Wiesława Młodawska

University of Agriculture in Krakow

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A. Okólski

University of Agriculture in Krakow

Joanna Kochan

University of Agriculture in Krakow

Agnieszka Nowak

University of Agriculture in Krakow

Wojciech Niżański

Wroclaw University of Environmental and Life Sciences

Sylwia Prochowska

Wroclaw University of Environmental and Life Sciences

Agnieszka Partyka

Wroclaw University of Environmental and Life Sciences

Józef Skotnicki

Ogród Zoologiczny w Krakowie

Marcin Pałys

Ogród Zoologiczny w Krakowie

Teresa Grega

Wrocław Zoo

Patrycja Mrowiec

University of Agriculture in Krakow

Cooperative Institutions

University of Agriculture in Krakow

Jagiellonian University

Wroclaw University of Environmental and Life Sciences

National Research Institute of Animal Production

Wroclaw Medical University

AGH University of Krakow

Physiologie de la Reproduction et des Comportements

University of Agriculture Faisalabad

Polish Academy of Sciences

University of Wrocław

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51 DEVELOPMENT OF EQUINE CLONED EMBRYOS DERIVED FROM IN VITRO-MATURED OOCYTES RECEIVING ADULT DERMAL FIBROBLAST CELL NUCLEI

Reproduction Fertility and Development (2008)

M. Skrzyszowska M. Samiec Wiesława Młodawska Joanna Kochan A. Okólski

The purpose of our study was to determine the in vitro developmental competences of equine NT embryos reconstructed with adult dermal fibroblast cells. Frozen/thawed fibroblast cells, whose mitotic cycle had been synchronized at G1/G0 stages through a contact inhibition of their migration and proliferative activity under total confluency, were used as a source of nuclear donor cells in the somatic cell cloning procedure. In vitro-matured oocytes were used as recipient cells for fibroblast cell nuclei. The compact cumulus–oocyte complexes (cpCOCs) were collected from abattoir-derived mare ovaries and selected for in vitro maturation. The cpCOCs were cultured in TC-199 medium supplemented with 5 mU mL–1 follicle-stimulating hormone (FSH), 10% fetal bovine serum (FBS) and 75 μg mL–1 kanamycin monosulfate (kanamycin A) for 30 h at 38.2°C in a 100% water-saturated atmosphere of 5% CO2 and 95% air. Cumulus-denuded in vitro-matured oocytes were incubated in the maturation medium supplemented with 0.4 μg mL–1 demecolcine for 40 min. The treated oocytes were subsequently transferred into TC-199 medium containing 4 mg mL–1 BSA-V and 5 μg mL–1 cytochalasin B. Metaphase chromosomes, which had been allocated into the chemically-induced protrusion of the plasma membrane, were removed microsurgically. The chemically-assisted enucleation was accomplished by gently aspirating the ooplasmic cone, which contained the condensed chromosome mass, with the aid of a beveled micropipette. The single nuclear donor cells were inserted into perivitelline space of previously enucleated oocytes. Fibroblast cell-ooplast couplets were fused with two consecutive DC pulses of 2.4 kV cm–1 for 30 μs. After a 1.5-h delay, nuclear transfer-derived oocytes were chemically activated by exposure to 5 μm L–1 calcium ionomycin for 5 to 7 min, followed by their incubation in B2 medium with addition of 2 mm L–1 6-dimethylaminopurine (6-DMAP) for 4 h. Reconstructed embryos were in vitro cultured in B2 medium for 2 days. Afterwards, cleaved embryos were co-cultured with Vero cells in B2 medium supplemented with 10% FBS for 5 to 6 days up to morula/blastocyst stages. From among 88 in vitro cultured cpCOCs, 55 (62.5%) acquired meiotic nuclear and cytoplasmic maturity state after reaching the Metaphase II stage. A total of 55 enucleated oocytes underwent reconstruction and 44/55 (80.0%) were successfully fused with nuclear donor cells. Out of 44 cultured NT embryos, 21 (47.7%) were cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages were 6/44 (13.6%) and 3/44 (6.8%), respectively. In conclusion, the cell nuclei of in vitro cultured adult dermal fibroblast cells, which had undergone the contact inhibition, were able to direct the preimplantation development of equine cloned embryos to morula and blastocyst stages. This work was supported by the Scientific Net of Animal Reproduction Biotechnology.

