Biomedical subjects

B Colenbrander

Publications and source records attributed to B Colenbrander.

At least 73 records · Page 4Linked to original sources

PubMed1998

Immunohistochemical localization and mRNA expression of activin, inhibin, follistatin, and activin receptor in bovine cumulus-oocyte complexes during in vitro maturation.

The aim of this study was to investigate whether bovine cumulus-oocyte complexes (COCs) synthesize activin A, inhibin, and follistatin and whether they contain activin receptor during in vitro maturation. Therefore, COCs obtained from small and medium-sized follicles were cultured in M-199 supplemented with 10% fetal calf serum (FCS) and gonadotropins for 24 hr. At 0, 6, 12, and 24 hr after the onset of culture, COCs were removed for immunohistochemical staining to detect the expression of activin A, inhibin, follistatin, and activin receptor type II proteins. At 0 and 24 hr, COCs were removed and prepared for reverse-transcriptase polymerase chain reaction (RT-PCR) to assess the presence of mRNA of these proteins. It appeared that cumulus cells and oocytes express activin, follistatin, and activin receptor proteins as well as their mRNA. While expression of inhibin mRNA was found exclusively in cumulus cells, the inhibin protein was present in cumulus cells and oocytes. Immunohistochemical study both in cumulus cells and in oocytes often showed a moderate and strong staining intensity for activin and follistatin, respectively. Activin staining underwent little or no change during culture except at 24 hr of maturation, where about 60% of the oocytes showed no staining. Follistatin immunoreactivity remained strong in the majority of COCs. At the onset of culture, a spotlike inhibin staining was observed in the oocyte, which increased after 12 hr and was absent at the end of culture. Activin receptor immunoreactivity in cumulus cell membranes and oolemma increased during oocyte maturation to maximum values at the end of culture in most of the COCs. It is concluded that the consistent presence of activin and the increase in activin receptor in cumulus cells and oocytes during in vitro maturation indicate a paracrine and/or autocrine action for activin on bovine oocyte maturation. This action may be modulated by inhibin and/or follistatin.

Activin Receptors↗

PubMed1998

The promotory effect of growth hormone on the developmental competence of in vitro matured bovine oocytes is due to improved cytoplasmic maturation.

In a previous study we have shown that the addition of growth hormone (GH) during in vitro maturation accelerates nuclear maturation, induces cumulus expansion, and promotes subsequent cleavage and embryonic development. The aim of this study was to investigate whether the promotory effect of GH on subsequent cleavage and blastocyst formation is due to an improved fertilization and whether this effect is caused by an improved cytoplasmic maturation of the oocyte. Therefore, bovine cumulus oocyte complexes (COCs) were cultured for 22 hours in M199 supplemented with 100 ng/ml bovine GH (NIH-GH-B18). Subsequently the COCs were fertilized in vitro. Cultures without GH served as controls. To verify whether the promoted fertilization is caused by the effect of GH on cumulus expansion or oocyte maturation, cumulus cells were removed from the oocytes after in vitro maturation (IVM) and denuded MII oocytes were selected and fertilized in vitro. Both IVM and in vitro fertilization (IVF) were performed at 39 degrees C in a humidified atmosphere with 5% CO2 in air. At 18 hours after the onset of fertilization, the nuclear stage of the oocytes was assessed using 4,6-diamino-2-phenylindole (DAPI) staining. Oocytes with either an metaphase I (MI) or MII nuclear stage and without penetrated sperm head were considered unfertilized; oocytes with two pronuclei, zygotes, and cleaved embryos were considered normally fertilized; and oocytes with more than two pronuclei were considered polyspermic. To evaluate cytoplasmic maturation, the distribution of cortical granules 22 hours after the onset of IVM, and sperm aster formation 8 hours after the onset of fertilization were assessed. In addition, to assess the sperm-binding capacity, COCs were fertilized in vitro, and 1 hour after the onset of fertilization the number of spermatozoa bound to the oocytes was counted. The addition of GH during IVM significantly (P < 0.001) enhanced the proportion of normal fertilized oocytes. Removal of the cumulus cells prior to fertilization and selection of the MII oocytes did not eliminate the positive effect of GH on fertilization. No effect of GH on the sperm-binding capacity of the oocyte was observed. In addition, GH supplementation during IVM significantly (P < 0.001) enhanced the migration of cortical granules and sperm aster formation. It can be concluded that the promotory effect of GH on the developmental competence of the oocyte is due to a higher fertilization rate as a consequence of an improved cytoplasmic maturation.

