bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of embryonic genes during preimplantation rat development
Authors: Zernicka‐Goetz Magdalena
Published: February 2005

Expression of the embryonic genome has been examined during preimplantation rat development. Proteins synthesized at different stages of embryogenesis were labelled with [35S]methionine and then separated by one‐dimensional gel electrophoresis. A major transformation in the pattern of protein synthesis has been observed between the two‐ and the four‐cell stages of embryonic development. Also the culture of embryos with an inhibitor of transcription (α‐amanitin) has shown that the first α‐amanitin‐sensitive events take place during the late two‐cell stage. However, inhibition of transcription does not arrest the embryo development up to the four‐cell stage. Taken together, the results indicate that in rats the initiation of embryonic gene activation occurs at the late two‐cell stage. However, the first two cleavage divisions can occur in the absence of transcription. © 1994 Wiley‐Liss, Inc.
Evidence for a strong paternal effect on human preimplantation embryo development and blastocyst formation
Authors: Janny Laurent , Menezo Yves J. R.
Published: February 2005

In human in vitro fertilization (I.V.F.), it was first assumed that all the embryos obtained had the same developmental potential whatever the quality of sperm. However, this has not been confirmed. We have used the coculture technique and determined the blastocyst formation rate in three groups of patients: group 1: patients with normal sperm count (>20 × 106/ml), motility (>30%), and morphology (>50%); group 2: patients treated by I.V.F. with frozen donor sperm; group 3: patients with severely impaired sperm quality (<3 × 106 forward motile and morphologically normal spermatozoa per ml). In group 1, we found a strong correlation between cleavage rate and blastocyst formation rate (P < 0.0001) with a blastocyst formation rate comprised between 40% and 50%. This was not true for the two other groups for which the overall number of blastocysts obtained and the number of patients having at least one blastocyst were severely reduced (P < 0.0001). These data are discussed in terms of DNA quality, timing of formation of the pronuclei, and delays in cell cycles at the time of genomic activation. These observations lead to a new approach to the study of fertilizing ability of poor quality sperm. It may help in the decision as to whether couples treated for male infertility should be excluded from I.V.F. protocols. © 1994 Wiley‐Liss, Inc.
Epididymal glycoprotein involved in rat sperm association
Authors: Fornes Miguel W. , Burgos Mario H.
Published: February 2005

Recently, a new head‐to‐head sperm association was described in the rat during epididymal transit. This association was called a rosette and a filamentous and PAS‐positive material was also described joining the sperm heads. The begining of rosette formation in the epididymis and the linking material between heads have remained unclear. Epididymides of adult rats were fixed by vascular perfussion and thin sections of the principal regions were studied by transmission electron microscopy (TEM). The first evidence of rosette formation was observed in the distal corpus. Rosettes were isolated from the distal corpus and processed for immunogold and immunofluorescence microscopy to detect an epididymal glycoprotein called DE. This glycoprotein is secreted by the corpus epididymis and appears to be involved in sperm maturation. Colloidal gold marks and fluorescence were observed in the linking material between the sperm heads. The results presented here show that rosettes begin to appear following the sites of DE secretion and permit us to postulate that DE is involved in rosette formation and constitutes another example of gamete‐epididymal interaction. © 1994 Wiley‐Liss, Inc.
Characterization of the main egg envelope proteins of the sea bass Dicentrarchus labrax L. (teleostea, serranidae)
Authors: Scapigliati Giuseppe , Carcupino Marcella , Taddei Anna Rita , Mazzini Massimo
Published: February 2005

Fish eggs are surrounded by a resistant acellular coat commonly called the chorion or zona radiata. This study characterizes the eggshell proteinaceous content of unfertilized eggs of the sea bass Dicentrarchus labrax, with a view to the prepration of immunogens. Solubilization of the purified eggshells was achieved in 8 M urea followed by one‐ and two‐dimensional gel electrophoresis. Glycoproteins were detected using concanavalin‐A in one and two‐dimensional gels, and the principal glycoproteins had a molecular weight of 47 kDa and 170 kDa. Partial purification of a few polypeptides in the 45 kDa to 55 kDa range was achieved by gel filtration chromatography. Although whole eggshells were relatively insoluble even in 8 M urea, partial purification of these polypeptides enable them to dissolve completely in solutions at low ionic strength. © 1994 Wiley‐Liss, Inc.
Storage of messenger RNA in eukaryotes: Envelopment with protein, translational barrier at 5′ side, or conformational masking by 3′ side?
Authors: Spirin Alexander S.
Published: February 2005

Messenger RNA can be stored in the cytoplasm of higher Eukaryotes in the form of masked messenger ribonucleoprotein particles (masked mRNPs, or informosomes). The typical example is the storage of mRNPs in germ cells (oocytes and spermatocytes). The masked mRNPs are inactive in translation, stable, i.e., protected against degradation, and unavailable for poly(A) tail processing, such as cytoplasmic polyadenylation and deadenylation. The major nonspecific mRNA‐binding protein forming mRNPs and belonging to a special p50 family of basic, glycine‐rich, phosphorylatable proteins seems to be necessary, but not sufficient for the masking. In some cases, mRNA‐specific repressor proteins bound to the 5′‐untranslated regions (5′‐UTR) of mRNAs may be involved. Interactions of the 3′‐untranslated regions (3′‐UTR) with sequence‐specific proteins seem to be of decisive importance for the masking of mRNPs. The hypothesis is proposed that the masking is achieved through a 3′‐UTR–induced conformational rearrangement of mRNP; closing into a circle and condensation of mRNP are considered plausible. © 1994 Wiley‐Liss, Inc.
Mouse oocyte maturation is affected by lithium via the polyphosphoinositide metabolism and the microtubule network
Authors: Pesty Arlette , Lefèvre Brigitte , Kubiak Jacek , Géraud Gérard , Tesarik Jan , Maro Bernard
Published: February 2005

