bluuebunny/crossref_metadata_2025 huggingface dataset:
Cleavage rate of haploid and diploid parthenogenetic mouse embryos during the preimplantation period
Authors: Henery C. C. , Kaufman M. H.
Published: February 2005

The lack of a paternal genome in parthenogenetic embryos clearly limits their postimplantation development, but apparently not their preimplantation development, since morphologically normal blastocysts can be formed. The cleavage rate of these embryos during the preimplantation period gives a better indication of the influence of their genetic constitution than blastocyst formation. Conflicting results from previous studies prompted us to use a more suitable method of following the development of haploid and diploid parthenogenetic embryos during this period.

Two classes of parthenogenetic embryos were analysed following the activation of oocytes in vitro with 7% ethanol: (1) single pronuclear (haploid) embryos and (2) two pronuclear (diploid) embryos. Each group was then transferred separately during the afternoon to the oviducts of recipients on the 1st day of pseudopregnancy. Control (diploid) 1‐cell fertilised embryos were isolated in the morning of finding a vaginal plug, and transferred to pseudopregnant recipients at approximately the same time of the day as the parthenogenones. Embryos were isolated at various times after the HCG injection to induce ovulation, from each of the three groups studied. Total cell counts were made of each embryo, and the log mean values were plotted against time. The gradient of the lines indicated that (1) the cell doubling time of the diploid parthenogenones was 12.25 ± 0.34 h, and was not significantly different from the value obtained for the control group (12.74 ± 1.17 h), and that (2) the cell doubling time of the haploid parthenogenones (15.25 ± 0.99 h) was slower than that of the diploid parthenogenones and the control diploid group. It appears from these findings that the ability of diploid parthenogenetic embryos to divide normally during the preimplantation period does not require the presence of a paternal genome. Diploid parthenogenones would, therefore, appear to be at a developmental advantage over haploid parthenogenones at the time of implantation, as their cell number is greater, and it is likely that this is related to the total number of cells that contribute to their inner cell mass.
Hemizona assay for measuring zona binding in the lowland gorilla
Authors: Lanzendorf S. E. , Holmgren W. J. , Johnson D. E. , Scobey M. J. , Jeyendran R. S.
Published: February 2005

The hemizona assay is a diagnostic test used to evaluate the binding potential of spermatozoa to zonae pellucida and has been used to predict fertilization potential in the human. In this study, frozen‐thawed gorilla spermatozoa were coincubated with human hemizonae to evaluate tight binding and to assess the use of human zonae in evaluating sperm fertility. Matching hemizonae were incubated with human sperm to serve as a control. For evaluation of binding studies in a homologous system, matching halves of gorilla hemizonae were coincubated with both gorilla and human sperm. Whole, intact zonae of both human and gorilla oocytes were also coincubated with heterologous sperm to determine of penetration into the perivitelline space could occur. This study found that gorilla sperm bound well to both gorilla and human hemizonae, with a mean of 112.5 and 81.0 tightly bound sperm, respectively. Human sperm also bound to gorilla (mean 229.5) and human (mean 236.5) hemizonae. Following incubation with intact gorilla zonae, motile human sperm were found within the perivitelline space. However, gorilla sperm were not visible within the perivitelline space of nonviable human oocytes. These findings demonstrate that the zonae of nonviable human oocytes can be used to assess sperm binding of gorilla sperm. Studies continue for optimizing assay condition and correlation of findings with the fertility potential of gorilla sperm.
Inhibitory effect of purines in meiotic maturation of denuded mouse oocytes
Authors: Shim Chanseob , Lee Dae Kee , Lee Chung Choo , Cho Wan Kyoo , Kim Kyungjin
Published: February 2005

The potential action of purines, such as hypoxanthine and adenosine, in meiotic arrest was examined using denuded mouse oocytes. The spontaneous meiotic maturation of denuded oocytes was significantly inhibited by hypoxanthine and/or adenosine in a dose‐dependent manner. Germinal vesicle breakdown (GVBD) was inhibited even at a low concentration (1 nM) of hypoxanthine, when hypoxanthine was microinjected into the cytoplasm of denuded oocytes. This inhibitory action was potentiated by co‐injection with allopurinol, a metabolic blocker of hypoxanthine that can block a metabolic pathway to uric acid. By contrast, a microinjection of adenosine was no longer effective in inhibiting GVBD. Inhibitory action of purines in meiotic maturation was correlated with sustaining intracellular cAMP levels. GVBD was resumed by econazole, one of the nitroimidazole derivatives which act as inhibitors of catalytic subunit of adenylate cyclase. This compound was effective in counteracting the effect of adenosine, but not the action of 3‐isobutyl‐1‐methylxanthine (IBMX) on GVBD, indicating that adenosine is probably exerted at the level of oocyte plasmalemma. These data suggest that the inhibitory action of hypoxanthine and adenosine in oocyte meiotic maturation may be involved in the regulation of cAMP metabolism in a differential manner.
Protein and nuclear changes in pig eggs at fertilization
Authors: Ding Jianchi , Clarke N. , Nagai T. , Moor R. M.
Published: February 2005

