bluuebunny/crossref_metadata_2025 huggingface dataset:
Human sperm injection into the perivitelline space (SI‐PVS) of hamster oocytes: Effect of sperm pretreatment by calcium‐ionophore A23187 and freezing‐thawing on the penetration rate and polyspermy
Authors: Lassalle B. , Testart J.
Published: February 2005

Calcium‐ionophore A23187 and freezing‐thawing were used as sperm treatments before human sperm injection into the perivitelline space (SI‐PVS) of hamster oocytes. The penetration rate (PR) was higher when SI‐PVS was performed with calcium‐ionophore‐treated (28%) or frozen‐thawed (51%) sperm than with untreated sperm (8%). Optimal PR occurred when five calcium‐ionophore‐treated (57%) or frozen‐thawed (71%) sperm were injected under the zona pellucida. When the sperm:egg ratio was 1:1, PR was higher for calcium‐ionophore‐treated (18.5%) or frozen‐thawed (27.8%) sperm than for untreated sperm (0.0%). Calcium‐ionophore sperm treatment had no effect on the polyspermic oocyte rate (POR) or the mean number of swollen sperm nuclei per penetrated oocyte (Pd) or per injected sperm (SR). This may result from premature oocyte activation induced by Ca‐ionophore. However, POR was higher with frozen‐thawed (74%) than with untreated (50%) or Ca‐ionophore‐treated (50%) sperm. Whatever the sperm treatment, there was a trend toward a lower SR as the number of injected sperm increased. Cytoplasmic regulation of polyspermy in the hamster oocyte is discussed.
Low molecular weight factor in bovine caudal epididymal fluid that stimulates calcium uptake in caput spermatozoa
Authors: Vijayaraghavan Srinivasan , Hoskins Dale D.
Published: February 2005

Secretions from the mammalian epididymis contain proteins that bind to developing sperm and are presumed to play a role in sperm maturation. The biochemical functions in sperm of most of these proteins are not known. In this report we describe the presence of a low molecular weight compound in bovine caudal epididymal luminal fluid (CF) that has a potent stimulatory effect on calcium (45Ca2+) uptake in immature caput epididymal spermatozoa. The studies were initially undertaken to characterize the effect of the protein caltrin, present in bovine seminal plasma (BSP), on calcium uptake into caput spermatozoa. Caltrin is known to block calcium influx into mature bovine sperm. Unexpectedly, the kinetics of calcium uptake into caput sperm showed a biphasic response when treated with BSP, namely, a stimulation of uptake at 1 to 5 min and inhibition of uptake after this time. Since caudal sperm do not show this biphasic response, we reasoned that BSP contained a factor derived from CF that must interact with developing sperm before the binding of caltrin to sperm can prevent further calcium uptake. We first demonstrated that preincubation of caput sperm with CF eliminated the biphasic calcium uptake effect induced in caput sperm by BSP and that caudal fluid alone had a potent stimulatory effect on calcium uptake in caput sperm. Half‐maximal stimulation (fivefold over control) occurred at a caudal fluid protein concentration of 0.27 mg/ml. Partial purification of the factor indicates that it is of low molecular weight (MW ∼ 1,000), but further chemical characterization has not been carried out and its epididymal site of origin is not known. The results indicate that the regulation of intracellular calcium levels in sperm differs in immature and mature bovine sperm in that an epididymal factor promotes calcium uptake during epididymal maturation, and the seminal fluid protein caltrin prevents it at ejaculation.
Cholesterol efflux from bovine sperm: II. Effect of reducing sperm cholesterol on penetration of zona‐free hamster and in vitro matured bovine ova
Authors: Ehrenwald Eduardo , Parks John E. , Foote Robert H.
Published: February 2005

Several reports have indicated that sperm capacitation includes loss of membrane cholesterol (Chol) with a concomitant decrease in the Chol‐to‐phospholipid (PL) ratio. Methods were developed for quantifiable removal of bovine sperm Chol, which predisposed sperm to induction of the acrosome reaction upon addition of lysophosphatidylcholine (LPC). The objective of this study was to evaluate the effect of Chol removal from bovine sperm on penetration of zona‐free hamster and intact bovine ova in vitro. Washed ejaculated bovine sperm were incubated (2 h, 39°C) in a modified Tyrode's solution (TALP) containing (1) Chol‐free liposomes (—Chol, 50 × 106 sperm and 600 nmol phospholipid/ml); (2) liposomes containing 30 mol% Chol (+ Chol, 2 × 108 sperm and 300 nmol total lipid/ml); or (3) no liposomes (Control). We have previously shown that net Chol efflux from sperm is 31% of the total sperm Chol with —Chol liposomes and less than 1% with control media. Sperm were then washed twice and challenged with LPC bound to bovine serum albumin (BSA) using celite as a carrier. Treated sperm (25 × 106) were incubated immediately with either zona‐free hamster ova (HO) or in vitro matured bovine ova (BO) in 50‐μl droplets of TALP under medical fluid in an atmosphere of 5% CO2 in air (3 h, 39°C). Ova were fixed in ethanol:acetic acid, stained with 1% orcein, and examined. Percent penetration (%P) of HO (X ± SEM) for 30 and 40 μg of LPC/mg BSA was 59.4 ± 5.3 and 82.9 ± 5.4; 38.5 ± 5.6 and 52.3 ± 4.7; and 16.0 ± 4.6; and 23.2 ± 5.6 for —Chol, Control, and + Chol treatments, respectively (n = 3). %P of BO (X ± SEM) for 30, 35, and 40 μg of LPC/mg BSA was 43.3 ± 5.4, 70.7 ± 7.5, and 81.5 ± 5.1 for —Chol and 16.4 ± 6.9, 36.2 ± 6.9, and 44.2 ± 8.6 for Control treatments, respectively (n = 3). In a second set of experiments %P of BO (X ± SEM) was 63.6 ± 6.8, 31.8 ± 4.9, and 10.5 ± 3.4 for —Chol, Control, and +Chol treatments, respectively, when 40 μg LPC/mg BSA was added (n = 2). %P and the number of sperm per fertilized ovum were consistently higher for the —Chol treatment for both HO and BO (P < .01). These results demonstrate that Chol removal from bovine sperm facilitates penetration of ova in vitro suggesting a potential role in bovine sperm capacitation.
Filipin‐labelled complexes are polarized in their distribution in the cytoplasm of meiotically mature mouse eggs
Authors: Allworth Ann , Ziomek Carol A.
Published: February 2005

