bluuebunny/crossref_metadata_2025 huggingface dataset:
Culture media for mouse oocyte maturation affect subsequent embryonic development
Authors: van de Sandt J. J. M. , Schroeder A. C. , Eppig J. J.
Published: February 2005

These experiments were done to determine whether the culture medium used for the spontaneous maturation of mouse oocytes can affect the subsequent capacity of the ova to become fertilized and complete preimplantation development in vitro and development to live young. Oocytes obtained from antral follicles of gonadotropin‐primed immature mice underwent spontaneous maturation in control medium, i.e. Eagle's Minimum Essential Medium (MEM) supplemented with 5% fetal bovine serum, or in one of eight different media which were also supplemented with serum. All of the ova were fertilized in Whitten's medium and were assessed for cleavage to the 2‐cell stage and for further preimplantation development to blastocysts during culture in Whitten's medium. Three of the eight media used for oocyte maturation improved the capacity of the ova to develop to the blastocyst stage when compared with the control: Waymouth MB 752/1, MEM with non‐essential amino acids, and MEM Alpha; Waymouth medium promoted the highest frequency of development of ova to the blastocyst stage. Moreover, the blastocysts derived from oocytes that matured in Waymouth medium contained more cells than blastocysts derived from oocytes that matured in control medium. Although BGJb medium promoted the cleavage of eggs to the 2‐cell stage when present during oocyte maturation, it had a detrimental effect on their subsequent preimplantation developmental capacity. Following transfer to foster mothers, more 2‐cell stage embryos developed to live young after oocyte maturation in Waymouth medium (21%) than in control medium (13%). It is concluded that the medium used for oocyte maturation in vitro can affect processes involved in the subsequent development of the eggs and that, of the media tested, Waymouth MB 752/1 promoted the highest capacity for embryo development of maturing mouse oocytes.
Evidence for an increased sensitivity to Ca2+ in the flagella of sperm from tw32/+ mice
Authors: Lindemann Charles B. , Goltz Jason S. , Kanous Kathleen S. , Gardner Tressa K. , Olds‐Clarke Patricia
Published: February 2005

The majority of sperm from mice carrying the tw32 haplotype undergo hyperactivation sooner than sperm from +/+ mice of the same strains (Olds‐Clarke, Dev Biol 131:475–482, 1989). To investigate the mechanism underlying this abnormal motility, the Ca2+ sensitivity of their flagellar apparatus was compared to that of age‐ and strain‐matched controls using Triton X‐100‐extracted sperm. Under these conditions, the curvature of the sperm flagellum is controlled by the free calcium concentration. Sperm from mice carrying the tw32 haplotype consistently exhibited a change in flagellar curvature at lower free calcium concentrations than controls. In addition, intact sperm from tw32/+ mice were much more likely than congenic control sperm to have a hook‐like bend in the midpiece, which persisted throughout most of the beat cycle. Sperm exhibiting the hooked middle piece could be converted to a more normal appearance by 2mM procaine, which immobilizes cytoplasmic calcium. Thus an increased sensitivity of the sperm motor apparatus to calcium could be the cause of the precocious hyperactivation of sperm from mice carrying the tw32 haplotype.
Effects of gonadotropins and granulosa cell secretions on the maturation and fertilization of rat oocytes in vitro
Authors: Vanderhyden B. C. , Armstrong D. T.
Published: February 2005

Fully grown germinal vesiclestage oocytes are induced to resume meiosis and acquire the capacity to undergo fertilization in response to a surge of gonadotropins. The present study examined possible direct and indirect roles of gonadotropins in the maturation and fertilization of rat oocytes by determining 1) the effect of exogenous administration of gonadotropins (priming) to immature rats prior to oocyte collection on the capacity of oocytes to undergo maturation and fertilization in vitro, 2) the effect of follicle‐stimulating hormone (FSH) in the maturation media on the resumption of meiosis and subsequent capacity of oocytes to undergo fertilization, and 3) the capacity of oocytes to undergo maturation and fertilization following culture in preovulatory follicular fluid or in conditioned media obtained from gonadotropin‐stimulated granulosa cell (GC) cultures. In the first experiment, oocytes from unprimed rats underwent spontaneous meiotic maturation in vitro and 17% underwent subsequent fertilization. Priming increased the proportion of oocytes undergoing fertilization. Maturation of oocytes in media supplemented with various concentrations of FSH or for various lengths of time (6–16 h) in medium with 500 ng FSH/ml indicated that FSH slowed the rate of meiotic maturation, but had no effect on the capacity of the oocytes to be fertilized. Oocytes obtained from primed animals and cultured in the presence of preovulatory follicular fluid were fertilized in proportions similar to those cultured in serum‐containing medium. In the third experiment, medium conditioned by FSH‐stimulated GC for 40 h slowed the rate of meiotic maturation; the addition of luteinizing hormone (LH) to the FSH‐stimulated cells produced a medium in which the rate of oocyte maturation was not different from that of control oocytes (in medium from unstimulated cells). Medium conditioned by FSH‐ or LH‐stimulated GC, but not fibroblasts, increased the proportions of oocytes undergoing fertilization following maturation in those media. FSH + LH stimulation of GC increased the fertilization of oocytes to proportions significantly higher than with either gonadotropin alone. These data suggest that GC respond to gonadotropin stimulation by providing a factor(s) that regulates the rate of oocyte maturation and promotes the capacity of oocytes to undergo fertilization.
In vitro fertilization of horse follicular oocytes matured in vitro
Authors: Zhang J. J. , Muzs L. Z. , Boyle M. S.
Published: February 2005

