bluuebunny/crossref_metadata_2025 huggingface dataset:
Energy metabolism of spermatozoa of the sea urchin Glyptocidaris crenularis
Authors: Mita Masatoshi
Published: February 2005

Energy metabolism in spermatozoa of the sea urchin Glyptocidaris crenularis was examined. The spermatozoa contained not only several kinds of phospholipids and cholesterol but also triacylglycerides (TG). Following dilution of the dry sperm in sea water, the TG content decreased rapidly. Other lipids, however, remained at constant levels, except for an increase in the level of free fatty acid. Oil red‐O staining of spermatozoa showed that TG was principally located in part of the sperm midpiece. Also, high lipase activity was demonstrated in the spermatozoa. In both intact cells and a cell‐free system, 14C‐labeled fatty acids were oxidized to 14CO2. It is thus concluded that G. crenularis spermatozoa use TG as a substrate for energy metabolism.
Ultrastructural study of the mature egg of the marine sponge Stelletta grubii (porifera demospongiae)
Authors: Sciscioli Margherita , Liaci Lidia Scalera , Lepore Elene , Gherardi Miriam , Simpson Tracy L.
Published: February 2005

Stelletta grubii is an oviparous demosponge, which, during its reproductive period from summer to autumn, has small eggs (80–90 μm) dispersed uniformly in the mesohyl. The nucleolated nucleus is surrounded by dictyosomes containing small vesicles, which contribute to form reserve material. Vesicles, numerous food vacuoles, and groups of mitochondria are observed in the granular cytoplasm. Electron‐dense yolk inclusions and lipids are found peripherally. The cortical portion of the egg cytoplasm possesses vacuoles with fibrillar contents. The egg forms pseudopodia, which could permit the capture of numerous bacteria present in the surrounding mesohyl. A thick layer of collagen fibrils, including lophocytes, separates the egg from the surrounding sponge mesohyl. Ultrastructural analysis has demonstrated the presence both of cellular components capable of autosynthetic activity (nutrient vesicles) and of phagocytosis mechanisms (pseudopod capture of bacteria) for the storage of nutrients by the egg.
Association of exogenous DNA with cattle and insect spermatozoa in vitro
Authors: Atkinson Peter W. , Hines Eric R. , Beaton Sandra , Matthaei Klaus I. , Reed Ken C. , Bradley Mark P.
Published: February 2005

Spermatozoa isolated from domestic cattle (Bos taurus), the Australian sheep blowfly (Lucilia cuprina) and the honeybee (Apis mellifera) are capable of binding exogenous radiolabeled linear DNA. Both motile and nonmotile bovine sperm exhibit four distinct patterns of DNA association. Following treatment with DNase I, the relative proportion of one of these patterns increases specifically in living sperm, suggesting that a small proportion of DNA that associates with bovine sperm may be sequestered within the sperm head.
Gamete self‐discrimination in ascidians: A role for the follicle cells
Authors: De Santis Rosaria , Pinto Maria Rosaria
Published: February 2005

Gamete self‐incompatibility in the hermaphrodite tunicate Ciona intestinalis is a useful system with which to study self‐nonself recognition. We have used in vitro fertilization of oocytes isolated from the gonad of Ciona intestinalis to identify the cellular source of self‐sterility elements present on the egg envelopes. Here we show for the first time that self‐discrimination, which occurs on the egg vitelline coat, is established there in late oogenesis and is contributed or controlled by products of the overlying follicle cells. The acquisition of self‐sterility by the oocyte is prevented by the ionophore monensin, which suggests that the follicle cell self‐sterility controlling factor is a glycoprotein.
Fertilizing capacity of epididymal and testicular spermatozoa microinjected under the zona pellucida of the mouse oocyte
Authors: Lacham Orly , Trounson Alan
Published: February 2005

Mouse spermatozoa were obtained from the testis and caput, corpus, and cauda epididymis incubated for 2 h in capacitation medium and a single spermatozoon from the capacitated samples microinjected into the pervitelline space of mature mouse oocytes. Spermatozoa from the testis were unable to fertilize oocytes and few spermatozoa from the caput were capable of fertilization (0–7% depending on the method of preparation). Similar fertilization rates (30–45%) were obtained with spermatozoa with forward progressive motility from the proximal and distal corpus and the cauda epididymis, but those that were vibratory or with local circular motility had significantly reduced fertilizing capacity (6–10%). The capacity of spermatozoa from the different regions of the testis and epididymis to bind to zona‐free oocytes followed the same pattern as fertilization rate after microinjection. There was a progressive increase in acrosome reactions after 2 h incubation in capacitation medium in samples obtained from the testis to the cauda epididymis. Maturation of the capacity to bind to the perivitelline membrane and to develop forward progressive motility, rather than the capacity to acrosome react, appears to govern the fertilization of oocytes in which the zona has been bypassed by microinjection. These characteristics are obtained in the proximal segment of the corpus. However, there was evidence that the embryo developmental capacity of oocytes fertilized by spermatozoa from the higher segments of the epididymis is reduced, particularly in oocytes fertilized by caput spermatozoa. These observations suggest that sperm microinjection may have only a limited benefit for improving fertilization rates for men with high epididymal obstructive azoospermia.
Nuclear transplantation in bovine embryo: Fine structural and autoradiographic studies
Authors: Kaňka Jiřia , Fulka Josef , Fulka Josef , Petr Jaroslav
Published: February 2005

