Oviduct fluid extracellular vesicles regulate polyspermy during porcine in vitro fertilisation.

High polyspermy is one of the major limitations of porcine invitro fertilisation (IVF). The addition of oviductal fluid (OF) during IVF reduces polyspermy without decreasing the fertilisation rate. Because extracellular vesicles (EVs) have been described as important OF components, the aim of this study was to evaluate the effect of porcine oviductal EVs (poEVs) on IVF efficiency compared with porcine OF (fresh and lyophilised). OF was collected from abattoir oviducts by phosphate-buffered saline flush, and poEVs were isolated by serial ultracentrifugation. Four IVF treatments were conducted: poEVs (0.2mgmL-1), OF (10%), lyophilized and reconstituted pure OF (LOF; 1%) and IVF without supplementation (control). Penetration, monospermy and IVF efficiency were evaluated. Transmission electron microscopy showed an EVs population primarily composed of exosomes (83%; 30-150nm). Supplementation with poEVs during IVF increased monospermy compared with control (44% vs 17%) while maintaining an acceptable penetration rate (61% vs 78% respectively) in a similar way to OF and LOF. Western blotting revealed poEVs proteins involved in early reproductive events, including zona pellucida hardening. In conclusion, our finding show that poEVs are key components of porcine OF and may play roles in porcine fertilisation and polyspermy regulation, suggesting that supplementation with poEVs is a reliable strategy to decrease porcine polyspermy and improve invitro embryo production outcomes.

Deciphering the oviductal extracellular vesicles content across the estrous cycle: implications for the gametes-oviduct interactions and the environment of the potential embryo.

BACKGROUND: The success of early reproductive events depends on an appropriate communication between gametes/embryos and the oviduct. Extracellular vesicles (EVs) contained in oviductal secretions have been suggested as new players in mediating this crucial cross-talk by transferring their cargo (proteins, mRNA and small ncRNA) from cell to cell. However, little is known about the oviductal EVs (oEVS) composition and their implications in the reproductive success. The aim of the study was to determine the oEVs content at protein, mRNA and small RNA level and to examine whether the oEVs content is under the hormonal influence of the estrous cycle. RESULTS: We identified the presence of oEVs, exosomes and microvesicles, in the bovine oviductal fluid at different stages of the estrous cycle (postovulatory-stage, early luteal phase, late luteal phase and pre-ovulatory stage) and demonstrated that their composition is under hormonal regulation. RNA-sequencing identified 903 differentially expressed transcripts (FDR < 0.001) in oEVs across the estrous cycle. Moreover, small RNA-Seq identified the presence of different types of ncRNAs (miRNAs, rRNA fragments, tRNA fragments, snRNA, snoRNA, and other ncRNAs), which were partially also under hormonal influence. Major differences were found between post-ovulatory and the rest of the stages analyzed for mRNAs. Interesting miRNAs identified in oEVs and showing differential abundance among stages, miR-34c and miR-449a, have been associated with defective cilia in the oviduct and infertility. Furthermore, functional annotation of the differentially abundant mRNAs identified functions related to exosome/vesicles, cilia expression, embryo development and many transcripts encoding ribosomal proteins. Moreover, the analysis of oEVs protein content also revealed changes across the estrous cycle. Mass spectrometry identified 336 clusters of proteins in oEVs, of which 170 were differentially abundant across the estrous cycle (p-value< 0.05, ratio < 0.5 or ratio > 2). Our data revealed proteins related to early embryo development and gamete-oviduct interactions as well as numerous ribosomal proteins. CONCLUSIONS: Our study provides with the first molecular signature of oEVs across the bovine estrous cycle, revealing marked differences between post- and pre-ovulatory stages. Our findings contribute to a better understanding of the potential role of oEVs as modulators of gamete/embryo-maternal interactions and their implications for the reproductive success.

Effect of interval-training exercise on subchondral bone in a chemically-induced osteoarthritis model.

