S. Maffei

Characterization of the Constitutive Pig Ovary Heat Shock Chaperone Machinery and Its Response to Acute Thermal Stress or to Seasonal Variations

ABSTRACT 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.

Beneficial effect of directional freezing on in vitro viability of cryopreserved sheep whole ovaries and ovarian cortical slices

STUDY QUESTION Does directional freezing improve the structural and functional integrity of ovarian fragments compared with conventional slow freezing and to whole ovary cryopreservation? SUMMARY ANSWER 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. WHAT IS KNOWN ALREADY 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. STUDY DESIGN, SIZE, DURATION 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. PARTICIPANTS/MATERIALS, SETTING, METHODS 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. MAIN RESULTS AND THE ROLE OF CHANCE Most studied parameters were significantly improved (P < 0.05) 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 (P < 0.05) 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. LIMITATIONS, REASONS FOR CAUTION 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. WIDER IMPLICATIONS OF THE FINDINGS 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. STUDY FUNDING/COMPETING INTEREST(S) 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.

Direct comparative analysis of conventional and directional freezing for the cryopreservation of whole ovaries

OBJECTIVE To compare conventional slow equilibrium cooling and directional freezing for cryopreservation of whole ovaries. DESIGN Experimental animal study. SETTING Academic research environment. ANIMAL(S) Adult ewes. INTERVENTION(S) Eighty-one ovaries were randomly assigned to fresh control, conventional freezing (CF), and directional freezing (DF) group. Ovaries of CF and DF groups were perfused via the ovarian artery with Leibovitz L-15 medium, 10% fetal bovine serum, and 1.5 M dimethyl sulfoxide for 5 minutes. Each ovary was inserted into a glass test tube containing 10 mL of the same solution and cooled to -100°C or -70°C, respectively. Ovaries were stored in liquid nitrogen for a minimum of 2 weeks. MAIN OUTCOME MEASURE(S) Structural integrity of cortical and medulla regions, vascular integrity, follicle in vitro development, cell proliferation, and DNA damage and repair. RESULT(S) All examined parameters indicate that the structure of DF ovaries remains largely intact and comparable to fresh controls, whereas significant damages were observed in CF ovaries. CONCLUSION(S) Directional freezing allows good preservation of whole ovaries, with most of the parameters taken into consideration almost identical to those recorded in fresh control samples. This encourages a reconsideration of the possible use of whole-ovary cryopreservation as a viable alternative to cortical fragments.

Parthenogenesis in non-rodent species: developmental competence and differentiation plasticity

An oocyte can activate its developmental process without the intervention of the male counterpart. This form of reproduction, known as parthenogenesis, occurs spontaneously in a variety of lower organisms, but not in mammals. However, it must be noted that mammalian oocytes can be activated in vitro, mimicking the intracellular calcium wave induced by the spermatozoon at fertilization, which triggers cleavage divisions and embryonic development. The resultant parthenotes are not capable of developing to term and arrest their growth at different stages, depending on the species. It is believed that this arrest is due to genomic imprinting, which causes the repression of genes normally expressed by the paternal allele. Human parthenogenetic embryos have recently been proposed as an alternative, less controversial source of embryonic stem cell lines, based on their inherent inability to form a new individual. However many aspects related to the biology of parthenogenetic embryos and parthenogenetically derived cell lines still need to be elucidated. Limited information is available in particular on the consequences of the lack of centrioles and on the parthenotes ability to assemble a new embryonic centrosome in the absence of the sperm centriole. Indeed, in lower species, successful parthenogenesis largely depends upon the oocytes ability to regenerate complete and functional centrosomes in the absence of the material supplied by a male gamete, while the control of this event appears to be less stringent in mammalian cells. In an attempt to better elucidate some of these aspects, parthenogenetic cell lines, recently derived in our laboratory, have been characterized for their pluripotency. In vitro and in vivo differentiation plasticity have been assessed, demonstrating the ability of these cells to differentiate into cell types derived from the three germ layers. These results confirmed common features between uni- and bi-parental embryonic stem cells. However data obtained with parthenogenetic cells indicate the presence of an intrinsic deregulation of the mechanisms controlling proliferation vs. differentiation and suggest their uni-parental origin as a possible cause.

