Influence of lineage of oocyte donor on the yield and morphology of oocytes recovered by ultrasound-guided follicular aspiration in nellore cows

The objective of this study was to investigate the influence of lineage of oocytes donors on the number and quality of oocytes obtained through ultrasound-guided follicular aspiration in Nellore breed cows derived from two lineages of bulls (Karvadi; K and Taj-Mahal; T). Both maternal (Km and Tm) and paternal (Kp and Tp) lineages, as well as their combinations were investigated. Oocyte aspirations were repeatedly performed with an aspiration interval of 15 days in 56 donor females. Recovered cumulus oocyte-complexes (COCs) were counted, morphologically examined and classified into seven categories (grades from I to VII) according to the number of layers of the cumulus oocyte and cytoplasm appearance. The mean number of oocytes retrieved from donors of lineage Tp-Tm was significantly higher (28.23±1.92, P<0.05) than those obtained from lineages Kp-Tm, Kp-Km, and Tp-Km (21.34±1.32, 21.28±1.73, and 16.72±1.31, respectively). There was no significant difference in the mean number of recovered oocytes between donors of lineages Kp-Km and Kp-Tm, whereas animals of lineage Tp-Km yielded the lowest number of oocytes. Higher mean number of grade III oocytes was recovered from donors of lineage Kp than lineage Tp (10.11±0.66 versus 8.79±0.58, respectively), with more grade III oocytes being obtained in both lineages as compared to the others. Paternal lineage did not influence the quality of recovered oocytes in any other category, but both Kp and Tp yielded a great mean number of oocytes graded as I, II, and III (3.14±0.21; 4.93±0.33, and 10.11±0.66 versus 3.19±0.21, 5.59±0.44, and 8.79±0.58, respectively) than those classified as IV, V, VI, and VII. However, when considering the data from the maternal lineage significantly more oocytes (P<0.05) of grade I, II and III were obtained from Taj-Mahal (11.67 ± 0.67, 5.9±0.42 and 3.64±0.25, respectively) than for lineage Karvadi, with similar results for oocytes of grades IV, V, VI, and VII. Similarly to the paternal lineage, the number of oocytes of grade III was superior (P<0.05) when compared to other categories for both lineages. In conclusion, we demonstrate here a direct influence of lineage of oocyte donor on the production and quality of oocytes obtained through ultrasound-guided follicular aspiration in Nellore cows.

A substituição do meio TCM pelo meio BARC, suplementado com SFB, BSA ou PVA, para maturação de oócitos bovinos in vitro, não incrementa a subsequente produção de blastocistos

This study was carried out to assess the influence of bovine embryo culture medium Beltsville Agriculture Research Center (BARC), supplemented with FCS, BSA or PVA, on the in vitro oocyte maturation, evidenced by cleavage rate and blastocysts production at different developmental stages. Three experiments were performed, as follows: exp.1: addition of FCS to BARC medium at concentrations of 0, 5 and 10%; exp. 2: addition of BSA to BARC medium at concentrations of 0, 4 and 8 mg/ml; exp. 3: addition of PVA to BARC medium at concentrations of 0, 0.5 and 1.0 mg/ml. TCM 199 supplemented with bicarbonate, pyruvate, gentamicin sulfate, FSH, LH and FCS was used as control group. Oocytes obtained from cow ovaries at slaughterhouse were selected in PBS, and then matured in BARC medium supplemented with FSH, LH and gentamicin sulfate, according to the experimental design. Percoll gradient was used for sperm selection and TALP medium for IVF. In vitro embryo culture was in SOF-m medium; a humidified atmosphere with 5% CO2, in air, at 38.7oC was used for all steps. The number of oocytes reaching blastocyst, expanded blastocyst, and hatched blastocyt stages was recorded, respectively at 72 and 168 h post-insemination. ANOVA and Bonferroni t test were used to determine differences among groups. Differences of P<0.05 were taken as significant. Higher percentage (P<0.05) of cleaved oocytes was observed in group TCM + FCS than for the other groups matured in BARC supplemented with FCS or BSA, regardless the concentration used. However, the cleavage rate was similar between groups BARC plus PVA with 1 mg/ml (85.7%) and TCM + FCS (90.8%). Significant difference was found among groups for the production of blastocysts, with the control group yielding a higher number of blastocysts (results ranging from 47.4 to 51.4%, in comparison with groups using BARC + FCS (4.1 to 19.7%), BSA (1.4 to 5.6%) and PVA (5.7 to 10.6%). In conclusion, BARC medium supplemented with different macromolecules did not promote a beneficial effect on in vitro oocyte maturation, resulting in lower rate of cleavage and blastocyst production when compared with TCM + FCS medium.

