Free Oocytes Research Articles

Detection of histamine receptors on mouse oocytes and their involvement in fertilization potential.

Histamine widely involves in local immune responses, physiological function in the gut, and acting as a neurotransmitter in the brain. Scientist also found the importance of histamine in the reproductive systems. The present study aimed to determine the existence of histamine receptor subtypes; H1R, H2R, H3R, and H4R on mouse oocytes through immunofluorescence (IF) staining and reverse transcription- polymerase chain reaction (RT-PCR). These further confirmed by the involvement of histamine receptor antagonists in in vitro fertilization (IVF). In IF staining, mouse oocytes were incubated with primary antibody against histamine receptor, followed by incubation with fluorescence conjugated secondary antibody. Then RT-PCR analysis was carried out for the undetected receptors during IF for confirmation. The RT-PCR used RNA extracted from mice COCs and cumulus free oocytes. In IVF, sperm was cultured in a group of treated histamine receptor antagonists oocytes. This investigation revealed the existance of H1R, H2R, and H3R on mouse oocytes in IF and RT-PCR analyses. The treatment of IVF with histamine receptor antagonists (H1R: pyrilamine; H2R: cimetidine; H3R: thioperamide) led to a significant reduction quantity of 2-cell embryos (4.61 ± 2.44%; 5.83 ± 4.65%; 3.83 ± 1.82%, respectively) as compared with the control group (22.50 ± 6.44%). Therefore, according to the results of this study, the presence of H1R, H2R, and H3R on mouse oocytes possibly will suggest the involvement of histamine in fertilization.

Toll-like Receptor 2 is Involved in Calcium Influx and Acrosome Reaction to Facilitate Sperm Penetration to Oocytes During in vitro Fertilization in Cattle.

Cumulus cells of ovulated cumulus-oocyte complexes (COCs) express Toll-like receptor 2 (TLR2), pathogen recognition receptors, to recognize and react to sperm signals during fertilization. Sperm also express TLR2, but its contribution to the sperm-oocytes crosstalk is still unclear. Here, we adapted the in vitro fertilization (IVF) model to characterize the potential relevance of sperm TLR2 in sperm-oocytes interactions during fertilization in bovine. The IVF results showed that the ligation of sperm TLR2 with its specific antagonist/agonist resulted in down/up-regulation of the cleavage and blastocyst rates either in COCs or cumulus-free oocytes, but not in zona pellucida (ZP)-free oocytes. The computer-assisted sperm analysis (CASA) system revealed that sperm motility parameters were not affected in TLR2 antagonist/agonist-treated sperm. However, fluorescence imaging of sperm-ZP interactions revealed that the blockage or activation of the TLR2 system in sperm reduced or enhanced both binding and penetration abilities of sperm to ZP compared to control, respectively. Flow cytometrical analysis of acrosome reaction (AR) demonstrated that the TLR2 system adjusted the occurrence of AR in ZP-attached sperm, suggesting that sperm TLR2 plays physiological impacts on the sperm-oocyte crosstalk via regulating ZP-triggered AR in sperm. Given that calcium (Ca2+) influx is a pre-requisite step for the induction of AR, we investigated the impact of the TLR2 system on the ionophore A23187-induced Ca2+ influx into sperm. Notably, the exposure of sperm to TLR2 antagonist/agonist reduced/increased the intracellular Ca2+ level in sperm. Together, these findings shed new light that the TLR2 system is involved in sperm AR induction which enables sperm to penetrate and fertilize oocytes during the fertilization, at least in vitro, in cows. This suggests that sperm possibly developed a quite flexible sensing mechanism simultaneously against pathogens as well as COCs toward fertilization with the same TLR2 of the innate immune system.

Lipofection-Mediated Introduction of CRISPR/Cas9 System into Porcine Oocytes and Embryos.