Unfertilized (germinal vesicle [GV] stage, superovulated and naturally ovulated) and fertilized mouse eggs were treated with the polyene antibiotic filipin, which complexes with unesterified sterols; specimens were observed by fluorescence microscopy and scanning electron microscopy (SEM).

In all oocytes examined, filipin fluorescence was localized to the plasma membrane and to subcellular structures of various sizes. In the unfertilized oocyte, polarity was observed both in the plasma membrane stain and in the pattern formed by the subcellular structures. SEM of filipin‐treated oocytes had several characteristic features including a specific distribution of heterogeneous microvilli that appears to have a spatial relationship with the fluorescent pattern of the filipin‐positive subcellular structures.

In GV stage and fertilized eggs the filipin‐positive subcellular structures were associated with the germinal vesicle and in fertilized eggs they were associated with the site of polar body abstriction.
Changes of elemental concentrations around and on the surface of fowl sperm membrane during maturation in the male reproductive tract and after in vitro storage
Authors: Ashizawa Koji , Ozawa Yoko , Okauchi Keizo
Published: February 2005

X‐ray microprobe analysis was performed to investigate the changes of elemental concentrations around or on the membrane of the head, midpiece, and principal piece regions of individual fowl spermatozoa during maturation in the male reproductive tract and after storage in vitro at 4°C. The pattern of change of elemental concentrations during maturation and postejaculation was, in general, similar in the three different subcellular regions; i.e., concentrations of sodium, potassium, chlorine, and calcium decreased gradually during sperm passage through the male reproductive tract and after storage. Phosphorus concentration remained almost constant in the male tract and decreased gradually after storage. In contrast, magnesium, zinc, and copper concentrations showed an interesting pattern: concentrations increased significantly during maturation to a maximum at ejaculation and decreased again after storage. The ratios of sodium to potassium in the midpiece region showed patterns similar to those of magnesium, zinc, and copper concentrations.
Sperm entry into zona‐free oocytes in the hamster oviduct: Implications for the mechanisms of acrosome reaction induction
Authors: Cuasnicu Patricia S. , Bedford J. M.
Published: February 2005

The possible importance of the zona pellucida for induction of the acrosome reaction (AR) and establishment of sperm/egg associations in the fallopian tube was investigated by instilling zona‐free eggs into the oviductal ampulla of hamsters that had been inseminated with epididymal spermatozoa 6–7 hours previously. The eggs were recovered only 60–90 minutes later because of increasing difficulty with time of collecting zona‐free eggs from the oviduct. In the zona‐free group, 41 (4%) of 1,101 transferred eggs were recovered, of which 20% contained spermatozoa with decondensing nuclei (mean 4.4/egg). A similar (22%) fertilization rate (mean 3.2 spermatozoa/egg) was found among intact (control) eggs recovered after instillation into the contralateral oviduct.

Mammalian spermatozoa are not incorporated even into zona‐free eggs before AR occurs. These results thus demonstrate that an AR in functional hamster spermatozoa in vivo and establishment of sperm/egg associations in vivo require no interaction with the zona pellucida nor with other products of ovulation.
Fertilization envelope assembly in sea urchin eggs inseminated in Cl− deficient sea water: I. Morphological effects
Authors: Lynn John W. , Goddard Russell L. , Glas Patricia , Green Jeffrey D.
Published: February 2005

Elevation and hardening of the fertilization envelope (FE) occur within 15 min following insemination of the sea urchin egg. When chloride ions were replaced in the media with various anion substitutes, including methyl sulfonate, nitrates, bromide, and isethionate, the fertilization envelope failed to harden and collapsed back to the surface of the egg of Lytechinus variegatus, L. pictus, and Strongylocentrotus purpuratus. At the light microscopy level, the collapse of the envelope was accompanied by a decrease in birefringence, compared with controls. When examined with electron microscopy, the FEs of eggs inseminated in reduced Cl− solutions failed to transform from an amorphous layer into the more robust laminar structure observed around eggs incubated in normal sea water. Furthermore, in the case of S. purpuratus, the I‐T transformation of the FE did not occur. When transfer of the inseminated eggs from the Cl−‐deficient sea water to normal sea water was carried out before 10 min elapsed, the envelope did not collapse, and the birefringence of the envelope was similar to that of controls. Partial envelope collapse was also observed in a dose‐dependent manner, varying with the concentration of the Cl− in the sea water solution. The results suggest that lack of Cl− in the media may interfere with proper fertilization envelope assembly. Possible mechanisms, including proper incorporation of the cortical granule exudate into the nascent envelope structure, are discussed.
Sperm‐engulfing response of sea urchin egg surfaces inseminated with acrosome‐reacted starfish sperm
Authors: Kyozuka Keiichiro , Osanai Kenzi
Published: February 2005