The nuclear restructuring that occurs between insemination and full pronuclear formation in pig eggs is accompanied by posttranslational changes to specific egg proteins. Sperm penetration begins in vitro at 3 hr postinsemination (hpi). By 5 hr, decondensing sperm heads and anaphase II plates are observed in 50% of eggs, and, by 8 hpi, both male and female pronuclei have formed. Three consistent changes to the pattern of newly synthesised proteins are triggered in this period; they affect the 46K, 25K, and 22K polypeptides. Changes are also triggered in the 180–200K polypeptides and in the 14K polypeptides, but these are highly variable. The same changes in the prefertilization pattern were observed when prelabelled eggs were used and new protein synthesis was suppressed. The first and most abrupt change involves the apparent catabolic elimination of a group of 46K unphosphorylated polypeptides (pl 7.3–6.4), whose synthesis was greatest before germinal vesicle breakdown but declined slowly in the final phase of maturation, then declined precipitously after activation. Ageing (beyond maturation) also leads to the disappearance of these polypeptides. The progressive disappearance of a set of 25K polypeptides and the concomitant appearance of a dominant 22K polypeptide is the most characteristic fertilization‐induced modification to porcine egg proteins. These modifications begin within 1 hr of sperm penetration or activation, are specific to the pig, and involve heavily phosphorylated polypeptides (25K, pl 6.7–6.0) whose synthesis is begun in the early metaphase I stage. Dual ([35S] and [32P]) labelling, protein blocking experiments, and use of alkaline phosphatase suggest that dephosphorylation selectively affects these 25K polypeptides and is mainly or wholly responsible for converting them (completely within 6 hr) to a single, new (22K, pl 7.6) species that is positively charged. The 25K/22K polypeptide modification has a close temporal relationship with the formation of the male and female pronuclei.
Human zona pellucida during in vitro fertilization: An ultrastructural study using saponin, ruthenium red, and osmium‐thiocarbohydrazide
Authors: Familiari Giuseppe , Nottola Stefania A. , Macchiarelli Guido , Micara Giulietta , Aragona Cesare , Motta Pietro M.
Published: February 2005

The human zona pellucida (ZP) and its changes during in vitro fertilization in oocytes at different maturational stages and polypronuclear ova at one‐ to four‐cells stages were studied by transmission electron microscopy (TEM) and correlative scanning electron microscopy (SEM). To define the microstructure of the ZP, its amorphous masking material was removed using a detergent (saponin), and its structural glycoproteins were stabilized with a cationic dye, ruthenium red, followed by osmium‐thiocarbohydrazide treatment. These methods allowed in all samples the clear visualization of variously arranged networks of filaments composing the outer and inner surfaces of the ZP. These filaments were straight or curved, 0.1–0.4 μ in length and 10–14 nm thick as seen via TEM or 22–28 nm thick as seen via SEM (the difference in thickness was due to the presence of the metal coating for SEM). The filament arrangement was remarkably different between the inner and outer surfaces of the ZP and among the various stages studied. The filaments of the outer surface of the ZP were basically arranged in “large” and “tight” meshed networks. Mature oocytes and fertilized (polypronuclear) ova had a regular alternating pattern of wide and tight meshed networks of filaments. On the other hand, immature and atretic oocytes displayed almost exclusively a tight meshed network of filaments. The inner surface filaments of the ZP of unfertilized oocytes at any stage were arranged in repetitive structures characterized by numerous short and straight filaments anastomosing with each other and sometimes forming at the intersections small, rounded structures. After fertilization, the inner surface of the ZP displayed numerous areas where filaments fused together. Collectively, these data clearly reveal that oocyte maturation and fertilization in humans are accompanied by changes of ZP filaments arrangement, which may be relevant in the processes of binding, penetration, and selection of spermatozoa. © 1992 Wiley‐Liss, Inc.
Differential expression of the TGF‐β isoforms in embryogenesis suggests specific roles in developing and adult tissues
Authors: Roberts Anita B. , Sporn Michael B.
Published: February 2005

The TGF‐β's are multifunctional, pleiotropic molecules with major effects in control of cellular migration, cellular proliferation, and elaboration of extracellular matrix. Thus far, five distinct isoforms of TGF‐β have been described, each approximately 65–85% homologous and containing the characteristic 9 positionally conserved cysteine residues. Although the actions of the activated mature forms of the different isoforms on cells are qualitatively similar in most cases, there are a few examples of distinct activities. For example, TGF‐β's 1 and 3, but not TGF‐β2, inhibit the growth of large vessel endothelial cells, and TGF‐β's 2 and 3, but not TGF‐β1, inhibit the survival of cultured embryonic chick ciliary ganglionic neurons. In addition, selective targeting of the latent forms of the TGF‐β's is suggested by the observation that latent TGF‐β2 is the prominent isoform found in body fluids such as amniotic fluid, breast milk, and the aqueous and vitreous humor of the eye; it is noteworthy in this regard that TGF‐β2 is unique among various isoforms in that it lacks a RGD integrin‐binding sequence in its precursor. The most dramatic differences in the TGF‐β isoforms are seen at the level of expression, where there is now a wealth of data demonstrating both spatially and temporally distinct expression of both the mRNAs and proteins in developing tissues, regenerating tissues, and in pathologic responses. Moreover, the post‐transcriptional regulation of TGF‐β expression by members of the steroid/retinoid family of nuclear receptors is also isoform‐specific; thus, treatment of keratinocytes with retinoids induces secretion of TGF‐β2, whereas treatment of breast cancer cells with gestodene, a synthetic progestin, induces secretion of TGF‐β1. Recent characterization of the 5′ regulatory regions of the human TGF‐β 1, 2, and 3 genes suggests that distinct features of the promoters, including the presence of TATAA boxes and transcription factor binding sites, form the basis for the observed differential transcription. © 1992 Wiley‐Liss, Inc.
Regulation of mammary growth and function by TGF‐β
Authors: Daniel Charles W. , Robinson Stephen D.
Published: February 2005