Nucleolar fine structure, “blebbing” activity of nuclear evelope, and activation of heterogeneous nuclear RNA (hnRNA) synthesis were studied in bovine reconstructed embryos obtained by electrofusion of a single eight‐cell blastomere with an enucleated oocyte. Developmental progress of nucleolar fine structure and hnRNA synthesis are arrested during three cell cycles following fusion. The activation of both appears during the eight‐cell stage of the reconstructed embryo, after the same number of cell cycles after fusion as in nonmanipulated bovine embryos after fertilization. “Blebbing” activity of nuclear envelope, which is already absent in original blastomeres, reappears after fusion and continues for the next two cell cycles. From the present results, it can be concluded that the donor nuclei are arrested after fusion in morphology and function. Their reactivation corresponds to the developmental pattern typical for normal bovine embryos.
Situs inversus in the developing mouse: Proteins affected by the iv mutation (genocopy) and the teratogen retinoic acid (phenocopy)
Authors: Van Keuren Margaret L. , Layton William M. , Iacob Ruxandra A. , Kurnit David M.
Published: February 2005

To decipher genes that are important in the determination of laterality, we compared two‐dimensional protein gels from wild‐type C57BL/6J mice and C57BL/6J mice that carried the iv mutation, which confers random determination of visceral situs. To span the time period(s) during which laterality determination occurs, we compared computer‐analyzed two‐dimensional protein gels from wild‐type mouse embryos and iv/iv mouse embryos at 7.5, 8.0, and 8.5 days post‐coitum. One polypeptide that was expressed only on day 8.0 of development and only in wild‐type embryos represents a particular candidate for determination of laterality. Day 8.5 postcoitum represents the earliest time in murine development that laterality is manifest. Two‐dimensional gels were compared from 8.5 day embryos that were C57BL/6J wild‐type, C57BL/6J iv/iv, or C57BL/6J wild‐type and exposed to the teratogen retinoic acid late on day 7. Reproducible alterations of protein synthesis were observed in both the iv genocopy and retinoic acid phenocopy, yielding abnormal laterality determination. The intersection of these peptide changes identifies a protein likely to play a role in the determination of laterality.
Cross‐linking a maturation‐dependent ram sperm plasma membrane antigen induces the acrosome reaction
Authors: McKinnon Christine A. , Weaver Frances E. , Yoder Jeffrey A. , Fairbanks Grant , Wolf David E.
Published: February 2005

ESA152 is a highly hydrophobic 18 kDa sialoglycoprotein, which becomes expressed on ram sperm in the proximal cauda epididymis. ESA 152 is expressed on all regions of the sperm surface, most strongly on the posterior region of the head, most weakly on the anterior region of the head. In this paper, we show that induction of the acrosome reaction with Ca2+ ionophore causes ESA152 to be redistributed from the posterior to the anterior region of the head plasma membrane. Cross‐linking ESA152 with bivalent antibody causes similar redistribution and induces the acrosome reaction, Induction of the acrosome reaction with ESA152 antibody requires Ca2+ but is insensitive to (10 ng/ml) pertussis toxin.
Induction of c‐fos transcripts in early postimplantation mouse embryos by tgf‐α, EGF, PDGF, and FGF
Authors: Nielsen Loretta L. , Werb Zena , Pedersen Roger A.
Published: February 2005

The activity of growth factor receptors in the early postimplantation mouse embryo was studied by analyzing changes in expression of mRNA transcripts of an early response gene, c‐fos, after binding of specific ligands. Reverse transcription of mRNA coupled with the polymerase chain reaction was used to detect gene transcription in single embryos after exposure to growth factors. Postimplantation embryos (at 7.5 days of gestation) had physiologically active receptors for transforming growth factor‐α (TGF‐α), epidermal growth factor (EGF), human platelet‐derived growth factor (PDGF), recombinant PDGF‐AA homodimer, and basic fibroblast growth factor (FGF), as indicated by induced expression of c‐fos mRNA. c‐fos expression was not induced in untreated embryos or in embryos incubated with active recombinant PDGF‐BB homodimer. These results show that growth factor receptors are functional during early mammalian embryogenesis.
In vitro maturation of the potential for movement of carp spermatozoa
Authors: Redondo‐Müller C. , Cosson M.‐P. , Cosson J. , Billard R.
Published: February 2005

Carp semen obtained from isolated fish after hormonal stimulation was highly variable in terms of volume of semen, osmotic pressure of the seminal plasma, and sperm capacity to move. Moreover, this last parameter was unstable when the spermatozoa were kept within the seminal plasma, and the present work was designed to investigate and possibly correct this phenomenon. Sperm potential movement was the major parameter studied and was measured by the percentage of motile cells in a final 3,000‐fold dilution in a medium of low osmotic pressure in which sperm movement is known to occur (Morisawa and Suzuki, Science 210:1145–1147, 1980). This was completed with occasional measurements of flagellar beat frequencies and demembranation‐reactivation of axonemal movement. The results showed that sperm potential movement was preserved upon dilution of the semen into cold 200 mM KCl medium and that semen of initially “poor” quality or spermatozoa that had lost their capacity to move during storage in the semen recovered gradually their potential movement during incubation at 2°C in the same medium. The K+ dependence for both the conservation and the regeneration of sperm capacity to move showed a minimal requirement of 50 mM KCl in media of high osmotic pressure. Na+ ions had similar properties but not divalent cations. The K+ activation was not pH dependent between pH 9.03 and 6.04. Whatever the functional state of live spermatozoa, demembranation‐reactivation occurred in ATP‐Mg2+. It is concluded that, with dilution of the semen in appropriate conditions, carp spermatozoa retain or acquire potential movement and therefore are a lower vertebrate spermatozoa model available year‐round. In addition, obtaining potentially nonmotile sperm and reversion in vitro might be useful to study the control of in vitro maturation.
Thiol status in human sperm
Authors: Rufas Onit , Fisch Benjamin , Seligman Judith , Tadir Yona , Ovadia Jardena , Shalgi Ruth
Published: February 2005

