Morphometric analysis of the zona pellucida and ooplasm of squirrel monkey (Saimiri sciureus) eggs

The objective of this study was to correlate the thickness of the zona pellucida (zona) with egg (oocyte) maturity determined through a widely-used method of assessing oocyte maturation namely, the evidence for cumulus and corona cells expansion. Measurements of the zona of cultured oocytes were recorded at 0, 3, 6, 24, and 48 hr after laparoscopic oocyte retrieval from hormonallystimulated squirrel monkeys. The results indicated that at the time of oocyte retrieval, oocytes that were classified as mature had thicker zonae compared with immature oocytes. The zona layer of the mature oocyte expanded to a maximum after 6 hr of incubation while the zona layer of the immature oocyte became compressed. The diameter of the mature oocyte (minus the zona and perivitelline space) became smaller with time while the immature oocyte diameter remained relatively unchanged. The correlation between the maturational state of the oocyte and the thickness of the zona layer suggest the possible application of zona morphometric evaluations as an indicator of oocyte maturation.     

Development of an in vitro model for the multi-parametric quantification of the cellular interactions between Candida yeasts and phagocytes

We developed a new in vitro model for a multi-parameter characterization of the time course interaction of Candida fungal cells with J774 murine macrophages and human neutrophils, based on the use of combined microscopy, fluorometry, flow cytometry and viability assays. Using fluorochromes specific to phagocytes and yeasts, we could accurately quantify various parameters simultaneously in a single infection experiment: at the individual cell level, we measured the association of phagocytes to fungal cells and phagocyte survival, and monitored in parallel the overall phagocytosis process by measuring the part of ingested fungal cells among the total fungal biomass that changed over time. Candida albicans, C. glabrata, and C. lusitaniae were used as a proof of concept: they exhibited species-specific differences in their association rate with phagocytes. The fungal biomass uptaken by the phagocytes differed significantly according to the Candida species. The measure of the survival of fungal and immune cells during the interaction showed that C. albicans was the more aggressive yeast in vitro, destroying the vast majority of the phagocytes within five hours. All three species of Candida were able to survive and to escape macrophage phagocytosis either by the intraphagocytic yeast-to-hyphae transition (C. albicans) and the fungal cell multiplication until phagocytes burst (C. glabrata, C. lusitaniae), or by the avoidance of phagocytosis (C. lusitaniae). We demonstrated that our model was sensitive enough to quantify small variations of the parameters of the interaction. The method has been conceived to be amenable to the high-throughput screening of mutants in order to unravel the molecular mechanisms involved in the interaction between yeasts and host phagocytes.     

Zona pellucida resistance to sperm penetration before the completion of human oocyte maturation

Human oocytes exposed to capacitated spermatozoa in vitro when at metaphase of the 1st meiotic division (metaphase I) were not penetrated, even though some subsequently progressed to metaphase of the 2nd meiotic division (metaphase II). When the non-penetrated oocytes that had reached metaphase II during the incubation with spermatozoa were freed from the zona pellucida and reinseminated, two or more pronuclei developed in most of them. By contrast, no penetration was observed when the oocytes were reinseminated in the zona-intact state. When compared with metaphase II oocytes, metaphase I oocytes had a similar zona-binding capacity for spermatozoa, but fewer spermatozoa were found within the zona. These results indicate that the zona pellucida of human oocytes undergoes important maturational changes during the transition from metaphase I to metaphase II. Ultrastructural and previous histochemical findings suggest that these changes involve secretions from both the oocyte and cumulus cells and that the increased zona resistance at metaphase I may be due to relative insufficiency of cumulus cell-secreted 'softening' factors. If the integrity of the cumulus oophorus is disturbed at this stage, this condition becomes irreversible.     

