Oocyte morphometry in female dogs (Canis familiaris, Linnaeus, 1758)

In the present study, the ovaries of 15 healthy bitches divided into three groups according to age were analysed by histology and morphometry: group 1 (1-3 years), group 2 (> 3-5 years) and group 3 (> 5-7 years). After ovariosalpingohysterectomy, the ovaries were fixed, routinely processed for embedding in paraffin and stained with haematoxylin-eosin. The following morphometric parameters were analysed: maximum and minimum diameter (mu m), perimeter (mu m), area (mu m(2)) and roundness of the cytoplasm and nucleus of oocytes from different types of follicles. Significant differences in the cytoplasmic and nuclear parameters of follicular oocytes between the experimental groups were determined by ANOVA and the Tukey test (5%). For the biotechnology of reproduction, the present results showed that in vitro maturation yielded the best performance for oocytes from primordial follicles in group 3 females and for oocytes embedded in secondary and tertiary follicles in group 2 females. In addition, the present findings will help in the diagnosis and prognosis of diseases affecting bitches, and will especially contribute to a better understanding of these cells by researchers in the field of histology and canine reproduction.

NDACC/SAOZ UV-visible total ozone measurements: improved retrieval and comparison with correlative ground-based and satellite observations

Accurate long-term monitoring of total ozone is one of the most important requirements for identifying possible natural or anthropogenic changes in the composition of the stratosphere. For this purpose, the NDACC (Network for the Detection of Atmospheric Composition Change) UV-visible Working Group has made recommendations for improving and homogenizing the retrieval of total ozone columns from twilight zenith-sky visible spectrometers. These instruments, deployed all over the world in about 35 stations, allow measuring total ozone twice daily with limited sensitivity to stratospheric temperature and cloud cover. The NDACC recommendations address both the DOAS spectral parameters and the calculation of air mass factors (AMF) needed for the conversion of O-3 slant column densities into vertical column amounts. The most important improvement is the use of O-3 AMF look-up tables calculated using the TOMS V8 (TV8) O-3 profile climatology, that allows accounting for the dependence of the O-3 AMF on the seasonal and latitudinal variations of the O-3 vertical distribution. To investigate their impact on the retrieved ozone columns, the recommendations have been applied to measurements from the NDACC/SAOZ (Systeme d'Analyse par Observation Zenithale) network. The revised SAOZ ozone data from eight stations deployed at all latitudes have been compared to TOMS, GOMEGDP4, SCIAMACHY-TOSOMI, SCIAMACHY-OL3, OMI-TOMS, and OMI-DOAS satellite overpass observations, as well as to those of collocated Dobson and Brewer instruments at Observatoire de Haute Provence (44 degrees N, 5.5 degrees E) and Sodankyla (67 degrees N, 27 degrees E), respectively. A significantly better agreement is obtained between SAOZ and correlative reference ground-based measurements after applying the new O-3 AMFs. However, systematic seasonal differences between SAOZ and satellite instruments remain. These are shown to mainly originate from (i) a possible problem in the satellite retrieval algorithms in dealing with the temperature dependence of the ozone cross-sections in the UV and the solar zenith angle (SZA) dependence, (ii) zonal modulations and seasonal variations of tropospheric ozone columns not accounted for in the TV8 profile climatology, and (iii) uncertainty on the stratospheric ozone profiles at high latitude in the winter in the TV8 climatology. For those measurements mostly sensitive to stratospheric temperature like TOMS, OMI-TOMS, Dobson and Brewer, or to SZA like SCIAMACHY-TOSOMI, the application of temperature and SZA corrections results in the almost complete removal of the seasonal difference with SAOZ, improving significantly the consistency between all ground-based and satellite total ozone observations.

