bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytological analysis of nuclear migration and dissolution during steroid‐induced meiotic maturation in vitro of follicle‐enclosed oocytes of the goldfish (Carassius auratus)
Authors: Lessman Charles A. , Kavumpurath Soosamma
Published: February 2005

The effect of progestogens on nuclear or germinal vesicle dissolution (GVD) was tested in goldfish (Carassius auratus) follicle‐enclosed oocytes in vitro. The results indicated the following hierarchy of GVD activity: 17α20β dihydroxy progesterone (DHP) > 17α hydroxy progesterone (HP) > progesterone (P). Further studies with DHP indicated that nuclear migration (GVM) preceded GVD, and that no significant GV size changes (ie, diameter in animal‐vegetal pole axis) occurred during GVM. The in situ GV visualization technique used (ie, acetic acid yolk clearing with concurrent nuclear precipitation) was verified using standard paraffin sectioning procedures. The two techniques yielded highly correlated results and indicated that GVM is initiated between 12–18 hr, after DHP addition at 15°C. The goldfish oocyte in vitro system as described here is a useful model for the study of hormonally induced GVM and GVD.
Ultrastructural aspects of in vivo fertilization in the cow
Authors: Crozet Nicole
Published: February 2005

In vivo fertilization of cow eggs has been studied by electron microscopy. Eggs were recovered from intracervically inseminated heifers 30 to 42 hr after the onset of oestrus. The corona cells remained attached to 4 out of the 15 eggs studied, but no sign of sperm phagocytosis was noted.

Spermatozoa close to the zona pellucida, but not in contact with it, were not acrosome reacted. In contrast, all sperm penetrating the zona pellucida had completed the acrosome reaction. Vesiculated products of the reaction were present at the zona surface of every penetrated egg, indicating that in this species, the acrosome reaction occurs at the surface of the zona pellucida.

During sperm passage through the zona pellucida, the equatorial segment overlaid by its plasma membrane remained intact.

Soon after penetration into the ooplasm, the sperm nucleus decondensed; at the same time, the female chromosomes resulting from the second meiotic division aggregated in a few masses of condensed chromatin. A nuclear envelope started to form around the condensed female chromatin, while it was not yet present around the decondensing male nucleus.

After swelling, the two pronuclei presented similar ultrastructural morphology; they contained small, compact, agranular nucleoli with a large fibrillar center and unevenly distributed chromatin. The pronuclear envelope contained pores and presented characteristic blebbing. The endoplasmic reticulum was closely apposed to the nuclear envelope and large Golgi structures were proximal to the pronuclei.
Chloral hydrate effects on oogenesis in Marsilea vestita Hook. et Grev
Authors: Kermarrec Odette , Tourte Yves
Published: February 2005

Chloral hydrate [3.5 × 10−2 M] applied to archegonium of Marsilea vestila during oogenesis modifies the asymmetry of the gametic mitoses and the nuclear migrations. Multinucleate female gametes are obtained whose polarity is disturbed. At the ultrastructural level, the distribution of the organelles is changed profoundly. The ‘fertilization cone’ disappears, and there is increased vacuolation associated with myelin figures in the cytoplasm and the appearance of filament bundles. The nuclei also contain vacuoles and myelin figures.
Spontaneous oocyte maturation in Rana pipiens: Estrogen and follicle wall involvement
Authors: Lin Yu‐Wai Peter , Schuetz Allen W.
Published: February 2005

Immature (germinal vesicle stage) Rana pipiens oocytes typically remain arrested in prophase I of meiosis even after extended periods of in‐vitro culture, if not stimulated with hormones. We have, however, sporadically observed “spontaneous” occurrences of oocyte maturation in vitro without the addition of hormones. This study documents some of our observations on this phenomenon and presents experimental results concerning the effects and possible involvement of estrogen and follicle wall components in regulating spontaneous oocyte maturation. Estrogen was found to inhibit spontaneous oocyte maturation (GVBD) in a dose‐dependent fashion. Follicles in which spontaneous maturation was inhibited by estrogen retained their responsiveness (GVBD) to both frog pituitary homogenate (FPH) and progesterone stimulation. Inhibitory effects of estrogen on spontaneous maturation, however, were not reversed following incubation of washed follicles in plain culture medium without added hormones. Possible involvement of progesterone synthesis in spontaneous oocyte maturation was ascertained by simultaneously monitoring endogenous progesterone synthesis and the occurrence of spontaneous GVBD over the course of the maturation process. In spontaneous maturing follicle there was a gradual increase in basal levels of progesterone synthesis that preceded GVBD. Significantly, addition of estrogen abolished both the spontaneous progesterone production and spontaneous oocyte maturation. When FPH was added to follicles exhibiting spontaneous oocyte maturation, progesterone production was augmented and the time course of oocyte maturation was greatly accelerated. Involvement of ovarian components in the maturation process was investigated by selective removal of various follicle layers by microdissection. Removal of follicle epithelium and theca layer (defolliculation) markedly decreased spontaneous and FPH‐induced maturation, whereas removal of the entire follicle wall (denudation) completely blocked it. Our results suggest that both spontaneous and FPH‐induced maturation involve an estrogen sensitive process in the follicle wall. Thus, somatic follicle cells appear to serve as a common mediator for both types of maturation, which are linked by some intrafollicular mechanism involving steroidogenesis. Hence, estrogen may play an important role as an endogenous intrafollicular regulator of oocyte meiotic maturation.
The kinetics of human sperm binding to the human zona pellucida and zona‐free hamster oocyte in vitro
Authors: Singer Sherry L. , Lambert Hovey , Overstreet James W. , Hanson Frederick W. , Yanagimachi Ryuzo
Published: February 2005

In these experiments the mean number of sperm bound to the zonae pellucidae of immature human oocytes and to the vitelli of zona‐free hamster oocytes were counted after centrifuging the oocytes through a discontinuous dextran gradient into a fixative. Sperm suspensions (107 sperm/ml) were preincubated in BWW medium containing 35 mg/ml of human serum albumin for 0 to 20 hr and coincubated with oocytes for 4 hr.