The incubation of mechanically denuded mouse oocytes in medium containing LiCl delayed both germinal vesicle breakdown (GVBD) and polar body extrusion in a dose‐dependent and reversible manner. When myo‐inositol alone was added to the culture medium, we observed that it accelerated GVBD and increased the rate of polar body extrusion, whereas, when combined with LiCl, the normal timing of GVBD was recovered. In the same way, when inositol trisphosphate (InsP3) was microinjected into the ooplasma, we observed an important improvement of the rate of GVBD, as compared to control oocytes, and prevention of lithium inhibition. However, neither myo‐inositol nor InsP3 were able to rescue totally the oocytes from the negative effect of lithium on polar body extrusion. Moreover, lithium induced some important changes in microtubule and chromosome organizations. Before extrusion of the first polar body, the reduction of the spindle size or the appearance of short individualized chromosomes dispersed around a large aster of microtubules were often observed, whereas, after polar body extrusion, the spindle appeared smaller and chromosomes were often trapped in the midbody. Thus lithium affects mouse oocyte maturation at two different levels: GVBD and polar body extrusion. Whereas the former seems to be affected via polyphosphoinositide turnover, the latter is InsP3‐independent and seems to be influenced negatively via underdevelopment of microtubular structures. © 1994 Wiley‐Liss, Inc.
Fertilization current in the human oocyte
Authors: Gianaroli Luca , Tosti Elisabetta , Magli Cristina , Iaccarino Mariano , Ferraretti Anna Pia , Dale Brian
Published: February 2005

In this report we show that the first event of activation in the human oocyte, the Fertilization current (FC), is a slow transient outward current of 300 pA, which induces a gradual hyperpolarization of the plasma membrane from –20mV to –60mV, 60–120 min after insemination, followed by a repolarization to –20mV. Activation currents (AC) of 600‐2,500 pA, induced by exposure to the calcium ionophore A23187 or by microinjection of InsP3 into the cytosol, are also outward. The AC are inhibited by preloading oocytes with EGTA suggesting they are calcium dependent. Since AC are 2–10‐fold the amplitude of the FC the fertilizing spermatozoon in the human only activates a portion of the primary elements stored in the oocyte for triggering metabolic depression. Oocyte activation in the human resembles that in the hamster rather than other mammals or invertebrates studied to date. © 1994 Wiley‐Liss, Inc.
Mechanism for procaine‐mediated hyperactivated motility in guinea pig spermatozoa
Authors: Mújica Adela , Neri‐Bazan Leticia , Tash Joseph S. , Uribe Salvador
Published: February 2005

Hyperactivated motility was studied in guinea pig spermatozoa. In the presence of the local anesthetic procaine, a high number of sperm cells (64%) showed hyperactivation when incubated in minimal culture medium with pyruvate, lactate, and glucose. Hyperactivated motility was dependent on glucose in the medium. Sperm ATP concentration was increased twofold in hyperactivated sperm when compared to procaine‐treated nonhyperactivated cells. cAMP levels were also higher in hyperactivated cells than in control spermatozoa. Thus, in living spermatozoa high levels of ATP appear to be needed to generate hyperactivation. cAMP is present at a high concentration in hyperactivated spermatozoa, therefore a role of cAMP in hyperactivation cannot be excluded. Depletion of external Ca2+ did not inhibit procaine‐induced hyperactivated motility. Hence, procaine canceled the requirement of external Ca2+ for sperm to express hyperactivated motility. © 1994 Wiley‐Liss, Inc.
Identification of two human sperm populations using flow and image cytometry
Authors: Pasteur Xavier , Métézeau Philippe , Maubon Ivan , Sabido Odile , Kiefer Héléagne
Published: February 2005

Flow cytometric studies of human sperm from fertile men display a constant and characteristic bimodal nonartifactual DNA pattern confirming the existence of two distinct populations. The main population is represented by a peak followed by a shoulder (“marginal population”). The appearance of this marginal population fluctuates with either freezing and thawing or with Percoll gradient centrifugation.

We have analyzed both the main and marginal sperm populations by flow cytometry after cell sorting, laser scanning cytometry, light microscopic evaluation, and their sensitivity to DNase digestion.

We have observed that the marginal population detected in fertile men represents a sperm group altered in the nuclear condensation, yielding unstable chromatin which appears more stainable with propidium iodide. © 1994 Wiley‐Liss, Inc.
DFP‐sensitive multicatalytic protease complexes (proteasomes) involved in the control of oocyte maturation in the toad, Bufo japonicus
Authors: Takahashi Mika , Tokumoto Toshinobu , Ishikawa Katsutoshi
Published: February 2005

The inhibition of progesterone‐induced oocyte maturation by diisopropylfluorophosphate (DFP), a typical serine protease inhibitor, was investigated in oocytes of the Japanese toad Bufo japonicus for the first time. Oocytes to which DFP was externally applied did not undergo germinal vesicle breakdown (GVBD), which is an early signal of oocyte maturation, in response to progesterone. The more inhibitory period was found to be 0–0.5 GVBD50 on a relative time scale [when the time at which 50% of the oocytes had completed GVBD (GVBD50) was set at 1.0], namely, before the beginning of GVBD. DFP‐sensitive proteases, which seem to be multifunctional nonlysosomal protease complexes (proteasomes), may already be present in the cytosol of premature oocytes. Peptide hydrolyzing activity, as reflected by proteasome activity, was found to be regulated before and after GVBD. In addition, immunoblotting regarding the native electrophoretic protein profile of the proteasomes throughout the maturational process demonstrated that they undergo alterations in mobility dependent upon the maturational process.

