PubMed Health⌕ Search

Biomedical subjects

Katrin Hinrichs

Publications and source records attributed to Katrin Hinrichs.

10 recordsLinked to original sources
PubMed2006

Equine cloning.

Equine cloning is now in use as a clinical technique. It is available commercially, and its efficiency seems to be increasing. The foals produced by cloning may differ in some phenotypic and behavioral traits from the original animal but should produce offspring that reflect those that the original donor animal would have produced. This is especially true in the case of male animals, where the mitochondrial DNA is not passed to the progeny. Results of pregnancies due in 2006 should add significantly to our understanding of the factors affecting production of viable cloned foals and of the similarities and differences among cloned foals and between these foals and the donor animals.

Animals↗
PubMed2005

Update on equine ICSI and cloning.

Intracytoplasmic sperm injection (ICSI) has recently become efficient enough to be considered for clinical use. With ICSI, one spermatozoa is injected into a mature oocyte. Harvesting of an oocyte ex vivo, followed by ICSI and transfer of the fertilized oocyte to the oviduct, may be applicable when semen quality is insufficient for standard insemination. Sperm injection, followed by in vitro embryo culture to the blastocyst stage, may be used in cases where multiple oocytes are to be fertilized (e.g. when oocytes are collected post-mortem). Nuclear transfer (cloning) of horses is possible but still inefficient; however, commercial companies currently will culture and store cells from privately owned animals for a reasonable fee. Horse owners are beginning to realize the potential of cloning for salvaging valuable equine genetics that may otherwise be lost.

Animals↗
PubMed2003

Patterns of intracellular calcium oscillations in horse oocytes fertilized by intracytoplasmic sperm injection: possible explanations for the low success of this assisted reproduction technique in the horse.

In all species studied, fertilization induces intracellular Ca2+ ([Ca2+]i) oscillations required for oocyte activation and embryonic development. This species-specific pattern has not been studied in the equine, partly due to the difficulties linked to in vitro fertilization in this species. Therefore, the objective of this study was to use intracytoplasmic sperm injection (ICSI) to investigate fertilization-induced [Ca2+]i signaling and, possibly, ascertain problems linked to the success of this technology in the horse. In vivo- and in vitro-matured mare oocytes were injected with a single motile stallion sperm. Few oocytes displayed [Ca2+]i responses regardless of oocyte source and we hypothesized that this may result from insufficient release of the sperm-borne active molecule (sperm factor) into the oocyte. However, permeabilization of sperm membranes with Triton-X or by sonication did not alleviate the deficient [Ca2+]i responses in mare oocytes. Thus, we hypothesized that a step downstream of release, possibly required for sperm factor function, is not appropriately accomplished in horse oocytes. To test this, ICSI-fertilized horse oocytes were fused to unfertilized mouse oocytes, which are known to respond with [Ca2+]i oscillations to injection of stallion sperm, and [Ca2+]i monitoring was performed. Such pairs consistently displayed [Ca2+]i responses demonstrating that the sperm factor is appropriately released into the ooplasm of horse oocytes, but that these are unable to activate and/or provide the appropriate substrate that is required for the sperm factor delivered by ICSI to initiate oscillations. These findings may have implications to improve the success of ICSI in the equine and other livestock species.

Animals↗
PubMed2003

Activation of equine nuclear transfer oocytes: methods and timing of treatment in relation to nuclear remodeling.

Early development of embryos produced by transfer of equine nuclei to bovine cytoplasts is superior to that of intraspecies equine nuclear transfer embryos. This may be related to differences in chromatin remodeling or efficiency of activation between the two oocyte types. The pattern of donor nucleus remodeling was examined in equine-equine and equine-bovine reconstructed oocytes. Chromosome condensation occurred in equine cytoplasts by 2 h but was not seen in bovine cytoplasts until 4 h. We investigated the effect of activation of equine-equine reconstructed oocytes at <30 min or at 2 h after reconstruction. Four activation treatments were evaluated at each time point: injection of sperm extract alone, or in combination with 6-dimethylaminopurine (6-DMAP), cytochalasin B, or 1% dimethylsulphoxide. There was no significant difference in normal cleavage rate or average nucleus number of embryos between equine oocytes activated <30 min or at 2 h after reconstruction. The combination of 6-DMAP with sperm extract significantly (P < 0.01) improved cleavage rate compared with the other three treatments. Activation with sperm extract and 6-DMAP 2 h after donor nucleus injection gave the highest cleavage (79%) and the highest cleavage with normal nuclei (40%). Sperm extract and 6-DMAP also effectively activated oocytes parthenogenetically, yielding 83% cleavage and 73% cleavage with normal nuclei. These results indicate that although nuclear remodeling occurs rapidly in equine cytoplasts, early activation does not improve embryonic development after reconstruction.