The kinetics of sperm binding and sperm penetration were clearly different for the two types of oocytes in this system. Sperm binding to the zona pellucida appeared to be associated with zona penetration; when the zona was penetrated, many sperm were tightly bound and vice versa. A similar association between human sperm binding to the zonafree hamster oocyte and hamster oocyte penetration was not so apparent. Transmission electron microscopy revealed that all sperm which were “bound” to the hamster vitellus were acrosome reacted. On the surface of the human zona pellucida acrosome intact and acrosome reacted sperm were observed.

These results suggest that tight binding of spermatozoa to the zona pellucida may be an important preliminary step in human fertilization. If this is true the activity of the sperm's “receptor for zona” may not be detected by the assay with zona‐free hamster oocytes.
Caproyl esterase from rat testis: Purification and action on cumulus cells
Authors: Waibel R. , Granet R. , Ficsor G. , Ginsberg L.
Published: February 2005

Rat caproyl esterase (E.C.3.1.1.1), extracted from testis with Tween 80, was purified by cation exchange and lectin affinity chromatography. The 104‐fold purified enzyme had an activity of 840 μmol/hr per mg protein.

The purified esterase did not contain any hyaluronidase or N‐acetyl‐glucosaminidase activity. Electrophoresis on sodium dodecyl sulfate polyacrylamide gels revealed a single band of approximately 60,000 molecular weight. The esterase had an isoelectric point of 5.1. Inhibition experiments showed high sensitivity of the enzyme to sulfhydryl agents and complete inactivation by sodium aurothiomalate.

The purified caproyl esterase was shown to digest the cumulus matrix from mouse ova.
Further characterization of boar sperm plasma membrane proteins with affinity for the porcine zona pellucida
Authors: Peterson R. N. , Henry L. , Hunt W. , Saxena N. , Russell L. D.
Published: February 2005

Boar sperm plasma membrane proteins (PMPs) with affinity for the zona pellucida were partially purified from columns of dextran sulfate using a linear salt gradient and a buffered detergent that retained their ability to block directly the binding of uncapacitated and capacitated sperm to isolated porcine oocytes. PMPs that bound most strongly to dextran sulfate (fraction IV) were also most effective in blocking sperm binding to porcine oocytes. These tightly bound proteins also bound to isolated zonae to a greater extent than other fractions. Monovalent antibodies to fraction IV PMPs completely blocked sperm binding to isolated eggs. Fraction IV PMPs lost the ability to inhibit directly the binding to eggs when treated with chaotropic agents and trypsin; the fraction also displayed a tendency to aggregate in the absence of high salt. This property and the affinity of proteins in this fraction for sulfated polysaccharides indicate that specific hydrophilic interactions may play a significant role in sperm‐zona attachments.
“Spontaneous” hardening of the zona pellucida of mouse oocytes during in vitro culture. II. The effect of follicular fluid and glycosaminoglycans
Authors: De Felici M. , Salustri A. , Siracusa G.
Published: February 2005

We have previously reported that when isolated mouse oocytes are cultured in vitro their zonae pellucidae (ZP) become increasingly resistant to solubilization by chymotrypsin (“spontaneous” ZP hardening).

In the present paper we report that follicular fluid contains factors that totally prevent such hardening. Furthermore, medium conditioned by granulosa cells partially prevents hardening.

The protection against ZP hardening offered by follicular fluid may be ascribed, at least in part, to its content in sulfated glycosaminoglycans heparin and chondroitinsulfate B which, when added to the culture medium at physiological concentrations, show a marked antihardening effect.
Altered sex ratio of live young after transfer of fast‐ and slow‐developing mouse embryos
Authors: Tsunoda Y. , Tokunaga T. , Sugie T.
Published: February 2005

We cultured eight‐cell mouse embryos to blastocyst stage, divided them into three groups according to the time of blastocoel formation, and transferred them separately into recipients. The proportion of live young from the fast‐developing embryos was slightly high (49%) but not significantly different from those of other embryos (38% and 39%). However, the sex ratio of live young from the fast‐ and slow‐developing embryos was significantly shifted toward the male (71%) and to the female (80%), respectively.
The human pronuclear ovum: Fine strcture of monospermic and polyspermic fertilization in vitro
Authors: Sathananthan A. H. , Trounson A. O.
Published: February 2005

The fine structure of pronuclear ova (monospermy and polyspermy) and one‐cell embryos has been investigated in our IVF programme. Sixteen oocytes were collected at laparoscopy after appropriate hormonal stimulation and were matured and fertilized in vitro by methods that have given rise to normal pregnancies.

Pronuclear ova showing monospermic fertilization had two vesicular pronuclei surrounded by aggregations of cellular organelles. The male pronucleus was closely associated with a sperm axoneme, while the female pronucleus was dismantling its envelope and condensing its chromatin ahead of its counterpart in late pronuclear ova. Each pronucleus had dispersed chromatin, dense compact nucleoli, and intranuclear annulate lamellae. Smooth endoplasmic reticulum, annulate lamellae, Golgi complexes, and mitochondria formed a conspicuous part of the perinuclear ooplasm. The one‐cell embryos were either in syngamy or in the process of undergoing first cleavage. Positive evidence of cortical granule release and second polar bodies were detected in the perivitelline space. A block to polyspermy seemed to operate at the level of the inner zona.

