bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunohistochemical localization of prostaglandin H synthase in the embryo and uterus of the mouse from ovulation through implantation
Authors: Marshburn P. B. , Shabanowitz R. B. , Clark M. R.
Published: February 2005

Prostaglandins (PGs) in the embryo and endometrium are involved in processes that are important for implantation. Although the presence of PGs (PGE2, PGF2α, PGI2) in decidualized endometrium has been widely reported, less is known about the capacity of the pre‐implantation embryo to synthesize PGs. Prostaglandin H (PGH) synthase is necessary for the production of PGs. Using an immunohistochemical method, PGH synthase was localized in the mouse embryo and uterus from superovulation through embryo implantation. No PGH synthase was detected in oocytes at the time of ovulation or in single‐cell embryos 1 day post‐fertilization (PF). Circular areds of immunostaining became evident in the cytoplasm of blastomeres at the morula stage (day 3 PF). After implantation (day 5 PF), a low level of PGH synthase reactivity was observed in embryonic cells; no PGH synthase was detected in the embryo by day 7 PF. The endometrial glands exhibited maximal immunostaining by day 3 PF, and after implantation, PGH synthase appeared in decidual cells along the border of placentation. Low levels of PGH synthase reactivity were detected in myometrial cells during the period after superovulation through day 7 PF. This is the first demonstration of PGH synthase in the mouse embryo prior to apposition with glandular endometrial epithelium, supporting the hypothesis that the embryo has the potential to produce PGs that may mediate autocrine and/or paracrine responses at the time of nidation.
Cytological effects of the microinjection of antibody to ras p21 in early cleavage Xenopus embryos
Authors: Miron Marie‐José , Lanoix Joël , Paiement Jacques
Published: February 2005

The presence of two ras‐related proteins (22 and 23 kDa) was demonstrated in Xenopus embryonic extracts by selective immunoprecipitation using anti‐ras monoclonal antibodies 142‐24EO5 and Y13‐259. We further describe the cytological effects of the microinijection of anti‐ras monoclonal antibody Y13‐259 into early cleavage blastomeres of Xenopus embryos. Injection of the antibody into a blastomere at the two‐, four‐, or eight‐cell stage caused cleavage arrest in the descendants of the injected blastomere. Light microscopy (LM) of cleavage‐arrested cells revealed extensive deformation of the cells as well as heterogeneity of distribution of yolk platelets and pigment granules. LM analysis of serial sections of cleavage‐arrested cells revealed the presence of multiple nuclei. Although the nuclei expressed similar morphological properties, indicating that they were probably in the same stage of the nuclear cycle, they revealed highly variable chromatin densities. Electron microscope (EM) analysis of the cytoplasm of cleavage‐arrested cells revealed the accumulation of vesicles and large membranous elements coincident with cleavage arrest. Further‐more, endoplasmic reticulum (ER) existed in two forms, as closed, circular profiles and as long, linear arrays. Mitochondria were characteristically aligned in single file on both sides of the two types of ER cisternae. EM analysis of nuclei confirmed variations in chromatin organization and suggested the occurrence of unique nuclear envelope fusion among micronuclei in cleavage‐arrested cells. Cleavage arrest and changes in cytological features were not observed in the cytoplasm of cells microinjected with normal rat lgG. Thus the immunochemical data and microinjection experiments suggest that ras‐like or ras antigenicity exists within rapidly replicating Xenopus blastomeres and may be involved in the organization of a number of its cytoplasmic elements.
Modulation of mammalian sperm motility by quercetin
Authors: Nass‐Arden Lea , Breitbart Haim
Published: February 2005

The flavonoid quercetin inhibits collective motility of ejaculated ram spermatozoa in the first 2 hr of incubation; during the next 3–4 hr motility is stimulated. To explain this interesting effect, we followed the influence of quercetin on sperm glycolysis, extracellular pH, ATP content, mitochondrial respiration, and lipid peroxidation. The collective motility of untreated cells is decreased to about 40% of the original motility during two hours of incubation. During this time, the rate of glycolysis is constant, respiration rate is increasing, there is no change in ATP content, the rate of lipid peroxidation is very slow, and the extracellular pH became very acidic (pH 5.5). It is concluded that motility is decreased due to this acidification. This acidification is prevented to some extent by quercetin, which indirectly inhibits glycolysis. Quercetin inhibits motility due to the inhibition of the plasma membrane calcium pump, as we showed previously (Breitbart et al., J Biol Chem 260:11548–11553, 1985). The motility of untreated cells is arrested after 3.5 hr of incubation, whereas quercetin‐treated cells show high motility, which continues for additional 2–3 hr. After 3.5 hr, the control cells show no glycolytic activity, ATP content and respiration rates are decreased, and rate of lipid peroxidation is highly increased. At this time, quercetin‐treated cells show no glycolytic activity, only a small decrease in ATP content and respiratory rate, and a very low rate of lipid peroxidation. Based on these data it is concluded that sperm motility after 3.5 hr of incubation is dependent mainly on mitochondrial respiration. It is also concluded that lipid peroxidation activity has a major role in loss of sperm motility during time of incubation.
Lectin‐induced abnormalities of mouse blastocyst hatching and outgrowth in vitro
Authors: Dealtry Gillian Barbara , Sellens Martin Howard
Published: February 2005

