bluuebunny/crossref_metadata_2025 huggingface dataset:
Electrofusion‐induced intracellular Ca2+ flux and its effect on murine oocyte activation
Authors: Rickords L. F. , White K. L.
Published: February 2005

These experiments were designed to monitor influx of extracellular Ca2+ into the murine ooplasm following a 1.56 kV · cm−1 direct current (DC) electrofusion pulse and subsequently to determine its effect on rate of activation. Pulse media consisted of nonelectrolyte (0.3 M mannitol) and electrolyte (phosphatebuffered saline; PBS) media each containing 0.0, 0.05, or 0.9 mM Ca2+ (groups T1–T3 and T4–T6, respectively). Cumulus‐free oocytes were incubated in 100 μl drops of PBS containing 2 μM of the calcium indicator fluo‐3/AM for 60 min at 37°C. Fluo‐3/AM‐loaded oocytes were equilibrated for 7 min in assigned treatment media (T1–T6) prior to application of DC pulse. Change in fluorescent intensity was monitored for 6.5 min after DC pulse by photon counting spectrofluorometry. Fluorometric measurements demonstrate a dramatic rise in intracellular free Ca2+ (Ca2+i) following DC pulse is associated with Ca2+ ion concentration in the pulse medium. Significantly (P < 0.01) higher Ca2+i levels were observed when 0.9 mM Ca2+ was added to the pulse medium (T3 and T6) compared with pulse medium containing lower Ca2+ ion concentrations (T1, T2, T4, and T5; P > 0.05). Differences (P < 0.01) were observed in peak Ca2+i levels 18 sec after pulse with mean percent change in fluorescence of 5.1%a, 33.9%b, 112.7%c, 1.2%a, 9.3%a, and 99.9%c for T1–T6, respectively (values with different superscripts are significantly different at P < 0.01). Increased oocyte membrane permeability to Ca2+ ion after DC pulse was observed for a minimum of 5 min after delivery of the 1.56kV · cm−1 pulse. Nonloaded oocytes receiving the same pulse treatments were cultured in 100 μl drops of Whitten's medium for 8 h prior to evaluation of oocyte activation. Activation was defined as the formation of one pronucleus and one polar body, two or more pronuclei, or two cells each containing a nuclear structure. Activation rates of 28.2%b, 31.1%b, 70.4%d, 35.6%b, 57.3%c, 71.8%d 8.7%a (P < 0.01) were observed for T1–T6 and control nonpulsed oocytes, respectively. These data demonstrate that supplementation of 0.9 mM of Ca2+ ion to pulse medium results in a dramatic rise in Ca2+i after DC pulse. This influx of Ca2+ ion has a positive effect on oocyte activation rate.
Sex‐related differences in developmental rates of bovine embryos produced and cultured in vitro
Authors: Xu K. P. , Yadav B. R. , King W. A. , Betteridge K. J.
Published: February 2005

The classical concept of sex determination in mammals is that a Y chromosomal gene controls the development of the indifferent gonad into a testis. Subsequent divergence of sexual phenotypes is secondary to this gonadal determination. The most likely candidate gene is SRY (sex‐determining region Y) in humans, and Sry in mouse. However, several lines of evidence indicate that sexual dimorphism occurs even before the indifferent gonad appears. Here we present evidence that bovine male embryos generally develop to more advanced stages than do females during the first 8 days after insemination in vitro. Corresponding relationships between both cell numbers and mitotic indices and sex were also seen. Although it is not clear whether this phenomenon involves factors originating before or after fertilization, these findings suggest that sex‐related gene expression affects the development of embryos soon after activation of the embryonic genome and well before gonadal differentiation.
Expression cloning of TGF‐β receptors
Authors: Lin Herbert Y. , Wang Xiao‐Fan
Published: February 2005

Using a powerful expression cloning method in COS cells, we have cloned the TGF‐β types II and III receptors. The type III TGF‐β receptor is a membrane‐bound proteoglycan with a core protein of about 110 kDa. Stable expression of the type III receptor in L6 myoblasts leads to an apparent increase in the ability of the type II receptor to bind iodinated TGF‐β1. The cloned type II receptor has a predicted protein core of about 60 kDa with a cysteine‐rich extracellular domain, a single transmembrane domain, and a functional serine/threonine kinase domain that is homologous to the activin receptor and to the C. elegans protein daf‐1. These results implicate serine/threonine phosphorylation as an important mechanism of TGF‐β action. © 1992 Wiley‐Liss, Inc.
Negative regulation of gene expression by TGF‐β
Authors: Matrisian Lynn M. , Ganser Gail L. , Kerr Lawrence D. , Pelton Ron W. , Wood Lauren D.
Published: February 2005

Stromelysin gene expression is transcriptionally activated by a number of growth factors (e.g., EGF and PDGF), tumor promoters (e.g., TPA), and oncogenes (e.g., ras, src) through an AP‐1‐dependent mechanism. TGF‐β repression of stromelysin induction is mediated at the level of transcription by an element located at position −709 in the rat stromelysin promoter referred to as the TGF‐β inhibitory element (TIE). A TIE‐binding protein complex is induced by treatment of rat fibroblasts with TGF‐β. This protein complex contains the protooncogene c‐fos, and induction of c‐fos by TGF‐β is required for the repressive effects of TGF‐β on stromelysin gene expression. Interestingly, c‐fos induction is also required for stimulation of stromelysin expression by EGF in rat fibroblasts. Preliminary studies suggest that differential regulation of members of the jun family of early‐response genes may explain this apparent paradox and determine whether stromelysin is induced or repressed by growth factors. TGF‐β stimulation therefore initiates a cascade of events that results in a specific pattern of gene expression: the direct stimulation of early‐response genes can lead to subsequent induction or repression of other genes.