Expression of α6 integrin subunit in bovine oocyte and its potential role during fertilization

Fertilization in mammals requires the successful completion of a sequence of steps, starting with the transport of gametes in the reproductive tract and ending with sperm-egg membrane fusion to produce a zygote. Although some integrin subunits are known to be associated with the plasma membrane of some mammalian oocytes and spermatozoa, the presence of α6 integrin on bovine oocytes with intact zona pellucida has not been reported. The present study was undertaken to evaluate the expression of α6 integrin subunit in bovine oocyte and to determine if in vitro binding to the zona pellucida and fertilization were affected by treating oocytes with α6 integrin subunit antibody. The α6 integrin subunit was identified on the bovine oocyte by immunocytochemistry. In vitro fertilization was significantly decreased when in vitro matured bovine oocytes were pre-incubated with α6 integrin subunit antibody at concentration 5 and 20 μg/mL, and spermoocyte binding increased. These studies demonstrated the presence of α6 integrin subunit on bovine oocyte, and its importance in fertilization and polyspermy.

Heat stress induced alteration in bovine oocytes: functional and cellular aspects

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

Gene Expression in Bovine Oocytes and Cumulus Cells After Meiotic Inhibition with the Cyclin-Dependent Kinase Inhibitor Butyrolactone I

Contents The aim of this study was to determine the effect of temporary inhibition of meiosis using the cyclin-dependent kinase inhibitor butyrolactone I (BLI) on gene expression in bovine oocytes and cumulus cells. Immature bovine cumulusoocyte complexes (COCs) were assigned to groups: (i) Control COCs collected immediately after recovery from the ovary or (ii) after in vitro maturation (IVM) for 24 h, (iii) Inhibited COCs collected 24 h after incubation with 100 mu m BLI or (iv) after meiotic inhibition for 24 h followed by IVM for a further 22 h. For mRNA relative abundance analysis, pools of 10 denuded oocytes and respective cumulus cells were collected. Transcripts related to cell cycle regulation and oocyte competence were evaluated in oocytes and cumulus cells by quantitative real-time PCR (qPCR). Most of the examined transcripts were downregulated (p < 0.05) after IVM in control and inhibited oocytes (19 of 35). Nine transcripts remained stable (p > 0.05) after IVM in control oocytes; only INHBA did not show this pattern in inhibited oocytes. Seven genes were upregulated after IVM in control oocytes (p < 0.05), and only PLAT, RBP1 and INHBB were not upregulated in inhibited oocytes after IVM. In cumulus cells, six genes were upregulated (p < 0.05) after IVM and eight were downregulated (p < 0.05). Cells from inhibited oocytes showed the same pattern of expression regarding maturation profile, but were affected by the temporary meiosis inhibition of the oocyte when the same maturation stages were compared between inhibited and control groups. In conclusion, changes in transcript abundance in oocytes and cumulus cells during maturation in vitro were mostly mirrored after meiotic inhibition followed by maturation.

Protein diets promote the maturation of oocytes and spawning of Piaractus mesopotamicus kept in cages

The objective of this study was to evaluate the effects of balanced diets on the maturation of oocytes and the reproductive performance of P. mesopotamicus in cages. A completely random design with 224 fish in 16 cages measuring 5 m(3) was employed for this purpose. The treatments consisted of diets containing 18, 24, 30, and 36% crude protein (CP) provided ad libitum. The external and internal morphological characteristics of the specimens were examined, as well as: the position of the germinal vesicle, the distribution of oocyte diameters, the fertilization and hatching rates, the number of oocytes released, the total number of oocytes, the remaining weight and total weight of the ovaries, the gonadosomatic index, the condition factor (K), and the histology of the oocytes and ovaries post-spawning and during ovarian regression. The diameters of the oocytes collected before the first hormonal application displayed a unimodal distribution for the lowest protein content and a polymodal distribution for the other treatments. A similar situation was seen during spawning. The lowest fertilization and hatching rates were found as a consequence of the treatment with 30% CP (P < 0.05). The greatest hatching rate occurred in the females fed 18% CP. The greatest total oocyte weight was found in the specimens that received between 30 and 36% CP. The lowest K index was found in the females fed 36% CP. In conclusion, a diet containing 18% CP satisfies the reproductive requirements of females adapted to this system.

