bluuebunny/crossref_metadata_2025 huggingface dataset:
Attachment of the ascidian sperm surface egg receptor N‐acetylglucosaminidase to the cell membrane
Authors: Downey John C. , Lambert Charles C.
Published: February 2005

Ascidian sperm bind to vitelline coat N‐acetylglucosamine groups of the egg bvia sperm surface N‐acetylglucosaminidase. This sperm surface egg receptor remains anchored throughout penetration. Localization to the sperm surface was verified by biotinylation of intact sperm followed by solubilization in Triton X‐100 and binding to streptavidin agarose. The enzyme was determined to be an integral membrane protein as judged by resistance to release by KI and high pH. Linkage of the enzyme to the sperm surface was probed through differential solubilization followed by measuring released enzymatic activity with a fluorogenic substrate. Nonionic detergents released 90% of the activity. Proteases released about 40%. No activity was released by a phosphatidyl–inositol specific phospholipase C. This finding, combined with the similarity of release level by all the detergents, including triton X‐114 phase separation experiment. This observation, coupled with the finding of release by nonionic detergents, suggests that the protein is hydrophilic once released from the membrane. Thus, although clearly an integral membrane protein, the enzyme has limited bydrophobicity such as would be present in a single transmembrane sequence or extensive glycosylation. © 1994 Wiley‐Liss, Inc.
Cumulus cells secrete a meiosi‐inducing substance by stimulation with forskolin and dibutyric cyclic adenosine monophosphate
Authors: Guoliang Xia , Byskov Anne Grete , Andersen Claus Yding
Published: February 2005

The role of the cumulus cells in initiating the resumption of meiosis after exposure to forskolin and dbcAMP was studied in the mouse. The resumption of meiosis was monitored by the percentage of germinal vesicle breakdown (GVBD) and polar body formation (PB). The cumulus‐enclosed oocytes (CEO) and denuded oocytes (DO) were cultured with and without hypoxanthine (HX) in the culture medium.

Three types of experiments were performed: (1) Effect of forskolin on spontaneous resumption of meiosis, i.e. cultures without HX, and two experiments in which HX is present throughout the culture: (2) Effect of transient exposure to forskolin or dibutyric‐cyclic adenosinemonophosphate (dbcAMP) on GVBD prior to continued culture without forskolin or dbcAMP (oocyte priming). (3) Priming of CEO with forskolin for 2 hr, separation of cumulus cells and oocytes, followed by coculture of rejoined cumulus cells and oocytes, or coculture of the cumulus cells and new, unprimed DO.

(1) Forskolin inhibited a spontaneous resumption of meiosis in a dose‐dependent manner during the first 5 hr of culturing. After 22 hr all controls and CEO resumed meiosis, whereas only half of the DO did.

(2) At least 1 hr of priming the CEO with forskolin is needed to induce GVBD and PB formation, but forskolin inhibited the resumption of meiosis when present for 24 hr. Similar results were obtained with a high concentration of dbcAMP.

(3) A separation and rejoining of oocytes and cumulus cells after priming induced the resumption of meiosis in a significantly greater number of oocytes than in the control oocytes which were not primed. The GVBD of unstimulated DO also increased significantly when cocultured with cumulus cells from primed CEO. The percentage of GVBD in unprimed DO and in DO isolated from primed CEO was the same.

We suggest that within 1–2 hr, forskolin and cAMP stimulate cumulus cells to produce a diffusible meiosis‐inducing substance which overcomes HX‐inhibition and induces oocyte maturation, including both GVBD and PB formation. The CEO must be primed for more than 2 hr before the resumption of meiosis in DO isolated from such CEO is induced. Oocyte‐cumulus connections are crucial as far as initiating the production of a meiosis‐inducing substance is concerned. Oocyte‐cumulus connections are not needed for transferring this substance to the oocyte. © 1994 Wiley‐Liss, Inc.
Role of fibroblast growth factors as inducing agents in early embryonic development
Authors: Slack Jonathan
Published: February 2005

To assess the potential role of a molecule in development we need to know three things: 1) what are the biological activities of the molecule, 2) what is its expression pattern, and 3) what are the consequences of removing it from the embryo?

In the case of the FGF family in Xenopus embryos we have quite a lot of information about all three questions. Most members of the family can induce mesoderm from isolated animal caps, thus mimicking the natural “ventral vegetal” inducing signal operative in the blastula. This activity can be exerted on isolated, disaggregated cells and does not involve a change in division rate. When overexpressed from injected mRNA, the activity of FGFs depends largely on whether or not they possess a signal sequence, showing the importance of secretion in the inductive process. In addition to the mesoderm‐inducing activity, there are effects of overexpression on whole embryos which lead to a suppression of anterior structures.

Three types of FGF have so far been cloned from Xenopus: direct homologs of each of the mammalian types FGF‐2 and FGF‐3, and eFGF (“embryonic FGF”), which is equidistant in sequence from mammalian FGF‐4 and FGF‐6. Attempts to find homologs of mammalian FGF‐5 and FGF‐7 in Xenopus have proved unsuccessful. All three types of Xenopus FGF are expressed in early development. FGF‐2 and eFGF are present in the oocyte and fertilized egg, and are thus both available at the time of mesoderm induction. FGF‐3 and eFGF are both expressed from the embryonic genome during gastrulation and concentrated in the forming mesoderm. FGF‐2 is expressed from the embryonic genome during neurulation in the brain, and a little later in the branchial arch mesenchyme and in the forming myotomes. These expression patterns suggest that there are several functions for the FGFs.