In vitro fertilizing ability of stallion spermatozoa was assessed using horse follicular oocytes matured in vitro. After collection, stallion spermatozoa were either: 1) washed and incubated in TALP medium with 3 mg/ml bovine serum albumin (BSA) and 10 μg/ml heparin for 4 h, 2) washed and incubated in TALP with 3 mg/ml BSA for 3 h and cultured for a further 1 h with 1 mM caffeine and 5 mM dbcAMP, 3) washed and incubated in TALP medium with 3 mg/ml BSA at pH 7.9–8.2 for 2–4 h, or 4) diluted and incubated in TALP medium with 10 mg/ml BSA and 7.14 μM calcium ionophore A 23187 for 5–10 min followed by washing. After a given pretreatment, suspensions were diluted into B2 medium to a concentration of 5 × 106 sperm/ml and co‐incubated with oocytes for 12 h or 24–48 h. In the ionophoretreated group, 18 of 54 oocytes (33%) were fertilized by 12 h, and 11 of 45 (24%) cleaved by 24–48 h. Evidence of fertilization was not found in the oocytes incubated with spermatozoa from other treatment procedures.
Changes in calmodulin compartmentalization throughout capacitation and acrosome reaction in guinea pig spermatozoa
Authors: Trejo Raquel , Mújica Adela
Published: February 2005

Calmodulin has been postulated as a mediator in the calcium‐dependent processes that culminate in the acrosome reaction. Changes in calmodulin compartmentalization as a consequence of the increased permeability to extracellular calcium during capacitation and acrosome reaction have been suggested. In the present study the temporal localization of calmodulin in guinea pig spermatozoa was studied during in vitro capacitation and acrosome reaction by indirect immunofluorescence. Capacitation was achieved by incubation in Tyrode medium supplemented with pyruvate, lactate, and glucose in the presence and in the absence of calcium. Acrosome reaction was elicited in three different conditions: 1) by transfer to minimal culture medium containing pyruvate and lactate (MCM‐PL) after in vitro capacitation, 2) by 0.003% Triton‐X 100 treatment, and 3) by A 23187 addition to sperm samples incubated in MCM‐PL. During capacitation, calmodulin was observed both in the acrosome and in the flagellum; this localization seemed to be independent of the presence of extracellular calcium and of exogenous substrates. Throughout the acrosome reaction, different stages of calmodulin compartmentalization were observed. It became clustered around the equatorial region just before or a little after the acrosome reaction had occurred. Later, it was observed around the postacrosomal region in the acrosome‐reacted sperm. The changes in calmodulin distribution were found to be dependent on the stage in the acrosome reaction.
Acrosome reaction of stallion spermatozoa evaluated with monoclonal antibody and zona‐free hamster eggs
Authors: Zhang J. , Boyle M. S. , Smith C. A. , Moore H. D. M.
Published: February 2005

The acrosome of the stallion spermatozoon was visualized by indirect immunofluorescence with monoclonal antibody (18.6) which recognized an integral acrosomal membrane component. Localization was confirmed by electron microscopy using peroxidase labelled antibody. In fresh semen samples (n = 19), 73.9 ± 9.1% of the spermatozoa from five fertile stallions displayed a uniform bright fluorescence over their acrosome region. In two semen samples from an infertile stallion only 28% and 35% of spermatozoa showed the same pattern of fluorescence. Spermatozoa from fertile stallions incubated for up to 12 hours in TALP medium maintained motility and exhibited a significant progressive loss of acrosomes as detected by immunofluorescence. Alternatively, a similar loss of acrosomes could be induced with calcium ionophore A23187 over a 90 minute incubation. Ultrastructural observations and incubation with zona‐free hamster eggs indicated that only with ionophore treatment was immunofluorescent acrosome loss correlated with a physiological acrosome reaction, while prolonged sperm incubation led to degenerative membrane changes. It was concluded that, if carefully validated, immunofluorescent localization of the acrosome of stallion sperm with monoclonal antibody could be used to monitor the acrosome reaction. Furthermore, definitive acrosome visualization would be valuable in assessing semen quality.
Cell‐cycle aspects of growth and maturation of mammalian oocytes
Authors: Motlík Jan , Kubelka Michal
Published: February 2005

In this review, recent data concerning growth and maturation of nonmammalian and mammalian female germ cells are compiled with regard to the increased understanding of somatic cells mitotic cycles, from yeast to human tissues. These data allow us to conclude that growing oocytes of nonvertebrates, lower vertebrates, and mammals resemble somatic cells in the G1 phase of the mitotic cycle in their metabolic and cell cycle behavior. Transcriptional and translational activity of growing oocytes and G1 somatic cells is not compatible with the activation of maturation promoting factor (MPF), with chromatin condensation or with nuclear membrane disintegration. Growing oocytes, even when they are in the dictyate stage of the first meiotic division, promptly inactivate MPF introduced into their cytoplasm by fusion or microinjection, just as do somatic interphase cells.