Complete replacement of the mitochondrial genotype in a Bos indicus calf reconstructed by nuclear transfer to a bos taurus oocyte

Due to the exclusively maternal inheritance of mitochondria, mitochondrial genotypes can be coupled to a particular nuclear genotype by continuous mating of founder females and their female offspring to males of the desired nuclear genotype. However, backcrossing is a gradual procedure that, apart from being lengthy, cannot ascertain that genetic and epigenetic changes will modify the original nuclear genotype. Animal cloning by nuclear transfer using host ooplasm carrying polymorphic mitochondrial genomes allows, among other biotechnology applications, the coupling of nuclear and mitochondrial genotypes of diverse origin within a single generation. Previous attempts to use Bos taurus oocytes as hosts to transfer nuclei from unrelated species led to the development to the blastocyst stage but none supported gestation to term. Our aim in this study was to determine whether B. taurus oocytes support development of nuclei from the closely related B. indicus cattle and to examine the fate of their mitochondrial genotypes throughout development. We show that indicus:taurus reconstructed oocytes develop to the blastocyst stage and produce live offspring after transfer to surrogate cows. We also demonstrate that, in reconstructed embryos, donor cell-derived mitochondria undergo a stringent genetic drift during early development leading, in most cases, to a reduction or complete elimination of B. indicus mtDNA. These results demonstrate that cross-subspecies animal cloning is a viable approach both for matching diverse nuclear and cytoplasmic genes to create novel breeds of cattle and for rescuing closely related endangered cattle.

Case-based system: Indexing and retrieval with fuzzy hypercube

In some applications with case-based system, the attributes available for indexing are better described as linguistic variables instead of receiving numerical treatment. In these applications, the concept of fuzzy hypercube can be applied to give a geometrical interpretation of similarities among cases. This paper presents an approach that uses geometrical properties of fuzzy hypercube space to make indexing and retrieval processes of cases.

The ultrastructural aspects of vitellogenesis or oocyte secondary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Serrasalminae)

Oocyte secondary growth in S. spiloleura corresponds to the period in which different vesicular structures are formed, including the cortical alveoli and the yolk granules. The oocytes with cortical alveolus formation show vesicular structures with filamentous content in the cortical cytoplasmic region, which are the cortical alveolus precursors. In these oocytes, electron-dense vesicles of heterogenous content are dispersed in the inner cytoplasmic region and their nuclei are irregular, showing many nucleoli of different sizes. The oocytes in vitellogenesis are filled with many vesicles. The cortical alveolus precursors are in the peripheral region, and electron-dense granules are seen near to the nucleus. These fuse and form yolk granules. The oocytes in vitellogenesis show a very irregular nucleus that has nucleoli of different sizes. In the oocytes in final vitellogenesis, the yolk granules are scattered throughout the cytoplasm, displacing the cortical alveoli toward cell periphery. The nucleus is similar to the other stages.

Correlação entre a taxa de fertilização e o fuso meiótico em oócitos humanos: Uma meta-análise

Objective: To investigate the relationship between the presence of meiotic spindle in human oocytes and fertilization rates in ICSI cycles. Methods: Search strategies included online surveys of databases from 1990 to 2008. Nine trials fulfilled the inclusion criteria Results: According to the meta-analysis, the results showed statistically significant higher fertilization rate (p<0.0001) when the meiotic spindle was viewed than when it was not. There was heterogeneity in this comparison. Conclusion: The presence of a birefringent meiotic spindle in human oocytes can predict a higher fertilization rate. This observation has clinical relevance mainly in countries where there is a legal limit on the number of oocytes to be fertilized. Copyright - SBRA - Sociedade Brasileira de Reprodução Assistida.

A human retrieval study of plasma-sprayed hydroxyapatite-coated plateau root form implants after 2 months to 13 years in function

Calcium phosphate-based bioactive ceramics in various physical and chemical formulations have been extensively utilized as biomaterials for bone regeneration/conduction. However, the determination of their in vivo temporal behavior from the short to long term in humans has been a challenge due to the lack of physical reference for morphologic and morphometric evaluation. The present study evaluated bone morphology and morphometry (bone-to-implant contact [BIC]) around plasma-sprayed hydroxyapatite (PSHA)-coated endosseous implants that were retrieved due to prosthetic reasons while successfully in function at the posterior region of the jaws from as early as 2 months to ~13 years after a 6-month healing period after placement. Bone morphology was evaluated by light microscopy, and BIC was determined using computer software. Irrespective of the time in vivo, lamellar bone was observed in close contact with the implant PSHA-coated surface and between plateaus. BIC ranged from ~35-95%, was highly directional, and Haversian-like osteonic morphology between plateaus was observed for most implants. The PSHA coating was present with little variation in thickness between the samples retrieved regardless of time in vivo. © 2010 by Begell House, Inc.

