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Biomedical subjects

G Brem

Publications and source records attributed to G Brem.

At least 91 records · Page 5Linked to original sources
PubMed1998

Laparoscopic recovery of pronuclear-stage goat embryos.

The oviducts of 16 Saanen does, superovulated with follicle-stimulating hormone (FSH) and synchronised with prostaglandin F2 alpha were flushed 75 to 86 hours after the injection of prostaglandin. The mean (sd) ovulation rate was 13.7 (3.9). The flushings were directed orthograde through a flexible intravenous catheter, which was introduced into the oviduct via the infundibulum. The flushing medium was recovered by a balloon-catheter, which was placed in the uterine lumen near the uterotubal junction. Five does were flushed unilaterally either because they had one blocked oviduct or because they had ovulated on only one ovary. The overall embryo recovery rate was 72 per cent. Nine weeks later 11 of the donor ewes were examined laparoscopically and no adhesions of the reproductive organs were observed. Eight of these does were synchronised with progestagen-vaginal sponges, superovulated with FSH and their oviducts were flushed again. Their mean ovulation rate was 16.0 (4.3) and 86 per cent of the embryos were recovered. The optimal time to obtain pronuclear stage embryos was 75 to 78 hours after the injection of prostaglandin. All the embryos recovered within this period were at the pronuclear stage whereas 28 per cent of those recovered one to six hours later were at the two- or four-cell stage.

Animals↗
PubMed1998

Secretion of cumulus expansion-enabling factor (CEEF) in porcine follicles.

The objective of this study was to find out whether porcine cumulus and mural granulosa cells can secrete cumulus expansion-enabling factor (CEEF). Culture drops of M-199 medium were conditioned with denuded porcine oocytes (1 oocyte/microliter), cumulus cells from oocytectomized complexes (1 OOX/microliter), pieces of mural granulosa isolated from preantral to preovulatory follicles (1000 cells/microliter), or oviductal cells (1000 cells/microliter) for 24 hr. The production of CEEF was assessed by the addition of mouse OOX and follicle-stimulating hormone (FSH) (1 microgram/ml) to microdrops of the conditioned medium. After 16-18 hr, expansion of the mouse OOX was scored on a scale of 0 to 4 by morphologic criteria. Mouse OOX did not expand in nonconditioned FSH-supplemented medium. Immature porcine oocytes produced +3 to +4 expansion of the mouse OOX. Granulosa cells isolated from preantral and early antral follicles and cumulus cells isolated from all states of follicle development constitutively secreted CEEF under in vitro conditions. Mural granulosa cells of small, medium, and preovulatory (PMSG) follicles also secreted CEEF in vitro; however, FSH or leutenizing hormone (LH) stimulation was essential for this secretion. Hormonally induced secretion of CEEF was accompanied by expansion of the mural granulosa itself. Granulosa cells isolated from follicles of gilts 20 hr after PMSG and human chorionic gonadotropin (hCG) administration did not produce CEEF and did not expand in response to FSH and LH in vitro. CEEF activity also was found in the follicular fluid of small antral follicles, was reduced in medium follicles, and was not detectable in PMSG-stimulated follicles. However, CEEF activity was reestablished in the follicular fluid of preovulatory follicles by hCG injection, conceivably due to increased production of CEEF by cumulus cells. We conclude that (1) porcine cumulus and mural granulosa cells are capable of CEEF production in vitro and (2) autocrine secretion of CEEF by cumulus cells is involved in regulation of porcine cumulus expansion both in vitro and in vivo.

Animals↗
PubMed1998

A detailed analysis of pronucleus development in bovine zygotes in vitro: cell-cycle chronology and ultrastructure.

The aim of the present experiment was to analyze the chronology of pronucleus development and DNA synthesis, as well as the ultrastructure of intranuclear bodies, in bovine zygotes produced in vitro. Bovine oocytes were matured and fertilized in vitro, and sperm penetration and pronucleus development were examined. DNA synthesis was investigated by sequential incubation with [3H]- and [14C]thymidine followed by autoradiography on semithin sections. Ultrathin sections for transmission electron microscopy were prepared from the same zygotes. Sperm penetration was noted for the first time at 4 hr after in vitro insemination and reached a maximum at 6 hr. Pronucleus formation was initiated at 4 hr, and up to at least 11 hr the maternal pronucleus was more developed than its paternal counterpart. DNA synthesis was initiated at 14-15 hr, and the S-phase lasted for 8-10 hr. The most prominent ultrastructural entities of the pronuclei were the nucleolus precursor bodies (NPBs). During the S- and G2-phases, the NPBs spatially associated with clusters of interchromatin-like granules. The two components were firmly attached to each other by an electron-dense reticulum. During the late G2-phase, the NPBs were apparently detached from the interchromatin-like granules and the electron-dense reticulum again. The interaction between the intranuclear bodies and granules appears to be comparable with the situation previously described for in vivo-produced bovine zygotes (J Laurincík et al., Mol Reprod Dev 43:62-69, 1996), except for the lack of vacuolization of the NPBs during the S-phase in vitro.