The passage of spermatozoa along the epididymis is characterized by a gradual stabilization of intracellular organelles mainly through the oxidation of thiol groups. In this study, we examined the relationship between the thiol–disulfide status of human spermatozoa (using a specific fluorescent probe, monobromobimane) and routine semen analysis parameters. Fluorescence intensity was measured by spectrofluorimeter and its frequency distribution within samples, using a fluorescence‐activated cell sorter. The mean proportion of reactive thiols SH/(SS + SH) in 29 semen samples was 29.8% ± 2.5%. When comparing thiol labeling patterns, oligozoospermic samples differed from normozoospermic ones (P < 0.05). However, within the normozoospermic group, no correlation was found between thiol‐labeling patterns and routine sperm parameters or fertilizing capacity in vitro. No difference in thiol labeling patterns was found between “swim‐up” and “whole semen” preparations.
Alterations in distribution of surface and intracellular antigens during epididymal maturation of rat spermatozoa
Authors: Phillips David M. , Jones Roy , Shalgi Ruth
Published: February 2005

The surface memrane of mammalian spermatozoa is known to undergo considerable conformational and organizational changes during epididymal maturation. However, much less is known about remodelling of intracellular membranes. In this communication we have used specific immunological markers to study the behavior of several antigens both on and within rat spermatozoa as they mature in the epididymis. Four monoclonal antibodies (McAbs) designated 5B1, 1B5, 2D6, and 1B6 were used to probe testicular and caput and cauda epididymal spermatozoa by indirect immunofluorescence and immunogold labeling techniques. None of the McAbs bound to testicular spermatozoa; in all cases, they became reactive only on spermatozoa which had reached the caput epididymis. McAb 5B1 was restricted to the outer acrosomal membrane (OAM) of the acrosomal cap domain. The epitope first appeared on antigen(s) with molecular mass (Mr) of ∼200 kDa in immature spermatozoa, but later in mature spermatozoa, but later in mature spermatozoa the antigen(s) had Mr of ∼160 kDa. The antigen(s) recognized by 1B5 McAb on the other hand was initially distributed over the OAM of the entire acrosomal domain (cap + equatorial segment), but during maturation it became progressively more restricted in area until in cauda spermatozoa only the anterior tip of the OAM bound the McAb. McAb 2D6 also bound to the entire OAM and acrosomal contents of caput spermatozoa, but, unlike 5B1 and 1B5 McAbs, reactivity was transient. That is, staining was first detected in caput spermatozoa but then disappeared in corpus and cauda spermatozoa. In contrast to all of the above, 1B6 McAb bound to the surface membrane overlying the entire head domain of caput spermatozoa, but during maturation it became restricted to the postacrosomal domain. These results indicate that, in addition to remodeling of the surface membrane during epididymal maturation, extensive processing of intracellular membrane antigens also takes place and that it is very active within the acrosome. The nature of these intracellular processing events remains to be elucidated, but they may have important consequences for membrane fusion and cell recognition phenomena during fertilization.
Distribution of lectin receptors sites in the zona pellucida of follicular and ovulated rat oocytes
Authors: Shalgi R. , Maymon R. , Bar‐Shira (Maymon) B. , Amihai D. , Skutelsky E.
Published: February 2005

Carbohydrates of the zona pellucida (ZP) in mammals are believed to have a role in sperm‐egg interaction. We have characterized the biochemical nature and distribution of the carbohydrate residues of rat ZP at the light (LM) and electron microscope (EM) levels, using lectins as probes. Immature female rats were induced to superovulate and cumulus‐oocyte complexes were isolated from the oviduct, fixed with glutaraldehyde, and embedded in araldite for LM and LR‐Gold for EM histochemistry. For examination of follicular oocytes, rat ovaries were fixed with glutaraldehyde and embedded in paraffin. The araldite or paraffin sections were deresined or deparaffinized, respectively, labeled with biotin‐tagged lectins as probes, and avidin‐biotin‐peroxidase complex as visualant. For EM examination, thin LR‐Gold sections were labeled with RCA‐I colloidal gold complex (RCA/G) and stained with uranyl acetate.

LM analyses indicate that in ovulated oocytes the ZP intensely binds peanut agglutinin (PNA); succinylated wheat germ agglutinin, (S‐WGA), Griffonia simplisifolia agglutinin‐I (GS‐I) and soybean agglutinin (SBA), and to a lesser extent, lectins from Ricinus communis (RCA‐I), Concanavaia ensiformis (Con A), Ulex europoeus (UEA‐I), and wheat germ agglutinin (WGA). The neighboring cumulus cells are considerably less reactive and exhibit membrane staining only with Con A, WGA, and PNA. EM analysis of RCA/G binding revealed intensive binding to the inner layer region of the ZP and moderate binding to cytoplasmic vesicles of the cumulus cells. The ZP of follicular oocytes exhibits a different lectin binding pattern, expressed in staining strongly with PNA and S‐WGA, and in a tendency of the lectin receptors to occur in the outer portion of the ZP. The results clearly reflect a change in both the content and distribution of sugar residues of the ZP following ovulation, which may have significance in fertilization.
Effect of 6‐dimethylaminopurine on germinal vesicle breakdown of bovine oocytes
Authors: Fulka J. , Leibfried‐Rutledge M. L. , First N. L.
Published: February 2005