Growth factor regulation of matrix metalloproteinases appears to play a role in embryonic development in the morphogenesis of the murine lung. Treatment of embryonic lungs in organ culture with the growth factors EGF or TGF‐β results in stimulation of growth and inhibition of branching morphogenesis. A similar inhibition of branching was observed when these lung rudiments were treated with the matrix metalloproteinase collagenase. Most interestingly, the effects of EGF and TGF‐β can be completely reversed by the tissue inhibitor of metalloproteinases, TIMP. TGF‐β has the opposite effect on growth of murine lung rudiments—growth is inhibited in a dose‐dependent manner. This example illustrates a potential role for growth factor regulation of matrix‐degrading metalloproteinases in complex developmental processes. © 1992 Wiley‐Liss, Inc.
TGF‐β3‐Mediated tissue interaction during embryonic heart development
Authors: Runyan Raymond B. , Potts Jay D. , Weeks Daniel L.
Published: February 2005

A critical process during early heart development is the formation of mesenchymal cells which will contribute to valves and septa of the mature heart. These cells arise by an epithelial‐mesenchymal transformation of endothelial cells in the atrioventricular (AV) canal and outflow tract areas of the heart. Adjacent endothelial cells in the atrium and ventricle remain epithelial. A three‐dimensional collagen gel culture system has been exploited to examine the interactions that mediate this transformation. The AV canal myocardium produces a stimulus that is transmitted through an intervening extra‐cellular matrix to the AV canal endothelium. This interaction is regionally specific, such that ventricular myocardium does not provide an adequate stimulus and ventricular endothelium does not respond to the AV canal myocardial stimulus. Exogenous TGF‐β1 (or TGF‐β2) can complement ventricular myocardium to produce transformation by AV canal endothelium. A blocking antibody, effective against several TGF‐β, prevents cell transformation. To identify the specific member of the TGF‐β family that functions in situ, antisense oligonucleotides for each of the numbered TGF‐β were topically added to AV canal explant cultures. Only the oligonucleotide targeted to TGF‐β3 was an effective inhibitor of mesenchymal cell formation. Studies have been undertaken to localize specific mRNas by in situ hybridization and RNase protection assays. These assays have concentrated on the regional and temporal appearance of TGF‐β2 and 3. Surprisingly, RNase protection assays with a TGF‐β3 sense probe showed the presence of a transcript complementary to TGF‐β3. Further analysis of this tissue interaction included the testing of a variety of signal transduction mechanisms including kinases, G‐proteins, and intracellular calcium. Tissue interaction in the heart is a complex interaction in which regulation of the induction process occurs in both the inducing and target tissues. © 1992 Wiley‐Liss, Inc.
Marsupial fertilization: Some further ultrastructural observations on the dasyurid Sminthopsis Crassicaudata
Authors: Breed W. G. , Leigh C. M.
Published: February 2005

Various morphological aspects of in vivo egg maturation and sperm—egg interaction were investigated in the Australian marsupial Sminthopsis crassicaudata with the transmission and scanning electron microscopes. Cortical granules invariably occurred in primary oocytes, with the number increasing after resumption of the first meiotic division. They generally occurred close to the oolemma, including the region near the oocyte nucleus. After mating, spermatozoa with intact acrosomes, which had a homogeneous electron‐dense matrix, were found on the outer zona surface, but loss of acrosomal contents had occurred by the time of zona penetration. Sperm incorporation into the egg took place at the metaphase II stage of meiosis, and, at this time, cortical granules disappeared from the egg cortex. Sperm heads with condensed chromatin in the egg cytoplasm had an electron‐dense layer of subacrosomal material over part of the dorsal nuclear surface, but no membranes were present around these incorporated spermatozoa. Sperm chromatin decondensation resulted in an elevation of egg cytoplasm, and the cell membrane over this area lacked microvilli. The pronuclear envelope was not laid down until after chromatin decondensation had occurred. By this time the fertilized egg had reached the uterus, and a smooth, electron‐dense, shell membrane had been deposited. These observations, together with our previous findings, indicate that some of the processes of sperm—egg interaction are similar to those in eutherian mammals, whereas others appear highly divergent.
drop out: A third chromosome maternal‐effect locus required for formation of the Drosophila cellular blastoderm
Authors: Galewsky Samuel , Schulz Robert A.
Published: February 2005

drop out (dop) is a recessive maternal‐effect locus identified in a screen for female‐sterile mutations in Drosophila polytene region 71C‐F. Phenotypic analyses of the dop mutation indicate that the gene is required for proper formation of the cellular blastoderm. In embryos derived from either homozygous or hemizygous dop mothers, cytoplasmic clearing, nuclear migration and division, and pole cell formation appear normal. However, developmental defects are observed prior to and during cellularization of the blastoderm. At the beginning of nuclear cycle 14, the distinct separation of the internal yolk mass and the cortical cytoplasm breaks down. Subsequently, a population of somatic nuclei located at the periphery of the syncytial blastoderm becomes irregularly spaced and nonuniform in their distribution. Despite a somewhat regular formation of the cortical actin network, cellularizaiton in mutant embryos is extremely variable. Such embryos fail to gastrulate normally and produce variable amounts of defective cuticle. Overall, our analyses suggest that the dop gene functions in maintaining the separation of yolk and cortical cytoplasm and in stabilizing the distribution of somatic nuclei in the Drosophila syncytial blastoderm.
Isolation of epiblast‐specific cDNA clones by differential hybridization with polymerase chain reaction‐amplified probes derived from single embryos
Authors: Varmuza Susannah , Tate Peri
Published: February 2005