Effect of superstimulatory treatments on the expression of genes related to ovulatory capacity, oocyte competence and embryo development in cattle.

Multiple ovulation (superovulation) and embryo transfer has been used extensively in cattle. In the past decade, superstimulatory treatment protocols that synchronise follicle growth and ovulation, allowing for improved donor management and fixed-time AI (FTAI), have been developed for zebu (Bos indicus) and European (Bos taurus) breeds of cattle. There is evidence that additional stimulus with LH (through the administration of exogenous LH or equine chorionic gonadotrophin (eCG)) on the last day of the superstimulatory treatment protocol, called the 'P-36 protocol' for FTAI, can increase embryo yield compared with conventional protocols that are based on the detection of oestrus. However, inconsistent results with the use of hormones that stimulate LH receptors (LHR) have prompted further studies on the roles of LH and its receptors in ovulatory capacity (acquisition of LHR in granulosa cells), oocyte competence and embryo quality in superstimulated cattle. Recent experiments have shown that superstimulation with FSH increases mRNA expression of LHR and angiotensin AT(2) receptors in granulosa cells of follicles >8 mm in diameter. In addition, FSH decreases mRNA expression of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in oocytes, but increases the expression of both in cumulus cells, without diminishing the capacity of cumulus-oocyte complexes to generate blastocysts. Although these results indicate that superstimulation with FSH is not detrimental to oocyte competence, supplementary studies are warranted to investigate the effects of superstimulation on embryo quality and viability. In addition, experiments comparing the cellular and/or molecular effects of adding eCG to the P-36 treatment protocol are being conducted to elucidate the effects of superstimulatory protocols on the yield of viable embryos.

Effect of ricinoleic acid esters from castor oil (Ricinus communis) on the oocyte yolk components of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

Rhipicephalus sanguineus are bloodsucking ectoparasites, whose main host is the domestic dog, thus being present in urban areas and closely located to people. Eventually, this tick species parasitize humans and can become a potential vector of infectious diseases. Methods to control this type of pest have been the focus of many research groups worldwide. The use of natural products is increasingly considered nowadays, due to the low toxicity levels to the host and low waste generation to the environment. This study tested the effect of ricinoleic acid esters from castor oil (as an potential acaricide) on the reproductive system of R sanguineus females, more specifically on the vitellogenesis process. For this, two groups were established: the control group (CG) and the treatment group (TG) with five rabbits in each (New Zealand White), used as hosts. NaCl and ester were added to rabbits' food and offered to the hosts. After full engorgement, the females were collected and had their ovaries extracted. The ticks ovaries were submitted to histochemical techniques so the effects of esters could be observed over polysaccharides, proteins and lipids yolk. Changes in the deposition of yolk components were observed. This caused modifications on elements of polysaccharide origin and on glycoprotein compounds, interfering in the final yolk synthesis and compromising the development of the future embryo. © 2012.

Physiology and endocrinology symposium: Influence of cattle genotype (Bos indicus vs. Bos taurus) on oocyte and preimplantation embryo resistance to increased temperature

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bone morphogenetic protein 15 and fibroblast growth factor 10 enhance cumulus expansion, glucose uptake, and expression of genes in the ovulatory cascade during in vitro maturation of bovine cumulus-oocyte complexes