Animals↗

PubMed1998

Glycolipids as potential binding sites for HIV: topology in the sperm plasma membrane in relation to the regulation of membrane fusion.

Although human sperm cells can bind human immunodeficiency virus (HIV-1), they lack CD4, galactoceramides (GalCer) and sulfogalactoceramides (SGalCer) as gp120 receptors. However, sperm specific glycolipids (sulfogalactosylalkylacylglycerol (SGalAAG) and galactosylalkylacylglycerol (GalAAG)) are structurally closely related to SGalCer and GalCer as predicted by computer simulated molecular modelling. SGalAAG and GalAAG are exclusively localized in the outer leaflet of the human sperm plasma membrane, and therefore we tested whether they could serve as alternative receptors for the gp120. Purified SGalAAG and GalAAG had similar affinities to recombinant gp120 as the hydroxy fatty acid (HFA) SGalCer and HFA-GalCer respectively. However, nonhydroxy fatty acid forms of (S)GalCer, galactosyldiacylglycerol and the deacylated (sulfo)galactosyllipids did not recognize recombinant gp120. Data obtained by surface pressure experiments revealed that the lipid monolayers that contained HFA-GalCer or GalAAG resulted in a similar significant penetration of recombinant gp120 in the monolayer. The penetration was a factor of two lower in monolayers with HFA-SGalCer or SGalAAG. The binding of recombinant gp120 to human sperm cells colocalized with GalAAG and could be blocked with monoclonal antibodies against galactolipids. The possible relevance of gp120 binding to glycolipids for HIV entry in sperm cells is discussed.

Cell Membrane↗

PubMed1998

Progesterone in mare follicular fluid induces the acrosome reaction in stallion spermatozoa and enhances in vitro binding to the zona pellucida.

The aim of this study was to investigate whether mare follicular fluid (FF) induces the acrosome reaction (AR) in stallion spermatozoa and, if so, to identify the component in FF responsible for it. Furthermore, the effect of this component on sperm-zona binding and the subsequent AR was studied. Pooled FF, aspirated from the preovulatory follicles of mares in oestrous, was used and aliquots of the fluid were treated with charcoal to remove steroids (CFF). Charcoal treatment reduced the progesterone concentration in FF from 153 to < 2 ng/mL. Spermatozoa from fertile stallions collected by a swim-up procedure were preincubated in modified Tyrode's medium for 5 h and then incubated for 30 min at 37 degrees C with either (1) 50% FF + 50% CFF, (2) 50% FF + 50% CFF + 150 ng/mL progesterone, (3) 50% CFF + 150 ng/mL progesterone, (4)150 ng/mL progesterone or (5) modified Tyrode's medium alone. The sperm-hemizona assay was applied: (a) to compare the number of spermatozoa bound to a hemizona in the presence and absence of 1.5, 15 or 150 ng/mL progesterone after 1 h co-incubation of spermatozoa and hemizonae, (b) to compare the incidence of the AR in sperm-hemizona complexes incubated for 1 h in the presence and absence of 1 microgram/mL progesterone. Both spermatozoa in suspension and bound to a hemizona were treated with the supravital dye Ethidium homodimer and fixed. Their plasma membranes were permeabilized, and the outer acrosomal membranes were labelled with FITC-PNA. Viable spermatozoa without the outer acrosomal membrane were considered as physiologically acrosome-reacted. Results showed that (1) FF induced a higher percentage of AR than did CFF or modified Tyrode's medium, (2) addition of 150 ng/mL progesterone to CFF restored 77% of the AR-inducing activity and (3) CFF and modified Tyrode's medium both induced the AR to a similar extent when supplemented with 150 ng/mL progesterone. Neither FF nor progesterone treatment affected sperm viability severely. The number of spermatozoa bound to a hemizona in the presence of 15 and 150 ng/mL progesterone was significantly higher (p < 0.05) than the number of spermatozoa bound in the absence of progesterone. A higher incidence of the AR was found in sperm-hemizona complexes incubated in the presence of progesterone (55.6 +/- 3.4% vs. 27.1 +/- 4.3%, in the presence and absence of progesterone, respectively) (n = 15, p < 0.05). It is concluded that mare FF can induce the AR in stallion spermatozoa. Progesterone is the physiological component responsible for this AR-inducing capacity. Progesterone enhances sperm-zona binding activity and exerts an additive effect on the zona-induced AR.