Animals↗
PubMed1998

Effect of cryoprotectants and their concentration on post-thaw survival and development of expanded mouse blastocysts frozen by a simple rapid-freezing procedure.

Experiments were conducted to develop a simple rapid-freezing protocol for expanded mouse blastocyst-stage embryos. The effect of type of cryoprotectant (ethylene glycol and propylene glycol) and its concentrations (4.5, 6.0 and 7.0 mol/L each with 0.5 mol/L sucrose) on morphological survival and development in vitro were studied. The survival and development of embryos frozen with best concentration of each cryoprotectant pre-exposed to either a low concentration (1.5 mol/L with 0.25 mol/L sucrose) of the respective cryoprotectant or ascending concentrations of sucrose were also compared. The in vivo development of embryos frozen with best protocol (pre-exposure to 1.5 mol followed by 7.0 mol ethylene glycol) was compared with nonfrozen embryos. The rate of re-expansion and hatching was influenced by the type and concentration of the cryoprotectant. A significantly higher re-expansion and hatching rate was achieved at 7.0 mol of both cryoprotectants compared with 4.5 and 6.0 mol of the respective cryoprotectants. When comparing 2 cryoprotectants, a higher (P < 0.05) rate of hatching was obtained with ethylene glycol at 7.0 mol compared with a similar concentration of propylene glycol. The highest re-expansion (91%) and hatching (86%) of expanded blastocysts was achieved with pre-exposure of embryos to a low concentration of ethylene glycol followed by freezing in the same cryoprotectant at 7.0 mol. The transfer of embryos frozen using this protocol resulted in the development of live fetuses. The proportion of live fetuses in the pregnant recipients with frozen-thawed embryos were not different from those transferred nonfrozen embryos (49 vs 57%). It may be concluded that simple rapid-freezing with dehydration in ascending sucrose concentrations or pre-equilibration in a low concentration of ethylene glycol or propylene glycol followed by exposure to the respective cryoprotectant at 7.0 mol resulted in high survival and development of expanded blastocysts. Ethylene glycol at 7.0 mol with pre-equilibration is, however, most effective for cryopreservation of this stage in the mouse.

Animals↗
PubMed1998

OMEC II: a new ovine mammary epithelial cell line.

We have established three independent ovine mammary epithelial cell lines which arose from primary cultures of ovine mammary epithelial cells by spontaneous immortalization. One of them, OMEC II, was characterised in greater detail. The cells grow on plastic dishes in medium containing 10% FCS without any requirement for additional growth factors or hormones. Immunofluorescence staining of this cell line showed expression of cytokeratin (46 kDa) and ZO-1, a tight-junction associated protein, but negative immunostaining for an anti-vimentin antibody. In confluent cell monolayers 'domes' because visible indicating the development of a polarised phenotype and the ability of directed secretion. When grown in collagen gels typical ducts with end-buds were observed. Treatment with lactogenic hormones increased the frequency of dome formation, but no expression of beta-lactoglobulin was found. To our knowledge this is the first report on an ovine mammary epithelial cell line.

Animals↗
PubMed1998

Primary culture of porcine PGCs requires LIF and porcine membrane-bound stem cell factor.

We studied the effect of murine leukaemia inhibitory factor (LIF), human basic fibroblast growth factor (bFGF) and porcine stem cell factor (SCF) on the survival and/or proliferation of porcine primordial germ cells (PGCs) obtained from 27-day-old embryos in vitro. PGCs were cultured in embryonic stem cell (ESC) medium supplemented with or without either LIF (1000 IU/ml) alone or LIF together with bFGF (10 ng/ml). They were seeded on mitotically inactivated feeder cells, either STO or transfected STO cells (STO#8), expressing the membrane-bound form of porcine SCF. PGCs were identified by their alkaline phosphatase (AP) activity and counted after 1, 3 and 5 days in culture. After 1 day of culture, PGCs cultured on STO#8 cells showed significantly higher survival than PGCs cultured on STO cells (p < 0.05). The combined effect of SCF and LIF caused a significant increase in PGC number by day 3 of culture when PGCs were cultured on either STO cells (p < 0.01) or STO#8 (p < 0.001). When SCF and LIF were used together with bFGF no increase in the PGC number was observed. Our results suggest that the membrane-bound form of porcine SCF plays a pivotal role in the primary culture of porcine PGCs and that bFGF is not required in vitro.