Ultrastructural studies of developing goat oocytes in vitro

The structure and distribution of organelles within developing goat oocytes at various stages of incubation were studied. In oocytes with 5 or more layers of cumulus cells, at 0 h of incubation, the zona pellucida had developed although zonation was not evident. Lipid bodies were present but no mitochondria were observed. At 20 h, the zona pellucida had differentiated into thicker and thinner regions. Clusters of membrane-bound electron-transparent bodies were present in the perivitelline space. The mitochondria were fully developed, distributed evenly and usually in close proximity with dilated endoplasmic reticula. Cortical granules were distributed at the periphery. At 40 h of incubation, a number of mitochondria was hooded. In oocytes of 2 to 4 layers of cumulus cells at 0 h, the zona pellucida was penetrated by cumulus cell processes, and the mitochondria were not well developed. However, in 20-h incubated oocytes, fully developed mitochondria, many of which were hooded, could be observed. Clusters of membrane-bound electron-transparent bodies were also observed, while cortical granules were at the periphery. In cumulus-free oocytes, zonation within the zona pellucida was indistinct. Very few vesicles and lipid bodies were observed. At 20 h, mitochondria were sparsely distributed and were not well developed and lacked cristae. At 40 h, the zona pellucida was less compact, and the membrane-bound electron-transparent bodies were less numerous compared with those of the other groups. Endoplasmic reticula were not dilated, and cortical granules were few and had no definite pattern of distribution.     

The effect of a mannose binding protein on macrophage interactions with Candida albicans.

A soluble mannose binding protein (MBP), obtained from rabbit serum, was found to inhibit phagocytosis of Candida albicans by bone marrow derived, cultured murine macrophages. During in vitro incubation of yeast with lymphocyte-free macrophage populations uptake of the yeast was significantly reduced at MBP concentrations of 5 micrograms/ml. A similar reduction in yeast phagocytosis was produced by dextrose, d-fucose, l-fucose, d-mannose and alpha-methyl-d-mannoside but required saccharide concentrations of 25-50 mg/ml. Inhibition of phagocytosis of the yeast also resulted from pretreatment of either the macrophages or the yeasts with MBP followed by washing. As expected, the addition of mannan to the assay medium blocked the inhibitory effect of MBP for uptake of C. albicans. These findings suggest that both cell bound and soluble mannose receptors may be important modulators of macrophage-Candida interactions.     

Quantitative evaluation of macrophage phagocytosing capacity by a fluorometric assay

This paper reviews sensitive and simple quantitative evaluation of macrophage phagocytosing capacity by applying fluoresecin-labeled Sacharomyces cerevisiae cells. Yeast cells were conjugated with fluoresceinisothiocyanate (FITC) and used as fluorescent particles. A time course analysis within this method showed that phagocytosis of yeast cells was temperature dependent and that the number of that ones ingested by macrophages increased rapidly during the initial 60 min of incubation at 37 degrees C. Free fluorescent cells can be effectively removed by aspiration from the well. Furthermore, yeast cells required preopsonization with serum to achieve optimal uptake of the cells. The uptake of nonopsonized yeast cells by macrophages was significantly lower than that of opsonized cells (P < 0.05). We propose that about 50% of mouse macrophages can carry functionally active FcR responsible for phagocytosis.     

Acrosome intact and acrosome-reacted human sperm can initiate binding to the zona pellucida

Mammalian sperm must be acrosome reacted before penetrating the zona pellucida. In some species the sperm undergo the acrosome reaction before binding to the zona pellucida and in other species only acrosome intact sperm can initiate binding to the zona. In this study we addressed the question of acrosomal status and sperm-zona binding with human gametes. Sperm acrosome reactions were induced by treatment with human follicular fluid or N-(6-amino-hexyl)-5-chloro-naphthalene sulfonamide (W-7). The sperm suspensions, containing various percentages of acrosome-reacted sperm, were then incubated with human oocytes for 1 min. The acrosomal status of the sperm population bound to the zona was similar to the acrosomal status of the population of sperm in suspension (R2 = 0.77), regardless of the treatment to induce acrosome reactions. Our interpretation of these results is that both acrosome intact and acrosome-reacted human sperm can initiate binding to the zona pellucida. However, we reported earlier (N. L. Cross, P. Morales, J. W. Overstreet, and F. W. Hanson, 1988, Biol. Reprod. 38, 235-244) that the human zona pellucida is able to induce acrosome reactions. Thus, to exclude the possibility that sperm had undergone the acrosome reaction on the zona within 1 min of binding, sperm were suspended in a nominally calcium-free Tyrode's medium (0 Ca-mTyr) before incubation with oocytes (this medium was supplemented with SrCl2 and spermine to support sperm motility and zona binding). In 0 Ca-mTyr, the proportion of acrosome-reacted sperm on the zona was still highly correlated with the proportion of reacted sperm in suspension, indicating that the sperm were reacted before binding. Evidence that 0 Ca-mTyr effectively inhibited acrosome reactions induced by the zona pellucida was derived from experiments in which sperm were treated with human follicular fluid or control medium and the suspensions were diluted with either 0 Ca-mTyr or control medium.4+ Human oocytes were added for 1 min (pulse) at which time some oocytes were fixed and other oocytes were transferred to sperm-free medium and incubated for 35 min (chase) before fixation. Sperm diluted in control medium, pretreated with either human follicular fluid or control medium, showed a similar increase (40%) in the percentage of acrosome reactions among the zona-bound sperm after the chase. Sperm diluted in 0 Ca-mTyr did not show an increase in the percentage of acrosome-reacted sperm on the zona pellucida after the chase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Capacity of boar spermatozoa to bind zona pellucida proteins in vitro in relation to fertilization rates in vivo