We have previously shown that TGF‐β1 rapidly and reversibly inhibits ductal growth in vivo when administered by miniature slow‐release plastic implants. A possible role for endogenous TGF‐β1 was suggested by the observation that the normal gland displayed substantial, developmentally regulated levels of TGF‐β1 transcripts and protein. These studies have now been extended to include the other two mammalian TGF‐β isoforms. When tested with slow‐release plastic implants, TGF‐β2 and TGF‐β3 also caused disappearance of the proliferating mammary stem cell layer, with rapid involution of ductal end buds and cessation of glandular growth. None of the isoforms was active in inhibiting alveolar morphogenesis. We conclude that under the conditions of these tests, the three mammaliian isoforms are functionally equivalent. However, striking differences in patterns of gene expression and in the distribution of immunoreactive peptides suggest that TGF‐β2 was expressed only at low levels, and mainly during pregancy. TGF‐β3 was expressed in ductal stroma and epithelium, and was the only isoform detected in myoepithelial cells. Developing alveolar tissue and its associated ducts displayed striking TGF‐β3 gene expression and immunostaining, which were greatly reduced during lactation. We are now investigating the possibility that the observed high levels of TGF‐β expression in pregnancy, particularly of TGF‐β3, and the absence of substantial expression of any isoform during lactation, may indicate a role for the TGF‐β in regulating functional differentiation or the onset of milk secretion. © 1992 Wiley‐Liss, Inc.
Localization of ras proto‐oncogene expression during development in Xenopus Laevis
Authors: Andéol Yannick , Méchali Marcel , Hourdry Jacques
Published: February 2005

The expression of the ras protooncogene was investigated in Xenopus laevis, throughout development, by in situ hybridization using a 35S‐labelled antisense RNA probe. During oogenesis, the ras RNA was strongly expressed in the cytoplasm of previtellogenic oocytes and further diluted between yolk platelets; no specific localization of transcripts was observed. The signal density was particularly weak over embryo sections until the tailbud stage. On the other hand, a high level of ras RNA expression was detected on sections through the young tadpoles. An intense labelling was observed in several areas, including the branchial apparatus, gut, somites, nervous system, and lens. It is noteworthy that the heterogeneity of labelling increases as tadpoles grow older. Together, these results are discussed in relation to cellular events appearing throughout the early development.
Transcription of the H19 gene in differentiating cytotrophoblasts from human placenta
Authors: Rachmilewitz J. , Gileadi O. , Eldar‐Geva T. , Schneider T. , De‐Groot N. , Hochberg A.
Published: February 2005

Placental differentiation is closely correlated with the appearance of specific proteins, yet factors regulating cytotrophoblast differentiation are unknown. One strategy employed to search for such factors makes use of differential screening of cDNA libraries. For this purpose, cytotrophoblasts were isolated from human term placentae and cultured for 24 and 120 hr. cDNA libraries were constructed from the cell's RNA, and differential screening resulted in the isolation of three identical clones highly expressed after 120 hr. A DNA sequencing of 139 bp at the 3′ end of these clones and a search of the data bank revealed that the sequence was identical to the parallel domain in the human H19 gene. This highly conserved gene is unusual in that it may not encode a protein. In the mouse, its RNA was shown to accumulate to high levels in embryonic tissues of endodermal and mesodermal origin. Our present findings imply that, in humans, the H19 gene is efficiently expressed in placental tissue and differentiated cytotrophoblasts, which are of ectodermal origin. RNA blot hybridization revealed a unique bimodal pattern of expression for the H19 gene in cultured cytotrophoblasts. The modulation in expression of H19 during cytotrophoblast growth was not due to the increase in the number of multinuclear cells. Size fractionation of cytotrophoblasts by centrifugal elutriation revealed that H19 expression is correlated with the stage of cell differentiation. We therefore propose that H19 transcripts might play a regulatory role in the process of cytotrophoblast differentiation.
Microtubule distribution during fertilization in the rabbit
Authors: Yllera‐Fernandez Maria Del Mar , Crozet Nicole , Ahmed‐Ali Michèle
Published: February 2005

The distribution of microtubles was studied during fertilization of the rabbit oocyte by immunofluorescence microscopy after staining with an anti‐α‐tubulin antibody. In ovulated oocytes, microtubules were found exclusively in the meiotic spindle. At fertilization, the paternal centrosome generated sperm astral microtubules. During pronclear development, the sperm aster increased in size, and microtubules extended from the male pronucleus to the egg center and towards the female pronucleus. These observations indicate that microtubules emanating from the sperm centrosome were involved in the movements leading to the union of the male and female pronuclei. At late pronuclear stage, microtubules surrounded the adjacent pronuclei. The mitotic spindle that emerged from the perinuclear microtubules contained broad anastral poles.
A rat sperm flagellar surface antigen that originates in the testis and is expressed on the flagellar surface during epididymal transit
Authors: Toshimori K. , Tanii I. , Araki S. , Oura C.
Published: February 2005