We have examined the role of cell surface glycoconjugates during mouse blastocyst maturation, hatching, attachement, and outgrowth by monitoring the influence of six lectins on blastocyst development in vitro. Two lectins, concanavalin A and wheat germ agglutinin were toxic to blastocysts at the concentrations used. Bandierea simplicifolia lectin 1 (BSL‐1) induced abnormal growth, developmental arrest at the hatching stage, and some disruption of cell contacts. Culture with Lotus tetragonolobus lectin‐1 (LTA‐1) also disrupted cell contacts and caused developmental arrest. The remaining lectins, Dolichos biflorus agglutinin (DBA) and Ulex europaeus agglutinin (UEA), retarded balstocyst hatching and outgrowth but did not induce any major defects, although differentiation of the inner cell mass was limited by both. This study demonstrates that very low concentrations of lectins can disrupt blastocyst development, suggesting that exposed surface saccharide moieties may be involved in interactions between blastomeres and their enviroment.
Development of cortical vesicles in Sicyonia ingentis ova: Their heterogeneity and role in elaboration of the hatching envelope
Authors: Pillai Muralidharan C. , Clark Wallis H.
Published: February 2005

In the marine shrimp Sicyonia ingentis, ova lack cortical vesicles at spawning. Previous ultrastructural studies suggested that two different populations of cortical vesicles (dense vesicles and the ring vesicles) appear within 30 min post‐spawning. These vesicles undergo sequential exocytosis (exocytosis of the dense vesicles followed by exocytosis of the ring vesicles) that leads to the formation of a hatching envelope around the ovum (see Pillai and Clark: Tissue & Cell 20:941–52, 1988). In the present study, lectins were used as molecular probes to study the development of cortical vesicles subsequent to spawning and the role of these vesicles in formation and elaboration of the hatching envelope. Isolated envelopes were screened with 11 different lectins to determine what group(s) were specific to the envelope glycoconjugates; Concanavalin A (Con A), Griffonia simplicifolia (GS II), Lens culinaris (LCA), and wheat germ agglutinin (WGA) bound to the envelopes. FITC‐lectin studies of sectioned ova (fixed at various time points after spawning) utilizing WGA and LCA showed different labelling patterns. Data obtained at the light microscopical level indicated that WGA was specific to the dense vesicles and the outer portion of the envelope, while LCA exhibited specificity for the ring vesicles and the inner portion of the envelope. At the ultrastructural level, gold‐LCA labelling was seen associated with the cisternal elements (containing ring‐shaped structures), ring vesicles, and the inner layer of the fully formed envelope. These data demonstrated that (1) the ring vesicles are formed by fusion of cisternal elements containing ring‐shaped structures; (2) the two species of cortical vesicles are chemically heterogeneous; and (3) the components of each type of vesicle contribute to different integral parts (the outer and inner layers) of the hatching envelope.
Rat RIβ isoform of type I regulatory subunit of cyclic adenosine monophosphate‐dependent protein kinase: cDNA sequence analysis, mRNA tissue specificity, and rat/mouse difference in expression in testis
Authors: Massa John S. , Fellows Robert E. , Maurer Richard A.
Published: February 2005

A rat complementary DNA (cDNA) for the RIβ isoform of type I cyclic adenosine monophosphate (cAMP)‐dependent protein kinase regulatory subunit was cloned and sequenced and was found to contain the entire protein coding and 3′‐untranslated regions, with a single (ATTAAA) poly‐adenylation site. The largest open reading frame was preceded by a short outof‐phase open reading frame, which is not seen in the corresponding mouse RIβ cDNA due to a single base substitution. The rat RIβ cDNA clone was 2,374 bases long and detected a rat mRNA of approximately 2.8 kilobases. Rat RIβ mRNA was abundant in adult rat brain and testis but was undetectable in other rat tissues. The rat RIβ cDNA also detected RIβ mRNA in mouse brain, but not mouse testis, from 10‐week‐old BALB/c or 10‐ and 6‐week‐old Swiss Webster mice. Thus, despite a 96% nucleotide identity in the coding region of RIβ in rat vs. mouse, there are at least two differences in these closely related species. First, there is a short open reading frame, which precedes the coding region in the rat but not the mouse. Second, unlike the mouse testis, the rat testis contains abundant levels of RIβ mRNA.
Carbohydrates and fertilization in animals
Authors: Miller David J. , Ax Roy L.
Published: February 2005

A frequently used mechanism for sperm–egg recognition in many species involves complementary protein–carbohydrate interaction. The usual paradigm includes complex glycoconjugates in reproductive tract fluids or on the eggs which are recognized by carbohydrate‐binding proteins on the sperm surface. Various glycocojugates are utilized in the steps of sperm capacitation, sperm binding to the egg extracellular matrix and vitelline membrane and induction of the acrosome reaction. Several types of complex glycoconjugates are involved in these processes, including proteoglycans, lactosaminoglycans, sulfated fucose‐containing glycoconjugates, and glycoproteins. There appear to be some structural similarities between active glycoconjugates; they are large in molecular weight and complex, and they are often sulfated, fucosylated, and attached to a protein through serine or threonine residues. In some species, the protein core of the glycoconjugates also participates in the interaction by limiting the binding of carbohydrates to sperm only of the relevant species, likely by providing the proper steric arrangement for the interaction. In other cases the protein core seems to serve more as a crosslinker of the carbohydrate moieties. This review discusses the types of glycoconjugates implicated in fertilization and the complementary lectin‐like proteins found on sperm.
The effect of cytochalasin D on protein synthesis in Xenopus laevis oocytes
Authors: De Sousa Paul A. , Masui Yoshio
Published: February 2005