Simple SummaryLiposome-mediated gene transfer has become an alternative method for establishing a gene targeting framework, and the production of mutant animals may be feasible even in laboratories without specialized equipment. However, whether blastocyst genome editing can be performed by treatment with lipofection reagent, guide RNA, and Cas9, without performing electroporation or microinjection, remains unclear. In this study, we demonstrated that lipofection treatment successfully induced mutation into zygotes during in vitro fertilization and in embryos at the 2- and 4-cell stages. Although liposome-mediated gene editing is a feasible system for use with zona-pellucida-free oocytes/embryos, several challenges must be overcome.Liposome-mediated gene transfer has become an alternative method for establishing a gene targeting framework, and the production of mutant animals may be feasible even in laboratories without specialized equipment. However, how this system functions in mammalian oocytes and embryos remains unclear. The present study was conducted to clarify whether blastocyst genome editing can be performed by treatment with lipofection reagent, guide RNA, and Cas9 for 5 h without using electroporation or microinjection. A mosaic mutation was observed in blastocysts derived from zona pellucida (ZP)-free oocytes following lipofection treatment, regardless of the target genes. When lipofection treatment was performed after in vitro fertilization (IVF), no significant differences in the mutation rates or mutation efficiency were found between blastocysts derived from embryos treated at 24 and 29 h from the start of IVF. Only blastocysts from embryos exposed to lipofection treatment at 29 h after IVF contained biallelic mutant. Furthermore, there were no significant differences in the mutation rates or mutation efficiency between blastocysts derived from embryos at the 2- and 4-cell stages. This suggests that lipofection-mediated gene editing can be performed in ZP-free oocytes and ZP-free embryos; however, other factors affecting the system efficiency should be further investigated.

Brooding in the deep-sea sea anemone Actinostola crassicornis (Hertwig, 1882) (Cnidaria: Anthozoa: Actiniaria) from the southwestern Atlantic Ocean

Fil: Lauretta, Daniel Marcelo. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales Bernardino Rivadavia; Argentina

196 ELEVATED TEMPERATURE DURING BOVINE OOCYTE MATURATION ALTERS MITOCHONDRIAL ACTIVITY

Exposure of maturing cumulus-oocyte complexes to a physiologically relevant heat stress altered the transcriptome in oocytes, especially certain transcripts important for mitochondrial function. To determine if perturbations are coincident with changes in mitochondrial activity, generation of reactive oxygen species (ROS), glutathione and ATP content were examined in maturing oocytes experiencing heat stress. In the first study, ROS content was assessed after 6 h at 38.5, 41, or 42°C using 37 µM 6-carboxy-2′7′-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) or 60 µM dihydrofluorescein diacetate for staining cytoplasmic and mitochondrial ROS, respectively. In a second study, cumulus-oocyte complexes were matured for 0, 12 or 24 h at 38.5°C (control) or 41°C for 12 h (heat stress; 38.5°C thereafter). Total glutathione (GSSG + GSH) and reduced glutathione (GSH) and ATP levels were assessed in cumulus denuded-zona free oocytes as per manufacturer (GSH-Glo Glutathione assay, Promega, Madison, WI, USA; 1 to 25 pmol standards) and ATP determination kit (Life Technologies, Carlsbad, CA, USA; 0.078 to 10 pmol standards). Next, ATP content was assessed in control and heat-stressed oocytes matured for 24 h and in embryos resulting after IVF of control or heat-stressed oocytes (i.e. 2-, 4-, and 8-to-16 cell and blastocyst-stage embryos). Data were analysed as a randomised block design with fixed effects of maturation temperature (and hours of maturation where appropriate), blocking on replicate, using PROC MIXED (SAS 9.2, SAS Institute, Cary, NC, USA). Culture at 41°C for the first 6 h in vitro-matured (IVM) reduced levels of cytoplasmic ROS compared to non-stressed controls (1.02 v. 0.84 fluorescent ratio for 38.5 v. 41°C, SEM = 0.16, P < 0.0001), whereas levels after 42°C were similar to controls (0.94). Mitochondrial ROS were higher after 41°C (1.42) than after 38.5 (0.98) or 42°C (0.98, SEM = 0.14, P < 0.0001). Heat stress exposure increased total glutathione content at 12 h (4.4 v. 5.2 pmol for 38.5 v. 41°C) but levels were decreased by 24 h (6.6 v. 5.9 pmol for 38.5 v. 41°C; SEM = 1.26, P = 0.0002). A similar pattern was found for GSH (P = 0.0001); GSSG was similar across treatments (P = 0.50). Levels of ATP increased during maturation (1.10, 1.20, and 1.65 pmol for 38.5°C at 0, 12, and 24 h) and were increased by heat stress at 24 h (1.65 and 2.01 pmol for 38.5 and 41°C; SEM = 0.31, P = 0.001). In embryos, ATP content was higher in 8-to-16 cell embryos derived from heat-stressed oocytes than in those from control oocytes (P = 0.03); levels were similar in blastocyst-stage embryos (P = 0.21) regardless of origin. In conclusion, application of a physiologically relevant heat stress during maturation altered mitochondrial activity in bovine oocytes. Carryover of effects to the early embryo may explain some of the reductions in embryo development experienced by heat-stressed oocytes. This research was supported in part by USDA National Institute of Food and Agriculture, Hatch Project No. 227701, the state of Tennessee through University of Tennessee AgResearch, Department of Animal Science, and East Tennessee Research and Education Center.