The purpose of this study was to determine variation among boars in the percentage of sperm in an ejaculate that express enhanced binding of zona pellucida proteins during treatment for capacitation in vitro, and to determine whether this relates to fertilizing ability in vivo. Ejaculates (n=35) were collected from 12 boars. A sample of each ejaculate was treated for capacitation in vitro. During incubation, the zona binding ability of spermatozoa was assessed at regular intervals with fluorescein-conjugated solubilized zona pellucida proteins (FITC-sZP) and propidium iodide, using a flow cytometer. After incubation, a percentage of the sperm had enhanced FITC-sZP binding. The percentage of viable sperm with enhanced FITC-sZP binding, expressed as a percentage of the total sperm population, increased rapidly over the first 60 min and thereafter reached a plateau after 120-180 min. Averaged over all ejaculates, the percentage at 180 min was 46% (range 27-61%); this percentage was significantly different among boars. However, the variation between ejaculates within a boar was relatively small. There was no significant boar effect on the rate at which the percentage of viable cells with enhanced FITC-sZP binding reached the maximum. In ejaculates (n=14) from four boars (selected from the group of 12), we investigated the increase in the percentage of viable sperm with enhanced sZP binding during treatment for capacitation in vitro in relation to the ability to fertilize in vivo. Sows (n=44) were inseminated 4 h after ovulation with a suboptimal insemination dose (0.5x10(9) spermatozoa). Time of ovulation was determined using transrectal ultrasonography and sows were killed at 120 h after ovulation. The percentage of fertilized oocytes, embryo development, and numbers of accessory spermatozoa were determined. The percentage of spermatozoa that were viable and showed enhanced sZP binding after 180 min of incubation was 48 +/- 12% (range 28-56%). The percentage of fertilized oocytes was 85 +/- 27% and 64% of the sows had 100% fertilized oocytes. The percentage of sows with 100% fertilized oocytes correlated well (P< or =0.05, R2=0.98) with the percentage of viable spermatozoa with enhanced FITC-sZP binding after capacitation in vitro.     

The transfer of foreign DNA by rabbit spermatozoa into hamster eggs

Capacitated rabbit sperm was incubated with pCMVlacZ plasmid and then used to fertilize hamster oocytes liberated from zona pellucida. After treatment with DNase I, these oocytes were examined for the presence of exogenous DNA by PCR. DNA of pCMVlacZ was not found in oocytes after simple incubation with male gametes. The presence of DNA was recorded in the experiments where the sperms were treated with DMSO and subjected to heat shock prior to fertilization.     