Acrosome↗

PubMed1998

Fertility of Shetland pony stallions used in different breeding systems: a retrospective study.

In horses reproductive performance is usually expressed as the foaling rate. This rate ranges from 40% to 80%. Three major factors contribute to this variation namely, the stallion, the mare and management. In this study, the performance of Shetland ponies kept in three different breeding systems was investigated retrospectively. In one breeding system, the stud farmer travelled with his stallion (n = 9) to the mare (system 1) while in another system, the stallion (n = 3) stayed at the stud farm and the mares came to the stallion (system 2). The last system was pasture breeding (system 3; n = 9). Each stallion participated in only one system. The average number of cycles per mare used for breeding did not differ significantly between systems 1 and 2. However, the number of matings per cycle was higher in system 2 than in system 1. The average number of mares serviced per stallion was 91, 50, and 17 for systems 1, 2 and 3, respectively. Mares mated in pasture had a 2.8-fold higher chance (p < 0.05) of having a foal the next season than the mares mated under systems 1 and 2. The foaling rate per season was 58%, 48%, and 80% for systems 1, 2 and 3, respectively. Management aspects play an important role in the relatively low foaling percentages of systems 1 and 2.

Animal Husbandry↗

PubMed1998

Progesterone-induced acrosome reaction in stallion spermatozoa is mediated by a plasma membrane progesterone receptor.

The aim of the present study was to investigate whether the induction of stallion sperm acrosome reaction (AR) by progesterone is mediated by binding of progesterone to a receptor on the sperm plasma membrane or to an intracellular progesterone receptor. Progesterone-BSA conjugate labeled with fluorescein isothiocyanate (P-BSA-FITC) in combination with a vital stain, ethidium homodimer, was applied to visualize the presence of the progesterone receptor on living spermatozoa. Alternatively, an indirect immunofluorescence technique employing a monoclonal antibody (C-262) against human intracellular progesterone receptor was conducted to validate the presence of the progesterone receptor. Immunogold labeling techniques enabled ultrastructural localization of P-BSA-FITC or C-262 with transmission electron microscopy. The dynamic changes in labeling patterns were monitored for sperm cells, using fluorescence microscopy and flow cytometry during a 5-h capacitation period. An increasing number of viable cells showed affinity for P-BSA-FITC or C-262 at the acrosomal plasma membrane region of the sperm head, while a decreasing number of viable cells were not labeled. In contrast, almost all deteriorated cells were labeled in the cytosol of the postequatorial region of the sperm head. Incubation with P-BSA-FITC resulted in the induction of AR but to a lesser extent than that for sperm incubated with free progesterone. Therefore, coupling of progesterone to its receptor on the sperm plasma membrane appears to be an important step in the induction of the AR.

Acrosome Reaction↗

PubMed1998

Use of lectins to characterize plasma membrane preparations from boar spermatozoa: a novel technique for monitoring membrane purity and quantity.