Blastomeres aggregation as an efficient alternative for trophoblast culture from porcine parthenogenetic embryos

Zona pellucida free (ZPF) oocytes were cultured after electrical activation to allow blastomeres aggregation and compared to ZP intact (ZPI) oocytes. In feeder-dependent conditions, the trophoblast attachment and primary outgrowths were significantly higher in ZPF than in ZPI groups. In feeder-free conditions, trophoblast attachment and typical morphological trophoblast primary outgrowths were observed in ZPF group. The primary colonies derived from the ZPF embryos in both culture conditions were able to establish secondary and tertiary colonies and showed mRNA expression of CDX2, TEAD4 and KRT8 as trophoblast markers, while outgrowths from the ZPI embryos could not grow beyond primary colonies.

Developmental potential of zona pellucida–free oocytes obtained following mild in vitro fertilization

To determine the developmental potential of oocytes in which the zona pellucida was damaged and subsequently removed, producing "zona-free" (ZF) oocytes that were cultured until the blastocyst stage. ZF eggs from cycles with more than one oocytes retrieved (n = 97) were compared with zona-intact (ZI) oocytes originating from the same patient. Private infertility clinic. Infertile patients (n = 135) undergoing minimal ovarian stimulation or natural-cycle in vitro fertilization treatment during 2010-2012. ZF oocytes undergoing intracytoplasmic sperm injection (ICSI) fertilization, blastocyst culture, elective vitrification, and subsequent single vitrified-thawed blastocyst transfer (SVBT). Rate of fertilization, cleavage, and blastocyst development. Live birth rate and neonatal outcome in subsequent SVBT cycles. There were no significant differences in fertilization (77% vs. 77%), cleavage (75% vs. 75%), or blastocyst development rates (39% vs. 32%) between the internally controlled ZF and ZI groups, respectively. Survival after thawing (90% vs. 100%) and live birth rates (37% vs. 36%) per thawed blastocyst were also similar. Newborns originating from all ZF and ZI oocytes had a similar gestational age at delivery (38.3 ± 3.7 wk vs. 39.5 ± 1.5 wk) and birth weight (3,115 ± 946 g vs. 3,010 ± 441 g). Our retrospective comparative study suggests that ZF eggs could be as successfully fertilized and cultured until the blastocyst stage as ZI control eggs without adversely affecting subsequent pregnancy rates and basic neonatal outcome.