The most successful strategy for inhibition of the FGF system has been the use of a dominant negative receptor construct introduced by Kirschner and colleagues. Overexpression of this construct can abolish the FGF responsiveness of animal caps. In whole embryos, the absence of FGF signaling causes a reduction, although not a total ablation, of mesoderm formation. There is also a severe effect on axis formation in which formation of the posterior parts is reduced consequent on an inhibition of invagination and elongation of the dorsal mesoderm.

Thus, the present evidence suggests that the FGF system contributes to, although is not solely responsible for, mesoderm induction in vivo. It is also necessary for normal gastrulation movements, particularly in the dorsal mesoderm, and is likely to have several later functions, particularly in development of the central nervous system and the myotomes. © 1994 Wiley‐Liss, Inc.
Structure of the bovine follicle‐stimulating hormone receptor complementary DNA and expression in bovine tissues
Authors: Houde Alain , Lambert Anick , Silversides David W. , Lussier Jacques G. , Saumande Jean
Published: February 2005

We report the complementary DNA structure obtained by reverse transcription and polymerase chain reaction amplification encoding the complete amino acid sequence for the bovine follicle‐stimulating hormone receptor (bFSHr). The deduced amino acid sequence for the cDNA revealed a mature polypeptide consisting of 678 amino acids (theoretical weight of 76.4 kDa) and a 17 amino acid putative leading signal peptide. The receptor consists of a large NH2‐terminal extracellular membrane domain of 349 aa with 3 potential N‐linked glycosylation sites, a transmembrane domain (264 aa) consisting of 7 putative membrane spanning segments, and an intracytoplasmic COOH‐terminal domain (65 aa). Four potential phosphorylation sites were found in the transmembrane domain and the COOH‐terminal domain. The amino acid sequence is 97%, 89%, and 88% homologous to the ovine, human, and rat FSHr respectively, with complete conservation of the 22 cysteine residues in the whole protein and the 3 N‐linked glycosylation sites on the extracellular membrane domain. Northern blot analysis of total mRNA in bovine tissues revealed a major mRNA transcript of 2.55 kb for the bFSHr in the ovary without corpus luteum, and in the testis. No expression was found in other tissues analyzed. Total RNA from bovine granulosa cells collected from pregnant mare serum gonadotropin (PMSG)‐treated prepubertal heifers showed 2 major mRNA transcripts of 6.8 and 2.55 kb, and 3 minor transcripts of 3.8, 3.3, and 1.6 kb. Bovine granulosa cells cultured with porcine FSH (0, 2, 10 ng/ml) for 4 days showed a decrease in the steady state level of the FSHr mRNA. This decrease was shown to be independent of the size of the transcript. Therefore, expression of the bovine FSHr by bovine granulosa cells is downregulated at the message level when exposed to constant concentrations of FSH. ©1994 Wiley‐Liss, Inc.
Production of germline chimeric chickens, with high transmission rate of donor‐derived gametes, produced by transfer of primordial germ cells
Authors: Naito Mitsuru , Tajima Atsushi , Yasuda Yoshiaki , Kuwana Takashi
Published: February 2005

Germline chimeric chickens were produced by transfer of primordial germ cells from White Leghorn to Barred Plymouth Rock, and vice versa. Blood was collected from stage 13–15 embryos and primordial germ cells were concentrated by Ficoll density gradient centrifugation. Approximately 200 primordial germ cells were injected into the bloodstream through the dorsal aorta of stage 14–15 recipient embryos from which blood had been drawn via the dorsal aorta prior to the injection. Intact embryos were also prepared as recipients for White Leghorns only. The manipulated embryos were cultured in recipient eggshells until hatching. Germline chimerism of the chickens reaching maturity was examined by mating them with Barred Plymouth Rocks and donor‐derived offspring were identified based on their feather color. The efficiency of production of germline chimeras was 95% (19/20). When primordial germ cells were transferred from White Leghorn to Barred Plymouth Rock, the average frequency of donor‐derived offspring was 81% for three male chimeras (96% for one female chimera), and it was ∼3.5 times higher for transfer in the opposite direction (23% for 6 male chimeras). Removing blood from recipient embryos prior to primordial germ cell injection enhanced the frequency of donor‐derived offspring by 10% in resulting male chimeras. Male chimeras produced donor‐derived offspring more frequently (∼3.8 times) than female chimeras. Increases, decreases, or no changes were observed in the frequency of donor‐derived offspring from the germline chimeras with increasing age. Male to female ratio of the offspring derived from the donor primordial germ cells did not deviate significantly in male and female chimeras, suggesting that primordial germ cells that have different sex from recipient embryos could not differentiate into functional gametes. The technique for primordial germ cell transfer employed in this experiment is simple to perform and resulted in the efficient production of germline chimeras with high transmission rates of donor‐derived gametes. This system provides a powerful tool for avian embryo manipulation. © 1994 Wiley‐Liss, Inc.
Follicular wall maintains meiotic arrest in bovine oocytes cultured in vitro
Authors: A.M. De Loos F. , Zeinstra E. , M. Bevers M.
Published: February 2005

Cumulus oocyte complexes (COCs) were cocultured with parts of the follicular wall. Coculture conditions were such that the COCs were 1) in continuous contact with the follicular wall (FWC), 2) separated from the follicular wall at collection but in contact with it during culture (FWR), and 3) separated from the follicular wall, but cultured in its vicinity (FWNR). Oocytes cultured for 24 hr under FWC conditions maintained the germinal vesicle stage. Under FWR conditions the germinal vesicle stage was not maintained, but an arrest at metaphase I of meiosis occurred in mostof the oocytes. When COCs were cultured in the vicinity of the follicular wall (FWNR), meiosis was resumed and similar numbers of oocytes progressed to metaphase II of meiosis as compared to cultures of COCs without coculture with parts of the follicular wall. When COCs were isolated from the follicular wall after 24 hr of culture and additionally cultured for another 24 hr, the oocytes showed the same capability of resuming meiosis as fresh, isolated cumulus oocyte complexes.