In mammals, the LH surge induces “de novo” RNA and protein synthesis in granulosa cells. This metabolic change in granulosa cells abolishes their inhibitory activity, and meiosis in fully grown oocytes in preovulatory follicles is then resumed. Resumption of meiosis requires an activation of pre MPF molecules within oocytes. This can be achieved either without (mouse, rat, and rabbit) or with (pig, sheep, and cow) an active protein synthesis by the oocytes. The species specificity is probably dependent on the presence or absence of cyclin‐like and/or mos‐like molecules in fully grown oocytes. Both major events during GVBD, chromatin condensation, and nuclear envelope disintegration require protein phosphorylation. Experimentally, these two phosphorylation activities can be separated one from another. The active MPF molecules are amplified autocatalytically in amphibian and starfish oocytes. However, an increase of MPF activity in mouse and pig oocytes, similarly as in Rana pipiens and sturgeon oocytes, requires an active protein synthesis.
Development of the normal XY male and sex‐reversed XXSxr pseudomale mouse epididymis
Authors: Lebarr Deborah K. , Blecher Stan R. , Moger William H.
Published: February 2005

XXSxr pseudomale mice (chromosomally XX animals “sex‐reversed” by the Sxr factor) develop testes and produce sufficient androgens for masculinization as assessed at the macroscopic level. However, adult XXSxr pseudomales lack the epididymal initial segment (I.S.) In this study prenatal and postnatal epididymal development was examined histologically and biochemically, and it was found that XXSxr pseudomales are indistinguishable from normal XY males up to day 21 of postnatal life. By 25 days postnatally, before the onset of the pubertal androgen surge, the I.S. precursor is evident in normal animals but absent in XXSxr mutants. No major abnormalities were seen in other segments of the XXSxr epididymis. Our data suggest that androgen levels in testis and epididymis are not higher in normal XY males than in XXSxr pseudomale mice of the same age. Inadequate availability of androgens at the target site is unlikely to be the cause of the epididymal abnormality in XXSxr pseudomale mice.
Dynamics of paternal chromatin changes in live one‐cell mouse embryo after natural fertilization
Authors: Adenot Pierre G. , Szöllösi Maria S. , Geze Marc , Renard Jean‐Paul , Debey Pascale
Published: February 2005

Using video‐enhanced fluorescence microscopy, we describe in live mouse zygotes the paternal chromatin changes undergone after fertilization. We focus on the sperm recondensation process and the formation of the paternal pronucleus, in relationship with the progression of maternal chromatin. Chromatin is labeled with the vital fluorophore Hoechst 33342. Our conditions of dye concentration and irradiation allow a continuous following of the dynamics of changes without major perturbation. We combine these observations with ultrastructural analysis performed by electron microscopy of the same eggs fixed at chosen stages. We show that the highly recondensed state corresponds to the appearance of the nuclear envelop and therefore the beginning of the pronuclear stage.
The Binding Characteristics of Cholinergic Sites in Rabbit Spermatozoa
Authors: Young R. J. , Laing J. C.
Published: February 2005

Binding of neurotrophic ligands to rabbit spermatozoa was studied. Nicotinic cholinergic antagonists, [3H]α‐bungarotoxin and [3H]dihydro‐β‐erythroidine (DE), bound with high affinity to different sites in the tails of rabbit spermatozoa with the former binding to 10,207 sites/cell and the latter to 562 sites/cell. α‐Bungarotoxin and DE sites resemble nicotinic sites in brain in binding affinity and specificity. [3H]Quinuclidinyl benzilate (QNB), a muscarinic cholinergic antagonist, also bound with high affinity to a single class of sites located in the heads and tails of rabbit spermatozoa. The binding characteristics of the sperm muscarinic site are similar to muscarinic sites in both innervated and noninnervated cells. Rabbit spermatozoa incubated for 16–18 h in a medium which supported motility for an extended period possessed fewer binding sites than nonincubated spermatozoa for [3H]α‐bungarotoxin and [3H]QNB and the KD for the latter ligand was also lower. Ligands specific for the kappa and delta opiate receptors showed no affinity for rabbit spermatozoa.
Galactosyltransferase activity is restricted to the plasma membranes of equine and bovine sperm
Authors: Fayrer‐Hosken Richard A. , Caudle A. B. , Shur Barry D.
Published: February 2005

β1, 4‐Galactosyltransferase (GalTase) is localized to the plasma membrane of mouse sperm, in which it mediates the binding of sperm to glycoconjugate residues in the egg zona pellucida. In this study, the presence and subcellular distribution of sperm GalTase were determined in two other mammalian species that yield sufficient sperm for subecellular fractionation. Equine and bovine semen were collected, and the plasma membranes (PM), outer acrosomal membranes (OAM), and inner acrosomal membranes (IAM) were sequentially removed. The purities of the isolated membrane preparations were determined by transmission electron microscopy and found to be ≥90%, 96%, and 98% for equine PM, OAM, and IAM, respectively, and ≥80%, 94%, and 97% for bovine PM, OAM, and IAM, respectively. GalTase activity was assayed under optimal conditions in all membrane preparations and was preferentially localized to the isolated PM both in equine and in bovine spermatozoa. The selective localization of GalTase to the sperm PM in two other species suggest that it may serve as a generalized gamete receptor during initial sperm–egg binding in mammals.
Nuclear transfer in the bovine embryo: A comparison of 5‐day, 6‐day, frozen‐thawed, and nuclear transfer donor embryos
Authors: Westhusin M. E. , Pryor J. H. , Bondioli K. R.
Published: February 2005