Adenine↗
PubMed2003

Meiotic competence of equine oocytes and pronucleus formation after intracytoplasmic sperm injection (ICSI) as related to granulosa cell apoptosis.

Follicle atresia and granulosa cell apoptosis may be related to oocyte meiotic and developmental competence. We analyzed the relationships among granulosa cell apoptosis, initial cumulus morphology, oocyte nuclear maturation in vitro, and pronucleus formation after intracytoplasmic sperm injection (ICSI) in the horse. For each follicle, the size was measured and granulosa cells were used for DNA laddering analysis. Oocytes were evaluated for cumulus morphology, cultured for in vitro maturation, and submitted to ICSI. Apoptosis was categorized as absent, intermediate, or advanced according to the relative concentrations of two DNA fragments at 900 and 360 base pairs (bp). In 98 oocyte-follicle pairs, 52 oocytes were classified as expanded (Exp), 39 as compact (Cp), and 7 as having a partial (P) cumulus. Advanced apoptosis was detected in 55% (54/98) of follicles; 37% (36/98) of follicles showed an intermediate level of apoptosis; and 8 follicles (8%) were nonapoptotic. Follicle size was not significantly correlated with granulosa cell apoptosis (P > 0.05). Significantly more Exp than Cp oocytes originated from follicles with advanced apoptosis (P < 0.001). The proportion of oocytes maturing in vitro was significantly higher in oocytes issuing from apoptotic follicles than in oocytes issuing from healthy follicles (P < 0.05). The proportion of normally (two pronuclei) or abnormally fertilized oocytes (one or greater than two pronuclei, or partially decondensed sperm) did not differ in relation to granulosa cell apoptosis. We conclude that, in the mare, granulosa cell apoptosis is related to cumulus expansion and an increase in oocyte meiotic competence but has no effect on the proportion of meiotically competent oocytes that activate after ICSI. These results provide selection criteria for horse oocytes used in assisted reproductive techniques so that embryo production may be maximized.

Animals↗
PubMed2003

Effect of ovary storage and oocyte transport method on maturation rate of horse oocytes.

Two experiments were conducted to determine the effects of storage on equine ovaries or isolated oocytes. Ovaries were collected at an abattoir and were maintained at room temperature during collection and transport (3-9h total). After arrival at the laboratory, ovaries were divided into three groups: immediate oocyte collection (control), storage at room temperature overnight (15-18 h) before oocyte collection, or storage at 4 degrees C overnight before oocyte collection. Collected oocytes were cultured in maturation medium for 24h. There was a significant increase in the proportion of oocytes classified as having compact cumuli in the two storage groups when compared with the controls. For oocytes originally having expanded cumuli, the rate of maturation to MII was significantly higher in the control group (72%) than in either storage group, and the maturation rate for oocytes from ovaries stored at room temperature (27%) was significantly higher than that for ovaries stored at 4 degrees C (10%). A similar trend was seen for oocytes originally having compact cumuli (24, 11, and 3% in MI-II for control, room temperature, and cold groups, respectively). In Experiment 2, we evaluated the effect of different packaging systems on the maturation of horse oocytes within a portable incubator. Use of 1 ml of equilibrated maturation medium in a 1 ml glass vial was associated with maturation equivalent to that for standard incubation.

Abattoirs↗
PubMed2003

Effects of gas conditions, time of medium change, and ratio of medium to embryo on in vitro development of horse oocytes fertilized by intracytoplasmic sperm injection.

This study was conducted to evaluate the effects of two different gas conditions (5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2), mixed gas), time of medium change (Day 3 or 4) and ratio of medium to embryo (2, 5 or 10 microl per presumptive zygote) on the development of horse oocytes fertilized by intracytoplasmic sperm injection and cultured in G1.2/2.2 medium. Oocytes from slaughterhouse-derived ovaries were matured in vitro for 24 h and fertilized by injection of frozen-thawed sperm using micromanipulation with a Piezo drill. Presumptive zygotes were randomly assigned to 5% CO(2) in air or mixed gas and fixed after 96 h of culture. Cleavage rates between two gas conditions were similar (67 and 63%), but the mean nucleus number of embryos in the mixed gas treatment was significantly (P<0.05) higher than that of embryos cultured in 5% CO(2) in air (15.2 versus 7.0, respectively). Further experiments were done with mixed gas incubation. Development of embryos was compared after change from G1.2 to G2.2 medium at Day 3 or 4. There was no significant difference in cleavage rate (56 and 65%, respectively) or development to the blastocyst stage after 7 days of culture (5% and 46%, respectively) between embryos changed on different days. To evaluate the effect of the ratio of medium to embryo, zygotes were cultured at a ratio of 2, 5 or 10 microl medium per zygote. There were no significant differences among ratio treatments in rates of cleavage or development to blastocyst.

