bluuebunny/crossref_metadata_2025 huggingface dataset:
Ultrastructure of the sperm and spermatogenesis in five South African species of the trochid genus Oxystele (Mollusca, Prosobranchia)
Authors: Hodgson A. N. , Heller J. , Bernard R. T. F.
Published: February 2005

Sperm of Oxystele are of the primitive type, which is typical of molluscs using external fertilization. They comprise a head, which is composed of a barrel‐shaped nucleus and large conical acrosome; a midpiece of five or six spherical mitochondria; and a tail, with a 9 + 2 arrangement of microtubules. The morphology of the sperm broadly resembles that of the sperm of other trochids as well as that of the sperm of the Pleurotomariidae, Haliotidae, and Fissurellidae but differs considerably from that of the Patellidae. Each of the five species examined was found to have a different sperm morphology. Our results support the suggestion of Heller and Dempster (in preparation) that Oxystele variegata Anton actually consists of two sympatric species. The morphological changes that occur during spermatogenesis are very similar in all species and resemble descriptions given for other molluscs with primitive sperm.
Antigens on rat spermatozoa with a potential role in fertilization
Authors: Shalgi R. , Matityahu A. , Gaunt S. J. , Jones R.
Published: February 2005

Eight monoclonal antibodies (McAbs), directed against antigens on rat cauda epididymal spermatozoa, were tested for their capacity to interfere with fertilization in vitro as a means of identifying molecules a potential role in sperm‐egg recognition and fusion. Antigens recognized by the McAbs were visualized on live spermatozoa by indirect immunofluorescence (IIF) and characterized by immunoblotting. Five McAbs (designated 1B5, 2C4, 4B5, 5B1, and 8C4) recognized antigens specifically on the sperm acrosome and three (designated 2B1, 2D6, and 6B2) bound to the flagellum. Of the eight McAbs investigated, three (2B1, 2C4, and 6B2) were effective in blocking fertilization in vitro when added as culture supernalants to mixtures of sperm and eggs. McAb 6B2 was inhibitory due to its ability to agglutinate spermatozoa. McAbs 2B1 and 2C4 did not agglutinate capacitated spermatozoa, had no observable effect on motility, and yet blocked fertilization in a dose‐dependent manner. McAb 2C4 did not give a reaction on immunoblots, but the 2B1 antigen was identified as an Mr 40 kD glycoprotein. McAb 2B1 appeared to block fertilization at the level of zona binding, whereas the effects of 2C4 were directed more against zone penetration and/or fusion with the vitellus. When sperm‐egg complexes were stained with 2C4 or 2B1 McAbs and viewed by IIF, all spermatozoa that were attached to the zona showed fluorescence on the head. These results suggest that different antigens on the rat sperm head participate in different aspects of the fertilization process and that during capacitation there is either exposure of these antigens or else they migrate to their site of action from the flagellum.
Changes in nuclear structure during eupyrene spermatogenesis in Murex brandaris
Authors: Amor M. J. , Durfort M.
Published: February 2005

Changes in nuclear structure during eupyrene spermatogenesis of Murex brandaris have been studied using light and electron microscopy. In the first phases, spermatogonia show round nuclei, with several electrodense masses of chromatin and a thin layer of heterochromatin associated with the nuclear membrane. Primary spermatocytes possess larger nucleii, with less condensed chromatin, and the synaptonemal complexes are apparent. During spermiogenesis, chromatin becomes lamellar, and the nucleus twists about its principal axis while it elongates. The nuclear twisting is accompanied by a progressive chromatin condensation, which causes a highly electrodense nucleus at the end of the process.
Oogenesis: Chromatin and microtubule dynamics during meiotic prophase
Authors: Mattson Britta A. , Albertini David F.
Published: February 2005

Changes in the organization of germinal vesicle chromatin in mouse oocytes have been analyzed by fluorescence microscopy with respect to progressive stages of follicular development and the disposition of oocyte cytoplasmic microtubules. Four discrete patterns of chromatin organization exist in germinal vesicle (GV)‐stage oocytes isolated from the ovaries of 21–25‐day‐old gonadotropin‐primed mice. Analysis of ovarian cryosections stained with the DNA‐binding fluorochrome Hoechst 33258 indicates that sequential changes in GV chromatin occur duing folliculogenesis that result in the formation of a continuous perinucleolar chromatin sheath at the time of antrum formation. Specific alterations in the cytoplasmic microtubule complex of GV‐stage oocytes were observed that correlate with chromatin patterns. The extensive cytoplasmic microtubule complex seen in oocytes of preantral follicles initially localizes to perinuclear areas of the ooplasm. This is followed by a progressive reduction in cytoplasmic microtubules and the appearance of prominent microtubule‐organizing centers at the nuclear periphery. Coordinated nuclear and microtubular alterations also occur under in vitro conditions prior to progression of meiosis to prometaphase‐1. The results are discussed with respect to the ongoing differentiation of the oocyte nucleus and the microtubule cytoskeleton during folliculogenesis in preparation for the resumption of meiosis.
Protein synthesis and secretion in the human epididymis and immunoreactivity with sperm antibodies
Authors: Ross Peter , Kan Frederick W. K. , Antaki Pierre , Vigneault Normand , Chapdelaine Alcide , Roberts Kenneth D.
Published: February 2005

