Extrusion Of First Polar Body Research Articles

Mitochondrial Ca2+ Overload Leads to Mitochondrial Oxidative Stress and Delayed Meiotic Resumption in Mouse Oocytes.

Overweight or obese women seeking pregnancy is becoming increasingly common. Human maternal obesity gives rise to detrimental effects during reproduction. Emerging evidence has shown that these abnormities are likely attributed to oocyte quality. Oxidative stress induces poor oocyte conditions, but whether mitochondrial calcium homeostasis plays a key role in oocyte status remains unresolved. Here, we established a mitochondrial Ca2+ overload model in mouse oocytes. Knockdown gatekeepers of the mitochondrial Ca2+ uniporters Micu1 and Micu2 as well as the mitochondrial sodium calcium exchanger NCLX in oocytes both increased oocytes mitochondrial Ca2+ concentration. The overload of mitochondria Ca2+ in oocytes impaired mitochondrial function, leaded to oxidative stress, and changed protein kinase A (PKA) signaling associated gene expression as well as delayed meiotic resumption. Using this model, we aimed to determine the mechanism of delayed meiosis caused by mitochondrial Ca2+ overload, and whether oocyte-specific inhibition of mitochondrial Ca2+ influx could improve the reproductive abnormalities seen within obesity. Germinal vesicle breakdown stage (GVBD) and extrusion of first polar body (PB1) are two indicators of meiosis maturation. As expected, the percentage of oocytes that successfully progress to the germinal vesicle breakdown stage and extrude the first polar body during in vitro culture was increased significantly, and the expression of PKA signaling genes and mitochondrial function recovered after appropriate mitochondrial Ca2+ regulation. Additionally, some indicators of mitochondrial performance—such as adenosine triphosphate (ATP) and reactive oxygen species (ROS) levels and mitochondrial membrane potential—recovered to normal. These results suggest that the regulation of mitochondrial Ca2+ uptake in mouse oocytes has a significant role during oocyte maturation as well as PKA signaling and that proper mitochondrial Ca2+ reductions in obese oocytes can recover mitochondrial performance and improve obesity-associated oocyte quality.

The Effect of Epigenetic Changes on the Extrusion of the First Polar Body in Pig Oocytes During In Vitro Maturation.

The present study was designed to investigate the comprehensive function of maternal factors of primordial germ cell 7 (PGC7) and POU5F1-POU class 5 homeobox 1 (OCT4), as well as the epigenetic modification roles on the mitosis for the extrusion of first polar body (PB1) in pig maturated oocytes. First, the common distribution of histone modifications, including H3K4me2, H3K27me3, H3K9me2, and H4K12ac and DNA methylation, were detected at the high level in the nucleus. However, only one part of the chromosome was higher methylated or acetylated when the mitosis happened to extrude the PB1. When the mitosis was inhibited by the cytochalasin B (CB) treatment, the expression of PGC7, OCT4, DNA methyltransferase1 (DNMT1), DNA methyltransferase3b (DNMT3b), tet methylcytosine dioxygenase 1 (TET1), tet methylcytosine dioxygenase 2 (TET2), and tet methylcytosine dioxygenase 3 (TET3) could be inhibited (p < 0.01), and no concentrated expression of the PGC7 and OCT4 was observed on the chromosome, but the levels of H3K9me2 and H4K12ac were higher. In addition, when the trichostatin A was performed on the in vitro maturation, the extrusion of the PB1 was inhibited too. And the histone methylation (H3K9me2 and H3K27me3) could be detected all the time with relative higher level and no demethylation could be observed. However, the expression of PGC7 and OCT4 was lower in the chromosome. It might indicate that the maternal factor of PGC7 and histone modification that included H4K12ac and H3K9me2 could regulate the extrusion of the PB1 and play an important role in the maturation of pig oocytes.