A short sperm-oocyte incubation time ZBA in the dog

The fertilising capacity of a semen sample can be predicted by evaluation of spermatozoa with in vitro tests. The zona pellucida binding assay (ZBA) accounts for several parameters and interprets the interaction between the spermatozoa and the oocyte. The present study was made in two parts. The aim of the first experiment was to evaluate whether the sperm binding capacity of oocytes varies between different oocyte pools. Each zona binding was made with oocytes from different bitches, using pooled frozen-thawed semen from the same two dogs. The sperm-oocyte complexes were incubated for 1h. There was a significant difference between the six replicates in the number of sperm bound to the zona pellucida (ZP), which indicates that the sperm binding capacity of the ZP differs between oocyte pools. The aims of the second experiment were to evaluate the effects of five different treatments of the spermatozoa on the ZBA, and to evaluate two different incubation times of the sperm-oocyte complexes. ZBAs were made with: fresh semen; semen kept chilled for 1 or 2 days prior to the ZBA; and with semen that had been frozen with or without Equex. The oocytes and spermatozoa were incubated for 1 or 4h. For fresh semen and for semen frozen without Equex, incubation for 1h resulted in a higher number of bound spermatozoa per oocyte than incubation for 4h (P<0.0001). When the effect of the different sperm treatments on the number of spermatozoa bound to the ZP was evaluated, it was found that this number was higher for fresh spermatozoa than for chilled or frozen-thawed spermatozoa both after 1 and 4h of co-incubation (P<0.0001). After 1-h incubation of the sperm-oocyte complexes, spermatozoa chilled for 1 day showed better zona binding capacity than spermatozoa chilled for 2 days, and spermatozoa frozen without Equex had a better zona binding capacity than spermatozoa frozen with Equex. Sperm motility and sperm plasma membrane integrity were higher in fresh than in chilled and frozen-thawed semen. The acrosome integrity was high in all groups of treated semen. In conclusion, 1-h incubation of the sperm-oocyte complexes seems to be sufficient for fresh and chilled semen. Further studies are required to establish the optimal incubation time for sperm-oocyte complexes when frozen-thawed semen is evaluated, as a comparison between semen frozen with Equex and semen frozen without Equex gave different results depending on whether the incubation time was 1 or 4h (in the present study), or 6h [Str?m Holst B, Larsson B, Linde-Forsberg C, Rodriguez-Martinez H. Evaluating chilled and frozen-thawed dog spermatozoa using a zona pellucida binding assay.     

Reversible changes in dissolution of the zona pellucida of immature bovine oocytes

The influence of holding immature bovine oocytes in in vitro bovine oviducts on the dissolution of zona pellucida in 0.1% pronase, the role of cumulus cells in this process and the possibility of reversing the process were examined. For the study, 1,045 oocytes were obtained from 2 to 6 mm ovarian follicles. Cumulus-free oocytes were placed in isolated bovine oviducts at 37 degrees C. The average dissolution time of the zona pellucida increased in proportion to the holding time of oocytes in oviducts: 9.9, 13.8, 48.3, 239.3 and 788.3 min after 5, 20, 40, 80 and 120 min in the oviduct, respectively. For the control group, only 4.6 min were required for dissolution of the zona. When cumulus-free and cumulus enclosed oocytes were held for 120 min, no differences were seen in the average lytic time of the zona pellucida of cumulus enclosed oocytes compared with the control group. When cumulus-free oocytes were held in vitro for 120 min and then immersed in follicular fluid from 30 min to 18 h, there was a significant reversal in the sensitivity of the zona pellucida to proteolysis.     

Schistosoma mansoni modulation of phagocytosis in Biomphalaria glabrata

Both short-term (3 hr) exposure of Biomphalaria glabrata snails (M-line and 13-16-R1) to Schistosoma mansoni (PR1) miracidia and in vitro incubation of parasite sporocysts with host hemolymph components altered host phagocytic ability. Hemocytes obtained from susceptible (M-line) snails that had been exposed to parasite miracidia for 3 hr showed reduced levels of phagocytosis of yeast cells in vitro compared to hemocytes from unexposed individuals. Incubation of whole hemolymph with sporocysts in vitro also reduced yeast phagocytosis in this susceptible strain. In contrast, resistant (13-16-R1) hemocytes showed increased levels of yeast phagocytosis after in vitro incubation with the parasite, and the opsonic properties of 13-16-R1 plasma were greater after exposure of snails to miracidia. These strain-specific effects of S. mansoni on host hemocyte phagocytosis and plasma opsonization were seen only when both plasma and hemocytes were present at the time of exposure to the parasite.     