The object of this study was to develop a method to quantify the amount of outer acrosomal membrane material in isolated plasma membranes from boar sperm cells. The cells were fractionated by nitrogen cavitation, and plasma membranes were isolated by subsequent differential centrifugation steps. Marker enzyme measurement showed that the plasma membrane isolates were enriched in plasma membrane markers and did not contain nuclei, inner acrosomal membranes, or mitochondria. Since there is no marker enzyme known for the outer acrosomal membrane, lectins were used for the detection of this membrane. The membrane specificity of a number of lectin conjugates was tested with fluorescence microscopy and transmission electron microscopy. Membrane binding of these lectin conjugates was quantified with flow-cytometry and an enzyme-linked lectin binding assay. Wheat germ agglutinin was specific for the plasma membrane while peanut agglutinin was specific for the outer acrosomal membrane. The use of these lectins made it possible for the first time to discriminate between these two membranes. The isolated plasma membrane fraction was enriched more than 10-fold (17-fold after further purification by a sucrose gradient) in plasma membrane material compared to outer acrosomal membrane material. Highly purified sperm plasma membranes should prove to be useful for research on primary sperm-zona interactions.

Acrosome↗

PubMed1997

Relationship between sperm-zona pellucida binding assays and the 56-day nonreturn rate of cattle inseminated with frozen-thawed bull semen.

Assays based on sperm-zona pellucida binding have been developed as diagnostic tests to predict the fertilizing potential of mammalian spermatozoa. Recently, we reported on the development of a sperm-zona pellucida binding assay (SZBA) for bull spermatozoa. The aim of the present study was to develop a hemi-zona assay (HZA) for bull spermatozoa and to investigate the relationship between SZBA and HZA outcomes and in vivo fertility. Frozenthawed semen samples from 8 fertile Swedish Red and White bulls (one ejaculate per bull) designated as the test semen samples and a single ejaculate from a fertile Holstein-Friesian bull designated as the control semen sample were used in this study. In the SZBA, 2 groups of 20 oocytes per semen test sample and in the HZA a minimum of 6 matching pairs of hemizonae were used for comparison of sperm binding with control semen. Sperm binding to matching hemi-zonae of individual semen samples was equal, and clearly demonstrated the feasibility of the HZA for cattle. A significant correlation was found between the SZBA and the HZA indices obtained from the different semen test samples (r = 0.42, P < 0.001; n = 67). There was no significant relation between the SZBA indices and the 56-d nonreturn rate of the test samples. However, the HZA indices of the semen test samples and the 56-d nonreturn rate were significantly correlated (r = 0.46, P < 0.0001; n = 67). It is concluded that HZA can be regarded as a potential assay for predicting the fertilizing ability of bovine semen samples. However, further studies using more semen samples are necessary to confirm this view.

Journal Article↗

PubMed1997

Epidemiologic aspects of Taylorella equigenitalis.

Contagious equine metritis (CEM) is a sexually transmissible disease in mares. Although the disease is commonly diagnosed by culturing the causative bacterium Taylorella equigenitalis (T. equigenitalis) . false negative results do occur. A recently developed Polymerase Chain Reaction (PCR) assay, however, appeared to be much more sensitive, with initial results indicating an unexpected high incidence of the agent in selected horses. In this study, samples from 107 randomly selected mares with no clinical signs of CEM submitted for conventional culture were all negative for T. equigenitalis . but in the PCR-assay 54 (49%) were positive for Taylorella -DNA. Positives in the PCR-assay were found in all breeds tested, even in horses imported from the isolated population in Iceland. These findings suggest that T. equigenitalis was present long before it was first isolated in 1977, The high incidence of Taylorella in horse populations without apparent clinical signs of CEM, the occurrence of incidental clinical case and the known variability between strains, all indicate that Taylorella is endemic in the horse population. In order to explore whether the organism is present in species other than the horse, we also used the PCR-assay on clinically health donkeys (n = 14), zebras (n = 15), Przewalski horses (n = 2) and cows (n = 21). All the animals showed negative results except one of the Przewalski horses, and one cow that was repeatedly found to give positive reaction. We also found that the fertility of 7 stallions with cultures positive for Taylorella (6 used in an AI-program and 1 by natural breeding) was not affected, as shown by the normal range of foaling rates in mares inseminated or bred by these stallions. The overall results may be interpreted to mean that Taylorella is of limited significance in horse breeding.