Perivitelline space

Cytoplast

Cytochalasin B

In vitro maturation

10.1071/rdv21n1ab51

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Developmental competence of cat (Felis domesticus) oocytes and embryos after parthenogenetic stimulation using different methods

Zygote (2018)

Joanna Kochan Agnieszka Nowak Wojciech Niżański Sylwia Prochowska Anna Migdał

Summary The aim of this study was to compare the effects of various activating factors on feline oocytes. The study included activation within the ovary (natural), activation during in vitro maturation (spontaneous activation), chemical activation (ionomycin + 6-DMAP), activation by spermatozoa and injection (ICSI) and mechanical activation (sham ICSI). According to our results, parthenogenetic embryos could emerge at every step of in vitro embryo production (IVP) procedures. After oocyte collection, 6% of parthenogenetic embryos were observed, mainly at the 2–4-blastomere stages. After 24 h of in vitro maturation, parthenogenetic activation was observed in 7% of oocytes. Using ionomycin and 6-DMAP to artificially activate oocytes, 53% of cleaved embryos were obtained. The results after ICSI (54% cleaved embryos) were not significantly different from the results in Group III using chemical activation (53% cleaved embryos). But only after ICSI were blastocysts obtained (5/73.7%) as a result of in vitro culture. Moreover, embryos after ICSI were of the best morphological quality with minor levels of fragmentation evident in the embryos. After sham mechanical activation, ‘sham ICSI’, 8% of cleaved embryos were noted. Therefore, it is advised to maintain a negative control in parallel with each step of IVP techniques, to avoid misleading results. Chemical methods for artificial activation of feline oocytes are the most promising for application to the cloning and production of parthenogenetic embryos for experimental studies.

Oocyte activation

Blastomere

Parthenogenesis

In vitro maturation

Ionomycin

10.1017/s0967199418000011

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Citations (6)

Ultrastructural changes in feline oocytes during ovary storage for 24- and 48-hours

Theriogenology (2022)

Małgorzata Ochota Michał Kulus Wiesława Młodawska Marta Kardasz-Kamocka Katarzyna Haczkiewicz-Leśniak

Organelle

Lipid droplet

10.1016/j.theriogenology.2022.11.025

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Citations (5)

Equine oocyte‐cumulus morphology as affected by follicular size

Equine Veterinary Journal (1997)

Wiesława Młodawska A. Okólski

From the ovaries of 256 slaughtered mares a total of 1713 follicles were isolated from which 1641 (95.8%) oocytes were recovered (6.4/mare). A total of 564 follicles and oocytes were evaluated for the degree of vascularisation of the follicle wall, the appearance of the follicular fluid and the location and morphology of the cumulus-oocyte-complex. Follicles with a diameter of >10 mm displayed more numerous, well branched and more pronounced blood vessels than the smaller ones (4-10 mm diameter) and most of them contained clear, yellowish fluid with few granulosa cells. The percentage of oocytes with compact cumuli increased significantly with an increasing diameter of the follicle, being 233%, 43.9%, 55.6% and 64.2% (P<0.01) for the follicles with diameters of 4-10, 11-15, 16-20 and 21-35 mm, respectively. The percentage of oocytes attached to the follicle wall also increased with increasing follicle size, being 48.0%, 59.6%, 81.5% and 90.1% (P<0.01), respectively. On the contrary, the percentage of oocytes floating in the follicular fluid decreased with increasing follicle diameter, from 52.0% in the smallest follicles to 9.9% in the biggest ones. A significantly greater percentage of oocytes found on the follicular wall than in the follicular fluid had a compact cumulus (56.6 versus 21.3%; P<0.01). For in vitro culture were accepted 30.4%, 54.3%, 60.7% and 77.8% (P<0.01) of oocytes from the follicles with diameters of 4-10, 11-15, 16-20 and 21-35 mm, respectively. After culture for 28-40 h in TCM 199 medium, 90 of a total of 165 (54.5%) oocytes reached the metaphase II stage of maturation.

