bluuebunny/crossref_metadata_2025 huggingface dataset:
Aspartate and glutamate transport in unfertilized pig oocytes and blastocysts
Authors: Prather R. S. , Peters M. S. , Van Winkle J.
Published: February 2005

Amino acid transport is facilitated by specific transporters within the plasma membrane of the cell. Mediated Na+‐independent transport of L‐glutamate can be easily detected in mouse oocytes, but it is nearly undetectable in blastocyst‐stage embryos. In contrast, the Na+‐dependent transport of L‐aspartate is not detectable in oocytes, but it is detectable in eight‐cell embryos and reaches relatively high levels by the blastocyst stage. It is believed that the amino acid transporters responsible are systems x−c and X−AG, respectively. Here we report the detection of Na+‐dependent L‐aspartate transport, which increased as pig blastocysts developed, although Na+‐dependent aspartate transport was not detected in pig oocytes. Mediated Na+‐independent L‐glutamate transport was not detected in pig oocytes, in contrast to the mouse, nor in early or hatched pig blastocysts. Thus, while the developmental regulation of system X−AG is similar in both the pig and the mouse, system x−c was not detectable in pig oocytes or blastocysts. Elucidation of the molecular mechanisms controlling amino acid transport and other gene expression in early embryos should contribute to an understanding of whether and even why some aspects of developmental regulation of gene expression may need to differ among species. © 1993 Wiley‐Liss, Inc.
Competent mouse oocytes isolated from antral follicles exhibit different chromatin organization and follow different maturation dynamics
Authors: Debey Pascale , Szöllösi Maria S. , Szöllösi Daniel , Vautier Dominique , Girousse Anne , Besombes Didier
Published: February 2005

After labelling DNA with the specific vital fluorophore Hoechst 33342, oocytes, isolated by puncture from antral follicles in adult mice, have two essentially different configurations of their nuclear fluorescence images. These have been called SN (where the nucleolus is not surrounded by chromatin) and NSN (where the nucleolus is not surrounded by chromatin). Intermediate configurations are also found, although with a lower frequency. The proportion of each class is on the average equal and depends neither on the presence of cumulus cells nor on the age of the mouse. Electron microscopy confirms several ultrastructural differences between these two nuclear configurations, namely, the structure of the nucleolus, which is vacuolated in NSN‐type and compact in SN‐type oocytes. Using video‐enhanced fluorescence microscopy at low level of excitation light, we could follow directly in vitro the meiotic maturation of both classes, without impairing their viability. We show that in germinal vessicle (GV) state, the chromatin does not change from one configuration into the other and that both classes are able to mature to metaphase II, although the maturation has slightly different characteristics. © 1993 Wiley‐Liss, Inc.
Mitogen‐activated protein kinase (MAP kinase) activation in Xenopus oocytes: Roles of MPF and protein synthesis
Authors: Haccard Olivier , Jessus Catherine , Rime Helene , Goris Jozef , Merlevede Wilfried , Ozon Rene
Published: February 2005

Mitogen‐activated protein kinase (MAP kinase) is a serine/threonine kinase whose enzymatic activity is thought to play a crucial role in mitogenic signal transduction and also in the progesterone‐induced meiotic maturation of Xenopus oocytes. We have purified MAP kinase from Xenopus oocytes and have shown that the protein is present in metaphase ll oocytes under two different forms: an inactive 41‐kD protein able to autoactivate and to autophosphorylate in vitro, and an active 42‐kD kinase resolved into two tyrosine phosphorylated isoforms on 2D gels.

During meiotic maturation, MAP kinase becomes tyrosine phosphorylated and activated following the activation of the M‐phase promoting factor (MPF), a complex between the p34cdc2 kinase and cyclin B. In vivo, MAP kinase activity displays a different stability in metaphase l and in metaphase II: protein synthesis is required to maintain MAP kinase activity in metaphase I but not in metaphase II oocytes. Injection of either MPF or cyclin B into prophase oocytes promotes tyrosine phosphorylation of MAP kinase, indicating that its activation is a downstream event of MPF activation. In contrast, injection of okadaic acid, which induces in vivo MPF activation, promotes only a very weak tyrosine phosphorylation of MAP kinase, suggesting that effectors other than MPF are required for the MAP kinase activation. Moreover, in the absence of protein synthesis, cyclin B and MPF are unable to promote in vivo activation of MAP kinase, indicating that this activation requires the synthesis of new protein(s). © 1993 Wiley‐Liss, Inc.
Production, characterization, and immunocytochemical applications of monoclonal antibodies to human sperm protamines
Authors: Le Lannic G. , Arkhis A. , Vendrely E. , Chevaillier P. , Dadoune J. P.
Published: February 2005

Three monoclonal antibodies against human protamines were obtained by immunization with total human basic nuclear proteins or purified protamine HP3. The specificity of antibodies was assessed by enzyme‐linked immunosorbent assay (ELISA) and Western blot. They recognized three distinct epitopes: One was specific for the protamine P1 family, another was specific for the protamine P2 family and the third was common to both families. All were specific for the human species. Antibodies were used to detect protamines in germ cells by indirect immunofluorescence and by immunoelectron microscopy. Protamines appeared in spermtid nuclei at steps 4–5 of spermiogenesis, i.e., during the chromatin condensation process, and were not accumulated in the cytoplasm before entering the nucleus. © 1993 Wiley‐Liss, Inc.
Differential expression of insulin‐like growth factor‐II in specific regions of the late (post day 9.5) murine placenta
Authors: Redline Raymond W. , Chernicky Cheryl L. , Tan Hui‐Qing , Ilan Joseph , Ilan Judith
Published: February 2005