Alkaline Phosphatase↗
PubMed1998

Stable production of human insulin-like growth factor 1 (IGF-1) in the milk of hemi- and homozygous transgenic rabbits over several generations.

One transgenic rabbit line was generated carrying a fusion gene consisting of the cDNA for human IGF-1 fused to a mammary gland specific expression cassette derived from bovine alpha-S1-casein sequences. Transgene expression was shown to be strictly tissue and lactation period specific. The transgenic rabbit line was bred for six generations. All transgenic animals showed stable production of biologically active IGF-1 over the generations and no apparent effect on the physiological or reproductive performance was observed. The absence of adverse effects on homozygous transgenic rabbits suggested the absence of insertional mutagenesis. Eight hemizygous transgenic offspring analysed produced on average 363 +/- 12 micrograms/ml (ranging from 223 +/- 61 to 484 +/- 39 micrograms/ml) mature human IGF-1 in their milk, whereas three homozygous animals produced on average 543 +/- 41 micrograms/ml (ranging from 360 +/- 15 to 678 +/- 80 micrograms/ml). Homozygous hulGF-1 females clearly showed a significantly increased production performance of the recombinant protein.

Animals↗
PubMed1998

Multiple mating in wild Drosophila melanogaster revisited by microsatellite analysis.

The occurrence of multiple mating in Drosophila melanogaster is of particular interest to evolutionary biologists, as seminal fluid has some toxic effects for females. Thus, it has been predicted that the number of matings per females should be low. We have tested this prediction with seven highly polymorphic microsatellite loci in inseminated females from a Viennese D. melanogaster population. In contrast to the predicted low number of matings and previous studies in natural populations, we identified the genotypes of four to six different males fertilizing the offspring of each female tested. Potential causes and consequences are discussed.

Animals↗
PubMed1998

High mutation rate of a long microsatellite allele in Drosophila melanogaster provides evidence for allele-specific mutation rates.

Within recent years, microsatellite have become one of the most powerful genetic markers in biology. For several mammalian species, microsatellite mutation rates have been estimated on the order of 10(-3)-10(-5). A recent study, however, demonstrated mutation rates in Drosophila melanogaster of at least one order of magnitude lower than those in mammals. To further test this result, we examined mutation rates of different microsatellite loci using a larger sample size. We screened 24 microsatellite loci in 119 D. melanogaster lines maintained for approximately 250 generations and detected 9 microsatellite mutations. The average mutation rate of 6.3 x 10(-6) is identical to the mutation rate from a previous study. Most interestingly, all nine mutations occurred at the same allele of one locus (DROYANETSB). This hypermutable allele has 28 dinucleotide repeats and is among the longest microsatellite reported in D. melanogaster. The allele-specific mutation rate of 3.0 x 10(-4) per generation is within the range of mammalian mutation rates. Future microsatellite analyses will have to account for the dramatic differences in allele-specific mutation rates.

Alleles↗
PubMed1998

Conservation of locus-specific microsatellite variability across species: a comparison of two Drosophila sibling species, D. melanogaster and D. simulans.

Fifteen microsatellite loci were studied in Drosophila melanogaster and Drosophila simulans, two closely related sibling species which split 2-3.5 MYA. Within-species variances in repeat number were found to differ up to 1,000-fold among individual microsatellite loci. A significant correlation of log variances between both species indicated a locus-specific mutation rate of microsatellites. Hence, locus-specific effects are apparently among the major forces influencing microsatellite variation and deserve more consideration in microsatellite analysis.

Africa↗
PubMed1998

A method for endoscopic embryo collection and transfer in the rabbit.

Thirty-two rabbits were used for endoscopic embryo collection and transfer. For embryo collection midventral laparoscopy and transcervical endoscopy were combined for orthograd flushing of the oviducts and uterine horns. Transfer was performed transcervically under laparoscopic control. The mean number of corpora lutea counted in nine donors was 13.3 +/- 8.4. A total of 72 morulae/blastocysts were obtained. No embryos were recovered when only the uterine horns were flushed. 291 embryos were transcervically transferred to 23 recipients which resulted in 10 pregnant animals at day 12. Two of three slaughtered recipients showed together 4 implantation sites. Four animals delivered 12 pups.

Animals↗
PubMed1998

Transgenic approaches to the increase of disease resistance in farm animals.