A mouse day 7.5 embryonic ectoderm cDNA library containing 2 × 106 clones was screened by differential hybridization with polymerase chain reaction (PCR)‐amplified probes derived from a single embryo. Day 7.5 ectoplacental cone and embryonic ectoderm served as the source of mRNA to make minus and plus probes, respectively. In a limited screen of fewer than 2,000 clones, 23 up‐regulated clones were identified by the difference in hybridization singal with the two probes. DNA sequence analysis revealed the nature of some, but not all, of the clones. Northern blot and in situ hybridization with a subset of the clones confirmed the utility of the approach, since the differential signal was also observed in these experiments. This approach may prove useful for identifying genes that play a role during development.
Prefertilization treatment of mice with platelet activating factor affects pregnancy
Authors: Podsiadly B. T. , Adamson L. M. , Stanger J. D. , Smart Y. C. , Roberts T. K.
Published: February 2005

Embryo‐derived platelet activating factor (EPAF) is thought to be either biologically similar to platelet activating factor (PAF) or responsible for PAF liberation in vivo. We have previously shown that premating PAF treatment in the mouse renders the platelets nonresponsive to EPAF, leading to a reduced implantation rate in these animals. In this study, we have shown that females, injected with PAF before mating, show altered embryo development in vivo on day 4 postfertilization. This is manifested as an interruption of compaction, a reduced cell number per embryo, and reduced embryo number per mouse. Results suggest that EPAF represents an early pregnancy signal that supports embryo development. The most likely mechanism is via platelet activation, since only those mice that showed thrombocytopenia after fertilization were found to have normal embryos on day 4 postmating.
Purification and characterization of vitellogenin and lipovitellins of the sand crab Emerita asiatica: Molecular aspects of crab yolk proteins
Authors: Tirumalai R. , Subramoniam T.
Published: February 2005

In the mole crab Emerita asiatica, the main yolk proteins consist of two slow moving lipovitellins (Lv I and Lv II) of glycolipoprotein nature. Lv I cleaves into subunits (MW: 109,000 and 105,000) and Lv II gives rise to six subunits (MW: 65,000, 54,000, 50,000, 47,000, 44,000, and 42,000) in SDS‐PAGE (with β‐mercaptoethanol). In order to observe the stability of Lv II as well as to achieve better resolution of the proteins, two different buffer systems (Phosphate buffered saline and tris‐buffered saline), 40% sucrose, and glass distilled water were used as homogenizing media. Among them, better resolution was achieved with tris‐buffered saline and 40% sucrose, and tris‐buffered saline seems to be the ideal medium for elution of Lv II. The analysis of biochemical constituents of the major Lv II reveals a percentage composition of 69.325, 27.927, and 2.753 respectively for protein, lipid, and bound sugars. In the I stage embryo, protein comprises about 67.276%, lipid 29.65%, and bound sugars 3.015%. Vitellogenin (Vg) electrophoretically corresponding to the Lv I and Lv II was present in the female haernolymph during the entire period of embryogenesis. The number of subunits(8) of Vg in all stages remained unaltered and their approximate molecular weights were Vg1, 91,000; Vg2, 87,000; Vg3, 83,000; Vg4, 61,000; Vg5, 58,000; Vg6, 45,000; Vg7, 42,000; and Vg8, 38,000. Different proteins present in the embryos (I and IV stage) and the serum obtained from the animal carrying the I stage embryo were separated by gel‐filtration in high performance liquid chromatography (HPLC). Sephadex (G‐200) gel filtration chromatography was used to purify the Lv II in large quantity.

Total lipid extracted from Lv II as well as the embryos belonging to different stages of development were separated into their constituent neutral, glycolipids, and phospholipids, using silicic acid column chromatography. Thin layer chromatography (TLC) was used to isolate the different phospholipids purified from various stages of embryos and Lv II. As many as seven different phospholipids were separated from Lv II and I and IX stage embryos; and whereas thin layer chromatogram of V and VI stage embryos showed six different phospholipids, embryos of VII and VIII stage contained four phospholipid species. Cholesterol, glycolipids, and individual phospholipids isolated from the Lv II and I stage embryo were quantified spectrophotometrically and the results were discussed. © 1992 Wiley‐Liss, Inc.
Recovery of penetration ability in protease‐treated zona‐free mouse eggs occurs coincident with recovery of a cell surface 94 kD protein
Authors: Kellom Theresa , Vick Angela , Boldt Jeffrey
Published: February 2005

Previous studies have demonstrated that protease treatment of zona‐free mouse eggs impairs sperm‐egg interaction (Boldt et al.: Biol Reprod 39:19–27. 1988) and causes modification of a 94 kD egg plasma membrane protein (Boldt et al., Gamete Res 23:91–101, 1989), In this report, the ability of eggs to recover penetration ability following protease treatment was examined. Zona‐free mouse eggs were isolated and treated with either trypsin or chymotrypsin (1 mg/ml, 20 min), then cultured for 0, 3, or 6 hr before insemination. Eggs cultured for 3 or 6 hr displayed significantly higher penetration levels than eggs inseminated immediately after protease treatment, indicating a recovery of penetration ability during the 3 or 6 hr incubation period. The recovery of penetration ability was not blocked by inclusion of cyclohexamide (50 μg/ml) during the 3 or 6 hr culture period, indicating that protein synthesis was not required for recovery of fusion ability. Cell surface radiolabeling studies with 125I revealed that a 94 kD cell surface protein was lost immediately following trypsin or chymotrypsin treatment but was found on the egg surface after the 3 or 6 hr recovery period. Recovery of the 94 kD egg surface protein occurred in the presence of cyclohexamide, and metabolic radiolabeling studies with 35S‐methionine confirmed that synthesis of a 94 kD protein was blocked by cyclohexamide. These results suggest that the recovery of penetration ability after protease treatment of zona‐free eggs is due to recovery of the 94 kD cell surface protein, providing further evidence for the involvement of the 94 kD protein in sperm‐egg interaction. © 1992 Wiley‐Liss, Inc.
Subzonal insemination of a single mouse spermatozoon with a personal computer‐controlled micromanipulation system
Authors: Kobayashi Kazuhiko , Kato Keisuke , Saga Masahiko , Yamane Masami , Rothman Cappy M. , Ogawa Shyoso
Published: February 2005