These findings raise the possibility that the activities of some endogenous DFP‐sensitive proteasomes play distinct, essential roles in oocyte maturation triggered by progesterone in Bufo. © 1994 Wiley‐Liss, Inc.
Surface alterations of the bovine oocyte and its investments during and after maturation and fertilization in vitro
Authors: Suzuki Hiroyuki , Yang Xiangzhong , Foote Robert H.
Published: February 2005

Surface characteristics of the bovine oocyte and its investments before, during, and after maturation, and fertilization in vitro were evaluated by scanning electron microscopy (SEM). Oocyte diameters were also measured during SEM analysis of the oocyte. The cumulus cells manifested a compact structure with minimal intercellular spaces among them in the immature oocytes. These became fully expanded with increased intercellular spaces after maturation in vitro, but contracted again after fertilization. The zona pellucida (ZP) showed a fibrous, open mesh‐like structure in the maturing and matured oocytes. The size and number of meshes on the ZP decreased dramatically after fertilization. The vitelline surface of immature oocytes was characterized by distribution of tongue‐shaped protrusions (TSPs) varying in density. After 10 and 22 hr of maturation incubation, oocyte surface microvilli (MV) increased to become the predominant surface structure, and TSPs decreased substantially. The vitelline surface of fertilized oocytes (at 6 and 20 hr) was similar to that of the matured oocytes, but unfertilized oocytes had less dense MV than did fertilized oocytes (at 20 hr). The diameter of the oocytes decreased from 99 to 80 μm during maturation and increased to 106 μm after insemination (P < 0.05). Membrane maturation was characterized by surface changes from a TSP‐predominant pattern to a MV‐predominant pattern. Thus, the bovine oocyte maturation process was found to involve the expansion of cumulus cells and the maturation of the ZP, which changes dramatically upon fertilization. Also, volumetric changes occurred in ooplasm processed for SEM following oocyte maturation and insemination. © 1994 Wiley‐Liss, Inc.
Oocyte structure and follicular steroid concentrations in superovulated versus unstimulated heifers
Authors: Assey R. J. , Hyttel P. , Roche J. F. , Boland M.
Published: February 2005

A highly variable yield of viable embryos in superovulated cattle is a major hindrance to the embryo transfer industry. To trace the cause of this problem, investigations were carried out on the intrafollicular steroids and structure of oocytes originating from follicles of follicular stimulating hormone (FSH)‐stimulated (superovulated) and unstimulated heifers.

Unstimulated heifers were slaughtered at midcycle, or administered cloprostenol (PG) at midcycle and slaughtered after 24, 48, or 72 hr, while superovulated heifers were administered 4 injections of pFSH (2 injections per day) and slaughtered 12 hr later, or administered 6, 7, or 8 injections of FSH in combination with PG at the 5th and 6th injection, and slaughtered 24, 36, or 60 hr, respectively, after the first PG injection. The follicular fluid from the largest (presumptive dominant) follicle of the unstimulated heifers and from potentially ovulatory follicles (≥8 mm in diameter) of the superovulated heifers were assayed for estradiol‐17β (E2) and progesterone (P4), while the oocyte cumulus complexes from such follicles were processed for transmission electron microscopy.

The mean E2 and especially P4 concentrations of the potentially ovulatory follicles of the superovulated heifers were lower than similar follicles of the unstimulated animals (83.7 ± 76.7 ng/ml vs. 208.1 ± 357.0 ng/ml, P > 0.05 and 31.1 ± 38.7 ng/ml vs. 150.3 ± 202, P < 0.05, respectively). The unstimulated oocytes had, in general, spherical oocyte nuclei and compact nucleoli before PG administration, while after PG, undulation of the nuclear envelope and nucleolus vacuolization was characteristic. The superovulated oocytes, in comparison, displayed the following deviations: premature perivitelline space formation, lack of nucleolar vacuolization, reduced amount of lipid droplets and lack of lipid‐mitochondria association, enlarged rough endoplasmic reticulum compartment, and increased condensation of chromatin and elongation, i.e., expansion of some cumulus cells. Degenerative oocytes were only found in the superovulated group. It is concluded that FSH‐stimulation is associated with reduced intrafollicular E2 and P4 concentrations and subcellular deviations in the oocytes that are established early in the superovulatory process. These deviations may contribute to the reduced developmental competence of superovulated oocytes. © 1994 Wiley‐Liss, Inc.
Correlation of zona‐binding with oocyte maturation and sperm motility in rhesus monkeys by hemizona assay
Authors: Pu X. C. , Ji W. Z. , Yang S. C. , Chen J. C. , Zou R. J. , Shang E. Y.
Published: February 2005