Micromanipulation and electrofusion were utilized for nuclear transfer in bovine embryos. Embryonic blastomeres from 5‐day (estrus = day 0), 6‐day, frozen‐thawed 5‐day, and first‐generation nuclear transfer embryos (embryos were themselves a product of nuclear transfer with the original donor being a 5‐day embryo) were transferred into bisected bovine oocytes by electrofusion. The percentage of donor cells fusing with the recipient oocytes was compared between different types of donor embryos. The percentage of embryos developing normally into morula or blastocysts following 6 days culture in the sheep oviduct was also recorded and compared between different donor embryo types. No significant differences were found between donor blastomeres for the percent successfully fused to oocytes: 5‐day, 294 of 513 (57.3%); 6‐day, 252 of 405 (62.2%); frozen‐thawed 5‐day, 111 of 144 (77.1%); nuclear transfer, 142 of 223 (63.7%); or the percent developing normally following nuclear transfer: 5‐day, 92 of 444 (20.7%); 6‐day, 84 of 357 (23.5%); frozen‐thawed 5‐day, 32 of 127 (25.2%); nuclear transfer, 31 of 199 (15.6%). These data suggest that a variety of donor embryos can successfully be utilized for bovine embryo cloning. Also, development of blastomeres from frozen‐thawed 5‐day donors and from donors that are themselves the product of nuclear transfer suggest that the production of multiple identical offspring is possible by frozen storage of seed stock and serial recloning.
Two major acrosomal proteins act on different parts of the oocyte vitelline coat in the abalone, Haliotis discus
Authors: Usui Noriko , Haino‐Fukushima Kazu
Published: February 2005

Two proteins of molecular weights 20,000 (20K) and 15,500 (15.5K) are the major soluble substances released from the acrosomal vesicle of the abalone, Haliotis discus, spermatozoon. A crude preparation of them has been shown to possess lytic activity on the oocyte vitelline coat (VC). To elucidate the role(s) of each acrosomal protein (AP) in VC lysis, oocytes were examined after treatment with various AP preparations. The VC, which is about 1 μm thick, is composed of thin outer and inner electron‐dense layers and a thick main layer of a fine filamentous feltwork. When oocytes were treated with a crude preparation containing both APs, the outer layer disappeared and the feltwork of the main layer loosened extensively. A preparation containing predominantly the 20K AP dissolved the outer layer completely and the main layer to some extent, whereas another preparation containing predominantly the 15.5K AP caused loosening of the main layer without alteration of the outer layer, suggesting that the 20K AP acts on the outer layer, whereas the 15.5K AP acts on the main layer. However, when purified, each AP by itself failed to dissolve the VC, although lysis occurred in a 1:1 mixture of these preparations. Moreover, when the oocytes were pretreated with the 20K AP and thoroughly washed, the 15.5K AP alone could induce lysis. These results suggest that the lysis of the outer layer requires both APs but not simulataneously. The 15.5K AP, which is located posteriorly in the acrosomal vesicle, must be released to act on the VC following the action of the 20K AP.
The effects of α‐amanitin and cycloheximide on nuclear progression, protein synthesis, and phosphorylation during bovine oocyte maturation in vitro
Authors: Kastrop P. M. M. , Hulshof S. C. J. , Bevers M. M. , Destrée O. H. J. , Kruip Th. A. M.
Published: February 2005

Bovine cumulus oocyte complexes were cultured for various periods and either denuded and orcein stained or radiolabeled with 35S‐methionine or 32P‐orthophosphate. Specific inhibitors were added to the culture medium to investigate mRNA and protein synthesis requirements for both nuclear and cytoplasmic changes during maturation in vitro. Inhibition of mRNA synthesis by α‐amanitin during the first 2 h of culture prevented the phosphorylation of some specific proteins preceding GVBD and decreased the occurrence of GVBD from 97% to 27%. In addition, in oocytes that had undergone GVBD, only part of the changes in protein synthesis after GVBD were observed. Addition of α‐amanitin after 3 h of culture had no effect on meiotic maturation. When cumulus oocyte complexes were cultured in the presence of cycloheximide, the phosphorylation of specific proteins was also blocked and only 5% of the oocytes underwent GVBD. Addition of cycloheximide after 4, 6, or 8 h of culture resulted in an increasing percentage of GVBD, but the oocytes became arrested in metaphase I. When cycloheximide was added from 12 h of culture onwards, nuclear progression to metaphase II was increasingly restored.

It is concluded that after the onset of culture, both mRNA and protein synthesis are necessary for the phosphorylation of specific proteins and for GVBD. Further‐more, transcription during the first hours of culture is needed for the synthesis of new proteins after GVBD.
Two‐dimensional polyacrylamide gel electrophoresis characterization of apz, a sperm protein involved in zona binding in the pig and evidence for its binding to specific zona glycoproteins
Authors: Peterson R. N. , Campbell P. , Hunt W. P. , Bozzola John J.
Published: February 2005

A boar sperm integral plasma membrane protein (APz) involved in the adhesion of uncapacitated and capacitated sperm to the porcine zona pellucida (ZP) has been characterized by two‐dimensional polyacrylamide gel electrophoresis (PAGE) and tested for its ability to bind to various zona glycopeptides. APz shows microheterogeneity and focuses over a wide pH range, with predominant forms focusing above pH 7. The protein, when excised from nonreducing polyacrylamide gels, inhibited sperm‐egg binding and bound heat‐solubilized zonae preventing these zonae from blocking sperm binding to eggs. In an indirect assay, a polyclonal monovalent antibody, which blocks sperm‐egg binding and which is absorbed by APz, was used to determine the ability of zona glycopeptides to prevent the sperm‐egg blocking activity of the antibody from being absorbed by intact sperm. When whole heat‐solubilized ZP was added to sperm at doses that block sperm‐egg binding and the excess ZP was removed, the sperm‐egg blocking activity of the antibody was not absorbed by these sperm, and antibody‐containing supernatants blocked the binding of untreated sperm to eggs as effectively as antibody that was not mixed with fresh sperm. When alpha ZP3 was used in the same manner, sperm‐egg blocking activity again was not absorbed by antibody‐treated cells. Beta ZP3, however, failed to block sperm‐egg binding and failed to absorb the sperm‐egg blocking activity of the antibody. These findings support the argument that the action of APz is physiologically significant and involves specific binding sites on the ZP3 component of the ZP.
Expression of ras‐like proteins in embryonic and adult cells of Xenopus laevis
Authors: Hanocq‐quertier Jacqueline , Hanocq Françoise
Published: February 2005