The synthesis and secretion of proteins in the different regions of the human epididymis were studied in vitro. Epididymal tissues obtained from patients undergoing castration for prostatic carcinoma or from cadavers were incubated in the presence of [35S]methionine, and the resulting radiolabeled proteins were analysed on SDS‐PAGE. The corpus region was found to be the most active segment in total protein synthesis. Significant qualitative and quantitative changes were observed in the pattern of proteins secreted from the different epididymal regions. To establish those epididymal proteins that interact with maturing sperm, the secreted products were immunoreacted with antibodies raised against a Triton X‐100 extract of ejaculated human sperm heads. The antibodies react mainly with the head region of ejaculated spermatozoa as judged by indirect immunofluorescence. Protein A‐gold labeling of freeze‐fracture images showed gold particle distribution on the sperm plasma membrane. Western blot analysis of the secreted proteins revealed four bands (66, 37, 32, and 29 kDa) in the proximal regions and six additional bands 80, 76, 48, 27, 22, and 17 (kDa) in the distal part of the epididymis. Immunoprecipitation of the secreted proteins with these antibodies revealed six radioactive bands of 170, 80, 76, 60, 48, and 37 kDa, which indicates that certain proteins of epididymal origin bind to the sperm plasma membrane.
Characterization of the antiviral activity constitutively produced by murine conceptuses: Absence of placental mrnas for interferon alpha and beta
Authors: Cross J. C. , Farin C. E. , Sharif S. F. , Roberts R. M.
Published: February 2005

Antiviral activity has been found in conceptus and placental tissues in numerous species, including mice, pigs, sheep, cattle and humans. In sheep and cattle, the antiviral activity is due to an interferon alpha (IFN‐α), but in other species the nature of the protein(s) responsible for placental activity is unknown. The objectives of this study were to determine if the constitutive antiviral activity associated with the mouse conceptus is produced as early as the peri‐implantation period, and to determine if the activity is due to an IFN‐α or ‐β. Conceptus and placental tissue explants released antiviral activity from Day 4 through at least Day 16 of gestation as measured in an agar overlay bioassay employing CHO cells challenged with vesicular stomatitis virus. This activity was neutralized by antiserum against MuIFN‐α/β. The same antiserum failed, however, to immunoprecipitate radiolabeled proteins from medium collected from Day 4 blastocysts cultured in the presence of L‐[35S]‐methionine. S1 nuclease analysis of placental RNA and screening of ectoplacental cone and extraembryonic ectoderm cDNA libraries with MuIFN‐α and ‐β probes failed to detect IFN related mRNAs, even under relatively non‐stringent conditions of hybridization. Thus, while antiviral activity is produced by peri‐implantation conceptuses in several diverse mammalian species, it does not appear to be due to a conserved type of IFN in all these species.
Antispectrin antibodies stain the oocyte nucleus and the site of fertilization channels in the egg of Discoglossus pictus (Anura)
Authors: Campanella Chiara , Carotenuto Rosa , Gabbiani G.
Published: February 2005

In Discoglossus pictus eggs, only the dimple contains ionic channels active at fertilization; in particular, chloride channels are found in the central protion of the dimple, which is also the site of sperm penetration. Moreover the dimple hosts an imposing cytoskeleton, consisting of a cortical network and bundles of microfilaments extending from the microvilli. Since spectrin cross links actin and is connected through ankyrin to anion transporters in the plasma membrane of erythrocytes as well as to anion channels in other cells, we studied, in D. pictus egg, the relationship between the localization of spectrin and the high polarization of ionic channels and cytoskeletal organization. By means of immunocytochemistry, we localized spectrin exclusively in the egg dimple. In an attempt to trace back the source of spectrin localization, we immunostained sections of D. pictus ovary and localized spectrin in the nuclei of previtellogenic oocytes, where actin is also present. Antispectrin staining remained until germinal vesicle breakdown. By contrast, a cortical localization was found only when the oocytes divided into two hemispheres and into the germinative area (GA), which, after germinal vesicle breakdown, gives rise to the dimple. At this stage the antispectrin signal was particularly strong in the GA. Using Rho‐pialloidin, we also established that spectrin is generally present where F‐actin is found. However, spectrin and F‐actin do not have the same pattern of fluorescence. In conclusion, our data suggest that spectrin may play a role in oocyte and egg polarity. In eggs, it could be instrumental in anchoring to the cytoskeleton membrane proteins such as receptors and ionic channels, including chloride‐permeable channels.
Effects of cold shock and phospholipase A2 on intact boar spermatozoa and sperm head plasma membranes
Authors: Robertson L. , Bailey J. L. , Buhr M. M.
Published: February 2005

Head plasma membranes (HPM) isolated from cryopreserved boar spermatozoa show an excessive fluidization (Buhr et al., Gamete Res 23:441–449, 1989), which might be involved in the loss of fertility. The current study assessed the ability of cold shock (5°C) and phospholipase A2 (PA2) to duplicate these effects on membrane structure and to affect 45Ca2+ uptake and gross morphological characteristics of whole, fresh boar sperm. The HPM from cold‐shocked sperm showed a significantly greater rate of fluidization over time than did HPM from control sperm. Addition of PA2 (bee or snake venom, 0.1 or 10.0 ng/ml) to HPM from control sperm caused fluidization similar to cold shocking, but to a lesser degree (P < 0.05). Cold‐shocked intact sperm exhibited severe acrosomal disruption, loss of motility, and increased 45Ca2+ uptake relative to control sperm. Addition of PA2 (bee or snake venom, 0.1, 1.0., 10.0, and 1,000 ng/ml) to control sperm had not effect on gross morphology or motility while maintaining or increasing sperm extrusion of 45Ca2+. Therefore, although PA2 can, to some extent, duplicate the effects of cold shock on HPM molecular organization, its lipid hydrolytic action is insufficient to cause all the gross disruptions of severe thermal shock. Both PA2 and cold shock disrupted HPM structure, but only cold shock increased 45Ca2+ uptake, suggesting that cold shock may be increasing 45Ca2+ uptake in areas other than the head. Cold shock disrupts sperm on three levels; membrane molecular organization, intracellular Ca2+ regulation, and gross morphology/motility.
Antibodies to sperm surface fertilization antigen (FA‐1): Their specificities and site of interaction with sperm in male genital tract
Authors: Naz R. K. , Bhargava K. K.
Published: February 2005