Nucleoporin35 is a novel microtubule associated protein functioning in oocyte meiotic spindle architecture

Nucleoporins (Nups) are a large and diverse family of proteins that mediate nucleocytoplasmic transport at interphase of vertebrate cells. Nups also function in mitosis progression. However, whether Nups are involved in oocyte meiosis progression is still rarely known. In this study, we delineated the roles and regulatory mechanisms of Nucleoporin35 (Nup35) during oocyte meiotic maturation. The immunofluorescent signal of Nup35 was localized in the nuclear membrane at germinal vesicle (GV) stage, the microtubules and spindle at pro-metaphase I (pro-MI), metaphase I (MI), and metaphase II (MII), but to the spindle poles at anaphase I (AI) and telophase I (TI). The dynamic localization pattern of Nup35 during oocyte meiotic maturation implied its specific roles. We also found that Nup35 existed as a putatively phosphorylated form after resumption of meiosis (GVBD), but not at GV stage, implying its functional switch from nuclear membrane to meiotic progression. Further study uncovered that knockdown of Nup35 by specific siRNA significantly compromised the extrusion of first polar body (PBE), but not GVBD, with defects of spindle assembly and chromosome alignment and dissociated some localization signal of p-ERK1/2 from spindle poles to cytoplasm. A defective kinetochore - microtubule attachment (K-MT) was also identified in oocytes after knockdown of Nup35, which activates spindle assembly checkpoint. In conclusion, our results suggest that Nup35 is putatively phosphorylated and released to the cytoplasm after resumption of meiosis, and regulates spindle assembly and chromosome alignment.

Abce1 orchestrates M-phase entry and cytoskeleton architecture in mouse oocyte.

ATP-binding cassette E1 (ABCE1) is a member of the ATP-binding cassette transporters and essential for diverse biological events regulating abroad range of biological functions including viral infection, cell proliferation, anti-apoptosis, translation initiation and ribosome biogenesis. Here, we discovered that Abce1 also plays indispensable roles in mouse oocyte meiotic progression. In the present study, we examined the expression, localization, and function of Abce1 during mouse oocyte meiotic maturation. Immunostaining and confocal microscopy identified that Abce1 localized as small dots in nucleus in germinal vesicle stage. After germinal vesicle breakdown, it dispersedly localized around the whole spindle apparatus. During the anaphase and telophase stages, Abce1 was just like a cap to localize around the two pole region of spindle but not the midbody and chromosome. Knockdown of Abce1 by specific siRNA injection delayed the resumption of meiosis (GVBD) and affected the extrusion of first polar body. Moreover, the process of spindle assembly and chromosome alignment were severely impaired. Abce1-RNAi led to the dissociation of γ-tubulin and p-MAPK from spindle poles, thus caused mounts of spindle morphology abnormities and chromosome alignment defects, leading to high incidence of aneuploidy. Our findings refresh the knowledge of Abce1 function by exploring its role in oocyte meiotic resumption, spindle assembly and chromosome alignment.

Spontaneous Exit from Diplotene Arrest Associates with the Increase of Calcium and Reactive Oxygen Species in Rat Oocytes Cultured In Vitro

Several signal molecules modulate the physiology of mammalian oocytes. Changes in the level of various signal molecules and their downstream impact on maturation promoting factor (MPF) during the achievement of meiotic competency remain ill understood. Therefore, the present study aimed to analyze levels of various signal molecules and MPF during the achievement of meiotic competency in rat oocytes. For this purpose, cumulus oocyte complexes (COCs) were collected from animals treated with 20 IU pregnant mare's serum gonadotropin (PMSG) for 48 h, followed by 20 IU human chorionic gonadotropin (hCG) for 14 h. The morphological changes, meiotic status and oocyte competency, cytosolic free calcium (Ca2+), total reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS), adenosine 3′,5′-cyclic monophosphate (cAMP), guanosine 3′,5′-cyclic monophosphate (cGMP), cell division cycle 25B (Cdc25B), calcium-calmodulin-dependent protein kinase II (CaMKII), Wee1, specific phosphorylation status of cyclin-dependent kinase 1 (Cdk1), and cyclin B1 expression levels were analyzed. Our data suggest that the culture of denuded diplotene-arrested oocytes resulted in spontaneous exit from diplotene arrest (EDA) in a time-dependent manner. The supplementation of hCG (2 IU) did not show any additive effect over spontaneous EDA in vitro. The hCG (20 IU) surge induced achievement of meiotic competency as most of the ovulated oocytes showed metaphase-II (M-II) arrest with extrusion of first polar body (PB-I). The spontaneous EDA was associated with a moderate increase of cytosolic free Ca2+ as well as total ROS levels. Furthermore, iNOS, cAMP, cGMP, Cdc25B, Thr161 phosphorylated Cdk1, as well as cyclin B1 levels were significantly decreased, while CaMKII, Wee1, and Thr14/Tyr15 phosphorylated Cdk1 levels were increased significantly during spontaneous EDA. These changes were reversed in ovulated oocytes that showed M-II arrest. The above results suggest that the oscillation of signal molecules modulates MPF destabilization during the achievement of meiotic competency in rat oocytes cultured in vitro.