A polyclonal antibody raised against v‐Ha‐ras p21 was purified and its specificity was checked on Ha‐ras transformed cell lines. It was used to immunoprecipitate p21 from different Xenopus laevis cell types: brain cells, blood cells, and embryonic material. By one‐dimensional Western blot analysis, we show that ras p21 is synthesized very early in oogenesis and accumulates throughout vitellogenesis. The ras p21 content, estimated to be 1.1 ng in the full‐grown oocyte, remains constant during oocyte maturation and egg cleavage. Increase in the amount of ras p21 occurs at the beginning of neurulation. Two‐dimensional Western blot patterns reveal the presence of multiple molecular forms of p21 in all Xenopus cell types studied. The numerous resolved polypeptides were ascribed to the expression of at least two different ras genes. Furthermore, specific charge modification of the ras polypeptides are observed in brain, blood, and embryonic cells. During oogenesis and early embryonic development, differences in two‐dimensional patterns mainly concern variations in the relative amounts of the different polypeptides. The results are discussed in relation to the well documented synthesis activities of the growing oocyte and of the early developing embryo.
Involvement of superoxide radicals in the mouse two‐cell block
Authors: Noda Yoichi , Matsumoto Hisashi , Umaoka Yoh , Tatsumi Kenichi , Kishi Junji , Mori Takahide
Published: February 2005

The effect of oxygen toxicity on the development of mammalian embryos was asssessed by the use of superoxide dismutase (SOD), a potent scavenger of superoxide radicals. Mouse pronuclear embryos recovered 17 h after human chorionic gonadotropin (hCG) were cultured in medium BWW at 37°C under an atmosphere of 5% CO2 in air. Culture of mouse pronuclear embryos in the presence of Cu ∣ Zn‐SOD (500 μg/ml) significantly increased the blastulation rate (44.6%) when compared with the control culture system (4.2%). Essentially the same effects were observed in SOD containing either Mn or Fe in the catalytic center. Heat treatment of the SOD preparation, and the addition of anti‐SOD antibodies to the culture medium, significantly reduced the attenuation of the two‐cell block by SOD, indicating that this effect is SOD dependent. SOD activity was detected in rabbit oviduct fluid (3,675 ± 3,084 mlU/mg protein) by electron spin resonance. These results suggest that active oxygen is involved in the two‐cell block phenomenon in mouse embryos exposed to air and that SOD in the oviduct may play an important role in the protection of embryos from superoxide radicals.
Preimplantation development of rabbit embryos after transfer of embryonic nuclei into different cytoplasmic environment
Authors: Modlinski Jacek A. , Smora̧ Zdzisław
Published: February 2005

The development of nuclear‐transfer oocytes and zygotes was tested in the rabbit. Metaphase II oocytes and zygotes in the early pronuclear stage were treated with a cytoskeletal inhibitor (cytochalasin D), enucleated, and subsequently fused either with single blastomeres from eight‐ and 16‐cell stages (oocytes and zygotes) or with pronuclei‐containing karyoplasts (zygotes only). Also, nonenucleated zygotes were fused with 1/8 blastomeres. Fusion was performed by means of an electric field. Development of reconstituted embryos was monitored mainly in vitro, but a certain number of embryos developed from oocytes and zygotes receiving nuclei from eight‐cell stages were also transferred into pseudopregnant does. Development of nuclear‐transfer oocytes was distinctly better than that of nuclear‐transfer zygotes, since 16.9% and 9.5% oocytes vs. 8.1% and 3.7% zygotes carrying eight‐ and 16‐cell nuclei, respectively, developed to the blastocyst stage. Two advanced but already dead fetuses were found after transfer of 27 four‐cell embryos obtained after fusion of oocytes with 1/8 blastomeres. No implantations were observed after transfer of 25 four‐cell embryos developed from enucleated zygotes receiving eight‐cell nuclei. These findings indicate that, in the rabbit, some nuclei from 16‐cell embryos are still capable of promoting at least preimplantation development. Comparison between the developmental abilities of oocyte‐ and zygote‐derived nuclear‐transfer embryos also suggests that the cytoplasmic environment of recipient cell is more crucial for the development of reconstituted embryos than the stage of introduced nuclei (at least up to the 16‐cell stage). The majority of pronuclear exchange embryos (69.9%) and 40% of nonenucleated zygotes receiving eight‐cell nuclei were able to develop to the blastocyst stage. This latter observation indicates, similarly as with mouse, a supporting role of residual pronuclei for participation of an eight‐cell nucleus in the development of reconstituted zygotes.
In vitro and in vivo association of an oviduct estrus‐associated protein with bovine zona pellucida
Authors: Wegner Carole C. , Killian Gary J.
Published: February 2005

