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The present study was designed to investigate the relationship between pre-mating nutrition and the relative amounts of a panel of developmentally relevant genes in ovine oocytes and granulosa cells. Cast age ewes were fed a ration providing 0.5× (0.5 M) or 1.5× (1.5 M) live weight maintenance requirements for 2 weeks before slaughter. The ewes were synchronized and superovulated with FSH and pregnant mares serum gonadotropin. At slaughter, oocytes and granulosa cells were aspirated from follicles >2 mm in diameter and the relative abundance of 8 and 17 transcripts in oocytes and granulosa cells respectively were analyzed by semi-quantitative RT-PCR. In the oocytes, no differences between groups were observed for five transcripts (GDF9,BMP15, c-kit, glucose transporter 1 (SLC2A1), and hexokinase 1), but a lower amount of glucose transporter 3 (SLC2A3), sodium/glucose cotransporter 1 (SLC5A1), and Na+/K+ATPase mRNAs was detected in the 0.5 M group. Increased expression ofPTGS2,HAS2, and the leptin receptor long form was observed in granulosa cells from the 0.5 M group. No differences between groups were observed for the other transcripts (early growth response factor-1, estrogen receptor-α, LH and FSH receptors, gremlin 1, pentraxin 3, KIT ligand, glucose transporters 1, 3, and 8,IGF1, IGF1 receptor, leptin receptor, and tumor necrosis factor-stimulated gene 6). Expression of leptin and sodium/glucose cotransporter 1 was not detected in both groups. The present data indicate that pre-mating nutrition is associated with alteration in the mRNA content in oocytes and surrounding follicle cells in ewes, which may account for the reduced reproductive performance typical of ewes that are fed a restricted ration for a short period of time before mating.

Embryonic stem cells (ESCs) represent a promising tool for cell therapy, regenerative medicine and tissue repair. At the same time they constitute an invaluable model for basic investigations in developmental biology, nuclear reprogramming and differentiation process. ESCs are very unique due to their unlimited self-renewal ability and high plasticity that allow them to differentiate into all embryonic tissues. However, these properties have been so far only demonstrated in the mouse and, to a lesser extent, in man. Assessment of ESC capabilities in species different from the mouse is an ongoing topic of interest and is crucial in view of their potential use as experimental models in pre-clinical applications. The mouse model is not adequate when long-term effects of cell replacement need to be evaluated. The pig has been considered for a long time among the best models for pre-clinical development of therapeutic approaches and represents an innovative model due to its morphological and functional affinity with man; therefore, pig ESCs are attracting renewed interest. However, a number of open questions need to be addressed since no validated protocols for the derivation and maintenance of pig ESCs have yet been established. In the present paper data from the literature will be presented together with experimental evidence recently obtained in our laboratory. We will discuss aspects related to the timing of isolation, the initiation of primary cultures, the use of different culture conditions and cytokines. The identification of pluripotency-related molecular markers in the pig will also be examined. Finally, the ability to respond to specifically formulated medium with spontaneous as well as induced differentiation will be assessed.

This chapter illustrates the physiological characteristics of oocytes and ovarian follicles in relation with their possible exposure to environmental contaminants. The total amount of oocytes present in the adult ovary is established shortly before birth. As soon as the primordial follicle store is established, follicle recruitment begins and it continues without halting for the rest of life or until the ovary is depleted. Between recruitment and ovulation, the oocyte goes through a deep transformation in order to become able to sustain embryonic development. The permanent nature of the oocyte population together with its essential role in supporting embryonic development makes it a sensitive target for the adverse effects of environmental contaminants. Amongst the different environmental contaminants we focussed our analysis on a range of chemicals known as endocrine disrupters (EDs) because of their widespread diffusion and the potential hazard they represent for reproductive health. Their sites of action include the hypothalamus-hypophyseal system, resulting in disruption of the normal pattern of gonadotropin secretion, and the ovary, resulting in destruction of the oocyte. In turn, oocyte destruction can result from directly impairing oocyte viability or from indirect mechanisms involving alterations within the follicular wall. Recent work performed in our laboratory on bovine and pig, has begun to elucidate some of the cellular and molecular mechanisms involved in EDs negative effects on oocyte developmental competence. Our results indicate that EDs perturb maternal mRNA stability and disrupt the physiological remodelling of the cytoplasm, taking place during oocyte maturation.

The progression of oocyte meiosis is accompanied by major changes in the ooplasm that play a key role in the completion of a coordinate nuclear and cytoplasmic maturation. We review evidence from the literature and present data obtained in our laboratory on different aspects of pig oocyte cytoplasm compartmentalization during maturation and early embryo development. In particular, we will discuss the changes in adenosine triphosphate (ATP) concentration and distribution taking place during the maturation process and their possible significance for oocyte developmental competence. We describe two important aspects of cytoplasmic streaming: mitochondrial distribution patterns in oocytes and early embryos and the complex rearrangements of cytoplasmic microtubule networks, while discussing their possible correlations with ooplasm compartmentalization. Recent evidence indicates that the cytoskeleton is used to shuttle not only organelles but also mRNAs to specific sites within the oocyte cytoplasm. Localization is driven by specific molecular motors belonging to the kinesin superfamily and requires the involvement of the RNA targeting molecule Staufen. We present recent experimental evidence, obtained in our laboratory, on the pig orthologues for kinesin KIF5B and Staufen, describe their expression patterns and discuss their possible role in oocyte maturation.

Embryonic stem cells (ESCs) hold great promise for therapeutic use and represent a unique tool for investigating the process of self-renewal and differentiation. The properties that make ESCs unique are their capacity of unlimited self-renewal coupled with the property of re-entering the developmental process if returned inside a blastocyst. Such plasticity enable ESCs to form all embryonic tissues including germ cells. However, these remarkable properties, at present, have been demonstrated only for mouse ESCs even if cells with somehow more limited capacities have been derived in many different species including humans. The isolation of pluripotent embryonic cells lines from human embryos marked a crucial change of perspective in evaluating the properties defining an embryonic stem cell lines moving the focus from the generation of a germ-line chimera, obviously not feasible nor desirable in human, to the capacity of these cells to differentiate both in vivo and in vitro in fully mature and functional cell types of all kinds. Therefore, ESCs properties in species different from the mouse are being reassessed and re-evaluated, in view of their potential use as experimental models for the development of clinical applications. Among the species that may play a useful role in this field, the pig has a long-standing history as a prime animal model for pre-clinical biomedical applications and therefore, pig ESCs are attracting renewed interest. In this review, we will summarize the current knowledge on this topic and will contrast the relatively limited data available in this species with the much larger wealth of information available for mouse and human ESCs, in an attempt to assess whether or not pig ESCs can actually become a useful tool in the fast growing field of cell therapy.

Currently, approximately 60 chemicals have been identified as endocrine disruptors (EDs): exogenous agents that interfere with the synthesis, secretion, transport, metabolism, binding, action, or elimination of natural blood-borne hormones. Farm animals ingest these substances with food and drinking water. Their stability and lipid solubility has led to increased concern that these substances may compromise the reproductive health of both humans and animals. Oocytes are a permanent cell population established before birth which is exposed to environmental stimuli for a period that, in farm animals, can be as long as several years. Oocyte competence is acquired within the ovary during the developmental stages that precede ovulation and its role is critical during the interval between fertilization and the so-called maternal to embryonic transition, when the transcriptional activity of the embryonic genome becomes fully functional. Any perturbation of these delicate process is likely to reduce oocyte developmental competence and, therefore, to cause an arrest of embryonic development at any given stage. A critical analysis of the doses and time of exposure is presented together with a description of the effects of different EDs on farm animal oocytes and early embryonic development. Finally some of the mechanisms mediating EDs effects on the oocytes will be described. In particular the role of arylhydrocarbon receptor, maternal mRNA stability and cytoplasmic remodelling during oocyte maturation will be discussed in some details.