Dispermic and polyspermic ova had 3–16 pronuclei resembling those of monospermic ova and had sperm tails in the ooplasm. Sperm were also seen penetrating the inner zona and were occasionally found in the perivitelline space. Incomplete cortical granule release and early signs of cytoplasmic fragmentation were noted in polyspermic ova.

Both normal and abnormal features of these ova are reported and compared with pronuclear structure in vivo and in vitro.
Effect of demecolcine (colcemid) on goldfish oocyte meiosis in Vitro
Authors: Habibi Hamid R. , Lessman Charles A.
Published: February 2005

Germinal vesicle migration (GVM) and dissolution (GVD) were studied in goldfish oocytes treated with 17‐α,20–β‐dihydroxyprogesterone (DHP) and/or demecolcine (DE; a colchicine derivative also known as colcemid) in vitro. DE (100 μg/ml) in the presence of DHP, enhanced steroid‐induced GVM, after both 24 and 48 hr of incubation and significantly reduced the DHP ED50 value for GVM. Similarly, administration of DE alone elicited a significant, dose‐related increase in GVM after 24 or 48 hr of incubation. The presence of DE, either alone or in combination with DHP, was without effect on GVD. The effect of DE was also tested on ooplasmic viscoelasticity in goldfish follicles subjected to a centrifugal force (160g for 1 min). Preincubation (24 hr) of goldfish follicles in DE significantly influenced the direction and the extent of the centrifugally induced GV movement along the axis of centrifugal force in a dose‐related fashion. The present results provide support for the hypothesis that cytoskeletal components, such as microtubules that are sensitive to DE, are involved in the mechanism of GVM in goldfish oocytes.
Effects of the calcium channel blocker diltiazem on the excitability of mouse oocytes
Authors: Yoshida Shigeru
Published: February 2005

Effects of the organic Ca antagonist diltiazem on Ca channels were studied in ovulated and unfertilized oocytes of the mouse by using intracellular recording techniques. The resting potential was not affected by diltiazem. The threshold level of the Ca action potential shifted slightly toward positive voltages with diltiazem concentration, but the shift was not statistically significant. The overshoot and maximum rate of rise of the Ca action potential were inhibited by the drug in a dose‐dependent manner, but higher amounts of diltiazem were necessary to cause similar blocking effects on Ca channels in mouse oocytes than in other differentiated cells. Increases of external concentration of Ca2+ antagonized the degree of diltiazem inhibition. However, the sequence of block of Ca2+, Sr2+, and Ba2+ currents was different for diltiazem vs Cd2+. It is suggested that diltiazem inhibition can not be explained by simple competitive scheme, ie, antagonism between diltiazem and permeant cations does not occur at the same binding site associated with the Ca channel in mouse oocytes.
Study on oocyte maturation and activation of the common prawnpalaemon serratus(Pennant): Relationship between oocyte maturation and the molt cycle cytological aspects
Authors: Cledon Philippe
Published: February 2005

A detailed chronology of the cytological events related to maturation that take place within the reproduction molt cycle has been established. It has been shown that oocytes, initially arrested at prophase I, resume meiosis when approaching stage D1‴ of the molt cycle, ie, 4–5 days before molting. The following steps characterize this premolt period of oocyte maturation: nuclear envelope folding, nucleolar dissociation, condensation of the chromosomes, and beginning of the breakdown of the nuclear envelope (GVBD). At the ultrastructural level, it has been confirmed that GVBD actually takes place at the D1‴−D2stage transition, when the germinal vesicle still occupies a central position in the oocyte. The migration of the chromosome takes only a few hours and begins approximately 4 hr before molting. It is only 1–2 hr before molting that the divalent chromosomes that are not yet organized in a metaphase plate become visible at the surface of the oocyte. They lay in a nucleoplasmic area no longer limited by the nuclear envelope. Metaphase I is reached a few minutes after molting. A second meiotic block appears at this stage, which persists until spawning, ie, for about 24 hr. Fertilization occurs at the moment of spawning. In vitro fertilization experiments demonstrated that fertilization normally triggers the release of the second meiotic block. Extrusion of the two polar bodies can be easily observed using a method for clearing and staining the oocytes in toto.
Lectin binding sites on the plasma membrane of epididymal and ejaculated chimpanzee sperm
Authors: Young Leona G. , Gould Kenneth G. , Hinton Barry T.
Published: February 2005

Lectins have been used to analyze variations in the distribution and density of exposed saccharides of the sperm plasma membrane during physiologic maturation and after ejaculation. Studies have been conducted in a number of nonprimate species but have been conducted to only a limited extent in nonhuman primates. In this study, pure suspensions of chimpanzee sperm from the caput and cauda epididymis and from the ejaculate were labeled with lectins conjugated to fluorescein isothiocyanate in order to visualize changes in the distribution of exposed membrane glycocomponents. The lectins used were Con A, DBA, RCA‐I, and WGA. Con A binding showed minimal change during epididymal transit, with an increased binding to the flagellum after ejaculation. DBA binding was relatively constant in all specimens. RCA‐I showed distinct changes in binding pattern between epididymal and ejaculated sperm. On ejaculated sperm strong fluorescence was limited to the posterior head and to the midpiece. WGA binding increased during epididymal passage and decreased after ejaculation. There appears to be a wide variety of saccharide groups available for lectin binding on the surface of epididymal and ejaculated chimpanzee sperm. The general similarity in binding patterns of caput and cauda epididymal chimpanzee sperm exposed to Con A and DBA might reflect the fact that sperm morphology does not change during epididymal transit in this species, thus implying a more stable membrane structure than is present in other primates so far studied.
Sperm‐egg interactions in the shrimp Rhynchocinetes typus
Authors: Barros C. , Dupré E. , Viveros L.
Published: February 2005