Moderate increase of reactive oxygen species triggers meiotic resumption in rat follicular oocytes

The mammalian ovary generates reactive oxygen species (ROS) on an extraordinary scale; however, the role of ROS during meiotic cell cycle progression in follicular oocytes remains poorly understood. The present study was aimed to determine whether a moderate increase of ROS level in the ovary is beneficial for meiotic resumption from diplotene arrest in follicular oocytes. Cumulus oocyte complexes were collected from the ovaries of female rats that had been treated with either: (i) pregnant mare's serum gonadotrophin; or (ii) pregnant mare's serum gonadotrophin + human chorionic gonadotrophin. We analyzed morphological changes, ROS and hydrogen peroxide levels, catalase activity, 3',5'-cyclic adenosine monophosphate and 3',5'-cyclic guanosine monophosphate levels, Thr14/Tyr15, Th-161, total cyclin-dependent kinase 1 (Cdk1) and cyclin B1 levels. Human chorionic gonadotrophin treatment induced meiotic resumption from diplotene arrest and extrusion of first polar body in cumulus oocyte complexes collected from ovaries and cultured for 3h in vitro. Meiotic resumption from diplotene arrest was associated with increased ROS and hydrogen peroxide levels but decreased 3',5'-cyclic adenosine monophosphate as well as 3',5'-cyclic guanosine monophosphate levels. The reduced cyclic nucleotide levels were associated with decreased Thr161 phosphorylated Cdk1 and cyclin B1 level but increased Thr14/Tyr15 phosphorylated Cdk1 level leading to maturation promoting factor destabilization. Destabilized maturation-promoting factor triggered meiotic resumption from diplotene arrest and progression to metaphase-I as well as metaphase-II stage in follicular oocytes. Our findings suggest that a moderate increase of ROS in the ovary is beneficial for meiotic resumption from diplotene arrest and extrusion of first polar body in follicular oocytes.

Effect of hormones, follicular fluid, serum and media on in vitro maturation of porcine oocyte

The present study was conducted to evaluate the effect of hormones, follicular fluid, serum and media on in vitro maturation (IVM) of porcine oocyte. The procedures of IVM could be used for basic research purposes or commercial utilization. The IVM of oocyte is a critical step in in-vitro embryo production and it needs to be carried out in a precise manner under optimal conditions for subsequent fertilization and embryo development. For this purpose ovaries were collected from slaughtered pigs from an abattoir. Ovaries (1,232) were aspirated in 52 trials for IVM and fertilization for this study. Dulbecco Modified Eagle's medium (DMEM) or tissue culture medium (TCM-199) supplemented with gentamycin (50μl/ml), sodium pyruvate (100 mmol) and 10% serum, viz. estrus sow serum (ESS) or fetal calf serum (FCS) with or without porcine follicular fluid (PFF) and hormones such as pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) were used. The maturation was assessed by loosening of cumulus cells, enlargement of peri-viteline space or extrusion of first polar body and examination of metaphase II by aceto-orcein stain. The results revealed that DMEM with ESS yielded 53% while TCM+ESS yielded 45% maturation rates. The replacement of ESS with FCS resulted in 48.50 and 35.33% maturation in DMEM and TCM-199 medium, respectively. The addition of PFF in DMEM+ESS improved the maturation rate and was found better (74.55%) than TCM-199+ESS (65.35%). However, the replacement of ESS by FCS in either DMEM orTCM-199+PFF did not differ significantly. It can be concluded from the present study that use of DMEM+ESS+PFF along with hormones PMSG + luteinizing hormone (LH) for first 20-24 h followed by hormone free medium for next 20-22 h i.e. 42-44 h gave optimum (81.5%) in-vitro maturation rates in pigs. DMEM with ESS and PFF with hormone (PMSG/HCG) yielded highest maturation (81.50%) rate than TCM-199 with ESS, PFF and hormone (PMSG/HCG) which showed maturation rate of 76.59%.