The fertiization antigen (FA‐1) isolated from murine testes demonstrated its dimeric form of 49,000 ± 2,000 molecular weight (M.W.) or a monomer of 23,000 M.W. on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The FA‐1 was immunogenic in all three female rabbits tested and raised a high‐titer antisera [enzyme‐linked immunosorbent assay (ELISA) titers; 1:1,024 to 1:4,096]. The rabbit anti‐FA‐1 antisera predominantly recognized the dimeric form of 49,000 ± 2,000 M.W. on the Western blot of lithium diiodosalicylate (LIS)‐solubilized murine testes. None of the antisera reacted with any somatic tissue, indicating germ‐cell specificity of FA‐1. To determine the cellular localization of the immunoreactive FA‐1, a novel ultrasensitive immunogold‐silver staining (IGSS) procedure was developed. The anti‐FA‐1‐lgG showed intense staining in the luminal region of the seminiferous tubules containing spermatids and spermatozoa. No reaction was observed in the peripheral area of the tubules containing Sertoli cells, spermatogonia, leptotene, and zygotene spermatocytes. The biodistribution studies of 125I‐labeled anti‐FA‐1 lgG in mice revealed that the antibodies do not bind to somatic tissues such as blood cell, liver, heart, kidney, muscle, and gastrointestinal tissue and do not transudate into testes nad seminal vesicle. However, the antibodies preferentially transudate into epididymis (especially corpus or cauda regions) and vas deferens to bind to sperm cells. In conclusion, our data indicate that FA‐1 can induce an immune response that is germ cell‐specific, directed against later stages of spermatogenesis. The antibodies to FA‐1 interact with sperm after penetration through epididymis (especially corpus and cauda regions) and vas deferens rather than through testes and seminal vesicles.
Changes in protein phosphorylation associated with compaction of the mouse preimplantation embryo
Authors: Bloom Theodora , McConnell Josie
Published: February 2005

In order to investigate the role of protein phosphorylation in the early differentiative events of mouse preimplantation development, timed groups of embryos of various stages were incubated in medium containing [32P]orthophosphate and harvested immediately after labelling or following a chase period. The phosphoproteins obtained were separated by electrophoresis in one and two dimensions. While some of the phosphoproteins found were common to all the stages examined, the detection of many depended on both the combination of pulse‐labelling and chase periods used and on the developmental stage examined. Some phosphoproteins were only found in compacted 8‐cell embryos, a correlation which suggests a possible link with the post‐translational mechanisms which underlie compaction.
Analysis of protein synthesis in morphologically classified bovine follicular oocytes before and after maturation in vitro
Authors: Kastrop P. M. M. , Bevers M. M. , Destrée O. H. J. , Kruip Th. A. M.
Published: February 2005

Bovine cumulus oocyte complexes (COCs) were isolated from antral ovarian follicles (4–8 mm). Immature COCs were classified into four categories, based on the homogeneity and clearness of the ooplasm and the transparency and compactness of the cumulus investment. In this study, the incorporation of TCA‐precipitable 35S‐methionine and the protein synthesis patterns of oocytes of these four categories were examined. Before maturation in vitro, similar incorporation rates and identical protein synthesis patterns were observed between oocytes of categories 1–3. Immature oocytes of category 4 showed reduced incorporation rates and exhibited aberrant protein synthesis patterns. After maturation in vitro, the patterns of category 4 oocytes were identical with the patterns of those in categories 1–3. The incorporation of 35S‐methionine into in vitro matured oocytes was lower (P < .001) in all categories.

Based on these results, it is concluded that the initial classification of oocytes into four categories can be reduced to two categories.
Hyaluronic acid synthesis and gap junction endocytosis are necessary for normal expansion of the cumulus mass
Authors: Chen Lin , Wert Susan E. , Hendrix E. Michael , Russell Paul T. , Cannon Michelle , Larsen William J.
Published: February 2005

The application of a quantitative videographic technique has provided an opportunity to compare the quantitative volumetric expansion of cultured oocyte complexes (COCs) to quantitative changes in gap junction down‐regulation and hyaluronic acid synthesis and to investigate the effects of physiological agents that influence these processes. Results of these experiments support the idea that the down‐regulation of cumulus gap junctions is required for the initial phase of cumulus cell disaggregation and confirm earlier reports that hyaluronic acid synthesis plays a major role in additional expansion of the cumulus. These studies also provide evidence that the degree of expansion observed in culture lacking substrates of hyaluronic synthesis is significantly attentuated when compared with expansion occurring in vivo and that the failure of cultured complexes to expand maximally can be overcome by the addition of substrates of hyaluronic acid synthesis to the culture medium.
Subzonal transfer of multiple sperm (MIST) into early human embryos
Authors: Ng S. C. , Sathananthan A. H. , Bongso T. A. , Ratnam S. S. , Tok V. C. N. , Ho J. K. C.
Published: February 2005