Insulin‐like growth factor‐II (IGF‐II) expression has been implicated as a major determinant of fetal size during murine pregnancy. It remains unclear whether expression in the fetus, the placenta, or both is the overriding factor controlling growth. To gain further understanding of the placental contribution, we mapped IGF‐II expression in the fetal vascular and trophoblastic portions of the late murine placenta (day 9.5–18.5). We found that, as in the fetus itself, vasculogenic mesenchyme, in this case derived from the allantois, was the strongest expressor of IGF‐II. Trophoblast, on the other hand, while expressing somewhat less IGF‐II, showed a dynamic pattern of IGF‐II expression, which reflected its continuing differentiation during late pregnancy. Initially (days 9.5 and 12.5), the spongiotrophoblast, which is homologous to the cytotrophoblast columns and shell in early human pregnancy, strongly expressed IGF‐II. Later, expression in the spongiotrophoblast was down‐regulated as a new population, the 30‐called glycogen cells, emerged within the spongiotrophoblast (day 12.5–15.5) and went on to invade the mesometrial decidua. Glycogen cells, which are homologous to human intermediate trophoblast, strongly expressed IGF‐II. Trophoblast lining the area of maternalfetal exchange, the labyrinth, on the other hand, maintained a constitutive lower level of IGF‐II expression throughout late pregnancy. © 1993 Wiley‐Liss, Inc.
Anion channels in the sea urchin sperm plasma membrane
Authors: Morales Ezequiel , De La Torre Lucia , Moy Gary W. , Vacquier Victor D. , Darszon Alberto
Published: February 2005

Ionic fluxes in sea urchin sperm plasma membrane regulate cell motility and the acrosome reaction (AR). Although cationic channels mediate some of the ionic movements, little is known about anion channels in these cells. The fusion of sperm plasma membranes into lipid bilayers allowed identification of a 150 pS anion channel. This anion channel was enriched from detergent‐solubilized sperm plasma membranes using a wheat germ agglutinin Sepharose column. Vesicles formed from this preparation were fused into black lipid membranes (BLM), yielding single channel anion‐selective activity with the properties of those found in the sperm membranes. The following anion selectivity sequence was found: NO3− > CNS− > Br− > CI−. This anion channel has a high open probability at the holding potentials tested, it is partially blocked by 4,4′‐diisothiocyano‐2,2′ ‐stilbendisulfonic acid (DIDS), and it often displays substates. The sperm AR was also inhibited by DIDS. © 1993 Wiley‐Liss, Inc.
Surface‐associated glycosyltransferase activities in rat sertoli cells in vitro
Authors: Raychoudhury Samir S. , Millette Clarke F.
Published: February 2005

We have previously demonstrated fucosyltransferase (FT) activity on mouse germ cell surfaces at different stages of spermatogenesis. To complement these findings, here we report FT activity on the Sertoli cell (SC) surface. SC isolated and cultured from 20‐day‐old rat testes displayed FT activity with a Vmax of 12.5 pmoles/mg protein/min and a Km of 22 μM, while purified Sertoli cell plasma membranes (SCPM) showed FT activity with a Vmax of 10 pmoles/mg protein/min and a Km of 18.2 μM for GDP‐[14C]‐L‐fucose. Fucosyltransferase activities were 16.7 and 2.6 pmoles/mg protein/min in SC and SCPM, respectively; 16% of FT activity is, therefore, on the cell surface. To test whether the expression of FT activity in SC was regulated by hormones and growth factors, SC were cultured in serum‐free medium supplemented with insulin, transferrin, sodium selenite, and epidermal growth factor (medium 4F) or in 4F plus follicle‐stimulating hormone, testosterone, hydrocortisone, and vitamin E (medium 8F). We found that FT activity in SC is not modulated by these hormones or growth factors (4F or 8F). For comparison with FT, galactosyltransferase (GalTase) activities in SC and SCPM were also determined. SC displayed GalTase activity with a Vmax of 50 pmoles/mg protein/min and a Km of 38.5 μM, while SCPM showed GalTase activity with a Vmax of 25 pmoles/mg protein/min and a Km of 20.8 μM for UDP‐[3H]‐galactose. Galactosyltransferase activities were 29.2 and 9.6 pmoles/mg protein/min in SC and SCPM, respectively. Therefore, ∼33% of the total cell GalTase activity was detected on the surface membranes of rat Sertoli cells. These results suggest that cell surface glycosyltransferases may be involved in Sertoli cell function during mammalian spermatogenesis. © 1993 Wiley‐Liss, Inc.
Uptake and metabolism of pyruvate and glucose by individual sheep preattachment embryos developed in vivo
Authors: Gardner David K. , Lane Michelle , Batt Paul
Published: February 2005

The uptake of pyruvate and glucose by individual sheep oocytes and preattachment sheep embryos at each state of development up to the hatching blastocyst was determined using a microfluorescence technique. After an initial increase at fertilization, pyruvate uptake was relatively constant (˜15 pmol/embryo/h) from the zygote through to the morula. Upon blastocyst formation and hatching, there were significant increases in uptake (39 pmol/embryo/h, P < 0.001; and 53 pmol/embryo/h, P < 0.001, respectively). In contrast to that of pyruvate, glucose uptake was very low (˜1 pmol/embryo/h) up to the time of genome activation (eight‐ to 16 cell stage), after which there were significant increases in uptake at each successive stage of development. By the hatching blastocyst stage, glucose uptake had reached 54 pmol/embryo/h. The ability of day‐7 hatching blastocysts to oxidize pyruvate and glucose was determined indirectly by measuring the production of lactate when either substrate was present as the sole energy source. Unlike the mouse blastocyst, which has a considerable oxidative capacity for both pyruvate and glucose, the day‐7 sheep blastocyst showed limited ability to oxidise either substrate. Rather, in the sheep blastocyst, 65% of pyruvate and 98% of glucose taken up could be accounted for as lactate. Such low levels of substrate oxidation appear to be inconsistent with the energy requirements of the proliferating preattachment ruminant blastocyst. The utilization of alternative substrates at the blastocyst, such as amino acids, is proposed. © 1993 Wiley‐Liss, Inc.
Calcium channels play a pivotal role in the sequence of ionic changes involved in initiation of mouse sperm acrosomal exocytosis
Authors: Fraser Lynn R.
Published: February 2005