Sperm‐egg interaction was examined in two interclass combinations of sea urchin (Strongylocentrotus nudus and Hemicentrotus pulcherrimus) eggs and starfish (Asterina pectinifera and Asterias amurensis) sperm. Cross‐fertilization was unsuccessful between these combinations. When the vitelline coat‐free sea urchin eggs were mixed with acrosome‐reacted starfish sperm, the elongated microvilli on the egg surface wrapped the sperm head. This sperm‐engulfing response observed on the denuded egg surface was induced only in sperm immediately after initiation of the acrosome reaction. Further fertilization events, such as gamete membrane fusion or discharge of cortical granules, did not proceed. These observations suggest that acrosome‐reacted sperm can induce a local response on the heterologous egg surface, that is independent of gamete membrane fusion and egg activation.
Different outcomes of sperm competition in right and left sides of the female reproductive tract revealed by thymidine‐3H‐labeled spermatozoa in the rat
Authors: Špinka M.
Published: February 2005

We attempted to record directly the results of sperm competition between two male rats in samples from female reproductive tracts. Gametes of 7 male rats were labeled in vivo by repeated injections of thymidine‐6‐3H. 33 females were mated successively with one of the injected males and with a noninjected male, or vice versa. Samples of the resulting sperm mixtures were recovered from both the uterine horns and the oviducts of the females. Pure samples of the injected males' sperm were obtained from uterine horns of females mated solely to them. Smears of samples were evaluated autoradiographically. Except for one male, the injections resulted in a broad peak of sperm labeling with maximum intensity on days 56–58 postinjection. This labeling enabled the assessing of contribution of either male to uterine and tubal sperm in the competitive matings. The injections did not influence competitive ability of male gametes in terms of sperm transport to horns or oviducts. The outcomes of sperm competition were significantly different in the right and left sides in most female subjects. Nevertheless, the outcomes were usually similar in a horn and a tube on the same side of the body. In vivo sperm labeling appears to be a useful method in the study of rodent sperm competition.
Production of chimeric rabbits from morulae by a simple procedure
Authors: Yang Xiangzhong , Foote R. H.
Published: February 2005

Experiments were conducted to develop a simple and reliable technique to produce chimeric rabbits from morula stage embryos. In Experiments 1 and 2, an in‐vitro test of viability was initially performed by culturing embryos to the blastocyst stage. Ninety‐three percent of the “chimeric” embryos developed to the blastocyst stage compared to 94% for controls when embryos were manipulated soon after collection (Exp. 1). Eighty‐one percent chimeric embryos and 78% control embryos developed to blastocyst stage when embryos were held at room temperature for 4 hr (Exp. 2). In Experiment 3, enough morula‐stage embryos were available from true breeding Dutch‐belted and albino rabbits to form potentially 67 diverse “color” pairs. These micromanipulated pairs of morulae were successfully combined to produce 64 chimeric embryos (96%, 64/67). They were transferred to the uteri of seven recipient does and three became pregnant producing 13 young. Four of the young exhibited substantial overt chimerism (31%) and one more was a possible chimera.
Role for fucose‐sulfate‐rich carbohydrates in the penetration of zona‐pellucida‐free hamster eggs by hamster spermatozoa
Authors: Dravland J. E. , Mortimer D.
Published: February 2005

The interaction of acrosome‐reacted hamster spermatozoa and zona‐pellucida‐free hamster eggs was investigated by incubating gametes in the presence of a variety of simple and complex carbohydrates. Significant inhibition of gamete fusion was achieved only in the presence of fucoidan and ascophyllin, two algal polysaccharides containing fucose sulfate. These compounds did not interfere with sperm motility, capacitation, or acrosome reactions. It is concluded that these two compounds share common structural features with putative cell‐surface carbohydrates involved in sperm‐oolemmal interaction.
Hemizona assay using salt‐stored human oocytes: Evaluation of zona pellucida capacity for binding human spermatozoa
Authors: Franken Daniel R. , Burkman Lani J. , Oehninger Sergio C. , Coddington Charles C. , Veeck Lucinda L. , Kruger Thinus F. , Rosenwaks Zev , Hodgen Gary D.
Published: February 2005

Human oocytes were stored (25°C) in 1.5 M MgCl2 for 6–30 days, then utilized in the new hemizona assay (HZA) for tight binding of human spermatozoa [Burkman et al.: Fertil Steril 49:688–697, 1988]. We have compared 1) the ability of matching salt‐treated hemizonae or dimethylsulfoxide (DMSO)‐treated hemizonae to distinguish between sperm from semen having normal versus subnormal characteristics and, 2) the kinetics of fertile sperm binding to salt‐treated or DMSO‐treated hemizonae. After sperm preparation, one salt‐treated hemizona was incubated with normal spermatozoa and the matching hemizona was placed with sperm from the subnormal group. As a control, DMSO‐treated hemizonae were incubated in additional sperm droplets. After 4 hours, the number of sperm tightly bound to each hemizona was counted. Within the normal semen group, there was equivalent binding to salt‐ or DMSO‐treated hemizonae (54.0 ± 12 and 49 ± 14, respectively, mean ± SEM). Similarly, tight binding of sperm from the subnormal group was not affected by the zona storage method (21 ± 8 and 17 ± 5, respectively). For either storage approach, binding of subnormal sperm was significantly less (P < 0.01) compared with the number of normal sperm attached to the matching hemizona. For the kinetics study, the hemizona binding of proven fertile spermatozoa was followed throughout 8.5 hours. The shape of the binding curve was the same for zonae stored by either method and was consistent with our published kinetics data. Salt storage offers a simple and inexpensive means for accumulating and transporting human zonae pellucida; the resulting hemizonae function effectively in the HZA for estimating sperm binding potential.
Analysis of the chromosome complement in outbred mouse sperm fertilizing in vitro
Authors: Martin‐DeLeon Patricia A.
Published: February 2005

