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Cellular and molecular mechanisms mediating the effect of polychlorinated biphenyls on oocyte in vitro maturation: Polychlorinated biphenyls (PCBs) are stable, lipophilic compounds that accumulate in the environment and in the food chain. Recent studies provide evidence that exposure to PCBs can cause reproductive problems. PCBs have been identified in the ovarian follicle of women and other mammals and many data in the literature clearly indicate that both follicles and oocytes are particularly susceptible to these pollutants. In the present review we describe the multifaceted effects of PCBs on mammalian oocyte maturation in detail. Published studies clearly indicate that PCB congeners, both singly or as complex mixtures, disrupt mammalian oocyte maturation and subsequent embryo development. Specifically, data point out to the ability of PCBs to interfere with the organization of the microtubules cytoplasmic network resulting in an altered compartmentalization of the ooplasm. Furthermore, a critical role of cumulus cells in mediating PCB ovotoxicity has been observed, most likely related to a disregulation in intracellular communication between the germinal and the somatic compartment. Finally, since coplanar PCBs, induce gene expression via a ligand-dependent transactivating factor, the aryl hydrocarbon receptor, this signalling pathway is also reviewed with respect to understanding the toxic mechanisms of these compounds.

The purpose of this work was to determine the mechanisms regulating the acquisition of cytoplasmic maturation and embryonic developmental competence in pig oocytes. The presence or the absence of porcine follicular fluid (pff; 25% or 0%) in the maturation medium was used as a means to achieve complete nuclear maturation accompanied or not accompanied by cytoplasmic maturation. ATP content, active mitochondria relocation, and microtubule distribution were analyzed at different times during in vitro maturation (IVM). While nuclear maturation did not differ among the two groups, parthenogenetic embryonic development was significantly higher (41.5%) in the 25% pff group than in the 0% pff group (19.0%) with blastocysts that had a significantly higher number of blastomeres (76.1 ± 6.3, and 47.2 ± 6.5, respectively). Oocyte ATP content increased significantly during IVM, but at the end of maturation no significant differences were observed between high- and low-competence oocytes. An extensive relocation of mitochondria to the inner cytoplasm during IVM together with the formation of a well-developed mesh of cytoplasmic microtubules was observed only in the high-competence oocyte group. However, no differences in the formation of microtubules associated with the meiotic spindles were observed between high- and low-competence groups. We conclude that low developmental competence is associated with the lack of a microtubule cytoplasmic network, which prevents correct relocation of mitochondria and is likely to reflect a more generally altered compartmentalization of the ooplasm. This can be independent from the formation of the microtubule machinery required for the completion of chromosome disjunctions and does not affect the overall ATP content.

Summary The efficiency of breeding schemes is dependant on the high fecundity of the selected individuals. Reproductive technologies are constantly pushing the physiological limits, but while the male reproductive potential is almost fully exploited, female reproductive physiology is the subject of constant research. Since the number of offspring that a female can bring to term each pregnancy cannot be changed, the ideal approach is to remove the potential offspring at the beginning of development and to transfer them to recipients of lesser genetic value. The earlier the collection takes place, the higher the number of descendants that a female can generate, so that now, the number of available oocytes becomes the limiting factor. This article will describe how detailed studies on oocyte physiology are beginning to unravel the complex sequence that transforms a small primordial follicle into a large ovulatory follicle containing a mature oocyte. Progressively, the limits to oocyte manipulation have been recognised and gradually overcome with adequate hormonal treatments in vivo and with specific media supplementation in vitro. This has lead to the development of highly efficient reproductive technologies and the promise of even greater advances in the future. Surprising new findings, such as ovarian stem cells that can replenish the follicle population or long term embryonic stem cell lines that can differentiate into oocytes, are rapidly changing our expectations.

The present study was designed to address the physiological role played by cAMP on gap junction (GJ) mediated communications between oocyte and cumulus cells during in vitro maturation. Cyclic AMP was stimulated by different collection and maturation media known to induce different rates of nuclear maturation and developmental competence as well as different levels of cumulus expansion. Cumulus-oocyte complexes (COCs) were matured for 0, 3, 7, 12, 18, and 24 h in the absence of stimulation or in the presence of serum and gonadotropins (fetal bovine serum+human menopausal gonadotropins [FCS+hMG]) or 0.01 μg/ml of invasive adenylate cyclase (iAC). For each time point, intracellular cAMP concentration ([cAMP]i) was determined either in the whole COC or oocyte after cumulus cell removal. GJ functional status was analyzed by microinjection of Lucifer yellow fluorescent dye in cumulus-enclosed oocytes and by immunohistochemical localization of connexin 43 (Cx43). In the absence of stimulation, [cAMP]i ...

Alkylphenolic compounds are a widespread family of xenoestrogens. High concentrations of these substances are present in sewage sludge that is spread on arable land and pasture as fertilizer. Because of their known endocrine system-disrupting activity, alkylphenols represent a potential risk for the reproductive health of farm animals. In this study, the impact of p-tert-octylphenol (OP) on the developmental competence of bovine oocytes was evaluated. Endocrine activity of OP was investigated for its effect on estrogen receptors α and β (ERα and ERβ) and progesterone receptor (PR) mRNA levels. Cumulus-oocyte complexes (COCs) were exposed during in vitro maturation to serial concentrations of OP (1–0.0001 μg/ml) and were compared with vehicle-treated controls and a group of COCs treated with 17β-estradiol (E2). A dose-related decrease in the percentage of oocytes that completed maturation after 24 h and in oocyte fertilization competence was observed at doses of OP as low as 0.01 μg/ml. Groups tre...

To date, approximately 60 chemicals have been identified as endocrine disruptors: exogenous agents that interfere with various aspects of natural hormone physiology. The potential reproductive and health hazards of these environmental chemicals have recently generated concern among the scientific community, policy makers and general public. The present review presents and discusses the available evidence that environmental chemicals are causing ovarian toxicity in various species, with particular attention to farm animals. The impact of chronic exposure to endocrine disruptors via food and drinking water cannot be neglected when studying fertility problems in these species. This review focuses attention on the superfamily of organochlorine chemicals, persistent organic pollutants (POPs), because of their persistence in the environment, ability to concentrate up the food chain, continued detection in environmental matrices and ability to be stored in the adipose tissue of animals and humans. Published data clearly indicate that POPs disrupt mammalian oocyte maturation and follicle physiology in every species studied so far, including farm animals. However, as most of the data available still derive from experiments performed on laboratory species or in vitro models, great care should be taken when extrapolations to other species or environmental situations are attempted.