Cytoplasm replacement following germinal vesicle transfer restores meiotic maturation and spindle assembly in meiotically arrested oocytes

Both the cytoplasmic and nuclear compartments are essential for the acquisition of meiotic competence. This study assessed the role of the cytoplasm in meiosis resumption in meiotically arrested oocytes at the germinal vesicle (GV) stage. Mouse oocytes at GV stage were meiotically arrested with 3-isobutyl-1-methylxanthine (IBMX). GV transfer was performed between IBMX-treated and non-treated (control) mouse oocytes, and between control mouse and human GV oocytes. Extrusion of first polar body (PB) was examined as an indication of nuclear maturation. Meiotic spindle assembly and chromosome alignment were examined by immunostaining. Results indicated that oocytes arrested with IBMX for 24 and 48 h exhibited reduced ability for meiotic maturation and for extruding the first PB when compared with controls (P < 0.01). IBMX-treated oocytes reconstituted with cytoplasm, but not GV, of control oocytes restored the assembly of meiotic spindle and meiotic maturation. Mouse oocytes reconstituted with GV of human oocytes underwent meiosis similar to that observed in mice, but not humans. Additionally, human oocytes reconstituted by mouse GV underwent meiosis similar to that observed in humans, but not mice. These findings suggest that cytoplasm replacement by GV transfer could represent a potential therapeutic option for women who do not produce mature oocytes during IVF.

Sub-zonal versus intracytoplasmic injection produces a higher rate of cloned caprine-bovine interspecies blastocysts

The production of cloned-caprine embryos using the intraspecies somatic cell nuclear transfer (SCNT) technique is limited by the low source of caprine oocytes as the recipient cytoplasts in certain countries. Therefore, using bovine oocytes as recipient cytoplasts in interspecies somatic cell nuclear transfer (iSCNT), is an alternative approach to produce a large number of cloned-caprine embryos and subsequently offspring at a rapid rate. The aim of this research was to compare the effect of nuclear transfer methods on iSCNT cloned embryos’ developmental competence, involving: (a) an intracytoplasmic injection (ICI), and (b) the sub-zonal injection with electrofusion (SUZI), using fetal fibroblast cells as donor karyoplasts. The bovine ovaries were collected from local abattoir and transported to the laboratory within 2–3h in NaCl (0.9%). The oocytes (n=725) were recovered by checkerboard slicing of the entire surface of the ovary, inside a culture dish, using a razor blade. After slicing, the cumulus–oocyte complexes (COCs) were recovered and selected under a stereomicroscope. Oocytes with several compact layers of cumulus cells were selected and cultured in in vitro maturation (IVM) medium for 20–22h. After maturation, COCs were denuded in hyaluronidase (0.1%) to remove the cumulus cells. The matured oocytes (with extrusion of first polar body) were selected for enucleation to remove the spindle. Caprine-fetal fibroblast cells (donor karyoplasts) were harvested and transferred to enucleated bovine oocytes, by using either an intracytoplasmic injection or sub-zonal injection, with electrofusion. The injected/fused oocytes were activated and the reconstructed couplets were cultured in KSOM medium for in vitro embryo development in a CO2 (5%) incubator, at 38.5°C in a humidified atmosphere for 8–9days. The culture medium was changed every 2days of IVC. The percentage of cleaved embryos and blastocyst formation following sub-zonal injection, with electrofusion was higher than for oocytes which underwent intracytoplasmic injection (60.2% vs. 54.1% and 12.1% vs. 4.5%, respectively). In summary, the nuclear transfer using both methods of sub-zonal injection and intracytoplasmic injection showed satisfactory results – with the former method being apparently higher in in vitro developmental competence in both cases. In conclusion, using caprine-bovine iSCNT to produce caprine embryos and offspring may offer a new approach to increase genetically superior goat populations at a rapid rate to meet the goat meat and milk demand for the industry – especially in the developing countries.