It is concluded that maintenance of contact with the follicular wall is necessary to maintain meiotic arrest. When COCs restore a physical contact with the follicular wall during culture, an arrest at metaphase I occurs. © 1994 Wiley‐Liss, Inc.
T‐cell functional regions of the human IL‐3 proximal promoter
Authors: R. Ryan G. , A. Vadas M. , F. Shannon M.
Published: February 2005

The human interleukin‐3 (IL‐3) gene is expressed almost exclusively in activated T cells. Its expression is regulated at both the transcriptional and post‐transcriptional level. We have previously shown that treatment of Jurkat T cells with phytohemaglutinin (PHA) and the phorbol ester, PMA, activated transcription initiation from the lL‐3 gene. To define the regions of the gene required for transcription activation, we generated a series of reporter constructs containing different regions of the IL‐3 gene 5′ and 3′flanking sequences. Both positive and negative regulatory elements were identified in the proximal 5′ flanking region of the lL‐3 gene. The promoter region between –173 and –60 contained the strongest activating elements. The transcription factor AP‐1 could bind to this positive activator region of the promoter. We also examined the function of the lL‐3 CK‐1/CK‐2 elements that are present in many cytokine genes and found that they acted as a repressor of basal level expression when cloned upstream of a heterologous promoter but were also inducible by PMA/PHA. © 1994 Wiley‐Liss, Inc.
Leukaemia inhibitory factor and the regulation of pre‐implantation development of the mammalian embryo
Authors: L. Stewart Colin
Published: February 2005

This paper reviews the evidence that certain growth factors, particularily leukaemia inhibitory factor (LIF), play a crucial role in regulating the development of the pre‐implantation mammalian embryo. LIF was originally implicated in regulating the early development of the mouse embryo because it inhibited the differentiation of embryonic stem (ES) cells, pluripotential cells derived from the inner cell mass of the blastocyst. Subsequent studies on its role in vivo revealed, surprisingly, that it is essential for the growth rather than the differentiation of the blastocyst. In vivo, overtly normal blastocysts can be produced in a LIF‐deficient environment that are capable of forming viable fertile adults. However, in the absence of LIF, they fail to implant and enter into a state resembling that exhibited by blastocysts undergoing delayed implantation, which is characterized by a cessation of cell proliferation. This failure to implant occurs because the principle sites of LIF production are the endometrial glands of the uterus. These synthesize and secrete LIF at implantation, with LIF synthesis essential for implantation. Preliminary evidence indicates that LIF synthesis is required both by the uterus for it to undergo decidualization and by the blastocyst for implantation. These data indicate that the maternal environment plays a crucial role in the development and growth of the pre‐implantation embryo, by supplying factors that regulate these processes in the embryo. © 1994 Wiley‐Liss, Inc.
Relationships between biochemical markers for residual sperm cytoplasm, reactive oxygen species generation, and the presence of leukocytes and precursor germ cells in human sperm suspensions
Authors: Aitken John , Buckingham Donna , Krausz Csilla
Published: February 2005

In this study, we have examined the relationship between creatine phosphokinase (CPK), a biochemical measure of human sperm quality (Huszar et al., 1988 a, b, 1990; Huszar and Vigue, 1994), and a marker for the presence of residual cytoplasm in human spermatozoa, glucose‐6‐phosphate dehydrogenase (G6PDH). We then determined whether the diagnostic potential of these enzymes was related to the capacity of the sperm suspensions to generate reactive oxygen species (ROS) and/or the presence of leukocytes and precursor germ cells. Across the data set as a whole, G6PDH and CPK were highly correlated with each other and, to a lesser extent, with the generation of ROS. Contamination of the sperm suspensions with leukocytes might have contributed to these associations, since the presence of such cells was also significantly correlated with CPK, G6PDH, and ROS. However, even after the leukocytes had been carefully removed, G6PDH was still highly correlated with CPK (r= 0.794), indicating that both criteria were providing similar information on the cytosolic component of human sperm suspensions. In the absence of leukocyte contamination, CPK and G6PDH activities were also correlated with the presence of precursor germ cells, and this association may, in part, explain the diagnostic value of these criteria. An additional component of their prognostic value may be reflected in the statistically significant association observed between G6PDH activity and ROS generation. A possible mechanism for such an association is suggested, which should be amenable to experimental verification. © 1994 Wiley‐Liss, Inc.
Putative rat sperm lipid‐binding protein: Isolation and partial characterization
Authors: Xu Wenhao , Moore Alison , M. Ensrud Kathy , W. Hamilton David , L. Pryor Jon
Published: February 2005