In mammals, the study of gene expression in the preimplantation embryo has been difficult because the standard procedures used to quantify mRNA generally require large amounts of starting material. The development of protocols using different quantitative strategies generally involving the polymerase chain reaction (PCR) has provided new tools for exploration of gene expression in preimplantation embryos. However, the use of an internal standard, often referred as a housekeeping gene, is essential to normalize the mRNA levels. RNA levels of eight housekeeping genes were quantified using real time PCR throughout the preimplantation period of the bovine embryo to find the most suitable gene to be used as standard. Histone H2a was the best internal standard because the transcript levels were constant across the preimplantation period. Linear amplification of antisense RNA using the T7 promotor for in vitro transcription of the entire RNA pool was evaluated as a suitable way to preamplify the starting material prior to quantification and was effective in providing accurate RNA abundance profiles throughout the preimplantation period. However, the amplification appears to be template dependent because the amplification factors were higher for some genes.

Development and cell differentiation are driven by complex epigenetic mechanisms that regulate chromatin structure and specific gene transcription programs. We recently demonstrated that it is possible to modify the epigenetic signature of terminally differentiated cells, switching their phenotype into one of higher plasticity, through the use of molecules that remove epigenetic marks from DNA and histones (Pennarossa et al. 2013 Proc. Natl. Acad. Sci. 110, 8948–8953; Brevini et al. 2014 Stem Cell Rev. 10, 633–642). Here we drive mammalian fibroblasts into a high plasticity state using the epigenetic eraser, 5-aza-cytidine (5-aza-CR), and investigate whether the simultaneous use of a micro-bioreactor culture system is able to promote three-dimensional (3D) cell rearrangement, boost the induction of high plasticity, and stably maintain it. To this purpose, fibroblasts were either plated on plastic dishes (Group A) or encapsulated in a liquid marble micro-bioreactor (polytetrafluoroethylene powder; Sigma 430935, St. Louis, MO; Group B). Both groups were erased with 5-aza-CR and cultured in embryonic stem cell medium for 28 days. Morphological analysis was carried out for the entire length of the experiment. The OCT4, NANOG, and REX1 expression levels were assessed by real-time PCR at different time points. Exposure to 5-aza-CR induced a dramatic change in morphology in Group A fibroblasts. Cells became rounded, with larger and granulated nuclei and retained a monolayer distribution for the entire length of the experiment. The same changes in cell and nuclear morphology were observed also in cells encapsulated in liquid marble (Group B). In addition, these cells formed 3D spherical structures that were stably maintained until Day 28. These morphological rearrangements were accompanied by the active expression of the pluripotency markers, OCT4, NANOG, and REX1, in both groups. However, while Group A cells progressively down-regulated their expression by Day 6, Group B cells steadily transcribed these genes until Day 28, when cultures were arrested. Altogether, the data confirm that epigenetic erasing induces a high plasticity state in terminally differentiated fibroblasts with the expression of pluripotency related genes. Striking morphological changes accompanied the removal of epigenetic marks. These were influenced by the use of an adequate 3D in vitro culture system, with the induction of distinctive cell rearrangements and the formation of spherical structures that boosted and maintained cell plasticity. These results suggest a correlation between the mechanotransduction pathways induced by the micro-bioreactor culture system and the epigenetic regulation of cell phenotype. Study was supported by Carraresi Foundation. Authors are members of the COST Actions FA1201, BM1308 and CM1406.

Different cell types have been suggested as candidates for use in regenerative medicine. Embryonic pluripotent stem cells can give rise to all cells of the body and possess unlimited self-renewal potential. However, they are unstable, difficult to control and have a risk of neoplastic transformation. Adult stem cells are safe but have limited proliferation and differentiation abilities and are usually not within easy access. In recent years, induced pluripotent stem (iPS) cells have become a new promising tool in regenerative medicine. However, the use of transgene vectors, commonly required for the induction of iPS cells, seriously limits their use in therapy. The same problem arising from the use of retroviruses is associated with the use of cells obtained through transdifferentiation. Developing knowledge of the mechanisms controlling epigenetic regulation of cell fate has boosted the use of epigenetic modifiers that drive cells into a ‘highly permissive’ state. We recently set up a new strategy for the conversion of an adult mature cell into another cell type. We increased cell plasticity using 5-aza-cytidine and took advantage of a brief window of epigenetic instability to redirect cells to a different lineage. This approach is termed ‘epigenetic conversion’. It is a simple, direct and safe way to obtain both cells for therapy avoiding gene transfection and a stable pluripotent state.

In recent years, cryo and marine biologists started new approaches to prevent extinction of underwater species. Great efforts have been addressed in order to set up and optimize protocols for freezing corals in liquid nitrogen. In particular, results have been described to freeze corals from the Great Barrier Reef and Hawaii. In this chapter, we report coral cryopreservation techniques and some recently published data.

The preservation of biological material is a fundamental requirement in basic and medical science and has generated great interest in the scientific community since 1949.

Polychlorinated biphenyls (PCBs) are one of the most persistent and widespread group of endocrine disrupting compounds in the ecosystem. High concentrations of these substances are known to be present in sewage sludge from industrial, agricultural, and domestic origin that is spread in increasing amounts on arable land and pasture as fertilizer and is found in water, representing an increasing risk for the reproductive health of farm animals. Objective of this study was to determine the impact of PCBs on maturation and developmental competence of cattle oocytes. Since PCBs are a family of 209 molecules present in the environment as a mixture, Aroclor-1254, a pool of more than 60 congeners, was used in these experiments as its composition is considered to be environmentally relevant. Cumulus-oocytes complexes were exposed during IVM to serial concentrations of Aroclor-1254 (between 1 microg/ml and 0.0001 microg/ml) and compared with control groups. Aroclor decreased the percentage of oocytes that reached metaphase II stage after 24 hr, at doses as low as 0.01 microg/ml. Groups treated with 0.001 microg/ml or above, showed an impaired fertilization rate and a dramatic increase of polyspermy. Moreover, exposure during maturation resulted in a reduced proportion of oocytes that cleaved and developed until blastocyst stage although no differences in embryo cell numbers were observed. The present study indicates that very low PCBs concentrations are sufficient to disrupt bovine oocyte maturation, its fertilization, and developmental competence. These results also provide a set of reference data for the assessment of the risk posed by these substances to animal reproductive health, though further work will be necessary to equate in vitro doses to in vivo exposures.