We identified a rat sperm flagellar surface antigen using an IgG1 monoclonal antibody (MC31) against rat epididymal sperm. Avidin‐biotin‐peroxidase immunohistochemistry demonstrated that the antigen was first expressed in the cytoplasm of early primary spermatocytes, then gradually became restricted to the principal piece of the sperm flagellum during spermatogenesis. However, when the sperm reached the corpus epididymidis, the antigen was expressed on the surface of both the principal piece and the midpiece of the flagellum. The epithelial cells of the epididymis were not stained with MC31. Immunogold electron microscopy showed that the antigen was present on the surface of the sperm flagellar plasma membrane. Immunoblotting of Triton X‐100 extracts of epididymal sperm after one‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) under nonreducing conditions demonstrated that MC31 detected a major antigen of 26,000–28,000 daltons (26–28K). Two‐dimensional isoelectric focusing and SDS‐PAGE indicated that the 26–28K antigen had an isoelectric focusing point (pl) of 5.8‐5.3; minor antigens were also detected from 26K (pl 5.8) to 35K (pl 5.0). These results indicate that the antigen recognized by MC31 is an acidic 26–35K protein that originates in the testis, is integrated into the sperm flagellar plasma membrane of the principal piece during spermatogenesis, and then is expressed on the entire flagellar surface during epididymal transit. © 1992 Wiley‐Liss, Inc.
Extraplacental human fetal tissues express mRNA transcripts encoding the human chrorionic gonadotropin‐β subunit protein
Authors: Rothman Paula A. , Chao Victor A. , Taylor Mark R. , Kuhn Robert W. , Jaffe Robert B. , Taylor Robert N.
Published: February 2005

The glycoprotein hormone human chorionic gonadotropin (hCG) is synthesized in large quantities by the developing placenta, reaching peak concentrations in maternal blood during the late first trimester and early midtrimester of pregnancey. In general it is believed that the β‐subunit of this dimeric hormone is expressed in pituitary gonadotropes, thyrotropes, and trophoblasts, while the β‐subunit is expressed exclusively by trophoblasts. Studies from our laboratory and other laboratories have shown that some midtrimester human fetal tissues, in addition to the placenta, can synthesize proteins that appear to be very similar to the β‐subunit of hCG. To define precisely the nature of this putative hCG‐β‐subunit in extraplacental fetal tissues, we have examined the mRNA from a variety of human fetal and adult tissues using nucleic acid hybridization and reverse transcription‐polymerase chain reaction (PCR) methods. Our results demonstrate that midtrimester fetal kidney and adrenal tissues contain hCG‐β mRNA transcripts at concentrations comparable to that of placenta, while fetal lung, brain, muscle, and adult adrenal contain only trace to undetectable levels of hCG‐β mRNA. By restriction endonuclease mapping of PCR fragments from fetal tissue cDNAs, we show that the hCG‐β transcript expressed in midtrimester human fetal organs is a bone fide copy of hCG‐β gene No. 5 of the β‐subunit gene family located on chromosome 19. © 1992 Wiley‐Liss, Inc.
Effects of protein synthesis on maturation, sperm penetration, and pronuclear development in porcine oocytes
Authors: Ding Jianchi , Moor Robert M. , Foxcroft George R.
Published: February 2005

In vitro matured porcine oocytes were used to test the importance of protein synthesis for sperm penetration, the second meiotic division, and pronuclear development. Experiments were carried out to measure rates of protein synthesis inhibitors (35 μM or 350 μM cycloheximide or a combination of inhibitors) (study 1); to test for sperm penetration and pronuclear development when protein synthesis was inhibited during fertilization (study 2); to test for oocyte meiosis, sperm penetration, and female and male pronuclear development when protein synthesis was inhibited during maturation (oocyte maturation in vitro with addition of inhibitor at 0, 24, or 36 hr of culture) (study 3); and to analyze the changes in the pattern of protein synthesis during these phases. Sperm penetration, oocyte meiosis, and female pronuclear development were not affected by the total inhibition of protein synthesis during fertilization. By contrast, inhibiting protein synthesis during maturation severely impaired the completion of meiosis and pronuclear development. Although inhibition of protein synthesis after 36 hr of maturation culture did not totally block male pronuclear development (MPN), the rate of MPN formation was lower than for controls (52% vs. 72%, P < 0.05). However, protein synthesis was absolutely essential between 24 and 36 hr for the formation of MPN after decondensation. This period of maturation coincided with the dominant phase of protein reprogramming in the oocyte. © 1992 Wiley‐Liss, Inc.
F‐Actin in acrosome‐reacted boar spermatozoa
Authors: Castellani‐Ceresa L. , Brivio M. F. , Radaelli G.
Published: February 2005

Biochemical and immunoelectron microscopic methods have been used to analyze the distribution of actin in boar spermatozoa and its state of aggregation before and after acrosome reaction. F‐actin was detected on sperm head and tail by electron microscopy using an improved phalloidin probe: incubation with a fluorescein–phalloidin complex and an anti‐fluorescein antibody, followed by labeling with protein A‐gold complex. Gold particles, indicating the presence of F‐actin, were localized on the sperm surface of the acrosome‐reacted spermatozoa. Specific labeling was localized (1) between the outer acrosomal membrane and the plasma membrane in the equatorial region, (2) between the outer surface of the fibrous sheath and the plasma membrane in the postacrosomal region, (3) around the connecting piece and the neck region, and (4) on the external surface of the fibrous sheath in the principal piece of the tail. Furthermore, after NP‐40 extraction, the SDS‐PAGE revealed a difference in solubility between reacted and unreacted boar spermatozoa, reflecting actin polymerization. We conclude that most actin in the acrosome reacted boar spermatozoa is polymeric. © 1992 Wiley‐Liss, Inc.
Genetic manipulation of mammalian dictyate oocytes: Factors affecting transient expression of microinjected DNA templates
Authors: Bevilacqua Arturo , Kinnunen Leann H. , Mangia Franco
Published: February 2005