Objective To compare the efficiency of equilibrium cooling and vitrification for cryopreservation of human ovarian tissue and to determine the best experimental model for developing new protocols. Design Experimental prospective study. Setting An academic research environment. Patient(s) and Animal(s) Human ovarian biopsy specimens were obtained from three women undergoing operative laparoscopy for ovarian cyst enucleation. Adult cow and pig ovaries, collected at the abattoir. Intervention(s) Ovarian tissue fragments of three individuals for each species were cryopreserved by using two protocols, either for equilibrium cooling or vitrification. Main Outcome Measure(s) Comparison between fresh and cryopreserved tissue of primordial, primary, and secondary follicle morphology, graded in three classes. Result(s) Human and bovine follicles responded in the same way to the two equilibrium cooling protocols, whereas pig tissue was more cryoresistant. Both vitrification protocols caused extensive damage to the tissue of all species. Human tissue showed a response to vitrification that was different from that of both animal species. Conclusion(s) Bovine is a good animal model for the development of human ovarian tissue cryopreservation protocols by equilibrium cooling procedures. Vitrification is less efficient than equilibrium cooling, and at present, neither bovine nor pig can be considered relevant animal models for human tissue.

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.

Horse is a seasonal breeder and information on oocyte quality outside the breeding season is very limited. Ovaries obtained at the slaughterhouse are a convenient but often limited source of oocytes in this species. As the low quantity of ovaries leads to an intensive use of all available material, it would be useful to know whether ovaries collected during the non-breeding season are suitable for in vitro maturation (IVM). In an attempt to characterize the effect of season on oocyte quality, we investigated the permeability of the gap junctions (GJ) present between cumulus cells and oocytes because of their important role in oocyte growth and maturation. We also compared the effect of supplementing the maturation medium with bovine serum albumin (BSA) or oestrus mare serum (EMS). A total of 645 oocytes isolated from 158 and 154 ovaries collected during the breeding and the non-breeding season, respectively, were used in this study. Oocytes were matured for 30 h in TCM 199 supplemented either with 10% EMS or with 4 mg/ml BSA. The presence of permeable GJs between cumulus cells and oocytes was investigated with the injection of a 3% solution of the fluorescent dye Lucifer yellow into the ooplasm. No differences in efficiency of oocyte retrieval or oocyte meiotic competence were detected between oocytes collected during the breeding and non-breeding season. The vast majority (90%) of the oocytes collected during the breeding season had fully functional communications with their surrounding cumulus cells but such communications were completely interrupted in 55.3% of the oocytes collected during the non-breeding season. During the non-breeding season, the proportion of oocytes whose communications with cumulus cells were classified as closed or intermediate at the end of maturation was lower in the group matured with BSA than with EMS (71.4 vs 97.7, p < 0.05). The same trend, although not statistically significant, was observed during the breeding season also. The presence of BSA caused an incomplete cumulus expansion during both seasons. Our data indicate that oocytes collected during the non-breeding season do not show any meiotic deficiency but lack active communication with the surrounding cumulus cells at the time of their isolation from the ovary. No data are available at present for determining the consequences on the developmental competence even if data from other species suggest that this is likely.

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 microg/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 in COC and oocyte was lower than in other groups, and communications declined after 3 h of culture. In the FCS+hMG group, [cAMP]i increased significantly in COC, with a peak between 3 and 7 h that was temporally correlated with the beginning of the cumulus expansion process, which occurred only in this group and with the termination of the communications. COC matured in the presence of iAC showed a moderate increase of [cAMP]i during all of the maturation times as well as a prolongation of oocyte-cumulus cell communications. The immunohistochemical localization of Cx43 confirmed the delay in connexons protein turnover in iAC-treated COCs. Our results show that cumulus expansion and oocyte developmental competence are induced by different levels of cAMP and that its intracellular concentration may affect cell coupling between oocyte and cumulus cells. We hypothesize that the higher developmental competence of COCs matured in the presence of iAC could be achieved through a moderate increase of intracellular cAMP, which in turn determines a prolongation of communications between the two cell types.

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 alpha and beta (ERalpha and ERbeta) and progesterone receptor (PR) mRNA levels. Cumulus-oocyte complexes (COCs) were exposed during in vitro maturation to serial concentrations of OP (1-0.0001 microg/ml) and were compared with vehicle-treated controls and a group of COCs treated with 17 beta-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 microg/ml. Groups treated withor =0.001 microg/ml OP showed impaired embryo development. No adverse effects of E2 were observed. In the E2-treated COCs, ERalpha mRNA was decreased but PR mRNA was upregulated compared with controls. Treatment with 0.001 and 0.0001 microg/ml OP induced a decrease in ERalpha mRNA, but ERbeta and PR mRNA were not affected. Treatment with 0.01 microg/ml OP did not produce changes in the expression of any of the mRNAs studied. OP impairs meiotic progression and developmental competence of bovine oocytes without demonstrating clear estrogen-mimic activity.

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.

Interspecific hybrid embryos are useful models for the study of maternal-fetal interactions, transmission pattern of species-specific markers and parental contributions to growth and developmental potential of pre-attachment embryos. In an attempt to investigate the possibility of producing hybrid embryos of domestic cattle (Bos taurus) and water buffalo (Bubalus bubalis), cattle oocytes were exposed to buffalo sperm and buffalo oocytes were exposed to cattle sperm and the cleavage rate and the post-fertilisation features of hybrid embryos up to the blastocyst stage were compared with those of buffalo and cattle embryos. The cleavage rate in buffalo oocytes exposed to cattle sperm was low (40.8%), with only 8.8% of these hybrid embryos reaching the blastocyst stage. Cattle oocytes exposed to buffalo sperm showed 86.3% cleavage, while 25.9% of these attained the blastocyst stage. The speed of development of both types of hybrids was intermediate between that of cattle and buffalo embryos, with hatching occurring on day 7.5 in hybrid embryos, day 8-9 in cattle and day 7 in buffalo. The proportions of cells contributing to the trophectoderm and the inner cell mass were closer to those of the maternal species in both types of hybrid embryos. Our results indicate that cattle-water buffalo hybrid embryos produced using interspecies gametes are capable of developing to advanced blastocyst stages and that their in vitro fate, and developmental potential, are influenced by the origin of the oocyte.

Cryopreservation and retransplantation of ovarian tissue is a real option to preserve fertility in young cancer patients. However, a high risk of retransmission of malignancy exists in several tumours. In these patients, cryopreserved whole ovaries could provide an appealing source of oocytes to be grown and matured in vitro. The aim of this study was to develop a perfusion system for ex vivo culture of fresh and cryopreserved whole ovaries. Upon arrival to the laboratory, all ovaries were perfused via the ovarian artery with Ringer's solution and 10 UI L–1 of heparin for 10 min. Ovaries to be frozen were subsequently perfused with cryoprotectant solution [L-15 medium, 10% FBS, and 1.5 M dimethyl sulfoxide (DMSO)] and then frozen using Multi Thermal Gradient freezing technology (Core Dynamics Ltd., Ness Ziona, Israel), pushing the samples along the thermal gradient (4 to –70°C) at 0.01 mm s–1, resulting in a cooling rate of 0.3°C min–1. Samples were thawed at 37°C and immediately perfused with L-15 medium supplemented with decreasing sucrose concentrations (0.25, 0.125, and 0 M). In a closed-circuit perfusion system, 100 mL of recirculating medium (M199, 25 mM HEPES, 1% BSA, 2 mM glutamine, and antibiotic/antimycotic) was pumped into the ovarian artery using a peristaltic pump. The flow rate through the ovary was maintained between 1 and 1.5 mL min–1. Whole sheep ovaries were cultured at 38.5°C for 1 or 3 days. After culture, ovaries were fixed with 10% formaldehyde. Statistical analysis was performed using Student's t-test (SPSS 20, IBM Corp., Armonk, NY, USA). Morphological analysis showed that the rate of intact follicles was inversely related to the days of culture but was not affected by cryopreservation. In fact, the percentage of morphologically normal follicles in fresh and frozen ovaries cultured for 1 day (87 ± 3.4 and 83 ± 3.2%, respectively; P = 0.058) was higher (P = 0.048) than in ovaries cultured for 3 days (75 ± 2.9 and 71 ± 2.8%, respectively; P = 0.053). Cell proliferation, measured as Ki67-positive stromal cells, decreased during culture (P = 0.028) and was affected by cryopreservation both on Day 1 (13 ± 7 v. 15 ± 4%; P = 0.047) and Day 3 (10 ± 4 v. 12 ± 6%; P = 0.039). Similar results were observed for the apoptotic index that increased during culture both in fresh and cryopreserved ovaries (P = 0.028). The number of apoptotic cells per millimeter squared was lower (P = 0.031) in fresh (23 ± 10%) than in frozen ovaries (27 ± 15%) both on Day 1 and on Day 3 (30 ± 14 v. 33 ± 20%, respectively; P = 0.03). Cell viability and active endocrine function during culture is confirmed by steroid secretion, which is conserved in both fresh and cryopreserved ovaries for up to 3 days. Our results show that it is possible to culture both fresh and cryopreserved whole ovaries for up to 3 days. Although fresh ovaries, on average, did better than cryopreserved ones, we observed large individual variations, with positive and negative results overlapping between fresh and frozen samples. Further studies are in progress to explain the reason of such variations. Supported by AIRC IG 10376, Carraresi Foundation, and by Legge 7 (R.A.S).