Sperm‐egg interaction in Rhynchocinetes typus was studied with the phase‐contrast and scanning electron microscopes. R typus spermatozoa present in the vas deferens have the shape of a round‐headed nail. After contact with seawater it is possible to observe the unfolding of the rays or stellate arms, giving the spermatozoon the appearance of an inverted umbrella. From the center of the flat face of the umbrella emerges a spike with longitudinal striations. Ovarian eggs and spermatozoa were mixed in vitro by agitating them for two minutes in Millipore‐filtered seawater. The first gamete contact was established by the spermatozoon through the tip of the spike, which exerted a lytic action on the egg envelopes. After the rigid spike was completely inside the egg, the rays became aligned parallel to each other and began to enter the eggs. Toward the final stages of ray entry, it was possible to observe fusion of the ray membranes with one another, and later the fusion process continued toward the tip of the radial spines. Concomitantly, the egg surface that surrounds the sperm swelled in a circular fashion and formed a fertilization cone. After the spermatozoon entry was complete, a scarlike mark appeared at the place on the egg surface through which penetration occurred. The whole penetration process was completed within 45‐60 minutes.
Estimation and manipulation of glutathione levels in prepuberal mouse ovaries and ova:Relevance to sperm nucleus transformation in the fertilized egg
Authors: Calvin Harold I. , Grosshans Kurt , Blake Emily J.
Published: February 2005

Glutathione (GSH), the major low‐molecular‐weight thiol in mammalian cells, is believed to be a necessary factor for the transformation of the disulfide‐stabilized sperm nucleus into the male pronucleus after fertilization. Its concentration in mouse ova, isolated from the ampulla of the oviduct after hormone‐induced superovulation of 3–4‐week‐old mice, has been determined by an enzymic cycling microassay. The level found was 1.80 pmol per ovum. Mean ovum diameter was estimated as 71–72 μm, indicating a GSH concentration of 9–10 mM in the mouse egg. Administration of L‐buthionine S, R‐sulfoximine, an inhibitor of GSH biosynthesis, during the 2 days preceding ovulation, reduced ovum GSH content below 0.20 pmol (<1.0 mM). The mean GSH concentration of the hormone‐stimulated ovaries was reduced from 3.2 mM to 0.2 mM under these conditions.

It has also been demonstrated that measurement and manipulation of ovum and ovarian levels of GSH can aid in studying its function in ovaries, ova, and early embryos. Hormone‐induced superovulation was achieved in BSO‐treated prepuberal C57B1/6 X SJL mice whose ovaries contained less than 10% of control levels of GSH. Over 50% of the isolated ova were fertilized in vitro. However, abnormal one‐cell embryos resulted in which the maternally derived pronucleus coexisted with an unchanged sperm nucleus, thus confirming that adequate levels of GSH are necessary for initiating transformation of the fertilizing sperm nucleus.
Cell surface markers of mouse primordial germ cells defined by two monoclonal antibodies
Authors: Hahnel A. C. , Eddy E. M.
Published: February 2005

Monoclonal antibodies EMA‐1 and EMA‐6 bind to cell surface antigens of mouse primordial germ cells between 7 and 14 days of development. Primordial germ cells can be identified by alkaline phosphatase activity, and EMA‐1 and EMA‐6 apparently recognize this same population of cells prior to 14 days of development. EMA‐1 labels primordial germ cells in 8‐ to 13‐day mouse embryos and EMA‐6 in 8‐ to 10‐day embryos. A difference in the cells recognized by EMA‐1 and the lectin Dolichos biflorus agglutinin in early embryos suggests that these probes may be useful for studying primordial germ cells in situ or for isolating them from mouse embryos.
Immunological evidence that a 305‐kilodalton vitelline envelope polypeptide isolated from sea urchin eggs is a sperm receptor
Authors: Acevedo‐Duncan Mildred , Carroll Edward J.
Published: February 2005

Direct isolation of the sea urchin egg vitelline envelope with intact sperm receptors is difficult because the envelope is firmly attached to the egg plasma membrane. We now report a method for producing an inseminated egg preparation in Strongylocentrotus purpuratus (using soybean trypsin inhibitor [STI] and Ca2+, Mg2+‐free seawater) that contains an elevated vitelline envelope (VE*‐STI). The VE*‐STI is devoid of cortical granule material, and supernumerary sperm do not detach postinsemination, suggesting that the VE*‐STI contains active sperm receptors. VE*‐STIs contain a 305‐kD polypeptide and additional components that range from 225 to 31 kD, whereas the 305‐kD polypeptide was considerably reduced in VE*s. Electrophoresis of sperm receptor hydrolase digests of VE*‐STIs showed that the 305‐kD polypeptide and several other envelope polypeptides are protease substrates. Univalent Fab fragments against VE*s, VE*‐STIs, and 305 and 225‐kD polypeptides blocked sperm binding and fertilization in an Fab concentration‐dependent manner. The 305 and 225‐kD polypeptides were localized in the VE*‐STI using indirect immunofluorescence. Enzyme‐linked immunosorbent assays showed that the 305 and 225‐kD polypeptides share determinants, suggesting that the 225‐kD polypeptide may be derived from the 305‐kD polypeptide by the proteolysis that occurs at the cell surface during fertilization. Fab fragments against S purpuratus VE*‐STI antigens neither bound to nor blocked homologous sperm binding and fertilization of Lytechinus variegatus eggs. Cross fertilizability occurred to the extent of 5% or less between L variegatus and S purpuratus, therefore, we conclude that the 305 kD‐polypeptide isolated from S purpuratus is a species‐specific vitelline envelope sperm receptor.
Equine zona pellucida and capsule: Some physicochemical and antigenic properties
Authors: Bousquet Daniel , Guillomot Michel , Betteridge Keith J.
Published: February 2005