Follicle development is the result of a balanced ratio between cell proliferation and cell death. Previous studies demonstrated differential mitotic responses to insulin-like growth factor (IGF)I and epidermal growth factor (EGF) of cumulus cells (CC) and mural granulosa cells (MGC). Because cell-to-cell contact seems to modulate the occurrence of programmed cell death, the present experiments investigated the role of cell association in mediating apoptosis and the mitogenic responses to these growth factors of CC and MGC. Cumulus cells were cultured either as intact cumulus-oocyte complexes (COC) or after dissociation with EGTA 1 sucrose, in the presence of 50 ng/ml IGF-I, 5 ng/ ml EGF, or both. Mural granulosa cells from the same follicles were similarly cultured either as cell aggregates or as dissociated cells. Synthesis of DNA was assessed by measurement of [ 3 H]thymidine incorporation during the last 6 h of a 24-h culture in TCM199. Percentages of cells undergoing apoptosis were determined immunohistochemically in intact COC and GC aggregates, before and after dissociation as well as after the culture period. Epidermal growth factor and IGF-I stimulated DNA synthesis in both cell types; however, EGF inhibited the action of IGF-I in intact COC but not in MGC. Compared to nondissociated cells, dissociation resulted in a reduction of the mitogenic response of CC to both growth factors and of MGC to EGF. Unlike the response of intact COC to combined treatment with the two growth factors, dissociated CC displayed additive responses to the two growth factors in combination. Addition of denuded oocytes to cultures of dissociated CC enhanced both basal and growth factor-stimulated DNA synthesis but did not restore the inhibitory effect of EGF on the IGF-I response characteristic of intact COC. A significant proportion of intact MGC aggregates underwent apoptosis after 24 h of culture, while no increase of apoptotic cells was observed in intact COC. A dramatic increase in the percentage of apoptotic cells was observed in both CC and MGC when cell-cell contact was interrupted, and EGF and IGF-I were able to partially prevent its occurrence. Taken together these data showed that CC and MGC exhibit qualitatively and quantitatively different responses to IGF-I when cultured in the presence of EGF both in terms of DNA synthesis and onset of apoptosis. Moreover, the disruption of

The idea of using a sperm cell for introducing exogenous DNA into an oocyte at the time of fertilization would be brilliant if only we were sure that it can be done. Since 1989, contradictory reports have appeared in the literature and, at present, no consensus has been reached on the topic. Given the potential impact of this method for the generation of transgenic animals, for both mammalian and non-mammalian species, this review summarizes what has been achieved in this field. While some aspects, such as the binding of DNA molecules to spermatozoa, have now a solid experimental base, others, such as the generation of real transgenic individuals, are still based on disputed evidence. A critical analysis of the most relevant data will be presented in order to provide the tools for an objective evaluation of the efficiency of this method.

Serum, gonadotrophins, growth factors, and steroid hormones stimulate the in vitro maturation (IVM) of competent oocytes, acting, directly or indirectly, upon the adenylate cyclase pathway to produce the intracellular messenger, cAMP. The intracellular levels of cAMP in cattle cumulus-oocyte complexes (COC) were manipulated by adding to the collection and maturation media invasive adenylate cyclase (iAC), a toxin produced by the bacterium, Bordetella pertussis. High concentrations of iAC (1 or 5 microgram/ml) in the maturation medium inhibited the resumption of meiosis, while low concentrations (0.1 or 0.01 microgram/ml) resulted in high rates of maturation to the MII stage (92.6 +/- 2.5 and 98.5 +/- 1.4% respectively). The same low concentrations of iAC in the maturation medium resulted in rates of development to the blastocyst stage 8 days post insemination (30.1 +/- 4.2 and 45.1 +/- 3.9%, respectively), which were either not different, or significantly better, than those obtained after IVM in medium supplemented only with serum and gonadotrophins (36.1 +/- 2.9%). Finally, the addition of 0.1 microgram/ml iAC and 0.5 mM 3-isobutyl 1-methylxanthine (IBMX) in the collection medium significantly improved the blastocyst rate when IVM was performed in control medium or medium supplemented with 0.01 microgram/ml iAC (31.9 +/- 5.5 vs. 12.1 +/- 1.6 and 45.5 +/- 2.9 vs. 19.1 +/- 2.3% respectively). It is concluded that the maintenance of an optimal intracellular concentration of cAMP before and during IVM ensures a high developmental competence of bovine oocytes matured in medium without serum and hormones. Mol. Reprod. Dev. 54:86-91,1999.

We describe isolation and characterization of the bovine ortholog of POU5F1 (bPOU5F1) encoding octamer-binding transcription factor-4 (Oct-4). The organization of bPOU5F1 is similar to its human and murine orthologs, and it shares 90.6% and 81.7% overall identity at the protein level, respectively. Transient transfection of luciferase reporter constructs in murine P19 embryonal carcinoma cells demonstrated that bPOU5F1 has a functional promoter and contains two enhancer elements, of which one is repressed by retinoic acid. bPOU5F1 was mapped to the major histocompatibility complex on chromosome 23. bPOU5F1 mRNA was detected by nested reverse transcriptionpolymerase chain reaction in immature oocytes and in in vitroproduced preattachment-stage embryos. Oct-4 in oocytes and in vitro-produced preattachment-stage embryos was demonstrated by indirect immunofluorescence. Confocal laser scanning microscopy revealed Oct-4 in both the inner cell mass and trophoblast cells of the blastocyst until Day 10 of development. Immunofluorescence performed on the outgrowths formed at Day 13 postfertilization from in vitro-produced Day 8 blastocysts showed Oct-4 staining in all cells. This expression pattern suggests that bPOU5F1 acts early in bovine embryonic development but that its expression is not restricted to pluripotent cells of the blastocyst.

To determine possible causes of reported differences between developmental competence of oocytes isolated from prepubertal (10- to 14-week-old calves) and adult cows, three parameters were analysed, comparatively, during in vitro maturation (IVM): (1) oocyte diameter, (2) oocyte energy metabolism, and (3) protein synthesis of oocytes and cumulus cells. Cumulus-oocyte complexes were isolated from follicles of 3-5 mm in diameter in both age groups. Mean oocyte diameter was smaller (P < 0.02) in calves than in cows (118.04 +/- 1.15 versus 122.83 +/- 0.74 microns). During the first 3 hr of IVM, calf oocytes metabolised glutamine and pyruvate at lower rates than adult oocytes, but after 24 hr of culture, both molecules were metabolised at the same rate as for adult oocytes. A significant decrease in protein synthesis, as measured by [35S]methionine and [35S]cysteine incorporation was recorded after 9 hr of IVM in calf oocytes, while in adult oocytes a significant decrease in protein synthesis was detected only after 24 hr. After the first 3 hr of maturation, proteins of 130, 26, and 24 kDa were more abundant in adult than in calf oocytes, while a protein of 55 kDa was more visible in calf than in adult oocytes. At the same time, among proteins newly synthesised by cumulus cells, molecules of 405, 146, 101, and 77 kDa were more abundant in adults than in calves. In conclusion, calf oocytes and cumulus cells showed several differences when compared with their adult counterparts, which are consistent with their reported lower developmental competence.

The effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on the maturation and subsequent development of cattle oocytes in vitro were evaluated in three experiments. Cumulus\p=n-\oocytecomplexes (COC) were collected from cattle ovaries and matured for 20\p=n-\24h in control medium or in medium containing 50 ng EGF ml \m=-\1, 100 ng IGF-I ml\m=-\1, EGF + IGF-I, or 10% (v/v) fetal calf serum plus 0.1 i.u. human menopausal gonadotrophin ml \m=-\ 1 (hMG). In Expt 1, treatment with EGF + IGF-I stimulated cumulus expansion, the metabolism of pyruvate and glutamine, and nuclear maturation. In Expt 2, only the metabolic measurements from oocytes that reached metaphase II were considered, and EGF + IGF-I stimulated pyruvate metabolism to the same extent as serum + hMG. In Expt 3, the oocytes were fertilized after maturation culture, and the resultant embryos cultured for up to 8 days. The cleavage was greater in the EGF and EGF + IGF-I groups than in the controls but less than in the serum + hMG group. Moreover, the number of blastocyst cells at 7 days after insemination and the proportion of cleaved embryos that developed to the blastocyst stage by day 8 was greater in the serum + hMG group than in the control group indicating that maturation treatment can affect early embryonic development. In conclusion, EGF + IGF-I can stimulate cumulus expansion, oxidative metabolism, nuclear maturation and cleavage after fertilization of bovine oocytes in vitro. The relative effects of the treatments on oocyte pyruvate metabolism in Expts 1 and 2 generally paralleled their effects on cleavage and subsequent development in Expt 3, suggesting that mitochondrial function is related to developmental potential. Further investigation is required to determine which component(s) of serum or gonadotrophin treatment is responsible for the effects on subsequent embryonic development.

The idea that sperm cells could be used as an effective tool for introducing exogenous DNA into an oocyte at fertilization is generally regarded with scepticism. However, in recent years, several investigators have been working on different aspects of this intriguing research topic. In the present review, their results are summarised and discussed. Sections have been dedicated to the way DNA molecules bind to spermatozoa of different species, to the events regulating such binding, to the fate of the DNA within sperm cells, and to the attempts made to produce transgenic animals with this method. The data available on the interaction between DNA and spermatozoa begin to explain how this event takes place and how it is regulated. However, the stable integration of exogenous genes into the genome of adult animals mediated by sperm cells is a very rare event, although several reports describe forms of partial success. Available evidence suggests that changes to the DNA molecules, oc curring mostly within the oocyte, represents the limiting step in the production of transgenic animals using spermatozoa as vectors of exogenous genes. At present there are not enough data to understand what happens to sperm-associated DNA upon its entrance into the oocyte at fertilization. Therefore, it has not yet been resolved whether sperm-mediated gene transfer is a possible way to manipulate the genome or if evolution has imposed some unsurpassable barriers to its use

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.

Co-culture remains a common method to support the development of bovine embryos, derived from IVM/IVF procedures. However, the mechanism by which somatic cells confer their benefit to the developing embryo remains undetermined. This study therefore analysed the changes made to the culture medium TCM-199, used in bovine embryo co-culture systems, by somatic cells and determined the effects of specific changes in medium composition on bovine embryo development in culture. Bovine oviduct epithelial (BOE), Buffalo rat liver (BRL) and fibroblast (3T3) cells were compared. The concentrations of glucose, L-lactate, pyruvate, amino acids, NH4+, H+ and the gas tensions of O2 and CO2 were measured in TCM-199 supplemented with 10% fetal calf serum (FCS) prior to and directly following 48 h incubation periods with each cell type. All three somatic cell types modified the carbohydrate composition of the media in a similar manner with the greatest changes made by the BOE cells. Notable alterations were an increase in the levels of L-lactate and pyruvate and a reduction in glucose concentration, which in the case of the BOE cells, fell from 5.55 mM to 2.67 mM. In order to determine the relevance of such changes in carbohydrate concentrations on bovine embryo development, modifications were made to carbohydrate levels in synthetic oviduct fluid (SOF) medium and their effect on blastocyst development in vitro assessed. In SOF medium supplemented with amino acids and BSA (SOFaa), significantly more zygotes developed to the blastocyst stage (64%; P < 0.01) than in SOFaa medium with the concentrations of glucose, D/L-lactate and pyruvate equivalent to those in TCM-199 (11%). Interestingly, when the levels of carbohydrates in SOFaa mimicked those present in TCM-199 following a 48 h incubation with BOE cells, 57% of zygotes reached the blastocyst stage. This improvement was ascribed to the reduction in glucose and increases in D/L-lactate and pyruvate concentrations in the culture system. Results from this study demonstrate that BOE cells create an environment favourable to embryonic development. The analysis of media samples by enzymatic methods meant that only the biologically active L-isomer of lactate was quantified. However, in SOFaa, both the L-isomer and inactive D-isomer are present in equimolar amounts. As such, culture media in which D/L-lactate syrup is used actually contain only 50% biologically active lactate meaning that all D/L-lactate concentrations are reported at twice the effective concentration. Therefore the effect of D/L-lactate concentration on blastocyst development was subsequently determined in this study. Blastocyst development was poor (24-36%) until the total D/L-lactate was present in the culture system at concentrations equal to or greater than 0.82 mM. However, blastocyst cell numbers remained low (60.1 +/- 6.9 - 78.5 +/- 6.6) until a total D/L-lactate concentration of 3.3 mM. This data reinforces that embryo morphological appearance is not sensitive enough to be used as the sole criterion for assessing embryo development.