The hemizona assay (HZA) in Rhesus monkeys was employed to study the correlation of zona‐binding ability with sperm motility or with naturally developing oocytes at various maturational stages. Oocytes from unstimulated ovaries were retrieved within 2 hr from monkeys sacrificed for vaccine production (in reproductive season, but with their menstrual cycles not determined). Oocytes were divided into four groups based on their morphological maturation: 1) Oocytes surrounded by more than one cumulus layer (MC); 2) Oocytes retaining intact germinal vesicle nuclei (GV); 3) Oocytes with germinal vesicle breakdown showing distinct perivitelline space (PVS); and 4) Oocytes extruding the first polar body (PBI). The mean numbers of sperm bound to hemizona for PB1, PVS, GV, and MC groups were 132.9 ± 12.0, 71.5 ± 10.1, 36.1 ± 4.0, and 20.1 ± 2.9 (Mean ± SE), respectively. The four groups showed significant differences from each other in sperm/egg binding ability (P < 0.01). The number of bound sperm significantly increased with oocyte maturation. The present study also showed that zona‐binding ability was also affected by sperm motility. For sperm with 67.7% motility and sperm with 31.2% motility, the average numbers of bound sperm were 43.5 ± 2.2 and 25.3 ± 2.9 (Mean ± SE), respectively. There was significantly higher binding ability for sperm with higher motility (P < 0.01). The results suggest that: 1) The rhesus monkey model can serve as a very sensitive model for studying sperm/egg interaction by HZA; 2) Sperm motility positively correlated with sperm/egg binding; and 3) Sperm/egg binding ability increases with oocyte maturation. The binding ability is highest when oocytes matured to the PB1 stage, which is also the best opportunity for fertilization. This is strong evidence for the “zona maturation” hypothesis. © 1994 Wiley‐Liss, Inc.
Targeted disruption of int‐2 (fgf‐3) causes developmental defects in the tail and inner ear
Authors: Mansour Suzanne L.
Published: February 2005

The int‐2 gene (also designated fgf‐3) was originally identified because its transcription is activated by the nearby integration of mouse mammary tumor virus in virus‐induced tumors. Molecular analyses have revealed that the int‐2 gene produces at least four mRNAs, all of which encode a protein that has 40–50% amino acid sequence similarity with the fibroblast growth factors (FGFs). Int‐2 gene expression is localized to a small number of discrete sites in the developing mouse, but has not been detected in any nonneoplastic adult tissue. The expression data and the amino acid sequence similarity with the FGFs suggested several potential roles for int‐2 during normal development.

To evaluate these possibilities, we initiated a genetic analysis of int‐2. A gene‐targeting protocol was used to generate embryonic stem (ES) cells that are heterozygous for an insertion of the neor gene into the first proteincoding exon of int‐2. These cells were used to establish a line of mice that carry the gene disruption. Animals that are heterozygous for the int‐2neo allele are normal and fertile. Homozygous mutants survive embryonic development and can be visually identified after 12.5 days of gestation by a short, initially dorsally curled tail. This defect is potentially due to the disruption ofint‐2 expression in the primitive streak/tail bud. Most of the homozygous mutants die at orsoon after birth, but several have survived to adulthood. In addition to the tail phenotype, the surviving homozygotes show, to varying extents, symptoms characteristic of innerear abnormalities. The development of these defects could be attributed to the disruption of normal int‐2 expression in the hindbrain rhombomeres thought to induce inner ear development and/or the otocyst (precursor of the inner ear) itself. Other sites of int‐2 expression are not affected by the mutation. Although the tail phenotype is 100% penetrant, the inner ear phenotype caused by this mutation shows reduced penetrance and variable expressivity.

A second int‐2 allele was generated in ES cells by using homologous recombination to introduce a IacZ reporter gene into the int‐2 locus. This allele has also been established in a line of mice. The pattern of β‐galactosidase activity in carriers of the int‐2lacZ allele has been examined in embryos from 8.5–14.5 days of gestation and in neonatal heads using whole mount X‐gal staining. Expression of the lacZ gene mirrors the expected pattern of int‐2 mRNA. Several new sites int‐2 of expression not identified in the initial in situ hybridization studies of int‐2 mRNA have also been identified. Compound heterozygotes (int‐2neo/int‐2lacZ) were found to have the same defects in tail and innerear development as homozygous int‐2neo mice. After staining for β‐galactosidase activity, these mutants should allow an analysis of the int‐2 mutant phenotype at the cellular level. © 1994 Wiley‐Liss, Inc.
Studies on FGF‐2: Nuclear localization and function of high molecular weight forms and receptor binding in the absence of heparin
Authors: Rifkin Daniel B. , Moscatelli David , Roghani Monireh , Nagano Yutaka , Quarto Natalina , Klein Sharon , Bikfalvi Andreas
Published: February 2005