Micronsemination sperm transfer (MIST) is a technique whereby sperm are transferred into the perivitelline space (PVS) with the aid of a micromanipulator. MIST is now used to investigate whether blastomere membranes of early human embryos are capable of fusing with the sperm as in the metaphase II oocyte. Between 10 and 30 sperm were transferred into 11 donated human embryos between pronuclear and 16 cell stage. After culture for 6–24 hr in vitro, the embryos were fixed for transmission electron microscopy (TEM). Both acrosome‐intact and acrosome‐reacted sperm were located in the PVS and between blastomeres. Sperm in the PVS were sometimes penetrating the inner regions of the zona. Sperm‐blastomere membrane fusion was not observed, but sperm tail incorporation by phagocytosis was occasionally evident. Sperm heads incorporated into blastomeres were often located in membrane‐bound vesicles. Both acrosome‐intact and acrosome‐reacted sperm heads were found in vacuoles. Acrosome‐reacted sperm heads were lying passively in vacuoles or were undergoing degenerative changes at their surfaces. Sperm chromatin decondensation was not observed in any of the sperm heads that were detected in the blastomeres. The evidence presented clearly shows that sperm heads are incapable of expanding their chromatin to form typical male pronuclei following MIST into early human embryos.
Functions of maternal mRNA in early development
Authors: Dworkin Mark B. , Dworkin‐Rastl Eva
Published: February 2005

In this review, the types of mRNAs found in oocytes and eggs of several animal species, particularly Drosophila, marine invertebrates, frogs, and mice, are described. The roles that proteins derived from these mRNAs play in early development are discussed, and connections between maternally inherited information and embryonic pattern are sought. Comparisons between genetically identified maternally expressed genes in Drosophila and maternal mRNAs biochemically characterized in other species are made when possible. Regulation of the meiotic and early embryonic cell cycles is reviewed, and translational control of maternal mRNA following maturation and/or fertilization is discussed with regard to specific mRNAs.
Maturation‐associated changes in the rat zona pellucida
Authors: Rufas Onit , Shalgi Ruth
Published: February 2005

Rat follicular oocytes, arrested at prophase I, cannot be fertilized in vitro. This capacity is acquired following resumption of meiosis and a series of changes involving both the oocyte and the cumulus cells surrounding it. Oocytes exposed to sperm at different hours before ovulation show a gradual increase in the permeability of their zona pellucida (ZP). Our study examined whether the ZP, in response to the physiological stimulus for maturation and concomitant with the other oocyte—cumulus components, undergoes maturational changes. Two ZP characteristics were assessed, sensitivity to proteolysis and sperm binding. ZP surrounding oocytes and eggs were collected from five sources: 1) germinal vesicle (GV)‐intact oocytes, 2) preovulatory eggs, 3) ovulated eggs isolated from oviducts of immature females, 4) fertilized eggs, 5) ovulated eggs isolated from oviducts of mature females. All ZP surrounding oocytes/eggs from groups 1–5 were dissolved by trypsin. When solubility by pronase and α‐chymotrypsin was examined, a large variation between groups was found. All ZP from group 2 were dissolved by 0.001% pronase, compared to 0% solubility in group 4. Only 10% of the ZP surrounding GV‐intact oocytes (group 1) were dissolved by this enzyme, compared to 82% in group 3. Solubility in 0.01% α‐chymotrypsin showed a similar pattern. Capacitated sperm were incubated with eggs from groups 1 and 3. The number of sperm binding to ZP in group 3 was repeatedly higher than that in group 1. In both tests it was found that the ZP surrounding the mature eggs differ in their characteristics from ZP of GV‐intact oocytes. These variations may reflect a series of events in the ZP initiated by the GV breakdown and completed with the production of a mature fertilizable egg.
Spermiogenesis in Xenopus laevis: From late spermatids to spermatozoa
Authors: Bernardini Giovanni , Podini Paola , Maci Rosalba , Camatini Marina
Published: February 2005

Spermatogenesis is a complex morphogenetic process in which microfilaments and microtubules have been shown to play an important role. The last steps of Xenopus spermatogenesis, i.e., the corkscrew shaping of the sperm head, have been followed to study actin and microtubule distribution by conventional and immunoelectron microscopy. During sperm head morphogenesis, actin is absent in the elongating spermatids, but it is present in the Sertoli cells where results localized at the periphery of their cytoplasm that surrounds the developing germ cells. Sertoli cell actin and microtubules may assist the elongation and the shaping of the spermatids and function in maintaining the Sertoli‐spermatid association.
EGF receptor activities in mammalian development
Authors: Adamson Eileen D.
Published: February 2005

The receptor for epidermal growth factor (EGF) and its analog transforming growth factor alpha (TGFα) is ubiquitous, implying quite general roles for EGF/TGFα in cell viability and tissue maintenance in adult tissues. There is also evidence that the EGF receptor is active in promoting wound healing and tissue regeneration in adult organs, such as skin, liver, and intestinal epithelium. It is likely that EGF receptors have more specific roles during the gestation period. For example, we have detected EGF receptors on the 3.5‐day blastocyst (trophectoderm) surface and since TGFα‐like mRNA sequences and peptides have been detected at this time (Rappolee et al., Science 241:1823, 1988), there is a strong implication for autocrine stimulation in pre‐ and peri‐implantation stage embryos. Paracrine stimulation between the embryo and maternal tissues is also likely since both receptors and TGFα are present in decidual cells. Therefore EGF receptors may take part in growth regulation of the early embryo and in the process of implantation. Other examples where EGF receptors may play specific roles during embryonic development are discussed.
Potential of hypertonic medium treatment for embryo micromanipulation: II. Assessment of nuclear transplantation methodology, isolation, subzona insertion, and electrofusion of blastomeres to intact or functionally enucleated oocytes in rabbits
Authors: Yang X. , Zhang L. , Kovács A. , Tobback C. , Foote R. H.
Published: February 2005

