The process of ovarian aging: it is not just about oocytes and granulosa cells.

Ovarian age is classically considered the main cause of female reproductive infertility. In women, the process proceeds as an ongoing decline in the primordial follicle stockpile and it is associated with reduced fertility in the mid-thirties, irregular menstruation from the mid-forties, cessation of fertility, and, eventually, menopause in the early fifties. Reproductive aging is historically associated with changes in oocyte quantity and quality. However, besides the oocyte, other cellular as well as environmental factors have been the focus of more recent investigations suggesting that ovarian decay is a complex and multifaceted process. Among these factors, we will consider mitochondria and oxidative stress as related to nutrition, changes in extracellular matrix molecules, and the associated ovarian stromal compartment where immune cells of both the native and adaptive systems seem to play an important role. Understanding such processes is crucial to design treatment strategies to  slow down ovarian aging and consequently prolong reproductive lifespan and, more to this, alleviaingt side effects of menopause on the musculoskeletal, cardiovascular, and nervous systems.

Silica encapsulation of ZnO nanoparticles reduces their toxicity for cumulus cell-oocyte-complex expansion.

BACKGROUND: Metal oxide nanoparticles (NPs) are increasingly used in many industrial and biomedical applications, hence their impact on occupational and public health has become a concern. In recent years, interest on the effect that exposure to NPs may exert on human reproduction has grown, however data are still scant. In the present work, we investigated whether different metal oxide NPs interfere with mouse cumulus cell-oocyte complex (COC) expansion. METHODS: Mouse COCs from pre-ovulatory follicles were cultured in vitro in the presence of various concentrations of two types of TiO2 NPs (JRC NM-103 and NM-104) and four types of ZnO NPs (JRC NM-110, NM-111, and in-house prepared uncoated and SiO2-coated NPs) and the organization of a muco-elastic extracellular matrix by cumulus cells during the process named cumulus expansion was investigated. RESULTS: We show that COC expansion was not affected by the presence of both types of TiO2 NPs at all tested doses, while ZnO NM-110 and NM-111 induced strong toxicity and inhibited COCs expansion at relatively low concentration. Medium conditioned by these NPs showed lower toxicity, suggesting that, beside ion release, inhibition of COC expansion also depends on NPs per se. To further elucidate this, we compared COC expansion in the presence of uncoated or SiO2-coated NPs. Differently from the uncoated NPs, SiO2-coated NPs underwent slower dissolution, were not internalized by the cells, and showed an overall lower toxicity. Gene expression analysis demonstrated that ZnO NPs, but not SiO2-coated ZnO NPs, affected the expression of genes fundamental for COC expansion. Dosimetry analysis revealed that the delivered-to-cell mass fractions for both NPs was very low. CONCLUSIONS: Altogether, these results suggest that chemical composition, dissolution, and cell internalization are all responsible for the adverse effects of the tested NPs and support the importance of a tailored, safer-by-design production of NPs to reduce toxicity.

Cumulus Extracellular Matrix Is an Important Part of Oocyte Microenvironment in Ovarian Follicles: Its Remodeling and Proteolytic Degradation.

The extracellular matrix (ECM) is an essential structure with biological activities. It has been shown that the ECM influences gene expression via cytoskeletal components and the gene expression is dependent upon cell interactions with molecules and hormones. The development of ovarian follicles is a hormone dependent process. The surge in the luteinizing hormone triggers ovulatory changes in oocyte microenvironment. In this review, we discuss how proteolytic cleavage affects formation of cumulus ECM following hormonal stimulation; in particular, how the specific proteasome inhibitor MG132 affects gonadotropin-induced cytoskeletal structure, the organization of cumulus ECM, steroidogenesis, and nuclear maturation. We found that after the inhibition of proteolytic cleavage, gonadotropin-stimulated oocyte-cumulus complexes (OCCs) were without any signs of cumulus expansion; they remained compact with preserved cytoskeletal F-actin-rich transzonal projections through the oocyte investments. Concomitantly, a significant decrease was detected in progesterone secretion and in the expression of gonadotropin-stimulated cumulus expansion-related transcripts, such as HAS2 and TNFAIP6. In agreement, the covalent binding between hyaluronan and the heavy chains of serum-derived the inter-alpha-trypsin inhibitor, essential for the organization of cumulus ECM, was missing.