OBJECTIVES: The role of subchondral bone in osteoarthritis (OA) development is well admitted. Cross-talk between subchondral bone and cartilage may be disrupted in OA, leading to altered subchondral bone remodeling. Osteocytes are involved in bone remodeling control and could play a key role in OA progression. Our purpose of this study was to evaluate the preventive effect of interval-training exercise on subchondral bone and osteocyte in monosodium iodoacetate (MIA) model of experimental OA. METHODS: At baseline, 48 male Wistar rats (8 weeks old) were separated into two groups: interval-training exercise or no exercise for 10 weeks. After this training period, each group was divided into two subgroups: MIA-injected knee (1 mg/100 µl saline) and saline-injected knee. Four weeks later, rats were sacrificed and carefully dissected. Evaluated parameters were: cartilage degeneration by OA scoring, bone mineral density (BMD) by Dual energy X-ray Absorptiometry (DXA), trabecular subchondral bone microarchitecture by micro-computed tomography (µCT), cortical subchondral bone lacunar osteocyte occupancy (by Toluidine Blue staining) and cleaved caspase-3 positive apoptosis (by epifluorescence). RESULTS: Our results showed deleterious effects of MIA on cartilage. OA induced a decrease in proximal tibia (PT) BMD which was prevented by exercise. Exercise induced increase in full osteocyte lacunae surface and osteocyte occupancy (+60%) of cortical subchondral bone independently of OA. Osteocyte apoptosis (<1%) in cortical subchondral bone was not different whatever the group at sacrifice. CONCLUSION: Our results suggest that preliminary interval-training improved BMD and osteocytes lacunar occupancy in subchondral bone. Our interval-training did not prevent MIA-induced cartilage degeneration.

Breast-cancer anti-estrogen resistance 4 (BCAR4) encodes a novel maternal-effect protein in bovine and is expressed in the oocyte of humans and other non-rodent mammals.

STUDY QUESTION: Does BCAR4 have a role in mammalian embryo development? SUMMARY ANSWER: Expression, localization and functional data support that BCAR4 is a maternal-effect protein in non-rodent mammals. WHAT IS KNOWN ALREADY: BCAR4 was previously identified as an oocyte-specific gene in cattle, and as a marker of certain breast tumors in humans. STUDY DESIGN, SIZE, DURATION: Human oocytes were obtained from patients undergoing IVF, but had failed to mature after ovarian stimulation. Dog oocytes were obtained from ovariectomized bitches. Pig, horse and bovine ovaries were obtained from commercial slaughterhouses for extraction of immature oocyte-cumulus complexes. In vivo matured bovine matured oocytes were obtained after ovulation induction and ovulation inducing treatment of Montbeliard heifers. MATERIALS, SETTING AND METHODS: Expression at the RNA level was analyzed by reverse transcription coupled to polymerase chain reaction. Western blot and immunolabeling coupled to confocal or electronic microscopy were used to analyze bovine protein expression and intracellular localization. For the functional approach, short-interfering RNA were microinjected into mature bovine oocytes, followed by IVF; cleavage and embryo development were recorded. MAIN RESULTS AND THE ROLE OF CHANCE: The BCAR4 gene is conserved in mammalian species from various orders and has been lost in rodents after divergence with lagomorphs. The transcript is expressed in the oocytes of humans and domestic species. We bring the first experimental evidence of the BCAR4 protein in mammals. In cattle, the protein is not detected in immature oocytes but starts to be synthesized during maturation, increases in the zygote and persists until the morula stage. The protein is detected throughout the cytoplasm in mature oocytes, concentrates in and around the pronuclei in the zygote, and appears to shuttle in and out of the nuclei starting in the 2-cell embryo; BCAR4 is also present at the junctions between blastomeres from 2-cell to morula. In our functional approach, targeting the BCAR4 transcript by small-interfering RNA significantly compromised development to the morula or/and blastocyst stages (P < 0.05, logistic regression). LIMITATIONS, REASONS FOR CAUTION: As indicated above, protein expression and function were investigated in cattle and mostly in vitro matured oocytes were used. WIDER IMPLICATIONS OF THE FINDINGS: This study provides a novel candidate gene whose mutation or deregulation may underlie certain cases of unexplained female infertility.