Observations on the ecology and reproductive biology of the sipunculan wormAspidosiphon muelleriin temperate waters

A population of the sipunculan wormAspidosiphon muelleri, located in temperate waters of the western Mediterranean Sea, was monitored monthly for a year. Some aspects related to its ecology and reproductive biology are shown in the present work. The sex-ratio for this population was close to 1:1 (54% females vs 46% males), thus indicating a dioecious reproduction, although showing a lack of sexual dimorphism. Oocytes were detected in females from 4–5 mm; this size might be reached byA. muelleriin a few months. The population density increased notably during the summer (June–August). The spawning event date was indirectly estimated from the average size of the oocytes and the percentage of females with free oocytes in the coelom. Both variables were significantly correlated to shallow water temperature (Pearson correlation;P = 0.003 andP = 0.001, respectively). Oocyte size was also significantly correlated to average irradiance level (Pearson correlation;P = 0.044). Spawning took place between August and September (when the water temperature is getting close to its annual maximum) and may last only a few weeks in these temperate waters. The abundance ofA. muelleridecreased drastically in September, likely as a consequence of the spawning event effort. Bivalves of the speciesEpilepton clarkiaewere collected together with specimens ofA. muelleriliving inside the polychaete tubes used as shelters by the sipunculans, with a prevalence of 11.64%. To a lesser extent some Foraminifera were also reported attached to the body of the sipunculans.

Calreticulin from suboolemmal vesicles affects membrane regulation of polyspermy

This study was designed to determine whether calreticulin (CRT), a chaperone protein, is present in in vitro-matured (IVM) pig oocytes and to study its potential role in the block to polyspermy. Western blot analysis, using an anti-CRT antibody, of oocyte lysate showed an immunoreactive band of ∼60 kDa. Simultaneous labeling of IVM oocytes with anti-CRT antibody and peanut agglutinin lectin (PNA lectin, a porcine cortical granules (CG)-specific binding lectin) revealed localization of CRT in the subplasmalemmal region with a 27.7% colocalization with PNA staining. After IVF, PNA labeling was not observed and anti-CRT labeling decreased significantly in zygotes and disappeared in two-cell embryos. Western blot analysis of oocyte exudate obtained from zona pellucida (ZP)-free oocytes activated with calcium ionophore confirmed the presence of a band that reacted with an anti-CRT antibody. Anti-CRT antibody and PNA labeling were not observed in activated oocytes despite being detectable in non-activated oocytes. The presence of CRT in vesicles located under the oolemma was demonstrated using immunogold cytochemistry at the ultrastructural level. To study the role of CRT in fertilization, ZP-enclosed and ZP-free oocytes were incubated with exogenous CRT and then inseminated. Whereas ZP-free oocytes showed fewer penetrating sperm and lower polyspermy rates than untreated oocytes, the opposite effect was observed in ZP-enclosed oocytes. In conclusion, CRT is confined to subplasmalemmal vesicles partially overlapping with CG contents. Its exocytosis after the oocyte activation seems to participate in the membrane block to polyspermy in pigs but is not involved in the ZP block.

Effect of multiple vs. single preovulatory follicle on oocyte quantity and quality andin vitromaturation of goat oocytes