In vivo versus in vitro oviductal glycoprotein (OGP) association with the zona pellucida (ZP) in the hamster and baboon

The goal of this study was to determine if differences exist between in vivo vs. in vitro OGP association with the ZP and to quantitate those differences. Ovarian oocytes were harvested 12.5 or 27 hr post-hCG from hyperstimulated hamsters or baboons, respectively. Hamster and baboon ovarian oocytes were incubated in vitro in media +/- homologous OGP (100 or 200 microg/100 microl) or in some studies with 100 microl oviductal fluid for 3, 6, or 24 hr at 37 degrees C. Some of the baboon ovarian oocytes were transferred immediately after harvesting to the ampulla of both oviducts using a tom cat catheter and retrieved after a 3 hr in situ incubation. Hamster oviductal oocytes were collected 3, 6, and 24 hr following ovulation. After incubation or oocyte retrieval from the oviduct, cumulus cells were removed, oocytes were washed extensively and binding of OGP to the ZP was examined by immunofluorescence. Fluorescence intensity was quantified using densitometric scanning of photographic negatives with the background of each negative as an internal control. In all studies, OGP association with the ZP was significantly greater in vivo than in vitro (P < 0.05). In vitro OGP association with the ZP did not significantly increase with incubation time or OGP concentration; however, a small nonsignificant increase in OGP association with the ZP in the oviduct was detected over time. Differences did not appear to be due to depletion of OGP from the in vitro incubation media, since Western blot analysis of the media showed that OGP was still present. Although OGP concentration in vivo is unknown, Western blots showed similar intensity comparing 100 microg of OGP media and oviductal fluid. Immunolocalization of OGP using laser confocal microscopy showed regional differences in OGP binding. The outer half of the zona pellucida had significantly more OGP bound than the inner half on oviductal oocytes. No regional differences were detected for in vitro incubated oocytes. In conclusion, OGP association with the ZP is greater in vivo vs. in vitro, suggesting that one must be cautious in designing and evaluating in vitro studies of OGP function.     

Ultrastructural observations on gamete interactions using micromanipulated mouse oocytes

Cumulus-free mouse oocytes were subjected to zona opening by cracking with microhooks (ZC) or acid drilling (ZD) and fixed 30–90 min after insemination (10⁵ pre-capacitated motile sperms/ml). Ultrastructural observations were made on serially thin-sectioned oocytes: 15 ZC and 12 ZD. The zona lesion in ZC oocytes was a clean cut, whereas in ZD oocytes it formed a patchy area of partial zona loss, with reduced microvillar height on the underlying oocyte surface. Spermatozoa were observed within the perivitelline space and partially fusing with the oocyte after 30 min in both situations. Only acrosome-reacted sperm heads were observed to fuse: acrosome intact forms were generally in contact with the zona pellucida, either with the inner or outer surface. Acrosome-intact spermatozoa were also observed deeply embedded in the zona matrix, possibly indicating surface enzyme activity preceding the membrane fusion events of the acrosome reaction proper. The observations are consistent with the need for spermatozoa to make contact preferentially with the zona pellucida during the course of the acrosome reaction.     

In vitro incubation of golden (syrian) hamster ovarian oocytes and human sperm with a human oviduct specific glycoprotein

The objective of this study was to determine if human oviduct specific glycoprotein (huOGP) would associate with hamster ovarian oocytes and human sperm during in vitro incubation. The huOGP used in these studies was partially purified from human hydrosalpinx fluid. Hamster ovarian oocytes and human sperm samples were incubated in culture medium with and without huOGP. Association of huOGP was assessed by indirect immunofluorescence assay using a polyclonal antibody prepared against huOGP. Intense fluorescence of the zona pellucida, and bright but uneven fluorescence of the perivitelline space, were observed in hamster ovarian oocytes following incubation in the presence of huOGP. A similar but more uniform pattern of fluorescence was observed when hamster oviductal oocytes (positive controls) were incubated in culture medium alone. Fluorescence was absent when oocytes were assayed with preimmune serum. The association of huOGP with the zona pellucida and perivitelline space appeared to be specific since thyroglobulin, a large molecular weight glycoprotein, and human serum albumin, the major protein in oviduct fluid, did not associate with the hamster oocytes nor inhibit huOGP association when included in the culture medium. Fluorescence was absent when human sperm incubated with huOGP were assayed with antiserum to huOGP. However, human sperm fluoresced when incubated with a uterine glycoprotein, CUPED, which had previously been shown to bind to cat sperm during in vitro incubation. Sperm also fluoresced brightly when human sperm antibody was used as a positive control. Solubilization of sperm membrane proteins postincubation and analysis of these proteins by 1-D SDS-PAGE followed by immunoblotting also failed to show an association of huOGP with human sperm. Electron microscopy of sperm both pre- and postsolubilization confirmed that the sperm membranes were removed by this process. In conclusion, the association of huOGP with hamster oocytes in vitro suggests that huOGP may associate with human oocytes in vivo, whereas that may not be true for human sperm in vivo. The association of huOGP with oocytes may serve to facilitate the process of fertilization and early embryonic development within the oviduct. © 1994 Wiley-Liss, Inc.     