The interaction between the bovine egg zona pellucida and a 97 kDa estrus‐associated protein produced by the oviduct was examined in vitro and in vivo. In vitro matured bovine eggs were incubated with oviduct fluid recovered throughout the estrous cycle from separate indwelling cannulae placed in the ampulla and isthmus of the same oviduct. Immunofluorescence techniques and a polyclonal antiserum against the 97 kDa protein were used to localize this protein on washed eggs previously incubated with oviduct fluid. Intensity and distribution of immunofluorescence varied with stage of cycle and to a lesser degree with region of oviduct from which the oviduct fluid was obtained. The most intense fluorescence was observed on the zonae pellucidae of eggs incubated with oviduct fluid pooled from days near estrus and ovulation compared to fluid pooled from luteal stage days. The immunofluorescence of isthmus‐derived oviduct fluid was more intense than was ampulla‐derived oviduct fluid collected near estrus. The zonae pellucidae of 7‐day‐old embryos flushed from the uterus displayed immunofluorescence comparable to that observed on the zonae pellucidae of eggs incubated in vitro with peri‐estrus oviduct fluid. No immunofluorescence was observed associated with the perivitelline space, egg cytoplasm, or blastomeres. The apparent uptake of a 97 kDa estrus‐associated protein by the zonae pellucidae of eggs in vitro and embryos in vivo may indicate that this protein functions in fertilization and/or early embryo development.
Proteasome (multicatalytic proteinase) of sea urchin sperm and its possible participation in the acrosome reaction
Authors: Matsumura Kiyotaka , Aketa Kenji
Published: February 2005

The egg jelly‐induced acrosome reaction of the sea urchin, Strongylocentrotus intermedius, was inhibited by succinyl‐Leu‐Leu‐Val‐Tyr‐4‐methylcoumaryl‐7‐amide (Suc‐Leu‐Leu‐Val‐Tyr‐MCA), but not by Suc‐Ala‐Ala‐Pro‐Phe‐MCA. The proteases with hydrolytic activity toward the former were purified from sperm extract by DEAE‐Sephacel and hydroxylapatite chromatographies, Sephacryl S‐300 gel filtration, and heparin‐Sepharose CL‐6B chromatography. Two types of protease were separated, and the molecular weights were estimated to be 65 and 700 kDa, respectively, by gel filtration. The former was accompanied by hydrolytic activity toward Suc‐Ala‐Ala‐Pro‐Phe‐MCA, which was not hydrolyzed by the latter. Polyacrylamide gel electrophoresis of 700 kDa protease gave a single protein band under nondenaturing conditions and at least eight bands in the range of 22–33 kDa in the presence of sodium dodecyl sulfate (SDS). The substrate specificity and the inhibitor sensitivity of 700 kDa protease indicate that it contains two types of the activity, one is chymotrypsin‐type and the other trypsin‐type. The former activity was enhanced by poly‐L‐lysine or SDS. These properties of 700 kDa protease are similar to those of proteasomes (multicatalytic proteinases) isolated from various eukaryotic sources. We had previously shown that inhibitors of chymotrypsin‐like proteases inhibit the increase of intracellular Ca2+ concentration by egg jelly, resulting in the inhibition of the acrosome reaction of St. intermedius (Matsumura and Aketa, Gamete Res 23:255–266, 1989). Bringing these findings together, we suggest that the chymotrypsin‐like activity of sperm proteasome participates in the onset of the acrosome reaction of St. intermedius.
Maturation of guinea pig sperm in the epididymis involves the modification of proacrosin oligosaccharide side chains
Authors: Anakwe Onyeama O. , Sharma Sunita , Hoff Henry B. , Hardy Daniel M. , Gerton George L.
Published: February 2005

Proacrosin from guinea pig cauda epididymal sperm has a lower molecular weight compared with the testicular zymogen. In this study, we have examined the structural basis of this change and where the conversion in proacrosin molecular weight occurs during sperm maturation. Immunoblotting of trifluoromethane‐sulfonic acid‐deglycosylated testicular and cauda epididymal sperm extracts with antibody to guinea pig testicular proacrosin demonstrated that the polypeptide backbones of proacrosins from the testis and cauda epididymal sperm had the same molecular weights (∽44,000). Keratanase, an endo‐β‐galactosidase specific for lactosaminoglycans, partially digested testicular proacrosin but had no effect on proacrosin from cauda epididymal sperm. In extracts of testis, caput epididymis, and corpus epididymis analyzed by immunoblotting, anti‐proacrosin recognized a major antigen with an apparent molecular weight (Mr) of 55,000, although a 50,000‐Mr minor antigen began to appear in the corpus epididymis. By contrast, extracts of cauda epididymis, vas deferens, and cauda epididymal sperm had the 50,000 Mr protein as the only immunoreactive antigen. By enzymography following electrophoresis, the major bands of proteolytic activity in extracts of testis, caput epididymis, and corpus epididymis had 55,000 Mr. A band of protease activity with 55,000 Mr also appeared in extracts of the corpus epididymis. However, the most prominent bands of proteolytic activity in cauda epididymis, vas deferens, and cauda epididymal sperm had 50,000 Mr. In addition, two other major protease activities were detected with 32,000 and 34,000 Mr; the relationships of these proteases to proacrosin are unclear. From these results, we conclude that the oligosaccharides of proacrosin are altered during epididymal transit and that this modification occurs in the corpus epididymis. Such structural alterations may be functionally significant, since sperm acquire the ability to fertilize eggs during epididymal maturation.
Oviduct function in pigs, with particular reference to the pathological condition of polyspermy
Authors: Hunter R. H. F.
Published: February 2005

Because the exceptionally high incidence of polyspermic fertilisation has been emphasised as a major defect in systems of in vitro fertilisation in pigs, the aetiology of the condition has been analysed in a series of experiments in vivo in the search for a common underlying cause and possible means of mitigation. Whereas the defense mechanism against polyspermy in pig oocytes is classically viewed as a zona reaction, more recent evidence suggests a secondary block at the vitelline surface. Both blocks may be compromised in situations leading to polyspermy, although deleterious influences seem to be expressed principally in an inadequate zona block, as judged by the presence of perivitelline spermatozoa.