Animals↗
PubMed2002

Effect of co-culture with theca interna on nuclear maturation of horse oocytes with low meiotic competence, and subsequent fusion and activation rates after nuclear transfer.

We conducted this study to examine whether or not co-culture with theca cells improves the maturation rate of horse oocytes with compact cumuli and to evaluate the cytoplasmic competence of oocytes after maturation by assessing fusion, activation and cleavage rates after nuclear transfer. We collected oocytes by scraping follicles from slaughterhouse-derived ovaries and classified them as having an expanded or a compact cumulus. Expanded oocytes were matured in M199 supplemented with 10% FBS and 5 microU/ml FSH for 24 h: compact oocytes were cultured in the same medium, or they were co-cultured in the same medium with theca interna explants, for 24 or 42 h. Oocytes were held with or without 10 microg/ml cytochalasin B, before washing and micromanipulation. and they were fused with donor fibroblasts by electrical pulse. Fused oocytes were activated with Ca ionophore/cycloheximide, cultured for 5 days, and stained with Hoechst to assess nuclear development. We considered oocytes with an enlarged nucleus, or having cleavage with multiple nuclei, to be activated. There was no significant difference in overall maturation rate between compact oocytes cultured with theca and compact controls. When these two groups were combined, there was a significant increase in the proportion of oocytes in MII between 24 and 42 h (P < 0.05). Expanded oocytes had a significantly higher rate of maturation than did compact oocytes (64% versus 25-30%; P < 0.001). There were no significant differences in rates of successful enucleation, fusion, activation or cleavage between compact control and compact + theca oocytes, nor between compact and expanded oocytes; however, expanded oocytes treated with cytochalasin B had a significantly higher survival rate after enucleation than did untreated expanded oocytes (P < 0.05). Three embryos developed from recombined oocytes, with maximum cleavage to 10 cells. The results of this study indicate that co-culture with theca cells does not increase either nuclear or cytoplasmic maturation of compact oocytes. Cytochalasin B is helpful in increasing survival of horse oocytes during enucleation. In vitro matured equine oocytes have the potential to develop into embryos after nuclear transfer; this is the first full report of production of cloned embryos in this species.

Animals↗
PubMed2002

Suppression of meiosis by inhibitors of m-phase proteins in horse oocytes with low meiotic competence.

Germinal vesicle (GV)-stage horse oocytes with diffuse chromatin are meiotically incompetent and degenerate in culture, whereas horse oocytes having condensed chromatin within the GV are meiotically competent. Degeneration of incompetent oocytes in culture may be related to premature GV breakdown, which could possibly be prevented by inhibition of m-phase protein activity. We examined the effects of 6-dimethylaminopurine (6-DMAP), butyrolactone and roscovitine on GV-stage horse oocytes. Culture in the presence of 2 mM 6-DMAP for 24 h suppressed meiosis (2% MI or MII compared with 38% for untreated oocytes). The proportion of GV-stage oocytes having condensed chromatin was not different between 6-DMAP culture and directly fixed controls; however, the proportion of oocytes with diffuse chromatin was significantly lower, and more oocytes with diffuse chromatin had atypical chromatin than did controls (p < 0.01). Culture with butyrolactone at 100 microM suppressed meiosis (5% MI + II). Again, this treatment maintained GV-stage oocytes having condensed chromatin, but the proportion of oocytes with diffuse chromatin was significantly reduced compared with directly fixed controls (p < 0.05). Culture with roscovitine at 25 microM was also effective in maintaining GV-stage oocytes having condensed chromatin; however, culture with 100 microM roscovitine did not suppress meiosis or maintain oocytes in the GV stage. These results indicate that meiosis in GV-stage horse oocytes having condensed chromatin may be suppressed by inhibitors of m-phase protein activity; however, oocytes originally having diffuse chromatin appear to degenerate in culture even in the presence of these inhibitors.

4-Butyrolactone↗