Versican G1 Fragment Establishes a Strongly Stabilized Interaction with Hyaluronan-Rich Expanding Matrix during Oocyte Maturation.

In the mammalian ovary, the hyaluronan (HA)-rich cumulus extracellular matrix (ECM) organized during the gonadotropin-induced process of oocyte maturation is essential for ovulation of the oocyte-cumulus complex (OCC) and fertilization. Versican is an HA-binding proteoglycan that regulates cell function and ECM assembly. Versican cleavage and function remain to be determined in ovarian follicle. We investigated versican expression in porcine ovarian follicles by real-time (RT)-PCR and western blotting. The aims of the present work were to determine whether 1) versican was produced and cleaved by porcine OCCs during gonadotropin stimulation; 2) these processes were autonomous or required the participation of mural granulosa cells (MGCs); and 3) versican cleavage was involved in the formation or degradation of expanded cumulus ECM. We demonstrate two cleavage products of G1 domain of versican (V1) accumulated in the HA-rich cumulus ECM. One of them, a G1-DPEAAE N-terminal fragment (VG1) of ~70 kDa, was generated from V1 during organization of HA in in vivo and in vitro expanded porcine OCCs. Second, the V1-cleaved DPEAAE-positive form of ~65 kDa was the only species detected in MGCs. No versican cleavage products were detected in OCCs cultured without follicular fluid. In summary, porcine OCCs are autonomous in producing and cleaving V1; the cleaved fragment of ~70 kDa VG1 is specific for formation of the expanded cumulus HA-rich ECM.

Relevance to investigate different stages of pregnancy to highlight toxic effects of nanoparticles: The example of silica.

Amorphous silica nanoparticles (SiO2NPs) have been recognized as safe nanomaterial, hence their use in biomedical applications has been explored. Data, however, suggest potential toxicity of SiO2 NPs in pregnant individuals. However, no studies relating nanoparticle biokinetic/toxicity to the different gestational stages are currently available. In this respect, we have investigated the possible embryotoxic effects of three-size and two-surface functionalization SiO2NPs in mice. After intravenous administration of different concentrations at different stages of pregnancy, clinical and histopathological evaluations, performed close to parturition, did not show signs of maternal toxicity, nor effects on placental/fetal development, except for amino-functionalized 25 nm NPs. Biodistribution was studied by ICP-AES 24 h after administration, and demonstrates that all particles distributed to placenta and conceptuses/fetuses, although size, surface charge and gestational stage influenced biodistribution. Our data suggest the need of comprehensive toxicological studies, covering the entire gestation to reliably assess the safety of nanoparticle exposure during pregnancy.

Cyclic AMP-elevating Agents Promote Cumulus Cell Survival and Hyaluronan Matrix Stability, Thereby Prolonging the Time of Mouse Oocyte Fertilizability.

Cumulus cells sustain the development and fertilization of the mammalian oocyte. These cells are retained around the oocyte by a hyaluronan-rich extracellular matrix synthesized before ovulation, a process called cumulus cell-oocyte complex (COC) expansion. Hyaluronan release and dispersion of the cumulus cells progressively occur after ovulation, paralleling the decline of oocyte fertilization. We show here that, in mice, postovulatory changes of matrix are temporally correlated to cumulus cell death. Cumulus cell apoptosis and matrix disassembly also occurred in ovulated COCs cultured in vitro. COCs expanded in vitro with FSH or EGF underwent the same changes, whereas those expanded with 8-bromo-adenosine-3',5'-cyclic monophosphate (8-Br-cAMP) maintained integrity for a longer time. It is noteworthy that 8-Br-cAMP treatment was also effective on ovulated COCs cultured in vitro, prolonging the vitality of the cumulus cells and the stability of the matrix from a few hours to >2 days. Stimulation of endogenous adenylate cyclase with forskolin or inhibition of phosphodiesterase with rolipram produced similar effects. The treatment with selective cAMP analogues suggests that the effects of cAMP elevation are exerted through an EPAC-independent, PKA type II-dependent signaling pathway, probably acting at the post-transcriptional level. Finally, overnight culture of ovulated COCs with 8-Br-cAMP significantly counteracted the decrease of fertilization rate, doubling the number of fertilized oocytes compared with control conditions. In conclusion, these studies suggest that cAMP-elevating agents prevent cumulus cell senescence and allow them to continue to exert beneficial effects on oocyte and sperm, thereby extending in vitro the time frame of oocyte fertilizability.