Previous work has identified a prominent 22–24‐kD protein that is present in rat male reproductive tissues, including epididymis and testis (Brooks, 1985; Jones and Brown, 1987; Moore et al., 1987). Using a monoclonal antibody (designated mAb‐B109) against this 24‐kD antigen (referred to as B109), we have isolated the protein using a combination of chromatofocusing and electroelution from SDS–PAGE gels, and reverse phase HPLC. B109 (pl = 4.8) is amino‐terminal blocked. To obtain internal amino acid sequences, the isolated protein was cleaved either with cyanogen bromide in 70% formic acid or with TLCK‐treated chymotrypsin. With cyanogen bromide treatment, two peptides, 17.8 kD and 11.9 kD, were isolated and partial amino acid sequences obtained. Chymotryptic peptides were isolated by reverse‐phase HPLC and two were chosen for sequence analysis A computer search for sequence homology through the protein identification resource (PIR) matched B109 to a basic 21‐kD cytosolic protein (pl = 7.4) found in bovine brain (> 80% homology). When peptide sequence differences obtained in the present study were substituted into the 21‐kD cytosolic protein sequence obtained from the PIR using IntelligeneticsTM software, the calculated pl dropped from 7.4 to 5.8, suggesting that pl differences between the bovine and rat molecules are the result of amino acid substitutions in the testis protein and not tissue‐specific posttranslational processing. It has been postulated that the 21‐kD bovine brain protein is associated with phospholipid transport, although the function of B109 is unknown. © 1994 Wiley‐Liss, Inc.
Vitronectin is an intrinsic protein of human spermatozoa released during the acrosome reaction
Authors: M. Fusi Francesco , Lorenzetti I. , Mangili F. , C. Herr John , J. Freemerman Alex , Gailit James , A. Bronson Richard
Published: February 2005

Evidence has been presented that oolemmal integrins and their ligands on spermatozoa may play a role in gamete interactions leading to fertilization. We previously demonstrated that vitronectin (Vn) could be extracted from fresh human spermatozoa and detected in Western blots, and Vn was observed on the surface of living, capacitated sperm by indirect immunofluorescence. In the present experiments, messenger RNA encoding Vn was detected in human testis poly (A+) RNA using Northern analysis, and Vn was localized within the acrosomal region of ejaculated sperm by immunoperoxidase and immunofluorescence staining. During the acrosome reaction, induced in capacitated spermatoza by lonomycin, Vn was released into the medium in a calcium‐dependent manner. Vn appears to be a specific product of intratesticular spermatozoa that is secreted during the acrosome reaction. These findings suggest that Vn is positioned to play a strategic role in gamete interactions leading to fertilization. © Wiley‐Liss, Inc.
Developmentally regulated protease expression during sea urchin embryogenesis
Authors: Vafa Omid , Nishioka David
Published: February 2005

A temporal study of protease expression employing the technique of SDS‐PAGE gelatin substrate zymography revealed a definitive appearance of proteases during early development in the sea urchins, Lytechinus pictus and Strongylocentrotus purpuratus. The levels of these proteases increase substantially during gastrulation in each species. The two major proteases with relative molecular masses of 57 and 50 kDa were found to be inhibited by the zinc chelator, 1,10‐phenanthroline, the more nonspecific metal chelator, EDTA, and the reducing agent, dithiothreitol. The serine protease inhibitor, benzamidine, exerted no effects on the activities of these proteases, and both enzymes exhibited activity in the neutral to slightly basic pH range. Treatment of embryos with actinomycin D, an inhibitor of transcription, beginning up to 9 hr after fertilization, inhibited the subsequent appearances of the two proteases 48 hr after fertilization, as well as any morphological changes associated with gastrulation. Treatments beginning 15 and 21 hr after fertilization resulted in increased levels of proteases that correlate with arrests at successively more advanced stages of gastrulation. SDS‐PAGE zymographic analyses of five different embryo fractions indicated that the 57‐ and 50‐kDa proteases are localized in the blastocoel, and blastocoelic protease activity was further confirmed microscopically by in situ zymography. Hence, the 57‐ and 50‐kDa proteases are characterized as metalloproteases. Their expression is dependent on transcription of the embryonic genome, and their spatiotemporal appearance suggests an involvement in blastocoelic matrix remodeling during gastrulation. © 1995 Wiley‐Liss, Inc.
Persistence of protamine precursors in mature sperm nuclei of the mouse
Authors: Debarle Michel , Martinage Arlette , Sautiere Pierre , Chevaillier Philippe
Published: February 2005

During mouse spermiogenesis, two protamines, mP1 and mP2, are synthesized in replacement of histones. One of them (protamine mP2, 63 residues) appears at first in elongating spermatid nuclei as a pro‐protamine of 106 residues (pmP2) with an amino‐terminal extension that is progressively excised. The two protamines were previously described as the only proteins associated with DNA in sperm chromatin.

This paper shows that the nuclear proteins of mouse spermatozoa are indeed heterogeneous: at least six minor polypeptides in addition to protamines can be identified. The primary structure of four of them has been established. They are intermediate in the maturation of the precursor of protamine mP2 and correspond to polypeptides pmP2/11, pmP2/16, pmP2/20, and pmP2/32, characterized previously in mouse testis. Therefore, these intermediates of proteolysis generated from pmP2 inside spermatid nuclei persist in mature sperm, whereas the largest precursors, pmP2 and pmP2/5, disappear. These findings clearly indicate that limited proteolysis events still occur outside of the testis. © 1995 Wiley‐Liss, Inc.
Role of sulfated glycoprotein‐1 (SGP‐1) in the disposal of residual bodies by sertoli cells of the rat
Authors: Igdoura Suleiman A. , Morales Carlos R.
Published: February 2005