Mammalian differentiation is obtained through epigenetic regulations that shape the genome, which is identical in all cells, to distinct phenotypes and tissue specific identities. The differentiated state of mature cells in an adult organism is therefore acquired through epigenetic restrictions that lead to a gradual loss of differentiative potency. In agreement with this, recent experiments demonstrate that terminally differentiated cells can be induced to de-differentiate in vitro and increase their plasticity in response to epigenetic modifiers that are capable of reverting cells from their lineage commitment to a more plastic state. Here we describe experiments where we prepared porcine skin fibroblasts and granulosa primary cultures and exposed them to an inhibitor of DNA methylation, the 5-aza-cytidine (5-aza-CR), to increase cell plasticity. Taking advantage of the obtained increased permissivity window, we investigated the ability of 5-aza-CR treated cells to respond to specific differentiation conditions and be re-addressed to a different cell lineage either within the same germ layer or to a different germ layer. Cells were evaluated for their morphological changes and assessed using RT-PCR and immunocytochemical studies during the treatment. Following the exposure to 5-aza-CR the phenotype of both cell types changed. Treated cells displayed an oval or round shape, and appeared smaller with larger nuclei and granular and vacuolated cytoplasm. This was accompanied by an active expression of the main pluripotency-related genes OCT4, NANOG, SOX2, and REX1, originally undetectable in untreated fibroblasts and granulosa cells. 5-aza-CR treated granulosa cells cultured with recombinant human vascular endothelial growth factor to induce myogenic specification (different lineage within the same germ layer) suppressed the expression of granulosa specific marker (Cytokeratin) as well as of the pluripotency genes, and expressed MYOD, MYF5, and MYOG (earliest myogenic markers that are involved in the coordination of skeletal muscle development or myogenesis). In order to trans-differente 5-aza-CR treated fibroblasts to cells of a different germ layer, they were exposed to activin A to promote endoderm commitment. Cells down-regulated Vimentin (fibroblast marker) as well as pluripotent gene expression and transcribed Nestin (transiently involved in multi-lineage progenitor cell differentiation), SOX17, FOXA2 (induction of definitive endoderm), and HNF4A, HNF1 (primitive gut tube specific genes). Altogether these results suggest that it is possible to obtain a direct inter-lineage conversion by removing epigenetic restriction, using demethylating agents such as 5-aza-CR, and avoiding a stable pluripotent state. This novel approach may represent a promising tool for regenerative medicine because it does not involve the use of any transgenic modifications, retroviral transfection, or both. Supported by Network Lombardo iPS (NetLiPS) Project ID 30190629.

Polychlorinated biphenyls (PCBs) can interfere with normal reproductive functions acting as endocrine disruptors. Aroclor-1254 (A-1254), is a pool of more than 60 congeners used for in vitro studies because its composition is representative of PCBs environmental pollution. We previously demonstrated that the exposure of bovine oocytes to A-1254 during in vitro maturation (IVM) was detrimental not only to the maturation process but also induced a significant increase of polyspermy and a reduction of developmental competence. Therefore, we investigated whether A-1254 acts on two processes that occur during IVM and may be related with its negative effects: maternal mRNA polyadenylation and cortical granules (CGs) migration and exocytosis. Bovine cumulus-oocyte complexes (COCs) were exposed to 0.1 microg/ml of A-1254 during IVM, a level of exposure known to affect oocyte maturation, fertilization, and developmental competence. Oocyte exposure to A-1254 altered the poly(A) tail length of 5 out of 10 genes examined. PCBs effect on mRNA polyadenylation was different depending on the gene considered and resulted either in a shorter or in a longer poly(A) tail. At the end of maturation, Aroclor treated oocytes presented clustered CG in a significantly higher percentage than the control group. In addition, CG exocytosis after 8 hr of fertilization occurred at significantly lower extent in zygotes derived from the exposed group compared to control. Our results indicated that the lower developmental competence of oocytes exposed to PCBs during IVM can be related to the interaction of these contaminants with mechanisms regulating maternal mRNA storage in the ooplasm and normal CGs function.

Glucose is readily been taken up and utilized by preimplantation embryos from different species. However, a comprehensive analysis of the glucose transporter expression throughout preimplantation development is still missing. Here, we have investigated the expression of facilitative glucose transporters (Glut1-5 and 8) and sodium-dependent-glucose transporter (SGLT-I) in bovine oocytes and preimplantation embryos up to d16 of development, using RT-PCR and immunohistochemistry. The embryos were produced in vitro by IVM-IVF. Glut1, Glut3, Glut8, and SGLT-I were expressed in all stages studied. Glut4 transcripts were first detected at the blastocyst stage. Glut2 expression was restricted to the period of blastocyst elongation at d14 and d16. Transcription of the fructose transporter Glut5 started at the 8-/16-cell stage. Our results show a distinct expression pattern for glucose transporters during bovine embryo development in vitro indicating specialized functions for these isoforms at different developmental stages in bovine embryos. Mol. Reprod. Dev. 60:370-376

This study was designed to evaluate the effects of pretreatment with various forms of cAMP before or during bovine oocyte maturation on the acquisition of embryonic developmental competence. The objective of the 4 experiments was to induce differentiation of the early maturing oocyte in conditions of maintained meiotic arrest or normal maturation. To promote differentiation, different forms of cyclic AMP-dependent protein kinase (PKA) pathways were investigated. The factors studied included follicular fluid, invasive adenylate cyclase (iAC), dibutyryl-cAMP (dbcAMP) and 3-isobutyl-1-methyl-xanthine (IBMX) with or without cycloheximide (CHX). High concentrations of iAC pretreatment were beneficial to the oocyte competence in BSA-iAC maturation while harmful in normal maturation. Also, after 2 to 3 h IBMX-iAC pretreatment, another 6 h of CHX treatment with or without iAC was harmful to the embryonic developmental competence of fertilized oocytes even though it did not have any effect on cleavage rate. Experiment 4 was to assess the role of cAMP in acquisition of oocyte developmental competence before meiotic resumption. Results supported that the intracellular cAMP concentration during the interval between oocyte isolation from the follicle and the beginning of in vitro maturation is critical for requiring optimal developmental competence.

RNA molecules and proteins are accumulated in the oocyte cytoplasm during its growth phase and are used to sustain the early phases of embryonic development before embryo DNA transcription begins. This makes the oocyte a very special cell, quite different from somatic cells where RNA and proteins usually undergo a rapid turnover. To enable the storage and timely use of such stored molecules, various mechanisms are effective in the oocyte and are gradually being elucidated. Our understanding of such mechanisms is important for constantly improving therapy for human and animal reproductive disorders as well as for understanding the process of nuclear reprogramming during cloning procedure or stem cell generation. This review focuses on the various aspects of these regulatory processes in an attempt to give an overview of the present knowledge on post-transcriptional and post-translational mechanisms taking place during oocyte maturation and early development. Mechanisms such as cytoplasmic regulation of the poly(A) tail, RNA localization and protein phosphorylation are described in some detail. Because most data are available from lower species these are presented together with appropriate reference to the mammalian oocyte when data are known, or when important differences have been described.