The objective of this research was to study efficiency of embryo development following transfer of blastomeres into the perivitelline space of oocytes. Single blastomeres from 8‐, 16‐, and 32‐cell embryos were obtained following mucin coat and zona pellucida removal by combined treatments with pronase and acidic phosphate‐buffered saline (PBS, pH = 2.5). Blastomeres were separated by pipetting with a fire‐polished micropipette following incubation in Ca++‐free PBS for 15 min at 39°C. This procedure resulted in over 97% blastomere separation. For ease of blastomere insertion, oocytes were placed in droplets of 0.5 M sucrose in PBS (SPBS) during micromanipulation. To functionally enucleate oocytes some were stained with Hoechst 33342 DNA stain and irradiated. A single 8‐ or 16‐cell blastomere was aspirated into an injection pipette (35 μm or 25 μm at the tip, respectively) and inserted into the perivitelline space of an irradiated or non‐irradiated oocyte, but not fused with the oocyte. This micromanipulation procedure did not affect development of individual blastomeres into blastocysts or trophectoderm vesicles when compared with cultured control single blastomeres (P > .05). When the inserted blastomere was induced to fuse with an intact non‐irradiated oocyte under an electric field, 56–57% were fused and 39–45% of the fused and activated oocytes developed to morulae or blastocysts. When an inserted blastomere (from 8–32‐cell embryos) was induced to fuse with a functionally enucleated oocyte treated by Hoechst 33342 staining, followed by washing and UV‐light irradiation, 63–66% of them were fused, but only 15–22% developed to the morula or blastocyst stage. This research demonstrated that the use of hypertonic medium treated oocytes greatly improved the ease and success rate of blastomere subzona insertion, but the value of functionally enucleated oocytes as recipient cells for nuclear transfer requires further investigation.
Effect of microinjection and two types of electrical stimuli on bovine sperm‐hamster egg penetration
Authors: Rickords L. F. , White K. L. , Wiltbank J. N.
Published: February 2005

These experiments were designed to test the effects of an electrofusion and an electroporation pulse on bovine sperm‐hamster egg development. In experiment 1, single motile sperm were injected into the perivitelline space of each egg. A 4,500 V/cm, 30 μsec fusion pulse (FP) was applied while sperm‐egg membrane contact was maintained. It was observed that single motile sperm were rendered immotile immediately after FP application whereas nonpulsed single motile sperm remained motile for up to 36 h postinjection. In addition, both motile and sanicated spermatozoa were injected directly into the ooplasm prior to receiving an FP to determine whether the FP was detrimental to sperm viability. In experiment 2, to induce the acrosome reaction, an 1,150 V/cm electroporation pulse was applied to washed bovine sperm suspended in TALP medium containing 5 mM Ca2+. Treated and nontreated sperm were coincubated with zona‐free hamster ova, and sperm‐penetrating ability was measured. Results from experiment 1 indicate that FP failed to induce sperm‐egg fusion (0/69). FP did not, however, inhibit decondensation or pronuclear formation of sperm injected into hamster egg ooplasm. Single motile sperm injected into the ooplasm resulted in development of both pulsed (19/28) and nonpulsed (21/28) groups. Sonicated tail‐free sperm heads injected into the ooplasm resulted in no detectable difference between treated (18/30) and nontreated (19/30) groups. In experiment 2, treatment of sperm with electroporation pulse +5 mM Ca2+ increased zona‐free hamster ova penetration scores over nontreated sperm within bulls (P < .05). These results indicate that an electroporation pulse in conjunction with high Ca2+ rather than an electrofusion pulse facilitates sperm penetration and early development.
Localization and characterization of the acrosomal antigen recognized by GB24 on human spermatozoa
Authors: Fénichel Patrick , Dohr Gottfried , Grivaux Catherine , Cervoni Florence , Donzeau Michèle , Hsi Bae‐Li
Published: February 2005

GB24, a mouse monoclonal antibody, recognizes a trophoblast–leukocyte cross‐reactive antigen (TLX), which is likely identical to the membrane cofactor protein (MCP), a complement regulatory protein. GB24 reacts also with a human acrosomal sperm antigen (Fénichel et al.: J Reprod Fertil 87:699–706, 1989). By immunofluorescence or immunoperoxidase, testicular, epididymal, and ejaculated spermatozoa were found to be positive after fixation by acetone. Motile, suspended spermatozoa became positive only through conditions known to induce acrosome reaction (A23187, follicular fluid, contact with oocytes). Ultrastructural studies with immunogold staining localized this protein on the inner acrosome membrane and in the acrosomal content. By SDS‐polyacrylamide gel electrophoresis, GB24 immunoprecipitated a unique protein of 48 kDa from capacitated and A23187–induced spermatozoa under reducing conditions. No cross‐reactivity was found with mouse, boar, or ram spermatozoa. Localization of this human sperm antigen recognized by GB24 and its similarity with the TLX‐MCP family antigens would suggest a possible role of this molecule during fertilization in sperm–egg binding or immune protection.
Fluorescence In situ hybridization to Y chromosomes in decondensed human sperm nuclei
Authors: Wyrobek Andrew J. , Alhborn Thomas , Balhorn Rod , Stanker Larry , Pinkel Daniel
Published: February 2005