The sequence of ionic changes involved in initiation of acrosomal exocytosis in capacitated mouse spermatozoa was investigated. Earlier studies demonstrated that a large influx of Na+ is required for exocytosis, this Na+ apparently being associated with an increase in intracellular pH (pHi) via an Na+‐H+ exchanger. This rise in pHi may in turn activate calcium channels and permit the influx of extracellular Ca2+ needed to trigger acrosomal exocytosis. In the present study, the dihydropyridine voltage‐dependent calcium channel antagonist nifedipine was able to inhibit significantly exocytosis in sperm cells treated in various ways capable of stimulating acrosomal loss. The monovalent cation ionophore monensin can promote Na+ entry required for both capacitation and acrosomal exocytosis, as demonstrated by using chlortetracycline to monitor changes in sperm functional potential. In the presence of 10 nM nifedipine, monensin treatment accelerated capacitation but was unable to trigger exocytosis. The requirement for internalization of a high concentration of Na+ can be bypassed by the addition of 25 mM NH4CI to raise the pHi of cells capacitated in 25NH4CI to raise the pHi of cells capacitated in 25 mM Na+ (insufficient Na+ to support exocytosis under usual conditions). Again, introduction of nifedipine was able to inhibit exocytosis. In a third experimental approach, amiloridestimulated exocytosis in capacitated cells was significantly inhibited by nifedipine. In contrast to these treatments directed at specific mechanisms, the ability of the Ca2+ inophore A23187 to promote more general entry of Ca2+ and thereby to accelerate capacitation and exocytosis was not inhibited by nifedipine. Finally, monensin‐treated cells exhibited a rise and then a fall in 45Ca2+ uptake, the time course of which paralleled stimulation of acrosomal exocytosis in similarly treated cells. Nifedipine significantly reduced this uptake. The fact that nifedipine can block exocytosis induced by a variety treatments strongly suggests that voltage‐dependent calcium channels play a pivotal role in the response. These results are consistent with the following sequence of ionic changes in capacitated cells leading to acrosomal exocytosis: [Na+]i ↑ → [H]i↓ → pHi ↑ → activation of calcium channels → [Ca2+]i ↑ → exocytosis. Given that zona‐induced exocytosis is reportedly an indirect response, mediated by voltage‐dependent calcium channels, and that the Na+‐H+ exchanger in somatic cells can be activated by receptor‐mediated mechanisms, we suggest that sperm‐zona inter action promotes an influx of Na+ by activating an Na+‐H+ exchanger and thereby initiating the above sequence of changes. © 1993 Wiley‐Liss, Inc.
Porcine zona pellucida ZP3α glycoprotein mediates binding of the biotin‐labeled Mr 55,000 family (ZP3) to boar sperm membrane vesicles
Authors: Yurewicz Edward C. , Pack Beverley A. , Armant D. Randall , Sacco Anthony G.
Published: February 2005

The two Mr 55,000 glycoproteins, ZP3α and ZP3b̃, of porcine zona pellucida copurify as a preparation designated ZP3. Gamete binding assays have implicated ZP3α, but not ZP3b̃, as participating in sperm‐zona recognition events. We now report that boar sperm contain membrane‐associated binding sites with specificity for ZP3α. Biotin‐labeled (b‐) preparations of ZP3 bind to intact boar sperm in a saturable manner, with localization on the anterior head region. Membrane vesicles obtained from capacitated sperm by nitrogen cavitation retain b‐ZP3 binding sites as determined by an enzyme‐linked method employing alkaline phosphatase‐conjugated strepavidin. In competitive binding assays using b‐ZP3 (0.1μg/ml) as probe, heat‐solubilized zonae and ZP3 were effective competitors, whereas the nonzona molecules fetuin and fucoidin were not. Digestion of ZP3 with endo‐b̃‐galactosidase, an enzyme that trims polylactosamines, enhanced its affinity for membrane receptors. In contrast treatments such as chemical deglycosylation, pronase digestion, or disruption of disulfide bonds abolished the ligand activity of ZP3. Finally, purified ZP3α was an at least 100‐fold better antagonist than purified ZP3b̃. The results demonstrate that binding of b‐ZP3 to isolated boar sperm membranes is mediated by sperm receptors with specificity for the ZP3α macromolecular component and reveal a complex contribution of both carbohydrate and protein moieties toward the ligand activity of this sperm adhesive zona molecule. © 1993 Wiley‐Liss, Inc.
Sequence, expression, and chromosomal assignment of a human sperm outer dense fiber gene
Authors: Gastmann Oliver , Burfeind Peter , Güunther Eberhard , Hameister Horst , Szpirer Claude , Hoyer‐Fender Sigrid
Published: February 2005