Reprogramming Human Female Adipose Mesenchymal Stem Cells into Primordial Germ Cell-Like Cells.

In the last two decades, considerable progress has been made in the derivation of mammalian germ cells from pluripotent stem cells such as Embryonic Stem Cells (ESCs) and induced Pluripotent Stem Cells (iPSCs). The pluripotent stem cells are generally first induced into pre-gastrulating endoderm/mesoderm-like status and then specified into putative primordial germ cells (PGCs) termed PGC-like cells (PGCLCs) which possess the potential to generate oocytes and sperms. Adipose-derived mesenchymal stromal cells (ASCs) are multipotent cells, having the capacity to differentiate into cell types such as adipocytes, osteocytes and chondrocytes. Since no information is available about the capability of female human ASCs (hASCs) to generate PGCLCs, we compared protocols to produce such cells from hASCs themselves or from hASC-derived iPSCs. The results showed that, providing pre-induction into a peri-gastrulating endoderm/mesoderm-like status, hASCs can generate PGCLCs. This process, however, shows a lower efficiency than when hASC-derived iPSCs are used as starting cells. Although hASCs possess multipotency and express mesodermal genes, direct induction into PGCLCs resulted less efficient.

Activation of cumulus cell SMAD2/3 and epidermal growth factor receptor pathways are involved in porcine oocyte-cumulus cell expansion and steroidogenesis.

Several lines of evidence suggest that in mice the activation of SMAD2/3 signaling by oocyte secreted factors, together with epidermal growth factor receptor (EGFR) activation, is essential to induce cumulus expansion. Here we show that inhibition of EGFR kinase in follicle stimulating hormone (FSH)-stimulated porcine oocyte-cumulus cell complex (OCCs) strongly decreases hyaluronan (HA) synthesis and its retention in the matrix, as well as progesterone synthesis. Although porcine cumulus cells undergo expansion independently of oocytes, we use biochemical and gene expression analyses to show that they do require activation of SMAD2/3 for optimal stimulation of HA synthesis and proteins involved in the organization of this polymer in the expanded matrix. Furthermore, FSH-induced progesterone synthesis by porcine cumulus cells was increased by blocking SMAD2/3 activation. In conclusion, these results support the hypothesis that an FSH-EGF autocrine loop is active in porcine OCCs, and provide the first evidence that the SMAD2/3 signaling pathway is induced by paracrine/autocrine factors in porcine cumulus cells and is involved in the control of both cumulus expansion and steroidogenesis.

Human adipose-derived stromal cells transplantation prolongs reproductive lifespan on mouse models of mild and severe premature ovarian insufficiency.

BACKGROUND: Although recent studies have investigated the ability of Mesenchymal Stromal Cells (MSCs) to alleviate short-term ovarian damage in animal models of chemotherapy-induced Premature Ovarian Insufficiency (POI), no data are available on reproductive lifespan recovery, especially in a severe POI condition. For this reason, we investigated the potential of MSCs isolated from human adipose tissue (hASCs), since they are easy to harvest and abundant, in ameliorating the length and performance of reproductive life in both mild and severe chemotherapy-induced murine POI models. METHODS: Mild and severe POI models were established by intraperitoneally administering a light (12 mg/kg busulfan + 120 mg/kg cyclophosphamide) or heavy (30 mg/kg busulfan + 120 mg/kg cyclophosphamide) dose of chemotherapy, respectively, in CD1 mice. In both cases, a week later, 1 × 106 hASCs were transplanted systemically through the tail vein. After four additional weeks, some females were sacrificed to collect ovaries for morphological evaluation. H&E staining was performed to assess stroma alteration and to count follicle numbers; immunofluorescence staining for αSMA was used to analyse vascularization. Of the remaining females, some were mated after superovulation to collect 2-cell embryos in order to evaluate their pre-implantation developmental capacity in vitro, while others were naturally mated to monitor litters and reproductive lifespan length. F1 litters' weight, ovaries and reproductive lifespan were also analysed. RESULTS: hASC transplantation alleviated ovarian weight loss and size decrease and reduced alterations on ovarian stroma and vasculature, concurrently preventing the progressive follicle stockpile depletion caused by chemotherapy. These effects were associated with the preservation of the oocyte competence to develop into blastocyst in vitro and, more interestingly, with a significant decrease of chemotherapy-induced POI features, like shortness of reproductive lifespan, reduced number of litters and longer time to plug (the latter only presented in the severe POI model). CONCLUSION: Human ASC transplantation was able to significantly reduce all the alterations induced by the chemotherapeutic treatment, while improving oocyte quality and prolonging reproductive functions, thus counteracting infertility. These results, strengthened by the use of an outbred model, support the potential applications of hASCs in women with POI, nowadays mainly induced by anticancer therapies.