Multiple forms of FGF‐2 have been shown to exist in many cell types. These different species of molecular masses of 18, 21.5, 22, and 24 kDa are all translated via the use of alternate initiation codons. The three forms of HMW FGF‐2 initiate at CUGs codons, whereas the 18 kDa form initiates at an AUG codon. The entire 18 kDa sequence is contained within the larger forms of HMW FGF‐2 as the AUG codon is 3′ to the CUG codons. Although the 18 kDa form FGF‐2 is localized primarily in the cytosol, a significant fraction of the HMW FGF‐2 has a nuclear location. The nuclear localization of HMW FGF‐2 is determined by amino acid residues in the amino‐terminal extended sequence. The residues required for nuclear localization appear to be RG repeats that are found at multiple sites within the amino‐terminal extension of HMW FGF‐2. The nuclear localization of HMW FGF‐2 suggested that these species may have unique properties. By selecting permanent transfectants of 3T3 cells expressing HMW, 18 kDa FGF‐2, or all forms of FGF‐2, we have found that HMW FGF‐2 can endow cells with a phenotype different from that of cells expressing 18 kDa FGF‐2. These cells are transformed by what appears to be the intracellular action of HMW FGF‐2. The interaction of FGF‐2 with heparin has also been examined. Contrary to other reports claiming that FGF‐2 required heparin or heparan‐sulfate for interaction with its high‐affinity receptor, we have found that FGF‐2 binds to its receptor in the absence of glycosaminoglycans, and that this binding activates the receptor. © 1994 Wiley‐Liss, Inc.
Transcriptional regulation of the murine k‐fgf gene
Authors: Rizzino Angie , Rosfjord Edward
Published: February 2005

Embryonal carcinoma (EC) cells provide a useful model system for studying the roles of growth factors during early mammalian development. In 1988, we determined that EC cells express a member of the fibroblast growth factor (FGF) family that cannot be detected after EC cells undergo differentiation. Attempts to understand how differentiation regulates the production of FGFs led to the finding that EC cells express the fibroblast growth factor k‐FGF (FGF‐4), whereas there is a large decrease in the steady state levels of k‐FGF mRNA when EC cells differentiate. This suggested that transcription of the k‐fgf gene is repressed when EC cells differentiate. To investigate this possibility, we prepared a series of reporter gene constructs containing various regions of the murine k‐fgf gene. These constructs were transfected into two mouse EC cell lines and one mouse embryonic stem (ES) cell line. We determined that the mouse 5′ flanking region cannot support expression of the reporter gene. In both EC and ES cell lines, expression of the reporter gene is elevated greatly by the addition of a 316 bp region from the third exon of the murine k‐fgf gene. Sequence analysis of the 316 bp region identified one and possibly two conserved octamer binding motifs. These sequences are likely to be involved in regulation of the k‐fgf gene, because differentiation of EC cells is known to reduce the expression of octamer binding proteins, including Oct‐3. To test the possible role of octamer binding proteins, we examined the expression of our reporter gene constructs in F9‐differentiated cells and in PYS‐2 cells. In these cells, the expression of our k‐fgf/reporter gene constructs is very low. However, expression of the reporter gene is elevated significantly when these cells are cotransfected with a construct that contains an Oct‐3 cDNA under the control of a strong viral promoter. To test further the importance of the octamer motifs found in the 316 bp enhancer‐like region, we replaced the 316 bp region in our constructs with a smaller region (48 bp) that contains the downstream octamer motif and flanking sequences. Like the 316 bp region, this 48 bp region elevated the expression of the reporter gene, but it does so at a much lower level. Thus, it appears that the downstream octamer motif may be involved in the regulation of the k‐fgf gene, but that at least one other cis‐regulatory element present in the 316 bp enhancer is likely to be involved in the expression of the k‐fgf gene in EC cells. Lastly, we have identified two potentially important regulatory regions upstream of the transcription start site. One appears to regulate positively the expression of the murine k‐fgf gene in EC cells and in ES cells, and the other appears to regulate negatively the expression of the k‐fgf gene in these cells. © 1994 Wiley‐Liss, Inc.
Modulation of gamete and embryonic microenvironments by oviduct glycoproteins
Authors: H.F. Hunter R.
Published: February 2005

Studies on protein molecules in oviduct luminal fluid are viewed historically, and then in terms of more recent studies on a possible involvement of unique glycoproteins in embryonic development. As a caution, however, it is noted that incorporation of such molecules into the vitellus may be nonspecific. The question is raised as to whether oviduct glycoproteins could be acting primarily in a physical sense to stabilize differing chemical environments along the oviduct. Equally or more importantly, glycoproteins might be acting as carrier molecules to present cations and metabolic substrates at appropriate concentrations to the vitelline membrane. This latter possibility is examined in some detail and could be tested by manipulating the composition of the perivitelline fluid. Glycoproteins may also be critically involved in regulating the physiological competence of spermatozoa in the pre‐ and peri‐ovulatory oviduct, in maintaining a coordinated pattern of cilial beat, and in immunosuppressive functions within the oviduct, not least in those associated with the masking of paternal antigens on both spermatozoa and embryos. © 1994 Wiley‐Liss, Inc.
The biochemical role of SRY in sex determination
Authors: R. Harley V. , N. Goodfellow P.
Published: February 2005

The human sex‐determining gene on the Y chromosome, termed SRY, has recently been isolated by positional cloning; compelling evidence now exists equating SRY with the testis‐determing factor, TDF. The SRY gene product is an HMG box protein whose DNA‐binding activity is vital for testis formation as sex‐reversed patients with SRY mutations lack this activity in vitro. The in vivo DNA target for SRY, however, remains elusive. Here, we show, by gel retardation analysis, that SRY recognises specific DNA sequences and that such sequences exist upstream of the AMH promoter, a potential downstream target for SRY. We also describe the DNA bending and cruciform DNA‐binding functions of SRY and propose a model for the potential action of SRY in the “HMG‐1‐rich” mammalian nucleus. © 1994 Wiley‐Liss, Inc.
Regulation of gene expression by transcription factors Ets‐1 and Ets‐2
Authors: J. Tymms Martin , Kola Ismail
Published: February 2005