Journal Article↗

PubMed1997

[Equine arteritis virus: clinical symptoms and prevention].

Sero-epidemiological surveys have revealed that equine arteritis virus (EAV) is prevalent in most European countries. The virus causes sporadic cases of respiratory disease and abortion in horses, the incidence of which has increased in recent years. Mares and geldings eliminate virus after acute infection, but 30% to 60% of stallions become persistently infected. In these animals, EAV is maintained within the reproductive tract and is shed continuously in the semen. Persistent infection with EAV in stallions has no negative consequences for fertility but mares inseminated with virus-contaminated semen can have an acute infection. These mares shed large amounts of virus in respiratory secretions and urine, leading to lateral spread of the virus to other susceptible horses. Acute infection at later stages of gestation can lead to abortion. Effective control of the spread of EAV infection depends on the identification of virus-shedding stallions. Persistently infected stallions should not be used for breeding or should be bred only to seropositive mares. Mares bred to shedding stallions should be isolated from other animals for a period of 3 weeks following insemination to prevent the lateral spread of EAV.

Abortion, Veterinary↗

PubMed1997

Stimulatory effect of growth hormone on in vitro maturation of bovine oocytes is exerted through cumulus cells and not mediated by IGF-I.

A previous study reported that the addition of bovine growth hormone (bGH) during in vitro maturation of bovine oocytes accelerates nuclear maturation and stimulates subsequent embryonic development. The aim of this study was to investigate whether bovine cumulus oocyte complexes (COCs) contain growth hormone receptor (GHR), and whether the stimulatory effect of GH on oocyte maturation is cumulus-dependent and mediated by insulin-like-growth factor (IGF-I). The expression of growth hormone receptor mRNA in mural granulosa cells, in cumulus cells, and in the oocyte was studied using reverse transcriptase polymerase chain reaction (RT-PCR). To investigate the importance of cumulus cells for GH-promoted maturation, COCs and denuded oocytes were cultured for 16 hr in M199 with or without bGH s(NIH-GH-B18). To investigate whether GH action is mediated by IGF-I, COCs were cultured in 1) 100 ng/ml bGH, 2) 100 ng/ml bGH plus anti-IGF-I, 1:100 dilution, 3) 100 ng/ml h-IGF-I, 4) 100 ng/ml h-IGF-I plus anti-IGF-I, 1:100 dilution, and 5) anti-IGF-I, 1:100 dilution. Culture was performed at 39 degrees C in a humidified atmosphere with 5% CO2 in air, and the nuclear stage of oocytes was assessed using 4,6-diamino-2-phenyl-indole (DAPI) staining. PCR on cDNA of mural granulosa cells, cumulus cells, and oocytes revealed that mRNA for GHR was present in all cell types. Addition of GH (100 ng/ml) to the culture medium of denuded oocytes did not affect the number of metaphase II oocytes after 16 hr, while a significant (P < 0.001) increase was observed, when COCs were cultured in the presence of GH. Addition of the antibody against IGF-I to the culture medium completely suppressed the stimulatory effect of IGF-I on oocyte maturation and cumulus expansion, while stimulation by GH was not affected by the antibody. It is concluded that bovine cumulus cells, mural granulosa, and oocytes express mRNA for the GH receptor. The stimulatory effect of GH on bovine oocyte maturation is dependent on the cumulus cells and is not mediated by IGF-I.

Animals↗

PubMed1997

Influence of thawing method on motility, plasma membrane integrity and morphology of frozen-thawed stallion spermatozoa.