The present study was conducted to investigate the effects of multiple vs. single preovulatory follicle on oocyte quantity and quality and in vitro maturation of goat oocytes. Slaughtered goat ovaries were collected and ranked into 2 types according to the number of preovulatory follicles on their surface: Type I (with multiple preovulatroy follicles, ≥ 2 follicles), Type II (with single preovulatory follicle). The number of goat oocytes retrieved from follicles (2–6 mm O) was recorded for each ovarian type. The released immature goat oocytes were scored for cumulus - oocyte cell adhesion into one of 3 grades (C+; cumulus - enclosed oocytes, C+/-; cumulus partially enclosed oocytes, C−; cumulus - free oocytes). Grade C+ and grade C+/ were matured in tissue culture media (TCM -199) supplemented with 10% Fetal Calf Serum for 30 h in CO2 incubator at 38.5oC and 95% humidity. The results indicated that a greater number of aspirated oocytes were found in type I than type II. The number of Grade C+ and Grade C+/- oocytes in Type I was significantly higher (P < 0.05) than type II. The in vitro maturation rate of oocytes recovered from goat ovaries was non significantly different in both types but the number of cumulus - full expanded oocytes (CE+) appeared to be higher in ovaries having multiple than single preovulatory follicle. In conclusion, higher quantity, quality and maturation rate of oocytes may produce in multiple preovulatory follicles of goat ovaries.

Endoplasmic reticulum protein 29 (ERp29), a protein related to sperm maturation is involved in sperm-oocyte fusion in mouse

BackgroundSperm-oocyte fusion is a critical step in fertilization, which requires a series of proteins from both spermatozoa and oocyte to mediate membrane adhesion and subsequent fusion. A rat spermatozoa membrane protein is endoplasmic reticulum protein 29 (ERp29), which significantly increases on the sperm surface as well as in the cytoplasm of epididymal epithelia from caput to cauda as the sperm undergo epididymal maturation. Moreover, ERp29 facilitates viral infection via mediating membrane penetration. We determined if in addition to promoting sperm maturation ERp29 may also play a role in facilitating gamete fusion during the fertilization process.MethodsLaser scanning confocal microscopy (LSCM) and Western blot analysis were employed to probe for ERp29 protein in BALB/c mouse epididymal and acrosome-reacted spermatozoa. We prepared rabbit polyclonal antibodies against mouse recombinant ERp29 (rERp29) to characterize: 1) fertilization rate (FR); 2) fertilization index (FI); 3) sperm motility and 4) acrosome reaction (AR).ResultsConfocal microscopy indicated that ERp29 was partially localized at the sperm head of the epididymal caput as well as over the whole head and part of the principal piece of the tail region from the epididymal cauda. However, when the acrosome reacted, ERp29 remained in the equatorial and post-acrosomal regions of the sperm head, which is the initial site of sperm-oocyte membrane fusion. Such localization changes were confirmed based on the results of Western blot analysis. Furthermore, the antibodies against mouse rERp29 inhibited the spermatozoa from penetrating into the zona pellucida (ZP)-free oocytes. The functional blocking antibodies reduced both mouse sperm-oocyte FR and FI at concentrations of 100 and 200 micro g/ml compared with pre-immunized rabbit IgG or with anti-mouse recombinant bactericidal/permeability-increasing protein (BPI, a sperm surface protein unrelated to sperm-oocyte fusion) antibodies (100 micro g/ml), but they had no effect on sperm motility and AR.ConclusionThis study demonstrates that ERp29 on mouse spermatozoa membrane changes during epididymal transit and AR. Accordingly, in mice this protein may be one of the important factors involved in sperm fertilization by facilitating sperm-oocyte membrane fusion.

ORIGINAL ARTICLE: Investigation of Recombinant Mouse Sperm Protein Izumo as a Potential Immunocontraceptive Antigen

ProblemTo determine if the recombinant mouse Izumo (mIzumo) could be used as a potential immunocontraceptive antigen.Method of studyThe recombinant mIzumo fused with 6His tag (6His‐mIzumo) was purified by immobilized Ni2+ affinity chromatography. Enzyme‐linked immunosorbent assay and Western blot were used to detect anti‐6His‐mIzumo activities of serum from the mice immunized with 6His‐mIzumo. Inhibition of the anti‐6His‐mIzumo antibody on mouse sperm–egg fusion in vitro was performed using the zona free oocytes and acrosome reacted sperm. Fertility of the 6His‐mIzumo immunized male and female mice was compared with control mice.ResultsThe recombinant mIzumo was successfully produced. Female and male mice inoculated with 6His‐mIzumo developed a specific serum antibody and the highest antibody titer lasted at least 6 weeks. The serum anti‐6His‐mIzumo antibody almost completely blocked mouse sperm–egg fusion in vitro. However, there was no significant reduction in fertility for both male and female mice immunized with 6His‐mIzumo compared with control mice.ConclusionThe circulated anti‐mIzumo antibody can block mouse sperm–egg fusion in vitro but has no effect on fertility in vivo. It seems that application of Izumo as a candidate antigen in development of contraceptive vaccine needs further investigation.