Outer dense fibers (ODFs) are located on the outside of the axoneme in the midpiece and principal piece of the mammalian sperm tail and may help to maintain the passive elastic structures and elastic recoil of the sperm tail. We have identified and describe here a human gene that is homologous to the Mst(3)CGP gene family of Drosophila melanogaster and encodes an ODF protein of 241 amino acids. The transcribed region has a size of ˜lkb and contains two exons of 416 bp and 406 bp, respectively, not including the 3′ untranslated region. The gene is expressed in testis but not in human spleen, kidney, or brain and resembles in this respect the expression of the Drosophila Mst(3)CGP gene family in the male germline. An antiserum raised against a synthetic peptide derived from the N‐terminus of the encoded sequence identified a protein of ˜ 32 kDa in an extract of human sperm flagella. By Southern‐blot analyses and in situ hybridization, the ODF gene was localized to band q22 of chromosome 8. The isolation of a human gene encoding a sperm tail protein may provide the ability to identify and investigate, on the molecular level, possible reasons for human male infertility that are dependent on flagellar disturbances. © 1993 Wiley‐Liss, Inc.
Zinc, iron, and copper contents of Xenopus laevis oocytes and embryos
Authors: Nomizu Tsutomu , Falchuk Kenneth H. , Vallee Bert L.
Published: February 2005

Zinc is essential for vertebrate development; its deficiency results in multiple congenital malformations. Knowledge of the zinc biochemistry that underlies embryologic development is very limited. This has led us to investigate the zinc, iron, and copper contents of Xenopus laevis oocytes and embryos. Stage 1–6 oocytes, isolated from ovaries, and stage 1–40 embryos, obtained by in vitro fertilization techniques, were washed in metalfree water prior to digestion by 70% ultrapure HNO3. The metal content of the digests was analyzed by atomic absorption spectrometry. Stage 6 oocytes contain 65.8 4, 31.1 ± 3, and 0.68 ± 0.2 ng of zinc, iron and copper, respectively. The corresponding concentrations are 1, 0.5, and 0.01 mM in 1 μl eggs. The metal content varies as a function of egg maturation. The zinc content increases from 3–7 to >60 ng by stages 3 and 6, respectively. A similar pattern is noted for iron, which increases from 2–5 to 30 ng at analogous stages. In contrast, the copper content remains virtually unchanged in oocytes undergoing maturation. Importantly, the total of all three metals does not vary throughout the first 50 stages of development, when all tadpole organs are forming. Hence, the full complement of zinc, iron, and copper needed for incorporation into apoproteins during development is already present at a time when oocyte maturation is completed. The specific metalloproteins that store, donate, and accept these metals during induction and organogenesis and the alterations caused by metal deficiency can now be identified. © 1993 Wiley‐Liss, Inc.
Mitogenic and cytogenetic evaluation of transforming growth factor‐β on murine preimplantation embryonic development in vitro
Authors: Lim Joyce , Bongso Ariff , Ratnam Shan
Published: February 2005

Slow cleavage rate has been a major contributory factor influencing embryo morphology in in vitro fertilization (IVF) programs. The role of transforming growth factor‐b̃ (TGFb̃1) in improving this characteristic was evaluated using the murine model. Replicate batches of eight‐cell compacting embryos from superovulated mice were divided into three groups. Group A were treated with 0.3 ng/ml TGFb̃1 at the initial compacting stage, followed by a second treatment of 0.1 ng/mL 22 h later at the cavitating stage; group B received 0.3 ng/ml TGFb̃1 at the cavitating stage; group C were controls. The percentages of treated embryos reaching fixed embryonic stages, total cell number (TCN), mitotic index, and incidence of chromosome anomalies were monitored. The percentage of embryos reaching the cavitating, expanded, hatching, and hatched stages in both treatment groups were not significantly different from control (96.6% ± 4.2% to 37.7% ± 12.7% vs. 95.3% ± 7.3% to 47.0% ± 3.5%; P > 0.05). Values between the two treatment groups were also not significantly different. Embryos in groups A and B produced significantly greater TCN at expanded blastocyst and hatching stages compared to controls (Group A: 107.0 ± 18.9 vs. 89.9 ± 17.4, P < 0.05 and 125.5 ± 16.4 vs. 113.9 ± 12.1, P < 0.05; Group B: 107.9 ± 14.0 vs. 89.9 ± 17.4, P < 0.05 and 124.9 ± 17.4 vs. 113.9 ± 12.1, P < 0.05). Values, however, were not significantly different between treatment groups. The mean mitotic index for eight‐cell compacting embryos treated with a single dose of 0.3 ng/ml TGFb̃1 was significantly greater than control (0.1944 ± 0.1376 vs. 0.1282 ± 0.2573, P < 0.05). No significant increase in the incidence of chromosome anomalies was observed in embryos exposed to TGFb̃1. The results demonstrate that TGFb̃1 had a tremendous mitogenic effect on late murine embryonic stages and may thus be useful to improve embryo morphology in IVF programs and to produce adequate metaphases from biopsied embryos for preimplantation cytogenetic diagnosis. © 1993 Wiley‐Liss, Inc.
Effect of follicle size on bovine oocyte quality and developmental competence following maturation, fertilization, and culture in vitro
Authors: Lonergan P. , Monaghan P. , Rizos D. , Boland M. P. , Gordon I.
Published: February 2005