Transcription of exogenous DNA templates in mouse ovarian oocytes was investigated by microinjecting constructs encoding for the Escherichia coli lacZ gene under control of promoters from: (1) the mouse hsp68 gene; (2) the human β‐actin gene; and (3) simian virus 40 (SV40) early genes. Various amounts of circular or linear DNA constructs were injected into dictyate oocyte nuclei at different stages of follicle growth, and the β‐galactosidase activity was then cytochemically evaluated in single cells. In middle‐sized growing oocytes, expression of circular constructs was observed with amounts of DNA ranging from 50 to 103 plasmid copies/nucleus and was first observed 10–12 hr after injection. Maximal expression levels were reached by 17 hr after injection and were specific for the constructs used. Circular constructs containing the hsp68 and early SV40 promoters were expressed at similar levels in small‐ and middle‐sized growing oocytes, while the construct carrying the β‐actin promoter was expressed only in small‐sized cell. In contrast to growing oocytes, these constructs were never expressed in fully grown oocytes. DNA linearization depressed construct activity regardless of the site of cleavage. These results show that: (1) lacZ is a valuable reporter gene in the analysis of eukaryotic promote activity in dictyate mouse oocytes; (2) transient construct expression requires the injection of DNA in circular form; and (3) the expression efficiency of different DNA templates is dependent on the presence of a specific promoter and on the differentiation stage of oocytes analyzed. © 1992 Wiley‐Liss, Inc.
A comparison of two autoradiographic methods for detecting radiolabeled nucleic acids in embryos
Authors: Plante Louise , Pollard John W. , King W. Allan
Published: February 2005

Two methods for preparing embryos for autoradiographic study of newly synthesized nucleic acids are described and compared. The first method consists of rapidly fixing radiolabeled embryos with acetic acid:methanol, spreading them on glass slides and exposing them for 8 days with a photographic emulsion. The second method consists of fixing, embedding in resin, and sectioning the embryos before their exposure with the emulsion for 3 weeks. Both techniques have many applications in studies of early embryonic activity, but the spread technique is very sensitive, simpler, and faster. © 1992 Wiley‐Liss, Inc.
Unexpected position‐dependent expression of H‐2 and β2‐microglobulin/lacz transgenes
Authors: Cohen‐Tannoudji M. , Morello D. , Babinet C.
Published: February 2005

In order to study sequences in volved in the developmentally regulated and tissue‐specific expression of the class I Major Histocompatibility Complex (MHC) genes, we have constructed several H‐2/lacZ transgenic lines in which the 5′ regulatory sequences of the H‐2Kbgene are linked to theEscherichia coliβ‐galactosidase (lacZ) gene. In five H‐2/lacZ lines, the pattern of lacZ expression, detected histochemically varied greatly from line to line. None of the H‐2/lacZ transgenes were transcribed in cells normally expressing a high level of endogenous H‐2 molecules, although these H‐2 regulatory sequences have been shown to be sufficient to drive tissuespecific expression of other reporter genes. Interstingly, when constructs containing 5′ β2‐microglobulin (β2m) regulatory sequences linked to lacZ were used to derive transgenic lines, similar results were obtained. A survey of lacZ labeling in H‐2/lacZ and β2m/lacZ transgenic mice strongly suggests that these transgenes are very sensitive to position effect, lacZ expression being controlled by endogenous chromosomal regulatory elements specific for each insertion site. Here we describe the complex pattern of lacZ expression in the different transgenic lines during development; we discuss the unusual properties of these transgenes and underline their potential use for developmental studies and characterization of genomic sequences involved in spatiotemporal gene expression. © 1992 Wiley‐Liss, Inc.
Structural changes in sperm from the fiddler crab, Uca tangeri (crustacea, brachyura), during the acrosome reaction
Authors: Medina Antonio , Rodriguez Antonio
Published: February 2005

The acrosome reaction (AR) was induced in sperm from the brachyuran crustacean Uca tangeri either by mixing male and female gametes in filtered seawater or by treating the spermatozoa with the divalent cation ionophore A23187. This latter method provided a sufficient number of reacted spermatozoa to allow a detailed ultrastructural study of the AR. The process consists of two separate phases: (a) initial release of the acrosomal vesicle contents, and (b) further elongation of the acrosomal filament, which causes reversal of the rigid capsule limiting the acrosomal vesicle contents. The elongate acrosomal filament consists of an apical perforatorium and a basal columnar structure called here the proximal piece. The former derives from the perforatorium of the uninduced sperm stage with only small ultrastructural changes. The proximal piece forms from myelin‐like membrane layers which are initially distributed all around the subacrosomal region and then accumulate in a column at the perforatorial base, thus promoting a sudden forward projection of the perforatorium. The AR in brachyurans is thought to be a passive mechanism that utilizes the negative pressure exerted on the nucleus—caused by emptying of the acrosomal vesicle—for an organized accumulation of membrane‐rich material immediately behind the perforatorium, with the final result of the raising of a 3 μm long acrosomal filament. © 1992 Wiley‐Liss, Inc.
Analysis of the expression of a large number of novel genes isolated from mouse prepubertal testis
Authors: Starborg Maria , Brundell Eva , Höög Christer
Published: February 2005