Ovarian tissue cryobanking is proposed as an effective option for preserving female fertility in cancer patients. At present 2 options are available: cryopreservation of ovarian cortical fragments or of the whole ovary. The use of whole ovary reduces ischemic insult. However, the larger the sample volume, the more difficult it is to introduce the cryoprotective agents and to ensure an adequate cooling rate that minimizes tissue damage. For this reason, we used the multi-thermal gradient method, based on running the sample through a temperature gradient. This allows a homogeneous cooling rate through the whole sample independently from its volume. The aim of the study was to determine whether multi-thermal gradient freezing allows a substantial reduction of the damages induced by cryopreservation of large samples by comparing the viability of cortical fragments versus whole ovaries after thawing and grafting in nude mice. Sheep ovaries were collected at the local abattoir and randomly divided into 3 groups: A) ovaries frozen as cortical fragments, B) ovaries frozen as whole organs, and C) fresh ovaries immediately processed for further analysis (control). Ovarian fragments (10 × 5 × 1 mm) were sliced from the cortical region and immersed into cryoprotectant solution (Leibovitz L-15 medium, 10% FCS, and 1.5 M dimethyl sulfoxide), while whole ovaries were perfused with the same solution. Samples were placed into glass freezing tubes 16 mm in diameter filled with cryoprotectant solution. Samples were frozen with the multi-thermal gradient freezing apparatus (Core Dynamics, Ness Ziona, Israel) progressing along the thermal gradient at a rate of 0.01 mm s–1, resulting in a cooling rate of 0.3°C min–1. Two weeks later, samples were thawed by plunging the tubes into a 37°C water bath with gentle shaking. Whole ovaries were perfused with 10 mL of HEPES-Talp medium, 0.5 M sucrose, and 10 IU mL–1 of heparin and their cortical region was cut into fragments. These fragments and those derived from group A were rehydrated in L-15 medium with decreasing sucrose concentrations. Fragments (2 × 2 × 1 mm) were xenografted in the dorsal region of 6 nude mice for each group. Mice were killed after 8 weeks and grafts were collected for analysis. Cryopreserved samples were compared with each other and fresh controls (group C). Morphologically normal follicles at primordial, primary, and secondary stages were visible in all samples. Cell proliferation was assessed measuring Ki-67 mRNA and counting immunohistochemically positive cells. The FSH receptor and GDF9 gene expression were used to evaluate tissue viability. No significant differences for any of these parameters were measured amongst the groups. We conclude that directional freezing is an effective method for ovarian tissue cryopreservation independently from the sample volume, thus overriding the limitations usually associated with whole-organ banking. Supported by AIRC IG 10376 and by Carraresi Foundation.

Maternal transcripts are accumulated during oocyte growth and drive early embryonic development; therefore, their characterisation is a relevant factor for predicting fertility. DNA microarrays have been the method of choice for transcriptional profiling, but this method has some limitations when applied to domestic species because it relies upon existing knowledge about genome sequence and offers a limited quantitative evaluation. These limits are overcome by next-generation sequencing technology. The aim of the work was to define a reference standard for bovine fertility determining the list and the level of transcripts stored in fully grown oocytes collected from heifers (H) and to compare this pattern with that of adult repeat breeders (RB). Oocytes were collected by ovum pick-up (OPU) from 5 Italian Dappled Red heifers of 11 to 15 months of age that became pregnant at the following oestrus and from 4 adult cows of the same breed 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. In both groups, oocytes were aspirated from follicles of 4 to 6 mm in diameter. Each oocyte was carefully denuded and immediately snap frozen in liquid nitrogen. Oocytes from each animal were pooled together (range 4 to 11) and analysed as a single sample. Total RNA extraction was performed by RNeasy Micro Kit (Qiagen, Valencia, CA, USA). Amplified cDNA, from both mRNA and non-polyadenylated transcripts, was prepared starting from total RNA using the Ovation RNA-Seq System V2 (NuGEN Inc., San Carlos, CA, USA). Purified cDNA was ligated directly into an Illumina sequencing library using TruSeq DNA Sample Prep kit (Illumina Inc., San Diego, CA, USA). Sequencing was performed on Illumina HiSEqn 2000 in the 50-bp long single-read set-up, at a 4-plex of multiplexing level, producing 30 to 40 million reads per sample. Data were annotated using the cDNA ENSEMBL UMD 3.1.67 database. On average, the number of transcripts present in each sample was 15 438 ± 766 in H and 15 624 ± 768 in RB oocytes. Nineteen thousand one hundred sixty-one transcripts were detected at least in one sample, and 12 174 were detected in all samples. The comparison between H and RB showed that 598 transcripts out of 19 161 (3.12%) and 437 out of 12 174 (3.59%) are expressed at a significantly different level (P

Recent studies have shown that the use of specific inhibitors for signalling pathways known to drive murine embryonic stem cell (ESC) differentiation may represent a tool to derive and maintain pluripotent cell lines. The application of this novel approach could provide a new strategy to overcome the limitations still existing for the derivation of ESC in large animal species. These molecules, also known as 3i factors, include CHIR99021 (GSK3 inhibitor), PD173074 (FGF inhibitor) and PD0325901 (MAPK/ERK kinase or MEK inhibitor). However only scattered information are available on the involvement of these pathways in the maintenance of pluripotency in domestic animals. The aim of this study was to investigate the presence of receptors for these inhibitors in porcine inner cell mass (ICM) and to isolate and culture porcine pluripotent lines in a serum-free medium supplemented with the 3i factors, without any additional growth factor. Ovaries were collected at the local abattoir and cumulus–oocyte complexes (COC) were aspirated from antral follicles. In vitro maturation was then performed for 46 h. Frozen–thawed spermatozoa were purified and live spermatozoa were co-cultured with denuded oocytes for 24 h. Putative embryos were cultured in NCSU-23 medium until the blastocyst stage and then subjected to immuno-surgery. Isolated ICM were analysed by RT-PCR. Poly(A)+RNA was extracted using Dynabeads® mRNA DIRECT Micro-kit (Invitrogen, Carlsbad, CA, USA) and immediately reverse-transcribed with Superscript™ II Reverse Transcriptase (Invitrogen). Specific primers were designed for FGF4, FGFR-1, FGFR-2, FGFR-4, GSK3, and MEK genes. The PCR amplified products were sequenced and aligned using ClustalW. The RT-PCR results showed that porcine ICM actively transcribe for GSK3, MEK, FGFR-2, FGFR-4, and FGF4 genes, whereas no signal was detectable for FGFR-1. Based on these observations, IVF-derived ICM were plated onto inactivated STO feeder cells and cultured in N2B27 medium supplemented with 3 µM CHIR99021, 100 nM PD173074, and 0.4 µM PD0325901. Outgrowth formation was monitored and cells were passaged to a new STO monolayer every 7 days, as previously described (Brevini et al. 2010 Stem Cell Rev.). Assessment of pluripotency markers was carried out both by RT-PCR and immunocytochemical analysis at every passage for up to 15 passages. The results obtained indicate that porcine cells cultured in 3i medium, without additional growth factors, expressed pluripotency markers; namely OCT4, NANOG, SOX2, and REX1, preserving their pluripotent state over time. Our data indicate that porcine ICM express 3i factor target molecules. In agreement with this, the use of 3i medium allows the establishment and proliferation of pluripotent cell lines. Together, these findings suggest the involvement of the GSK3, FGF, and MEK pathways in porcine pluripotency network and advocate the use of the 3i medium as an efficient tool for ESC derivation in porcine. Supported by NetLiPS Project ID: 30190629.