The chromosome complements in a population of mouse sperm from random‐bred ICR donors were analyzed at first‐cleavage metaphase after in vitro fertilization (IVF) of oocytes from females of the same strain. The sperm were aged as donations occurred within an average of 31 days, either since last mating or at arrival at the animal facility in the case of virgin males. Of a total of 598 sperm complements studied from 22 sexually mature males aged 10–26 weeks old, there was one diploid complement (0.17%). The frequencies of hyperhaploidy and structural aberrations that were studied in 338 complements were 4.4% and 3.6%, respectively, giving an overall frequency of 8.0%. The hyperhaploid complements consisted of n + 1, n + 2, n + 3, and n + 7 counts, while the structural abnormalities were of the chromosome type and included large and small fragments and a possible translocation. This is the highest frequency of sperm chromosome abnormalities reported for mouse sperm obtained from males under physiological conditions and fertilized in vitro or in vivo. Sperm aging, strain, and/or technique differences are among the factors that may be responsible for this high frequency. Since the 8.0% frequency of hyperhaploidy and structural abnormalities is similar to the frequency reported for human sperm after IVF, the outbred murine in vitro fertilization system may be a useful model to study the origin of human sperm chromosome abnormalities.
Meiotic resumption and gap junction modulation in the cultured rat cumulus‐oocyte complex
Authors: Wert Susan E. , Larsen William James
Published: February 2005

Intercellular communication within the ovarian follicle has been implicated in the control of meiotic arrest and maturation in the mammalian oocyte. We have shown that a rapid down‐regulation of cumulus cell gap junctions is correlated temporally with meiotic resumption in the intact rat follicle [Larsen et al., Dev Biol, 113:517–521]. Here this relationship has been analyzed further by incubating isolated rat cumulus‐oocyte complexes (COCs) with agents known to influence germinal vesicle breakdown (GVBD) or that have been shown to modulate gap junction turnover in vitro. Quantitative freeze‐fracture analysis revealed that cumulus cell gap junction membrane decreased significantly prior to the initiation of GVBD in COCs incubated in medium lacking serum or other additives. The addition of serum and follicle‐stimulating hormone, an experimental condition that delayed GVBD, accelerated and augmented gap junction loss at both the cumulus cell and oocyte surface. The continuous elevation of cyclic adenosine monophosphate levels, which stimulates gap junction formation in other systems, maintained meiotic arrest but did not interfere with gap junction loss. Conversely, the complete inhibition of junctional loss by a microfilament destabilizing agent, dihydrocytochalasin B, did not alter the course of GVBD normally seen in its absence. Subsequent freeze‐fracture analysis and dye coupling experiments confirmed that cumulus and oocyte gap junctions in these preparations were intact and functional during the period of meiotic resumption. These findings suggest that factors other than cumulus and oocyte gap junction turnover are required for the control of meiotic arrest and maturation in the isolated COC. However, these results do support our earlier suggestion that gap junction loss within the cumulus oophorus is instrumental in isolating the oocyte from the regulatory influence of its underlying membrana granulosa cells during meiotic maturation in the intact preovulatory follicle [Larsen et al., Dev Biol, 122:61–71].
Human antral follicles: Oocyte nucleus and the karyosphere formation (electron microscopic and autoradiographic data)
Authors: Parfenov Vladimir , Potchukalina G. , Dudina L. , Kostyuchek D. , Gruzova M.
Published: February 2005

The functional organization of the nucleus in the oocytes from human antral follicles was examined by morphological and autoradiographic analysis methods at the light and electron microscopic level. According to the position of the nucleus, the level of its transcriptional activity, and the pattern of distribution of structures in it, oocytes fall into two groups. In the first one, the oocytes with the nucleus in the central position are characterized by the distribution of numerous structures all over the nucleus or by a different extent of aggregation of chromatin around the nucleolus. The nuclei of these oocytes are characterized by [3H]uridine incorporation, the label being localized over purely fibrillar, agranular nucleoli and over dispersed fibrillar chromatin adjacent to either the regions of densely packed chromatin or fibrillar‐granular material of the nucleolus‐like bodies. The latter, the same as condensed chromatin, do not incorporate [3H]uridine. In the second group, the nuclei are displaced towards the oocyte's periphery, and chromosomes surround the nucleolus as a continuous mass closely adjacent to its surface, thus forming a karyosphere. The karyosphere formation takes place on the background of cessation of nuclear transcriptional activity. A fully formed karyosphere represents a complex of closely associated inactivated structures: Nucleolus, chromosomes, and nucleolus‐like bodies. The karyosphere nucleolus bears no granules and consists of densely packed finely fibrillar material (fibrils 3 nm thick). Two zones (central and peripheral) can be distinguished in a nucleolus. Nucleolus‐like bodies, consisting of granules 20 nm in diameter embedded in finely fibrillar material, are often associated with chromosomes.