The analysis of genes expressed in a restricted temporal and spatial manner during spermatogenesis has given insights into different gene‐regulatory mechanisms active in germ cells. However, very few genes have so far been described that are predominantly active in spermatogonia and the early meiotic cell types of testis. To isolate a battery of such genes, more than 100 different mRNA molecules were isolated from a mouse prepubertal testicular cDNA library, and their expression patterns in different tissues analyzed. Thirty mRNAs, 26 of them previously not described in the literature, were found to be predominantly expressed in mouse testis. A detailed analysis of their expression patterns identified a number of mRNA molecules differentially expressed in testicular cell types, including both germ cells and somatic cell types. Characterization of these mRNAs also revealed five distinct temporal phases of gene expression during prepubertal germ cell development. Three different genes, mainly active in the spermatogonial and the early meiotic cell types of testis, were isolated and will be used to characterize further stage‐specific gene expression during germ cell differentiation. © 1992 Wiley‐Liss, Inc.
Identification of a mouse B‐type cyclin which exhibits developmentally regulated expression in the germ line
Authors: Chapman Deborah L. , Wolgemuth Debra J.
Published: February 2005

To begin to examine the function of cyclins in mammalian germ cells, we have screened an adult mouse testis cDNA library for the presence of B‐type cyclins. We have isolated cDNAs that encode a murine B‐type cyclin, which has been designated cycB1. cycB1 was shown to be expressed in several adult tissues and in the midgestation mouse embryo. In the adult tissues, the highest levels of cycB1 transcripts were seen in the testis and ovary, which contain germ cells at various stages of differentiation. The major transcripts corresponding to cycB1 are 1.7 and 2.5 kb, with the 1.7 kb species being the predominant testicular transcript and the 2.5 kb species more abundant in the ovary. Examination of cDNAs corresponding to the 2.5 kb and 1.7 kb mRNAs revealed that these transcripts encode identical proteins, differing only in the polyadenylation signal used and therefore in the length of their 3′ untranslated regions. Northern blot and in situ hybridization analyses revealed that the predominant sites of cycB1 expression in the testis and ovary were in the germinal compartment, particularly in early round spermatids in the testis and growing oocytes in the ovary. Thus cycB1 is expressed in both meiotic and postmeiotic cells. This pattern of cycB1 expression further suggests that cycB1 may have different functions in the two cell types, only one of which correlates with progression of the cell cycle. © 1992 Wiley‐Liss, Inc.
Mediation of the actions of insulin and insulin‐like growth factor‐1 on preimplantation mouse embryos in vitro
Authors: Harvey Mark B. , Kaye Peter L.
Published: February 2005

Previous studies showed that both insulin and insulin‐like growth factor‐1 (IGF‐1) stimulate metabolism and growth of preimplantation embryos. Because the effects of insulin occur with very low doses, it was suggested that its effects were mediated by its own receptors. However, the effects of IGF‐1 occurred at higher doses, suggestive of cross reaction with the insulin receptor but still in the range for mediation via its own receptor. The aim of this study was to investigate the mediation of the metabolic and growth effects of insulin and IGF‐1 using a specific insulin receptor antagonist. The antagonistic B‐10 Fab fragment (B‐10f) completely blocked stimulation of protein synthesis by both insulin and IGF‐1, indicating that the insulin receptor mediates this action of both hormones. Alternately, only insulin's stimulation of inner cell mass mitogenesis and morphological development was inhibited by the B‐10 Fab fragment. This showed that growth stimulation by insulin and IGF‐1 was mediated via different receptors, insulin through its own receptor and IGF‐1 through some other receptor. However, mediation via the IGF‐2 receptor is not excluded since IGF‐1 stimulates compaction when there is evidence for only the presence of the IGF‐2 receptor. In summary, insulin or IGF‐1 at physiological concentrations stimulates preimplantation mouse embryos, suggesting an important role for both these growth factors in early development. © 1992 Wiley‐Liss, Inc.
Phosphatidylcholine and phosphatidylinositol‐specific phospholipases C of bull and rabbit spermatozoa
Authors: Hinkovska‐Galchev Vania , Srivastava Prakash N.
Published: February 2005

Acrosomal reaction is an essential prerequisite to fertilization. The changes in lipid composition of sperm membranes cause fusion of the plasma and outer acrosomal membranes that results in the exocytosis of acrosomal contents. We report that both bull and rabbit spermatozoa contain a phosphatidylcholine‐specific phospholipase C (PC‐PLC) that hydrolyzes L‐α‐dipalmitoyl‐(choline‐methyl‐14C‐153.0 Ci/mmol and a phosphatidyl‐inositol‐specific phospholipase C (Pl‐PLC)) that hydrolyzes L‐α‐(Myo)‐lnositol‐2‐3H (N)‐5.2 Ci mmol. Pl‐PLC from bull sperm acrosome has been purified 568 × fold with a specific activity 6.25 ± 0.6 nmol/min/mg protein, km 0.004 mM, and Vmax 12 nmol/min/mg protein. Both enzymes had optimum at pH 7.5. The activity of PC‐PLC remained unaffected by varying concentrations of Ca2+, whereas Pl‐PLC activity was significantly increased. The bulk of Pl‐PLC was found to be associated with inner acrosomal membrane of bull and rabbit sperm, while PC‐PLC was found in the outer acrosomal membranes in the bull sperm and the plasma membrane of the rabbit sperm. Both enzymes are compartmentalized in sperm cell. © 1992 Wiley‐Liss, Inc.
Biosynthesis of prostaglandins by human spermatozoa in vitro and their role in acrosome reaction and fertilization
Authors: Roy Ashim C. , Ratnam Shan S.
Published: February 2005