Different thawing methods are used for stallion semen, however, it is unclear which method is the optimal one. To determine if the thawing temperature has an effect on semen quality, we compared 2 thawing temperatures, 75 degrees C and 37 degrees C. The following parameters were used to measure sperm quality: sperm motility, sperm viability, plasma membrane integrity and sperm morphology. Twenty-three ejaculates from 10 Dutch Warmblood stallions were thawed either at 37 degrees C for 30 sec or at 75 degrees C for 7 sec. Sperm motility was evaluated by a Hamilton Thorn Motility Analyser. Plasma membrane integrity and sperm viability were evaluated by using a live/dead fluorescein stain containing a calcein AM probe and ethidium homodimer-1 probe. The eosinaniline blue staining method was used to evaluate the percentage of live and dead cells, as well as sperm morphology. There was no significant difference (P = 0.84) between sperm motility after thawing at 37 degrees C and 75 degrees C. There was also no significant difference (P = 0.053) between the percentage of live spermatozoa using the calcein AM/ethidium homodimer stain after thawing at 37 degrees C and 75 degrees C. There was, however, a significant difference (P = 0.032) between the percentage of live spermatozoa using the eosin-aniline blue stain after thawing at 37 degrees C compared with that at 75 degrees C. In conclusion, our laboratory results indicated that stud farms using frozen semen should thaw the straws at 37 degrees C instead of 75 degrees C. The lower temperature is easier to work with, as thawing at the higher temperature requires special equipment and has to be timed very carefully to avoid damage to the spermatozoa.

Journal Article↗

PubMed1997

Acrosome-intact boar spermatozoa initiate binding to the homologous zona pellucida in vitro.

The acrosome reaction is a prerequisite for zona pellucida penetration by mammalian spermatozoa. 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 the present investigation, we addressed the question whether acrosome-intact or acrosome-reacted boar sperm initiate binding to the pig zona pellucida by studying the acrosomal status of sperm bound to zonae pellucidae. Our approach was to vary the percentage of acrosome-intact sperm in suspension by long preincubation before incubation with hemizonae for 1 min. We hypothesized that if only acrosome-intact sperm are able to initiate binding to the zona pellucida, the majority of the sperm on the zona surface would be acrosome-intact regardless of the percentage of acrosome-reacted sperm in suspension. Fluorescein isothiocyanate-conjugated peanut agglutinin (Arachis hypogaea; FITC-PNA) in combination with optical sectioning by confocal laser-scanning microscopy was used to study the acrosomal status of sperm bound to the hemizona. Electron microscope studies showed that the FITC-PNA binding site is mainly limited to the outer acrosomal membrane of boar sperm, thus validating the use of FITC-PNA as an accurate probe for studying boar sperm acrosome reaction. Over 90% of the sperm bound to a hemizona were acrosome-intact irrespective of whether the majority of sperm in the suspension were acrosome-intact, acrosome-reacting, or acrosome-reacted. There was a significant difference (Kruskal-Wallis test, p < 0.05) in the mean +/- SEM number of sperm bound to the outer side, inner side, and edge of a hemizona (48 +/- 8, 14 +/- 3, and 7 +/- 2; n = 58; respectively). The acrosomal status of sperm bound to the various surfaces of hemizonae was similar. Taking the respective zona pellucida surface area into consideration, it was calculated that an average of 1.9 +/- 0.3, 1.0 +/- 0.2, and 1.5 +/- 0.3 spermatozoa were bound per 1000 microm2 of outer side, inner side, and edge of a hemizona, respectively (mean +/- SEM, n = 38). These observations indicate that acrosome-intact boar spermatozoa initiate binding to the pig zona pellucida. A gradient of sperm binding sites also exists, decreasing from the outside to the inside of the zona pellucida.

Acrosome↗

PubMed1997

Stimulatory effect of growth hormone on in vitro maturation of bovine oocytes is exerted through the cyclic adenosine 3',5'-monophosphate signaling pathway.