Postovulatory aging of mammalian oocytes prior to sperm penetration leads to polyspermy, as can be demonstrated in pig eggs. The primary lesion may concern the cortical reaction, owing to a delayed and incomplete exocytosis of the vesicular contents. Eggs ovulated after gonadotrophin treatment during the luteal phase of the cycle show a high incidence of polyspermic penetration (60.6%), as do those shed at estrus in animals treated with progesterone systemically (40%) or by local microinjections in the oviduct wall (32.3%). Whereas progesterone may be modifying interactions of the gametes and responses of the egg organelles in all four above experimental situations, enhanced numbers of spermatozoa ascending a more patent isthmus appear to be the principal cause of polyspermy. Support for this hypothesis comes from isthmus resection with re‐anastomosis of remaining portions of the oviduct (32.4% polyspermy), and surgical insemination directly into the oviducts (33.8% polyspermy). Recent experiments draw attention to a role for oviduct epithelial glycoproteins in modulating the extent of polyspermy in vitro. The role of such oviduct macromolecules in gamete interactions in vivo is considered.
Human α‐fetoprotein purified from amniotic fluid enhances growth factor‐mediated cell proliferation in vitro
Authors: Keel Brooks A. , Eddy Kevan B. , Cho Sechin , May Jeffrey V.
Published: February 2005

Using a primary monolayer culture of porcine granulosa cells (pGC) as an in vitro cell proliferation assay, we have examined the growth‐promoting activity of α‐fetoprotein (AFP) purified from term cord blood and midtrimester amniotic fluid. Increasing concentrations (2.5–20%) of crude human cord blood (CB) increased pGC proliferation, while identical concentrations of crude amniotic fluid (AF) were ineffective. When the cell system was maximally stimulated, AF dose dependently decreased cell proliferation. AFP purified from AF and CB (1.25–5.0 μg/ml) was not mitogenic alone, but, in the presence of epidermal growth factor (EGF) + insulin‐like growth factor (IGF‐I) (10 ng/ml each), AFP dose dependently increased cell proliferation to nearly double that of EGF + IGF‐I alone. The response of pGC to the proliferative effects of AF‐AFP and CB‐AFP were identical at each dose of AFP tested. These results indicate that although crude, pooled midtrimester AF does not display the mitogenic activity seen in cord blood, AFP purified from pooled AF significantly synergizes with growth factors to increase cell proliferation markedly.
Analysis of protamines isolated from alcohol preserved epididymides
Authors: Lee Catherine , Mazrimas Joe , Balhorn Rod
Published: February 2005

Protamines 1 and 2 have been isolated from the sperm of frozen and isopropanol preserved Syrian hamster (Mesocricetus auratus) epididymides and analyzed by gel electrophoresis, high‐performance liquid chromatography (HPLC), and amino acid analysis and sequencing. The results show that alcohol preservation does not alter the primary structure of the two sperm nuclear proteins and that the preservation of mammalian reproductive organs in alcohol is a viable alternative to freezing tissues collected in the field. Sperm were isolated from tissues fixed in isopropanol for as long as 7 months without detectable adverse effects on either the isolation of sperm or the primary structure of the protamines.
Activation and subsequent degradation of proacrosin is mediated by zona pellucida glycoproteins, negatively charged polysaccharides, and DNA
Authors: Eberspaecher Uwe , Gerwien Jens , Habenicht Ursula‐Friederike , Schleuning Wolf‐Dieter , Donner Peter
Published: February 2005

Boar proacrosin (E.C. 3.4.21.10, Mw 53 kD) was isolated by a modified method and subjected to autoactivation. Previously described molecular intermediates of 49 and 43 kD and a stable form (β‐acrosin, 35 kD) were identified by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Autoactivation was expedited in the presence of either zona pellucida glycoproteins, fucoidan, or DNA. The end point of this accelerated conversion was the complete degradation of otherwise stable β‐acrosin via the formation of a characteristic active intermediate protein of 30 kD. All intermediate molecular forms observed during proacrosin activation/conversion exhibited the N‐terminal sequence of the boar acrosin heavy chain, indicating a C‐terminal processing mechanism. Hence zona pellucida glycoproteins stimulate proacrosin activation as well as acrosin degradation. Such a mechanism of proenzyme activation and degradation is to our knowledge described here for the first time and points to a previously unrecognized role of zona pellucida during gamete interaction.
Developmental potential and chromosome constitution of strontium‐induced mouse parthenogenones
Authors: O'Neill G. T. , Rolfe L. R. , Kaufman M. H.
Published: February 2005