p75 neurotrophin receptor is involved in proliferation of undifferentiated mouse embryonic stem cells.

Neurotrophins and their receptors are known to play a role in the proliferation and survival of many different cell types of neuronal and non-neuronal lineages. In addition, there is much evidence in the literature showing that the p75 neurotrophin receptor (p75(NTR)), alone or in association with members of the family of Trk receptors, is expressed in a wide variety of stem cells, although its role in such cells has not been completely elucidated. In the present work we have investigated the expression of p75(NTR) and Trks in totipotent and pluripotent cells, the mouse pre-implantation embryo and embryonic stem and germ cells (ES and EG cells). p75(NTR) and TrkA can be first detected in the blastocyst from which ES cell lines are derived. Mouse ES cells retain p75(NTR)/TrkA expression. Nerve growth factor is the only neurotrophin able to stimulate ES cell growth in culture, without affecting the expression of stem cell markers, alkaline phosphatase, Oct4 and Nanog. Such proliferation effect was blocked by antagonizing either p75(NTR) or TrkA. Interestingly, immunoreactivity to anti-p75(NTR) antibodies is lost upon ES cell differentiation. The expression pattern of neurotrophin receptors in murine ES cells differs from human ES cells, that only express TrkB and C, and do not respond to NGF. In this paper we also show that, while primordial germ cells (PGC) do not express p75(NTR), when they are made to revert to an ES-like phenotype, becoming EG cells, expression of p75(NTR) is turned on.

Colour-sensitive conjugated polymer inkjet-printed pixelated artificial retina model studied via a bio-hybrid photovoltaic device.

In recent years, organic electronic materials have been shown to be a promising tool, even transplanted in vivo, for transducing light stimuli to non-functioning retinas. Here we developed a bio-hybrid optoelectronic device consisting of patterned organic polymer semiconductors interfaced with an electrolyte solution in a closed sandwich architecture in order to study the photo-response of photosensitive semiconducting layers or patterns in an environment imitating biological extracellular fluids. We demonstrate an artificial retina model composed of on an array of 42,100 pixels made of three different conjugated polymers via inkjet printing with 110 pixels/mm2 packing density. Photo-sensing through three-colour pixelation allows to resolve incoming light spectrally and spatially. The compact colour sensitive optoelectronic device represents an easy-to-handle photosensitive platform for the study of the photo response of artificial retina systems.

Molecular organization and mechanical properties of the hyaluronan matrix surrounding the mammalian oocyte.

Successful ovulation and oocyte fertilization are essential prerequisites for the beginning of life in sexually reproducing animals. In mammalian fertilization, the relevance of the protein coat surrounding the oocyte plasma membrane, known as zona pellucida, has been widely recognized, while, until not too long ago, the general belief was that the cumulus oophorus, consisting of follicle cells embedded in a hyaluronan rich extracellular matrix, was not essential. This opinion was based on in vitro fertilization procedures, in which a large number of sperms are normally utilized and the oocyte can be fertilized even if depleted of cumulus cells. Conversely, in vivo, only very few sperm cells reach the fertilization site, arguing against the possibility of a coincidental encounter with the oocyte. In the last two decades, proteins required for HA organization in the cumulus extracellular matrix have been identified and the study of fertility in mice deprived of the corresponding genes have provided compelling evidence that this jelly-like coat is critical for fertilization. This review focuses on the advances in understanding the molecular interactions making the cumulus environment suitable for oocyte and sperm encounter. Most of the studies on the molecular characterization of the cumulus extracellular matrix have been performed in the mouse and we will refer essentially to findings obtained in this animal model.