227. Eggs with a surprise: the 'sperm-specific' protein SPRASA is also expressed in the oocyte

SPRASA is a newly identified protein which in silico analysis suggests is not expressed in other tissues. Antibodies reactive with SPRASA have been identified in some infertile men and an antiserum reactive with recombinant SPRASA prevented human sperm binding to hamster oocytes in vitro, indicating an important role in sperm/oocyte recognition. The aim of this study was to investigate the spatial and temporal expression of SPRASA in reproductive and other tissues. Brain, thymus, heart, spleen, kidney, liver and the reproductive organs from duplicate female and male Balb/C mice were collected at several postnatal timepoints. RNA was extracted, reversed transcribed and analysed by quantitative real time PCR for SPRASA expression. Abattoir-derived, in vitro matured, bovine oocytes were examined for SPRASA expression by fluorescent immunochemistry. To examine SPRASA binding sites on oocytes, matured bovine oocytes were exposed to biotinylated recombinant human SPRASA or biotinylated α-lactalbumin (control), then visualised by confocal microscopy using DTAF-conjugated streptavidin. We found SPRASA mRNA was expressed in the reproductive organs of both females and male mice from postnatal day 10. Fluorescent immunochemistry indicated SPRASA was expressed on the oolemmal membrane and in the few cumulus cells remaining attached to zona-intact oocytes. Control preimmune serum did not stain the oocytes or cumulus cells. Recombinant human SPRASA bound to the oolemmal membrane of both zona intact and zona free bovine oocytes. To date the expression of SPRASA has only been reported in the testes/sperm with an additional single EST identified in brain. Our quantitative real-time PCR analysis demonstrated SPRASA is also expressed in the female reproductive organs. This was confirmed by our immunoassays which show oocytes and possibly cumulus cells express SRPASA while the oolemmal membrane has the ability to bind (sperm-derived) SPRASA. That SPRASA expression is restricted sperm and oocytes confirms the likely function of this protein in reproduction.

Oral antioxidants counteract the negative effects of female aging on oocyte quantity and quality in the mouse.

This study aims to compare the effect of early and late onset administration of oral antioxidants on number and quality of oocytes retrieved from aged mice after exogenous ovarian stimulation. Control hybrid females were fed a standard diet supplemented or not supplemented with pharmacological doses of vitamins C and E either from the first day of weaning or from the age of 32 weeks until they were autopsied at the age 40-42, 50-52, or 57-62 weeks after exogenous ovarian stimulation. Analysis of chromosomal distribution, DNA organization and cellular morphology was performed in ovulated cumulus-enclosed and -free oocytes, ovarian non-germinal vesicle oocytes enclosed by or free of mucous cumulus cells and in vitro-matured ovarian germinal-vesicle oocytes. Both early and late onset administration of oral antioxidants counteracted the negative effects of female aging on number of ovarian oocytes and total percentage of oocytes retrieved from oviducts and ovaries exhibiting a normal distribution of chromosomes in the metaphase-II plate and/or morphological traits of apoptosis. Although both early and late onset administration of oral antioxidants can counteract the negative effects of female aging on number and quality of oocytes, transference of these results to human beings should be made with caution because of the potential side effects of high doses of vitamins on reproductive function as well as many other undesirable systemic disorders.