The reproducibility of the use of sperm cells as vectors of foreign DNA in the genome of pigs was verified in the present study and the effectiveness of four different procedures for sperm treatment was assessed. For each gilt, approximately 6 x 10(6) ejaculated boar spermatozoa were incubated for 30 min in 1 mL TALP medium containing 3 micrograms of linearized pSV2CAT plasmid DNA. Before incubation, spermatozoa were treated in four experimental groups: (1) cells were stored at 16 degrees C for 24 h and then washed three times in TALP; (2) cells from the fresh, undiluted sperm-rich fraction of an ejaculate were used immediately after collection, following the same procedure as (1); (3) cells were treated as in (2) with an extra wash; and (4) incubation with DNA was performed in TALP medium supplemented with 0.5 mg mL(-1) poly-L-lysine hydrobromide. As determined by immunolocalization, plasmid DNA molecules were found to be associated with 12-17.1% spermatozoa, depending on sperm treatment. Of 35 inseminated gilts, 20 gave birth to a total of 126 piglets. None of the piglets showed sign of exogenous DNA incorporation in any of the tissues tested, as assessed by the polymerase chain reaction and Southern blot. The potential of modifying the pig genome through 'transformed' spermatozoa was not confirmed by these experiments.

It is evident that members of several growth factor families are actively involved in embryogenesis from its earliest phases. Several reports also indicate the oviduct as a possible source of growth factors, suggesting an active role of this organ in mammalian embryonic development. The aim of this study was to investigate the presence of activin/inhibin subunits in bovine oviduct since activin is a well-characterised morphogen in amphibian development. The presence of transcripts for α. βA, and βB subunits was investigated by analysing oviduct epithelial cells mRNA with reverse transcription-polymerase chain reaction (RT-PCR). Moreover, antisera specific for the three subunits were used for the Western blot analysis of the proteins secreted by oviduct epithelial cells in vitro and for their immunohistochemical localisation in different oviductal regions. Oviduct epithelial cells expressed only the βA-subunit gene. Immunoreactive material was present among in vitro secreted proteins, indicating that the transcript is translated into a polypeptide that has been localised in the epithelium of both the ampullary and isthmic tract of the organ. Consistent with these results, the antisera for the α and βB subunits did not recognise any specific antigen either among secreted proteins or in the sections. These results indicate that βA subunit gene is expressed in bovine oviduct epithelial cells, and the protein is secreted in vitro and can be found along the whole extension of the organ. In the absence of α or βB subunits, this suggests that activin A is present in bovine oviduct. Such a finding would be consistent with an embry-otrophic activity of this organ, but definitive conclusions on the target tissue and the specific functions of oviductal activin require further studies. © 1995 Wiley-Liss, Inc.

We previously reported that parthenogenetic stem cells display abnormal centrosome and spindle formation that results in severe chromosome missegregation, with a high incidence of hypoploid karyotypes. Unexpectedly, this is not accompanied by a correspondingly high rate of apoptosis and, by contrast, parthenogenetic cells share the pluripotency, self-renewal and in vitro differentiation properties of their bi-parental counterparts. We hypothesise that this is possible through a series of adaptive mechanisms that include the presence of intercellular bridges similar to those that connect germ cells during spermatogenesis. This would provide a way for mutual exchange of missing cell products, thus alleviating the unbalanced chromosome distribution that would otherwise hamper normal cell functions. The presence of intercellular bridges was investigated in pig parthenogenetic embryonic stem cells (PESC) by transmission electron microscopy (TEM). Cultured cells were fixed in 2% glutaraldehyde and post-fixed in 1% osmic acid. After standard dehydration in ethanol series, samples were embedded in an Epon-Araldite 812 mixture and sectioned with a Reichert Ultracut S ultratome (Leica). Thin sections were stained and observed with a Jeol 1010 electron microscope. Pig PESC were also subjected to scanning electron microscopy (SEM). To this purpose, they were fixed and dehydrated as described above, covered with a 9-nm gold film by flash evaporation of carbon in an Emitech K 250 sputter coater (Emitech) and examined with an SEM-FEG Philips XL-30 microscope. To demonstrate functional trafficking activity through intercellular canals, fluorescent 10-kDa dextran was injected into the cytoplasm of a single cell with FemtoJet Microinjector (Eppendorf). Movement of the molecule from the injected cell to others was observed with a Nikon Eclipse TE200 microscope. Ultra-structural analysis of PESC demonstrated the existence of intercellular bridges that ensured cytoplasmic continuity among cells. These canals appeared variable in size and were characterised by the presence of stabilising actin patches. Furthermore, extensive movement of 10-kDa dextran among cells demonstrated functional intercellular trafficking through these communication canals, suggesting their use for transfer of mRNA, proteins and ribosomes among cells. Our results demonstrate that PESC present a wide network of functional intercellular bridges that may constitute an adaptive mechanism to support normal cell functions. This process is commonly observed in transformed cells and gives further support to the recent hypothesis that suggests the existence of common features and links between oncogenesis and self-renewal in pluripotent cell lines. Supported by AIRC IG 10376. PG was supported by INGM.

Decreased fertility during the hot season is a common problem in pigs. Maternal hyperthermia reduces oocyte fertilizability and increases embryonic mortality. Cell biochemical thermoprotection mechanisms involve members of the heat shock protein (Hsp) family. Hsp40, also known as Mammalian Relative of DnaJ (MRJ) protein, plays a pivotal role as co-chaperone after heat shocks. This protein binds Hsp70 and, through ATP hydrolysis, induces the conformational changes needed by Hsp70 to bind and release heat unfolded proteins. However, no information is available on the role of Hsp40 in mammalian ovary. Here we investigate a) the expression and localization of Hsp40 in pig ovaries; b) its response to heat stress in oocytes and cumulus cells. To identify Hsp40 in pig, we extracted RNA from ovaries, granulosa cells and from pools of 5 oocytes. cDNA was amplified using primers specifically designed for Hsp40, based on sequence data available in other species. The amplified products were sequenced and aligned using ClustalW. The nucleotide sequence showed an homology of 95% with the human, 92% with the bovine and 84% with the mouse orthologs. A polyclonal antibody was raised against the mouse GST/MSJ1(145–242) homologue protein in New Zealand rabbits and serum was affinity-purified using a GST-coupled Affigel-10 column. Whole ovary proteins were separated by SDS PAGE, immunoblotted and stained with the Hsp40 antibody. The protein displayed a MW of 38 KDa, in agreement with results obtained in other species. Immunofluorescence studies showed that Hsp40 is found in oocytes, granulosa and theca cells of follicle at all developmental stages. Pig ovaries, collected at the slaughterhouse, were exposed to 42°C for 1 h, to verify whether Hsp40 has a functional response to heat stress in this specie. Transcript level was compared with the control group, maintained at 38.5°C. mRNA was extracted from cumulus cells and pools of 5 oocytes, isolated from follicles of 3 to 5 mm. Semi-quantitative analysis was performed in the exponential phase of PCR amplification, using 28S as endogenous control. Data were analyzed with Quantity One (Bio-Rad) and Student-t statistical analysis was performed. Exposure of porcine ovaries to 42°C for 1 h resulted in a significant increase (P ≤ 0.05) of Hsp40 mRNA levels (2.4 ± 0.35 fold) in oocytes, while no significant raise was detected in cumulus cells. To our knowledge, this is the first demonstration that Hsp40 is expressed and responds to a thermal stress in pig ovary. Since this co-chaperone acts upstream to other heat shock protein-such as Hsp70- and it is specifically up regulated in the oocytes, our findings suggest that it may play an important protective role against heat stress infertility.