Human sperm nuclei were isolated with mixed alkyltrimethylammonium bromide and dithiothreitol (MATAB/DTT) and decondensed by treatments with lithium diiodosalicylate (LIS), sodium chloride, or Tris salts. Concentrations as low as 1 mM LIS induced measurable nuclear swelling compared to 600 mM required for the other two salts. As measured by image analyses, the projected nuclear area increased linearly up to approximately fivefold with LIS concentrations up to 10 mM. Swollen nuclei also maintained the elliptical shapes characteristic of the human sperm head. Expanded sperm nuclei of three men were hybridized with a fluorescently labeled 3.4 kb Y chromosome‐specific repetitive DNA probe; 50.1% of the nuclei of each semen sample showed fluorescent labeling over a part of the nucleus indicating presence of the Y chromosome. In comparison, unswollen sperm did not yield reliable hybridization signals. This procedure is suitable for determining the proportion of human sperm with Y chromosomes and can be used to evaluate sperm separation techniques. The availability of probes specific for most human chromosomes suggests that this procedure may find general application in studies of sperm chromosomal constitution.
Co‐culture of rabbit 2‐Cell embryos with rabbit oviduct epithelial cells and other somatic cells
Authors: Carney E. W. , Tobback C. , Ellington J. E. , Foote R. H.
Published: February 2005

Rabbit 2‐cell embryos were co‐cultured in Basal Synthetic Medium II + 10% fetal bovine serum with one of the following: primary cultures of rabbit oviduct epithelial cells (ROEC), a rabbit kidney epithelioid cell line (RK13), a rabbit epidermal epithelioid cell line (Sf1), or a rabbit skin fibroblast‐like cell line (RAB9). Embryos cultured in medium alone served as controls. After 4 d of culture at 39°C in 5% CO2in air, 77–93% of the rabbit embryos which were co‐cultured with somatic cells had reached the blastocyst stage, and 60–76% were hatching through their zonae pellucidae. These percentages, however, were not significantly different (P>.05) from those of embryos in medium alone, of which 90% had reached the blastocyst stage and 83% were hatching. Mean intrazonal embryo diameters also did not differ significantly among treatments (239–302 μm). Bovine 1–8‐cell embryos were also co‐cultured with ROEC. This stimulated 60% of these embryos to develop beyond the so‐called “16‐cell block” in vitro, whereas 0% of the embryos cultured in medium alone developed past this block. Evaluation of the ROEC cultures by light microscopy, immunocytochemistry, and gel electrophoretic analysis of conditioned medium, together with the positive results with bovine embryos, indicate that the ROEC culture partially simulates oviductal conditions in vivo. Therefore, our results suggest that oviduct epithelial cells may play a less pivotal role in regulating early development in the rabbit than in the cow. However, other factors besides those provided by the ROEC cultures must also be important for optimal development, as both rabbit and bovine embryos develop more slowly in co‐culture than in vivo.
Early embryo development in the Siberian hamster (Phodopus sungorus)
Authors: Nieder Gary L. , Caprio Terri L.
Published: February 2005

Development of preimplantation embryos of the Siberian hamster (Phodopus sungorus) in vivo and in vitro was examined. The timing of early development in vivo was found to be slower than that reported for the golden hamster. Progression through the cleavage stages, cavitation, and hatching from the zona pellucida occurred later, with blastocyst formation beginning on the afternoon of day 4 and uterine attachment occurring early on day 5. In vitro, morulae, and early blastocysts collected on day 4 and cultured in serum‐containing medium formed expanded blastocysts and some began to hatch from the zona pellucida. With extended culture, blastocysts attached and formed trophoblast outgrowths. Outgrowth was characterized by an initial migration of small cells from the blastocyst, followed by formation of a sheet of trophoblast giant cells. Differences in the morphology of outgrowth between the hamster and mouse suggest that further comparative studies with the Siberian hamster may be useful.
Nonisotopic in situ hybridization as a method for nondisjunction studies in human spermatozoa
Authors: Coonen Edith , Pieters Math H. E. C. , Dumoulin John C. M. , Meyer Henk , Evers Johannes L. H. , Ramaekers Frans C. S. , Geraedts Joep P. M.
Published: February 2005

Human spermatozoa were studied with a nonradioactive in situ hybridization method. Using a chemically modified DNA probe and immunocytochemical reactions for visualization, it was possible to obtain hybridization signals in 31 of 32 semen samples. Positive hybridization reactions, depending on cell accessibility, varied from 40% to over 90% for the different samples. Using a chromosome 1‐specific DNA probe, disomy for this chromosome was found in 0.67% of all accessible sperm cells.
Expression and activity of gamma‐glutamyl transpeptidase in the rat epididymis
Authors: Hinton Barry T. , Palladino Michael A. , Mattmueller Danette R. , Bard Dawn , Good Kyle
Published: February 2005