The capsule which surrounds the pre‐attachment equine embryo has been compared with the zona pellucida (zp) that it replaces, as well as with the rabbit blastocyst coverings, by means of physicochemical and immunological methods. Trypsin solution at pH varying between 7.5 and 9.0 completely solubilized the capsule, as did Na borohydride. However, solutions of pH 2.0 or 12.0, urea, high temperature (65°C, 60 min or 80°C, 30 min), mercaptoethanol and dithiothreitol were able to solubilize the zp but not the capsule at the concentrations used. Indirect immunofluorescence on cryostat sections and whole mounts of fresh or frozen‐thawed material showed that 1) common antigens are shared by equine, porcine and bovine zp; 2) day 7 to day 15.5 capsule reacted with anti‐capsule‐serum but not with anti‐zp‐serum except for a few patches on the surface of the capsule; 3) anti‐capsule‐serum, but not anti‐zp‐serum, reacted with the capsular material recovered along with a broken day 27.5 conceptus; 4) anti‐capsule‐serum does not react with rabbit blastocyst coverings; 5) anti‐capsule antibodies can be absorbed from the anti‐capsule‐serum by uterine proteins from either pregnant or non‐pregnant mares; and 6) the capsule does not contain mouse laminin‐like material.
A protein family immunorelated to a sperm‐binding protein and its regulation in human semen
Authors: Metafora S. , Lombardi G. , De Rosa M. , Quagliozzi L. , Ravagnan G. , Peluso G. , Abrescia P.
Published: February 2005

In human seminal plasma a family of proteins that is immunologically related to the RSV‐IV protein secreted under androgen control from the epithelium of the rat seminal vesicles was detected by a radioimmunoassay. Evidence for the origin of these antigens from human seminal vesicle is presented. Quantitative measurements of this family of proteins were performed in men with low levels of serum testosterone (idiopathic hypogonadotropic hypogonadism) and in individuals having serum testosterone in the normal range of values but carrying sex chromosome aberrations (Klinefelter's syndrome). In the first case we have found a marked decrease in the total amount of the RSV‐IV‐related proteins. An increase of about 40% in the total amount of these antigens was obtained in these subjects by gonadotropin treatment. A decreased amount of these proteins was also detected in the subjects affected by Klinefelter's syndrome. The possibility that some factor(s) under genetic control is involved, in addition to testosterone, in the regulation of this family of proteins is discussed.
Characterization of hatching‐associated changes in the sea urchin fertilization envelope
Authors: Uher Vera Irene , Carroll Edward J.
Published: February 2005

The embryo of the sea urchin Strongylocentrotus purpuratus hatches from the fertilization envelope (FE) via synthesis and secretion of a hatching enzyme and by ciliary activity. Although the basic characteristics of the hatching enzyme are known, little is understood about changes in the FE during hatching. We have studied the biochemical changes in FEs during hatching. Polyacrylamide gel analysis revealed an increasingly complex polypeptide spectrum of the extractable fraction of FEs isolated during development. Immunoblotting of these polypeptides (using antiserum against the soluble polypeptides extracted from FEs isolated at 30 minutes postinsemination) revealed a decrease in the soluble FE components during hatching. Immunochemical analysis of hatching medium showed a strong correlation between the soluble FE components released and the hatching interval. Immunoblotting of hatching media indicated the presence of soluble FE polypeptides of similar and lower molecular weights than those obtained for extracts of FEs. These results imply that the hatching‐associated changes in the FE of S purpuratus occur via proteolysis of FE components, which are derived from the paracrystalline protein fraction, a subset of cortical granule proteins.
Intracellular localization of argyrophilic proteins in the maturing oocyte, fertilized egg, and spermatozoa of the starfish, Asterina pectinifera
Authors: Amikura Reiko M. , Yamada Hisafumi , Hirai Setsuro , Nagano Hiroshi
Published: February 2005

Changes in intracellular localization of argyrophilic proteins visualized as silver‐stained particles by nuclear organizer region (NOR)‐silver staining were investigated in starfish oocyte maturation. The silver‐stained particles were localized in the germinal vesicle and nucleolus of immature oocytes and dispersed into the cytoplasm at the time of germinal vesicle breakdown (GVBD). In the mature egg cytoplasm, silver‐stained particles were distributed on yolk‐like granules with diameters of 0.3–1.0 μm. In spermatozoa, silver‐stained particles were detected heavily in the acrosome and centrosomes but few were detected in the nucleus, whereas they were present in the male pronucleus of fertilized eggs. The silver‐stained particles were removed by pretreatment of eggs with protease but not with nuclease. These results indicate that argyrophilic proteins disperse to the egg cytoplasm during GVBD and might be incorporated to the male pronucleus from the egg cytoplasm in fertilization. The morphological changes from chromosomes through chromosome vesicles to female pronucleus were also observed with light microscopy after NOR‐silver staining.
Effects of induced ovulation by pregnant mare's serum and human chorionic gonadotropin on the sex ratio of mouse fetuses
Authors: Sakai N. , Endo A.
Published: February 2005