Isolation, Characterization and Differentiation Potential of Cardiac Progenitor Cells in Adult Pigs

IntroductionCardiovascular disease (CVD) remains as the first cause ofdeath worldwide [1, 2]. It is known that a rising heart failureincidence is associated with unhealthy life styles and in-creasing life expectance. Current therapies are typicallysymptomatic and, even though they provide some survivalbenefit, they cannot reverse the loss of contractile cardiactissue due to ischemic injury. For this reason, high expect-ances are associated to the recent developments in stem cellbiology and regenerative medicine that promise to replacedamaged or lost cardiac muscle with healthy tissue and thusto improve the quality of life and survival in patients withvarious cardiomyopathies [3].The recent identification of different classes of cardiacprogenitor cells suggests that the heart, classically considereda terminally differentiated, post-mitotic organ, may rathercontain a stem cell compartment, responsible for both tissueturn-over and regeneration, which follows acute or chronicdamage to the cardiac tissue [4–6]. Several groups havealready reported the isolation of different types of cardiacstem-like cells based on distinct cell surface markers, suchas c-kit, Isl-1 and Sca-1 [7–11] or the ability to generategenuine cardiomyocytes and integrate into heart tissue asinduced pluripotent stem cells [12], or also in relation to theircapacity to influence the neovascularization of the ischemictissue, as satellite cells [13]. These cells are able to restorecardiac function after ischemic injury, although with variableefficiency [7, 14]. Another type of cells associated withthe heart was mesoangioblast, a class of vessel-associatedstem cells that can differentiate into various mesoderm celltypes.Theywereoriginallydescribedinthemouseembryonicdorsal aorta [15] and later similar cells have been identifiedand characterized from postnatal small vessels of humanskeletal muscle [16] and mouse and human heart [4, 17].However, the experiments carried out until now werepredominantly performed on mice and human. This restrictssignificantly the possibility to apply the results obtained inpreclinical studies that cannot be performed using the hu-man as a model and, at the same time, are limited by theevident differences between mouse and humans, such astheir size, mice heart rate and their general anatomy [18,19]. In particular, human and mouse hearts diverge in thecoronary architecture, the variations of which are muchbigger in humans compared to mice. As a consequence,whereas the size and location of the ischemic area are fairlyconstant in the mouse, a much larger variation exists in thehuman [20]. Differences can also be appreciated at thecellular level, as indicated by the higher capillary densityand the larger cross sectional area of the myocytes in hu-man, in comparison to the mouse [21, 22]. Consequently,extrapolation of murine systems, particularly after inductionof cardiovascular stress, must be meticulously monitored,when applied clinically, because of the obvious differences

Expression and intracytoplasmic distribution of staufen and calreticulin in maturing human oocytes

PurposeIn this study we hypothesized that the mRNA vector Staufen mediates RNA relocalization during meiotic maturation, and by virtue of its interactions with endoplasmic reticulum, provides a possible mechanism by which protein synthesis is regulated.MethodsWe assessed the expression of staufen (STAU) and calreticulin (CALR), the latter adopted as a marker of the endoplasmic reticulum, in human oocytes at different stages of maturation: GV, metaphase MI and MII. Oocytes were subjected to polymerase chain reaction in order to investigate the expression of STAU and CALR. The corresponding protein products were identified by immunofluorescence and confocal laser scanning microscopy.ResultsSTAU and CALR were constantly expressed and selectively localized during oocyte maturation. At the GV stage the both proteins displayed a dispersed distribution localization throughout the cytoplasm. Progressing to the MII stage, STAU tended to compartmentalize towards the cortical area of the oocyte clustering in granules of larger sizes. At the MII stage, CALR assumed a pattern reminiscent and possibly coincident with the position of the meiotic spindle.ConclusionsThe changing pattern of STAU distribution during meiotic maturation of human oocytes implicates a novel mechanism for the regulation of protein synthesis based on mRNA localization. Moreover, the unique disposition of CALR at the MII spindle uncovers a physical interaction with endoplasmic reticulum that may mediate cytoskeletal remodelling during oocyte maturation.

Extended ex vivo culture of fresh and cryopreserved whole sheep ovaries

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 72h with or without FSH; Groups CM96 and CM96-FSH (n=6 each), perfused with a complex medium for 96h with or without FSH; Group CM96-FSH-cryo, (n=3) cryopreserved and perfused for 96h 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 2.2mm and then for these follicles to reach the ovulatory size of 4mm or more. It could be used as a research tool or to complement current techniques for preserving female fertility.

Intercellular bridges are essential for human parthenogenetic cell survival

Parthenogenetic cells, obtained from in vitro activated mammalian oocytes, display multipolar spindles, chromosome malsegregation and a high incidence of aneuploidy, probably due to the lack of paternal contribution. Despite this, parthenogenetic cells do not show high rates of apoptosis and are able to proliferate in a way comparable to their biparental counterpart. We hypothesize that a series of adaptive mechanisms are present in parthenogenetic cells, allowing a continuous proliferation and ordinate cell differentiation both in vitro and in vivo. Here we identify the presence of intercellular bridges that contribute to the establishment of a wide communication network among human parthenogenetic cells, providing a mutual exchange of missing products. Silencing of two molecules essential for intercellular bridge formation and maintenance demonstrates the key function played by these cytoplasmic passageways that ensure normal cell functions and survival, alleviating the unbalance in cellular component composition.

Freezing and Freeze-Drying: The Future Perspective of Organ and Cell Preservation

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.

Brief Introduction to Coral Cryopreservation: An Attempt to Prevent Underwater Life Extinction

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.