Does directional freezing improve the structural and functional integrity of ovarian fragments compared with conventional slow freezing and to whole ovary cryopreservation?Compared with slow freezing, the use of directional freezing significantly improves all structural and functional parameters of ovarian fragments assessed in vitro and, overall, whole ovaries were better preserved than ovarian fragments.Directional freezing has been developed to provide an alternative way to cryopreserve large biological samples and it is known to improve the structural and functional integrity of whole ovaries. Conventional slow freezing of ovarian fragments is the procedure more widely used in clinical settings but it causes substantial structural damage that limits the functional period after transfer back into the patient.We performed a 2 × 2 factorial design experiment on a total of 40 sheep ovaries, divided into four groups (n = 10 ovaries per group): (i) directional freezing of whole ovary (DFwo); (ii) directional freezing of ovarian fragments (DFof); (iii) conventional freezing of whole ovary (CFwo); (iv) conventional freezing of ovarian fragments (CFof). An additional eight ovaries were used as fresh controls.Ewe ovaries were randomly assigned to one of the experimental groups and frozen accordingly. Upon thawing, ovarian tissue was examined morphologically and cultured in vitro for 7 days. Samples were analyzed for cell proliferation and apoptosis, for DNA damage and repair activity, and for the presence of a panel of heat shock proteins (HSPs) by immunohistochemistry.Most studied parameters were significantly improved (P0.05) in all samples cryopreserved with directional compared with slow freezing. The proportion of primordial follicles, which developed to the primary stage in whole ovaries (53 ± 1.7%) and in ovarian fragments (44 ± 1.8%) cryopreserved with directional freezing, was greater than with slow frozen whole ovaries (6 ± 0.5%, P = 0.001) or fragments (32 ± 1.5%, P = 0.004). After 7 days of culture, cell proliferation in DFwo (28 ± 0.73%) was the highest of all groups (P0.05) followed by DFof (23 ± 0.81%), CFof (20 ± 0.79%) and CFwo (9 ± 0.85%). Directional freezing also resulted in a better preservation of the cell capacity to repair DNA damage compared with slow freezing both in whole ovaries and ovarian fragments. Apoptosis and HSP protein levels were significantly increased only in the CFwo group. Direct comparison demonstrated that, overall, DFwo had better parameters than DFof and was no different from the fresh controls.The study is limited to an in vitro evaluation and uses sheep ovaries, which are smaller than human ovaries and therefore may withstand the procedures better.Improved integrity of ovarian morphology may translate to improved outcomes after transplantation. Alternatively, the particularly good preservation of whole ovaries suggests they could provide a source of ovarian follicles for in vitro culture in those cases when the presence of malignant cells poses a substantial risk for the patient.Supported by: Associazione Italiana per la Ricerca sul Cancro (AIRC) IG 10376, Carraresi Foundation and by Legge 7 Regione Autonoma Sardegna (R.A.S). There are no conflicts of interest.

The derivation of stem cells in domestic animal species represents a great improvement in developmental biology and could provide a powerful tool for regenerative medicine and genetic engineering. Despite the enormous efforts and work dedicated to the establishment of stem cell lines from large animals, no conclusive results have been obtained so far, and validated lines have yet to be established.

Regenerative medicine requires preclinical trials of new therapies before starting human studies. In this context, animal models play a fundamental role for investigating biological and functional activities of new cells and tissues. The rodent species is extensively used in studies related to stem cell biology, providing important information, but at the same time its use has important limitations for a variety of disease categories because of the different body size and physiology relative to humans. Large animal species, such as dogs, pigs, sheep, cattle, horses, and nonhuman primates, are better predictors of human responses than rodents, but in each case it will be necessary to choose the best model for a specific application.

In this chapter, all the main events taking place during the first phases of embryo development are examined. Syngamy and the first mitotic spindle formation are described, as well as the processes of cleavage, compaction, and blastulation. The importance of the integrity of the first mitotic spindle is highlighted because it is fundamental for normal and correct embryo development and cell commitment. Gastrulation and subsequent inner cell mass cell differentiation with the definition of the three germ layers and establishment of the body axis are also illustrated. In fact, each cell constituting an embryo is the target, as well as the source, of specific signals. These signals regulate cell differential gene expression and epigenetic restrictions that gradually limit cell potency to a phenotype-related expression pattern. The hypothesis according to which each cell present in the embryo maintains a memory of its own proliferation history and positional changes during development is also discussed.

Each oestrous cycle the ovary produces one or more oocytes, depending on the species considered, which are fully competent to sustain the development of a new individual and are defined as matured. Ovulation is the terminal phase of a lengthy and complex selection process which enables only a minute proportion of the oocytes present in the ovary to be ovulated and possibly fertilised. The use of exogenous gonadotrophins for the pharmacological stimulation of the ovaries has clearly indicated that oocytes naturally excluded by the selection process can also be matured and, if fertilisedin vivoorin vitro, can generate normal offspring.

Studies on embryonic development in vitro as well as observations in vivo, suggested that two aspects of oviduct physiology are important for early development. On one side has to be considered the oviduct "environment": temperature, pH, osmotic pressure, nutrients, oxygen tension, free radical scavengers, etc. On the other, the oviduct "active components": stimulatory and/or regulatory molecules, supposed to finely regulate the fertilisation process and the first differentiative steps. While the physical environment of the oviduct has been under investigation for some decades, studies on oviduct-specific molecules and their functions have only been developed much more recently. The amount of information on this topic, however, has rapidly reached the size that demands a summary. In this review the descriptive literature on oviduct specific proteins will be examined as a basis for illustrating the possible functions of these molecules. In particular their role in fertilisation and early embryonic cleavages will be analysed in some details. Finally a section is devoted to the presence and physiological significance of growth factors in oviduct fluid.

Gene expression in early stage embryos relies mostly on post-transcriptional control of maternal transcripts accumulated during oocyte maturation. However, while the building process to obtain a competent oocyte is now better understood, it is still not clear what transcriptome blueprint composes a competent oocyte. The aim of the study was to compare the mRNA expression pattern between oocytes collected from fertile heifers and repeat breeders by using RNAseq. Oocytes were collected by ovum pickup from 3 heifers that were 11–15 months of age and became pregnant at the following oestrus and from 4 adult cows with an age of 4 to 7 years, classified as repeat breeders after they failed to become pregnant for a minimum of 3 consecutive AI. To obtain oocytes from follicles with the same degree of development, at time 0 all follicles visible through transrectal ultrasound examination were removed by transvaginal aspiration. Five days later oocytes were collected by ovum pick up from the newly formed follicles with diameters >5 mm. Oocytes from each animal were pooled and sequenced as a single sample. Total RNA was extracted by RNeasy Micro Kit (Qiagen, Valencia, CA, USA). Amplified cDNA, was prepared starting from total RNA using the Ovation RNA-Seq System V2 (Nugen Technologies, San Carlos, CA, USA). After library preparation with TruSeq DNA Sample Prep kit (Illumina, Madison, WI, USA), sequencing was performed on an Illumina HiSEqn 2000. Galaxy and Chipster open web-based platforms were used to analyse the data. We identified 49 differentially expressed genes. Heifers’ oocytes mRNA pattern indicated greater potential to sustain cell division. In particular, oocytes expressed more Keratin 14 (a gene involved in cell proliferation) and kinesin family member 20B (a protein involved in cytokinesis). More competent oocytes also have a greater ability to repair single-strand breaks due to the high levels of endo/exonuclease (5′-3′), endonuclease G-like. This may reflect greater capacity to neutralise DNA damage and, therefore, greater ability to preserve and transmit high-quality DNA. Repeat breeders portray a different landscape; their greater expression of Jun oncogene, Heat shock protein 1, Stimulated by retinoic acid gene6, arylhydrocarbon receptor nuclear translocator, fibromodulin, and aryl-hydrocarbon receptor repressor suggest that these oocytes have been subjected to environmental stress during oocyte maturation. Their greater expressions of inhibin α, stearoyl-CoA desaturase, junctional adhesion molecule 2 have been previously shown to correlate with a reduced oocyte developmental potential. Furthermore, the Cannabinoid receptor protein 1 expression suggests a compromised ion function that can lead to a failed activation of the development program. Finally, the greater expression of Ubiquilin3 and Heat shock protein 1 led to high protein and mRNA degradation, respectively, suggesting that these oocytes are deprived of essential components to sustain embryo growth. In conclusion our data provide the first detailed snapshot of the mRNA pattern defining the differences between a competent oocyte and an incompetent oocyte in vivo. Study supported by PRIN 2008, 2009 and EU-Quantomics.