In this study, data obtained by observations on the loss of association between the oocyte (with karyosphere) and corona radiata cells are evaluated. The relation of the karyosphere formation to the atresia process and the duration of karyosphere existence in human antral follicles are also discussed.
Continuous live‐dead discrimination of ram sperm during freezing
Authors: Fiser P. S. , Marcus G. J.
Published: February 2005

Use of the dye amaranth (Color Index 16185) as a supravital stain for ram sperm is described. At a concentration of 0.4% in diluted semen, the dye was completely excluded by motile sperm and had no effect on sperm motility. The nuclei of immotile sperm were stained pink by amaranth. The decrease in sperm motility during 24‐h storage at 5°C was accompanied by a corresponding increase in stained sperm nuclei. The presence of the dye during freezing had no effect on sperm cryosurvival but tended to reduce sperm motility during post‐thaw incubation. Insemination of ewes with fresh semen containing amaranth or with semen frozen in the presence of amaranth resulted in pregnancies in 7/10 ewes in each group, compared to 6/9 in the case of ewes inseminated with fresh semen without dye.
Induction and inhibition of amphibian (Rana pipiens) oocyte maturation by protease inhibitor (TPCK)
Authors: Ishikawa Katsutoshi , Schuetz Allen W. , Francisco S. K. San
Published: February 2005

We report for the first time that oocyte nuclear and cytoplasmic maturation are triggered in vitro in non‐hormone‐treated amphibian (Rana pipiens) ovarian follicles following transient exposure to synthetic chymotrypsin inhibitor Nα‐tosyl‐L‐phenylalanine‐chloromethyl ketone (TPCK). The mechanism of action of TPCK in regulating oocyte maturation was investigated and compared to that induced by progesterone or pituitary hormone. Follicular oocytes failed to mature following continuous exposure to the same doses of TPCK in the presence or absence of progesterone. Continuous treatment of follicles with lower levels of TPCK occasionally induced GVBD in the absence of progesterone and augmented maturational effects of low levels of progesterone. TPCK induced maturation of intrafollicular oocytes without stimulating progesterone production and also induced maturation of naked oocytes. Stimulation of follicular progesterone synthesis following gonadotropin stimulation or addition of pregnenolone was inhibited by TPCK, indicating that TPCK affects metabolic processes in both the somatic and germinal components of the ovarian follicle. Oocyte maturation induced by either TPCK or progesterone was inhibited by cycloheximide, calcium‐deficient medium, and forskolin. Results suggest that TPCK induces oocyte maturation independent of steroidogenesis via mechanisms similar to those triggered by progesterone involving protein synthesis, formation of cytoplasmic maturation‐promoting factor (MPF), and changes in cAMP levels. Our data indicate that a chymotrypsin‐like protease plays a role(s) in regulating the oocyte meiotic maturation process.
Influence of serum and hormones on bovine oocyte maturation and fertilization in vitro
Authors: Younis A. I. , Brackett B. G. , Fayrer‐Hosken R. A.
Published: February 2005

Three approaches were investigated for improvement of in vitro maturation (IVM), in vitro fertilization (IVF), and early embryonic development in cattle. These were: 1) Selection of oocytes, 2) medium supplementation with fetal calf serum (FCS) and cow sera (DO, Dl, D10, and D20 to correspond with estrus, metestrus, diestrus, and proestrus, respectively), and 3) addition of follicle‐stimulating hormone (FSH), luteinizing hormone (LH), and estradiol‐17β (E2)during maturation. Greater proportions (percentage) of oocytes initially selected for their compact cumulus cells completed IVM and IVF when compared to unselected oocytes (P < .05). Proportions (percentage) of selected oocytes that matured and cleaved after in vitro insemination according to serum type used for IVM were: FCS: 110/175 (62.9%) and 37/110 (33.6%) and DO: 130/145 (89.7%) and 52/130 (40.0%); D1 127/130 (97.7%) and 41/127 (32.3%); D10 95/98 (96.9%) and 35/95 (36.8%); D20:113/116 (97.4%) and 49/113 (43.4%). A higher proportion (P < .05) of embryos resulting from the D20 group reached four‐ and eight‐cell stages. In FCS‐supplemented maturation media with no hormones added during maturation (control), results of IVM and IVF were 157/265 (59.2%) and 39/157 (24.8%), respectively. With E2 (1 μg/ml) and FSH (5 μg/ml), comparable results were 189/215 (87.9%) and 71/189 (37.6%); with E2 (1 μg/ml) plus LH (10 μml), 280/327 (85.6%) and 111/280 (39.6%). Added hormones improved IVM results (P < .05) and, when FSH or LH was added with E2, in vitro development to four‐ and eight‐cell stages was markedly enhanced (P < .05). Selection of oocytes, D20 serum, and added E2 and FSH or LH for IVM improved in vitro development of bovine embryos after IVF.
Incorporation and fate of specific sperm plasma membrane components following insemination as revealed by ultrastructural immunocytochemistry
Authors: Longo Frank J.
Published: February 2005

Studies have been carried out to 1) further characterize sperm specific plasma membrane polypeptides (33 and 35 kDa) that are recognized by a monoclonal antibody previously described (Longo, 1989) and 2) follow the incorporation and dispersal of these proteins within plasmalemmae of monospermic and polyspermic sea urchin (Arbacia punctuluta) eggs and oocytes utilizing immunocytochemical methods at the ultrastructural level of observation. Only sperm labeled when incubated with monoclonal antibody to the 33 and 35 kDa proteins followed by colloidal gold‐tagged second antibody. Colloidal gold label was observed on the egg plasma membrane immediately after gamete membrane fusion; the amount and extent of label, i.e., the distance from the site of sperm incorporation, increased with time postinsemination. By 20 min postinsemination approximately one hemisphere of the inseminated egg/oocyte was associated with label. The expanding distribution of colloidal gold label on inseminated eggs and oocytes vs. time reflects the free diffusion of 33 and 35 kDa sperm surface proteins among egg/oocyte plasma membrane components. Label was also found in forming endocytotic vesicles, suggesting that sperm plasma membrane proteins may be internalized.
Monospermy and polyspermy after partial zona dissection of reinseminated human oocytes
Authors: Matter Henry , Talansky Beth , Gordon Jon , Cohen Jacques
Published: February 2005