Five homogenates of human sperm cells were separately incubated with [14C]arachidonic acid in the presence of reduced glutathione, L‐tryptophan, and haematin as cofactors. The cyclooxygenase products of archidonic acid metabolism were extracted, separated, and measuréd for their radioactivity. The rate of formation of prostaglandin (PG)D2, PGE2, PGF2α, 6‐keto PGF1α, and thromboxane (TX)B2 were 18.0 ± 1.11, 10.9 ± 0.68, 5.8 ± 0.21, 3.9 ± 0.13 and 6.6 ± 0.52 pmol/106 cells/min, respectively. These results are discussed in relation to the hypothesis that cyclooxygenase metabolites of certain polyunsaturated fatty acids play an important part in the sperm acrosome reaction and fertilization. © 1992 Wiley‐Liss, Inc.
In vitro decondensation of mammalian sperm and subsequent formation of pronuclei‐like structures for micromanipulation
Authors: Montag Markus , Tok Veronica , Liow Swee‐Lian , Bongso Ariff , Ng Soon‐Chye
Published: February 2005

In this study, we describe an efficient protocol for the formation of in vitro developed pronuclei for micromanipulation techniques. Our approach involved incubation of demembranated or permeabilized mammalian sperm in a phosphate buffer supplemented with heparin and β‐mercaptoethanol. Under the prevailing conditions, we achieved a uniform and reliable synchronous decondensation of sperm nuclear DNA. This initial decondensation facilitated the removal of mammalian protamines upon subsequent incubation in an amphibian egg extract. The interchange of protamines for histones to stabilize the DNA structure is recognized as a prerequisite for pronuclear formation. Furthermore, immunocytochemical studies have revealed that pronuclear development is accompanied by the formation of a nuclear lamina with corresponding DNA synthesis. The method described gave a high yield of nuclei during pronuclear formation. Ultimately, our aim is to transfer the in vitro‐developed pronuclei into mammalian oocytes by micromanipulation. This novel procedure may prove useful in alleviating severe male factor problems especially in oligozoospermic cases in our in vitro fertilization center. (c) 1992 Wiley‐Liss, Inc.
Expression of estrogen receptor gene in mouse oocyte and during embryogenesis
Authors: Wu Tsung‐Chieh J. , Wang Lai , Wan Yu‐Jui Y.
Published: February 2005

Estrogen is required for oocyte maturation and embryonic development in vivo; however, the mechanism involved is not clear. Since the effect of estrogen is mediated through the estrogen receptor (ER), we examined the ontogeny and expression of the ER gene in mouse oocytes and embryos of various gestational stages using the highly sensitive reverse transcriptase‐polymerase chain reaction (RT‐PCR) technique. Total RNA, extracted from 40 ovulated oocytes, 2‐cell embryos, morulae, and blastocysts, was reverse transcribed into cDNA. A pair of primers flanking the 453‐bp region encoding the hormone‐binding domain of ER was used for 30 cycles of PCR. The identity of the amplified product was confirmed by sizing and Southern blot hybridization. The results indicated that ER gene is expressed in unfertilized oocytes and cumulus–oocyte complexes. The amount of ER mRNA decreases in 2‐cell embryos, coincident with degradation of maternal mRNA. No ER transcript can be detected in the morulae or blastocyst stage when the embryonic genome has been activated. Postimplantation embryos do not contain detectable ER mRNA until gestation day 8. The levels of ER mRNA increase from day 10 to day 18 of gestation. These data suggest that estrogen, secreted by granulosa cells, may directly influence oocyte growth and maturation in vivo. Since estrogen is known to stimulate the production of growth factors in mouse uteri, the absence of ER mRNA in periimplantation embryos suggests that the effects of estrogen on early embryogenesis may be indirect, i.e., through estrogen‐regulated growth‐promoting factors produced by the reproductive tract. In mid‐ and late‐post‐implantation embryos which contain ER mRNA, estrogen may affect embryonic development through the receptor‐mediated mechanisms. © 1992 Wiley‐Liss, Inc.
Human sperm proteins from testicular and epididymal origin that participate in fertilization: Modulation of sperm binding to zona‐free hamster oocytes, using monoclonal antibodies
Authors: Boue Franck , Lassalle Bruno , Duquenne Clotilde , Villaroya Svetlozara , Testart Jacques , Lefevre Anick , Finaz Catherine
Published: February 2005

In order to identify human sperm surface proteins involved in the gamete recognition process, mouse monoclonal antibodies were directed against human spermatozoa and screened with live spermatozoa by enzyme‐linked immunosorbent assay (ELISA). Immunoperoxydase staining of human testis showed the early presence of four corresponding proteins on germinal cells, while six were detected primarily in testis fluid. The presence of 17 proteins was evidenced in the epididymis. Eight were detected with a decreasing gradient from the begining to the end of the organ, including vasa efferentia for three of them. The other nine were observed in only one defined segment, usually the caput epididymis, which was found to be the most active region. Comparison of spermatozoa patterns from testis, vasa efferentia, and the three regions of epididymis pointed out a progressive coating. By contrast, three antibodies displayed a migration of spermatozoa surface domains in the course of epididymal transit. Six antibodies were found to inhibit human spermatozoa adherence to zona‐free hamster oocytes, while nine promoted it. Molecular weights of antigens corresponding to nine of the antibodies ranged from 11 to 215 kDa. No correlation could be established with previously described human proteins. These observations emphasize the role of epididymis in human sperm maturation. © 1992 Wiley‐Liss, Inc.
The cell biology of blastocyst development
Authors: Watson Andrew J.
Published: February 2005