Oocyte meiotic-stage-specific differences in spindle depolymerization in response to temperature changes monitored with polarized field microscopy and immunocytochemistry

Objective: To compare the polymerization status of mouse oocyte spindles exposed to various temperatures at various stages of meiosis. Design: Experimental animal study. Setting: University animal laboratory. Animal(s): CF1 mice. Intervention(s): Immature oocytes matured to metaphase I (MI), telophase I (TI), and metaphase II (MII) were incubated at 37 degrees C (control), room temperature (RT), or 4 degrees C for 0, 10, 30, and 60 minutes. Spindle analysis subsequently was performed using polarized field microscopy and immunocytochemistry. Spindles of TI and MII oocytes that underwent vitrification and warming were analyzed also by immunocytochemistry. Main Outcome Measure(s): Detection of polymerized meiotic spindles. Result(s): At RT, and after 60 minutes at 4 degrees C, a significant time-dependent decrease in the percentage of polymerized meiotic spindles was observed in MI and MII oocytes, but not in TI oocytes. The polymerization of TI spindles at 4 degrees C was similar to that of TI spindles at 4 degrees C that underwent vitrification and warming. Conclusion(s): Significant differences in the microtubule dynamics of MI, TI, and MII oocytes incubated at different temperatures were observed. In particular, meiotic spindles in TI oocytes exhibited less depolymerization than did metaphase spindles. (Fertil Steril (R) 2012; 97: 714-9. (C) 2012 by American Society for Reproductive Medicine.)

In Vitro Development and Cell Allocation After Aggregation of Syngeneic Wild Type and Fluorescence-Expressing Bovine Cloned Embryos

Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre- and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the first third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may influence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells. Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the. 2 or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student's test (P < 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were similar between one another. Cloned GFP embryos had lower in vitro development to the blastocyst stage than WT embryos, which had more TCN than parthenote or aggregated chimeric WT/GFP embryos. Aggregated GFP embryos had fewer cells than the other embryo groups. Discussion: The in vitro development of GFP cloned embryos was lower than WT embryos, with no effects on cell allocation in resulting blastocysts. Differences in blastocyst rate between groups were likely due to lower GFP-expressing cell viability, as GFP donor cells were at high population cell doublings when used for cloning. On a per embryo basis, embryo aggregation on Day 1 resulted in blastocyst development similar to non-aggregated embryos on Day 7, with no differences in cell proportion between groups. The use of GFP-expressing cells was proven a promising strategy for the study of cell allocation during embryo development, which may assist in the elucidation of mechanisms of abnormalities after in vitro embryo manipulations, leading to the development of improved protocols for the in vitro production (IVP) of bovine embryos.

Gonadal steroids levels and vitellogenesis in the formation of oocytes in Prochilodus lineatus (Valenciennes) (Teleostei: Characiformes)

The objective of this study was to obtain information about the possible mechanisms related to poor reproductive performance in tropical rheophilic fish. To that effect, cages (Cs) and earthen ponds (EPs) were used as experimental systems to provide unsuitable and suitable conditions, respectively, for curimbata (Prochilodus lineatus) breeders. Fish were maintained under experimental conditions for 18 months, and during this period females were randomly sampled every two months for biometric analysis (n=30), blood (n=5/sampling) and ovary (n=5/sampling). After this period EPs females (EPFs) and Cs females (CFs) were submitted to the induced breeding experiments. The results showed that rearing curimbata for such long time in a cage at this stocking density, reduces its growth, plasma E2 levels and vitellogenesis. During vitellogenesis, the mean plasma estradiol levels of CFs were three times lower than those of EPFs (P<0.01). CFs presented poorer results than EPFs for all the examined parameters of reproductive performance. Taken together these data showed that the reduced estradiol levels during vitellogenesis (and the consequently less intense transition from the previtellogenic to vitellogenic phase) and reduced amounts of yolk are mechanisms associated with the formation of low quality oocytes and shortened and delayed breeding season in this species. Moreover, our data showed that the onset of vitellogenesis (six months before the spawning season) must be considered as a key period related to the formation of oocytes of good quality, and adequate management should be provided throughout the year.