Molecular genetics and reproductive biology techniques enable the transfer of foreign DNA into mammals. Novel approaches to modify disease resistance or susceptibility in livestock are justified by economic and animal welfare concerns. Current research on the improvement of disease resistance by gene transfer focuses on three main strategies, as follows: a) somatic gene transfer, i.e., nucleic acid vaccines b) deletive germ-line gene transfer, i.e., gene knockout c) additive germ-line gene transfer. These strategies aim at either the transient or stable expression of components known to influence non-specific or specific host defence mechanisms, or the disruption of genes known to cause susceptibility to disease. Referring to the source of the effective agent and the site and mode of action, the strategies are termed 'genetic', 'congenital', 'intracellular' and 'extracellular' immunisation. Each gene transfer experiment has to be evaluated carefully with respect to the potential to create novel cases of pathogen resistance or to lose species barriers or cell-type restrictions.

Animals↗
PubMed1997

Endoscopic embryo collection and embryo transfer into the oviduct and the uterus of pigs.

We describe the first complete embryo transfer program, including flushing of embryos from the oviducts via the uterine horns, transfer of embryos into the Fallopian tubes or the uterine horns and recording of the number of piglets born live. The described procedure is minimally invasive and allows the use of pigs simultaneously for embryo collection and production of normal pregnancies. A 30 degrees forward oblique endoscope provided optimal visualization of the reproductive organs and free access to the organs for embryo flushing and transfer. In contrast to surgical and nonsurgical methods, endoscopy allows to pre-examine the genital tract for reproductive abnormalities and successful ovulation. A total of 95 prepuberal gilts or cyclic sows were used in this trial. Embryos or oocytes were collected from hormonally treated pigs via endoscopy(n = 17) on Day 3 and via laparotomy or post mortem after slaughter (control group, n = 38) on Day 3 and 6 after insemination. One (unilateral collection, n = 7) or both oviducts (bilateral collection, n = 10) were flushed endoscopically. We recovered 114 (average 16/pig) and 279 (average 28/pig) oocytes or embryos with fertilization rates of 89% and 72%, respectively. In the control group 834 oocytes or embryos were collected at Day 3 and 6 after insemination (fertilization rate 64%, total 534 embryos, 33 at 2-, 367 at 4-, 2 at 8-cell stage, 24 morulae and 108 blastocysts). Of 836 embryos recovered by endoscopy, surgery or slaughter 528 Day 3 embryos at 2- to 4-cell stage were transferred into (one) oviducts (n = 27 pigs, about 20/pig) resulting in 9 pregnant pigs diagnosed at Day 28 by sonography. Of the 9, 8 carried a total of 49 piglets to term. A total of 195 Day 6 embryos were transferred into uterine horns (n = 12 pigs, about 16/pig), resulting in 5 pregnant pigs carrying a total of 38 offspring to term. The use of endoscopy in assisted reproduction of pigs has the advantages of allowing easy access to the ovary, oviduct and uterus, clear view of the organ manipulation without exposure and exteriorization of viscera during surgery.

Journal Article↗
PubMed1997

Correction of retinal abnormalities found in albinism by introduction of a functional tyrosinase gene in transgenic mice and rabbits.

The factors that regulate normal retinal development remain obscure. However, it is known that elements in the retinal pigment epithelium are critical. When melanin is absent there is a reduction in rods, the central retina fails to develop fully and there is a systematic distortion in the chiasmatic projection to the brain. It has been demonstrated using transgenic mice that the chiasmatic abnormality is controlled by the tyrosinase gene, which is the key enzyme in melanin synthesis. Here we examine whether the two retinal deficits are regulated by this gene. We have examined the distribution of photoreceptors in an albino mouse strain in which a functional tyrosinase gene has been inserted and compared these transgenics with albino and wild type mice. In albinos, rod photoreceptors were reduced by approximately 30%, but were normal in the transgenics. Cone numbers were unchanged. Cell density in the ganglion cell layer was examined in transgenic rabbits, in which albinism had also been rescued with the tyrosinase gene. Normal rabbits have a steep gradient in cell density between central and peripheral retina. Cell density was abnormally low in the central retina in albinos, but normal in the transgenics. Hence, the tyrosinase gene is responsible for each of the retinal deficits associated with albinism. However, it is not clear whether this is due to the absence of melanin or whether the key agent is an associated cell product.

Albinism, Ocular↗
PubMed1997

Karyoplast-cytoplast volume ratio in bovine nuclear transfer embryos: effect on developmental potential.