A personal computer‐controlled micromanipulation system was developed for automatic injection of spermatozoa into the perivitelline space of mouse ova. A pair of three‐dimensional hydraulic micromanipulators driven by pulse motors was used for this automatic system. The pulse signals that regulate the motors are initiated by the computer program, and these signals cause the micromanipulator to move the microtool precisely. The computer program was designed to perform the most effective movements of the sperm injection needle used during manual micromanipulation. Prior to the manipulation, the computer locates the tip of the injection needle and the end of the egg‐holding pipette in the microscope field using image processing. The trajectory of the injection needle is determined according to these initial positions. Using this robotic system, subzonal insemination with a single mouse spermatozoon was attempted in a total of 143 ova. The sperm insertion was successfully completed in all cases without damaging any of the ova. Spermatozoa treated with ionophore A23187 and those without the treatment were used. The fertilization rate (68.8%) of the ova inseminated with treated sperm was significantly higher than that (37.5%) obtained with the nontreated sperm (P < 0.05). These findings suggest the feasibility and potential for further applications of a robotic microinsemination system and, in addition, that a higher fertility rate in the subzonal insemination of mouse ova can be achieved with the ionophore treatment of spermatozoa. © 1992 Wiley‐Liss, Inc.
Characterization of low Mr Zona Pellucida binding proteins from boar spermatozoa and seminal plasma
Authors: Parry R. V. , Barker P. J. , Jones R.
Published: February 2005

A group of low Mr (16 kDa ‐ 23 kDa) glycoproteins on ejaculated boar spermatozoa have been shown to have high affinity for homologous zona pellucida glycoproteins (ZPGPs). These ZPGP binding proteins are derived from seminal plasma as shown by their absence from epididymal spermatozoa and their presence in seminal plasma as identified by N‐terminal amino acid sequence analysis. They bind to ZPGPs by a polysulphate recognition mechanism similar to that found for proacrosin‐ZPGP interactions. The haemagglutination activity of boar seminal plasma is also associated with these low Mr glycoproteins. It is suggested that they play a role in regulating the rate of sperm capacitation and survival in the female reproductive tract. © 1992 Wiley‐Liss, Inc.
Developmental regulation ofan snRNP core protein epitope during pig embryogenesis and after nuclear transfer for cloning
Authors: Prather Randall S. , Rickords Lee F.
Published: February 2005

The appearance and stabilization of a core protein epitope of the snRNP is developmentally regulated during pig embryogenesis. The epitope recognized by the monoclonal antibody Y12 is present in the germinal vesicle of mature ooocytes and interphase nuclei of late 4‐cell stage (24 to 30 hours post cleavage to the 4‐cell stage) to blastocyst stage embryos. There was no antibody iocalization within pronuclei, or nuclei of 2‐cell or early 4‐cell stage embryos. Zygotes or 2‐cell stage embryos cultured in the presence of α‐amanitin to the late 4‐cell stage showed no immunoreactivity, whereas control embryos had immunoreactivity. Thus antibody localization was correlated with RNA synthesis and RNA processing that begins by 24 hours post cleavage to the 4‐cell stage. A final experiment showed no detectable immunoreactivity in 16‐cell stage nuclei that had been transferred to enucleated activated meiotic metaphase II oocytes. Since immunoreactivity is associated with active RNA synthesis and RNA processing, it suggests that the 16‐cell stage nucleus, which is RNA synthetically active, does not process RNA after nuclear transfer to an enucleated activated meiotic metaphase II oocyte. © 1992 Wiley‐Liss, Inc.
Purification and partial characterization of the 17 kDa sperm coating protein from boar seminal plasma
Authors: Moos JiřRí , Veselskýa Leopold , Pěknicov´ Jana , Drahoréd Josef
Published: February 2005

Sperm coating proteins of 16, 17, and 19 kDa have been purified from boar seminal plasma. The 17 kDa protein has been identified as an antigen recognized by monoclonal antibody ACR.3 and is thus identical to low molecular mass zona pellucida binding protein from boar spermatozoa (Moos et al., 1990). The 17 and 19 kDa proteins are glycosylated and tend to form hetero‐complexes. The 17 kDa ACR.3 antigen is sequentially released from the sperm cell surface during capacitation and, after induction of the acrosome reaction, the 16 kDa form was also observed. Immunocytochemical studies on boar reproductive tissues have suggested that the seminal vesicle epithelium may be the source of these proteins. © 1992 Wiley‐Liss, Inc.
Increasing carbon dioxide from five percent to ten percent improves rabbit blastocyst development from cultured zygotes
Authors: Hallden Kristine , Li Jianming , Carney Edward W. , Foote Robert H.
Published: February 2005