Sulfated glycoprotein‐1 (SGP‐1) is a polypeptide secreted by Sertoli cells in the rat. Sequence analysis revealed a 76% sequence similarity with human prosaposin produced by various cell types. Human prosaposin is a 70 kDa protein which is cleaved in the lysosomes into four 10–15 kDa polypeptides termed saposins A, B, C, and D. The function of lysosomal saposins is to either solubilize certain membrane glycolipids or to form complexes with lysosomal enzymes and/or their glycolipid substrate to facilitate their hydrolysis. The present investigation dealt with the delivery of SGP‐1 into the phagosomes of Sertoli cells; these phagosomes contain the residual bodies which detach from the late spermatids at the time of spermiation. Immunogold labeling with anti‐SGP‐1 antibody was found over Sertoli cell lysosomes, but was absent from phagosomes formed after phagocytosis of spermatid residual bodies in the apical Sertoli cell cytoplasm in stages VIII and early IX of the cycle of the seminiferous epithelium. The phagosomes found later in the basal Sertoli cell cytoplasm in stages IX and X of the cycle became labeled with the antibody as the components of the residual bodies rapidly underwent lysis and disappeared from the Sertoli cells. Sertoli cell lysosomes isolated by cell fractionation (estimated purity of 80%) were found to contain a 65 kDa form of SGP‐1 or prosaposin, as well as the 15 kDa polypeptides or saposins. Thus, it appears that this unique lysosomal form of SGP‐1 reached the Sertoli cell phagosomes and that their derived polypeptides, the saposins, must play a role in the hydrolysis of membrane glycolipids found in phagocytosed residual bodies. © 1995 Wiley‐Liss, Inc.
Calcium and meiotic maturation of the mammalian oocyte
Authors: Homa Sheryl T.
Published: February 2005

The role of calcium in the regulation of both the meiotic and mitotic cell cycles has been the subject of considerable investigation in the nonmammalian field. In contrast, the mechanisms for signalling meiotic maturation in the mammalian oocyte are not as well documented nor as clearly defined. In the mammalian oocyte, calcium is associated with both spontaneous and hormone‐induced meiotic maturation. A transient release of endogenously stored calcium precedes germinal vesicle breakdown and can override cyclic AMP maintained meiotic arrest; it thus may signal the resumption of meiosis. Additionally, extracellular calcium is apparently required for meiotic progression past metaphase I. The time sequence for meiotic resumption and progression is very varied between species. The timing of cell cycle protein synthesis during meiosis suggests that cyclins may be expressed in oocytes of some species much earlier in their development than in others. A generic model is proposed for the mechanism for triggering meiotic resumption in the mammalian oocyte. In this model, the critical components of meiotic resumption involve the temporal relationship of cyclin synthesis and the subsequent activation of the MPF complex by the calcium signal generated, which accounts for differences among species. © 1995 Wiley‐Liss, Inc.
Inhibition of rabbit sperm acrosomal enzymes by gossypol
Authors: Yuan Y. Y. , Shi Q. X. , Srivastava P. N.
Published: February 2005

The effect of gossypol on the activities of 10 acrosomal enzymes of the rabbit sperm was evaluated. Acrosin, Azocoll proteinase, neuraminidase, and arylsulfatase were significantly inhibited or completely inactivated by 12–76 μM gossypol. Hyaluronidase, β‐glucuronidase, and acid phosphatase were inhibited only at a higher concentration of gossypol (380 μM). Phospholipase C, alkaline phosphatase, and β‐N‐Acetyl glucosaminidase were not inhibited even at 380 μM gossypol. Gossypol was found to be a noncompetitive inhibitor of arylsulfatase with a Ki of 120 μM. The inhibition was reversible and dose‐dependent. As the acrosomal enzymes were more sensitive to the inhibition by gossypol compared to sperm enzymes involved in glycolysis or energy production, these assays may serve as a more reliable indicator for monitoring the occurence of gossypol‐induced sterility. © 1995 Wiley‐Liss, Inc.
Changes in dopamine uptake and developmental effects of dopamine receptor inactivation in the sea urchin
Authors: Carginale Vincenzo , Borrelli Lucia , Capasso Antonio , Parisi Elio
Published: February 2005

[3H]‐dopamine ([3H]‐DA) uptake was measured in the presence or absence of the catecholamine uptake inhibitor nomifensine in both unfertilized and fertilized eggs. Specific [3H]‐DA uptake depended on time and [3H]‐DA concentration; it was high in unfertilized eggs, declined 20–30 min after fertilization, and rose again during cleavage. Irreversible inactivation of dopamine receptors by N‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline (EEDQ) resulted in a complete loss of sensitivity of egg adenylate cyclase to dopamine stimulation. In fertilized eggs treated with EEDQ for 1 hr, restoration of adenylate cyclase activity sensitive to dopamine stimulation could be observed 4 hr after the end of treatment, thus suggesting the appearance of new dopamine receptors in cleaving eggs. Short‐term EEDQ treatment on unfertilized eggs, although not impairing fertilization, resulted in cleavage inhibition; the same treatment carried out soon after fertilization, on the other hand, elicited no effect on development. On the contrary, in embryos subjected to continuous treatment with EEDQ, development was impaired independent of the stage at which the treatment was started. © 1995 Wiley‐Liss, Inc.
The baboon expresses the calbindin‐D9k gene in intestine but not in uterus and placenta: Implication for conservation of the gene in primates
Authors: Jeung Eui‐Bae , Fan Nancy C. , Leung Peter C. K. , Herr John C. , Freemerman Alex , Krisinger John
Published: February 2005