Preimplantation development encompasses the “free”‐living period of mammalian embryo‐genesis, which culminates in the formation of a fluid‐filled structure, the blastocyst. Cavitation (blastocyst formation) is accompanied by the expression of a novel set of gene products that contribute directly to the attainment of cell polarity with the trophectoderm, which is both the first epithelium of development and the outer cell layer encircling the inner cell mass of the blastocyst. Several of these gene products have been identified and include the tight junction (ZO‐1), Na/K‐ATPase (α and β subunits), uvomorulin, gap junction (connexin43), and growth factors such as transforming growth factor‐α (TGF‐α) and epidermal growth factor (EGF). This review will examine the role(s) of each of these gene products during the onset and progression of blastocyst formation. The trophectodermal tight junctional permeability seal regulates the leakage of blastocoel fluid and also assists in the maintenance of a polarized Na/K‐ATPase distribution to the basolateral plasma membrane domain of the mural trophectoderm. The polarized distribution of the Na/K‐ATPase plays an integral role in the establishment of a trans‐trophectoderm Na+ gradient, which drives the osmotic accumulation of water across the epithelium into the nascent blastocoelic cavity. The cell adhesion provided by uvomorulin is necessary for the establishment of the tight junctional seal, as well as the maintenance of the polarized Na/K‐ATPase distribution. Growth factors such as TGF‐α and EGF stimulate an increase in the rate of blastocoel expansion, which could, in part, be mediated by secondary messengers that result in an increase in Na/K‐ATPase activity. Insight into the mechanism of cavitation has, therefore, directly linked blastocyst formation to trophectoderm cell differentiation, which arises through fundamental cell biological processes that are directly involved in the attainment of epithelial cell polarity. © 1992 Wiley‐Liss, Inc.
A major mRNA of the human epididymal principal cells, HE5, encodes the leucocyte differentiation CDw52 antigen peptide backbone
Authors: Kirchhoff Christiane , Krull Nora , Pera Ilka , Ivell Richard
Published: February 2005

HE5, a very abundant human epididymal gene product, was cloned by a differential screening procedure which employed testis as the primary negative control tissue. Sequencing of the HE5 cDNA showed it to be identical to that encoding the peptide backbone of the human leucocyte differentiation antigen CDw52. Since human genomic DNA contained only one cross‐hybridizing fragment, this implies that both products were transcribed from the same gene. Northern blot analysis and in situ transcript hybridization revealed a highly tissue‐ and cell‐type‐specific expression pattern for HE5, showing the gene product to originate from epithelial cells of the epididymal and deferent duct. The possible identity of the HE5 product with the CDw52 antigen suggests a link between the immune and reproductive systems. © 1993 Wiley‐Liss, Inc.
Region‐specific variation of gene expression in the human epididymis as revealed by in situ hybridization with tissue‐specific cDNAs
Authors: Krull Nora , Ivell Richard , Osterhoff Caroline , Kirchhoff Christiane
Published: February 2005

Three tissue‐specific gene probes that had been isolated by differential screening from a human epididymal cDNA library—HE1, HE2, and HE5—were employed to investigate regional specializations in the human epididymis. All 3 cDNAs were derived from major transcripts of the epithelial cells lining the epididymal duct. Each mRNA species, however, exhibited a discrete longitudinal pattern of hybridization with maxima in different regions of this organ, suggesting regional specializations of gene expression. The HE5 mRNA, which was recently shown to encode the peptide backbone of the human leukocyte differentiation antigen CDw52, showed maximum levels in the distal corpus epididymidis and in the vas deferens, whereas the HE2 mRNA was found predominantly in the caput and proximal corpus sections of the epididymis. HE1 mRNA was found in high amounts in all parts of the epididymis, displaying a 2‐peak expression pattern with maxima in the distal caput and distal corpus of the epididymal duct. © 1993 Wiley‐Liss, Inc.
Chromatin and microtubule organization in the first cell cycle in rabbit parthenotes and nuclear transplant embryos
Authors: Pinto‐Correia Clara , Collas Philippe , De Leon F. Able Ponce , Robl James M.
Published: February 2005

Artificial activation and nuclear transfer in rabbit oocytes have been used in past years in an attempt to develop viable techniques for cloning in cattle. The procedures established in our laboratory, using the rabbit as a model, consistently lead to high rates of development to the blastocyst stage. However, the rate of embryos developing to term is considerably lower. In the present study, we undertook a detailed immunocytochemical study of the patterns of both microtubules and chromatin during the first cell cycle of electrical pulse‐activated oocytes and of nuclear transfer embryos. Our goal was to investigate the responses of the cell to the different stimuli applied and to establish the sequence of events leading to first cleavage in the absence of normal fertilization. Our results show that, in both electrically activated oocytes and nuclear transfer embryos, although the initial development patterns are rather unusual, embryos become synchronized at the time of the formation of a pronuclear‐like structure, and then organize metaphase spindles and cleave. These spindles consistently present small defects, suggesting that problems in the formation of the mitotic apparatus during the first cell cycle may have a long‐term effect leading to embryo mortality. © 1993 Wiley‐Liss, Inc.