Defolliculated fully grown oocytes of Xenopus laevis were treated with cytochalasin D (10 μg/ml) and their protein synthesis was studied by labelling with S‐35 methionine. This treatment brought about an alteration in pigment pattern as well as a reduction in amino acid uptake by the oocytes. However, the radioactive amino acid taken by cytochalasin‐treated oocytes was incorporated into protein in the same proportion as in untreated oocytes. These results suggested that subcortical pigment distribution and amino acid uptake in fully grown oocytes were microfilament‐dependent processes, whereas protein synthesis in the oocyte was not.
Ultrastructural localization of silver‐staining nuclear proteins at the onset of transcription in early bovine embryos
Authors: Antalíková Libuše , Fulka J.
Published: February 2005

Argyrophilic nuclear proteins, known to be functionally associated with ribosomal genes, were localized, in four‐, eight‐, and 16‐cell bovine embryo blastomere nuclei using two different silver‐staining procedures. Within the eight‐cell cleavage stage by the process of embryonal nucleologenesis in the cow embryo the full‐capacity ribosome‐producing machinery is established. In the four‐cell embryo, many patches and islands of argyrophilic (Ag+) material were detected in the nucleoplasm. The nucleolus‐precursor bodies (NPBs), composed uniformly of a homogeneous compact mass, were completely devoid of any silver staining. On the other hand, clear‐cut localization of argyrophilic proteins was detected during the eight‐cell stage either inside the transforming NPBs or in the close vicinity, or in the already differentiated nucleolus. In compact, nonvacuolated NPB, an intensive Ag+ area was detected, in the form of a lenticle, at the periphery of the NPB. During and following vacuolation of the NPB, no Ag+ was detected inside these vacuoles. It was seen, however, in the dense fibrillar nucleolar component surrounding the smaller vacuoles formed at the time of the establishment of nucleolar structure. Ag+ areas were seen repeatedly in the vicinity of NPBs, probably a part of the nucleolus‐associated chromatin or, alternatively, representing the extranucleolar bodies. In blastomere nuclei of 16‐cell embryos, already possessing reticulated nucleoli known from intensively synthesizing somatic cells, the silver‐staining pattern corresponded to the usual situation in differentiated cells: slight staining of fibrillar centers, heavy labelling in the dense fibrillar component, and absence of silver deposits in the granular component. These observations confirm and expand existing evidence of the localization of the onset of rDNA transcription to the eight‐cell stage in the early cow embryo.
Mouse zygotes express endogenous lectins
Authors: Legge Michael , Sellens Martin H.
Published: February 2005

Cell interactions during mouse development have been shown to involve carbohydrate‐containing macromolecules (glycoconjugates). We have therefore used a series of fluorescein‐labelled synthetic glycoproteins to determine if mouse oocytes and zygotes also express sugar binding molecules (endogenous lectins) which might participate in such interactions. Unfertilised secondary oocytes did not express endogenous lectins at 4°C but a low level of expression of fucose, mannose, and galactose‐binding activity could be detected at 37°C. In contrast, the zygote clearly expressed three classes of endogenous lectins, with preferential binding for i) fucose or mannose, ii) glucose or galactose, and iii) lactose. The expression of these lectins was much reduced at 4°C and maximal binding at 37°C was achieved only after 2 h incubation. We therefore conclude that a low level of endogenous lectin expression in the mouse oocyte is greatly enhanced after fertilisation and that, at both stages, expression, or the detection of expression, is markedly temperature dependent.
Effect of oviduct cells on the incidence of polyspermy in pig eggs fertilized in vitro
Authors: Nagai T. , Moor R. M.
Published: February 2005

The consequences of interactions between porcine sperm, eggs, and oviduct cells before and during fertilization in vitro (IVF) has been examined with particular reference to the block to polyspermy. The pattern of polypeptides secreted by porcine oviduct epithelial cells has been determined and its effects on sperm both during pre‐fertilization co‐culture and during fertilization have been examined. In standard IVF procedures with no oviduct cell involvement, high rates of penetration (91%) were accompanied by equally high rates of multiple sperm penetration (91% of penetrated eggs). Fertilization on oviduct cell monolayers or a combination of 1 h co‐culture of sperm and oviduct cells before the addition of in vitro matured oocytes did not reduce polyspermy. However, a sperm‐oviduct cell co‐culture period of 2.5 h followed by IVF on oviduct cells selectively reduced the rate of polyspermy by 40% and 50% in two separate series of trials (United Kingdom and Japan, respectively): Overall fertilization rates after this treatment were high (95% or 84%, respectively). A 3.5 h period of pre‐fertilization co‐culture further reduced polyspermy to only 14% of penetrated eggs, but this treatment was accompanied by a sharp drop in the fertilization rate from an overall mean of 88% for all other groups to 19% after 3.5 h co‐culture.
Glycosaminoglycans in porcine follicular fluid promoting viability of oocytes in culture
Authors: Sato E. , Miyamoto H. , Koide S. S.
Published: February 2005

The viability of oocytes cultured in vitro was determined by the trypan blue exclusion test. Isolated porcine oocytes with or without cumulus cells cultured in modified Krebs‐Ringer medium undergo cell death after 48 h. The addition of glycosaminoglycans (GAGs) prepared from porcine follicular fluid (pFF) to the medium delayed or prevented the onset of cell death in vitro. GAGs at concentrations of 0.25 mg/ml or greater prevented cell death in a dose‐dependent manner. To identify the active factor, GAGs were purified from pFF by ethanol precipitation, chromatography on Dowex 1‐x2, and high performance liquid chromatography (HPLC) on TSK gel DEAE‐2 SW column. The fraction with a retention time nearly coincident with that of hyaluronic acid possessed high oocyte viability promoting activity. The present results suggest that the viability of oocytes in vitro is influenced by the presence of specific GAGs separated from follicular fluid.
Prediction of human sperm penetrating ability using computerized motion parameters
Authors: Fetterolf P. M. , Rogers B. J.
Published: February 2005