Expression of the Calbindin‐D9k (CaBP‐9k) gene was studied in the baboon. Northern blot analysis using a human CaBP‐9k cDNA probe detected expression in duodenum but not in uterus and placenta. Reverse transcription/polymerase chain reaction (RT/PCR) confirmed this expression pattern and indicated a high degree of identity between the baboon and human CaBP‐9k mRNAs. PCR was employed to amplify the intron A region of the baboon CaBP‐9k gene using human‐derived primers and baboon genomic DNA. The baboon intron was closely related to the human CaBP‐9k intron A, including the presence a complete Alu‐repetitive element. Most significantly, a 13 nucleotide long element at the 5′ end of the baboon intron matched exactly the human sequence. This element represents a nonfunctional variation of an estrogen response element found at the same location in the rat CaBP‐9k gene. The rat element functions as an enhancer and mediates uterine and possibly placental CaBP‐9k expression in the rat and probably most other mammals. The finding of a modified ERE in baboon as in human suggests that during primate evolution the expression of this mammalian‐specific gene has been eliminated in uterus and placenta. This scenario raises the question of the role of CaBP‐9k in these reproductive tissues. © 1995 Wiley‐Liss, Inc.
Identification of an Mr 60,000 polypeptide unique to the meiotic spindle of the mouse oocyte
Authors: Sobel J. Sabina , Pinto‐Correia Clara , Goldstein Elaine G.
Published: February 2005

The mouse oocyte expresses an Mr 60,000 (p60) polypeptide that is associated with the first and second meiotic spindles. Immunoreactive p60 was not detectable in the meiotic spindles of male germ cells or in mitotic spindles. P60 was identified with a polyclonal antibody whose predominant activity is directed against ankyrin. However, immunoadsorption experiments demonstrated that p60 is not an ankyrin isoform and represents a secondary activity of the polyclonal antibody. Circumstantial evidence suggest that p60 may be a micro‐tubule‐associated protein. Since the most obvious difference between the female meiotic spindle and other spindles is the long half‐life of the former, we hypothesize that p60 may function in the maintenance of the long‐lived female meiotic apparatus. © 1995 Wiley‐Liss, Inc.
Evidence that XR family interspersed RNA may regulate translation in Xenopus oocytes
Authors: Liu Chengyu , Smith L. Dennis
Published: February 2005

It has been shown that about two thirds of Xenopus oocyte or sea urchin egg cytoplasmic poly(A)+ RNA contains interspersed repetitive sequences. The functional significance of this interspersed RNA has remained unknown. Here the function of a subfamily of interspersed RNA (XR family; McGrew and Richter, 1989: Dev Biol 134:267–270) in Xenopus oocytes was studied. We found that the elimination of T7 XR (one of the two complementary strands of the XR repeat) interspersed RNA by complementary oligodeoxynucleotides significantly inhibited protein synthesis. On the other hand, the injection of in vitro synthesized T7 XR RNA stimulated translation. Moreover, the insertion of the T7 XR RNA sequence into globin mRNA repressed the translation of the globin mRNA. In order to explain these results, we analyzed interactions between the XR interspersed RNA and oocyte proteins. We found that the major XR RNA binding proteins were p56 and p60, which could be the known mRNA “masking” proteins that bind mRNA and inhibit translation. Further, a 42 kD protein has been identified that appears to bind T7 XR RNA relatively specifically, although it interacts with mRNA with a lower affinity. Based on all of these data, we have proposed that interspersed RNA may be involved in regulating translation by competing with mRNA to interact with certain proteins that can regulate translation. © 1995 Wiley‐Liss, Inc.
Intracellular pH of bovine sperm increases during capacitation
Authors: Vredenburgh‐Wilberg W. L. , Parrish J. J.
Published: February 2005

The effect of heparin‐induced capacitation on the intracellular pH (pHi) of individual bovine sperm was determined with image analysis. Sperm were loaded with the acetoxymethyl ester of the pH sensitive fluorescent indicator, 2′,7′‐bis(carboxyethyl)‐5(6)‐carboxy‐fluorescein (BCECF). The pHi of 5303 sperm was evaluated from a total of five bulls at .5, 2, 3, 4, and 5 h of incubation. The pHi did not differ between the sperm head and mid‐piece (P > 0.05). An increase in sperm head pHi was seen in heparin‐treated sperm at 3, 4, and 5 h of incubation relative to sperm incubated without heparin (control, P < 0.05). At 5 h of incubation, the pHi in heparin‐treated sperm was 6.92 ± 0.07, while control‐treated sperm pHi was 6.70 ± 0.03. Initially a normal frequency distribution was seen for sperm pHi in both heparin‐ and control‐treated sperm. As the incubation progressed, the frequency distribution began to skew towards higher pHi in both samples but was more dispersed for the heparin‐treated sperm. Following an NH4Cl‐induced alkaline load, the pHi of both control‐ and heparin‐treated sperm recovered toward the resting pHi with a half‐time of recovery of 1.5–1.7 min. The recovery of sperm pHi was not due to leakage of NH4+ into sperm because recovery also occurred with trimethylamine. The instantaneous velocity of the pHi recovery (vi) was dependent on pHi and decreased as pHi decreased. Capacitation by heparin was associated with an 81% decrease in vi at a pHi of 7.00, but there was no effect of capacitation on the proton buffering power of the sperm, which was 87 ± 8 mM/pH unit. Results demonstrate that both the regulation of pHi and resting pHi were altered during capacitation of bovine sperm by heparin. © 1995 Wiley‐Liss, Inc.
Isolation of cDNAs from maternal mRNAs specifically present during early development
Authors: Moreau Jacques , Iouzalen Nathalie , Mechali Marcel
Published: February 2005