To evaluate the effect of karyoplast-cytoplast ratio on the development of nuclear transfer embryos, karyoplasts from day 4, day 5, and day 6 embryos were transferred to oocytes enucleated with different volumes of cytoplasm: Type 1, removal of a small volume of cytoplasm equivalent to the first polar body, Type 2, removal of a volume of cytoplasm approximately equal to the volume of the respective karyoplast, and Type 3, removal of half of the oocyte volume. In addition, the effect of experimental reduction of karyoplast cytoplasm was investigated in day 4 and day 5 karyoplasts. Intact day 4 karyoplasts fused to Type 3 cytoplasts did not support development to blastocysts, whereas these karyoplasts yielded blastocysts in combination with Type 1 (7%) and Type 2 cytoplasts (12%). After experimental reduction of cytoplasmic volume in day 4 karyoplasts, blastocysts (10%) were also obtained after fusion with Type 3 cytoplasts, probably due to reduction of cytoplasmic chimerism. With day 5 karyoplasts, blastocyst rate was higher in combination with Type 2 (34%) than with Type 1 (19%) and Type 3 cytoplasts (16%; P < 0.05). The use of day 6 intact karyoplasts resulted in a significantly (P < 0.05) higher proportion of blastocysts when fused with Type 2 (38%) or Type 1 cytoplasts (34%) than with Type 3 cytoplasts (16%). These results suggest that enucleation of oocytes with a volume similar to that of the respective karyoplast creates better conditions for cell cycle interactions with all types of karyoplasts than enucleation with minimal or large volume of cytoplasm.

Animals↗
PubMed1997

Repeated endoscopic ovum pick-up in hormonally untreated ewes: a new technique.

Multiple ovulation and embryo transfer (MOET) are cumbersome and may not be used in all animals. An alternative method for obtaining embryos is to harvest ova and subject them to in vitro maturation/fertilization (IVM/IVF). We tested a modified endoscopic technique designed to allow repeated recovery of oocytes from donor ewes without any hormonal treatment. Seventeen randomly chosen Merino donors were used for ovum pick-up 5 times (OPU1 to OPU5) at 1-wk intervals. The OPUs were performed by ventral laparoscopy. The follicular fluid was aspirated through a needle (0.9 x 75 mm) connected to a 5 ml-syringe. A total of 385 oocytes was collected from 567 aspirated follicles for a collection rate of 67.9%. The number of follicles and oocytes per ewe and the collection rate did not differ significantly (P > 0.05) between collection periods; however, a large variability in the number of follicles and oocytes was observed among individual animals. The collection rate tended to be higher in donors which showed a higher number of follicles. Possible reasons for this are discussed. Oocytes were divided into 4 classes based upon morphological criteria. The portion of oocytes suitable for in vitro production (Classes I to III) were similar among collection periods (83% in OPU1 vs 84% in OPU5). The technique described in this study is useful for obtaining large numbers of oocytes from individual animals during a defined period. This method combined with established in vitro production programs provides a way to increase the number of offspring from genetically valuable animals even when they are affected by infertility due to adhesions or deformations of the oviduct and/or uterus.

Journal Article↗
PubMed1997

Correlations between chemical parameters, mitogenic activity and embryotrophic activity of bovine oviduct-conditioned medium.

To establish parameters predicting the quality of bovine oviduct epithelial cell-conditioned media, we compared media conditioned by oviduct cells from cows at Day 2 (n = 3) and Day 15 (n = 3) of the estrous cycle. In addition, we tested the influence of time of conditioning. Media were evaluated for their embryotrophic activity using a cumulus cell co-culture system as a control. The same media were tested for their mitogenic activity on NIH 3T3 cells and for chemical parameters, including total protein, and de novo synthesized protein as well as for concentrations of glucose, lactate and ammonium. Analysis of variance did not reveal a significant effect by stage of the estrous cycle on the embryotrophic activity of conditioned media. However, there was a significant effect by time of conditioning on the proportion of 5- to 8-cell embryos (P < 0.01) and of blastocysts and hatched blastocysts (P < 0.05). None of the conditioned media (19 to 31% blastocysts) was superior to the cumulus cell co-culture system (32% blastocysts). In the conditioned media, the proportion of 5- to 8-cell embryos correlated positively with mitogenic activity on 3T3 cells (r = 0.64; P < 0.05), whereas the proportion of blastocysts was not significantly correlated with this parameter. In summary, our results provide evidence for an effect of time of conditioning on embryotrophic activity of oviduct epithelial cell-conditioned media. The fact that mitogens for NIH 3T3 cells affect the proportion of 5- to 8-cell embryos but not of blastocysts suggests different culture requirements for early and late preimplantation stage development of bovine embryos.

Journal Article↗