The effect of 6‐dimethylaminopurine (6‐DMAP) on germinal vesicle breakdown (GVBD) and maturation in bovine oocytes was investigated in this study. This puromycin analog has been shown to be an inhibitor of phosphorylation. Whereas GVBD occurred in nearly all oocytes (96.8%; 120/124) in control medium, presence of 6‐DMAP (2 mM) blocked this process almost completely, irrespective of the presence (98.3% GV, 349/355) or absence (97.1% GV, 165/170) of cumulus cells. When lower concentrations of 6‐DMAP were used (100–500 μM), GVBD was observed in 87.9% of oocytes, but their maturation was arrested at late diakinesis‐metaphase I stage. The inhibition of GVBD was fully reversible, but most of the metaphase II plates were abnormal (80%). To assess whether the action of 6‐DMAP is different from the inhibitors of protein synthesis, metaphase II oocytes were exposed to either cycloheximide or 6‐DMAP, respectively. Whereas in cycloheximide‐supplemented medium approximately 80% of the oocytes were activated, parthenogenetic activation was much less frequent after incubation in 6‐DMAP (14.5%). Fusion studies showed that, even if GVBD occurs in 6‐DMAP supplemented medium, the level of the maturation‐promoting factor (MPF) is decreased. These experiments may indicate the importance of phosphorylation for GVBD in cattle oocytes.
In situ histochemical analysis of region‐specific gene expression in the adult rat epididymis
Authors: Garrett Susanne H. , Garrett James E. , Douglass James
Published: February 2005

The epididymal tubule is a dynamic structure, in which spermatozoa undergo distinct physiological and morphological changes. The epithelial cells lining the ductuli vary dramatically in their histochemical and cytological properties according to the region of the tubule in which they are located. Additionally, regional variation is observed regarding the biosynthetic, secretory, and absorptive properties of the epithelial cells. Using in situ histochemical analysis, we document here the region‐specific expression of a variety of genes that are transcriptionally active in the adult rat epididymis. Radiolabeled antisense riboprobes were used to localize, within the efferent duct/caput epididymis, transcripts encoding protein B/C, protein D/E (acidic epididymal glycoprotein), sulphated glycoprotein 1, sulphated glycoprotein 2, cellular retinol‐binding protein, and the neuroendocrine peptide precursor proenkephalin. Each species of mRNA exhibits a unique pattern of hybridization, revealing that gene transcription within the efferent duct/caput epididymis is also highly region specific. This observation may partially elucidate the molecular basis underlying the phenomenon of regional alternations in the composition of protein factors within the tubule lumen.
Zygotic expression of the connexin43 gene supplies subunits for gap junction assembly during mouse preimplantation development
Authors: Valdimarsson Gunnar , De Sousa Paul A. , Beyer Eric C. , Paul David L. , Kidder Gerald M.
Published: February 2005

De novo assembly of gap junctions begins during compaction in the eight‐cell stage of mouse development, and intercellular coupling mediated by gap junctions appears to be required for maintenance of the compacted state. We have begun to explore the expression of the family of genes encoding the connexins, the proteins that form the gap junction channels. We recently reported that a protein with antigenic and size similarity with connexin32, the rat liver gap junction protein, is inherited as an oogenetic product by the mouse zygote, but its gene appears not to be transcribed prior to implantation (Barron et al., Dev Genet 10:318–323, 1989). Here we report that another member of this gene family, connexin43, is transcribed by the embryonic genome from shortly after the time of genomic activation. As revealed by Northern blotting, connexin43 mRNA is absent from ovulated oocytes, becomes detectable in the 4‐cell stage, and accumulates steadily thereafter to reach a maximum in blastocysts. In contrast, no transcripts of connexin26 could be detected in any preimplantation stage. A protein with antigenic and size similarity with connexin43 from rat heart was found by Western blotting to accumulate from the four‐cell stage onward. Immunofluorescence analysis with embryo whole mounts was used to demonstrate that this protein is incorporated into punctate interblastomeric foci during compaction, consistent with its assembly into gap junction plaques. We conclude that connexin43 is one member of the connexin gene family whose zygotic expression is critical for preimplantation morphogenesis.
Birth of lambs after in vitro maturation, fertilization, and coculture with oviductal cells
Authors: Czlonkowska Maria , Eysymont Urszula , Guszkiewicz Andrzej , Kossakowski Maciej , Dziak Jacek
Published: February 2005

Control ovine oocytes matured and fertilized in vitro were transferred to intermediate recipient ewes. After 5 days, 59% of eggs were recovered. Thirty‐one (38%) reached morula/blastocyst stage. Twenty‐one embryos at the morula or blastocyst stage were transferred to six recipient ewes, resulting in five pregnancies, of which four were maintained. Nine lambs were born (43%). In the experiment, 72 ooctyes matured and fertilized in vitro were cocultured for 5 days with sheep oviductal epithelial cells. Thirty‐one eggs (43%) developed to the noncompacted morula stage. Transfer of 26 embryos to 11 recipient ewes resulted in two pregnancies (18%). Two male lambs were born. The result indicates that the coculture of in vitro matured and fertilized ovine eggs with sheep oviductal epithelial cells throughout the preimplantation period is compatible with further development to term.
Comparison of sperm binding potential of uninseminated, inseminated‐unfertilized, and fertilized‐noncleaved human oocytes under hemizona assay conditions
Authors: Franken Daniel R. , Windt M. L. , Kruger Thinus F. , Oehninger Sergio , Hodgen Gary D.
Published: February 2005