The aim of the present series of experiments was to investigate the effect of the size of follicle from which the oocytes originate on their subsequent in vitro developmental ability. Ovarian follicles were isolated and grouped according to size (2–6 mm, >6 mm). Primary oocytes were carefully liberated and grouped according to morphology into one of five categories: denuded; expanded; with two or three layers of cumulus; with four or five layers; and with many (six or more) layers. Following in vitro maturation (IVM), fertilization (IVF), and culture (IVC), more oocytes with many layers of cumulus (P < 0.01, 70.2%, 73/104 vs. 46.8%, 87/186, respectively) and a higher proportion of blastocysts were obtained from follicles > 6 mm compared to 2–6 mm follicles (P < 0.01, 65.9%, 60/91 from >6 mm follicles vs. 34.3%, 34/99 from 2–6 mm follicles, respectively). Use of follicular fluid (BFF) from follicles of different sizes in the IVM medium did not significantly increase the cleavage rate or blastocyst yield compared to controls. Administration of procine folliclestimulating hormone (pFSH) to donors prior to slaughter was investigated as a possible means of increasing the number of larger sized follicles in the ovaries and, thereby, the quality of the recovered oocytes. It was found that administration of six injections of pFSH beginning 3 days prior to slaughter resulted in a significant increase (P < 0.001) in the proportion of follicles >6 mm in diameter (31.6%) compared to that in nontreated controls (6.6%) and to animals that received only four injection groups (9.4%). The blastocyst yield from oocytes originating from >6 mm follicles, whether from unstimulated or from pFSH‐treated animals, was approximately double that of oocytes from 2–6 mm follicles (P < 0.01; 42.9%, 24/56 for >6 mm follicles vs. 22.8%, 21/92 for 2–6 mm follicles, respectively, for the 6 pFSH group; P > 0.05; 62.5%, 5/8 for >6 mm follicles vs. 32.8%, 22/67 for 2–6 mm follicles, respectively, for the control). © 1994 Wiley‐Liss, Inc.
Nuclear dynamics of parthenogenesis of bovine oocytes matured in vitro for 20 and 40 hours and activated with combined ethanol and cycloheximide treatment
Authors: Presicce Giorgio A. , Yang Xiangzhong
Published: February 2005

Bovine oocytes matured in vitro (IVM) for 20 hr vs. 40 hr were treated for activation with 7% ethanol in Dulbecco's phosphate‐buffered saline for 5 min followed by incubation in M199 + 7.5% fetal calf serum containing cycloheximide (10 μg/ml). TreatedIVM oocytes and the controls (no ethanol and cycloheximide exposures) were fixed after 0, 1, 2, 3, 4, 5, 7, 10, and 20 hr of incubation and stained 24 hr later with 1% acetoorcein to examine nuclear events. Different stages of nuclear development of the activated oocytes were identified on the basis of nuclear and chromosomal morphology. Pronuclear development was classified into four stages (PN I, II, III, and IV) according to pronuclear progression in chromatin decondensation, nucleoplasm appearance, and nuclear size. The results demonstrated that the combined activation treatment effectively drove the IVM oocytes, both young (20 hr) and aging (40 hr), out of metaphase arrest. The activation rates for young oocytes examined immediately after 0, 1, 2, 3, 4, 5, 7, 10, and 20 hr of incubation with cycloheximide were, respectively, 7%, 24%, 77%, 96%, 92%, 97%, 98%, 93%, and 98%. For aging oocytes (40 hr) the corresponding activation values at the same time intervals were 6%, 84%, 100%, 100%, 100%, 100%, 98%, 100%, and 100%, respectively. These values were significantly higher than those for the corresponding controls. The activated aging oocytes achieved peak activation response more rapidly than did young oocytes. In addition, nuclear events in aging oocytes proceeded faster than those in young ones. Spontaneous activation rates of the aging oocytes were also higher (6–57%) than those of the young ones (0–14%). © 1994 Wiley‐Liss, Inc.
Deficiency in fertilization by morphologically abnormal sperm produced by Azh mutant mice
Authors: Meistrich Marvin L. , Kasai Kenichi , Olds‐Clarke Patricia , Macgregor Grant R. , Berkowitz Albert D. , Tung Kenneth S. K.
Published: February 2005

Male mice homozygous for the azh mutation produce spermatozoa with abnormal head shapes and have significantly reduced fecundity, to between 5% and 10% that of wild‐type or heterozygous mice. Several possible causes of this infertility were investigated. No gross endocrine disorders in azh/azh male mice were observed, and they exhibited apparently normal mating behavior. In addition, their sperm were motile, were capable of hyperactivated motility, and did not show premature acrosome reactions. However, quantitative analysis revealed slight but significant reductions in several motility parameters. Analysis of embryos following mating of azh/azh males with superovulated females indicated a reduction in the number of fertilized eggs compared to control matings. In vitro, spermatozoa from azh/azh mice failed to fertilize cumulus‐intact/zona‐intact and cumulus‐free/zona‐intact ova, although they successfully fertilized zonafree ova. These results indicate that the primary defect in fertility of azh/azh male mice is a result of sperm quality, likely, in sperm morphology, and is manifest at the level of interaction with the zona pellucida. © 1994 Wiley‐Liss, Inc.
Unusual nuclear structure of the spermatozoon in a marsupial, Sminthopsis crassicaudata
Authors: Breed W. G. , Leigh C. M. , Washington J. M. , Soon L. L. L.
Published: February 2005

The organization of sperm chromatin in the dasyurid marsupial, Sminthopsis crassicaudata, was investigated using various morphological techniques. Transmission electron microscopy indicates two quite distinct chromatin regions became evident late in spermiogenesis with an outer globular region containing blocks of very electron‐dense chromatin. Fluorescent light microscopical studies after staining with DNA dyes and 7‐amino actinomycin D of testicular, caput, and cauda epididymal spermatozoa showed that this region fluoresced less brightly than the rest of the nucleus, indicating the presence of fewer DNA binding sites. Freeze fracture showed that the chromatin in most of the nucleus had randomly arranged particles of various sizes, but that of the outer region was composed entirely of small particles. This outer region was more resistant to low concentrations of the ionic detergent, SDS, whereas both guanidine hydrochloride and urea together with sodium chloride generally dispersed all the chromatin except that in the outer globular region and in a localized area of the nucleus beneath the acrosome. This study has thus revealed that the outer globular chromatin of these spermatozoa responds differently to ionic detergents and protein denaturing agents and has a different chromatin organization than most of the rest of the nucleus. The significance of these differences remains, however, to be determined. © 1994 Wiley‐Liss, Inc.
Contractile proteins and nonerythroid spectrin in oogenesis of Xenopus laevis
Authors: Ryabova L. V. , Virtanen I. , Wartiovaara J. , Vassetzky S. G.
Published: February 2005