Morphology

10.1111/j.2042-3306.1997.tb05097.x

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Citations (9)

Influence of Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) on the In Vitro Characteristics of Equine Gametes

Journal of Equine Veterinary Science (2017)

Agnieszka Nowak Joanna Kochan Wojciech Niżański Agnieszka Partyka Roland Kozdrowski

In vitro maturation

Gamete

10.1016/j.jevs.2017.11.002

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Mammalian Oocyte Analysis by MALDI MSI with Wet-Interface Matrix Deposition Technique

Materials (2023)

Anna Bodzoń‐Kułakowska Wiesława Młodawska Przemysław Mielczarek Dorota Lachowicz Piotr Suder

Oocytes are a special kind of biological material. Here, the individual variability of a single cell is important. It means that the opportunity to obtain information about the lipid content from the analysis of a single cell is significant. In our study, we present a method for lipid analysis based on the MALDI-based mass spectrometry imaging (MSI) approach. Our attention was paid to the sample preparation optimization with the aid of a wet-interface matrix deposition system (matrix spraying). Technical considerations of the sample preparation process, such as the number of matrix layers and the position of the spraying nozzle during the matrix deposition, are presented in the article. Additionally, we checked if changing the 2,5-dihydroxybenzoic acid (DHB) and 9-Aminoacridine (9AA) matrix concentration and their solvent composition may improve the analysis. Moreover, the comparison of paraformaldehyde-fixed versus nonfixed cell analysis was performed. We hope that our approach will be helpful for those working on lipid analyses in extraordinary material such as a single oocyte. Our study may also offer clues for anybody interested in single-cell analysis with the aid of MALDI mass spectrometry imaging and the wet-interface matrix deposition method.

Matrix (chemical analysis)

Deposition

Sample Preparation

Sample (material)

Interface (matter)

10.3390/ma16041479

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Citations (2)

Lipid content and G6PDH activity in relation to ooplasm morphology and oocyte maturational competence in the domestic cat model

Reproductive Biology (2024)

Wiesława Młodawska Bartosz Maliński Gabriela Godyń Beata Nosal

10.1016/j.repbio.2024.100927

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Changes in mitochondrial activity in horse oocytes and zygotes during maturation and fertilisation in vitro.

H. Torner H. Alm W. Kanitz Wiesława Młodawska A. Okólski

Fertilisation

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Zona pellucida-sperm binding assay for equine oocytes.

PubMed (2000)

Wiesława Młodawska E. Palmer Guy Duchamp A. Okólski J. Bézard

The binding of a spermatozoon to the zona pellucida is the first step in fertilization. The number of spermatozoa bound to a zona pellucida may reflect the functional status of both the oocyte and spermatozoa. The aim of the present study was to determine whether the stage of maturation of the equine oocyte affects the capacity of the zona pellucida to bind with spermatozoa. Cumulus-oocyte complexes (COCs) were collected from the ovaries of mares from abattoirs or were obtained in vivo by ultrasound-guided follicular aspiration. Oocytes were inseminated directly after collection or after 30 h of in vitro maturation (IVM) with semen from the same ejaculate. The results indicate that the immature equine oocytes collected from abattoirs and inseminated directly after collection bound fewer spermatozoa than did mature (metaphase I or II) oocytes inseminated after 30 h of IVM. A similar correlation was observed with the oocytes collected in vivo. The mean number of spermatozoa bound by mature oocytes collected from preovulatory follicles and inseminated directly after collection was 324 +/- 29, whereas the mean number of spermatozoa bound by immature oocytes from nonpreovulatory follicles inseminated without maturation was 222 +/- 26 (P < 0.05). This difference was not observed in the oocytes inseminated after maturation. It is postulated that immature equine oocytes bind fewer spermatozoa than do oocytes matured either in vivo or in vitro.

Spermatozoon

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Immunolocalization of androgen receptors and assesment of steroidogenic activity of the ovaries in prepubertal and pubertal mare

Journal of Equine Veterinary Science (2013)

Wiesława Młodawska A. Okólski

10.1016/j.jevs.2013.10.078

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Citations (1)