In this study, human oocytes obtained after ovarian hyperstimulation for in vitro fertilization (IVF) and gamete intrafallopian transfer (GIFT) were utilized to evaluate sperm/zona pellucida binding potential. Three groups of oocytes were evaluated: (1) uninseminated; (2) inseminated‐unfertilized; and (3) fertilized‐uncleaved. All oocytes had undergone germinal vesicle breakdown at the time of retrieval and were salt‐stored (pH 7.2) for not more than 30 days. Sperm binding was recorded under hemizona assay (HZA) conditions using spermatozoa from eight fertile men (HZA control) and from (1) four teratozoospermic (HZA test) and (2) four normozoospermic (HZA test) infertile men. First, the mean numbers (±SD) of sperm tightly bound for fertile controls and teratospermic men to hemizonae from uniseminated oocytes were 69.7 ± 16 and 14.5 ± 7, respectively (P = 0.02). Likewise, hemizonae from uninseminated oocytes bound 102.0 ± 19 and 114.0 ± 28, respectively, for fertile controls and normospermic men (P = 0.5). Second, hemizonae obtained from inseminated‐unfertilized IVF oocytes bound 44.2 ± 12 and 19.7 ± 6 for fertile controls and teratospermic men, respectively (P = 0.02). This category of oocytes bound 100.5 ± 7 and 108.5 ± 11 sperm, respectively, for fertile controls and normospermic semen (P = 0.3). Third, HZA results of fertilized but uncleaved oocytes showed a mean number of tightly bound sperm of 6.0 ± 4 compared with 65.0 ± 1 in control, uniseminated oocytes using fertile sperm. These results demonstrate that uninseminated and inseminated‐unfertilized human oocytes, salt‐stored under controlled pH conditions, give reliable information regarding sperm binding potential under HZA conditions.
In vivo secretion and association of clusterin (SGP‐2) in luminal fluid with spermatozoa in the rat testis and epididymis
Authors: Mattmueller Danette R. , Hinton Barry T.
Published: February 2005

Clusterin (sulfated glycoprotein‐2) is a heterodimeric glycoprotein synthesized and secreted by rat Sertoli cells. An antigenically similar form is synthesized and secreted by the epididymis. The goal of this study was to define the epididymal regions in which clusterin is present and the regions in which clusterin is secreted and interacts with developing spermatozoa. Seminiferous tubule (STF), caput, corpus, and cauda fluids were collected by micropuncture and/or microperfusion and two‐dimensional Western blot analysis was performed with a polyclonal antibody directed against Sertoli cell clusterin. Clusterin was found in both STF and epididymal fluid. STF contained predominantly the clusterin heavy chain (45 kd); however, a 70 Kd heterodimer was present under nonreducing conditions. Two subunits of clusterin with lower molecular weights (41 kd, heavy chain; 32 kd, light chain) and higher isoelectric points were present in the luminal fluid of all epididymal regions. The intraluminal levels of the heavy and light chains decreased from caput to cauda. Analysis by two‐dimensional gel electrophoresis of proteins secreted directly into the epididymal liminal fluid revealed that clusterin was secreted by caput epithelium and not by the corpus and cauda epithelium. Western blots of membrane extracts from testicular, caput, and cauda spermatozoa revealed that testicular clusterin was associated with testicular sperm and epididymal clusterin with predominantly caput sperm. Our findings suggest that clusterin is secreted into the caput epididymal lumen, where it binds to sperm and then dissociates from sperm to be endocytosed by cells of the distal epididymal epithelium.
Spermatogenesis in XY, XYSxra and XOSxra mice: A quantitative analysis of spermatogenesis throughout puberty
Authors: Sutcliffe Maxine J. , Darling Susan M. , Burgoyne Paul S.
Published: February 2005

Adult XYSxra mice exhibit varying degrees of spermatogenic deficiency but are usually fertile, while XOSxra mice have severe spermatogenic failure and are always sterile. The present quantitative spermatogenic analysis documents when these anomalies first appear during puberty. The results demonstrate that in XYSxra mice there was increased degeneration of pachytene spermatocytes and, to a lesser extent, meiotic metaphase stages. On average, there were only one‐half the number of spermatids compared with the XY controls. The defect in XOSxra mice appeared a little later, with an almost complete arrest and degeneration during the meiotic metaphases, so that the number of spermatids produced was only 3% of the control value. These results are discussed in relation to an hypothesis that links sex chromosome univalence during meiotic prophase with spermatogenic failure.
Expression of SV40‐lacZ gene in mouse preimplantation embryos after pronuclear microinjection
Authors: Takeda Satoshi , Toyoda Yutaka
Published: February 2005

In order to study the expression of an exogenous gene in developing mouse embryos during the preimplantation period, DNA carrying the SV40 early promoter fused with the Escherichia coli β‐galactosidase gene (lacZ) was microinjected into the pronucleus of fertilized mouse eggs. Expression of lacZ gene was detected by staining embryos with 5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactopyranoside (X‐gal) as a substrate at pH 7.2. The embryos expressing the lacZ gene showed various intensities of blue staining, all showing a mosaic pattern. The exogenous gene was expressed from the 4‐cell stage until the blastocyst stage. The proportion of embryos expressing the lacZ gene was maximal (38%) at the morula stage, and the expression was dependent on the presence of the SV40 promoter.
Isolation, composition, and biological activity of sugar chains of porcine oocyte zona pellucida 55K glycoproteins
Authors: Yurewicz Edward C. , Pack Beverley A. , Sacco Anthony G.
Published: February 2005