The distribution of contractile proteins, actin and myosin, and an actin‐binding protein, spectrin, was studied in oogenesis of Xenopus laevis. These proteins are present in oocytes already at the previtellogenic stages, which are characterized by their diffuse distribution. The localization of proteins changed with the beginning of vitellogenesis. At all vitellogenic stages, including the fully grown oocyte, animal–vegetal differences were noted in localization of actin and myosin: in the animal hemisphere they appear as fibrillar‐like structures, while in the vegetal one they are localized around the yolk platelets. By the end of the oocyte's growth, a cortical gradient appeared: predominant localization of actin and myosin in the cortical area. As the oocyte maturation proceeded, the distribution of actin and myosin again became diffuse and nonuniform, so that a cortical gradient appears. At the beginning of vitellogenesis spectrin is distributed as a network all over the ooplasm, while in the fully grown oocyte it is localized mostly in teh subcortical area of the animal hemisphere and, as individual inclusions, in other regions of the oocyte. No spectrin is found by the end of maturation. Actin, myosin, and spectrin are also present in the oocyte's nuclei. Changes in the distribution of contractile proteins and spectrin during oocyte maturation are discussed with respect to the development of cortical contractility, as well as to the changes in spatial distribution of yolk platelets and regional sensitivity of the maturing oocyte to cytochalasin B. © 1994 Wiley‐Liss, Inc.
Characterization of a cell surface glycoprotein associated with maturation of rat spermatozoa
Authors: Eccleston Eric D. , White Thomas W. , Howard James Bryant , Hamilton David W.
Published: February 2005

The principal galactose oxidase/NaB[3H]4‐labeled membrane protein of rat caudal epididymal spermatozoa was isolated by hydrophobic interaction chromatography. The protein is released from the membrane by the action of phosphatidylinositol specific phospholipase C, and thereby its properties are transformed from those of a protein anchored to the hydrophobic membrane to those of a hydrophilic solution protein. Because it is the only membrane‐associated protein released by the enzyme which did not absorb to a propylaspartate resin, a simple, single step purification procedure was devised.

Although the amino terminus of the protein is blocked to Edman degradation, the majority of the protein structure was determined from a series of tryptic peptides and from limited acid hydrolysis. Approximately 65% of the protein mass is carbohydrate which is primarily attached through O‐glycosidic bonds to the 18 threonines. The molecular weight of the glycoprotein was estimated to be 16,600, considerably smaller than the Mr = 26,000 to 37,000 previously determined by gel electrophoresis. The anomalous electrophoretic behavior is undoubtedly due to the large percentage of carbohydrate. The distribution of carbohydrate on the protein side chains suggests the protein may form a positively charged, specialized scaffolding for the presentation of the carbohydrate moieties. Because the appearance of the ability to label the protein with galactose oxidase is correlated with sperm maturation in the epididymis, the glycoprotein structures may be an important componetn in the fertilization process. The combination of linkage by glycosylphosphatidylinositol and low molecular weight mucin‐like structure indicates this may be a member of a new class of membrane proteins. © 1994 Wiley‐Liss, Inc.
Major human epididymis‐specific gene product, HE3, is the first representative of a novel gene family
Authors: Kirchhoff Christiane , Pera Ilka , Rust Werner , Ivell Richard
Published: February 2005

Differential screening of a human epididymal cDNA library led to the isolation and characterization of a major epididymis‐specific cDNA clone family, referred to as HE3. More detailed sequence and PCR analysis identified two different but homologous gene transcripts, HE3α and HE3β. The former represents an mRNA of ca. 1 kb, encoding a putative small secretory polypeptide of 14903 MW. The HE3β transcript was only found as incomplete 3′ fragments. Analysis of human genomic DNA by Southern blotting suggested the presence in the human genome of at least three independent HE3‐related genes. Isolation of genomic clones for the HE3α gene showed this to contain a single intron of 1.4 kb in the 5′ noncoding region. Although genomic clones corresponding to HE3β could not be found, a third highly homologous gene, HE3γ, was identified as a potential pseudogene. Neither nucleotide nor encoded amino acid sequences of the HE3 gene family are related to any other known sequence in the central databases, and thus represents a novel human gene family, with at least three nonallelic members. Northern hybridization analysis showed that HE3 gene products are specifically expressed in the human epididymis, and not in any other tissue examined. Furthermore, except for the pig, no other nonprimate species has been identified to express homologous sequences in the epididymis. RNase protection assays showed that both the HE3α and HE3β, but not the HE3γ genes, are expressed in the human epididymis. © 1994 Wiley‐Liss, Inc.
Low‐affinity nerve growth factor receptor is expressed during testicular morphogenesis and in germ cells at specific stages of spermatogenesis
Authors: Russo Mario A. , Odorisio Teresa , Fradeani Andrea , Rienzi Laura , De Felici Massimo , Cattaneo Antonino , Siracusa Gregorio
Published: February 2005

Nerve growth factor (NGF) is essential for neuronal development and differentiation. Recent reports have shown that its low‐affinity receptor (LNGFR) is expressed and developmentally regulated in a broad range of embryonic and adult tissues outside the nervous system, although the functions of the receptor in such tissues remain unknown. Recently, NGF and LNGFR have been detected in adult mouse, rat, and human testis.