The ETS family of transcription factors have a DNA‐binding domain in common that binds a core GGA(A/T) DNA sequence. A large number of proteins have now been identified that contain an ETS DNA‐binding domain (see review by Wasylyk et al., 1993). Ets‐1 was first described as the cellular homolog of v‐ets, which is translated as a 135‐kDa gag–myb—ets fusion protein from the replication‐deficient retrovirus E26 in chickens. Ets‐2 was subsequently described as a closely related protein that contains the highly conserved ETS DNA‐binding domain. This paper considers the manner by which the two closely related genes, Ets‐1 and Ets‐2, apparently play distinct roles in embryo development and in the immune system of adult mice. Although both Ets‐1 and Ets‐2 transform fibroblasts (Seth et al., 1989), the temporal and tissue‐specific expression patterns suggest that these two proteins play distinct biological roles and consequently transactivate different downstream cellular target genes. © 1994 Wiley‐Liss, Inc.
Discriminating translation of insulin‐like growth factor‐II (IGF‐II) during mouse embryogenesis
Authors: Newell Susan , Ward Andrew , Graham Chris
Published: February 2005

The problem is to discover which of the promoters of the insulin‐like growth factor‐II gene stimulate the transcription of mRNA which is translated into protein. Three alternative leader exons are attached to the coding sequences in RNA transcribed from this gene in other systems, and it is mainly the paternal allele which is expressed in mouse development. Transcripts bearing each of the three leader exons were found in the RNA from the chorio‐allantoic placenta, visceral yolk sac, and embryo, starting at 9.5 days. A varying proportion of one abundant transcript was disengaged from the polysomes at different days of development. This transcript was prefixed by the longest of the three alternative untranslated 5′ leader exons (exon 2), and it was consistently associated with polysomes in the choroid plexus and leptomeninges of the brain. Many exon 2 transcripts were abbreviated by endonucleolytic cleavage and lacked a poly(A) tail. In contrast, the transcripts with the shortest leader (exon 3) were mainly displayed on polysomes at all the stages of development which were examined. During mouse development, the production of IGF‐II protein must be partly controlled by the mechanisms which regulate translation. © 1994 Wiley‐Liss, Inc.
Characterization of two antigenically related integral membrane proteins of the guinea pig sperm periacrosomal plasma membrane
Authors: Anne Westbrook‐Case V. , P. Winfrey Virginia , E. Olson Gary
Published: February 2005

The periacrosomal plasma membrane of mammalian spermatozoa functions both in recognition and in binding of the egg's zona pellucida and in the acrosome reaction. This study characterizes two antigenically related proteins with molecular weights of 35 kD (PM35) and 52 kD (PM52) of the guinea pig sperm periacrosomal plasma membrane. Polyclonal antisera were prepared against electrophoretically purified PM35 or PM52. Each antiserum recognized both the 35‐kD and 52‐kD polypeptides on Western blots, indicating that they are structurally related. This conclusion was supported by peptide mapping experiments demonstrating comparably sized fragments of both PM35 and PM52. Both PM35 and PM52 behave as integral membrane proteins during phase‐separation analysis with Triton X‐114. Electron microscopic immunocytochemistry and differential fractionation of sperm membranes established that both PM35 and PM52 are exclusively localized to the periacrosomal plasma membrane. Three different antisera were used for ultrastructural studies, and each specifically bound the cytoplasmic but not the extracellular membrane surface. The electrophoretic mobilities of the PM35 and PM52 polypeptides were unchanged during sperm maturation and during the ionophore‐induced acrosome reaction. The localization of PM35 and PM52 suggests a potential role for these integral plasma membrane proteins in signal transduction or membrane fusion events of the acrosome reaction. © 1994 Wiley‐Liss, Inc.
Molecular identities of human sperm proteins that bind human zona pellucida: Nature of sperm–zona interaction, tyrosine kinase activity, and involvement of FA‐1
Authors: K. Naz Rajesh , Ahmad Khaliq
Published: February 2005

The present study was conducted to investigate the molecular identities, nature of interaction, and tyrosine phosphorylation activity of the spermzona pellucida binding proteins in humans. Sperm proteins belcnging to four major molecular regions, namely 95, 63, 51, and 14–18 kDa, reacted with zona pellucida proteins in the Western blot and immunoprecipitation procedures. In these procedures, zona pellucida protein that reacted strongest with the sperm proteins belonged to the molecular region of 55 kDa (ZP3), besides weakly reacting proteins in the 110‐kDa (ZP1/ZP2) and 14–18‐kDa molecular regions. The major forces involved in the sperm‐zona protein interactions were of hydrophobic and ionic in nature. Three (95, 51, and 14–18 kDa) of the four molecular regions of sperm proteins that bound to the zona pellucida proteins also seem to involve o‐phospho‐L‐tyrosine residues in their interaction, and these proteins demonstrated the presence of phosphotyrosine residues, and the 51‐kDa protein also showed autophosphorylating activity in the in vitro kinase assay. The sperm binding zona protein of 55 kDa also demonstrated autophosphorylating activity. Using specific monoclonal antibody to the well characterized sperm‐specific glycoprotein, designated FA‐1, and the competitive inhibition in the immunoprecipitation procedure, it was found that the 51 kDa protein is indeed FA‐1 antigen. Besides elucidating the molecular nature of the spermzona interaction, these antigens will find application in the development of a multivalent contraceptive vaccine, and may also help in specific diagnosis and treatment of infertility mediated through defective gamete (sperm or oocyte) function. © 1994 Wiley‐Liss, Inc.
Tissue‐Specific variation in the length of the 5′ untranslated region of the βa‐inhibin mRNA in sheep
Authors: Fleming J. S. , Galloway S. M. , Crawford R. J. , Tisdall D. J. , Greenwood P. J.
Published: February 2005