The Influence of Pentraxin 3 on the Ovarian Function and Its Impact on Fertility.

Follicular development is a highly coordinated process that in humans takes more than 6 months. Pituitary gonadotropins and a variety of locally produced growth factors and cytokines are involved in determining a precise sequence of changes in cell metabolism, proliferation, vascularization, and matrix remodeling in order to obtain a follicle with full ovulatory and steroidogenic capability. A low-grade inflammation can alter such processes leading to premature arrest of follicular growth and female reproductive failure. On the other hand, factors that are involved in inflammatory response as well as in innate immunity are physiologically upregulated in the follicle at the final stage of maturation and play an essential role in ovulation and fertilization. The generation of pentraxin 3 (PTX3) deficient mice provided the first evidence that this humoral pattern recognition molecule of the innate immunity has a non-redundant role in female fertility. The expression, localization, and molecular interactions of PTX3 in the periovulatory follicle have been extensively studied in the last 10 years. In this review, we summarize findings demonstrating that PTX3 is synthesized before ovulation by cells surrounding the oocyte and actively participates in the organization of the hyaluronan-rich provisional matrix required for successful fertilization. Data in humans tend to confirm these findings, indicating PTX3 as a biomarker of oocyte quality. Moreover, we discuss the emerging evidence that in humans altered PTX3 systemic levels, determined by genetic variations and/or low-grade chronic inflammation, can also impact the growth and development of the follicle and affect the incidence of ovarian disorders.

Thyroid hormone regulates protease expression and activation of Notch signaling in implantation and embryo development.

A clinical association between thyroid dysfunction and pregnancy complications has been extensively reported; however, the molecular mechanisms through which TH might regulate key events of pregnancy have not been elucidated yet. In this respect, we performed in vivo studies in MMI-induced hypothyroid pregnant mice, evaluating the effect of hypothyroidism on the number of implantation sites, developing embryos/resorptions and pups per litter, at 4.5, 10.5, 18.5 days post-coitum (dpc) and at birth. We also studied the expression of major molecules involved in implantation and placentation, such as the proteases ISPs, MMPs, TIMPs and Notch pathway-related genes. Our results demonstrate that hypothyroidism may have a dual effect on pregnancy, by initially influencing implantation and by regulating placental development at later stages of gestation. To further elucidate the role of TH in implantation, we performed in vitro studies by culturing 3.5 dpc blastocysts in the presence of TH, with or without endometrial cells used as the feeder layer, and studied their ability to undergo hatching and outgrowth. We observed that, in the presence of endometrial feeder cells, TH is able to anticipate blastocyst hatching by upregulating the expression of blastocyst-produced ISPs, and to enhance blastocyst outgrowth by upregulating endometrial ISPs and MMPs. These results clearly indicate that TH is involved in the bidirectional crosstalk between the competent blastocyst and the receptive endometrium at the time of implantation.

Physico-chemical properties mediating reproductive and developmental toxicity of engineered nanomaterials.

With the increasing production of engineered nanomaterials (ENMs) exploited in many consumer products, a wider number of people is expected to be exposed to such materials in the near future, both in occupational and environmental settings. This has raised concerns about the possible implications on public health. In particular, very recently the scientific community has focused on the effect that ENMs might exert on the reproductive apparatus and on embryonic development. Indications that ENMs might have adverse effects on cells of the germ line and on the developing embryos have been reported. In the present minireview we will perform a critical analysis of the published work on reproductive and developmental toxicity of the most commonly used nanoparticles with a major focus on mammalian models. We will place emphasis on the main physico-chemical characteristics that can affect NP behaviour in biological systems, i.e. presence of contaminants and nanoparticle destabilization, size, dosage, presence of functional groups, influence of the solvent used for their suspension in biological media, aggregation/agglomeration, intrinsic chemical composition and protein corona/opsonisation. The importance of this specific field of nanotoxicology is documented by the rapidly increasing number of published papers registered in the last three years, which might be a consequence of the growing concerns on the possible interference of ENMs with reproductive ability and pregnancy outcome, in a time in which reproductive age has increased and the possibility to bear children appears reduced.