The results of the present work demonstrate that LNGFR is expressed much before the onset of spermatogenesis in both mouse and rat testis. In situ hybridization shows that the mRNA for LNGFR is expressed in the peritubular cells of the embryonic mouse testis. Immunohistochemical analysis of the rat testis shows LNGFR‐expressing cells to be scattered in the intertubular compartment in the embryonic testis, and to become organized in a cellular layer that surrounds myoid cells of the seminiferous tubules during postnatal development. Furthermore, in peripuberal and adult mouse and rat testis we have identified the expression of an abundant and shorter mRNA of 3.2 kb that cross hybridizes to the low‐affinity NGF receptor transcript (3.7 kb). This shorter mRNA species, which appears at the beginning of spermatogenesis in the adult, has been identified by in situ hybridization and by Northern blot with RNA isolated from homogeneous populations of meiotic germ cells to be expressed by pachytene spermatocytes and round spermatids. Our results suggest a complex developmental role for LNGFR during testicular morphogenesis and identify the expression, at specific stages of spermatogenesis, of a new germ cell—specific transcript homologous to the receptor RNA. © 1994 Wiiey‐Liss, inc.
Polyadenylation and deadenylation of maternal mRNAS during oocyte growth and maturation in the mouse
Authors: Paynton Barbara V. , Bachvarova Rosemary
Published: February 2005

We previously showed that, during mouse oocyte maturation, specific maternal mRNAs (actins) are deadenylated, while others (hypoxanthine phosphoribosyltransferase:HPRT) are adenylated. As in other systems, these changes can be correlated with changes in transiational activities. Maturation‐specific polyadenylation in Xenopus depends on the presence of a U‐rich cytoplasmic polyadenylation element (CPE) close to the 3′ end of the RNA. RNAs that lack CPEs appear to be deadenylated by default when meiosis resumes. We show here that this default program also applies to maturing mouse oocytes. Microinjected β‐ and γ‐actin 3′ UTR (untranslated region) transcripts lacking CPEs but including polyA tails (100–200 N) behave as endogenous maternal actin mRNAs and are at deadenylated by maturing oocytes. “Nonsense” transcripts taht do not include CPEs, but that do contain polyA tails, are also deadenylated. β‐ and γ‐Actin 3′ UTRs with short polyA tails (50–80 N) are stable and exhibit no detectable change in adenylation when injected into growing, fullgrown, or maturing oocytes. In contrast, HPRT 3′ UTRs, which include the CPE UUUUAAAU and a short polyA tail (50 N), are polyadenylated during maturation. HPRT 3′ UTR transcripts with long polyA tails (100–200 N) are more extensively deadenylated by growing and full‐grown oocytes that retain germinal vesicles than by maturing oocytes. The presence of CPEs may be required for polyA tail shortening and translational inactivation of stable mRNAs during oocyte growth and subsequent selective readenylation and translation during meiotic maturation. We propose that the absence of CPEs on maternal mRNAs translationally active during oogenesis enables them to retain polyA tails of sufficient length for recruitment onto polysomes and later allows for their deadenylation by default and removal from polysomes after meiosis resumes. © 1994 Wiley‐Liss, Inc.
Immunolocalization of proacrosin/acrosin in rabbit sperm during acrosome reaction and in spermatozoa recovered from the perivitelline space
Authors: Valdivia M. , Yunes R. , Melendez J. , de Ioannes A. E. , Leyton L. , Becker M. I. , Barros C.
Published: February 2005

The participation of acrosin in mammalian sperm penetration through the zona pellucida has been amply debated. In this paper we report the immunolocalization—by silver enhanced immunogold technique using ACRO‐8C10 monoclonal antibody to human acrosin—of proacrosin/acrosin on ejaculated rabbit spermatozoa incubated in vitro in a capacitating medium and on spermatozoa recovered from the perivitelline space. After incubation in a capacitating medium, four different patterns were observed: (1) no labeling on acrosome intact spermatozoa; (2) labeling on the rim of the head; (3) labeling on the whole acrosome area; and (4) no labeling on acrosome reacted spermatozoa. At the start of incubation, spermatozoa with pattern 1 were the most abundant, whereas at the end of the 32 h incubation period, patterns 2 and 3 were the most frequent. On the other hand, 625 perivitelline spermatozoa were recovered from 17 fertilized rabbit eggs, of which 26% were labeled with the anti‐acrosin monoclonal antibody ACRO‐8C10 in two different areas: (1) only on the equatorial region; and (2) only on the postacrosomal area. These results are consistent with the idea that proacrosin/acrosin remains associated to the acrosome reacted spermatozoa for long periods of time, and that proacrosin/acrosin associated to perivitelline spermatozoa could be responsible for the second penetration of fresh rabbit eggs by perivitelline spermatozoa. © 1994 Wiley‐Liss, Inc.
Oocyte morphology in dominant and subordinate follicles
Authors: Assey R. J. , Hyttel P. , Greve T. , Purwantara B.
Published: February 2005

The structure of oocytes aspirated from the dominant and its subordinate follicles was investigated from the achievement of follicular dominance to ovulation. Ovulation was induced in 18 heifers and 5 cows by injection of cloprostenol at days 8–14 (day 0 = day of ovulation), and follicular development was monitored by ultrasonography. The animals were slaughtered at days 3–11, but animals slaughtered on days 8–11 were given a second injection of cloprostenol at day 7 to allow ovulation of the dominant follicle of the first follicular wave. Oocytes were aspirated from the dominant (largest) and two largest subordinat efollicles and processed for transmission electron microscopy, whereas the follicular fluids were analyzed for concentrations of estradiol‐17β (E2) and progesterone (P4).