Oocyte developmental competence is correlated with antral follicle count through ill-defined mechanisms. Oocytes from ovaries with fewer than 10 mid-antral follicles of 2 to 6 mm in diameter (low group) show reduced competence compared with those from ovaries with more than 10 follicles (high group). To unravel mechanisms underlying this phenomenon, this work explored the role of follicular fluid microRNAs (miRNAs, short non-coding RNAs regulating gene expression at the post-transcriptional level). A total of 3 pools of 300 µL of follicular fluid (FF) were collected from mid-antral follicles of low (L) and high (H) groups, respectively. Following miRNA extraction and library preparation, deep sequencing was carried out on Illumina HisEqn 2000 (Illumina Inc., San Diego, CA, USA). Differentially expressed miRNAs were identified with R package edgeR (http://bioconductor.org/packages/release/bioc/html/edgeR.html). Target genes of differentially expressed miRNAs were predicted with DIANA miRPath using homologous human miRNA and gene union options (P 0.05). We found that 27 miRNAs were differentially expressed (false discovery rate ≤0.001): 17 were up-regulated in L and 10 in H. Up-regulated miRNAs in L group were predicted to target 121 genes, 39 of which are specific for ovarian function (e.g. BCL2, FOXO3, KIT, TP53, and PTK2). The GO analysis indicated that these genes were kinases, anti-apoptotic and oncogenic factors, and enriched stress-activated MAPK cascade mediated by oxygen reactive species, G1/S transition checkpoint, cellular response to interleukin-1, and negative regulation of cellular adhesion. Overexpressed miRNAs in H group were predicted to target 92 genes, 22 of which (e.g. MAPK, APC, JNK, PKA) are involved in folliculogenesis. These genes were represented by kinases, apoptotic and cytoskeleton remodelling factors, and enriched very important ovarian processes such as cell cycle, ephrin receptor, smoothened and phosphatidylinositol 3-kinase activity GO processes. Only 7 target genes were common between the 2 groups, 2 of which were important in ovarian functionality (CDKN1A and ITGA5). Interestingly, overexpressed miRNAs in both groups regulate several genes involved in processes apparently not related to folliculogenesis. Finally, regulation can be exerted also by low levels of specific miRNAs such as miR-320, which was reduced in L group and is known to be associated with premature ovarian senescence in women and decreased developmental potential of mouse oocytes. Our results indicate that the different oocyte quality is associated with a different miRNA blueprint, which may alter the expression of several genes relevant for intra- and extra-ovarian processes. Further studies will be necessary to determine if FF miRNAs can act outside the ovary and if their levels can be detected in the bloodstream thereby becoming possible noninvasive, real-time markers to determine oocyte quality in living animals. This study was supported by FP7-KBBE-2012-FECUND-312097.

We describe an original perfusion system for the culture of whole ovine ovaries for up to 4 days. A total of 33 ovaries were divided into six groups: control (n = 6), not perfused and fixed; Groups SM72 and SM72-FSH (n = 6 each), perfused with a simple medium for 72 h with or without FSH; Groups CM96 and CM96-FSH (n = 6 each), perfused with a complex medium for 96 h with or without FSH; Group CM96-FSH-cryo, (n = 3) cryopreserved and perfused for 96 h with Group CM96-FSH medium. Depending on the medium used, morphological parameters of cultured ovaries differed from fresh organs after 72 (SM72, SM72-FSH) or 96 (CM96, CM96-FSH) h of perfusion. Oestradiol and progesterone were secreted in all groups but FSH had an effect only on Group CM96-FSH, stimulating continued oestradiol secretion 10 times higher than in all other groups. Morphological parameters and hormone secretion of cryopreserved ovaries were not different from fresh controls. This method enables the culture of whole ovaries for up to 4 days, the time required in vivo for 0.5-mm follicles to grow to 2.2 mm and then for these follicles to reach the ovulatory size of 4 mm or more. It could be used as a research tool or to complement current techniques for preserving female fertility.

Pluripotency and commitment are driven by transcription factors that act as molecular switches to activate or repress specific gene expression programs. Among these, the POU transcription factor OCT4 (encoded by POU class 5 homeobox 1, POU5F1) is known to be a regulator in pluripotent cells and it is essential for the maintenance of a high plasticity state in mammalian cells. In recent years, particular attention has been given to the importance of physical extracellular matrix properties in regulating pluripotent cell maintenance and differentiation. In particular, recent studies demonstrated that high plasticity cells are intrinsically soft and respond optimally to physical forces when cultured on a “low stiffness” substrate that matches their intrinsic softness. To investigate these aspects, here we used a protocol where terminally differentiated cells are driven into a high plasticity/OCT4-positive state through the use of the epigenetic eraser, 5-aza-cytidine (5-aza-CR) and we observed the effect of substrate stiffness on the maintenance of OCT4 expression. To this purpose, human skin fibroblasts were plated either on standard plastic dishes (group A) or on polyacrylamide gels (PAA) of low stiffness (1 kPa; group B). Cells were treated with 1 μM 5-aza-CR for 18 h and were subsequently maintained and cultured in embryonic stem cell medium for 14 days. Assessment of OCT4 was carried out both by real-time PCR and immunocytochemical analysis, at different time points: untreated fibroblasts (T0), after 5-aza-CR treatment, and on Days 2, 4, 6, 8, 10, and 14 of culture. The results obtained indicate that untreated fibroblasts (T0) were negative for OCT4, whereas, after 5-aza-CR exposure, cells actively expressed the pluripotency marker, in both the A and B groups. However, whereas group A cells progressively down-regulated OCT4 expression and eliminated its positivity by Day 6, group B cells steadily transcribed and were homogeneously immunopositive for the pluripotency factor until Day 14, when cultures were arrested. Overall, the data obtained suggest that a soft substrate, matching the intrinsic stiffness distinctive of high plasticity cells, may help in promoting their maintenance, via a biophysical mechanism of low-traction/low-stiffness-dependent manner. Study was supported by Carraresi Foundation.