The aim of this study was to investigate whether the stimulatory effect of growth hormone (GH) on the in vitro maturation and cumulus expansion of bovine oocytes is exerted through the cAMP or the tyrosine kinase pathway. Therefore bovine cumulus-oocyte complexes (COCs) were cultured in Medium 199 without fetal calf serum and gonadotropins, but supplemented with 100 ng/ml bovine GH (bGH; NIH-GH-B18) with or without 10 microM methyl 2,5-dihydroxycinnamate (erbstatin analogue), a specific tyrosine kinase inhibitor; 100 microM 2',3'-dideoxyadenosine (DDA), a specific adenylate cyclase inhibitor; or 10 microM H-89, a specific inhibitor of cAMP-dependent protein kinase A. Epidermal growth factor (EGF; 20 ng/ml) was added as a positive control for tyrosine kinase activation, and FSH (0.05 IU/ml) was added as a positive control for cAMP mediation during in vitro maturation in the absence or presence of the inhibitors. Culture was performed at 39 degrees C in a humidified atmosphere with 5% CO2 in air. To assess the effect on nuclear maturation, the proportion of oocytes in metaphase II stage after 16 h of culture was determined using 4,6-diamino-2-phenylindole staining. To determine the effect on cumulus expansion, the diameter of COCs at the onset and after 24 h of culture was measured. The stimulatory effects of GH on oocyte maturation and cumulus expansion were blocked by DDA and H-89 (p < 0.01). Similarly, FSH-induced cumulus expansion was abolished by DDA and H-89 (p < 0.05), while DDA did not block either EGF-induced oocyte maturation or cumulus expansion. Erbstatin analogue significantly blocked the stimulation of oocyte maturation and cumulus expansion by EGF (p < 0.02) but did not inhibit GH action on the COCs. It is concluded that the stimulatory effect of GH on oocyte maturation and cumulus expansion is mediated by the cAMP signal transduction pathway and not by JAK2 phosphorylation.

Adenylyl Cyclase Inhibitors↗

PubMed1997

Induction of acrosome reaction in dog sperm by calcium ionophore.

The sensitivity of the plasma membrane to calcium ionophore (A23187) challenge was studied in dog sperm using fluorescein lectin staining for the assessment of acrosomal status and viability. Second fraction ejaculates from 5 dogs were washed, resuspended in Ca(2+)-free (EDTA-treated), 50, 100, 500, 1000 and 2000 microM/l Ca(2+)-containing Sp-TALP medium and induced with 50, 250, 500, 1000, 2500 and 5000 nM/l calcium ionophore. Samples were collected from each aliquot after 30 and 60 min of induction to assess the percentage of acrosome reacted sperm cells (AR rate), viability and motility by fluorescein isothiocyanate conjugated peanut agglutinin (FITC-PNA) and ethidium-homodimer combined staining. On each slide, 200 sperm cells were assessed under epifluorescence microscope (x 1250) in a blind manner. The response to ionophore challenge (AR rate, viability, motility) varied with Ca2+ and ionophore concentration in the suspension. A significantly higher AR rate was detected in samples containing 100, 500, 1000 and 2000 microM/L Ca2+ (> 40%) than in that containing 50 microM/L. Acrosome reaction could not be successfully induced in the EDTA-treated sample and in any of the aliquots in which 50, 250 and 500 nM/L ionophore concentrations were used for induction. Motility decreased drastically in all of the treated samples and stopped in that sample where as significant AR rate could be detected. Viability remained high (> 75%) during the incubation and did not differ significantly in the treated and the control groups.

Acrosome↗

PubMed1996

In vitro maturation of bovine oocytes in the presence of bovine activin A does not affect the number of embryos.