Differentiation of osteoblast and osteoclast precursors on pure and silicon-substituted synthesized hydroxyapatites.

Calcium phosphate-based materials should show excellent bone-bonding and cell-mediated resorption characteristics at the same time, in order to be employed for bone replacement. In this perspective, pure (HAp) and silicon-substituted hydroxyapatite (Si-HAp, 1.4% wt) porous cylinders were prepared starting from synthesized powders and polyethylene spheres used as porogens, and investigated as supports for osteoblast and osteoclast progenitor differentiation. A systematic and detailed biological characterization is reported, in terms of cell adhesion, viability, proliferation, differentiation and bioresorption, aimed at proposing a complete and reliable picture of bone cell in vitro behavior, comprehensive of both the osteogenesis and the bone resorption processes. In order to achieve this purpose, cytocompatibility, differentiation and gene expression by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) were carried out using parietal bone-derived pre-osteoblasts obtained from neonatal mice and the bioresorption capability was assessed by seeding human peripheral blood monocytes, as osteoclast precursors. It resulted that both pure and Si-substituted HAps were able to promote differentiation of precursor cells in mature osteoblasts and osteoclasts. In particular, the Si-HAps enhanced the pre-osteoblast proliferation and showed higher osteoclast-mediated bioresorption capability, as supported by the presence of larger and more numerous resorption lacunae, whereas HAps promoted a more robust cell differentiation in terms of both osteocalcin gene expression by qRT-PCR and cell morphological evaluation by SEM analysis.

Implication of the oligomeric state of the N-terminal PTX3 domain in cumulus matrix assembly.

Pentraxin 3 (PTX3) plays a key role in the formation of the hyaluronan-rich matrix of the cumulus oophorus surrounding ovulated eggs that is required for successful fertilization and female fertility. PTX3 is a multimeric protein consisting of eight identical protomers held together by a combination of non-covalent interactions and disulfide bonds. Recent findings suggest that the oligomeric status of PTX3 is important for stabilizing the cumulus matrix. Because the role of PTX3 in the cumulus resides in the unique N-terminal sequence of the protomer, we investigated further this issue by testing the ability of distinct Cys/Ser mutants of recombinant N-terminal region of PTX3 (N(_)PTX3) with different oligomeric arrangement to promote in vitro normal expansion in cumuli from Ptx3-null mice. Here we report that the dimer of the N(_)PTX3 is unable to rescue cumulus matrix organization, and that the tetrameric assembly of the protein is the minimal oligomeric state required for accomplishing this function. We have previously demonstrated that PTX3 binds to HCs of IαI and TSG-6, which are essential for cumulus matrix formation and able to interact with hyaluronan. Interestingly, here we show by solid-phase binding experiments that the dimer of the N(_)PTX3 retains the ability to bind to both IαI and TSG-6, suggesting that the octameric structure of PTX3 provides multiple binding sites for each of these ligands. These findings support the hypothesis that PTX3 contributes to cumulus matrix organization by cross-linking HA polymers through interactions with multiple HCs of IαI and/or TSG-6. The N-terminal PTX3 tetrameric oligomerization was recently reported to be also required for recognition and inhibition of FGF2. Given that this growth factor has been detected in the mammalian preovulatory follicle, we wondered whether FGF2 negatively influences cumulus expansion and PTX3 may also serve in vivo to antagonize its activity. We found that a molar excess of FGF2, above PTX3 binding capacity, does not affect in vitro cumulus matrix formation thus ruling out this possibility. In conclusion, the data strength the view that PTX3 acts as a nodal molecule in cross-linking HA in the matrix.

Low doses of pristine and oxidized single-wall carbon nanotubes affect mammalian embryonic development.