Reduced oocyte competence causes the lower fertility reported in domestic sows during the warm months of the year. Somatic cells express heat shock proteins (HSPs) to protect themselves from damage caused by thermal stress. HSPs are classified as molecular chaperones and control the correct folding of newly synthesized or damaged proteins. The present work performed a comprehensive survey of the different components of the heat shock chaperone machinery in the pig ovary, which included the HSP40, HSP70, HSP90, and HSP110 families, as well as heat shock factors (HSF) 1 and 2. Pig ovarian follicles constitutively expressed different members of these families; therefore, we examined their ability to respond to heat stress. In order to take into account the role of the complex follicular architecture, whole pig ovaries were exposed to 41.5°C for 1 h. This exposure significantly disrupted oocyte maturation and determined the upregulation of the HSP70, HSP40, HSPH1, HSPA4, HSPA4L, HSF1, and HFS2 genes, whereas expression levels of HSP90A and HSP90B, as well as those of genes unrelated to heat stress were not altered. Unexpectedly HSP and HSF expression levels changed only in oocytes but not in cumulus cells. Cumulus-oocyte complexes isolated from ovaries collected in summer showed the same pattern as those collected in winter. We conclude that the HSP chaperone machinery is constitutively fully operational in the pig ovary. However, following thermal stimuli or seasonal variations, cumulus cell HS-related gene expression remains unchanged, and only oocytes activate a response, suggesting why this mechanism is insufficient to preserve their competence both in vitro and in vivo.

Reduced oocyte competence causes the lower fertility reported in domestic sows during the warm months of the year. Somatic cells express heat shock proteins (HSPs) to protect themselves from damage caused by thermal stress. HSPs are classified as molecular chaperones and control the correct folding of newly synthesized or damaged proteins. The present work performed a comprehensive survey of the different components of the heat shock chaperone machinery in the pig ovary, which included the HSP40, HSP70, HSP90, and HSP110 families, as well as heat shock factors (HSF) 1 and 2. Pig ovarian follicles constitutively expressed different members of these families; therefore, we examined their ability to respond to heat stress. In order to take into account the role of the complex follicular architecture, whole pig ovaries were exposed to 41.5°C for 1 h. This exposure significantly disrupted oocyte maturation and determined the upregulation of the HSP70, HSP40, HSPH1, HSPA4, HSPA4L, HSF1, and HFS2 genes, whereas expression levels of HSP90A and HSP90B, as well as those of genes unrelated to heat stress were not altered. Unexpectedly HSP and HSF expression levels changed only in oocytes but not in cumulus cells. Cumulus-oocyte complexes isolated from ovaries collected in summer showed the same pattern as those collected in winter. We conclude that the HSP chaperone machinery is constitutively fully operational in the pig ovary. However, following thermal stimuli or seasonal variations, cumulus cell HS-related gene expression remains unchanged, and only oocytes activate a response, suggesting why this mechanism is insufficient to preserve their competence both in vitro and in vivo.

Parthenotes have been proposed as a source of embryonic stem cells but they lack the centriole which is inherited through the sperm in all mammalian species, except for rodents. We investigated the centrosome of parthenotes and parthenogenetic embryonic stem cells using parthenogenetic and biparental pig pre-implantation embryos, human and pig parthenogenetic and biparental embryonic stem cells, sheep fibroblasts derived from post implantation parthenogenetic and biparental embryos developed in vivo. We also determined the level of aneuploidy in parthenogenetic cells. Oocytes of all species were activated using ionomycin and 6-dimethylaminopurine (6-DMAP). Over 60% of parthenogenetic blastomeres were affected by an excessive number of centrioles. Centrosome amplification, was observed by microscopical and ultrastructural analysis also in parthenogenetic cell lines of all three species. Over expression of PLK2 and down regulation of CCNF, respectively involved in the stimulation and inhibition of centrosome duplication, were present in all species. We also detected down regulation of spindle assembly checkpoint components such as BUB1, CENPE and MAD2. Centrosome amplification was accompanied by multipolar mitotic spindles and all cell lines were affected by a high rate of aneuploidy. These observations indicate a link between centrosome amplification and the high incidence of aneuploidy and suggest that parthenogenetic stem cells may be a useful model to investigate how aneuploidy can be compatible with cell proliferation and differentiation.

In mammals, embryonic development is the result of a delicate interaction between the genetic program in the chromosomes of each zygote and the female genital tract. Although an accurate formulation of the culture medium allows an increasing proportion of embryos to develop in defined conditions, they are often affected by various degrees of abnormalities. This indicates a persistent inadequacy of the culture systems both with or without somatic cells. Current studies on the physiology of preimplantation embryos and of the oviduct indicate that several factors are involved in this delicate period of cell proliferation and differentiation. These have here been divided in three groups: autocrine factors, mostly growth factors, generated by the embryo for sustaining its own development; paracrine factors, growth factors and specific proteins, generated by the oviduct which reach the conceptus; environmental factors, various substances - i.e. energy substrates, ions, amino acids, vitamins - which generate the “perfect” medium for an embryo to grow. Recent data suggest that successful embryonic development requires the balanced combination of all these factors and culture techniques based on this principle are giving very promising results.

Connection between mastitis and fertility is multifaceted; therefore, several aspects need more elucidation. In particular, the aim was to investigate if naturally occurring chronic mastitis has an effect on ovarian function. At the time of slaughter, a milk sample and both ovaries were collected from 68 cows. The presence and intensity of chronic mastitis was diagnosed by the combined evaluation of bacteriological examination and somatic cell count of the milk of each individual quarter according to the measures of the National Mastitis Council. Animals were divided into 4 groups characterized by a low (n=15), mild (n=14), intense (n=19), or severe (n=16) degree of infection. A count of visible follicles on each ovary was followed by a quantitative analysis of microscopic traits on a selected group of animals (n=16). The latter included the classification and count of the entire preantral follicle population, and the morphometric analysis of the vascular bed extension and connective stroma in the cortical region. Finally, the expression of growth and differentiation factor-9 (GDF-9) was studied. The number of follicles with diameters ranging from 1 to 3 mm and 4 to 7 mm was not affected by the degree of infection. A significant effect of the degree of udder infection was observed on the number of follicles with a diameter larger than 8 mm. Furthermore, the intensity of mastitis had no effect on the number of primordial and primary follicles, but severely affected cows showed a lower number of secondary follicles (0.5±0.1 vs. 0.2±0.03). Quantitative analysis demonstrated a decrease in the density of blood vessels (6.30±1.08 vs. 4.68±0.28) expressed as ratio of vascular bed/total area) and a higher incidence of fibrous stroma (1.60±0.99 vs. 6.04±3.08 expressed as ratio of connective tissue/total area) in the cortical area of the most affected animals. Finally, the level of GDF-9 protein within the oocytes of different follicle size was lower in the animals with the severe form of chronic mastitis (1.34±0.05 vs. 0.78±0.21 expressed as arbitrary units). In conclusion, decreased fertility of cows with chronic mastitis takes place through an effect on the ovary altering the dynamics of folliculogenesis. Within the ovary, this implies a reduction of the vascular bed and an increase in the fibrotic tissue together with a direct effect on oocyte-specific factors as GDF-9, all of which are essential regulatory elements of folliculogenesis.