The 5′ untranslated region (UTR) of βA inhibin mRNA was compared in a variety of sheep tissues, using primer extension. Considerable variation in the length and number of 5′ extended products were noted between tissues. Specific bands were noted in ovarian follicular RNA, which were also present in samples from corpora lutea, stroma, and placental cotyledon RNA. Other extended products were observed in RNA from corpora lutea, stroma, cotyledon, pituitary, bone marrow, frontal cortex, medial basal hypothalamus, adrenal, liver, and kidney, which were not present or weakly represented in follicular RNA, Additional tissue‐specific bands were noted in testis and bone marrow RNA. No specific differences in the lengths of the 5′ UTR of the βA inhibin mRNA were observed in sheep homozygous for the Booroola fecundity gene FecB, in any tissue studied. The coding region of ovine βA inhibin cDNA was sequenced and a genetic polymorphism confirmed within or close to the ovine βA inhibin gene.

We conclude that the βA inhibin gene is expressed widely in the sheep. Furthermore there is variation in the length of the 5′ UTR of βA inhibin mRNA between male and female gonads and other tissues, implying that expression of this gene is differentially controlled. However, the FecB mutation does not affect mRNA splicing events or the initiation site used in ovarian transcription. The mechanism by which the FecB mutation influences the amounts of βA inhibin mRNA, follicle‐stimulating hormone (FSH) secretion and ovulation rate has still to be elucidated. © 1995 Wiley‐Liss, Inc.
Endogenous substrate for energy metabolism and ultrastructural correlates in spermatozoa of the sea urchin Diadema setosum
Authors: Mita Masatoshi , Yasumasu Ikuo , Nakamura Masaru
Published: February 2005

Energy metabolism in spermatozoa of the sea urchin Diadema setosum of the order Diadematoida was examined. The spermatozoa contained not only several kinds of phospholipids and cholesterol, but also triglyceride (TG). Glycogen and glucose were present at extremely low levels. Following dilution of dry sperm and incubation in seawater, the TG content decreased rapidly. Other lipids, however, remained at constant levels, except for a slight increase in the level of free fatty acid. High lipase activity was demonstrated in the spermatozoa. 14C‐labeled fatty acid was oxidized to 14CO2. Ultrastructural study also showed that lipid globules were present at the bottom of the midpiece. After incubation in seawater, morphological changes in the lipid globules were observed and some vacuoles appeared. Thus, the results obtained strongly suggest that D. setosum spermatozoa obtain energy through oxidation of fatty acid from TG stored in the lipid globules at the midpieces. © 1995 Wiley‐Liss, Inc.
Vitelline coat of Unio elongatulus: II. Biochemical properties of the 220‐ and 180‐kD components
Authors: Focarelli R. , Santucci A. , Lampariello R. , Rosati Floriana
Published: February 2005

In a previous study we found that two glycoproteins with apparent molecular weights of 220 kD and 180 kD account for 80–90% of the material dissolved from the vitelline coat of the egg of the bivalve mollusk, Unio elongatulus (Focarelli and Rosati, 1993: Mol Reprod Dev 35:44–51). In this study we isolated and purified these glycoproteins by electroelution. The two proteins differ in many respects: the 180‐kD molecule is acidic in nature and highly heterogeneous, whereas the 220‐kD protein is neutral and less heterogeneous. Both seem to have O‐ and N‐linked oligosaccharide chains. The 180‐kD protein contains 13–16% carbohydrate, whereas the 220‐kD molecular contains only 7–8%. Amino acid analysis and peptide mapping also show that each protein represents a unique polypeptide chain. © 1995 Wiley‐Liss, Inc.
Molecular mechanisms of the antihormonal and antiimplantation effects of norethisterone and its a‐ring reduced metabolites
Authors: Castro Ivone , Cerbón Marco Antonio , Pasapera Ana Maria , GutiéRrez‐sagal Ruben , Garcia Gustavo A. , Orozco Carlos , Camacho‐Arroyo Ignacio , Anzaldua Rene , PéRez‐Palacios Gregorio
Published: February 2005

Norethisterone (NET) has been used as a contragestational postcoital agent. It is biotrans‐formed to 5α dihydro‐NET (5α‐NET) and 3β,5α tetrahydro‐NET (3β,5α‐NET) in target tissues. The participation of these metabolites in NET effects is unknown. We have examined the antiimplantation and antiprogestational effects of NET and its metabolites, in adult mated female rabbits, by assessing the number of implantation sites and the expression products of the uteroglobin (UTG) gene in the uterus, and by comparing them with those of RU‐486 and estradiol. Steroids were daily administered s.c. at several doses for 7 consecutive days, starting 24 hr after coitus. To assure that fertilization occurred in all animals, the presence of early pregnancy factor was determined. The results demonstrated that high doses (5 mg/kg) of NET reduced both implantation and the expression of the UTG gene. On the other hand, lower doses (1.5 mg/kg) of 5α‐NET produced an antiimplantation effect and suppressed UTG synthesis and its mRNA. These effects were similar to those of RU‐486. At lower doses (1 mg/kg), both estradiol and the estrogenic metabolite 3β,5α‐NET were also effective in inhibiting implantation and UTG gene expression. The overall results suggest that NET metabolites exert antiimplantation and antiprogestational effects through their interaction with progesterone and estrogen receptors, and provide an explanation for the molecular mechanisms involved in the postcoital contraceptive action of NET. © 1995 Wiley‐Liss, Inc.
In vitro viability and ultrastructural changes in bovine oocytes treated with a vitrification solution
Authors: Fuku Eiji , Liu Jianmin , Downey Bruce R.
Published: February 2005

