Factors defining developmental competence of bovine oocytes collected for in vitro embryo production†.

Despite the currently relatively low effectiveness of producing bovine embryos in vitro, there is a growing interest in applying this laboratory method in the field of reproduction. Many aspects of the procedure need to be improved. One of the main problems is the inferior developmental competence of in vitro matured oocytes that are collected using the ovum pick-up method. The mechanisms of oocyte capacitation and maturation, as well as the in vivo conditions in which they grow and mature, should be carefully analyzed. A deliberate application of the identified mechanisms and beneficial factors affecting the in vitro procedures seems to be essential for achieving higher developmental competence of the oocytes that are subjected to fertilization. The results may be improved by developing and employing a laboratory maturation protocol that corresponds with appropriate preparation of donors before the ovum pick-up, an optimized hormonal treatment program, the appropriate size of ovarian follicles at the time of aspiration, and a fine-tuned coasting period.

The many problems of somatic cell nuclear transfer in reproductive cloning of mammals.

In 1996, when Dolly the sheep was born, a new, utopian era was expected to begin. Science fiction and popular culture instantly threatened the public with shortly upcoming human clones, portraying it as a very easy and instant procedure. Practice has proven otherwise; it exposed how little is known about the early development of mammals and epigenetic reprogramming. Unfortunately, somatic cell nuclear transfer success rate in mammals has not changed much since its very beginning. It is not uncommon that hundreds of oocytes need to be reconstructed to obtain a single live birth. In this review we provide a brief summary of the progress and problems of the field; beginning with selection of the donor cells and their susceptibility to different methods of epigenetic reprogramming; methods of the later gene activation, placental abnormalities, and their possible causes; to health issues that such offspring is prone to.

Signaling mechanisms and their regulation during in vivo or in vitro maturation of mammalian oocytes.

In vitro fertilization (IVF) is currently one of the most effective methods of infertility treatment. An alternative to commonly used ovarian hyperstimulation can become extracorporeal maturation of oocytes (in vitro maturation; IVM). Fertilization and normal development of the embryo depends on the cytoplasmic, nuclear and genomic maturity of the oocyte. The microenvironment of the ovarian follicle and maternal signals, which mediate bidirectional communication between granulosa, cumulus and oocyte cells, influence the growth, maturation and acquisition of oocyte development capability. During oogenesis in mammals, the meiosis is inhibited in the oocyte at the prophase I of the meiotic division due to the high cAMP level. This level is maintained by the activity of C-type natriuretic peptide (CNP, NPPC) produced by granulosa cells. The CNP binds to the NPR2 receptor in cumulus cells and is responsible for the production of cyclic guanosine monophosphate (cGMP). The cGMP penetrating into the oocyte through gap junctions inhibits phosphodiesterase 3A (PDE3A), preventing cAMP hydrolysis responsible for low MPF activity. The LH surge during the reproductive cycle reduces the activity of the CNP/NPR2 complex, which results in a decrease in cGMP levels in cumulus cells and consequently in the oocyte. Reduced cGMP concentration unblocks the hydrolytic activity of PDE3A, which decreases cAMP level inside the oocyte. This leads to the activation of MPF and resumption of meiosis. The latest IVM methods called SPOM, NFSOM or CAPA IVM consist of two steps: prematuration and maturation itself. Taking into account the role of cAMP in inhibiting and then unblocking the maturation of oocytes, they have led to a significant progress in terms of the percentage of mature oocytes in vitro and the proportion of properly developed embryos in both animals and humans.

[Children born from frozen embryos stored for 10 years -- analysis of 5 cases]. / Dzieci urodzone z zarodków przechowywanych w stanie zamrozenia przez 10 lat. Analiza 5 przypadków.

Faced with a scientific and legal debate on human embryo cryopreservation in Poland we show 5 documented clinical cases of successful thawing and transfer of embryos cryopreserved for a long period of time (8-11 years), resulting in successful delivery by the biological or the recipient mother. Cases described include patients with different infertility diagnoses, subjected to different hormonal stimulation treatments. Different oocyte fertilization methods were performed, and the obtained embryos were frozen after 2, 3 or 4 days of in vitro culture using methods employing various cryoprotective agents and freezing curves. As a result of performed thawing and transfer procedures normal, healthy babies were born. Our results are consistent with the international reports on successful long-term storage of embryos, (including the longest known period of over 19 years) resulting in no detectable reduction of the developmental potential after thawing. In light of data shown here, we do not see any medical or biological reasons for legally-regulated limitation of the period of frozen embryo storage. Moreover, if frozen, long-term stored embryos are not threatened by destruction and if prenatal adoption is a real, clinically documented option, we fail to see any reason for legal limitations of embryo cryopreservation in human infertility treatment.