The sperm penetration assay (SPA) is used to assess male fertilizing potential but it is tedious and costly. Computer analysis could replace the need for the SPA in some cases, if computerized sperm motility parameters are highly predictive of SPA performance. The objective of this study was to determine whether computerized motility parameters from fresh semen samples could be used to predict SPA performance. Computer automated semen analysis (CASA; CellSoft, Cryo Resources) was used to quantitate sperm concentration (CONC), percent motility (MOT), curvilinear velocity (VEL), linearity of swimming trajectory (LIN), mean amplitude of lateral head displacement (ALH), and beat/cross frequency (B/CF). The SPA was performed using either Biggers, Whitten, and Whittingham's medium (BWW) or TEST‐yolk buffer (TYB). Patients were divided into three groups depending on SPA performance: group 1, BWW‐treated, 0% versus > 0% penetration; group 2, TYB‐treated, 0% versus > 0% penetration; and group 3, TYB‐treated, < 20% versus ≥ 20% penetration. SPA performance was highly correlated with CONC, MOT, VEL, and B/CF. CONC, MOT, VEL, and B/CF were significantly higher for patients who penetrated in the SPA than for those who failed to penetrate. Discriminant function analysis (DFA) successfully classified 76% of all patients treated with TYB (group 2) who penetrated and 86% of nonpenetrators based on their computerized motility parameters. For group 2 DFA predicted that 93 men would penetrate in the SPA with TYB. Of these, 90 (96%) successfully penetrated at least one egg. It is concluded that DFA of computerized motility parameters can accurately predict whether a patient will penetrate (> 0%) in the TYB‐treated SPA.
Immunoreactive inhibin in follicular fluid is related to meiotic stage of the oocyte during final maturation of the porcine follicle
Authors: Miller K. F. , Xie S. , Pope W. F.
Published: February 2005

The concentration and content of inhibin was determined in individual porcine follicles from gilts ovariectomized at various times after the onset of estrus. In one experiment, gilts (n = 5) were ovariectomized at 0, 10, or 20 hr after the onset of estrus and the follicular fluids from all large follicles individually aspirated. In a second experiment, gilts (n = 6) were ovariectomized at 21, 24, 27, 30, or 34 hr after the onset of estrus; follicular fluids were aspirated; and each oocyte was stained and evaluated for cytogenetic stage of meiotic maturation. Inhibin was determined in diluted follicular fluids with a radioimmunoassay based on a synthetic peptide replica of part of the α subunit of porcine inhibin. Inhibin values were expressed in terms of thousands of units (kU) of a World Health Organization inhibin standard (86/690). Concentration of inhibin did not vary among hours (overall mean 248 kU/ml). Total follicular content of inhibin also was not different among hours (overall mean 57 kU/follicle). When follicles were classified on the basis of the maturation of the oocyte, significant differences were found. Concentration of inhibin in follicles with a germinal vesicle‐stage oocyte was 138 kU/ml, whereas follicles with more mature oocytes had concentrations of between 204 and 254 kU/ml. Follicular content of inhibin showed a similar pattern with 34.9 kU/follicle at germinal vesicle stage, increasing to 42.5–56.1 kU/follicle at later stages. Quantities of inhibin were also negatively skewed and were positively correlated to follicular content of estradiol and dermatan sulfate.
Identification and characterization of the major components of the Oncorhynchus mykiss Egg Chorion
Authors: Brivio Maurizio F. , Bassi Rosaria , Cotelli Franco
Published: February 2005

The extracellular coat surrounding the fish egg, commonly called the chorion, is a primary envelope that confers biochemical and morphological identity typical of the species. Purified chorions can be easily isolated from either oocytes or ovulated eggs. The aim of this work was to analyze the macromolecular composition of the various chorion components in Oncorhynchus mykiss (Salmonids). SDS‐PAGE analysis of purified chorion showed a reproducible pattern of four major components (129, 62, 54, and 47 kD), representing about 80% of total chorion proteins. The 129 and 47 kD polypeptides were periodic‐acid Schiff (PAS) and concanavalin A positive. After chemical and enzymatic deglycosylation treatments only the 129 and 47 kD components proved to be glycosylated and to belong to the “asparagine‐linked” glycoprotein family. Furthermore, peptide mapping performed on isolated polypeptides showed comigrating fragments on SDS‐PAGE. These results suggest that the four main chorion polypeptides might share common structural features.
Fertilizing competency of multiple ovulated eggs in the domestic fowl (Gallus domesticus)
Authors: Nakanishi A. , Miyake M. , Utsumi K. , Iritani A.
Published: February 2005