Dominant follicular growth was associated with increase in the concentration of E2 and P4 in the follicular fluid, which was E2‐dominated. From days 3–7, the dominant oocytes had pronounced junctional contacts with the cumulus cells and a nonundulating nuclear envelope but showed an increase in the number of lipid droplets and a decrease in the size of Golgi complexes, the size of cortical granule clusters, and the number of microvilli stacks. After cloprostenol injection on day 7, but before the anticipated LH surge, the dominant oocytes showed a reduced oocyte cumulus contact, vacuolization of the nucleolus, undulation of the nuclear envelope, and dispersal of the mitochondrial clusters. The morphological alterations occurring in the dominant oocytes before the anticipated LH surge are suggested to be a prerequisite for the oocyte to achieve the competence to undergo final maturation. Subordinate follicles ceased growing at about days 3–4 and their follicular fluid had low E2:P4 ratio or was P4‐dominated. Subordinate oocytes displayed degenerative features in their cumulus investment and nuclear activation and maturation especially after day 5. The structural changes associated with oocyte degeneration showed similarities with the processes seen before and during final maturation of the dominant oocytes. © 1994 Wiley‐Liss, Inc.
Transcription of circular and noncircular forms of Sry in mouse testes
Authors: Zwingman Theresa , Fujimoto Hirokazu , Lai Li‐Wen , Boyer Timothy , Ao Asangla , Stalvey John R. D. , Blecher Stan R. , Erickson Robert P.
Published: February 2005

Although its expression in adult testis was immediately apparent, the role for Sry (sex determining region, Y) in testicular function remains elusive. We have performed transcriptional studies in an effort to elucidate potential roles of Sry by studying the time and location of its transcription in mouse testes. Northern analyses and more sensitive nuclease protection assays detected transcripts in 28‐day‐old testes and beyond. The highly sensitive technique of reverse transcription polymerase chain reaction (RTPCR) could not detect Sry expression in 14‐day testes when primers for the most conserved portion of the gene, the high mobility group (HMG) box, were used, but primers for the circular form detected Sry transcription at all postnatal stages studied. The same HMG box primers were able to detect expression of Sry in XX, Sxra or Sxrb testes. This suggested that Sry is expressed in cells other than germ cells, which was confirmed with studies on fractionated cells—RTPCR detected transcription of Sry in the highly pure interstitial cell fraction. However, Leydig cells and a Leydig cell tumor were negative for Sry expression. We performed in situ studies in an attempt to localize the expression of Sry in the testes. Abundant expression of an Sry cross‐hybridizing transcript was found in spermatogonia, in early spermatocytes, and in some interstitial cells with antisense probes to the HMG box or a more specific, 3′ region, whereas the sense probe gave little or no hybridization. It is probable that the circular transcripts, which are seen in reverse transcriptase positive (RT+) and RT− reactions by PCR because of the RT activity of Taq polymerase, are responsible for the hybridization seen in spermatogonia and spermatocytes, whereas linear and circular forms are detected later. Thus Sry is expressed in pre‐ and postmeiotic germ cells and in somatic cells of the testes. © 1994 Wiley‐Liss, Inc.
Isolation and identification of germ cells from fetal bovine ovaries
Authors: Lavoir M.‐C. , Basrur P. K. , Betteridge K. J.
Published: February 2005

Gonadal cell suspensions were made from bovine fetuses of 35–55‐, 56–80‐, and 80–130‐day age groups corresponding to the periods predominated by primordial germ cells (PGCs), oogonia, and meiotic cells, respectively. Germ cells identified on morphological criteria prior to their isolation from suspensions were compared histochemically and morphologically with cells in cryosections, impression smears, and semithin sections of similar gonads. Oocytes were distinguished by their chromosomal configurations in cell spreads.

In suspensions from 35–55‐day fetuses, cells considered to be PGCs stood out by their size, large nucleus, intracytoplasmic vesicles, and occasional blebbing. The somatic cells were smaller and contained little cytoplasm and few vesicles. In bovine gonads, in contrast to murine gonads, alkaline phosphatase (AP) activity was not specific enough to identify germ cells once they had entered the gonad. In ovaries from the 56–80‐day age group, cells similar to PGCs, but slightly larger and with more cytoplasmic vesicles, were identified as oogonia. The cytoplasmic vesicles stained positively for lipid. In ovaries of 80–130‐day fetuses, oogonia, oocytes, degenerating germ cells, and multinucleate germ cells were recognized. Degenerating germ cells exhibited a variety of morphological characteristics and were consistently positive for acid‐phosphatase activity. Binucleate germ cells appeared around day 85 of gestation, while multinucleate germ cells were seen from day 95. It was concluded that bovine mitotic germ cells can be isolated from gonadal cell suspensions and that the best time to recover them is between 50 and 70 days of gestation. © 1994 Wiley‐Liss, Inc.
Regulation of the fertilization current in ascidian oocytes by intracellular second messengers
Authors: Tosti Elisabetta , Dale Brian
Published: February 2005

Neomycin, injected into ascidian oocytes to a final concentration of 10–50 mM, inhibits both the fertilization current and the surface contraction, showing that phosphoinositide hydrolysis is required for these early activation events. Sperm‐activated fertilization currents are not inhibited in the presence of 100 μg/ml intracellular heparin, suggesting that these currents are not directly gated by InsP3. The sulfhydryl reagent thimerosal at 100 μM, in contrast, significantly increases the fertilization current presumably by sensitizing the channel receptor. Since heparin inhibits the surface contraction, InsP3 receptors are shown to play a role in the propagation of the activation response in ascidian oocyte. Depleting intracellular calcium stores by microinjecting 50 mM EGTA into oocytes does not activate fertilization channels; however, subsequent fertilization of these EGTA loaded oocytes leads to a significantly larger and faster fertilization current. Thus in contrast to somatic cells studied to date, second messenger operated plasma membrane channels in ascidian oocytes are not gated by calcium released from intracellular stores. © 1994 Wiley‐Liss, Inc.