Partial zona dissection (PZD), a zona drilling method that uses mechanical force to open the zona pcllucida while the oocyte is shrunken in a sucrose solution, was applied to 121 unfertilized 1‐day‐old mature human oocytes prior to reinsemination. The 115 surviving oocytes were divided into three groups in which the duration between sucrose addition and reinsemination was varied: I) Less than 20 minutes, II) 21 to 45 minutes, and III) longer than 45 minutes. There was a trend toward a reduced fertilization and polyspermy rate as the time between sucrose exposure and insemination in sucrose‐free medium increased. Moreover, there was a statistically significant reduction in the number of oocytes penetrated by more than four sperm in group III (0/41) versus group I (7/34), and in group III, parthcnogcnctic development was observed. The incidence of polyspermy was also increased in oocytes manipulated more than 25 hours after retrieval compared with those manipulated 21‐24 hours after recovery, supporting the idea that aged oocytes have a reduced ability to block polyspermy. Oocyte contraction in sucrose occurred in three different patterns: spherical, pear‐shaped, and crenated. Both the fertilization and polyspermy rates were significantly higher in the crenated group.

These results indicate that changes resembling activation occur following sucrose exposure and that sucrose activation can be used to reduce the risk of polyspermic fertilization in zona drilling procedures. In addition, the pattern of shrinkage in sucrose can be used as an indicator of oocyte receptivity to sperm penetration.
Glycerylphosphorylcholine diesterase activity of uterine fluid in conditions inducing secondary sex ratio change in the rat
Authors: Mitra Jayashree , Chowdhury Mridula
Published: February 2005

Food restriction or decreasing the ratio of [Na+, K+] to Ca++, Mg++J in the diet of female rats before conception favoured the production of female offspring. Seven days of food deprivation decreased uterine fluid GPC diesterase activity in female rats, whereas long‐term food restriction (21 days), rather than decreasing the enzyme activity, apparently stimulated it. Dietary Ca and Mg supplementation, likewise, produced significant decrease in GPC di esterase activity. A significantly positive correlation was observed between the levels of uterine GPC diesterase and secondary sex ratio change, which indicates that these dietary techniques of sex‐ratio manipulation may modulate the uterine fluid GPC diesterase activity and influence sex determination in utero.
Cumulus cell dispersion induced by estradiol in mouse oviduct in vitro
Authors: Roblero Luis S. , Riffo Marta D. , Croxatto Horacio B.
Published: February 2005

Dispersion of cumulus cells in nonmated mice is completed in the oviduct 15–20 h after ovulation. Oviducts, isolated 1 h after ovulation (13 h post‐human chorionic gonaditropin), were cultured in vitro for 40 h. In these oviducts, denuded oocytes were first seen at 30 h of culture, indicating that cumulus dispersion proceeded at a slower rate in vitro.

Oocyte denudation was accelerated in a dose‐dependent manner by the addition of estiadiol to the culture medium in which oviducts were incubated. The addition of progesterone or cycloheximide to the culture medium strongly inhibited oocyte denudation even in the presence of estradiol. When isolated cumuli were incubated in the absence of oviductal tissue, the rate of cell dispersion was slower than that of cumuli incubated inside the oviduct and the addition of estradiol to the culture failed to accelerate this process.

On the basis of these data, we propose that cumulus cell dispersion is accelerated by an estrogen‐dependent protein produced by the oviduct and that this effect of estrogen is antagonized by progesterone.
Acrosomal function of human spermatozoa with normal and abnormal head morphology
Authors: Fukuda Masaru , Morales Patricio , Overstreet James W.
Published: February 2005

In previous studies we have shown differences in the function of morphologically normal and abnormal sperm by evaluating their flagellar movements and swimming trajectories. In this study we have compared the capability of morphologically normal and abnormal human sperm to undergo an acrosome reaction after incubation with human follicular fluid. Semen samples were studied from 6 research donors and 21 semen evaluation patients. All men had normal semen by clinical criteria. Semen was prepared either by a two‐step Percoll gradient centrifugation or the sperm werediluted, washed, and centrifuged three times. Sperm suspensions were incubated for 24 hours in a modified Tyrode's medium, containing 2.6% bovine serum albumin, prior to dilution with human follicuiar fluid. The percentage of acrosome reactions among viable sperm was assessed after 15 minutes using the supra vital Hoescht stain and fluoresceinated pea lectin. Sperm head size was measured with an ocular micrometer and normal values were defined as length 3–5 μm and width 2–3 μm. At least 25 viable normal sperm, and 25 viable abnormal sperm were analyzed for acrosome reactions on each slide. With Percoll separation the percentage of acrosome reactions (mean ± sem) for normal sperm was 38i ± 3% vs. 22 ± 2% for abnormal sperm (P < 0.005). After washing, the comparable values were 12 ± 1 % vs. 5 ± 1 % (P < 0.005). The incidence of spontaneous acrosome reactions (24 hours of incubation, no follicular fluid) was also higher for normal sperm than abnormal sperm (9 ± 1 % vs. 4 ± 1 %, P < 0.01). These data demonstrate an association between normal sperm morphology and acrosomal function.
Heterogeneity of epididymal spermatozoa of the hamster
Authors: Sullivan Robert , Robitaille Gilles
Published: February 2005