Fertilizing competency of multiple ovulated eggs in the domestic fowl was examined by fertilization in vitro and early development in culture. Normal laying hens (White Leghorn) were treated with 75 IU of PMSG for 7 days followed by injection of anterior pituitary extracts from chickens (CAPE). Ovulation began to occur 7.5 h after injection of CAPE. These hens ovulated 1–7 ova but some premature ovulation of GV stage ova were observed. In vitro fertilization of the multiple ovulated ova was examined by inseminating 106–107 sperm onto the germinal disks in m‐Ringer's solution. The gamete or zygote nuclei were detected by DNA specific fluorescence using DAPI (4′,6′‐diamidino‐2‐phenylindole) in the histological section prepared from the germinal disk. Process of fertilization was examined in the eggs incubated for 4 h after insemination in DMEM+ liquid albumen at 41°C under the atmosphere of 5% CO2 in air. Fertilization rate of the total multiple ovulated eggs was 55% (11/20), in which 90% (9/10) and 10% (1/10) in the eggs recovered 7.5–8.5 h and 9.0–9.5 h after CAPE injection were obtained, respectively. Normal pronuclei were formed in five eggs of those recovered 7.5–8.5 h after CAPE injection. Early development after fertilization in vitro was also examined by incubation for 12 h in DMEM + liquid albumen at 41°C under the atmosphere of 5% CO2 in air. Although development in vitro was delayed compared to that in utero condition, normal development was observed in naturally and multiple ovulated eggs. The developmental rate of the multiple ovulated eggs was 67% (20/30) in total, in which 94% (15/16) and 5% (5/14) were obtained in the eggs recovered 7.5–8.5 h and 9.0–9.5 h after CAPE injection, respectively. Both fertilization and developmental rate of the eggs recovered 9.0–9.5 h after CAPE injection were significantly lower (P<0.01) than those recovered 7.5–8.5 h after CAPE injection or naturally ovulated. These results suggested that multiple ovulated fowl eggs recovered 7.5–8.5 h after CAPE injection had normal fertilizing and developmental ability in vitro.
In vitro phosphorylation of sea urchin sperm adenylate cyclase by cyclic adenosine monophosphate‐dependent protein kinase
Authors: Bookbinder Louis H. , Moy Gary W. , Vacquier Victor D.
Published: February 2005

Addition of [γ‐32P]ATP to a 2% Brij‐78 40,000g supernatant of sea urchin sperm results in the cAMP‐dependent phosphorylation of eight to ten proteins. One phosphoprotein of Mr 190 kD is sperm adenylate cyclase (AC). An antiserum to the AC immunoprecipitates the Mr 190 kD protein. Peptide maps of immunoprecipitates show that the AC is the only phosphoprotein present in the Mr 200 kD range. With respect to the in vitro phosphorylation of AC, the endogenous kinase has a Km for ATP of 5.2 μM and is maximally stimulated by 4–8 μM cAMP. The protein kinase inhibitors H8 (9 μM) and PKI (30 U/ml) inhibit the phosphorylation of the AC. The catalytic subunit of bovine cAMP‐dependent protein kinase phosphorylates the AC on the same peptides as the endogenous protein kinase. Cyanogen bromide generated peptide maps of the phosphorylated AC show a minimum of five sites of phosphorylation. No change in the Km or Vmax of the sperm AC resulted from the additional phosphorylation by bovine kinase. Calcium ions at submicromolar concentrations completely block the in vitro phosphorylation of the AC, suggesting the presence in the preparation of a Ca2+‐activated protein phosphatase. To our knowledge, this is the first report of the phosphorylation of an AC by cAMP‐dependent protein kinase.
Presence and localization of the 60 KD calicin in human spermatozoa presenting postacrosomal sheath defects: Preliminary results
Authors: Courtot Anne‐Marie
Published: February 2005

Using an indirect immunofluorescence technique, we studied the anticalicin antibody distribution in four teratozoospermic human sperm samples with a high rate of spermatozoa with incomplete postacrosomal sheath structure (PAS) detected at the ultrastructural level. The calicin distribution obtained in these samples is compared with the distribution obtained in two normal human sperm samples with a high proportion of normal PAS and in 3 human sperm samples without PAS (“round‐headed syndrome”). In contrast to the homogeneous calicin distribution found in the majority of the normal group, similar to that reported by Longo et al. (J Cell Biol 105:1105–1120, 1987) and Paranko et al. (Differentiation 38:21–27, 1988), and the absence of calicin in the three non‐PAS samples, we observed new patterns of calicin distribution in the four sperm samples studied. In the light of these preliminary results, the use of anticalicin antibody as a tool for human postacrosomal region evaluation is discussed.
Localization of microfilaments and a tubulin‐like protein in crustacean (Rhynchocinetes typus) spermatozoon
Authors: Perez C. , Roco M. , Castro A. , Dupre E. , Schatten G. , Barros C.
Published: February 2005

Sperm from the decapod crustacean Rhynchocinetes typus undergo dramatic shape changes as they pass from the vas deferens to seawater and interact with the oocyte envelopes. Using FITC‐phalloidin and antitubulin antibodies, we were able to localize microfilaments and a tubulin‐like protein in R. typus spermatozoon. Microfilaments and the tubulin‐like protein were associated with the sperm rays and spines, but were absent at the spike and at its base. Folded and unfolded spermatozoa display similar fluorescence patterns. SDS‐PAGE of whole spermatozoa and electrotransfer to nitrocellulose confirmed the presence of actin and two proteins at 97 kd and 120 kd that bind to tubulin antibodies (tubulin‐like proteins). These results demonstrate the presence of actin, but not tubulin, and localize microfilaments in these sperm. It is proposed that this cytoskeletal component is active in sperm during crustacean fertilization.
Artificial activation of porcine oocytes matured in vitro
Authors: Prather R. S. , Eichen P. A. , Nicks D. K. , Peters M. S.
Published: February 2005