The use of parthenotegenetic and IVF bovine blastocysts as a model for the creation of human embryonic stem cells under defined conditions

Clinical application of human embryonic stem cells will be possible, when cell lines are created under xeno-free and defined conditions. We aimed to establish methodologies for parthenogenetic activation, culture to blastocyst and mechanical isolation of the inner cell mass (ICM) using bovine oocytes, as a model for derivation and proliferation of human embryonic stem cells under defined xeno-free culture conditions. Cumulus-oocyte-complexes were in vitro matured and activated using Ca(2+)Ionophore and 6-DMAP or in vitro fertilized (IVF). Parthenotes and biparental embryos were cultured to blastocysts, when their ICM was mechanically isolated and placed onto a substrate of fibronectin in StemProA (R) medium. After attachment, primary colonies were left to proliferate and stained for pluripotency markers, alkaline phosphatase and Oct-4. Parthenogenesis and fertilization presented significantly different success rates (91 and 79 %, respectively) and blastocyst formation (40 and 43 %, respectively). ICMs from parthenogenetic and IVF embryos formed primary and expanded colonies at similar rates (39 % and 33 %, respectively). Six out of eight parthenogenetic colonies tested positive for alkaline phosphatase. Three colonies were analyzed for Oct-4 and they all tested positive for this pluripotency marker. Our data show that Ca2+ Ionophore, and 6-DMAP are efficient in creating large numbers of blastocysts to be employed as a model for human oocyte activation and embryo development. After mechanical isolation, parthenogetic derived ICMs showed a good rate of derivation in fibronectin and Stem-Pro forming primary and expanded colonies of putative embryonic stem cells. This methodology may be a good strategy for parthenogenetic activation of discarded human oocytes and derivation in defined conditions for future therapeutic interventions.

Nuclear Transfer with Apoptotic Bovine Fibroblasts: Can Programmed Cell Death Be Reprogrammed?

Cell death by apoptosis is considered to be irreversible. However, reports have indicated that its reversibility is possible if the cells have not yet reached the "point of no return.'' In order to add new information about this topic, we used cells at different moments of apoptotic process as nuclear donors in somatic cell nuclear transfer (SCNT) in order to test if programmed cell death can be reversed. Adult bovine fibroblasts were treated with 10 mu M of staurosporine (STP) for 3 h and analyzed for phosphatidylserine externalization (Annexin assay) and presence of active caspase-9. Annexin-positive (Anx +) and Caspase-9-positive (Casp-9 +) cells were isolated by FACS and immediately transferred into enucleated in vitro matured bovine oocytes. After STP treatment, 89.9% of cells were Anx + (4.6% in control cells; p < 0.01) and 24.9% were Casp-9 + (2.4% in control cells; p < 0.01). Fusion and cleavage were not affected by the use apoptotic cells (p > 0.05). Also, the use of Anx + cells did not affect blastocyst production compared to control (26.4% vs. 22.9%, respectively; p > 0.05). However, blastocyst formation was affected by the use of Casp-9 + cells (12.3%; p < 0.05). These findings contribute to the idea of that apoptosis is reversible only at early stages. Additionally, we hypothesize that the "point of no return'' for apoptosis may be located around activation of Caspase-9.

Single-cell expression analysis of BMP15 and GDF9 in mature oocytes and BMPR2 in cumulus cells of women with polycystic ovary syndrome undergoing controlled ovarian hyperstimulation

To detect expression of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) in oocytes, and their receptor type 2 receptor for BMPs (BMPR2) in cumulus cells in women with polycystic ovary syndrome (PCOS) undergoing in vitro fertilization (IVF), and determine if BMPR2, BMP15, and GDF9 expression correlate with hyperandrogenism in FF of PCOS patients. Prospective case-control study. Eighteen MII-oocytes and their respective cumulus cells were obtained from 18 patients with PCOS, and 48 MII-oocytes and cumulus cells (CCs) from 35 controls, both subjected to controlled ovarian hyperstimulation (COH), and follicular fluid (FF) was collected from small (10-14 mm) and large (> 18 mm) follicles. RNeasy Micro Kit (Qiagen(A (R))) was used for RNA extraction and gene expression was quantified in each oocyte individually and in microdissected cumulus cells from cumulus-oocyte complexes retrieved from preovulatory follicles using qRT-PCR. Chemiluminescence and RIA assays were used for hormone assays. BMP15 and GDF9 expression per oocyte was higher among women with PCOS than the control group. A positive correlation was found between BMPR2 transcripts and hyperandrogenism in FF of PCOS patients. Progesterone values in FF were lower in the PCOS group. We inferred that BMP15 and GDF9 transcript levels increase in mature PCOS oocytes after COH, and might inhibit the progesterone secretion by follicular cells in PCOS follicles, preventing premature luteinization in cumulus cells. BMPR2 expression in PCOS cumulus cells might be regulated by androgens.

Frog oocytes to unveil the structure and supramolecular organization of human transport proteins

Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.

Reduced pretreatment ovarian reserve in premenopausal female patients with Hodgkin lymphoma or non-Hodgkin-lymphoma--evaluation by using antimüllerian hormone and retrieved oocytes

To study whether the ovarian reserve in female lymphoma patients is already reduced before the start of chemotherapy.