Centrosome fine ultrastructure of the osteocyte mechanosensitive primary cilium.

The centrosome is the principal microtubule organization center in cells, giving rise to microtubule-based organelles (e.g., cilia, flagella). The aim was to study the osteocyte centrosome morphology at an ultrastructural level in relation to its mechanosensitive function. Osteocyte centrosomes and cilia in tibial cortical bone were explored by acetylated alpha-tubulin (AαTub) immunostaining under confocal microscopy. For the first time, fine ultrastructure and spatial orientation of the osteocyte centrosome were explored by transmission electron microscopy on serial ultrathin sections. AαTub-positive staining was observed in 94% of the osteocytes examined (222/236). The mother centriole formed a short primary cilium and was longer than the daughter centriole due to an intermediate zone between centriole and cilium. The proximal end of the mother centriole was connected with the surface of daughter centriole by striated rootlets. The mother centriole exhibited distal appendages that interacted with the cell membrane and formed a particular structure called "cilium membrane prolongation." The primary cilium was mainly oriented perpendicular to the long axis of bone. Mother and daughter centrioles change their original mutual orientation during the osteocyte differentiation process. The short primary cilium is hypothesized as a novel type of fluid-sensing organelle in osteocytes.

Ultrastructure of erythrocytic stages of avian Plasmodium spp. of the sub-genus Novyella and its "globule".

A globule frequently refractory, appearing blue, pale blue, or white with Giemsa stain, is characteristic of the intraerythrocytic stages of the type species and of most of the other species included at present in the subgenus Novyella. This globule is absent from the other Plasmodium sub-genera. An ultrastructural study has been performed on schizogonic stages of Plasmodium merulae from the blood of the blackbird, Turdus merula. In section the globule contains a finely granular substance suggesting a condensed or coagulated substance. It differs distinctly from a classical food vacuole by denser contents, and show in some sections (19, 23) a peripheral opaque rim with a radial arrangement of ribosomes. Except for the presence of the globule, in other details P. merulae do not diverge from the ultrastructure common to the intraerythrocytic stages of avian Plasmodium.

pEg7, a new Xenopus protein required for mitotic chromosome condensation in egg extracts.

We have isolated a cDNA, Eg7, corresponding to a Xenopus maternal mRNA, which is polyadenylated in mature oocytes and deadenylated in early embryos. This maternal mRNA encodes a protein, pEg7, whose expression is strongly increased during oocyte maturation. The tissue and cell expression pattern of pEg7 indicates that this protein is only readily detected in cultured cells and germ cells. Immunolocalization in Xenopus cultured cells indicates that pEg7 concentrates onto chromosomes during mitosis. A similar localization of pEg7 is observed when sperm chromatin is allowed to form mitotic chromosomes in cytostatic factor-arrested egg extracts. Incubating these extracts with antibodies directed against two distinct parts of pEg7 provokes a strong inhibition of the condensation and resolution of mitotic chromosomes. Biochemical experiments show that pEg7 associates with Xenopus chromosome-associated polypeptides C and E, two components of the 13S condensin.

[The centrosome--a riddle of the "cell processor"].

In the present review the description of history of the centrosome investigation is given and the current state of knowledge of this cellular structure in morphological, biochemical, and functional aspects is presented. Besides of the classical functions of the centrosome as a MT nucleating, MT ancoriging, and MT organizing center, the idea about the centrosome as a cellular regulating center and as a structural part of the mechanism operating dynamic morphology of a cell is discussed.

Identification of a functional destruction box in the Xenopus laevis aurora-A kinase pEg2.

Like for all aurora-A kinases, the Xenopus pEg2 kinase level peaks in G(2)/M and is hardly detectable in G(1) cells, suggesting that the protein is degraded upon exit from mitosis as reported for the human aurora-A kinase. We identified for the first time a sequence RxxL in the C-terminal end of the kinase catalytic domain. Mutation of this sequence RxxL to RxxI suppresses the ubiquitination of the protein as well as its degradation. The sequence RxxL corresponding to the pEg2 functional destruction box has been conserved throughout evolution in all aurora kinases including aurora-A, -B and -C.