In many animals the regulation of early embryonic development is under the control of mRNAs of maternal origin. We describe here the construction and characterization of a subtractive cDNA library to isolate genes whose expression is restricted to early development. Such genes might be potentially involved in developmental diseases, if accidentally expressed during adult life. Seven yet unknown genes preferentially expressed in the oocyte and present during early development were isolated from this library by differential screening. Their transcription patterns provide evidence for a selective expression during oogenesis and the existence of a gene family specifically implicated in functions linked to early embryogenesis. © 1995 Wiley‐Liss, Inc.
Oxytocin induced a biphasic increase in the intracellular Ca2+ concentration of porcine myometrial cells: Participation of a pertussis toxin—insensitive G‐protein, inositol 1,4,5‐trisphosphate—sensitive Ca2+ pool, and Ca2+ channels
Authors: Zhuge Ronghua , Li Siben , Chen Ter‐Hsin , Hsu Walter H.
Published: February 2005

This study investigated the underlying mechanisms of oxytocin (OT)‐induced increases in intracellular Ca2+ concentrations ([Ca2+]i) in acutely dispersed myometrial cells from prepartum sows. A dosedependent increase in [Ca2+]i was induced by OT (0.1 nM to 1 μM) in the presence and absence of extracellular Ca2+ ([Ca2+]e). [Ca2+]i was elevated by OT in a biphasic pattern, with a spike followed by a sustained plateau in the presence of [Ca2+]e. However, in the absence of [Ca2+]e, the [Ca2+]i response to OT became monophasic with a lower amplitude and no plateau, and this monophasic increase was abolished by pretreatment with ionomycin, a Ca2+ ionophore. Administration of OT (1 μM) for 15 sec increased inositol 1,4,5‐trisphosphate (IP3) formation by 61%. Pretreatment with pertussis toxin (PTX, 1 μg/ml) for 2 hr failed to alter the OT‐induced increase in [Ca2+]i and IP3 formation. U‐73122 (30 nM to 3 μM), a phospholipase C (PLC) inhibitor, depressed the rise in [Ca2+]i by OT dose dependently. U‐73122 (3 μM) also abolished the OT‐induced IP3 formation. Thapsigargin (2 μM), an inhibitor of Ca2+‐ATPase in the endoplasmic reticulum, did not increase [Ca2+]i. However, it did time‐dependently inhibit the OT‐induced increase in [Ca2+]i. Nimodipine (1 μM), a Voltage‐dependent Ca2+ channel (VDCC) blocker, inhibited the OT‐induced plateau by 26%. La3+ (1 μM), a nonspecific Ca2+ channel blocker, abrogated the OT‐induced plateau. In whole‐cell patch‐clamp studies used to evaluate VDCC activities, OT (0.1 μM) increased Ca2+ Current (Ica) by 40% with no apparent changes in the current‐voltage relationship. The OT‐induced increase in Ica reached the maximum in 5 min, and the increase was abolished by nimodipine (1 μM). These results suggested that (1) activation of OT receptors in porcine myometrium evokes a cascade in the PTX‐insensitive G‐protein–PLC‐IP3 signal transduction, resulting in an increase in [Ca2+]i; (2) the OT‐induced increase in [Ca2+]i is characterized by a biphasic pattern, in which the spike is predominately contributed by the intracellular Ca2+ release from the IP3‐sensitive pool, and to a lesser extent by Ca2+ influx, whereas the plateau is from increased Ca2+ influx; and (3) the influx is via VDCC and receptor‐operated Ca2+ channels. © 1995 Wiley‐Liss, Inc.
Transient appearance of CRES protein during spermatogenesis and caput epididymal sperm maturation
Authors: Cornwall Gail A. , Hann Stephen R.
Published: February 2005

In previous studies we identified an epididymal gene that exhibits homology to the cystatin family of cysteine protease inhibitors. The expression of this gene, termed CRES (cystatin‐related epididymal and spermatogenic), was shown to be highly restricted to the proximal caput epididymal epithelium with less expression in the testis and no expression in the 24 other tissues examined. In this report, studies were carried out to examine CRES gene expression in the testis as well as to characterize the CRES protein in the testis and epididymis. In situ hybridization experiments revealed that within the testis CRES gene expression is stage‐specific during spermatogenesis and is exclusively expressed by the round spermatids of Stages VII‐VIII and the early elongating spermatids of Stages IX and X. Immunohistochemical studies demonstrated that CRES protein was transiently expressed in both the testis and epididymis. Within the testis the protein was localized to the elongating spermatids, whereas within the epididymis CRES protein was exclusively synthesized by the proximal caput epithelium and then secreted into the lumen. Surprisingly, the secreted CRES protein had completely disappeared from the epididymal lumen by the distal caput epididymidis. Western blot analysis of testicular and epididymal proteins showed that the CRES antibody specifically recognized a predominant 19 kDa CRES protein and a less abundant 14 kDa form. These observations suggest that the CRES protein performs a specialized role during sperm development and maturation. © 1995 Wiley‐Liss, Inc.
Characterization of alternatively spliced human SP‐10 mRNAs
Authors: Freemerman Alex J. , Flickinger Charles J. , Herr John C.
Published: February 2005