Epididymal transit is involved in the acquisition of fertilizing ability by mammalian spermato‐zoa. The epididymis is implicated in the addition and/or modification of sperm surface proteins functionally involved in the processes leading to fertilization. In order to characterize these sperm components, we have studied the modifications of sperm membrane proteins during epididymal maturation. Hamster spermatozoa were collected from the caput, corpus, and proximal and distal cauda of the epididymis and submitted to a continuous Percoll gradient centrifugation. Independently of their epididymal origin, the spermatozoa were distributed into two bands of buoyant densities of 1,045 and 1,088 on the gradient. However, the proportion of more dense spermatozoa increased progressively along the epididymis. This proportion is 87–fold higher in the distal cauda compared to the caput. SDS‐PAGE electrophoresis demonstrated that although they originate from different regions of the epididymis, spermatozoa of the same density exhibited similar membrane protein patterns. In contrast, the electrophoretic patterns of spermatozoa with densities of 1,045 and 1,088 were very different One of these differences involves a 26 kD protein that is implicated in zona pellucida recognition (Sullivan and Bleau, Gamete Res 12.101–116,1985). This protein is present in dense sperm obtained from all regions of the epididymis but is absent in spermatozoa of low density. If we consider that this 26 kD protein is a ‘marker’ of surface changes occurring during sperm maturation, we can hypothesize that in the proximal segments of the epididymis a certain proportion of spermatozoa are indeed mature but that this proportion increases considerably along the epididymis.
Movement of cynomolgus and rhesus monkey spermatozoa collected from the lower female reproductive tract
Authors: Behboodi Esmail , Katz David F. , Overstreet James W. , Hendrickx Andrew G.
Published: February 2005

Postcoital (pc) cervical mucus was collected in 73 menstrual cycles of cynomolgus monkeys and in 43 cycles of rhesus monkeys at 2,6,10,30 hr pc. Videomicrography was used to analyze sperm numbers and movement in the mucus. Both cynomolgus and rhesus monkeys had comparable populations of motile sperm in the mucus at 2 hr pc. However, by 6 hr pc, cervical mucus from cynomolgus monkeys contained twice as many total sperm and motile sperm as mucus from rhesus monkeys (P <.05). Mean swimming speeds of the free‐swimming cervical sperm were similar for the two species at this time. No motile sperm were recovered in mucus from rhesus monkeys at 30 hr pc. In cynomolgus monkeys, however, 14 of the 26 animals examined at 30 hr pc had motile sperm in their mucus. These sperm exhibited lower percent molility, percent free‐swimming sperm, and swimming speed than those sperm observed at 6 hr pc. Uterine sperm were collected by transcervical or transuterine aspiration from cynomolgus monkeys. In the transcervical technique, sperm were successfully obtained in four of nine animals examined at 6 hr and in four of five animals at 30 hr pc. The percentage of motile sperm in the uterine fluid was high, 82% ± 4%, and the swimming speeds (86 ± 2μm/sec) were higher than those observed in cervical mucus. Approximately 5–10% of the uterine sperm exhibited swimming motions similar to the hyperactivated motility seen in most mammals. These findings indicate that the sperm cervical mucus interaction in vivo in cynomolgus monkeys has more similarities to the human situation than does the interaction in rhesus monkeys.
Effects of bovine mammary alpha‐lactalbumin on hyperactivation and sperm‐zona pellucida binding of mouse spermatozoa
Authors: De Geyter C. H. , Cooper T. G. , De Geyter M. , Nieschlag E.
Published: February 2005

The effects of bovine mammary alpha‐lactalbumin on the motility and zona‐binding characteristics of mouse sperm were investigated. Two properties of sperm associated with capacitation, hyperactivated motility, and the ability of sperm to bind to the zona pellucida of oocytes were shown to be suppressed by alpha‐lactalbumin. These inhibitory effects were not accompanied by changes in the percentage of motile cells or by differences in the velocity parameters of the hyperactivated and non‐hyperactivated spermatozoa. Bovine serum albumin prevented and reversed the alpha‐lactalbumin‐induced suppression of hyperactivation. Sperm‐zona pellucida binding was partially restored by lowering the alpha‐lactalbumin concentration in the medium in which sperm were allowed to bind to the zona pellucida. The results suggest that mouse sperm are decapacitated by bovine mammary alpha‐lactalbumin. The counteracting effect of bovine serum albumin to the suppressive action of alpha‐lactalbumin on the flagellum suggests the involvement of a mechanism different from the action of alpha‐lactalbumin on the sperm head inhibiting binding to the zona pellucida.
A pharmacophore‐based evolutionary approach for screening selective estrogen receptor modulators
Authors: Yang Jinn‐Moon , Shen Tsai‐Wei
Published: February 2005