These studies were undertaken to understand the biological basis of artificially induced activation of meiotic metaphase II oocytes and to develop a source of oocytes as recipients for cloning by nuclear transfer. In vitro matured porcine oocytes were pulsed with various voltages of electricity and evalulated for pronuclear formation. The percentage of eggs that activated was significantly greater for the higher voltages. The effect on activation of the temperature of the ovaries returning from the abattoir was evaluated and it was found that oocytes derived from ovaries returning at 29°C activated at lower rates (45.5%) than those returning at 36°C (78.9%). An experiment was designed to evaluate the pH of electroporation medium (EM) and the duration of exposure to EM on activation. Oocytes were placed in EM at various pHs for 5 minutes, pulsed, and immediately removed to TL‐Hepes or allowed an additional 2 minutes in EM prior to rinsing in TL‐Hepes. The results indicate an optimum activation rate at a pH of 7.0 and allowing the additional 2 minutes in EM. Additional glucosamine (5 mM) had no affect on development of the oocyte to metaphase but reduced the percent pronuclear formation from 61% and 47%. A final experiment evaluated the developmental competence of oocytes subjected to a optimum combination of the above treatments and illustrated that a significant portion of the activated oocytes can show limited signs of cleavage. Thus in vitro matured pig oocytes can be induced to activate at high rates.
Localization of wheat germ agglutinin and antibody binding sites on the plasma membranes of sea urchin sperm heads as revealed by label‐fracture and fracture‐flip
Authors: Ru‐Long Shen , Ward Rita D. , Da Silva Pedro Pinto , Nishioka David
Published: February 2005

Freeze‐fracture electron microscopy reveals that intramembrane particles are concentrated in a band encircling the posterior portion of the acrosome of Strongylocentrotus purpuratus sperm. Two colloidal gold labeling methods, label‐fracture and replicastaining fracture‐flip, were employed to show that the plant lectin wheat germ agglutinin, which recognizes a 210 kDa sperm surface glycoprotein, binds to this localized band of intramembrane particles. Monoclonal antibody J18/2, which also recognizes the 210 kDa surface glycoprotein, shows this localized binding in ≈20% of the sperm observed in this study. The majority of sperm displayed a uniform distribution of receptor sites for monoclonal antibody J18/2. Since wheat germ agglutinin and monoclonal antibody J18/2 are known to agglutinate Strongylocentrotus purpuratus sperm but not sperm of another sea urchin, Lytechinus pictus, similar determinations were made for the latter species. Lytechinus pictus sperm are not labeled with wheat germ agglutinin and are only sparsely labeled with monoclonal antibody J18/2. The acrosomal localizations of wheat germ agglutinin and monoclonal antibody J18/2 receptors in Strongylocentrotus purpuratus sperm are consistent with the involvement of the 210 kDa surface glycoprotein in an egg jelly‐induced sperm acrosome reaction. Low‐temperature post‐embed labeling of thin sections with wheat germ agglutinin and monoclonal antibody J18/2 show concentrations of label within the acrosomal vesicle of Strongylocentrotus purpuratus sperm, suggesting the presence of an intracellular storage site for the 210 kDa glycoprotein.
Intercellular communication in the early human embryo
Authors: Dale B. , Gualtieri R. , Talevi R. , Tosti E. , Santella L. , Elder K.
Published: February 2005

A preliminary study on intercecullar communicative devices in the early human embryo has been made using dye‐coupling techniques and electron microscopy (EM). Lucifer yellow injected into single blastomeres of embryos at the 4‐cell stage up to the late morula stage did not spread to neighbouring cells, indicating that gap junctions and cytoplasmic bridges are not significant pathways for information transfer. Dye spread was first observed in the blastocyst stage, where trophectoderm cells and inner mass cells were shown to be in communication through gap junctions. Studies at the EM level confirmed this finding. Tight junctions and desmosome‐like structures, apparent from the 6‐cell stage onward, were located both peripherally and centrally and were initially nonzonular. The role of intercellular devices in the primary differentiation of the human embryo is discussed.
Reduction of the fertilizing capacity of sea urchin sperm by cannabinoids derived from marihuana. I. Inhibition of the acrosome reaction induced by egg jelly
Authors: Schuel Herbert , Berkery Diana , Schuel Regina , Chang Michael C. , Zimmerman Arthur M. , Zimmerman Selma
Published: February 2005

Δ9‐Tetrahydrocannabinol (THC) and two other major cannabinoids derived from marihuana–cannabidiol (CBD) and cannabinol (CBN)–inhibit fertilization in the sea urchin Strongylocentrotus purpuratus by reducing the fertilizing capacity of sperm (Schuel et al., 1987). Sperm fertility depends on their motility and on their ability to undergo the acrosome reaction upon encountering the egg's jelly coat. Pretreatment of S. purpuratus sperm with THC prevents triggering of the acrosome reaction by solubilized egg jelly in a dose (0.1–100 μM) and time (0–5 min)‐dependent manner. Induction of the acrosome reaction is inhibited in 88.9±2.3% of sperm pretreated with 100 μM THC for 5 min, while motility of THC‐treated sperm is not reduced compared to solvent (vehicle) and seawater‐treated controls. The acrosome reaction is inhibited 50% by pretreatment with 6.6 μM THC for 5 min and with 100 μM THC after 20.8 sec. CBN and CBD at comparable concentrations inhibit the acrosome reaction by egg jelly in a manner similar to THC. THC does not inhibit the acrosome reaction artificially induced by ionomycin, which promotes Ca2+ influx, and nigericin, which promotes K+ efflux. THC partially inhibits (20–30%) the acrosome reaction induced by A23187, which promotes Ca2+ influx, and NH4OH, which raises the internal pH of the sperm. Addition of monensin, which promotes Na+ influx to egg jelly or to A23187, does not overcome the THC inhibition. Inhibition of the egg jelly‐induced acrosome reaction by THC produces a corresponding reduction in the fertilizing capacity of the sperm. The adverse effects of THC on the acrosome reaction and sperm fertility are reversible. These findings show that cannabinoids reduce the fertilizing capacity of sea urchin sperm by inhibiting the induction of the acrosome reaction by egg jelly. THC may affect events in the stimulus–secretion coupling mechanism before the opening of ion channels.
Reduction of the fertilizing capacity of sea urchin sperm by cannabinoids derived from marihuana. II. Ultrastructural changes associated with inhibition of the acrosome reaction
Authors: Chang Michael C. , Schuel Herbert
Published: February 2005