One‐cell rabbit zygotes were cultured at 39°C in basal synthetic medium II (BSM‐II) with 5%, 10%, or 15% CO2 and humidified air to determine the effect of CO2 concentration on development in vitro. After 4 days in culture, 37% of the embryos grown in 10% or 15% CO2 had reached the hatching blastocyst stage, but only 10% of the embryos were hatching when cultured under 5% CO2 (P = 0.01). Over all blastocysts, cell numbers were 207, 246, and 205 for the 5%, 10%, and 15% CO2 treatments, respectiveiy. In a second experiment to determine if there was a beneficial effect, particularly at the blastocyst stage, of a higher concentration of CO2, embryos were cultured 4 days in either 5% or 10% CO2 or for 2 days in 5% CO2 followed by 2 days in 10% CO2. The numbers of blastomeres per embryo and embryo diameter were greater (P > 0.05) in embryos cultured continuously in 10% CO2 or in 10% CO2 only during days 3 and 4 of culture than in embryos cultured continuously in 5% CO2. In a third experiment, one‐cell rabbit zygotes were cultured with 5% or 10% CO2 in a defined, protein‐free medium consisting of 1:1 RPMI 1640 and Dulbecco's modified Eagle's medium. The proportion of embryos hatching and cell counts were significantly greater (P > 0.01) when cultured in the presence of 10% CO2. These data indicate that a 10% CO2 atmosphere exerts a beneficial effect on the development of zygotes into expanding and hatching rabbit blastocysts in vitro. © 1992 Wiley‐Liss, Inc.
Adsorption of oviductal fluid proteins by the bovine sperm membrane during in vitro capacitation
Authors: McNutt Tamara , Rogowski Lee , Vasilatos‐Younken Regina , Killian Gary
Published: February 2005

125I‐labeled oviductal fluid (ODF) proteins and antiserum to ODF were used to determine whether ODF proteins associate with the sperm membrane during in vitro capacitation. Luteal and nonluteal pools of ODF were obtained from oviduct catheters during the estrous cycle. Washed sperm (50 × 106 sperm/ml) were incubated up to 4 h in a protein‐free modified Tyrode's medium (MTM), or MTM supplemented with 40% ODF, or 0.5 ng 125I‐labeled ODF proteins. Solubilized sperm membrane proteins and incubatin media containing ODF proteins were separated by gel electrophoresis. Membranes isolated from bovine sperm previously incubated with ODF, absorbed five 125I‐proteins: A doublet at 85–95 kDa, and others at 24, 34, 53, and 66 kDa. The amount of 66 kDA 125I‐protein associated with the sperm decreased during the incubation, whereas the amount of 85 to 95‐kDa protein did not. Western blot analyses also detected the presence of ODF proteins (53, 66, 85–95, and 116 kDa) in solubilized membranes from sperm incubated in ODF. The 85 to 95‐kDa protein in ODF decreased in ODF. The 85 to 95kDa protein in ODF decreased in apparent molecular weight by 5 kDa when associated with the sperm membrane. At 53 kDa ODF proteins which associated with sperm were transformed from two to three separate proteins. These studies indicate that the surface of sperm is modified by adsorption of ODF proteins ot the membrane during in vitro capacitation. (c) 1992 Wiley‐Liss, Inc.
Automated analysis of rabbit sperm motility and the effect of chemicals on sperm motion parameters
Authors: Young J. , Bodt B. A. , Iturralde T. G. , Starke W. C.
Published: February 2005

Appropriate software settings and optimum procedures were determined for the measurement of the motion parameters of rabbit spermatozoa by the CellSoft (Cryo Resources Ltd., Montgomery, NY) computer‐assisted digital image analysis system. The system was used to follow motion parameter changes occurring in spermatozoa incubated for 6 hr with or without exposure to chemicals. Mean amplitude of lateral head displacement (AALH) increased over the 6 hr period, while curvilinear velocity (Vc) first increased and then decreased. Values for linearity (Lin), or beat cross frequency (BCF), were unchanged. The majority of spermatozoa progressed linearly, with rapid rotation of the sperm head, but subpopulations of spermatozoa with different swimming patterns appeared after 1–3 hr of incubation. Percentage motile sperm and Vc were most sensitive to the action of the compounds (pyrogallol, hydroquinone, ammonium oxalate, triethyl phosphite, and pinacolyl alcohol), while BCF was least affected. The decline in percentage of motile sperm was dependent on duration of exposure and chemical concentration. Mean Vc of the sperm population decreased rapidly upon chemical exposure and remained at a low value until motility ceased. The initial decrease in Vc was dependent on the concentration of the added compound. Motion‐based indices—motility concentration (MCl50), motility time (MTl50), and velocity (VI)—were defined and used as toxicological endpoints. The rank order of these indices, the end point of the neutral red in vitro assay for cytotoxicity, and LD50 values for the five compounds were the same, suggesting that chemical inhibition of sperm motility may be useful as a method for the in vitro assessment of chemical cytotoxicity. © 1992 Wiley‐Liss, Inc.
Rat sertoli and spermatogenic cells express a similar gene, and its product is antigenically related to an outer dense fiber‐associated protein
Authors: Smith Frank F. , Mertz James R. , Krebs Ingeborg , Tres Laura L. , Chae Chi‐Bom , Zakeri Zahra , Engelhardt Jeff , Hoover Dennis , Tenniswood Martin , Kierszenbaum Abraham L.
Published: February 2005

We have previously reported that a heterodimeric protein secreted by rat Sertoli cells is antigenically related to a protein associated with outer dense fibers of the sperm tail. Therefore, we have explored the possibility that Sertoli and spermatogenic cells express a similar gene encoding a homologous protein. A Sertoli cell heterodimeric protein cDNA probe recognizes specific mRNA in pachytene and round spermatids fractionated by centrifugal elutriation; however, this specific mRNA was less prominent than in cultured Sertoli cells. In agreement with these observations, in situ hybridization experiments show that Sertoli cells are predominantly engaged in active heterodimeric protein mRNA synthesis, while meiotic prophase spermatocytes and spermatids also show significant but less abundant specific mRNA. Immunoblotting experiments demonstrate that, while Sertoli cells synthesize a heterodimeric protein consisting of two disulfidelinked components with molecular masses of 45 and 35 kD, both primary spermatocytes and round spermatids synthesize single 30 kD monomers not associated by disulfide linkage but recognized by antisera to Sertoli cell heterodimeric protein Immunoblotting and immunogold electron microscopic studies show that antisera to Sertoli cell heterodimeric protein recognize a protein associated with outer dense fibers. This immunoreactivity was abolished by a 5‐min pronase treatment, without affecting the integrity of outer dense fibers. Results of this study and previous studies demonstrate that both Sertoli and spermatogenic cells express a similar gene and that an antigenically related product encoded by this gene becomes associated with outer dense fibers during their assembly at spermiogenesis. © 1992 Wiley‐Liss, Inc.
Developmental expression of the S35‐S45/SGP‐2/TRPM‐2 gene in rat testis and epididymis
Authors: Zakeri Zahra , Curto Marcello , Hoover Dennis , Wightman Karen , Engelhardt Jeff , Smith Frank F. , Kierszenbaum Abraham L. , Gleeson Timothy , Tenniswood Martin
Published: February 2005