We developed a pharmacophore‐based evolutionary approach for virtual screening. This tool, termed the Generic Evolutionary Method for molecular DOCKing (GEMDOCK), combines an evolutionary approach with a new pharmacophore‐based scoring function. The former integrates discrete and continuous global search strategies with local search strategies to expedite convergence. The latter, integrating an empirical‐based energy function and pharmacological preferences (binding‐site pharmacological interactions and ligand preferences), simultaneously serves as the scoring function for both molecular docking and postdocking analyses to improve screening accuracy. We apply pharmacological interaction preferences to select the ligands that form pharmacological interactions with target proteins, and use the ligand preferences to eliminate the ligands that violate the electrostatic or hydrophilic constraints. We assessed the accuracy of our approach using human estrogen receptor (ER) and a ligand database from the comparative studies of Bissantz et al. (J Med Chem 2000;43:4759–4767). Using GEMDOCK, the average goodness‐of‐hit (GH) score was 0.83 and the average false‐positive rate was 0.13% for ER antagonists, and the average GH score was 0.48 and the average false‐positive rate was 0.75% for ER agonists. The performance of GEMDOCK was superior to competing methods such as GOLD and DOCK. We found that our pharmacophore‐based scoring function indeed was able to reduce the number of false positives; moreover, the resulting pharmacological interactions at the binding site, as well as ligand preferences, were important to the screening accuracy of our experiments. These results suggest that GEMDOCK constitutes a robust tool for virtual database screening. Proteins 2005. © 2005 Wiley‐Liss, Inc.
Non‐surgical management of live ectopic pregnancy with ultrasound‐guided local injection: a case series
Authors: Monteagudo A. , Minior V. K. , Stephenson C. , Monda S. , Timor‐Tritsch I. E.
Published: February 2005

Objectives

To describe a series of consecutive cases of live ectopic pregnancies managed with ultrasound‐guided local injection of methotrexate (MTX) or potassium chloride (KCl).

Methods

Eighteen consecutive women with live and unruptured, tubal, cornual or cervical ectopic pregnancies referred to our unit for evaluation and management underwent risk–benefit counseling. Under transvaginal ultrasound guidance, puncture and injection of the ectopic pregnancy was performed using an automated puncture device. Either MTX or KCl was injected, producing immediate cessation of fetal cardiac activity.

Results

Of the 18 ectopic pregnancies, 10 were cervical, four were tubal and four were cornual. The mean initial β‐hCG level was 33 412 IU and the mean gestational age was 6 + 6 weeks. Ten ectopic gestational sacs were injected with KCl and eight were injected with MTX. There was no difference in time to resolution of the ectopic pregnancies between those injected with KCl and those with MTX.

Conclusions

Unruptured live ectopic pregnancies of many types can be successfully managed without surgical intervention through local injection of KCl or MTX. Copyright © 2005 ISUOG. Published by John Wiley & Sons, Ltd.
Perinatal management of right aortic arch with aberrant left subclavian artery associated with critical stenosis of the subclavian artery in a newborn
Authors: Tschirch E. , Chaoui R. , Wauer R. R. , Schneider M. , Rüdiger M.
Published: February 2005

A right‐sided aortic arch with an aberrant left subclavian artery is a congenital vascular anomaly that is easily detectable in utero at the level of the three vessels and trachea view, but which is rarely symptomatic in the neonate. We present a newborn with prenatally diagnosed right‐sided aortic arch and aberrant subclavian artery who showed a clinically relevant stenosis of the subclavian artery during the first week of life. An intravascular stent was implanted into the stenosis of the aberrant left subclavian artery by catheterization. This case report demonstrates that a right‐sided aortic arch with an aberrant subclavian artery can be diagnosed prenatally, that in these patients a stenosis of the subclavian artery can occur in early infancy and requires awareness of the neonatologist or pediatrician, and that stent implantation represents a minimally invasive therapeutic approach. Copyright © 2005 ISUOG. Published by John Wiley & Sons, Ltd.
Measurement of nuchal skin fold thickness in the second trimester: influence of imaging angle and fetal presentation
Authors: Cho J. Y. , Kim K. W. , Lee Y. H. , Toi A.
Published: February 2005

Objective

To assess the effect of imaging angle and fetal presentation on the measurement of nuchal skin fold thickness (NFT) in the second trimester.

Methods

Fetal NFT was prospectively measured in 921 women at 18–21 weeks' gestation. The population was divided into two groups according to fetal presentation. Group A comprised 643 fetuses in cephalic or transverse presentation and Group B comprised 278 fetuses in breech presentation. To determine whether there was a difference in NFT depending on imaging angle, in Group A the NFT was measured on the standard axial horizontal images and then on images obtained after moving the transducer about 30° toward the occiput. In Group B an assessment was made as to whether NFT changed with fetal presentation. Initial measurements were made with the fetuses in breech presentation. Repeat NFT measurements were made after 15 min in 64 fetuses that had converted to cephalic or transverse presentation. Statistical analysis was performed using a paired t‐test.

Results

The mean NFT on the standard horizontal image was 5.2 mm, while that on the 30° occiput image was 4.0 mm. The difference was significant (P < 0.001). One hundred and twenty‐three (19.1%) fetuses had a NFT greater than 6 mm on the standard image, but only two (0.3%) had an NFT of > 6 mm on the 30° occiput image. There were no chromosomal or structural abnormalities in the 123 fetuses in which the NFT was > 6 mm. The mean NFT of 64 fetuses in the breech presentation was 4.4 mm, while it was 3.9 mm when they had converted to other presentations. The difference was statistically significant (P = 0.001). In breech presentation, 13/64 (20.3%) fetuses had a NFT greater than 6 mm compared to only one (1.6%) fetus in the other presentations. There were no structural or chromosomal abnormalities in any fetus with a NFT > 6 mm in either group.

Conclusions

In this study the NFT measurement varied with scan direction and fetal presentation. To avoid falsely thickened NFT measurements the scan should be directed to image the posterior surface of the occipital bone. This can be achieved by scanning from a 30° occipital direction. Also, NFT is greater in fetuses in a breech presentation. Attention to these details can reduce false‐positive NFT measurements. Copyright © 2005 ISUOG. Published by John Wiley & Sons, Ltd.