Recent studies have suggested an association between reproductive failure and mastitis in lactating dairy cows, but our understanding of how mastitis affects reproduction is still limited. In the present study we investigated the effects of naturally occurring chronic mastitis on the population dynamics of ovarian follicles. Ovaries and milk samples were collected from 74 cows at slaughter. Milk samples from each quarter, were analyzed following National Mastitis Council procedures. Based on the presence of major pathogens and somatic cell count results, animals were sorted in 9 groups, but only the 2 extremes were considered for further analysis: uninfected (n = 8) and affected by chronic mastitis (n = 9). Primordial, primary, and secondary follicles were counted and scored on similar surface area of ovary sections for each animal (mean ± SD = 5.65 ± 0.25 cm2). They were analyzed with Fisher’s exact test, and the association between health status and follicle number was estimated by odds ratios ± confidence limits. Vasculature area in the ovarian cortex of healthy and mastitic animals was identified using Bandeiraea simplicifolia-I lectin (BSL-I). Results were quantified with the dedicated software MacBiophotonics image J, NIH, USA, and subsequently analyzed with t-test for statistical significance. Follicles were further characterized by immunostaining with a GDF-9-specific antibody. The intensity of the staining was semi-quantified using a relative scale: 0, 1, and 2 for no, weak, and strong staining, respectively. Our results indicate no (P > 0.05) difference between the numbers of primordial and primary follicles in healthy and affected animals. In contrast, the number of secondary follicles was significantly lower in sick animals (odds ratio 10.50*; P

The oviduct provides the best environment in which a zygote can grow and it can also support the development of embryos from a different species. However, there is no clear explanation of its embryotrophic properties at present. In several species, oviduct epithelial cells synthesize and secrete glycosylated proteins that become associated with developing embryos. Although these macromolecules may have a functional role at the time of fertilization and early embryonic development, the nature of such a role remains to be elucidated. The aim of this work was to perform a comparative analysis of oviduct-specific glycoproteins in search of molecules common to different species since their phylogenetic conservation would imply biological significance. In previous studies, sheep oviduct-specific proteins were characterized and a monoclonal antibody (AFRC MAC 264) specific for the sheep oviduct protein 92 (sOP 92) was produced; hence, sheep was taken as the reference species. The degree of similarity between sheep glycoproteins and those of the cow, goat, pig, rabbit and mouse was determined on the basis of: the presence of carbohydrate side-chains, cross-reactivity with AFRC MAC 264, correspondence of molecular weight between cross-reacting molecules, and similarity of immunohistochemical localization. On this basis, proteins similar to sOP 92 were present in cow and goat oviduct. A more limited similarity was also observed in pigs. This indicates a certain degree of phylogenetic conservation and suggests that these molecules may play an important physiological role; however, their function remains to be determined.

The regulatory role of very low density lipoproteins (VLDL) in de novo protein synthesis and in natural killer (NK) mediated cytotoxicity against cancer cells was evaluated in IL-2 activated peripheral blood mononuclear cells (PBL). Supplementing of VLDL to the incubation medium induced a potent dose-related inhibitory effect on the synthesis and secretion of (35S) labelled proteins. The maximal inhibitory response was observed at 50 micrograms/ml cholesterol VLDL. The effect of VLDL on the IL-2 induced ability of lymphocytes to kill cancer cells and to interfere with target cell proliferation was then evaluated. Maximal cytotoxicity was observed at 50 micrograms VLDL/ml as cholesterol. This VLDL-dependent stimulatory effect was associated with a significant decrease in the proliferative effect of the medium conditioned by PBL. The possibility that VLDL mediate the regulation of immune functions by interacting with metabolic patterns expressed by IL-2 stimulated lymphocytes is discussed in light of these results.

a i l e c v In the interest of continuing to expand opportuniies for authors to communicate their work, and to ublish relevant and timely articles for our readers, e are pleased to announce a new initiative, Special eview Articles. We anticipate that these will inlude individual articles, as well as two or more rticles on a common theme. In that regard, this issue ontains the first review article on method agreement nalysis, with a commitment to publish future artiles on selected topics within the general area of tatistical analysis. It is our vision for this initiative not only to provide ital information of interest to our regular readers, but urthermore, that these articles will be widely used and eferenced by a much broader audience. Therefore, to ake these articles widely accessible, we plan to proide free access to the articles in their entirety via n-line databases.

Due to logistical and economic reasons, assisted reproduction of domestic animals has been based mostly on the use of oocytes isolated from ovaries collected at the slaughterhouse. In order to propagate valuable or rare genetic material, perform somatic cell nuclear transfer or generate genetically modified animals, it is essential to obtain fully competent oocytes that will allow full-term development of the in vitro-produced embryos. Such a need makes clear the crucial role played by oocyte quality. In fact, it is easy to compromise the oocyte’s developmental potential but it is impossible to restore once it has been lost. Almost three decades after the first cow, sheep, goat, horse and pig in vitro-generated offspring were born, a large body of information has accumulated on the mechanisms regulating oocyte competence and on how the latter may be preserved during all the required manipulations. The amount of knowledge is far from complete and many laboratories are actively working to further expand it. In this review we will highlight the aspects of the ongoing research in which we have been actively involved.

Mature oocytes can be activated in vitro, leading to the generation of parthenotes that will develop in culture forming blastocysts morphologically indistinguishable from those derived from fertilized eggs. Parthenotes have been used as a source of pluripotent cells that show the traditional features associated with their biparental counterpart: expression of totipotency markers, telomerase activity, embryoid body formation, in vitro differentiation and, in most cases, teratoma formation. However, many aspects still need to be elucidated and, in particular, little attention has been paid to the inci- dence of aneuploidy in these cells. Limited data available for parthenotes derived from different mammalian species indicate a high rate of aneuploidy, whichis consideredtobecaused by the lackofthe paternal contribution, because alterations of the centrosome are knowntolead to multipolar spindles that, in turn, cause aneuploid cells. In this study, we analyzed the rate of aneuploidy and centriole distribution (as a marker of centrosome anomalies) in pluripotent cell lines (pSC) previously derived in our laboratory from pig parthenogenetic embryos and in primary fibroblast cultures and sections obtained from sheep parthenogenetic fetuses (n = 3) that reached 24 days of development in vivo. This protocol was chosen to separate the effect related tooocyte activation from those of the procedures used to derive pSC lines. Centriole number and distribution were assessed both by immunocy- tochemical analysis using an anti-centrin-1 antibody (1 : 200, Abcam, Cambridge, UK) and an appropriate secondary antibody, and by ultrastructural evaluation of thin sections, using a Jeol 1010 EX electron microscope (Jeol, Tokyo, Japan). Karyotyping was performed on mitotically active cells. Metaphases were fully karyotyped under a Leica HC microscope (Wetzlar, Germany). Images were then captured with a Leica DC250 digital camera and cells karyotyped using the Leica CW4000 Karyo software. The results obtained indicate that cell lines of parthenogenetic origin have, in all examined cases, an incidence of aneuploidy significantly higher than that of their respective controls. In particular, although the diploid configuration represented the modal value, the majority of the cells displayed a consistently lower number of chromosomes, between 1N to

When mouse spermatozoa were briefly exposed in culture to radioactively labelled DNA (pSV2CAT plasmid), radioactivity could be detected by high-resolution autoradiography on the surface and within the nucleus of the spermatozoa. Spermatozoa from other mammalian species (boar, bull, man) could also bind foreign DNA. With the exclusion of human spermatozoa, which in most experiments showed very low labelling values, labelling percentages (evaluated by light microscope autoradiography) ranged between 39 and 78%. In all four species the DNA-binding ability was mainly confined to a specific region of the sperm head (equatorial segment and postacrosomal region), and the sperm-DNA association kinetics were rapid (maximum values were reached within 20-40 min). The data also indicate that factor(s) in seminal plasma might protect spermatozoa from accidental transfection by foreign DNA that may be present in the genital tracts from bacterial or viral sources.

Due to logistical and economical reasons assisted reproduction of domestic animals has been based mostly on the use of oocytes isolated from ovaries collected at the slaughterhouse. In order to propagate valuable or rare genetic material, perform somatic cell nuclear transfer, generate genetically modified animals it was essential to obtain fully competent oocytes that would allow full term development of the in vitro produced embryos. Such demanding need has soon made clearly evident the crucial role played by oocyte quality, how easy it is to compromise its developmental potential and the fact that it is impossible to restore it once it has been lost. Almost three decades after the first bovine, sheep, goat, horse and pig in vitro generated offsprings were born, a large body of information has accumulated on the mechanisms regulating oocyte competence and on how the latter may be preserved during all the required manipulations. The amount of knowledge is far from being complete and many laboratories are actively working to further expand it. In this review we will highlight the aspects of the ongoing research in which we have been actively involved.