Induction of acrosome reactions by the human zona pellucida

We have used two approaches to test the ability of the human zona pellucida to induce acrosome reactions in human sperm. First, nonviable human oocytes were incubated for 1 min in a suspension of capacitated sperm (of which fewer than 5% were acrosome-reacted) to allow binding of about 200 sperm per oocyte. Some of the oocytes were fixed immediately, and the remainder were fixed after a further 1-h incubation without free-swimming sperm. As determined by light microscopy, sperm on the zona were only 3 +/- 2% (avg. +/- SD) acrosome-reacted at 1 min, and the incidence increased to 46 +/- 15% during the next hour. Electron microscopy confirmed that most sperm on the zona at 1 min were acrosome-intact. A few sperm were in an early stage of the acrosome reaction. Acrosome reactions occurring on the zona during the subsequent hour appeared to be morphologically normal. Second, treatment of sperm in suspension with acid-disaggregated zonae (2 to 4 zonae/microliter) increased the incidence of acrosome-reacted sperm from 3 +/- 1% to 24 +/- 4%. We conclude that the human zona pellucida, or material intimately associated with it, can induce acrosome reactions in human sperm.     

Porcine sperm surface beta1,4galactosyltransferase binds to the zona pellucida but is not necessary or sufficient to mediate sperm-zona pellucida binding.

The binding of sperm to the zona pellucida is an integral part of the mammalian fertilization process, investigated most extensively in the mouse. Several sperm receptors for the murine zona pellucida have been studied (Snell WJ, White JM. 1996. Cell 85:629-637; Wassarman PM. 1999. Cell 96:175-183), but the most compelling evidence exists for beta-1,4-galactosyltransferase (GalTase). Considering that GalTase is present on the surface of porcine sperm (Larson JL, Miller DJ. 1997. Biol Reprod 57:442-453), we investigated the role of GalTase in porcine sperm-zona binding. Sperm surface GalTase catalyzed the addition of uridine diphosphate-[(3)H]galactose to the 55 kDa group of the porcine zona pellucida proteins implicated in sperm binding, demonstrating that GalTase binds the porcine zona. The functional importance of GalTase-zona pellucida binding was tested. Addition of uridine diphosphate galactose, a substrate that completes the GalTase enzymatic reaction and disrupts GalTase mediated adhesion, had no effect on binding of sperm to porcine oocytes. Furthermore, removal of the GalTase zona ligand by incubation of oocytes with N-acetylglucosaminidase had no effect on binding of sperm to oocytes. These results suggest that GalTase is not necessary for sperm to bind to the zona pellucida. Digestion of isolated porcine zona proteins with N-acetylglucosaminidase did not affect the biological activity of soluble porcine zona proteins in competitive sperm-zona binding assays, suggesting that GalTase alone is not sufficient to mediate sperm-zona attachment. From these results, it appears that, although GalTase is able to bind porcine zona proteins, its function in porcine sperm-zona binding is not necessary or sufficient for sperm-zona binding. This supports the contention that porcine sperm-zona binding requires redundant gamete receptors.     

Accelerated modification of the zona pellucida is the primary cause of decreased fertilizability of oocytes in the 129 inbred mouse strain

We investigated whether the small litter size in the 129 inbred mouse strain results from a reduction in oocyte fertilizability. Sensitivity of the zona pellucida to α-chymotrypsin was examined for oocytes collected at 14 h (shortly after ovulation), 17 h, and 20 h after hCG injection. Passage of spermatozoa through the zona pellucida (using an in vitro fertilization (IVF) technique) and the density of cortical granules were examined for oocytes collected at 14 and 17 h after hCG injection. The capability of the oolemma to fuse with the sperm plasma membrane was also evaluated by IVF using zona-free eggs. The zona pellucida became markedly resistant to the enzyme 17 h after hCG injection. IVF rates significantly decreased at this time. In addition, there was a significant reduction in the density of cortical granules. When zona-free oocytes were inseminated, high fertilization rates were obtained at both 17 and 14 h after hCG injection. These results indicate that accelerated modification of the zona pellucida primarily causes a decreased fertilizability of oocytes in 129 mice, resulting in the low reproductive performance of this strain.     