Testosterone‐repressed prostate message‐2 (TRPM‐2) was originally isolated and cloned from the regressing ventral prostate of the rat. In this tissue, and in other hormone‐dependent tissues such as the mammary gland, this gene is induced in the absence of the appropriate trophic hormone. Sequence analysis of the cDNA and genomic clones of TRPM‐2 have demonstrated that the coding sequence of this gene is identical to S35‐S45 (also known as SGP‐2 and clusterin), which is constitutively expressed by the Sertoli cells of the adult testis. Using Northern, slot blot, S1‐nuclease analysis, and in situ hybridization, we have investigated the regulation of TRPM‐2 expression in the testis and epididymis during development. Slot blot analysis of RNA extracted from the testis and epididymis of 7‐, 14‐, 28‐, 35‐, and 91‐day‐old rats demonstrates that the gene is induced to detectable levels between days 7 and 14 and that the relative level of expression does not change significantly after day 14. In situ hybridization using frozen sections of testis from day 2‐, 7‐, 14‐, 28‐, 35‐, and 91‐day‐old rats confirms that there is little expression of TPRM‐2 in the seminiferous epithelium of 7‐day‐old rats, but this increases considerably after 14 days, primarily in Sertoli cells but also in association with meiotic developing spermatogenic cells. However, TRPM‐2 mRNA is expressed in the rete testis at 2 days of age, reaches a peak at 35 days of age, and continues to be expressed in the adult. Slot blot analysis demonstrates that TRPM‐2 is also induced in the epididymis between 7 and 14 days of age, although, as has been demonstrated by in situ hybridization, TRPM‐2 mRNA is detectable in the epithelial cells in the head of the epididymis but is barely detectable in the midportion or tail regions. Northern analysis suggests that the size of the TRPM‐2 transcript in the testis also changes during development. In the early stages of testicular development, the TRPM‐2 transcript appears to be a broad band of approximately 1.5 kb, while the transcript in the adult appears to be approximately 1.8 kb in length. S1‐nuclease protection assays suggest that this increase in size is not due to differential splicing of the first exon of TRPM‐2/SGP‐2 and most probably reflects a difference in the polyadenylation of the mRNA in the testis at different times during development. © 1992 Wiley‐Liss, Inc.
Monoclonal antibody AG7 inhibits fertilization post sperm‐zona binding
Authors: Brucker Cosima , Sandow Bruce A. , Blackmore Peter F. , Lipford Grayson B. , Hodgen Gary D.
Published: February 2005

Monoclonal antibodies (mAbs) against sperm cells are currently being used in an effort to define spermatozoal antigens involved in the fertilization process. We have produced a number of anti‐human sperm mAbs by immunization of female mice with the 100,000 × g supernatant of octylglycoside‐solubilized washed human sperm. From a panel of mAbs, 1 antibody, AG7, was selected and characterized due to its fertilization‐inhibiting characteristics. MAb AG7 defines a sperm acrosome antigen‐1 (SAA‐1) located in the acrosomal region of human sperm as evaluated by indirect immunofluorescence. Staining of life sperm cells indicated that the antigen is present on the sperm surface. SAA‐1 was also found on sperm of several other mammalian species, implying evolutionary conservation of the antigen. SAA‐1 was first observed on testicular sperm and can be followed through epididymal transit, ejaculation, and capacitation. When applied in a mouse in vitro fertilization assay, mAb AG7 inhibits fertilization by greater than 95%, and inhibition is dose dependent, with half‐maximal inhibition at 0.8 μg/ml. The block to fertilization could not be attributed to sperm agglutination, inhibition of motility, interference with adhesion to the zona pellucida, or inhibition of fusion with the oocyte membrane. MAb AG7 was demonstrated to inhibit calcium influx in spermatozoa in vitro (measured using the fluorescent indicator fura 2), a prerequisite for the acrosome reaction. Initial biochemical characterization of the antigen suggests it is proteinlike in nature, with a molecular weight of approximately 220 kD. The results suggest that SAA‐1, identified by mAb AG7, is a sperm antigen crucially involved in the fertilization process, possibly an atypical steriod receptor or ion channel located within the sperm plasma membrane. © 1992 Wiley‐Liss, Inc.
Fertilization envelope in diapause eggs of Pontella mediterranea (crustacea, copepoda)
Authors: Santella L. , Ianora A.
Published: February 2005