The establishment of porcine pluripotent ES cell lines would be an exciting and novel tool for animal biotechnology, such as cloning and transgenesis. Furthermore, it would represent a useful model for biomedical research, cell therapy, xenotransplantation as well as developmental biology research. However, in spite of several studies, no conclusive results have been obtained and a number of technical questions are still to be answered in order to derive genuine ES cells in the pig. Here we report the results obtained in our laboratory aimed at comparing IVF v. parthenogenetic embryos as a source for the establishment of putative ES cells. Oocytes were divided in two groups and subjected to IVF or parthenogenetically activated with ionomicyn and 6-DMAP. They were cultured in NCSU for 7 days and then subjected to immuno-surgery. Inner cell mass were plated onto inactivated STO feeder cells and outgrowth formation was monitored. Cells were passaged to a new STO monolayer every 7 days. Assessment of pluripotency markers was carried out both by RT-PCR and immunocytochemical analysis at every passage for up to 22 passages. Telomerase activity was measured every 5 passages. The results indicate that parthenogenetic embryos, although less resilient than IVF embryos to immuno-surgery, have a significantly greater ability to generate outgrowths and stable cell lines. Moreover, 77% of the 39 parthenogenetic lines derived v. only 33% of the IVF ones expressed pluripotency markers and displayed high telomerase activity. Altogether our findings are consistent with data obtained in the human where the efficiency to derive hES cell lines from parthenogenetic blastocysts appears greater as compared with regular blastocysts from IVF embryos (Cheng L 2008 Cell Research 18, 215–217). Table 1. Supported by: Prin 2005, 2006.

Porcine pluripotent ES cell lines are a promising tool for biotechnology, biomedical and developmental biology studies. However, no conclusive results have been obtained to derive genuine ES cells in the pig. Here we compare derivation efficiency of putative ES cells from IVF versus parthenogenetic pig embryos. We describe proliferation ability and doubling time, we study pluripotency markers and telomerase activity (TA) of the cell lines obtained. Pig oocytes were either fertilized in vitro or parthenogenetically activated. Blastocysts were subjected to immuno-surgery. Inner cell mass were plated and outgrowth expansion was monitored daily. Self renewal molecules were studied by RT-PCR and/or immunocytochemistry for up to 42 passages. TA was measured every five passages. The results obtained indicate that stable cell lines can be generated from IVF and parthenogenetic embryos. The latter appeared less resilient to immuno-surgery but demonstrated a higher ability to produce outgrowths. 77% of the parthenogenetic lines vs only 33% of the IVF ones expressed pluripotency markers and displayed high TA. Regardless to their origin, colonies showed a latency growth period in the 48 hours after plating, they grew exponentially between day 3 and 6 and then, proliferation rate was greatly reduced. Doubling time was estimated to be 31.5 hours. In both IVF and parthenogenetic cell lines, positivity for Oct-4, Nanog, Sox-2, Rex-1, SSEA-4, Alkaline phosphatase, TRA-1-81 and STAT3 was detected; no signal for LIF-Receptor beta and gp130 was shown. These results indicate that the main pluripotency network related molecules are expressed in the porcine species, while a classical LIF-Receptor beta- gp130-STAT3 activation pathway does not appear to be involved in the maintenance of self renewal. Finally, every cell lines expressed high TA, which was turned down once cells were induced to differentiate, indicating a physiologically normal control of TA in these cells.

Leukemia inhibitory factor (LIF), its receptor heterodimer (LRβ-gp130), and the related signal transducer and activator of transcription-3 (STAT3) constitute a system controlling self-renewal and pluripotency of embryonic stem cells (ESC) in the mouse, where LIF withdrawal or direct inhibition of STAT3 causes ESC differentiation. By contrast, several studies have demonstrated that LIF is not required to maintain human ESC pluripotency. Scattered information is available in other species, and data on the role of LIF in pig ESC are scanty. The aims of the present study were (a) to characterize the expression profile of gp130, LRβ, and STAT3 in pig parthenogenetic cell lines (ppC), previously derived in our laboratory and shown to be positive for the main pluripotency related markers; (b) to evaluate the role of LIF pathway in maintaining the pluripotency of these cells. To this purpose, ppC were cultured as previously described (Brevini et al. 2007 Theriogenology 68, 206–214) and screened by RT-PCR for the two LIF receptor subunits (LRβ and gp130) and STAT3. Pig granulosa cells were used as positive controls. To better investigate the possible role of LIF in maintenance of pluripotency in ppC, the formation of embryoid bodies (EB) was induced in the presence or in the absence of the cytokine. Undifferentiated cells were cultured in hanging drops either with or without LIF for 12 days. The EB formation and the expression of molecular markers specific for the three germ layers was evaluated at the end of the differentiation period. Molecular analysis allowed us to detect transcription of STAT3, whereas no signal for LRβ and gp130 was detected in ppC. These results seem to indicate that LIF does not play a role in the maintenance of pluripotency in the pig. However, after removal of LIF, ppC routinely formed EB that expressed molecular markers specific for the three germ layers. On the other hand, when LIF was added to the differentiation medium, pig cells were unable to form EB. They kept proliferating in an undifferentiated state, and no expression of molecular markers specific for the three germ layers was detected. Moreover, when re-plated on inactivated feeder-layers, they formed distinct colonies that maintained expression of pluripotency markers. Our results show that a role of LIF in pluripotency maintenance through a classical LRβ-gp130 and STAT3 activation pathway is unlikely. However, interaction with an alternative nonclassical activation signaling pathway cannot be ruled out. Indeed, the presence of the cytokine in the medium used for differentiation experiments actively inhibited EB formation, indicating a possible role in preventing differentiation in the porcine species. Further studies are needed to elucidate these aspects. Supported by: PRIN2005; PRIN2006; First 2006; First2007.

Embryonic stem cells (ESCs) can be differentiated into many cell types in vitro, thus providing a potentially unlimited supply of cells for cognitive in vitro studies and cell-based therapy. However, suitable animal models are crucial prerequisites for the development of safe preclinical protocols in biomedical research. Pig has been proposed as an alternative to the mouse model, due to the morphological and functional affinity of the porcine species with the human. We have previously obtained pluripotent cell lines from pig parthenogenetic blastocysts (Brevini et al. 2007 Theriogenology 67, 54–63). These cells expressed pluripotency-related molecules, such as Oct-4, Nanog, SSEA-4, etc., and were negative for markers specific for ectodermal, endodermal, and mesodermal differentiation (Brevini et al. 2007 Theriogenology 68, 206–213). In this study, we investigated their ability to differentiate into the neural lineage. Since no information was available in the literature, we tested and compared two differentiation protocols. Neurospheres were obtained by culturing, for 2 days, undifferentiated parthenogenetic pluripotent cells in hanging drops of DMEM:Ham F10 medium (1:1), supplemented with knockout (KO) serum replacement (10%), fetal calf serum (5%), retinoic acid (10 µm), and Sonic Hedgehog (100 ng mL–1). In protocol A, neurospheres were disaggregated and plated in adherent cultures in neural progenitor basal medium supplemented with neural survival factor-1 (NSF-1, 2%) and brain-derived neurotrophic factor (BDNF, 20 ng mL–1) for 3 weeks. In protocol B, cells were cultured in hanging drops for a further 7 days. Neurospheres were then plated in monolayer and cultured for 3 weeks in DMEM:Ham F12 medium (70:30), supplemented with epidermal growth factor (EGF; 200 ng mL–1), basic fibroblast growth factor (bFGF; 200 ng mL–1), B27 (2%), and N2 (1%). At the end of the culture period, cells were either harvested and screened by RT-PCR for neural differentiation-related markers or fixed and subjected to immunocytochemical fluorescent staining. Protocol A was not suitable for neural differentiation of pig pluripotent cell lines and did not seem to induce any differentiation. In contrast, protocol B could efficiently drive cells toward ectodermal differentiation and neural lineage. Neurospheres showed expression of neurofilament H after culture in hanging drops. At the end of the 21-day culture in monolayer, approximately 18% of the cells originally plated differentiated into more mature cell types of the neural lineage, as shown both by morphology and by staining with nestin and β-tubulin III antibodies. Our results indicate that pig pluripotent parthenogenetic cells can be induced to differentiate into both early and more mature neural subpopulations. These experiments, however, also indicate that the direct use of culture conditions developed for mouse and human cells is not always appropriate for pig cells, and the identification of specifically targeted protocols and media formulations for pig cells are required. This work was supported by PRIN 2005 and FIRST 2006.