Preliminary results of hemizona assay (HZA) as a fertility test for canine spermatozoa

The Hemizona assay (HZA) is considered to be an effective test for predicting the fertilizing potential of spermatozoa. It is a functional test that distinguishes the zona-binding capacity of spermatozoa from fertile and infertile males. The objective of this study was to validate the HZA for canine spermatozoa, as a test for diagnosing canine male fertility status. Various parameters that affect binding capacity were examined: the presence of an adequate number of capacitated and motile spermatozoa for an HZA, the influence of fertility status, sperm-binding variability within fertile dogs over 60 d, variability in sperm-binding capacity of different oocytes, the lower limit number of spermatozoa binding to a zona from the fertile control, and evaluation of HZI to determine the fertilizing capacity of spermatozoa. Hemizonae were obtained from frozen oocytes of spayed bitches. The oocytes were manually cut into nearly equal halves. Spermatozoa were capacitated by swim-up and 1 h incubation at 37 degrees C in modified Ham's F10 medium. Spermatozoa and hemizonae were co-incubated in 100-microL drops at 37 degrees C for 1 h. Spermatozoa from 7 fertile and 3 infertile dogs were used for this study. The optimal sperm concentration for hemizona insemination was 1 x 10(6)/mL capacitated and motile spermatozoa. A significant difference (P < 0.001) was found the number of tightly zonabound spermatozoa between fertile and infertile dogs. Although there was a small difference in zona binding capacity between ejaculates of the same fertile dog (44 +/- 18.24), the main cause for the difference mentioned above was that of poor zona pellucida-binding capacity of spermatozoa from infertile dogs. We found a maximum of 14.28% bad oocytes when we compared sperm samples from 3 fertile and 3 infertile dogs in 56 HZA replicates. To avoid the effect of bad zona on sperm binding we calculated 37 (95% CI) bound spermatozoa from infertile dogs in 56 replicates. Thus, an HZA experiment in which a control dog had < 37 zona bound spermatozoa was repeated. Based on a minimum of 37 bound spermatozoa for fertile males (controls), a differential zona-binding capacity and hemizona index (HZI) between fertile and infertile dogs and between 2 fertile dogs was determined. The binding differential between fertile and infertile dogs was 64.92 +/- 24.29, while between 2 fertile dogs it was 22.38 +/- 10.02 (P < 0.001). According to the HZI values, a value equal to or less than 41.11 indicated an infertile test dog, while an HZI value equal to or greater than 57.95 indicated a fertile test dog. Any value between these two could indicate either fertility or infertility. The evaluation of fertilizing potential of spermatozoa can be improved using the HZA protocols described above.     

Mechanisms of host defense against Candida species. I. Phagocytosis by monocytes and monocyte-derived macrophages

We studied the biochemical basis of phagocytosis of Candida albicans, a serious pathogen, and Candida parapsilosis, which is rarely pathogenic, by human monocytes (Mo) and monocyte-derived macrophages (MDM). Optimal phagocytosis of both species by Mo required the presence of extracellular Ca2+ and opsonization through both the classic and alternative complement pathways. Serum-opsonized Candida were ingested equally by Mo and MDM; unopsonized Candida were phagocytosed only by macrophages, and uptake began slowly. This opsonin-independent phagocytosis required Ca2+ and could be blocked by yeast mannan or mannose-BSA conjugate, suggesting a role for the mannose receptor. Opsonized Candida elicited a vigorous increase in the concentration of [Ca2+]i in Mo and MDM, but no Ca2+ transient was detected in MDM stimulated with unopsonized Candida. Pretreatment of MDM with ionomycin to increase [Ca2+]i had no effect on phagocytosis of unopsonized Candida. Addition of 5 mM EGTA completely inhibited changes in [Ca2+]i in Mo and MDM, suggesting that the Ca2+ transient induced by opsonized Candida is due to an influx of extracellular Ca2+. Differences in pathogenicity between the two Candida species could not be explained by differences in any aspect of phagocytosis. Uptake mediated by the macrophage mannose receptor could play a role in clearance of Candida under opsonin-poor conditions.