Oocyte-secreted factors (OSFs) regulate differentiation of cumulus cells and are of pivotal relevance for fertility. Bone morphogenetic protein 15 (BMP15) and fibroblast growth factor 10 (FGF10) are OSFs and enhance oocyte competence by unknown mechanisms. We tested the hypothesis that BMP15 and FGF10, alone or combined in the maturation medium, enhance cumulus expansion and expression of genes in the preovulatory cascade and regulate glucose metabolism favouring hyaluronic acid production in bovine cumulus-oocyte complexes (COCs). BMP15 or FGF10 increased the percentage of fully expanded COCs, but the combination did not further stimulate it. BMP15 increased cumulus cell levels of mRNA encoding a disintegrin and metalloprotease 10 (ADAM10), ADAM17, amphiregulin (AREG), and epiregulin (EREG) at 12 h of culture and of prostaglandin (PG)-endoperoxide synthase 2 (PTGS2), pentraxin 3 (PTX3) and tumor necrosis factor alpha-induced protein 6 (TNFAIP6 (TSG6)) at 22 h of culture. FGF10 did not alter the expression of epidermal growth factor-like factors but enhanced the mRNA expression of PTGS2 at 4 h, PTX3 at 12 h, and TNFAIP6 at 22 h. FGF10 and BMP15 stimulated glucose consumption by cumulus cells but did not affect lactate production or levels of mRNA encoding glycolytic enzymes phosphofructokinase and lactate dehydrogenase A. Each growth factor increased mRNA encoding glucosamine:fructose-6-PO4 transaminases, key enzymes in the hexosamine pathway leading to hyaluronic acid production, and BMP15 also stimulated hyaluronan synthase 2 (HAS2) mRNA expression. This study provides evidence that BMP15 and FGF10 stimulate expansion of in vitro-matured bovine COCs by driving glucose metabolism toward hyaluronic acid production and controlling the expression of genes in the ovulatory cascade, the first acting upon ADAM10, ADAM17, AREG, and EREG and the second on downstream genes, particularly PTGS2. © 2013 Society for Reproduction and Fertility.

Effects of FSH on the expression of receptors for oocyte-secreted factors and members of the EGF-like family during in vitro maturation in cattle

FSH induces expansion of bovine cumulus-oocyte complexes (COCs) in cattle, which can be enhanced by oocyte-secreted factors (OSFs). In this study it was hypothesised that FSH stimulates COC expansion in part from direct stimulation of the epidermal growth factor (EGF)-like ligands amphiregulin (AREG), epiregulin (EREG) and betacellulin (BTC), but also in part through regulation of OSFs or their receptors in cumulus cells. Bovine COCs were cultured in defined medium with graded doses of FSH. In the absence of FSH, COCs did not expand. FSH caused cumulus expansion, and increased the abundance of AREG and EREG mRNA in a time- and dose-dependent manner, but decreased BTC mRNA levels. FSH had modest stimulatory effects on the levels of mRNA encoding the bone morphogenetic protein 15 (BMP15) receptor, BMPR1B, in cumulus cells, but did not alter mRNA expression of the growth and differentiation factor 9 (GDF9) receptor, TGFBR1. More interestingly, FSH dramatically stimulated levels of mRNA encoding two receptors for fibroblast growth factors (FGF), FGFR2C and FGFR3C, in cumulus cells. FSH also stimulated mRNA expression of FGFR1B, but not of FGFR2B in cumulus cells. Based on dose-response studies, FGFR3C was the receptor most sensitive to the influence of FSH. This study demonstrates that FSH stimulates the expression of EGF-like factors in bovine cumulus cells, and provides evidence that FSH differently regulates the expression of distinct receptors for OSFs in cumulus cells. © CSIRO 2013.

Heparin and penicillamine-hypotaurine-epinephrine (PHE) solution during bovine in vitro fertilization procedures impair the quality of spermatozoa but improve normal oocyte fecundation and early embryonic development

The presence of heparin and a mixture of penicillamine, hypotaurine, and epinephrine (PHE) solution in the in vitro fertilization (IVF) media seem to be a prerequisite when bovine spermatozoa are capacitated in vitro, in order to stimulate sperm motility and acrosome reaction. The present study was designed to determine the effect of the addition of heparin and PHE during IVF on the quality and penetrability of spermatozoa into bovine oocytes and on subsequent embryo development. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes and mitochondrial function, was diminished (P < 0.05) in the presence of heparin and PHE. Oocyte penetration and normal pronuclear formation rates, as well as the percentage of zygotes presenting more than two pronuclei, was higher (P < 0.05) in the presence of heparin and PHE. No differences were observed in cleavage rates between treatment and control (P > 0.05). However, the developmental rate to the blastocyst stage was increased in the presence of heparin and PHE (P > 0.05). The quality of embryos that reached the blastocyst stage was evaluated by counting the inner cell mass (ICM) and trophectoderm (TE) cell numbers and total number of cells; the percentage of ICM and TE cells was unaffected (P > 0.05) in the presence of heparin and PHE (P < 0.05). In conclusion, this study demonstrated that while the supplementation of IVF media with heparin and PHE solution impairs spermatozoa quality, it plays an important role in sperm capacitation, improving pronuclear formation, and early embryonic development. © 2013 The Society for In Vitro Biology.