Melatonin protects against clomiphene citrate-induced generation of hydrogen peroxide and morphological apoptotic changes in rat eggs

The present study was aimed to determine whether clomiphene citrate-induces generation of hydrogen peroxide in ovary, if so, whether melatonin could scavenge hydrogen peroxide and protect against clomiphene citrate-induced morphological apoptotic changes in rat eggs. For this purpose, forty five sexually immature female rats were given single intramuscular injection of 10 IU pregnant mare's serum gonadotropin for 48 h followed by single injections of 10 IU human chorionic gonadotropin and clomiphene citrate (10 mg/kg bw) with or without melatonin (20 mg/kg bw) for 16 h. The histology of ovary, ovulation rate, hydrogen peroxide concentration and catalase activity in ovary and morphological changes in ovulated eggs were analyzed. Co-administration of clomiphene citrate along with human chorionic gonadotropin significantly increased hydrogen peroxide concentration and inhibited catalase activity in ovary, inhibited ovulation rate and induced egg apoptosis. Supplementation of melatonin reduced hydrogen peroxide concentration and increased catalase activity in the ovary, delayed meiotic cell cycle progression in follicular oocytes as well as in ovulated eggs since extrusion of first polar body was still in progress even after ovulation and protected against clomiphene citrate-induced egg apoptosis. These results clearly suggest that the melatonin reduces oxidative stress by scavenging hydrogen peroxide produced in the ovary after clomiphene citrate treatment, slows down meiotic cell cycle progression in eggs and protects against clomiphene citrate-induced apoptosis in rat eggs.

Optimal timing of oocyte maturation and its relationship with the spindle assembly and developmental competence of in vitro matured human oocytes

To develop and test a simple, feasible approach to improve spindle assembly and developmental competence of human in vitro matured oocytes by parthenogenetic activation in maturation medium. Prospective, randomized study. University research laboratory-based assisted reproductive technology laboratory. Four hundred thirty-two patients with male factor infertility undergoing intracytoplasmic sperm injection (ICSI) cycles. In vivo failed-to-mature oocytes from the ICSI cycles were divided into four groups according to differing duration after the extrusion of first polar body (0-1, 2-3, 4-5, and 8-9 hours). Oocytes spindles in each group were immunostained for α-tubulin and chromosomes, and observed by confocal microscopy. Rate from pronuclear stage to blastocyst, embryo grading at the eight-cell and blastocyst stages, and spindle assembly. There was a statistically significantly higher rate of development at the eight-cell and blastocyst stages in the 2- to 3-hour and 4- to 5-hour groups. The grading results at the eight-cell and blastocyst stages also showed that the proportion of embryos of high quality was similar among the 2- to 3-hour and 4- to 5-hour groups, and in these groups it was statistically significantly higher than the 0- to 1-hour and 8- to 9-hour groups. The results of immunofluorescence demonstrated that there was a statistically significantly higher rate of normal spindle assembly in the 2- to 3-hour and 4- to 5-hour groups. Optimal timing of maturation benefits the development of competence of in vitro matured oocytes by promoting normal spindle assembly.

263 REGULATION OF SEVERAL TRANSCRIPTS DURING IN VITRO MATURATION IN PORCINE OOCYTES COLLECTED FROM DIFFERENT SIZE FOLLICLES

Oocyte-specific transcripts play important roles in oocyte maturation, fertilization, and early embryonic development. Currently, oocytes from medium-size follicles have been used for different assisted reproductive techniques after in vitro maturation (IVM). The aim of this study was to compare the mRNA expression level in porcine oocytes collected from medium (3–6 mm) and small (&lt;2 mm) size follicles. Genes were selected based on their described maternal effect (NALP9, HSF1), their identification as markers of oocyte maturation (AURK-A, AURK-B, MOS, and C-mos), their involvement in fertilization (ZP3, ZP4), and anti-apoptotic effect (Bcl-2). All transcripts were studied in oocytes just after collection [germinal vesicle (GV) stage] and after in vitro maturation (IVM; metaphase II stage). To ensure nuclear stage of immature oocytes, oocytes were mechanically denuded just after collection, centrifuged (10 000 rpm, 5 min, RT), and observed under the microscope (60×). Those oocytes with clear nucleolus and evident nuclear membrane were selected and stored (n = 10) until study. For metaphase II oocytes, only those exhibiting the extrusion of first polar body after IVM (n = 10) were selected. Total RNA was extracted from the pool of 10 immature and mature oocytes. One picogram of luciferase mRNA per oocyte was added as an exogenous standard. Total RNA was extracted from oocytes and cDNA was obtained and used as a template for quantitative PCR to analyse the level of different transcripts. The whole process was replicated 4 times. Data were normalized to the luciferase RNA and analysed by one-way ANOVA with maturational stage (GV or metaphase II) and follicle size (small or medium) as fixed factors. Results show that all transcripts were significantly decreased during IVM (P &lt; 0.05). Therefore, after IVM, NALP9, AURK-A, MOS, C-mos, ZP3, ZP4, and Bcl-2 transcripts were significantly reduced in matured oocytes compared with immature ones irrespective of follicle diameter. Transcripts of AURKAB and HSF1 decreased after IVM in oocytes from medium follicles or small follicles, respectively. A significant effect of follicular size was only detected in MOS transcripts in GV-stage oocytes because those collected from middle follicles had a higher amount than the ones from small follicles (Table 1). These results suggest that the variations in the maternal store of RNA during IVM are not related with follicle diameter for the studied genes. Further investigations are necessary to determinate the developmental competence of oocytes that came from different types of follicles (small and medium follicles). Table 1.Variation of transcripts during in vitro maturation in porcine oocytes collected from small and medium follicles This study was supported by Okayama University. R. Romar was given a grant by JSPS (Ref. S-09210).