Following Northern analysis, GGT mRNA was found predominantly within the caput epididymides and kidney. The size of mRNAs for kidney, caput, corpus, and ducts deferens were 2.2, 2.3, 2.2., and 2.3 kb, respectively, whereas cauda showed a doublet of 2.2 and 2.3 kb. GGT transpeptidation and hydrolytic activity within epididymal luminal fluids collected by micropuncture showed caput=corpus>cauda and corpus>caput>cauda, respectively. Caput luminal GGT transpeptidation activity was significantly inhibited by serine‐borate and was optimal at pH 8.0. The calculated Km and Vmax values for hydrolysis of GSH by caput luminal GGT were 0.06 μM and 2.19 nmoles/min/μl luminal fluid at pH 8.5 compared to 0.49 μM and 0.49 nmoles/min/μl luminal fluid, respectively, at the physiological pH 6.5 of caput fluid. These studies would suggest that the epididymis can control the activity of luminal GGT by pH. Lower Km (0.12 μM) and higher Vmax (1.13 nmoles/min/μl luminal fluid) values were also calculated when GSSG was used compared to GSH. Results from Triton X‐114 partitioning experiments suggest that luminal GGT probably exists in both membrane bound and nonmembrane bound forms. Western blot analysis of proteins within epididymal luminal fluids revealed both subunits of GGT in all epididymal regions studied. However, two lower molecular bands, approximately 22 kDa and 21 kDa, were also observed in cauda fluid. It is suggested that as GGT is transported along the epididymal duct it undergoes degradation, which accounts for its loss of activity in the distal epididymal regions. Epididymal GGT may not be involved in the transport of L‐glutamate since transport was not related to the degree of GGT mRNA expression along the epididymal duct.
Identification of spectrin and calmodulin in rabbit spermiogenesis and spermatozoa
Authors: Camatini Marina , Colombo Anita , Bonfanti Patrizia
Published: February 2005

Actin was localized in the four subacrosomal bulges (Camatini et al., Eur. J. Cell Biol. 45:276–281, 1987), which characterize the sperm head of rabbit spermatozoa (Phillips, J. Ultrastruct. Res. 38:591–604, 1972), and in the postacrosomal region (Welch and O'Rand, Dev. Biol. 109:411–417, 1985; Camatini et al., Eur. J. Cell Biol. 45:276–281, 1987). Specific antibodies and indirect immunogold labelling on testis Lowicryl K4M sections and spermatozoa cryosections were used to study the distribution of calmodulin and a spectrin‐like protein. This protein was also present close to the shaping membranes of the head. The results presented suggest a membrane‐cytoskeletal role of actin, spectrin, and calmodulin.
Response of porcine oocytes to electrical and chemical activation during maturation in vitro
Authors: Hagen D. R. , Prather R. S. , First N. L.
Published: February 2005

These studies were conducted to examine activation of in vitro‐matured porcine oocytes in response to an electrical stimulus or to an ionophore. Cumulus‐enclosed porcine oocytes were incubated in maturation medium supplemented with either FSH and LH (MM:Exp.1) or pregnant mare serum gonadotropin (PMSG; MMP: experiments 2–4) at 39°C in 5% CO2:95% air with high humidity. In experiment 1, groups of oocytes were stripped of cumulus and then shampulsed (control) or electrically pulsed with a Zimmerman Cell Fusion unit at 24, 31, 41, 48, and 65 h of incubation. Control oocytes were exposed to the activation medium for 20 sec, whereas oocytes to be pulsed were subjected to a single activation pulse (120 V, 30 μsec). Oocytes were cultured for an additional 24 h and then fixed and examined. For oocytes pulsed at 24, 31, 41, 48, and 65 h, the proportions which activated were 0, 0, 87, 88, and 83%, respectively. In experiment 2, oocytes were electrically or sham‐pulsed with a BTX 200 Embryomanipulation System at 24, 30, and 40 h of incubation and respective proportions of oocytes activating were 27%, 39%, and 72%. In experiment 3, oocytes were subjected to 0, 1, or 2 activation pulses after 41 h of incubation in MM‐P. Double‐pulsing halved the proportion of activated oocytes (P<.0001). In experiment 4, oocytes were subjected to 0, 25, 50, or 100 μM ionophore at 48 h of incubation. Proportions of oocytes activated by ionophore were greater than for control (P < .05), but activation was not increased by increasing dose of ionophore. These results indicate that porcine oocytes may respond to an activation stimulus by 24 h of incubation, long before maturation is normally completed. Activation can be achieved also with an ionophore, as described previosly for other species.
Transforming growth factor‐alpha augments meiotic maturation of cumulus cell‐enclosed mouse oocytes
Authors: Brucker Cosima , Alexander Nancy J. , Hodgen Gary D. , Sandow Bruce A.
Published: February 2005

Growth factors have been shown to play an important role in the regulation of ovarian function. In this study, we examined the effects of transforming growth factor‐α (TGF‐α) on the meiotic maturation of immature mouse oocytes in vitro. Cumulus cell‐enclosed oocytes were exposed to TGF‐α with or without the meiotic inhibitor hypoxanthine (HX), and oocyte maturation was assessed by germinal vesicle breakdown (GVBD). Likewise, mechanically denucded oocytes were examined for GVBD following exposure to HX and TGF‐α. When cumulus cell‐enclosed oocytes were exposed to TGF‐α (1 μg/ml) in the presence of HX (4 mM), an increrase in GVBD was observed first after 5 hours of culture. Maximal stimulation was reached at 24 hours when 70% of the oocytes underwent maturation in the presence of TGF‐α and HX as compared to 33% with HX only. Concentrations of TGF‐α as low as 0.1 ng/ml produced a similar stimulatory response after 24 hours of culture. Spontaneous maturation in the presence of TGF‐α, but without HX, was also enhanced. The stimulation of GVBD by TGF‐α showed an increase over time both with and without HX. When denuded oocytes were exposed to TGF‐α in the presence of HX, no effect was observed. Our results suggest that TGF‐α is a potent stimulator of mouse oocyte maturation in vitro and that is effect is mediated by the surrounding cumulus cells. In view of the established presence and action of TGF‐α in the intact ovary, these studies indicate that TGF‐α may be part of an intricate system regulating the latter stages of maturation of oocytes during follicular development in vivo.
Spontaneous and induced activation of rat oocytes
Authors: Zernicka‐Goetz Magdalena
Published: February 2005