ZP3, a preparation of the 55K families of porcine oocyte zona pellucida, possesses carbohydrate‐dependent ligand activity for boar sperm. The aim of the present study was to analyze ZP3 N‐ and O‐linked oligosaccharides with respect to size distribution, composition, and role in sperm‐zona recognition events. Digestion of denatured ZP3 with peptide N‐glycosidase F (PNGaseF) released the majority of N‐glycans which fractionated on Sephadex G‐75 resin as a polydisperse population with apparent molecular masses ranging from 1,900–8,200 Da. The higher molecular weight N‐glycans were characterized by the presence of strongly anionic sulfated/sialylated polylactosamine structures. Alkaline‐borohydride treatment of the PNGaseF‐digested core proteins liberated O‐glycans as a heterogeneous population of oligosaccharide alcohols, which were fractionated on a Sephadex G‐50 column. Compositional analyses indicated sulfated polylactosamine units associated with the higher molecular weight O‐glycans. Preincubation of boar sperm with ZP3 or purified O‐glycans, but not N‐glycans, inhibited subsequent attachment to zona‐encased oocytes. Purified O‐glycans were, however, 2 to 3 orders of magnitude less effective than ZP3 as competitive ligands. The results document the extreme heterogeneity of the ZP3 carbohydrate moiety, in large part attributable to a board spectrum of variably sized N‐ and O‐linked sulfated polylactosamines. Ligand competition bioassays suggest that O‐glycans mediate, at least in part, the sperm adhesive properties of ZP3 and strongly imply that high‐affinity interaction of ZP3 sugar chains with complementary sperm receptors is dependent upon their covalent association with core proteins.
Preparation of spermatogenic cell populations at specific stages of differentiation in the human
Authors: Blanchard Y. , Lavault M. T. , Quernee D. , Le Lannou D. , Lobel B. , Lescoat D.
Published: February 2005

Studying biochemical events in human spermatogenesis requires separated populations of spermatogenic cells. Dissociation of these cells was performed by a Trypsin‐DNAse method adapted from the technique used for rodents. Cell separation was performed by centrifugal elutriation. Seven populations were collected, one further purified by Percoll gradient centrifugation, giving nine different cell populations. The efficiency of the cell separation was evaluated by phase contrast microscopy, flow cytometric DNA analysis, and electron microscopy. Five populations were enriched in spermatids: two in round spermatids (87% and 73%), another in round (52%) and elongating (44%) spermatids, another constituted by 80% elongating spermatids, and the last by 90% elongated spermatids. Two of the four remaining populations were enrichied in primary spermatocytes (74% and 54%); another population was the upper part of the Percoll gradient and constituted cytoplasmic lobes and residual bodies (89%); the last population was made up of various cells, with no specific enrichment. Electron microscopic observations revealed good preservation of the separated cells; only the flagella from elongated spermatids were lost. Furthermore, an unusual pattern of nucleoplasm distribution during stages 2–4 of spermatid differentiation was observed and its signification is discussed with regard to the shape of the human spermatozoon.
Developmental expression of cathepsin L and c‐rasHa in the mouse placenta
Authors: Hamilton Richard T. , Bruns Kerry A. , Delgado Michael A. , Shim Jae‐Kyoung , Fang Yu , Denhardt David T. , Nilsen‐Hamilton Marit
Published: February 2005

An investigation is described of the expression of the cysteine proteinase cathepsin L during placental development. In addition, whether cathepsin L expression is linked to c‐rasHa expression in development, as it is in metastatic cells, is examined. Large amounts of cathepsin L and its transcript are present in the mouse placenta, more than six times more than in adult kidney and liver. Throughout gestation, cathepsin L and its transcript are located in the giant cells and spongiotrophoblasts of the placenta. Several forms of different mobility on denaturing gels are found in the placenta. Their apparent molecular weights, as determined from the gels, are 43,000, 39,000, 29,000, and 20,000. The 39‐kDa form is procathepsin L. The 29‐kDa and 20‐kDa forms are lysosomal cathepsin Ls. The 39‐kDa procathepsin L and the 20‐kDa mature cathepsin L are the most abundant species in the placenta and are present in about equal amounts throughout gestation. At any time during gestation, placental minces synthesize and secrete only procathepsin L. The amniotic fluid of the fetus contains the 43‐kDa form of cathepsin L and procathepsin L, but no detectable amounts of mature cathepsin L. By contrast, serum from nonpregnant or pregnant mice contains three forms of cathepsin L (i.e., the 43‐kDa form, procathepsin L, and mature cathepsin L). Cathepsin L and the rasHa oncogene are expressed in two coincident waves corresponding to periods during which the placenta is invasive and just before parturition. The presence of large amounts of cathepsin L in the placenta suggests that the proteinase has a significant function there. Expression of cathepsin L in the placenta is potentially under the control of the ras gene product p21; both are under developmental control.
Sperm selection capacity of the human zona pellucida
Authors: Menkveld Roelof , Franken Daniel R. , Kruger Thinus F. , Oehninger Sergio , Hodgen Gary D.
Published: February 2005