Pretreatment of Strongylocentrotus purpuratus sperm with Δ9‐tetrahydrocannabinol (THC) prevents the triggering of the acrosome reaction by egg jelly. Examination of THC‐treated sperm by transmission electron microscopy reveals that the membrane fusion reaction between the sperm plasma membrane and the acrosomal membrane is completely blocked. Electrondense deposits are present in the subacrosomal fossa and in the centriolar fossa. The nuclear envelope is fragmented in close proximity to the electron‐dense deposits. The electron‐dense deposits are not bound by a limiting membrane, stain positively for lipid with thymol and farnesol, and disappear from THC‐treated sperm that are extracted with chloroform:methanol (2:1) after glutaraldehyde fixation. The electron‐dense deposits are lipid in nature and may be a hydrolytic product of the nuclear envelope. Electron‐dense deposits are seen in sperm after 1–10 min treatment with 5–100 μM THC. The electrondense deposits disappear after removal of THC from the sperm by washing, but the fragmented nuclear envelope in the subacrosomal fossa persists. Cannabidiol (CBD) and cannabinol (CBN) also inhibit the triggering of the acrosome reaction by egg jelly and produce ultrastructural changes in the sperm identical to those elicited by THC. Enhanced phospholipase activity stimulated by THC, CBD, and CBN may be the cause of the accumulation of lipid deposits in the sperm. Metabolites derived from this modification of membrane phospholipids may prevent triggering of the acrosome reaction by egg jelly and thereby inhibit fertilization.
Hamster oocyte penetrability during preovulatory maturation
Authors: Cuasnicu Patricia S. , Bedford J. M.
Published: February 2005

In vitro fertilization techniques were used to analyze the penetrability of preovulatory hamster oocytes. The zonas of granulosa cell‐free primary (GV) oocytes were penetrated in vitro in 2–3 h as readily as those of ovulated secondary oocytes (80% vs. 88%), whether inseminated separately or as mixed oocyte groups. In fact, a significantly higher (P<0.05) mean number of perivitelline spermatozoa was present in immature (3.6) compared with secondary (1.9) oocytes, primarily reflecting a lack of the zona block to polyspermy in the immature population. By contrast, when granulosa cells remained around GV oocytes, zona penetration was low and more were penetrated, with more spermatozoa incorporated into the vitellus as a function of increasing time of oocyte recovery after hCG. We conclude, contrary to previous reports, that the zona pellucida of the hamster GV oocyte is readily penetrable by spermatozoa in vitro. However, the resumption of meiosis brings an increase in the penetrability of the granulosa cell vestment as well as the capacity for cortical granule exocytosis and the ability to decondense and transform the fertilizing sperm nucleus. The fact that the zona pellucida of the immature oocyte has proved to be penetrable in vitro and/or in vivo in all the mammals studied in this respect is discussed with particular reference to the situation in man.
Ribosomal gene amplification in oocytes of the lizard Podarcis sicula
Authors: Motta Chiara M. , Andreuccetti Piero , Filosa Silvana
Published: February 2005

In order to provide cytological evidence of amplification, Podarcis sicula oocytes were studied by cytophotometry, thymidine incorporation and in situ DNA‐DNA hybridization.

Our results show that DNA replication is completed during the preleptotene stage, the leptotene oocytes having the typical 4C nuclear DNA content. Between the zygotene and the mid‐pachytene stages further DNA synthesis occurs with consequent increase of the ribosomal nuclear DNA content.

These results and the variations in nucleolar organization observed during differentiation give clear evidence of the existence of ribosomal gene amplification in Podarcis sicula oocytes.
Embryonic transcription in in vitro cultured bovine embryos
Authors: Barnes F. L. , First N. L.
Published: February 2005

The rate of cleavage and the onset of embryonic transcription of bovine embryos cultured in vitro (IVC) has been investigated. Embryos were derived from in vitro matured, in vitro fertilized oocytes (IVM/IVF) to improve developmental synchrony. The rate of cleavage was assessed by morphological evaluation between the one‐ and eight‐ to 16‐cell stage. The rate of cleavage was found to be equivalent to that reported for in vivo recovered embryos. To assess the onset of embryonic transcription, embryos were cultured to the eight‐ to 16‐cell stage in the presence of α‐amanitin for various periods of time followed by two‐dimensional polyacrylamide gel electrophoresis. Embryos readily cleaved to the eight‐ to 16‐cell stage in the presence of inhibitor α‐Amanitin‐sensitive protein synthesis was first detected at 36–48 h post‐insemination (hpi) and continued up to 84 hpi. We conclude that bovine embryos produced by IVM/IVF/IVC are competent to initiate embryonic transcription at 36–48 h post‐insemination and suggest that in vitro‐induced cleavage arrest is not due to failure of the embryonic genome to initiate transcription.
Effects of pH on the reactivation of human spermatozoa demembranated with triton X‐100
Authors: Giroux‐Widemann V. , Jouannet P. , Pignot‐Paintrand I. , Feneux D.
Published: February 2005