Comparison of traditional and modified (VitMaster) methods of rabbit embryo vitrification.

In spite of their cryobiological efficacy, minimum-volume vitrification methods suffer from the risk of microbiological contamination and are technically and/or manually demanding. In this study, the effects of a traditional, slightly modified vitrification method and vitrification using supercooled liquid nitrogen (VitMaster) applied for rabbit morula-stage embryos were compared. Embryos were equilibrated in a solution containing 1,2-propanediol (2.72 M) and glycerol (1.36 M) for 7 min and vitrified in 0.25-ml insemination straws after 1-min exposure to a vitrification solution containing additionally 1.0 M sucrose. Cooling was performed in 'normal' or supercooled liquid nitrogen. Regardless of the cooling method applied, high in vitro survival and development rates of vitrified embryos were obtained. All embryos were intact after warming, and 61 out of 65 (93.8%) and 23 out of 24 (95.8%) embryos developed to the blastocyst stage after 48-h in vitro culture of embryos vitrified in 'normal' or supercooled liquid nitrogen, respectively. The results suggest higher developmental ability of embryos vitrified in supercooled liquid nitrogen (91.7% vs . 83.1% of embryos vitrified traditionally developed to more advanced, expanding and/or hatching blastocyst stages). In vivo survival rate, tested for the traditional vitrification system only, revealed that 36.8% of embryos developed to term. The results show promise for establishing a fully successful method for rabbit embryo vitrification.

Melatonin effect on bovine embryo development in vitro in relation to oxygen concentration.

Melatonin promotes mouse embryo development in vitro. An effect of melatonin on bovine embryo development is described here. Slaughterhouse derived oocytes were subjected to standard in vitro maturation and fertilization procedures. Presumptive zygotes were cultured for 2 days in CR1aaLA medium supplemented with melatonin (10(-4) m) or without melatonin (control). Culture was performed under two different gas atmospheres containing physiological (7%) or atmospheric (20%) oxygen concentrations (2x2 factorial analysis). After day 2, embryos from each treatment group developed to at least four-cell stage, were cultured without melatonin until day 10 at optimum 7% O2 atmosphere. Blastocyst formation rates of presumptive zygotes and of four-cell embryos were calculated for each group. Significant interactions between oxygen tension and the melatonin treatment were found. Out of four-cell embryos put into in vitro culture after initial incubation in medium containing melatonin, decreased blastocyst rate was observed in melatonin group (47.7%) compared with control (67.7%; P=0.0327) when lower oxygen concentration was applied. A beneficial effect of melatonin was observed in 20% O2: out of 61 embryos, 42 (68.9%) developed to the blastocyst stage after treatment in melatonin versus 32 of 63 (50.8%; P=0.0458) blastocysts that developed in control group. In conclusion, beneficial or harmful effects of melatonin on bovine embryo development in vitro were observed, depending on the oxygen tension during the treatment.

Banking of embryos of mutated, paralytic tremor rabbit by means of vitrification.

Cryopreservation enables banking of embryos for future use in medicine and in animal breeding. It also enables protection of germ plasm of endangered species and unique strains or lanes of laboratory animals. This paper describes an example of employing a vitrification method for banking of embryos of a unique lane of rabbit. The paralytic tremor (pt) rabbit is an X-linked recessive mutant lane of the Chinchilla breed characterized by hypomyelination of the central nervous system. In order to obtain a sufficient number of embryos, pt females were subjected to superovulation and surgical embryo collection. All suitable embryos were vitrified in 0.25 mL insemination straws in a modified EFS vitrification solution comprised of ethylene glycol (40%), Ficoll 70 (18%) and sucrose (0.3 M) in Hepes buffered TCM medium containing 20% fetal calf serum. In order to assess the efficiency of the vitrification procedure, a representative portion of vitrified embryos was warmed after a period of storage. Warmed embryos were subjected to in vitro culture for 72 h or were transferred to the uterus of synchronized recipients. The majority of the 141 warmed embryos survived vitrification and 100/141 (71%) developed to the blastocyst stage. Moreover, out of an additional 34 warmed embryos transferred to four recipients, eight (23.5%) developed to term and seven live pups were born. Six of the rabbit pups exhibited paralytic tremor symptoms typical for the pt lane. Although the overall efficiency of the vitrification method was lower compared with the effects usually achieved for 'healthy' embryos, results presented confirm the real possibility of the future restoration of the colony of pt rabbit, if sufficient number of embryos are cryopreserved.