Ovulated rat oocytes undergo spontaneous activation during in vitro culture. After extrusion of the second polar body, they do not enter interphase but are arrested again in next metaphase‐like stage (M III arrest). The present study demonstrates that puromycin and chloral hydrate can trigger transition to interphase of metaphase II and spontaneously (incompletely) activated rat oocytes. The response of oocytes to these activators depends on their stage at the time of application of a stimulus. Metaphase II oocytes enter interphase at 86.8% when treated with puromycin and in 28.7% after chloral hydrate activation. Oocytes activated with chloral hydrate at the time of spontaneously induced anaphase II enter interphase at 64.8%, but after reaching the stage of telophase II their capability to shift to interphase is again low (28.8%). Finally, M III oocytes cannot be forced to enter interphase by either chloral hydrate or puromycin treatment. This study shows that resumption of the second meiotic division and transition to interphase–‐the two processes that normally occur in succession as a response to oocyte activatin‐–can be experimentally separated.
Synthesis and developmental regulation of an egg specific mouse protein translated from maternal mRNA
Authors: Richoux Veronique , Renard Jean‐Paul , Babinet Charles
Published: February 2005

Proteins synthesized by DDK mice embryos were analyzed by 2D electrophoresis and a new egg‐specific polypeptide, D14, was identified. The protein is characterized by its high rate of synthesis and electrophoretic properties (MW 36,500, pl>8). The synthesis of D14 is strictly developmentally regulated: starting in the maturing oocyte in the few hours following germinal vesicle breakdown (GVBD), it remains high over the first cell cycle and decreases abruptly during the two‐cell stage. The arrest of D14 synthesis is triggered by egg activation and does not directly depend on transcription by the zygotic genome. Nevertheless, drugs that perturb the onset of zygotic transcription concomittently inhibit D14 arrest of synthesis. D14 is present in both cytoplasmic and nuclear compartments at the two‐cell stage; it is very stable and remains detectable at least until the eight‐cell stage in the preimplantation embryo. Embryos of wild strains of mice synthesized either D14 or a D14 related polypeptide at a rate comparable to that of DDK embryos, which was at least ten times greater than that found in other laboratory strains. Both the developmental regulation and the genetic variability in its rate of synthesis make D14 an interesting polypeptide for the study of regulation of maternal information in the very early stages of mouse embryo development.
Experimental manipulation of compaction of the mouse embryo alters patterns of protein phosphorylation
Authors: Bloom Theodora
Published: February 2005

Compaction, occurring at the eight‐cell stage of mouse development, is the process of cell flattening and polarisation by which cellular asymmetry is first established. Changes in the pattern of protein phosphorylation have been correlated with this early event of development (TL Bloom, J McConnell: Mol Reprod Dev 26:199–210, 1990). In the study reported here, groups of embryos were treated in ways known to affect particular features of compaction and were then labeled with [32P]orthophosphate; the phosphoproteins obtained were examined following electrophoresis in one and two dimensions. Four‐cell embryos were treated with protein synthesis inhibitors, which advance cell flattening. This treatment resulted in only minor differences from the phosphoprotein profile of untreated four‐cell embryos. Inhibition of protein synthesis at the eight‐cell stage has little effect on cell flattening or polarisation. However, some phosphoproteins that are observed normally in eight‐cell but not in four‐cell embryos were no longer detectable if labeling took place in the presence of protein synthesis inhibitors. Eight‐cell embryos incubated in phorbol 12‐myristate 13‐acetate, which disrupts various features of compaction, showed a relative increase in the phosphorylation of a group of phosphorprotein spots associated with the eight‐cell but not with the four‐cell stage. Embryos incubated in Ca2+‐free medium, which prevents intercellular flattening and delays polarisation, showed a relative decrease in the phosphorylation of the same group of phosphoprotein spots. The behaviour of these phosphoproteins may therefore be correlated with some of the features of compaction.
Heterologous cell contacts and metabolic coupling in bovine cumulus oocyte complexes
Authors: de Loos F. , Kastrop P. , van Maurik P. , van Beneden Th. H. , Kruip Th. A. M.
Published: February 2005

The integrity of the cumulus cell processes were studied in four categories of bovine cumulus oocyte complexes (COCs) selected on their morphological characteristics. Three different types of cumulus cell process endings (CCPEs) were identified, one penetrating the cortex, another not penetrating the cortex, and a third form was intermediate and more rare in appearance. The process endings that penetrated the cortex frequently made gap junctions with the oolemma. The division of the three types of CCPEs over the four different COC categories was specific for three of the four categories. The first‐category COC predominantly possessed the penetrating CCPE, the fourth‐category COC possessed predominantly the nonpenetrating CCPE, and the second and third categories had both types of CCPEs. The metabolic coupling of the cumulus‐oocyte contacts was assessed by means of incorporation of 3H‐choline into the oocyte. The majority of category 4 COCs transferred low levels of choline into the oocyte while the majority of the oocytes of the other three categories transferred high levels of choline into the oocyte. Category 4 includes a smaller proportion of oocytes capable of cleaving after fertilization than the other three categories. This reduced developmental capacity is probably due to the loss of metabolic coupling before the onset of culture.