Alternatively spliced mRNAs encoding the human intraacrosomal protein SP‐10 were sought by the reverse transcriptase polymerase chain reaction (RTPCR). Eleven RTPCR products were identified, characterized, and found to represent authentic alternatively spliced SP‐10 mRNAs. The 11 alternatively spliced SP‐10 mRNAs encoded proteins ranging from 81 to 265 amino acids. The 10 smaller variants all resulted from one or two in‐frame deletions in exons 2 and/or 3 of the SP‐10 genomic sequence. Quantitative competitive RTPCR showed that the four largest SP‐10 mRNAs represented the majority (>99%) of the SP‐10 message in testes from each of four men. The relative abundance of each of the four SP‐10 mRNAs varied between individuals, but the longest SP‐10 mRNA, SP10‐1, which encoded a 265 amino acid protein, was consistently the most abundant, comprising 53–72% of the total SP‐10 message. This was followed by the second largest SP‐10 mRNA, SP10‐2, which encoded a protein of 246 amino acids and comprised 15‐32%. The third and fourth largest SP‐10 mRNAs, SP10‐3 and SP10‐4, encoded proteins of 210 and 195 amino acids and accounted for 3.4–8.3% and 8.7–12.5% of the total SP‐10 messages, respectively. The remaining 7 SP‐10 mRNAs combined accounted for<1% of the total SP‐10 message. Within the low abundance group of mRNAs were two that deleted the entire third exon of SP‐10. The present study suggests that phenomena of cryptic splicing and exon skipping occur within the SP‐10 mRNA. Along with proteolysis, alternative splicing also helps to explain the heterogeneous forms of SP‐10 that have been observed on Western blots of human sperm extracts. © 1995 Wiley‐Liss, Inc.
Homeobox gene expression in human oocytes and preembryos
Authors: Verlinsky Yury , Morozov Gennedy , Gindilis Viktor , Strom Charles M. , Freidin Michael , Rechitsky Svetlana , Verlinsky Oleg , Ivakhnenko Victor , Zdanovsky Valery , Kuliev Anver
Published: February 2005

Homeobox gene expression in human preimplantation development has not been established. We used reverse transcriptase‐polymerase chain reaction (RT‐PCR) with intron spanning primer sets to investigate the presence of mRNA of homeobox genes in human oocytes and preembryos. RT‐PCR products obtained from normal and unfertilized oocytes, from cleaving normal and triploid embryos, and from morulae and blastocysts were cloned, sequenced, and analyzed for the presence of homeobox sequences. The presence of mRNA of homeoboxes HoxA4 and HoxA7 was demonstrated; HoxA4 was present in normal and unfertilized oocytes and also in a 4‐cell embryo. HoxA7 was present in normal oocytes and cleaving triploid embryos. © 1995 Wiley‐Liss, Inc.
Modulation of gene expression in the preimplantation mouse embryo by TGF‐α and TGF‐β
Authors: Babalola Gbolagade O. , Schultz Richard M.
Published: February 2005

The effect of growth factors on regulating gene expression in the preimplantation mouse embryo was examined, since results of previous experiments revealed a stimulatory effect of exogenously‐added growth factors on preimplantation development in vitro. Treatment of early cavitating blastocysts with either 250 pM TGF‐α or TGF‐β results in changes in the pattern of total protein synthesis as assessed by high‐resolution two‐dimensional gel electrophoresis. In some cases, the synthesis of a particular polypeptide is either up‐ or downregulated by each growth factor, whereas in other instances the synthesis of a polypeptide is modulated by one but not the other growth factor. Use of the mRNA differential display method permitted the identification of genes whose expression is either up‐ or downregulated by these growth factors. Treatment of mouse blastocysts with either TGF‐α or TGF‐β results in the increased expression of the b subunit of the F0 ATPase. TGF‐β also stimulates the expression of the DNA polymerase α. TGF‐α treatment results in the increase in expression of a gene homologous to the human HEPG2 cDNA, as well as in a decrease in expression of fibronectin. © 1995 Wiley‐Liss, Inc.
Differential constitutive expression of interferon genes in early mouse embryos
Authors: Riego Eileen , Pérez Aimeé , Martínez Rebeca , Castro Fidel O. , Lleonart Ricardo , de la Fuente José
Published: February 2005

Recent evidence suggests that several processes during mammalian embryogenesis may be regulated by IFNs or IFN‐like molecules. With the use of MAPPing, the simultaneous presence of transcripts homologous to IFN‐α, IFN‐β, IRF‐1, and IRF‐2 was examined in mouse embryos and in embryonal carcinoma (EC) P19 cells, which are equivalent to epiblast cells of the early postimplantation blastocyst. Transcripts for IFN‐α, but not for IFN‐β, were detected as maternal transcripts in the ovulated oocyte and persisted over early embryogenesis. IRF‐1 transcripts appeared only after the first cell cleavage in the two‐cell stage embryo. IRF‐2 transcripts were analyzed only in EC P19 cells and were found in both undifferentiated (D−) and differentiated (D+) cells. The IFN‐α transcripts present in (D−) P19 cells were cloned and the partial cDNA sequences determined. Mu IFN‐αA and a new Mu IFN‐α species (Mu IFN‐α12) were isolated from (D−) P19 cells. The presence of constitutive IFN‐α transcripts in early mouse embryos suggests a role for these molecules during embryogenesis. © 1995 Wiley‐Liss, Inc.
Monoallelic expression of IGF2 at the human fetal/maternal boundary
Authors: Ekström Tomas J. , Cui Hengmi , Nyström Anders , Rutanen Eeva‐Marja , Ohlsson Rolf
Published: February 2005

IGF2 is expressed in both placental and decidual tissues, enabling an analysis of the parental imprinting over the fetomaternal boundary. Evidence is provided that IGF2 is monoallelically expressed in both placenta and pregnant, as well as nonpregnant, endometrium. These observations suggest that the maternally derived IGF2 allele is inactivated during germline transmission. Comparison of promoter usage in decidua and placental samples shows that the P3 promoter appears to regulated independently of the others. These observations are discussed with respect to current models of IGF2 imprinting and the hypothesized conflict of parental reproductive interests which bears on the phenomenon of parental imprinting. © 1995 Wiley‐Liss, Inc.