Sox2 is a member of the Sox (SRY-related HMGbox) family. It acts to maintain developmental potential and marks the pluripotent lineage of the early mouse embryo; in particular, as in the case of Oct-4 and Nanog, Sox2 is expressed specifically in the inner cell mass (ICM) and in the epiblast of this species. Moreover, it plays an important role in the transcription network that maintains stem cell pluripotency, interacting with other factors such as Oct-4 and Nanog. Little information is available on this gene in bovine; therefore aims of the present study were: a) to identify and characterize the Sox2 expression profile in bovine oocytes and preimplantation embryos; and b) to investigate its expression pattern in ICM and trophectoderm (TE). Bovine oocytes and embryos were obtained by in vitro maturation and fertilization; blastocysts at Day 7 post-insemination underwent microsurgery to separate TE from ICM. mRNA was isolated from 3 pools, each consisting of 5 MII oocytes, 2-, 4-, 8-, and 16-cell embryos, morulae, blastocysts, ICMs, and TEs. Semi-quantitative analysis of Sox2 expression was performed in the exponential phase of PCR amplification using rabbit globin as exogenous control. Data were analyzed with one-way ANOVA, followed by multiple pairwise comparisons with Tukey test (SigmaStat 2.03, SPSS, Inc., Chicago, IL, USA). Values are presented as mean � SEM and differences of P ≤ 0.05 are considered significant. In order to rule out false negative results, PCR amplifications of isolated ICMs and TEs were extended to the plateau phase. Fragment identity was confirmed by sequencing. Comparison of bovine Sox2 cDNA sequence (EMBL AM774325) with databases revealed a 98%, 93%, and 87% homology with sheep, human, and mouse, respectively. Sox2 mRNA was detectable in oocytes as well as in embryos at the different developmental stages analyzed. Semi-quantitative expression studies revealed that Sox2 was present as both maternal and embryonic transcript; in particular, a statistically significant increase from the 8-cell stage, concomitant with embryo genome activation, was observed. Differently from the mouse, Sox2 was expressed in both bovine ICM and TE, resembling the profile previously shown for Oct-4 (van Eijk et al. 1999 Biol. Reprod. 60, 1093–1103), and suggesting that Sox2 expression might be regulated by Oct-4 also in bovine, as described in mouse and human. These findings also suggest that its expression may become restricted to the ICM only at the expanded hatched stage, as previously described for Oct-4 in pig embryos (Vejlsted et al. 2006 Mol. Reprod. Dev. 73, 709–718). This work was supported by PRIN 2006, FIRST 2005, TECLA-MIUR, and EUROSTELLS-ESF.

Previous studies on sheep have demonstrated effects of maternal nutrition on fetal development with effects being observed on reproductive function (Rae et al. 2002 Anim. Reprod. Sci. 72, 63–71), blood pressure (Gopalakrishnan et al. 2004 Am. J. Physiol. Regul. Integr. Comp. Physiol. 287, R12–R20), and behavior (Erhard et al. 2004 Behav. Brain Res. 151, 25–35) in the adult offspring. It is known that gene expression in developing oocytes and very early embryos is influenced by their environment and some of the effects of nutrition on reproductive and other systems are likely to be expressed through such early changes (Hyttel 2000 Anim. Reprod. Sci. 60–61, 49–60). Effects of nutrition on oocyte function (i.e. before ovulation and fertilization) and resultant embryo quality have been observed (Gonzalez-Bulnes et al. 2004 Reprod. Fertil. Dev. 16, 421–435), but effects on oocyte gene expression have not been investigated. The aim of the present study was to investigate the effects of premating nutrition on the expression of selected genes in sheep cumulus cells and oocytes. For this purpose, 16 cast age ewes of proven fertility were used. At the time of manipulation and study, all ewes had a condition score of approximately 2.5. During the 2 weeks before slaughter, ewes of one group were fed a ration providing 0.5-live weight (n = 10) maintenance requirements and the other group 1.5-live weight (n = 6) maintenence requirements. This treatment ensured that all animals had similar numbers of potentially ovulatory follicles but different patterns of ovarian follicle maturation and, accordingly, different numbers of follicles and oocytes undergoing the final stages of maturation. The ewes's reproductive cycles were synchronized using intravaginal pessaries inserted 2 weeks before slaughter. At slaughter, ovaries were recovered and oocytes and associated cumulus cells were aspirated from all follicles >2 mm in diameter. For each ovary, oocytes derived from follicles with a diameter 4 mm were pooled separately, and stored for further analysis. Associated cumulus masses were collected and stored singly for subsequent investigations. As expected from previous observations, the number of visible follicles, the ratio of small-to-large antral follicles, and the total numbers of oocytes per ewe were similar for control and underfed ewes. Furthermore, the expression levels of the markers for oocyte growth, GDF-9, BMP-15, and c-kit, were investigated. Data were analyzed by ANOVA followed by Duncan test. Results showed no major differences between groups, independent of follicle diameter. These data suggest that the nutritional status of donor ewes did not significantly affect the expression profiles of these genes. However, further analyses on a greater panel of genes is required to rule out premating nutrition effects on oocyte gene expression. Furthermore, effects on the cumulus cell gene expression profile remain to be investigated. This work was supported by the University of Milan, FIRST 2006; Scottish Executive Environment and Rural Affairs Department.

In the early blastocyst, lineage segregation depends on the expression of several key specific transcription factors. In the mouse, commitment to inner cell mass (ICM), lineage is positively regulated by Oct-4, a repressor of trophectoderm (TE) cell fate, and Nanog, which inhibits the formation of extra-embryonic and primitive endoderm. Cdx2, a caudal-type homeodomain protein, is specifically expressed in the nascent TE. The mechanisms that drive Cdx2 segregation to the outside cells are still unclear. However, the expression of Fgf Receptor 2 (FgfR2), restricted to the outside cells, and the role for its ligand, Fgf4, in promoting TE development, suggest that this signalling pathway may act upstream or in parallel with Cdx2. Little information is available on these genes in bovine; therefore the aims of the present study were as follows: (a) to identify and characterize the expression profiles of Cdx2 and FgfR2 variants (IIIc and IIIb) in bovine oocytes and pre-implantation embryos; and (b) to compare their expression patterns in ICM and TE with that of Oct-4 and Nanog. Bovine oocytes and embryos were obtained by in vitro maturation and fertilization; blastocysts at Day 7 post-insemination underwent microsurgery to separate TE from ICM. RNA was isolated from MII oocytes; 2-, 4-, 8-, and 16-cell embryos; morulae; blastocysts; ICMs; and TEs. Semi-quantitative analysis of Cdx2 and FgfR2 expression in oocytes and embryos was performed in the exponential phase of PCR amplification with rabbit globin as exogenous control. In order to exclude false negative results, PCR amplification in isolated TE and ICM was extended to the plateau phase for all genes considered. Fragment identity was confirmed by sequencing. Comparison of bovine Cdx2 cDNA sequence (EMBL AM293662) with databases revealed a 91% and 87% homology with human and mouse, respectively. Cdx2 expression was not detectable in MII oocytes, but increased in 2-cell embryos. Transcript levels decreased at the 4- and 8-cell stages and then increased again in the blastocyst. FgfR2 variants were present as both maternal and embryonic transcripts, because they were detectable throughout pre-implantation development. Cdx2 and FgfR2 IIIc and IIIb expression was restricted to TE cells. Nanog was detected only in ICM, whereas Oct-4 was expressed in both lineages, as previously described in bovine (van Eijk et al. 1999 Bio. Reprod. 60, 1093-1103). In conclusion, the expression profiles of Nanog, Cdx2, and FgfR2 in bovine pre-implantation embryos follow the pattern previously described in the mouse. Their differentially segregated expression is consistent with their role as selector factors of ICM vs. TE fates. The significance of Oct-4 ubiquitous distribution still remains to be elucidated. This work was supported by FIRB RBNE01HPMX_005, TECLA-MIUR, and EUROSTELLS-ESF.