The aim of this work was to study the role of different parameters involved in the motility of human spermatozoa. Human spermatozoa were totally demembranated with 0.05% Triton X‐100, and the demembranation was checked using electron microscopy. We have shown that, with a concentration of ATP‐Mg lower than 2 mM, a pH effect was observed with a dose‐dependent motility reactivation at pH 7.1, with 14% ± 2.0% motile cells at 1 mM ATP‐Mg and a straight line velocity (VSL) of 12.0 ± 1.4 μm/sec. However, at pH 7.8, more than 65% of the spermatozoa were reactivated with as low as 0.02 mM ATP‐Mg and 77.8% ± 2.5% of them were motile at 1 mM ATP‐Mg and had a VSL of 23.4 ± 3.9 μm/sec. The depletion of free calcium by the addition of 0.5 mM EGTA in the reactivation medium (RM) improved the percentage of motile cells and the VSL most markedly at low ATP‐Mg and low pH. If no MgSO4 was added in RM, cells were not motile at pH 7.8, but 30–40% reactivated at pH 7.1. If 5 mM Ca2+ was added to the RM, up to 88% of the cells became reactivated at both pHs, but the beat frequencies were very low, suggesting different mechanisms of reactivation when Mg2+ or when Ca2+ is present in the RM.
Protein synthesis and phosphorylation patterns of bovine oocytes maturing in vivo
Authors: Kastrop P. M. M. , Bevers M. M. , Destrée O. H. J. , Kruip T H. A. M.
Published: February 2005

To investigate protein synthesis and phosphorylation during bovine oocyte maturation in vivo, oocytes were collected at consecutive times after the preovulatory luteinizing hormone (LH) peak. Therefore, heifers treated for superovulation were ovariectomized between 3 and 20 h after the maximum of the LH peak. Subsequently, cumulus‐enclosed oocytes, selected from nonatretic follicles >10 mm, were radiolabeled with 35S‐methionine or 32P‐orthophosphate for 3 h and individually prepared for gel electrophoresis. Changes in the protein synthesis patterns were observed coinciding with germinal vesicle breakdown (GVBD). No changes were detected during the ensuing maturation period or coinciding with the extrusion of the first polar body. In addition, the protein phosphorylation patterns exhibited striking differences around GVBD. In particular, a phosphoprotein band of 19 kDa and the two heavily phosphorylated proteins with apparent molecular weights between 50 and 60 kDa were present in patterns of oocytes in the germinal vesicle stage. The results are discussed in relation to previous data obtained during maturation in vitro.
A monoclonal antibody, MN13, that recognizes specifically a novel substance between the postacrosomal sheath and the overlying plasma membrane in the mammalian sperm head
Authors: Toshimori K. , Tanii I. , Ōura C. , Eddy E. M.
Published: February 2005

Monoclonal antibody MN13 raised against mouse spermatozoa specifically recognizes the postacrosomal region of the sperm head in several mammalian species. Colloidal gold‐immunoelectron microscopy of demembranated mouse spermatozoa indicated that the antigen is associated with the outer layer of the periodic substructure apparently linking the postacrosomal sheath to the overlying plasma membrane. The antigen recognized by MN13 may cotribute to the intimate association of the postacrosomal sheath with the overlying plasma membrane.
Structure and expression of the nerve growth factor gene in Xenopus oocytes and embryos
Authors: Carriero Filomena , Campioni Nadia , Cardinali Beatrice , Pierandrei‐Amaldi Paola
Published: February 2005

A large part of the coding portion of the Xenopus nerve growth factor (NGF) gene has been identified and cloned by the use of a chicken cDNA probe and its sequence has been determined. Comparison of the derived amino acid sequence of mature Xenopus NGF with that of other species showed a high conservation, whereas comparison of the prepropeptide showed large divergent regions alternated with short conserved regions. Expression of the NGF gene was examined during development of oocytes and embryos. Surprisingly, NGF mRNA was found in the oocyte; it is present in small previtellogenic as well as in fully grown oocytes. NGF mRNA, passed to the embryo at fertilization, is degraded before the gastrula stage and starts accumulating again around the stage of the neurula. The association of NGF mRNA with polysomes is indicative of NGF synthesis during oogenesis. In fact, by using antibodies against mouse NGF it was possible to reveal NGF molecules present as precursors. These molecules accumulate during oogenesis and are maintained in the embryos up to the blastula stage; a very faint band corresponding to a smaller size peptide is sometimes detected. A maternal role for the NGF can be proposed, although a possible activity of NGF in the oocyte cannot be ruled out.
Rabbit blastocysts accumulate [3H]quinuclidinyl benzilate in vitro
Authors: Jones M. A. , Harper M. J. K.
Published: February 2005

Day‐6 rabbit blastocysts were able to accumulate [3H]quinuclidinyl benzilate (QNB) from their environment. This accumulation was reduced approximately 50% in the presence of 1.5 × 10−4 M atropine (an accepted antagonist for ligands which bind to muscarinic cholinergic receptors). The accumulation of QNB was sensitive to temperature and was apparently saturable. In the presence of 2 nM QNB, Day‐6 blastocysts accumulated 30.3 ± 2.0 fmoles per blastocyst. When the cellular elements alone were examined, lesser amounts of specific binding were detected. Owing to the complexity of this multicompartmental system, Scatchard analysis did not provide meaningful results. This accumulation appears higher than that reported for other tissues such as rabbit heart homogenates or rabbit uterine endometrial cells. This muscarinic cholinergic accumulation may have some role in blastocyst‐maternal recognition.