Previous hemizona assay (HZA) results have illustrated a positive and significant correlation between the percentage of morphologically normal spermatozoa in the semen and the number of spermatozoa tightly bound to the zona pellucida. The present study was designed to evaluate the morphologic features using strict criteria of spermatozoa tightly bound to the zona pellucida. Semen samples of 4 normozoospermic and 11 teratozo‐ospermic men were used to compare the percentage of normal spermatozoa in the semen with that found (1) after swim‐up separation and (2) bound to the zona under HZA conditions. The mean (± SEM) % normal forms for normozoospermic men in semen, after swim‐up and zona‐bound spermatozoa were 21.5 ± 1.6, 27.5 ± 2.9, and 44.8 ± 3.4, respectively. A significantly higher % of normal forms were found among zona‐bound sperm compared to swim‐up forms (p = 0.02) and seminal sperm (p = 0.02). The mean % of normal sperm forms present in semen, after swim‐up and zona pellucida‐binding for teratozoospermic men, were 3.7 ± 0.9, 5.8 ± 1.6 and 15.6 ± 3.1, respectively. Significant differences existed between the % of normal sperm forms found in the swim‐up and zona‐bound spermatozoa (P = <0.01 and P = <0.0003, respectively) compared to the original ejaculates. Results indicate that a selective process against abnormal spermatozoa occurs at the site of the zona pellucida. Spermatozoa classified as normal or slightly abnormal are selectively bound to the zona in favor of abnormal (bizarre) spermatozoa; particularly those sperm cells with acrosomal abnormalities are either actively excluded or simply cannot bind to the zona or do it with a low efficiency due to inherent defects. This newly identified human zona property of sperm selectivity points out another potential use of the HZA, i.e., selection of sperm to be used in assisted fertilization.
Early development and X‐chromosome inactivation in mouse parthenogenetic embryos
Authors: Tada Takashi , Takagi Nobuo
Published: February 2005

Early development and X‐chromosome inactivation were studied in ethanol‐induced mouse parthenogenones. About 24% of oocytes transferred to 0.5‐day pseudopregnat recipients successfully implanted. However, only 49%, 20%, and 16% of implanted parthenogenones survived 5, 6, and 7 days later, respectively. Abnormal development was evident in every parthenogenone as early as 5 days after activation with the degenerating polar trophectoderm. These embryos were destined to become either small disorganized embryos or embryonic ectoderm vesicles bounded by the visceral endoderm. Only 2 of 51 representative 6‐ to 8‐day parthenogenones sectioned had morphology of the normal egg cylinder, although growth retardation was evident. Spontaneous LT/Sv parthenogenones shared similar morphological features. In late blastocysts, the frequency of cells with an apparently inactivated X chromosome was lower in parthenogenones than in fertilized embryos. The failure of X‐inactivation in the trophectoderm seems to contribute to the defective development of parthenogenones.
Purification and characterization of a sperm motility‐dynein ATPase inhibitor from boar seminal plasma
Authors: Iwamoto Teruaki , Tsang Angus , Luterman Maynard , Dickson John , de Lamirande Eve , Gagnon Claude , Okuno Makoto , Mohri Hideo
Published: February 2005

A sperm motility inhibitor from boar seminal plasma was purified. The purification procedure included dialysis against 0.1 M Tris‐HCl containing 0.1 mM DTT and chromatographies on SP‐Sephadex C‐25 and Phenyl‐Sepharose CL‐4B. With this procedure, the seminal plasma motility inhibitor (SPMI) preparation was highly purified with a 18% recovery of inhibitory activity. The molecular weight of SPMI in native conditions has been estimated at 50,000 by molecular sieving, but 3 polypeptides with molecular weights of 14,000, 16,000 and 18,000 were observed following polyacrylamide gel electrophoresis in denaturing conditions. SPMI is a thermolabile basic protein that is stable between pH 6 and pH 11. The observations that SPMI effects on motility of demembranated spermatozoa are reversed by Mg.ATP and that SPMI inhibited bull dynein ATPase in a concentration‐dependent manner suggest that this protein blocks the motility of demembranated spermatozoa by interfering with dynein arm function.
Enhanced in vitro growth of murine fibroblast cells and preimplantation embryos cultured in medium supplemented with an amphipathic peptide
Authors: Reed W. A. , White K. L. , Enright F. M. , Holck J. , Jeffers G. W. , Jaynes J. M.
Published: February 2005

Preliminary studies on the proliferative effects of lytic peptides were carried out using NIH 3T3 murine fibroblast cells and human lymphocytes. Cells were cultured in various concentrations of three different amphipathic peptides (SB‐37, Shiva‐1, and Vishnu), and enhanced proliferation was determined by uptake of 3Hthymidine with treated cells compared with control cultures. Enhanced proliferation of 3T3 cells was observed in cultures containing 50 μM or less SB‐37. The primary study consisted of 263 four‐cell‐ to eight‐cell‐stage mouse embryos from naturally bred mice and incubated in Whitten's medium containing 0.2, 1, or 10 μM of the amino terminus of an amphipathic cecropin B analog (Vishnu) or in Whitten's medium alone. Embryos were cultured to the hatched blastocyst stage, and effect of treatment was determined by the rate of growth to that stage of development. Statistical analysis revealed that culture in all three levels of Vishnu significantly accelerated in vitro growth of these stages of preimplantation embryos compared with controls. These results indicate that Vishnu promotes increased cleavage rates of embryos in vitro. A growth factor receptor clustering mechanism of action is proposed. This peptide may have some potential as an embryo culture medium additive to enhance in vitro growth rate.
Importance of mammalian sperm metalloendoprotease activity during the acrosome reaction to subsequent sperm‐egg fusion: Inhibitor studies with human sperm and zona‐free hamster eggs
Authors: Díaz‐Pérez Eneida , Meizel Stanley
Published: February 2005