The sex ratio of the fetuses from mice treated with pregnant mare's serum (PMS)/human chorionic gonadotropin (HCG) to induce ovulation did not differ appreciably from that of spontaneously ovulated controls. Rather, an intriguing observation was that the sex ratio in the right uterine horns tended to be lower than that in the left horns in both spontaneously ovulated controls and PMS/HCG‐treated groups. We speculate on its possible relation to the observed right‐left asymmetry of horn sizes (number of fetuses in the horn).
Embryonic development in culture of two dasyurid marsupials, Sminthopsis crassicaudata (gould) and Sminthopsis macroura (spencer), during cleavage and blastocyst formation
Authors: Selwood Lynne
Published: February 2005

Embryos of Sminthopsis crassicaudata and Sminthopsis macroura were cultured for up to 96 hours during cleavage and early expansion of the blastocyst in Dulbecco's modified Eagle's medium (DMEG), DMEG containing 2.76 gm/liter sodium lactate (DMEGL), DMEG containing 3.5 gm/liter galactose (DMEGAL), DMEG containing 15 ng/ml progesterone (DMEGP) or 150 ng/ml progesterone (DMEGP10), and DMEGL containing 15 ng/ml progesterone (DMEGLP).

The disappearance of sperm was used to indicate the time of ovulation (day 0). Fertilized eggs were found in the uterus at the end of day 1, four‐cell stages at the end of day 2, and embryos completing the fourth division by the end of day 3 in S. macroura and day 4 in S. crassicaudata. Estimated developmental times in culture were similar to those obtained in vivo. In both species, the first two divisions take about 24 hours, cleavage is arrested for 24 hours or longer at the rounded four‐cell stage, and the third and fourth divisions take a further 24 hours. The blastocyst expands during the next 24 hours in which time the fifth and sixth divisions occur. It was possible to culture embryos from S. macroura but not S. crassicaudata over the four‐cell stage to early expanding blastocysts.

DMEGAL did not support cleavage in culture. DMEG, DMEGL, DMEGP, DMEGP10, and DMEGLP all supported culture during cleavage and early blastocyst expansion. Blastocyst expansion was slightly enhanced using media containing sodium lactate. More embryos completed the fifth division and formed expanding blastocysts in DMEG, DMEGL, and DMEGLP.
Sea urchin testicular cells evaluated by fluorescence microscopy of unfixed tissue
Authors: Simpson Marcia V. , Poccia Dominic
Published: February 2005

A procedure is presented for rapid, quantitative evaluation of cell and nuclear types present in the male gonad of the sea urchin. Vitally stained whole mounts of tissue fragments or dissociated cells are prepared, which reveal detailed 3‐dimensional chromatin patterns and enough cytoplasmic features to provide reliable markers for most of the somatic and germ line cell types. Representative cellular morphologies are described. Nuclear volume changes during spermatogenesis are quantified. Spermatid nuclei contain an apparently interconnected network of heterochromatin. Regions relatively devoid of chromatin decrease in size as nuclear condensation proceeds and spherical nuclear shape is maintained. The major decrease in nuclear volume occurs prior to the late spermatid stage. The volume of the spermatozoan nucleus is achieved by the smallest late spermatid nucleus before the change from spherical to conoid morphology. The relationship of this morphological transition to sperm histone dephosphorylation is discussed.
Impaired ability of human spermatozoa to penetrate zona‐free hamster oocytes: Is a postacrosomal sheath anomaly involved?
Authors: Courtot A. M. , Escalier D. , Jouannet P. , David G.
Published: February 2005

We selected 17 infertile men whose sperm ultrastructural study revealed at least 70% of spermatozoa with postacrosomal sheath (PAS) anomalies. Among the other sperm head defects, those affecting the nuclear shape were most frequently encountered and were highly correlated with PAS anomalies (r = +0.71; P < .01). PAS anomalies were also correlated with chromatin condensation defects (r = +0.67; P < .01) and acrosome anomalies (r = +0.53; P <.05). Those spermatozoa were tested for their ability to penetrate zona‐free hamster oocytes and were compared to a control sperm population. It was shown that sperm head morphological anomalies impaired the ability of spermatozoa to attach to and penetrate the oocyte. The highest significant and negative correlations were found between the penetration rate and 1) the percentage of spermatozoa with PAS anomalies (r = −0.81; P < .01) and 2) the percentage of spermatozoa with nuclear shape anomalies (r = −0.66; P < .01). The effect of PAS anomalies on human fertilization process are discussed.
Development of mouse embryos in vitro is affected by strain and culture medium
Authors: Dandekar Pramila V. , Glass Robert H.
Published: February 2005