The sequence and timing of morphological changes during envelope formation was followed in diapause eggs of Pontella mediterranea (Crustacea, Copepoda). The multilayer coat enveloping these eggs resulted from the exocytosis of 4 types of cortical vesicles that sequentially released their contents in the perivitelline space. These included small high‐density vesicles (hDV) with electron‐dense material, vesicles (V) with dense ring granules and a uniform matrix contained within the same compartment, large high‐density (HDV) vesicles, and large moderately dense (MDV) vesicles. All of these cortical vesicles were present in newly spawned, fertilized eggs. Their exocytosis resulted from egg activation. One of these cortical vesicles (V) was similar in morphology to the intracisternal granules precursors of endogenous yolk. Intracisternal granules, characteristic of previtellogenic oocytes of many crustaceans, were present in previtellogenic oocytes of P. mediterranea but disappeared in later stages of oocyte development once yolk formation was completed. We discuss the role of cortical vesicles in the formation of the complex extracellar coat enveloping copepod diapause eggs. © 1992 Wiley‐Liss, Inc.
Immunological evidence for a P2 protamine precursor in mature rat sperm
Authors: Stanker Larry H. , McKeown Cathy , Balhorn Rod , Lee Catherine , Mazrimas Joe , Goralka Michael , Wyrobek Andy
Published: February 2005

High molecular weight proteins in Rattus norvegicus that are immunoreactive with an antiprotamine 2 specific antibody but not with an anti‐protamine 1 specific antibody are described. These proteins were detected by coupling high‐performance liquid chromatography (HPLC) with an enzyme‐linked immunosorbent assay (ELISA). Briefly, following HPLC separation of rat sperm nuclear proteins, the HPLC fractions were probed with the antibodies. We estimate that the antibody probes are 100–1000 times more sensitive than UV absorbance measurements. Immunoblot analysis following acid‐urea electrophoretic separation of rat sperm nuclear proteins, and of the HPLC fractions, also detected putatie protamine 2 precursor proteins. The proteins reactive with the anti‐protamine 2 antibody are most likely not mature protamine 2, since they were detected in a region of the chromatogram where we would not expect protamine 2 to migrate based on the chromatographic locations of human and mouse protamine 2. Likewise, the immunoblotting experiments demonstrated that the anti‐protamine 2 antibody recognized proteins with slower electrophoretic mobilities than would be expected for a mature protamine 2. An anti‐protamine 1 monoclonal antibody, Hup1 N, that binds rat protamine 1 is also described. Hup 1 N allowed for identification of the HPlc fractions that contained rat protamine 1. Finally, we demonstrated that Hup 1 N binds protamine 1 from a large number of species, suggesting a conserved epitope for Hup 1 N. © 1992 Wiley‐Liss, Inc.
Molecular cloning of a novel mRNA sequence expressed in cleavage stage mouse embryos
Authors: Miyamoto Hiroshi , Matsushiro Aizo , Nozaki Masami
Published: February 2005

In an approach to study genes transcribed during early mouse development, a cDNA library was constructed from poly(A) RNA isolated from the 8‐cell morula. The cDNA library was differentially screened with labelled cDNA probes synthesized on poly(A) RNA isolated from the 8‐cell morula or unfertilized eggs. Six clones which increased in abundance in the 8‐cell morula were selected and further analyzed. Sequencing analyses showed that some of these clones corresponded to RNA transcripts from B1 and B2 repetitive sequences, as well as mRNA for cytochrome C oxidase I and NADH dehydrogenase III derived from the mitochondrial genome. One clone was not identical to any known sequences. The unidentified sequence (M025) was found at low levels in the unfertilized egg, but increased at the 2‐cell stage. The predicted amino acid sequence revealed that the M025 gene may encode a Ca2+ binding protein. © 1993 Wiley‐Liss, Inc.
Human male infertility may be due to a decrease of the protamine P2 content in sperm chromatin
Authors: Belokopytova Irina A. , Kostyleva Elena I. , Tomilin Alexey N. , Vorob'ev Vladimir I.
Published: February 2005

Basic chromosomal proteins were extracted from the sperm of fertile and infertile human males. The relative proportions of protamine 1, 2, and 3 were determined by scanning microdensitometry following electrophoresis of total protamine in polyacrilamide gels. The findings were as follows: (1) The proportion of protamine P(2 + 3) in sperm obtained from infertile males was lower than that in fertile males. (2) Protamine P(2 + 3) in infertile human males showed reduced affinity to DNA. The possibility that some cases of human male infertility may be due to mutation within the protamine P2 gene is discussed. © 1993 Wiley‐Liss, Inc.
Mechanistic studies of the plasma membrane block to polyspermy in mouse eggs
Authors: Horvath Peter M. , Kellom Theresa , Caulfield Jeanne , Boldt Jeffrey
Published: February 2005

The mechanisms responsible for the plasma membrane associated block to polyspermy in mouse eggs were studied. Reinsemination experiments using zona‐free eggs indicated that, after fertilization, the egg plasma membrane is altered such that sperm binding to the egg plasma membrane is blocked, except in the region of the second polar body. Activation of the egg with either ethanol or strontium chloride did not result in a block to polyspermic penetration, as artificially activated eggs displayed identical penetration levels as to nonactivated control eggs. The penetrability of activated eggs was not altered by the presence or absence of the zona pellucida during activation. Lectin staining for egg cortical granule material indicated that activation did cause cortical granule exocytosis; however, activated eggs remained penetrable. These data support the following conclusions: (1) an alteration in the ability of the egg plasma membrane to allow sperm adherence accounts for the block to polyspermy; (2) establishment of the plasma membrane block to polyspermy is sperm dependent, since artificial egg activation does not result in a block response; (3) the contents of the egg's cortical granules do not play a role in the establishment of the plasmalemma block response. © 1993 Wiley‐Liss, Inc.
Evidence of new antigens in the mouse cumulus oophorus during preovulatory cumulus expansion
Authors: Amiel M.‐L. , Moos J. , Tesarik J. , Testart J.
Published: February 2005