The brief exposure of recently ovulated mouse oocytes to M16 embryo culture medium supplemented with strontium chloride (M16 Sr2+) for 2–10 min was observed to induce a high incidence of parthenogenesis. A lower incidence of activation and a significant rate of oocyte degeneration was observed when oocytes were incubated in M16 Sr2+ medium for 20–60 min. The majority of the oocytes exposed to this agent for 2–10 min developed as single‐pronuclear haploid parthenogenones. The incidence of this parthenogenetic class was reduced as the duration of exposure to M16 Sr2+ was increased from 2 to 30 min. Under these conditions a greater proportion of the activated oocytes developed as two‐pronuclear diploid parthenogenones, due to failure of second polar body extrusion. The activation frequency and the proportionate incidence of the pathways of parthenogenetic development observed following the exposure of ovulated oocytes to calcium‐free M16 medium differed significantly from that induced by exposure to M16 Sr2+. Cytogenetic analysis of the single‐pronuclear haploid class of Sr2+‐induced parthenogenones at metaphase of the first‐cleavage mitosis has shown that this agent did not induce a significant increase in the incidence of chromosome segregation errors during the completion of the second meiotic division. Analysis of the developmental potential of the two‐pronuclear class of diploid Sr2+‐induced parthenogenones during the preimplantation stages of embryogenesis revealed that their cell number and rate of cell division were less than those of fertilised embryos retained either in vivo or in vitro. The novel methods of activating oocytes indicated in this study present new opportunities to improve the efficiency of embryo cloning techniques with the ruminant species.
Acrosome reaction induced by immunoaggregation of a proteinase inhibitor bound to the murine sperm head
Authors: Aarons David , Boettger‐Tong Holly , Holt Ginger , Poirier Gary R.
Published: February 2005

ZP3, a glycoprotein of the murine zona pellucida, functions both to bind acrosome intact sperm and to induce the acrosome reaction. Solubilized whole zonae as well as purified ZP3 are able to induce acrosome reactions in capacitated sperm. Pronase digests of whole zonae yield glycopeptides that bind to sperm but are unable to induce acrosome reactions. However, immunoaggregation of these glycopeptides results in the exocytosis of the acrosome in the majority of treated sperm. The data suggest that ZP3 triggers the acrosome reaction by the aggregation of ZP3 binding sites on the sperm head. If aggregation of ZP3 binding sites is important in the induction of the acrosome reaction, then it may be possible to induce the acrosome reaction in the absence of zona by immunoaggregation of the sites. This presentation deals with the immunoaggregation of a proteinase inhibitor of seminal vesicle origin (SVI) that binds to a site on the sperm head known to participate in zona binding. We show that capacitated murine sperm, pretreated with the SVI, will acrosome react, as determined by Coomassie brilliant blue staining, when incubated with rabbit antiinhibitor antiserum (anti‐SVI). The percentage of SVI‐treated sperm displaying an acrosome reaction is dependent on the concentration of the immune serum. Sperm stain positive for intact acrosomes when anti‐SVI Fab fragments or normal rabbit serum is substituted for the immune serum. However, when capacitated sperm, treated with both SVI and anti‐SVI Fab fragments, are incubated with goat antirabbit IgG, the majority of sperm acrosome react. The data suggest that the aggregation of SVI bound to the sperm surface, in the absence of zona glycoproteins, is sufficient to induce the acrosome reaction.
Production of transgenic mice from cryopreserved fertilized ova
Authors: Leibo S. P. , De Mayo Francesco J. , O'Malley Bert
Published: February 2005

Cryopreserved fertilized mouse ova were used to generate transgenic mice via micromanipulation. Five DNA constructions were injected into a total of 1,052 cryopreserved ova, of which 683 (65%) survived the injection and were transferred into recipients. Of 35 recipients, 66% became pregnant and littered a total of 88 pups. As controls, these DNA constructions were also injected into 1,123 fresh ova, of which 744 (66%) survived and were transferred. Of 42 recipients, 79% became pregnant and littered a total of 167 pups. That is, 22% of fresh ova that were transferred developed into live pups, whereas only 13% of cryopreserved ova did so.

Of the pups born, 42 of the 167 (25%) produced from fresh ova were transgenic, and 28 of the 88 (32%) produced from cryopreserved ova were transgenic. In terms of the injected ova that had been transferred, 5.6% of the 744 fresh and 4.1% of the 683 frozen ova developed into transgenic mice. These data indicate that the efficiency of production of transgenic mice from cryopreserved ova is close to that from fresh ova. That observation and the fact that cryopreserved ova allow more efficient utilization of animals suggest that cryopreserved ova can be used instead of fresh ova to produce transgenic mice.
Enhancement of acrosome reaction and subzonal insemination of a single spermatozoon in mouse eggs
Authors: Palermo Gianpiero , Van Steirteghem André
Published: February 2005

The acrosome reaction in mouse spermatozoa was induced by various means. These were 1) varying incubation time in T6 medium, 2) incubation in T6 medium with added A23187, 3) incubation in T6 medium with added dbcGMP and imidazole, 4) exposure to an electric field, and 5) a combination of incubation in a medium with dbcGMP and imidazole and electroporation. The mean percentages of acrosome‐free spermatozoa obtained by these various methods and assessed on the basis of both Bryan's stain and immunolocalization by FITC‐labeled monoclonal antibodies increased by steps from 36% to 67%, 73%, 86%, and 92%.

Individual spermatozoa from the various treatments were afterwards microinjected under the zona pellucida of a mouse oocyte. The fertilization rate for eggs microinjected with a spermatozoon treated with A23187, dbcGMP, and imidazole, by electroporation and by a combination of the last two methods also increased by steps from 17% to 34%, 36%, and 70%, respectively.

Ninety‐five percent of the fertilized oocytes reached the early blastocyst stage, thirty‐eight percent of these blastocysts implanted in pseudopregnant mice, and twenty‐eight percent developed to term. These results indicated the varying degrees of success of different ways of inducing acrosomal loss in spermatozoa and their subsequent success rates in fertilization and further in vitro and in vivo development.