We have previously shown that each of the metalloendoprotease (MEP) inhibitors phosphoramidon, diethylenetriaminepentaacetic acid, and carbobenzoxy‐L‐phenlalanine, when present only during the human sperm acrosome reaction (AR), will not inhibit the AR or sperm motility but will decrease the number of sperm that penetrate zona‐free hamster eggs. The present study was designed to investigate whether this inhibition of penetration is due to an effect on sperm binding to the egg plasma membrane and/or to an effect on the actual membrane fusion event. In these studies we used ionomycin to initiate the AR and assayed binding in a Ca2+‐free medium and fusion in Ca2+‐containing medium in the same experiment. Eggs were loaded with the fluorescent dye Hoechst 33342, and the appearance of fluorescence in a sperm head indicated that fusion had occurred. The three MEP inhibitors reduced binding only slightly but inhibited the actual fusion step by 50–60% (determined with an equation that corrected for any inhibition of fusion due to inhibition of binding). MEP inhibitors present only during gamete interactions had little or no effect on fusion. We also found that phosphoramidon‐inhibitable MEP activity was released during the ionomycin‐initiated AR. Incubation of AR supernatant containing MEP activity with previously acrosome‐reacted, phosphoramidon‐treated sperm resulted in a large reversal of the phosphoramidon‐inhibitory effect on sperm‐egg fusion. These results support the hypothesis that the acrosomal phosphoramidon‐inhibitable MEP released during the AR acts directly or indirectly during that event to increase the fusibility of the sperm plasma membrane region required for subsequent sperm‐egg fusion.
Insulin‐like growth factor‐1 stimulates growth of mouse preimplantation embryos in vitro
Authors: Harvey Mark B. , Kaye Peter L.
Published: February 2005

Because recent studies have particularly implicated the insulin growth factor family in early development, the effects of insulin‐like growth factor (IGF‐1) on the development of mouse embryos in vitro were investigated in detail. When added to the medium for culture of two‐cell embryos, IGF‐1 stimulated the number of cells in the resultant blastocysts after 54 hr, entirely by increasing the number of cells in the inner cell mass (ICM) (16.0 ± 0.5 vs. 12.6 ± 0.5 cells/ICM). This stimulation was also achieved when ICMs were isolated from blastocysts prior to culture for 24 hr with IGF‐1 (22.3 ± 1.0 vs. 17.5 ± 0.8 cells/ICM). There was no effect of IGF‐1 on trophectoderm (TE) cell proliferation. In morphology studies, IGF‐1 also increased the proportion of blastocysts (62% ± 3% vs. 49% ± 4%) while decreasing the number of embryos remaining as morulae (32% ± 3% vs. 38% ± 2%) or in the early cleavage stages (7% ± 3% vs. 13% ± 3%) after 54 hr culture from the two‐cell stage. All these effects were achieved with EC50s of approximately 60 pM IGF‐1, which is in the range for IGF‐1 receptor mediation; however, cross reaction with insulin, IGF‐2, or other unknown receptors is not excluded. Nonetheless, the results show that physiological concentrations of IGF‐1 (17–170 pM, 0.1‐1 ng/ml), which have been observed in the reproductive tract, affect the early embryo, suggesting a normal role for this factor in the regulation of growth of the developing conceptus before implantation.
Structural aspects of bovine oocyte maturation in vitro
Authors: de Loos F. , van Beneden T. , Kruip T. A. M. , van Maurik P.
Published: February 2005

Bovine cumulus‐oocyte complexes (COCS) were collected from 4–8 mm follicles and graded inot four categories on their morphological characteristics. These four categories were matured in vitro and processed for transmission electron microscopy at 24 h after the onset of culture. The morphology of the four groups of oocytes was analysed and compared with that of occytes that had matured in vivi and were collected 20–23 h after the preovulatory luteinizing hormone peak. After in vivo maturation, oocytes formed a homogeneous group with respect to their morphologicalo characteristics. After in vitro muturation, the oocytes formed a heterogeneous group with respect to their morphology between as well as within the four categories of oocytes. Oocytes from the first three categories showed the same morphology after in vitro maturation. The fourth category showed some specific characteristics: (1) vacuolization, (2) flattening of cumulus cells, and (3) almost complete lack of cortical granules in some category 4 oocytes. These characteristics are interpreted as signs of degeneration. Besides these aspects of degeneration, other deviations from normal development were seen: (1) retraction of cumulus cell process endings from the oocyte without the breaking down of these processes, (2) retardation of some aspects of the cytoplasmic maturation, and (3) incomplete cumulus expansion. It is concluded that oocytes capable of development in vitro show a large morphological variability before the onset of culture. In vitro maturation systems can support normal development, but many oocytes show signs of degeneration and deviant development after in vitro maturation.
Requirement for glucose during in vitro culture of sheep preimplantation embryos
Authors: Thompson J. G. , Simpson A. C. , Pugh P. A. , Tervit H. R.
Published: February 2005

Glucose utilization by sheep embryos was examined in 8‐cell (N = 36) and blastocyst (N = 36) stages, by measuring conversion of [5−3H]glucose to 3H2O. Fifty percent glucose utilization occurred at 0.79 ± 0.69 mM for 8‐cell embryos and ∼0.06 ± 0.15 mM for blastocysts. Development of 1‐ and 2‐cell sheep embryos (N = 264) was examined under different glucose concentrations (0, 1.5, 3, or 6 mM) and in the presence or absence of 0.33 mM pyruvate and 3.3 mM lactate (PL). Overall, the presence of glucose was detrimental (P < 0.001) to embryonic development. By contrast, the presence of pyruvate and lactate was beneficial (P<0.001) to development. An interaction was observed between the concentration of glucose and presence or absence of PL (P <0.05). An optimum level of glucose occurs at 0–3 mM in the presence of PL (P ±0.1). Development to the blastocyst stage was observed in medium when supplemented with amino acids and albumin alone. Thus, glucose metabolism is not critical for embryonic development, but beneficial at low concentrations. High concentrations can inhibit development, possibly by inhibiting the tricarboxylic acid (TCA) cycle. Sheep embryos may also be using amino acids as an energy source for development.