This study was carried out to investigate whether bovine recombinant activin A present during the in vitro maturation (IVM) of cumulus enclosed bovine oocytes affects the proportion of embryos that develop to the blastocyst stage. In addition, the effect of the presence of activin A during maturation and during embryo culture was studied. Therefore, bovine cumulus oocyte complexes were matured at 39 degrees C in a humidified atmosphere with 5% CO2 in air for 24 h in: (1) culture medium M199 supplemented with 10% foetal calf serum (FCS), luteinising hormone (LH) and follicle-stimulating hormone (FSH) and 10 ng ml-1 activin A; (2) M199 without FCS but supplemented with LH and FSH and 10 ng ml-1 activin A; (3) M199 without FCS, LH and FSH but supplemented with 10 ng ml-1 activin A. Cultures without activin served as controls. After IVF the embryos were cultured in M199 supplemented with 10% FCS on a monolayer of buffalo rat liver (BRL) cells. For the second part of the study, COCs were matured in vitro in M199 supplemented with LH and FSH and 10 ng ml-1 activin A, fertilized in vitro and the embryos were cultured (1) on a monolayer of BRL cells in M199 supplemented with 10% FCS and 10 ng ml-1 of activin A, and (2) in droplets of serum free BRL-conditioned medium supplemented with 10 ng ml-1 activin A. IVM in the presence of LH, FSH and 10 ng ml-1 activin A did not change the proportion of blastocysts present at Day 9 or the proportion of hatched blastocyst at Day 11. Activin present during maturation in the absence of serum and gonadotrophic hormones also did not alter the proportion of blastocysts or hatched blastocysts. In vitro culture of embryos on BRL cells or in BRL-conditioned medium in the presence of activin had no effect on embryonic development. It is concluded that IVM in the presence of bovine activin A has no effect on subsequent embryonic development.

Activins↗

PubMed1996

In vitro maturation of bovine oocytes in the presence of growth hormone accelerates nuclear maturation and promotes subsequent embryonic development.

Regulatory effect of GH on follicular growth and development in the cow is well documented. The aim of this study was to investigate the role of GH on in vitro bovine oocyte maturation. Therefore bovine cumulus oocyte complexes (COCs) were cultured in M199 without FCS and gonadotropins and in the presence of 10, 100, or 1,000 ng/ml bovine GH (NIH-GH-B18). The COCs were incubated at 39 degrees C in a humidified atmosphere with 5% CO2 in air and nuclear stage was assessed after 2, 4, 8, 16, 22, and 24 hr of incubation using DAPI staining. To assess the effect of GH on developmental capacity of the oocytes, COCs were incubated in the presence of GH for 22 hr, followed by IVF and in vitro embryo culture. Cultures without GH served as controls. For subsequent development, the embryos were cultured in M199 supplemented with 10% FCS on a monolayer of BRL cells. Embryos were scored morphologically and the efficiency of the culture system was evaluated as (1) the percentage of cleaved embryos 4 days after IVF, (2) the percentage of blastocysts on day 9 expressed on the basis of the number of oocytes at the onset of culture, and (3) the percentage of hatched blastocysts on day 11 expressed on the basis of the total number of blastocysts present at day 9. GH (100 and 1,000 ng/ml) significantly accelerated nuclear maturation (P < 0.001). At 4 and 8 h the percentage of oocytes in GV stage after GH treatment (54% and 19%) was significantly lower than the control (64% and 41%). Similarly at 16 and 22 h the percentage of oocytes in MII stage was significantly higher in the GH-treated group; (58% and 77%) and (46% and 62%) for GH and control respectively. The number of oocytes in MII beyond 22 hr of culture did not differ; 100 and 1,000 ng/ml GH induced significant cumulus expansion (P < 0.05), which was not observed in the absence of GH. Addition of 100 and 1,000 ng/ml GH during maturation significantly (P < 0.01) enhanced subsequent cleavage rate from (64% and 67%) in control to (75% and 81%) in GH-treated group; embryonic development in terms of day 9 blastocyst formation was also significantly increased in the presence of GH (29% and 34%) compared to the control (18% and 24%). The hatchability of the blastocysts was not influenced by GH. From the present data, it can be concluded that GH present during IVM has a beneficial effect on subsequent development.

Animals↗

PubMed1996

Equine arteritis virus: a review of clinical features and management aspects.

Abortion, Veterinary↗