Events of reproduction occurring from meiotic resumption of the immature oocyte up to its exit from the second meiotic block following activation will be revealed. Morphological modifications of the oocyte during maturation will be related to signal transduction mechanisms involving primary or secondary messengers. A detailed view will be addressed to genetic and epigenetic control of oocyte maturation. At fertilization, reciprocal gamete activation and novel knowledge related to sperm factor and cascade mechanisms occurring in the oocyte will be focused. A detailed description of ion currents occurring during fertilization will depict another point of view of oocyte activation mechanisms, further supporting for their complexity. Finally, all basic information related to this short time lapse will be considered in relation to clinical application of assisted reproductive technologies. New frontiers, such as stem cells and cloning technologies, will be analyzed and future applications and improvements will be hypothesised.

Full oocyte competence is the indispensable requisite for embryonic development and, at present, no ways are known to restore competence if, for any reason, it has been even slightly compromised. The same is not true for sperm cells that can initiate and sustain development even if severely abnormal or damaged as long as the DNA is intact. This points clearly to the uneven burden carried by the two gametes and indicates clearly the essential role of the oocyte. Parthenogenesis is the obvious consequence of such disparity with a number of lower species capable of giving birth to new individuals without any paternal contribution. Mammals are an exception to the rule due to epigenetic mechanisms limiting the parthenotes developmental potential. However, the blastocyst stage is easily reached and parthenogenetic stem cells can be generated whose differentiation potential seems to be much wider than that of whole parthenotes. Switching perspective, we move from stem cells originated from oocytes to oocytes originated from stem cells. Since embryonic stem cells can colonize the germ cell lines when chimeras are generated, it was not so surprising that oocytes can be obtained from stem cells. Although there is still a long way to go before full competence is reached it clearly opens the way to the hypothesis of having an unlimited source of oocytes. Finally, a recent and highly controversial set of results suggests that oocytes are not in such a limited supply as it is generally believed but post-natal oogenesis takes place at a surprisingly high rate.

Cardiovascular disease is the leading cause of death in developed countries and is one of the leading causes of disease burden in developing countries. Therapies have markedly increased survival in several categories of patients, nonetheless mortality still remains high. For this reason high hopes are associated with recent developments in stem cell biology and regenerative medicine that promise to replace damaged or lost cardiac muscle with healthy tissue, and thus to dramatically improve the quality of life and survival in patients with various cardiomyopathies. Much of our insight into the molecular and cellular basis of cardiovascular biology comes from small animal models, particularly mice. However, significant differences exist with regard to several cardiac characteristics when mice are compared with humans. For this reason, large animal models like dog, sheep and pig have a well established role in cardiac research. A distinct characteristic of cardiac stem cells is that they can either be endogenous or derive from outside the heart itself; they can originate as the natural course of their differentiation programme (e.g., embryonic stem cells) or can be the result of specific inductive conditions (e.g., mesenchymal stem cells). In this review we will summarize the current knowledge on the kind of heart-related stem cells currently available in large animal species and their relevance to human studies as pre-clinical models.

In recent years, the development of methods for the genetic manipulation of domestic species has generated a rapidly increasing demand for pre-attachment embryos. The limited prolificacy of these species makes superovulation and surgical recovery of embryos necessary. However, these techniques are too expensive and labour-intensive to be used routinely for supplying enough material for experimental or commercial applications. This has provided the thrust for an unprecedented effort to develop methods for the culture of embryos derived from in vitro maturation and fertilization of oocytes collected from slaughtered animals. Offspring generated in vitro have been obtained using cattle, goats, pigs and sheep, but the efficiency and reliability of the techniques and the quantity of the embryos vary between species. At present, the best results can be obtained in ruminants, while pig embryos have proved to be more difficult to generate. Although many obstacles have been overcome simply by empirical trials and observations, the availability of high numbers of easily accessible embryos has also led to a substantial advance in our knowledge of their physiology. This has therefore widened the range of experimental models that can effectively be used in developmental studies, especially since, in some cases, models using these species may be more relevant to human embryology than those using rodents.

To develop a procedure for the analysis of gene expression in cumulus cells during the interval between ovum pick up and insemination to select the best oocytes for fertilization.Five RNA extraction methods, three reverse transcription procedures followed by Real-time quantitative PCR and one single-step mRNA quantification kit were tested to measure the expression of five genes in cumulus cells.Two RNA extraction kits gave the best combination of efficiency and purity. One reverse transcription procedure gave the best speed and efficiency. The single-step kit required more biological material than would be available from single cumulus oocyte complexes (COCs).Our test identified a combination of RNA extraction and reverse transcription procedures that enables the level measurement of 5 selected cumulus cell transcripts within 4 h. Using this combination it was possible to obtain a reliable quantification of gene expression in 44 out of 46 individual COCs collected from seven patients.

During the interval between fertilization and the process of implantation, some form of embryonic signalling is necessary for maternal recognition of pregnancy to occur and to cause the hormonal changes to elicit the uterine transformation necessary for implantation. Moreover, somatic cells provide specific molecules which are involved in embryonic transport and development. In this review only the following potential signal substances, for which recent progress has been reported, will be described and discussed. The earliest characterized embryo-maternal interaction is represented by the embryo-derived platelet activating factor/early pregnancy factor system. Prostaglandin E 2 , produced by horse embryos, is involved in their selective transportation to the uterus leaving unfertilized ova in the oviduct. The autocrine and paracrine role of growth factors of embryonic and/or maternal origin are discussed, together with the various oviduct specific proteins which become associated with developing embryos in several species. It is concluded that increasing sensitivity of available techniques is revealing new mechanisms of embryo-maternal interaction, which, although far from completely elucidating this complex matter, will bring new insights to the delicate period which precedes implantation.