Chromosomal abnormalities in the first and second polar body

Aneuploidy free oocytes may be pre-selected by testing the first and second polar bodies removed from oocytes following their maturation and fertilization. We present here our experience on the application of the method in IVF cycles from patients of advanced maternal age. Overall, 5590 oocytes were obtained from 917 cycles and tested by polar body sampling and fluorescent in situ hybridization (FISH) analysis using specific probes for chromosomes 13, 16, 18, 21 and 22. FISH results were available in 4599 (82.2%) of 5590 oocytes studied, from which 2077(45.2%) were with aneuploidies. Thirty six point one percent of aneuploidies were of the first meiotic origin, and 29.3% of the second meiotic origin. Most errors in the first meiotic division were represented by chromatid errors. The transfer of embryos deriving from 2014 of 2520 aneuploidy free oocytes in 821 treatment cycles resulted in 182 (22.2%) clinical pregnancies and 140 healthy children born after confirmation of the polar body diagnosis. Polar body testing of oocytes provides an approach for pre-selection of aneuploidy free embryos, improving pregnancy rate in IVF patents of advanced maternal age.

Sexual reproduction of &lt;i&gt;Nausithoe aurea&lt;/i&gt; (Scyphozoa, Coronatae). Gametogenesis, egg release, embryonic development, and gastrulation

SUMMARY: The structure of the ovaries and testes of Nausithoe aurea, reared in the laboratory, is described to update the knowledge of coronate scyphomedusae gametogenesis and early development. The testis is similar to those of other scyphozoans. The organization of the ovary agrees with the description for other coronates, with free oocytes in the mesoglea. The oocytes develop in a limited region of the gastrodermis, and a maturation gradient is observed from this point on. Egg release, embryonic development, and gastrulation mode of Nausithoe aurea are also described. Egg production was continuous for 55 days, and the output of released eggs oscillated without observed cue. Cleavage was holoblastic and adequal, but after the 8-cell stage, the cleavage became pseudospiral. Gastrulation occurred through multipolar ingression and began 24 hours after fertilization.

Replication of cytopathic and noncytopathic bovine viral diarrhea virus in zona-free and zona-intact in vitro-produced bovine embryos and the effect on embryo quality.

The aim of the present study was to determine whether or not cytopathic (CP) and noncytopathic (NCP) bovine viral diarrhea virus (BVDV) are able to replicate within in vitro-produced embryos and to investigate whether inoculation of embryos with BVDV affects their normal development. Zona pellucida (ZP)-free oocytes, zygotes, 8-cell-stage embryos, morulae, and hatched blastocysts (HB) were incubated for 1 h in 1 ml of Minimal Essential Medium containing 10(6.00) tissue culture infectious dose (TCID)50/ml NCP BVDV isolate 22,146 or 10(6.25) TCID50/ml CP BVDV strain Oregon C24V. At 0, 12, 24, 36, 48, 60, and 72 h postinoculation (hpi), groups of embryos were collected for virus titration. A small amount of newly produced virus was detected in 8-cell embryos at 60 hpi (10(1.8) TCID50/100 cells), but only for CP BVDV. For ZP-free morulae and HB, maximal intracellular virus titers were, respectively, 10(1.47) and 10(2.33) TCID50/100 cells at 48 hpi for the CP biotype and 10(0.64) and 10(0.84) TCID50/100 cells at 72 hpi for the NCP biotype. Only an infection with CP BVDV had a significant inhibitory effect on further development of ZP-free morulae. It can be concluded that ZP-free in vitro-produced embryos are permissive to an infection with BVDV, with increasing susceptibility of the embryos in accordance with their developmental stage. In contrast to observations in ZP-free in vitro-produced embryos, no virus replication or signs of embryonic degeneration were detected in ZP-intact in vitro-derived embryos.

Lithium stimulates the first meiotic division in mouse oocytes.