Neurotoxic glutamate treatment of cultured cerebellar granule cells induces Ca2+ -dependent collapse of mitochondrial membrane potential and ultrastructural alterations of mitochondria.

Rhodamine 123 staining and electron microscopy were used to reveal a correlation between the ultrastructural and functional state of cultured cerebellar granule cells after short glutamate treatment. Glutamate exposure (15 min, 100 microM) in Mg2+-free solution caused considerable ultrastructural alterations in a granule cell: clumping of the chromatin, swelling of the endoplasmic reticulum and mitochondria, and disruption of the mitochondrial cristae. After glutamate treatment, the mitochondria of the neurons lost their ability to sequester rhodamine 123. Both the N-methyl-D-aspartate receptor channel blocker MK-801 (30 microM) and cobalt chloride (2 mM) prevented the deteriorative effects of glutamate. These data suggest that glutamate-induced Ca2+ overload of the neurons can lead to non-specific permeability of the inner mitochondrial membrane, resulting in neuronal death.

Cell cycle analysis and synchronization of the Xenopus laevis XL2 cell line: study of the kinesin related protein XlEg5.

Cell free extracts prepared from Xenopus eggs are one of the most powerful in vitro systems to analyze cell cycle-regulated mechanisms such as DNA replication, nuclear assembly, chromosome condensation, or spindle formation. Xenopus embryos can complete several synchronous cell cycles in the absence of transcription, consequently Xenopus extracts are very helpful to study the molecular level of cellular mechanisms. Many key cell cycle regulators like p34cdc2 and cdk2 have been discovered and characterized using those extracts, but their regulation during somatic cell cycles have only been studied in mammalian cultured cells. In this paper, we describe optimized conditions to obtain cell cycle arrested Xenopus XL2 cultured cells. Synchronization of XL2 cells at different stages of the cell cycle was achieved by serum starvation and drug treatments such as aphidicolin, nocodazole, and ALLN. The degree of synchronization was assessed by indirect fluorescence microscopy and FACS analysis. This method was used to study the cell cycle expression of the Xenopus kinesin-related protein, XlEg5, a microtubule-based motor protein involved in movement and cell division in early development. We found that the expression of the protein was maximum in mitosis and minimum in G1, which correlated with the expression of its messenger RNA. XL2 cultured cells were also used to analyze the ultrastructural sub-cellular localization of XlEg5. During mitosis, the protein was found around the centrosome in prophase, on the spindle microtubules in metaphase, and, interestingly, around the minus end of the midbody microtubules in telophase.

Role of the centrosome in mitosis: UV micro-irradiation study.

Ultraviolet micro-irradiation (UV-MI) of the PK (pig kidney embryo) cell centrosome (lambda max = 280 nm, spot diameter 1.6 mm, exposure time 5-15 s) at metaphase and anaphase resulted in functional damage of the centrosome. After UV-MI of the centrosome at early metaphase, chromosomes quickly (in 1-3 min) moved away from the irradiated pole and then encircled the non-irradiated pole. Within 10 min after UV-MI the spindle disassembled and chromosomes remained unseparated. The minimal dose inducing this effect in 90% of cells was accumulated in 5 s. After the same UV-MI at late metaphase, chromosomes shifted towards the non-irradiated pole; however, anaphase started and chromosome motion towards the non-irradiated pole continued normally. UV-MI of the centrosome at early anaphase for 5-15 s slowed down and then stopped chromosome motion towards the irradiated pole. This was a result of rapid (within 2-3 min) disorganization of the half-spindle. Chromosomes continued to move towards the opposite pole normally, while cytokinesis was significantly retarded. No visible lesion was revealed by electron microscopy after 5 s UV-MI, while 15 s irradiation resulted in the truncation of the microtubule bundles 1.5-2 microns from the centrosome. We concluded that UV-MI inactivates the centrosome and induces disaggregation of microtubule initiation sites. The critical point (checkpoint) in mitosis up to which this damage induces mitotic arrest is mid-metaphase.