Several in vitro and in vivo studies suggest local and systemic effects following exposure to carbon nanotubes. No data are available, however, on their possible embryotoxicity in mammals. In this study, we tested the effect of pristine and oxidized single-wall carbon nanotubes (SWCNTs) on the development of the mouse embryo. To this end, SWCNTs (from 10 ng to 30 µg/mouse) were administered to female mice soon after implantation (postcoital day 5.5); 10 days later, animals were sacrificed, and uteri, placentas, and fetuses examined. A high percentage of early miscarriages and fetal malformations was observed in females exposed to oxidized SWCNTs, while lower percentages were found in animals exposed to the pristine material. The lowest effective dose was 100 ng/mouse. Extensive vascular lesions and increased production of reactive oxygen species (ROS) were detected in placentas of malformed but not of normally developed fetuses. Increased ROS levels were likewise detected in malformed fetuses. No increased ROS production or evident morphological alterations were observed in maternal tissues. No fetal and placental abnormalities were ever observed in control animals. In parallel, SWCNT embryotoxicity was evaluated using the embryonic stem cell test (EST), a validated in vitro assay developed for predicting embryotoxicity of soluble chemical compounds, but never applied in full to nanoparticles. The EST predicted the in vivo data, identifying oxidized SWCNTs as the more toxic compound.

Synthesis of tumor necrosis factor alpha-induced protein 6 in porcine preovulatory follicles: a study with A38 antibody.

We have previously shown that the heavy chains (HCs) of inter-alpha-trypsin inhibitor (IalphaI) become covalently linked to hyaluronan (HA) during in vivo and in vitro expansion of porcine oocyte-cumulus cell complexes (OCCs). We have now studied by immunoblotting the synthesis of tumor necrosis factor alpha-induced protein 6 (TNFAIP6), which is essential for catalyzing this reaction in expanding mouse OCCs. Expanding OCCs were collected from preovulatory follicles of naturally cycling pigs and also after in vitro culture (24 or 42 h) in medium supplemented with FSH and pig serum. After isolation, OCCs were treated with Streptomyces hyaluronidase or Chondroitinase ABC. Matrix, cell pellet, and total extracts were analyzed by Western blotting. A band of about 35 kDa and a doublet of about 120 kDa, corresponding to the molecular weight of the native and HC-linked forms of TNFAIP6, respectively, were detected by a rabbit anti-human TNFAIP6 polyclonal antibody in matrix extracts of expanded cumuli. Moreover, we found by using a cell-free assay that porcine follicular fluid collected from follicles at 24 h after hCG stimulation contains HC-HA coupling activity. This activity was abolished by the rat anti-human monoclonal antibody A38, which has an epitope within the Link module domain of TNFAIP6. These experiments suggest that free TNFAIP6 protein was present in follicular fluid aspirated from porcine follicles 24 h after hCG stimulation. In contrast to mouse, we show that the A38 monoclonal antibody does not affect in vitro cumulus expansion of porcine OCCs.

PTX3 interacts with inter-alpha-trypsin inhibitor: implications for hyaluronan organization and cumulus oophorus expansion.

Pentraxin 3 (PTX3) and heavy chains (HCs) of inter-alpha-trypsin inhibitor (IalphaI) are essential for hyaluronan (HA) organization within the extracellular matrix of the cumulus oophorus, which is critical for in vivo oocyte fertilization and female fertility. In this study, we examined the possibility that these molecules interact and cooperate in this function. We show that HCs and PTX3 colocalize in the cumulus matrix and coimmunoprecipitate from cumulus matrix extracts. Coimmunoprecipitation experiments and solid-phase binding assays performed with purified human IalphaI and recombinant PTX3 demonstrate that their interaction is direct and not mediated by other matrix components. PTX3 does not bind to IalphaI subcomponent bikunin and, accordingly, bikunin does not compete for the binding of PTX3 to IalphaI, indicating that PTX3 interacts with IalphaI subcomponent HC only. Recombinant PTX3-specific N-terminal region, but not the PTX3-pentraxin C-terminal domain, showed the same ability as full-length protein to bind to HCs and to enable HA organization and matrix formation by Ptx3(-/-) cumulus cell oocyte complexes cultured in vitro. Furthermore, a monoclonal antibody raised against PTX3 N terminus, which inhibits PTX3/IalphaI interaction, also prevents recombinant full-length PTX3 from restoring a normal phenotype to in vitro-cultured Ptx3(-/-) cumuli. These results indicate that PTX3 directly interacts with HCs of IalphaI and that such interaction is essential for organizing HA in the viscoelastic matrix of cumulus oophorus, highlighting a direct functional link between the two molecules.