Cumulus enclosed oocytes, cumulus enclosed oocytes denuded of their cumulus and cumulus free oocytes from 21 day old unstimulated mice were cultured for 18 hours in control medium supplemented with lithium chloride, dbcAMP and forskolin at various concentrations. In control medium 66% of the cumulus enclosed oocytes, 93% of the denuded oocytes, and 94% of the cumulus free oocytes resumed meiosis (germinal vesicle breakdown), whereas the levels of polar body formation were 27%, 12% and 39%, respectively. In the presence of lithium significantly more cumulus enclosed oocytes and cumulus free oocytes resumed meiosis and formed a polar body, whereas lithium had no effect on the denuded oocytes. Forskolin and dbcAMP stimulated resumption of meiosis and cumulus expansion in the cumulus enclosed oocytes and inhibited resumption of meiosis in the cumulus free oocytes. Lithium more or less eliminated this inhibitory effect of both forskolin and dbcAMP in the cumulus free oocytes. The results indicate (i) that activation of the cAMP second messenger path in the cumulus cells induces them to synthesize a meiosis inducing substance(s) which stimulates the oocyte to resume meiosis, and (ii) that other second messenger systems than the cAMP pathway, e.g. the phosphatidylinositol cycle, are involved in resumption of meiosis and polar body formation. We conclude that lithium enhances the capability of mouse oocytes for resumption of meiosis and polar body formation.

A mechanical and enzymatic approach to recovery of ovarian follicular oocytes in rabbits.

In order to investigate the morphology of oocytes which were recovered from ovarian follicles at several developmental stages and to examine the in vitro maturity of recovered oocytes, rabbit ovarian oocytes were recovered by mincing the ovary with a pair of razors and/or cutting isolated follicles. Twenty four point six oocytes per ovary were recovered by mincing the ovary with a pair of razors, and 19.9% of them were surrounded by a thick layer of cumulus cells. The mass of ovarian tissue remnant was digested by a collagenase solution to isolate ovarian follicles at several developmental stages. A great number of ovarian follicles (304.9±114.0 per ovary) could be collected by enzymatic isolation, however, it is difficult to isolate follicles over 700μm in diameter. The oocytes from antral follicles which were isolated enzymatically ( ?? 300 Am in diame-ter) were obtained by cutting with a pair of razors, but oocytes could not be recovered from preantral follicles by the same procedure. Some oocytes obtained by cutting follicles were cultured for 20 hr in TCM 199 supplemented with 15% FBS for in vitro matu-ration. Oocytes with a thick layer of cumulus cells were obtained only from follicles over 700 Am in diameter. They emitted the first polar body in vitro at a higher rate as compared with the other types of oocytes, which were surrounded by a thin or partial cumulus cell layer or had no cumulus cells at all. All types of oocytes resumed meiosis in vitro, although high percentages of the oocytes from the smaller follicles and/or in the cumulus free oocytes degenerated. These results suggest that a combination of mincing ovaries and cutting follicles which were isolated enzymatically or mechanically is useful for recovering a great number of oocytes. However, in vitro maturity of those oocytes depends on the follicular size and/or cumulus cells' adhesion to the oocytes.

The presence of zonae pellucidae influences fusion rates between spermatozoa and zona free hamster oocytes.

Human zonae pellucidae were added to suspensions of capacitating spermatozoa. The zonae were prepared from oocytes that remained unfertilized in our in-vitro fertilization (IVF) programme. After capacitation, zona free hamster oocytes were added and the penetration rate of the hamster oocytes was determined. Controls without the addition of zonae were run in parallel. A distinct enhancement of the penetration rate was found when zonae were present during capacitation, using sperm of fertile donors. Sperm of several infertile men did not respond to the presence of zonae with higher penetration rates. It was concluded that the zona pellucida is involved in the induction of the acrosome-reaction of human spermatozoa. In addition the incubation system with zonae as described might be useful for the identification of infertile semen.