One‐cell and two‐cell embryos from three random‐bred strains of mice–CF1, Dub:(ICR), and CFW (Swiss‐Webster)–were cultured to the blastocyst stage in Spindle's, Earle's, Ham's F10, Whittingham's T6, or Hoppe and Pitts' medium. CFW embryos were more successful than CF1 and Dub:(ICR) embryos in developing to the blastocyst stage in all five media. Dub:(ICR) and CFW two‐cell embryos showed the best development in Spindle's, Whittingham's T6, and Hoppe and Pitts', whereas CF1 two‐cell embryos were most successful in developing in Hoppe and Pitts' medium. Similar results were obtained with one‐cell embryos, although fewer developed to the blastocyst stage, and T6 rather than Hoppe and Pitts' medium sustained the best development of CF1 one‐cell embryos. For all strains, the least successful development was in Ham's F10, but CFW embryos did show good development in this medium. In addition to the effects of various media on mouse embryo development, our results indicate that the strain of mouse used for the bioassay of media is of critical importance. Random‐bred CFW (Swiss‐Webster) mice are as suitable as a hybrid strain for this purpose.
Susceptibility of epididymal boar sperm to cold shock and protective action of phosphatidylcholine
Authors: Simpson A. M. , Swan M. A. , White I. G.
Published: February 2005

Rapidly cooling (cold shocking) washed cauda boar sperm irreversibly reduced motility and respiration and greatly increased the uptake of 45Ca2+; the plasma membranes were removed and the acrosomes detached from nuclei. The motility, respiration, and calcium uptake of the less mature caput sperm were largely unaffected; and there was little damage to the ultrastructure. This indicates that boar sperm becomes less resistant to cold shock as they mature in the epididymis.

The oxygen uptake, glucose breakdown, and lactic acid production of control caput sperm was less than that of cauda sperm. This suggest that the maturation of sperm in the epididymis of the boar involves an increase in both the glycolytic and oxidative phases of glucose metabolism.

The presence of 2.0 mg/ml phosphatidylcholine (lecithin) in the medium prevented ultrastructural damage to cauda sperm on cold shock, and motility and respiration were maintained at levels similar to those of control sperm. Although the presence of phospholipid reduced the large calcium influx following cold shock, it was still greater that that of control sperm.

The “protective” effect against cold shock was not maintained after rewashing the sperm free of phosphatidylcholine prior to cold shock, indicating a fairly “loose” interaction of the phospholipid with boar sperm membranes that was easily disrupted.
Sperm‐sperm associations in the loris epididymis
Authors: Phillips David M. , Bedford J. Michael
Published: February 2005

In a few mammals, the passage of maturing spermatozoa through the epididymis is characterized by development of persistent associations between one or more neighboring cells over the acrosomal region. The converse situation is described here in the loris, Nycticebus coucang, a prosimian primate. Loris spermatozoa released at spermiation enter the caput epididymidis as single cells and then become stacked in rouleaux of 2–8 spermatozoa there, the peri‐acrosomal plasmalemma of one being linked by a unique junctional complex to that of its neighbor. However, by the time the cauda is reached, all the spermatozoa have separated again to lie as single cells, which now display major aggregations of ordered material over the concave surface of the acrosome. The functional significance of these unusual sperm surface‐related phenomena in the loris epididymis is not clear.
Cytogenetic technique for mouse metaphase II oocytes
Authors: Mailhes John B. , Yuan Zhi Ping
Published: February 2005

The acquisition of cytogenetic data from mammalian oocytes has required considerable time and expense, since only a relatively small number of oocytes could be processed from three to four animals daily. The availability of a procedure that would facilitate fixation and preparation of air‐dried slides from 25–30 superovulated mice within a 3‐h period would enhance development of germ cell cytogenetic data by reducing technician time and animal maintenance expense. We present such a procedure for mouse metaphase II oocytes. Mice were superovulated and the oocytes collected were fixed en masse prior to making air‐dried slides. Chromosomes were subsequently C‐banded to enhance objective cytogenetic analysis. The reliability of the procedure was determined by harvesting 44,814 oocytes from 1,875 mice over a 9‐month period and calculating the proportion of cells cytogenetically analyzed to those not analyzed. Cytogenetic analysis of oocytes is an assay for chemicals (and other agents) capable of inducing numerical and structural chromosomal aberrations.
Ultrastructure of in vivo fertilization in the goat
Authors: Crozet N. , Théron M. C. , Chemineau P.
Published: February 2005

In vivo fertilization of goat eggs has been studied by electron microscopy. Eggs were recovered from superovulated or natural cyclic goats, 32 to 52 hours after the onset of oestrus; only eggs recovered between 46 and 52 hours were fertilized.

Spermatozoa penetrated the zona pellucida tangentially leaving vesiculated products of the acrosome reaction at the zona surface.

As sperm penetrated into the ooplasm, the second meiotic division completed and cortical granule exocytosis occurred. However a few unreacted cortical granules usually remained in the cortex of the two fertilized eggs, adjacent to the plasma membrane.

After swelling the two pronuclei presented similar ultrastructural morphology: they contained small, compact, agranular nucleoli and unevenly distributed chromatin. The cytoplasm in close vicinity to the apposed pronuclei contained large stacks of annulate lamellae, smooth endoplasmic reticulum, prominent Golgi complexes, as well as dense areas of unidentified material. The abundance of cytoplasmic organelles near the pronuclei might be the expression of intensive metabolic activity. Conversely, in the cortex of fertilized ova several large organelles‐free cytoplasmic areas were randomly distributed.
Development of the nucleolus in early goat embryos
Authors: Chartrain I. , Niar A. , King W. A. , Picard L. , St‐Pierre H.
Published: February 2005

