‐acetyl‐β‐D‐glucosaminidase activity in the cortical granules of Xenopus laevis eggs
Authors: Greve L. Carl , Prody G. A. , Hedrick Jerry L.
Published: February 2005

The glycosidase activities associated with Xenopus laevis eggs were determined using p‐nitrophenyl glycosides as substrates. The egg lysate contained significant amounts of α‐fucosidase, N‐acetyl‐β‐D‐galactosaminidase, and α‐mannosidase activities with smaller amounts of other glycosidase activities, including N‐acetyl‐β‐D‐glucosaminidase. A cortical granule exudate obtained from ionophore‐activated dejellied eggs contained predominantly glucosaminidase activity with only trace amounts of other glycosidase activities. Perivitelline space material obtained from activated or fertilized jellied eggs contained only glucosaminidase activity. Using cytochemical staining procedures, glucosaminidase activity was present in the perivitelline space and the inner aspect of the jelly coat after fertilization or activation of the egg, but not before. The rate of glucosaminidase activity released from activated eggs occurred with the same kinetics as the cortical reaction. The cortical granule and noncortical granule glucosaminidase activities had different electrophoretic mobilities as determined by disc gel electrophoresis. Thus, the Xenopus laevis egg has two N‐acetyl‐β‐D‐glucosaminidase activities, one associated with the cortical granules and the other associated with the noncortical granule compartment of the cell. The cortical granule enzyme released from the egg at fertilization may function in altering the egg's penetrability to supernumerary sperm.
Ovostatin, an endogenous trypsin inhibitor of sea urchin eggs: Purification and characterization of ovostatin from eggs of the sea urchin, Strongylocentrotus intermedius
Authors: Yamada Yuji , Aketa Kenji
Published: February 2005

A trypsin inhibitor, termed ovostatin, has been purified approximately 265‐fold with 82% yield, from unfertilized eggs of the sea urchin Strongylocentrotus intermedius, using trypsin coupled Sepharose 4B as an affinity column for chromatography. The isolated ovostatin is homogeneous in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 20K–21.5K. Ovostatin inhibits preferentially trypsin‐like endogenous protease purified from the eggs of the same species and bovine pancreatic trypsin and also bovine pancreatic chymotrypsin. Values of IC50 (amount causing 50% inhibition of enzymes) for trypsin‐like protease purified from eggs of the same species, bovine pancreatic trypsin, and bovine pancreatic chymotrypsin, are 0.91 ± 0.13 μg/ml (4.55 ± 0.65 × 10−8 M), 3.0 ± 0.28 μg/ml (1.5 ± 0.14 × 10−7 M), and 4.8 ± 0.2 μg/ml (2.4 ± 0.1 × 10−7 M), respectively, in the experimental condition used. Kinetic studies indicate that ovostatin is a noncompetitive inhibitor of trypsin. The inhibitor is relatively heat labile. NaCl (0.025–0.01 M) enhances the inhibitor activity, whereas KCl is inhibitory. Ovostatin requires a low concentration of Ca2+ for activity. The activity is higher in unfertilized eggs than in fertilized eggs; total activity and specific activity in unfertilized eggs is about 1.67‐fold and 1.85‐fold higher than those in fertilized eggs, respectively. We believe that ovostatin may regulate the function of the cortical granule protease and other trypsin‐like proteases that are activated in sea urchin eggs during fertilization.
Ultrastructural studies of the early events of the human sperm acrosome reaction as initiated by human follicular fluid
Authors: Yudin Ashley I. , Gottlieb Wayne , Meizel Stanley
Published: February 2005

It has been suggested that the morphology of the human sperm acrosome reaction (AR) is markedly different from that of other mammalian sperm. The present study examines the fine structural events of the early stages of the human sperm AR as initiated by preovulatory human follicular fluid. Human sperm, capacitated in vitro for 6 hr at 40°C (90% motility) were diluted with equal volumes of follicular fluid before fixing in cacodylate‐buffered glutaraldehyde at 5, 10, 15, 20, and 180 sec. Fixed sperm were treated with either tannic acid or thiocarbohydrazine and OsO4. Over 2,000 sperm were viewed. By 5 sec, 2% of the sperm had initiated the AR. By 15 sec, 8% of the sperm were in some stage of the reaction, and after 180 sec 40% of the sperm had completed the acrosome reaction. The initial stages of the human AR are characterized by a swelling or decondensation of the acrosomal matrix. The fusion between the plasma membrane and outer acrosomal membrane begins at the most anterior tip of the head and progresses toward the equatorial segment. Fusion and fenestration ended at the equatorial segment. With thiocarbohydrazine + OsO4 fixation the fused membranes are distinct hybrid vesicles with the outer acrosomal membrane being far more electron dense. The acrosomal matrix is retained by 20 sec, but by 180 sec the matrix is completely dispersed, even when viewed after tannic acid fixation. Also by 180 sec, vesicles were being progressively lost. It is therefore concluded that the morphology of the human sperm AR, as initiated by human follicular fluid, is not unique, but is similar to that described for other mammalian sperm.