[A comparative level of expression of some proteins in XL2 cell synchronized on different phases of cell cycle]. / Issledovanie sravnitel'nogo urovnia ékspressii belkov v sinkhronizirovannykh na razlichnykh stadiiakh kletochnogo tsikla kletkakh XL2.

Cells of cultured line XL2 (Xenopus laevis) were synchronized by a combine effect of serum deprivation, aphidicolin, nocodazole and ALLN treatments. Four fractions were prepared, with maximum percentage of cells being in G1, S and G2 phases of cell cycle, and in mitosis, respectively. Comparative levels of six different proteins (beta-tubulin, DNA topoisomerase IIa, Xenopus Aurora A kinase pEg2, kinesin-like motor protein X1Eg5, and two members of condensis family proteins pEg7 (XCAP D2) and XCAP E were detected by quantitative Western blot analysis of these fractions. We used a new method of mathematic processing of data that commonly provides a possibility to calculate a comparative quantity of proteins in hypothetically "clean" fraction composed of cells being in the same phase of the cell cycle. This method makes it possible to use even partly synchronized cell cultures for analysis of changes in protein quantity, provided a precede determination of cell population composition is made.

[The effect of UV microirradiation of the centrosome on cell behavior. I. The destruction of the mitotic spindle and disruption of cell division during irradiation in the metaphase]. / Vliianie UF-mikrooblucheniia tsentrosomy na povedenie kletok. I. Raspad mitoticheskogo veretena i narushenie deleniia kletki pri obluchenii v metafaze.

A 5 second UV microirradiation of the centrosome during the early metaphase leads to a rapid (within 5 minutes) chromosome shift towards the normal pole and disorganizes the spindle. The later metaphase plate is also disorganized, chromosomes being situated chaotically in the central part of the cell. Numerous (up to 10 or more) microtubule convergence centers are observed instead of the spindle. 2-4 hours after the microirradiation some cells may enter cytotomy. The microirradiation of chromosomes and cytoplasm in similar and greater doses (exposure up to 15 seconds) did not lead to disorganization of the spindle and did not effect the normal completion of mitosis. Sometimes the 5 second microirradiation in the middle metaphase also blocked anaphase, but the microirradiation within the last 5 minutes of the metaphase always failed to block anaphase and normal completion of division.

[The effect of centrosome UV microbeam irradiation on cell behavior. II. The radiation sequelae in the anaphase: the completion of division and the fate of the interphase cell]. / Vliianie UF-mikrooblucheniia tsentrosomy na povedenie kletok. II. Posledstviia oblucheniia v anafaze: zavershenie deleniia i sud'ba interfaznoi kletki.

Ultraviolet (280 nm) microbeam irradiation of the centrosome (spindle pole) in the early anaphase slows down and then stops chromosome movement towards the irradiated pole. This happens as a result of rapid (in 1-2 min) disorganization of the half-spindle. Chromosome movement towards the opposite pole continues normally. Irradiation of the centrosome also affects cystotomy--the residual body is formed later than in the normal cell. In some cases additional constrictions are formed or the cytoplasm starts blebbing. Immediately after division the microtubule network in two daughter cells (one of them with irradiated centrosome) is similar. Two hours later in the irradiated cell the amount of microtubules is often less than in the sister cell. Incubation with nocodazole (0.5-1.5 h, 0.15 microgram/ml) shows that in the irradiated cells microtubules radiating from the centrosome are practically absent. Irradiation of other regions of the cytoplasm does not cause any of the effects described above.

[The effect of the UV microirradiation of the centrosome on cell behavior. III. The ultrastructure of the centrosome after irradiation]. / Vliianie UF-mikrooblucheniia tsentrosomy na povedenie kletok. III. Ul'trastruktura tsentrosomy posle oblucheniia.