In vivo nucleologenesis was studied in goat embryos from the pronuclear stage to the blastocyst stage by light and electron microscopy. Ultrastructural changes of the nucleoli were characterized by the following progression: homogeneous electron‐dense fibrillar primary nucleoli in the pronucleus; small, dense fibrillar masses dispersed in clusters of chromatin at the two‐cell stage; ring‐shaped nucleoli made up of a fibrillar center surrounded by a layer of dense fibrillar components at the four‐cell stage; reticulated nucleoli composed of a three‐dimensional network of fibrillar components surrounded by small amounts of granular components at the eight‐cell stage; fully developed compact‐type nucleoli consisting of several fibrillar centers each surrounded by a layer of fibrillar componenets and abundant granular components in morulae and blastocysts. Moreover, it was concluded that activation of rRNA transcription, as evidenced by specific silver nitrate staining of the nucleolus organizer regions of metaphase chromosomes, occurs at the two‐to four‐cell stage and that the morphological changes accompanied a substantial increase in nucleolar transcriptional activity up to the blastocyst stage. This study provides evidence that a structure‐function relationship exists during nucleologenesis in goat embryos.
Mechanism of egg attachment stalk formation in the lobster, Homarus
Authors: Goudeau M. , Talbot P. , Harper R.
Published: February 2005

We have examined the formation of the egg attachment stalk in the lobsters Homarus americanus and H. gammarus. The formation of the stalk is similar in both species. Ovulated oocytes are surrounded by a single coat, envelope 1, composed of layers 1A and 1B. After passage through the gonopore and exposure to sea water, envelope 1 swells and becomes sticky. A second coat, envelope 2, forms between the oocyte and envelope 1 during a complex cortical reaction initiated after fertilization. Eggs pass over the ventral surface of spawning females to the region of the pleopods, where they stick by means of layer 1A to each other and to the ovigerous setae. Layers 1A and 1B are soft and pliable at this time. During egg attachment, the pleopods beat vigorously and cause envelope 1 to stretch and form attachment stalks. Beating probably also causes the attachment stalks to twist and wrap around the ovigerous setae. After the egg mass is secured to the ovigerous setae, envelope 1 of both the attachment stalk and egg coat condenses to form a tough material capable of securing the egg mass to the pleopods for intervals up to 16 months. After larvae hatch, portions of the egg coats and the attachment stalks are retained on the ovigerous setae until the female undergoes her next molt.
Ultrastructure of the spermatozoon of Lepidogalaxias salamandroides and its phylogenetic significance
Authors: Leung Luke K.‐P.
Published: February 2005

The spermatozoon of Lepidogalaxias salamandroides possesses an acrosome (putative), one or two perforatoria (putative) but no nine‐triplet centrioles. Two elongated mitochondria (12 μm long) are situated in parallel between the nucleus (20 μm long) and the axoneme (53 μm long). The above features are unique among other teleosts with internal fertilization. The presence of an “acrosome” in this primitive teleost supports the hypothesis that this structure has been secondarily lost in teleosts during evolution. The uncertainty of phylogenetic placement of this fish is reflected by its unique sperm ultrastructure.
Morphological observations on sperm–egg interactions during in vivo fertilization in the dasyurid marsupial Sminthopsis crassicaudata
Authors: Breed W. G. , Leigh C. M.
Published: February 2005

The aim of the present study was to determine the morphological changes that take place in the male and female gametes during in vivo fertilization in the Australian marsupial, the fat‐tailed dunnart, Sminthopsis crassicaudata. Plastic sections were cut of sperm and eggs recovered from the oviducts of recently mated individuals, and light microscopy of thick, and transmission EM of thin, sections was carried out. It was found that, before penetration of the zona, the spermatozoon came to lie along the outer surface with its rostral tip forming a depression in the zona substance. During penetration, zona material was packed tightly around the spermatozoon, and no large hole was formed. A spermatozoon within the perivitelline space had made contact with the oolemma by way of its apical tip. In a spermatozoon partly incorporated into the ooplasm, fusion appeared to have taken place between its plasma membrane and that of the oolemma. Mucoid coat material became deposited outside the zona at this time; its existence and/or the release of cortical granule content probably prevented polyspermy. Once inside the egg cytoplasm, the sperm head sometimes travelled a considerable distance before chromatin decondensation occurred. In addition, it appeared to rotate somewhat on its axis at this time. Finally, some membranous structures were found around two condensed sperm heads in the ooplasm, which may have been part of the pronuclear envelope. Thus this study on in vivo fertilization in the dunnart documents, for the first time, some aspects of fertilization in an Australian marsupial as seen with the transmission electron microscope; it indicates a few differences from those previously found for the American opossum.
Sperm head structure of a murid rodent from Southern Africa: The red veld rat Aethomys chrysophilus
Authors: Breed W. G. , Cox G. A. , Leigh C. M. , Hawkins P.
Published: February 2005

The morphology of spermatozoa from the red veld rat, Aethomys chrysophilus, of Southern Africa is described; two very different types were found, which came from animals from two separate, as‐yet‐undescribed, species. In individuals from South Africa the sperm head had a somewhat disc‐shaped nucleus and a large acrosome with a huge apical segment that, during epididymal transit, changed in form from initially projecting anteriorly to a highly complex structure that was flexed caudad and lay alongside part of the rest of the sperm head. In addition, the chromatin generally appeared to be not fully condensed. Spermatozoa from animals collected in Malawi were very different in morphology and had a head with a typical apical hook, a perforatorium, fully condensed chromatin, and a 4‐μm‐long ventral spur. Its sperm tail was also significantly longer. The time of divergence of these two groups of animals from a common ancestor is not known, but the present results show that a considerable morphological change in the sperm nucleus, acrosome, and subacrosomal space can evolve even between two, presumably closely related, species.