Abattoir‐derived oocytes were exposed to a concentrated cryoprotectant solution (DAP213: 2 M DMSO, 1 M acetamide, 3 M propanediol, and 10% FCS in TCM199) for 1.5 or 5 min at the germinal vesicle (GV) stage or after maturation in vitro (IVM). Their viability was assessed by in vitro fertilization (IVF) and culture (IVC) to blastocysts. To investigate the effect of DAP213 on the ultrastructure, GV and IVM oocytes were processed for transmission electron microscopy (TEM) before (control) or after exposure to the cryoprotectant. DAP213 induced profound ultrastructural modifications to the microvilli and mitochondria, resulted in large vesicle formation, and, most significantly, caused the premature release of the cortical granules (CG). In IVM oocytes exposed to the cryoproteclant for 5 min, exocytosis of CG into the perivitelline space was common and the IVF rate was reduced (P <.05). After exposure for 5 min, GV oocytes displayed clusters of CG comparable to controls, but after IVM‐IVF, polyspermy rate was increased (P <.05). Furthermore, treated GV oocytes showed a reduced rate of cleavage and blastocyst formation and an increased percentage of oocytes exhibiting alterations in organelles, whereas the viability and ultrastructure of IVM oocytes treated for 1.5 min was not different from controls. These observations demonstrate that (1) cortical granule kinetics is one of the key elements controlling fertilizability of bovine oocytes treated with cryoprotectant, and (2) GV oocytes are more sensitive to the cryoprotectant than those that have already been matured in vitro.
Characterization of the heat shock protein P70 in rat spermatogenic cells
Authors: Raab Linda S. , Polakoski Kenneth L. , Hancock Larry W. , Hamilton David W.
Published: February 2005

A number of hsp70‐like proteins are associated with developing male germ cells. One of these molecules, P70, is not sensitive to heat stress and is germ cell‐specific, and its expression is developmentally regulated. We have characterized the association of the rat P70(rP70) with differentiating germ cells in the testis and with posttesticular sperm. An antibody originally raised against human sperm proacrosin (designated C3; Siegel et al., 1987: J Reprod Immunol 11:307–319) was found to immunostain rP70 by immunoblot analysis and was used in subsequent studies of the rP70 molecule. The C3 antibody reacted with P70 isoforms in rat, human, mouse, guinea pig, boar, and rooster testicular homogenates. In the developing rat testis, abundant rP70 protein levels were first detected on postnatal day 22, with upregulation to adult levels occurring after postnatal day 28. Purified populations of adult rat pachytene spermatocytes, round spermatids, and elongating spermatids, isolated by unit gravity velocity sedimentation, all expressed rP70. Posttesticular sperm exhibited a loss of the rP70 molecule; caput epididymal sperm were weakly immunoreactive for rP70, but no immunoreactivity was observed in either cauda epididymal sperm or epididymal fluid. In contrast to human ejaculated sperm, rat ejaculated sperm did not express rP70. The loss of P70 from rat posttesticular sperm may reflect species‐specific differences in P70 functions, which are thought to include a role in the structural modifications that occur during germ cell differentiation. © 1995 Wiley‐Liss, Inc.
Vitelline coat of Unio elongatulus: III. Glycan chain analysis of the 220‐ and 180‐kD components by means of lectins
Authors: Focarelli R. , Leotta F. , Lampariello R. , Rosati Floriana
Published: February 2005

Lectins of different binding specificity were used to analyze the oligosaccharide chains of the 220‐ and 180‐kD proteins of the Unio elongatulus egg vitelline coat (vc). The lectins ConA and RCA1 reacted with both glycoproteins, and four other lectins reacted with one or other vc components. The lectin from Galanthus nivalis, which recognizes terminal mannose residues of N‐linked high mannose type oligosaccharide chains, bound specifically to the 180‐kD protein. Binding sites for this lectin were found throughout the vc of the differentiating oocyte and the mature egg. Lectins specific for the O‐linked oligosaccharide chains, such as AIA and PNA, reacted only with the 220‐kD protein species. Binding sites for these lectins were found only in the crater region. The presence of fucosyl residues on the glycan chains was investigated with lectins from Lotus tetragonolobus and Aleuria aurantia. The latter was positive on both glycoproteins, whereas LTA was only positive to the 220‐kD species. The binding sites of both these lectins were in the same areas as those of PNA and AIA. These results suggest that while the 180‐kD protein is part of the entire vc structure, the 220‐kD protein is prevalently accumulated in the crater region. Since this is where sperm recognition and interaction take place, it has been suggested the 220‐kD protein acts as a ligand molecule in the sperm‐egg interaction. © 1995 Wiley‐Liss, Inc.