One of the spindle poles of mitotic PK cells was irradiated with UV microbeam in metaphase or in anaphase. Electron microscopy showed that immediately after irradiation the microtubules around the centrosome were maintained, and that the ultrastructure of both irradiated and nonirradiated poles was similar. After microirradiation of the centrosome in metaphase, the mitotic halo around this centrosome was retained, but in due time the number of microtubules was getting less compared to that around the nonirradiated centrosome. When daughter cells with irradiated centrosomes are passing into the interphase, their centrioles are not separated from each other, no primary cilia are formed, and no replication of centrioles occurs. In the interphase cells with irradiated centrosomes, satellites are formed on the active centriole, but centrosome-attached microtubules are practically absent.

[The effect of the laser microirradiation of the cell center on neutrophil motility]. / Vliianie lazernogo mikrooblucheniia kletochnogo tsentra na podvizhnost' neitrofilov.

The cell center of human neutrophils spread on polylysine-coated coverslips was irradiated with an argon laser microbeam. After the cells were pretreated with acridine orange, the irradiation of the cell center in a dose of over 0.1 J completely and irreversibly suppressed the motility of neutrophils (both random migration and chemotaxis), even though the cells retained their polarization. The same dose, applied to the cell nucleus and the forward and backward edges of the cytoplasm, resulted in little, if any, effect on cell motility, and did not inhibit their movement toward the target. Electron microscopy of the cells with the irradiated center showed the microtubules to persist for no less than 30 minutes; no visible destruction was caused in the cell center structure. Consequently, the cell center directly controls (not through polymerization of microtubules) the motility of neutrophils.

[The effect of ultraviolet microbeam irradiation of the centrosome on cellular behavior. IV. Synthetic activity, cell spreading and growth with an inactivated centrosome]. / Vliianie ul'trafioletovogo mikrooblucheniia tsentrosomy na povedenie kletok. IV. Sinteticheskaia aktivnost', rasplastyvanie i rost kletok s inaktivirovannoi tsentrosomoi.

One of the spindle poles of mitotic PK cells was irradiated with UV microbeam at anaphase. After irradiation, cell division completed with a minor delay and two daughter cells were spreading synchronously. Later on, cells with irradiated centrosomes slightly shrunk, while their sister cells enlarged normally. Sister cells entered S-phase, some of them undergoing mitosis. In the cells with irradiated centrosomes the formation of nucleoli was disturbed and numerous primary nucleoli remained for 50 h (the maximum time of observation). RNA synthesis in the cells with irradiated centrosomes was twice less than in the sister cells, with ribosomal RNA synthesis being suppressed predominantly. Cells with irradiated centrosomes did not enter S-phase for as long as 24 h. The same irradiation of a portion of cytoplasm outside the spindle performed during anaphase did not change the pattern behaviour in daughter cell. In is concluded that the centrosome regulates progression throughout the cell cycle, and that centrosome irradiation induces specific and irreversible damage of interphase cells.

[Intracellular localization of XCAP-E protein in XL2 (Xenopus laevis) cells under normal conditions and during inhibition of pRNA transcription and processing]. / Issledovanie vnutrikletochnoi lokalizatsii belka XCAP-E v kletkakh linii XL2 (Xenopus laevis) v norme i pri ingibirovanii transkriptsii i protsessinga pRNK

The interaction of condensin subunit XCAP-E with various nucleolar subcompartments in XL2 cells was studied. In the interphase cells, XCAP-E was associated with a granular component of nucleoli (as shown by double staining with antibodies against B23) and with small nucleolus-like structures in the nucleoplasm. Inhibition of transcription by actinomycin D does not disrupt interaction of XCAP-E with the granular compartment of segregated nucleoli. Treatment with DRB 5,6-dichloro-1 beta-ribofuranozide-benzimidazole causes disintegration of nucleolar fibrillar complexes, but does not affect nucleolar localization of XCAP-E. The data suggest that nucleolar association of XCAP-E is independent on the functional state of the nucleolus, and imply a possible role of this protein in rRNA processing and pre-fibosome assembly.