The effects of an antiserum (anti‐COC) against ovulated mouse cumulus‐oocyte complexes (COC) on in vitro fertilization of mouse oocytes were studied. Preincubation of ovulated COC with various concentrations of anti‐COC led to dose‐dependent impairment of fertilization rates as well as to a decrease in the number of spermatozoa attached to the zona pellucida. Anti‐COC was used to probe Western blots of cumulus proteins. These cumuli were obtained from 2 experimental groups of mice corresponding to 2 different maturational stages (preovulatory immature COC or preovulatory mature COC). Two antigens (70 and 80 kDa) present in cumulus intercellular matrix from mature COC were only found as traces in matrix from immature COC. In addition, the protein pattern of the cumulus intercellular matrix was different from that of cumulus cells, whatever the COC maturational stage. These results indicate the appearance of new proteins in the cumulus oophorus during preovulatory expansion and are consistent with the contraceptive action of anti‐COC. © 1993 Wiley‐Liss, Inc.
Cloning and sequencing of baboon and cynomolgus monkey intraacrosomal protein SP‐10: Homology with human SP‐10 and a mouse sperm antigen (MSA‐63)
Authors: Freemerman Alex J. , Wright Richard M. , Flickinger Charles J. , Herr John C.
Published: February 2005

In this study, cDNAs encoding the intraacrosomal protein SP‐10 were cloned and sequenced from baboon (Papio papio) and macaque (Macaca fasicularis) testis libraries and the sequence compared to that of human SP‐10. Two alternatively spliced SP‐10 cDNAs were obtained from both baboon and macaque testis libraries. The two cDNAs in each species contained open reading frames encoding proteins of exactly 285 and 251 amino acids. A 98% homology between baboon and macaque SP‐10 was found at the protein and DNA levels. An 85% and 89% homology between baboon and macaque SP‐10 and human SP‐10 was present at the protein and DNA level, respectively. A mouse intraacrosomal protein, MSA‐63, considered to be an SP‐10 homologue, exhibited an overall 53% homology to nonhuman primate SP‐10 and a 60% homology to human SP‐10 at the protein level. Polymerase chain reaction analysis of testis mRNA confirmed the existence of two alternately spliced SP‐10 mRNAs in both nonhuman primates. Primer extension analysis indicated a common major transcriptional start site in baboon, macaque, and human SP‐10 67 nucleotides 5′ to the ATG codon. The amino acid sequence data for nonhuman primate SP‐10s suggest that antibodies generated by vaccinating baboons and macaques with human SP‐10 will likely recognize nonhuman primate SP‐10, supporting the testing of an SP‐10 contraceptive vaccine based on human SP‐10 in these nonhuman primate models. © 1993 Wiley‐Liss, Inc.
Differential sensitivity of progesterone‐ and zona pellucida‐induced acrosome reactions to pertussis toxin
Authors: Tesarik Jan , Carreras Alfonso , Mendoza Carmen
Published: February 2005

Pertussis toxin‐sensitive guanine nucleotide‐binding regulatory proteins (G proteins) have previously been shown to mediate the zona pellucida‐induced acrosome reaction in mammalian sperm. In this study we compared the inhibitory effect of pertussis toxin on the zona‐induced acrosome reaction in human spermatozoa with that on the reaction induced by progesterone, another physiological acrosome reaction‐promoting stimulus associated with the ovulated oocyte. Up to the concentration of 1 μg/ml, pertussis toxin did not produce any direct effects on the acrosome reaction frequency nor did it influence sperm movement and viability. However, preincubation of spermatozoa with the toxin at a concentration of 100 ng/ml completely abolished the increase in the acrosome reaction frequency upon subsequent exposure to solubilized zona pellucida material. In contrast, the same treatment did not impair the ability of spermatozoa to initiate the acrosome reaction in response to progesterone. Moreover, the preincubation with pertussis toxin did not modify the changes in the intracellular concentration of calcium ions occurring after progesterone addition. These data suggest that different physiological stimuli may utilize different signal transduction pathways to induce the human sperm acrosome reaction. © 1993 Wiley‐Liss, Inc.
Cellular localization of the mRNAs of the somatic and testis‐specific cytochromes c during spermatogenesis in the rat
Authors: Morales Carlos R. , Hake Laura E. , Hecht Norman B.
Published: February 2005

During mammalian spermatogenesis, two forms of cytochromes c, cytochromes cs and ct, are present in male germ cells. During meiosis, cytochrome ctbegins to replace cytochrome cs. At least four size classes of cytochrome cs mRNA are expressed in all somatic cells and in early stages of male germ cells. In addition, a cytochrome cs transcript of 1.7 kB has been detected in rodent testes and is abundant in post meiotic male germ cells. Here we utilize “in situ” hybridization to define the cellular sites where the four ubiquitous cytochrome cstranscripts, the 1.7 kB cytochrome cs transcripts and the testis‐specific cytochrome ct transcripts are expressed in the rat. Low levels of cytochrome cs mRNAs are detected in Leydig cells, myoepithelial cells, Sertoli cells, all types of spermatogonia, and during meiotic prophase. The 1.7 kB cytochrome cs mRNA is first detected in late stages of meiotic prophase and reaches its highest levels in steps 1 to 9 spermatids. No cytochrome cs mRNAs are detected in spermatids between steps 10 to 19. Low levels of cytochrome ct mRNAs, initially detected in zygotene spermatocytes, reach maximal levels in round spermatids. For all three probes the majority of the silver grains are localized randomly throughout the cytoplasm, suggesting that neither the translating nor non‐translating (the 1.7 kB mRNA) forms of cytochrome cs mRNA nor the cytochrome ct mRNAs are sequestered during spermatogenesis. The absence of cytochrome cs or ct mRNAs in steps 10–19 spermatids suggest that the cytochrome ct protein does not turn over rapidly in late stage male germ cells. © 1993 Wiley‐Liss, Inc.