Non invasive imaging of spindle of in vivo matured oocytes from infertile patients with endmetriosis and controls

OBJECTIVE: Some studies suggest lower fertilization, implantation and pregnancy rates in women with endometriosis, possibly due to impaired oocyte quality. For a mature oocyte to be prepared for fertilization, the meiotic spindle must maintain its integrity. Polarization microscopy (polscope) permits the analysis of spindles in live oocytes without compromising ART outcomes. In this regard, we evaluated the presence and position of meiotic spindle in in vivo matured oocytes from endometriosis and control patients.DESIGN: Prospective, controlled study.MATERIALS AND METHODS: Ninety four consecutive cycles of 87 infertile patients (32 with infertility related to endometriosis and 55 with male or tubal causes of infertility – controls) undergoing stimulated cycles of oocyte retrieval for ICSI were selected. Oocytes showing the extrusion of first polar body were imaged by polarization microscopy immediately before ICSI. The presence and position of meiotic spindle was assessed in 394 oocytes (133 from endometriosis and 261 from control group) by the same operator.RESULTS: Meiotic spindles were imaged in 352 (89.3%) oocytes. There were no differences between endometriosis and control groups with respect to the percentage of telophase I (4 oocytes, 3% and 3, 2.3%, respectively) and proportion of oocytes with the meiotic spindle visible (125/133 oocytes, 94% and 227/261 oocytes, 87%, respectively). The majority of oocytes had the spindle directly beneath or adjacent to the first polar body (angle between the spindle and the first polar body varying from 0 to 60°) in both groups (117/125, 93.6% and 212/227, 93.4%, respectively for endometriosis and controls).CONCLUSIONS: Non invasive analysis of the presence and position of spindles of in vivo matured oocytes from stimulated cycles demonstrated similar results between endometriosis and controls. However, data regarding the proportion of meiotic abnormalities in live oocytes with spindle visible are not available. OBJECTIVE: Some studies suggest lower fertilization, implantation and pregnancy rates in women with endometriosis, possibly due to impaired oocyte quality. For a mature oocyte to be prepared for fertilization, the meiotic spindle must maintain its integrity. Polarization microscopy (polscope) permits the analysis of spindles in live oocytes without compromising ART outcomes. In this regard, we evaluated the presence and position of meiotic spindle in in vivo matured oocytes from endometriosis and control patients. DESIGN: Prospective, controlled study. MATERIALS AND METHODS: Ninety four consecutive cycles of 87 infertile patients (32 with infertility related to endometriosis and 55 with male or tubal causes of infertility – controls) undergoing stimulated cycles of oocyte retrieval for ICSI were selected. Oocytes showing the extrusion of first polar body were imaged by polarization microscopy immediately before ICSI. The presence and position of meiotic spindle was assessed in 394 oocytes (133 from endometriosis and 261 from control group) by the same operator. RESULTS: Meiotic spindles were imaged in 352 (89.3%) oocytes. There were no differences between endometriosis and control groups with respect to the percentage of telophase I (4 oocytes, 3% and 3, 2.3%, respectively) and proportion of oocytes with the meiotic spindle visible (125/133 oocytes, 94% and 227/261 oocytes, 87%, respectively). The majority of oocytes had the spindle directly beneath or adjacent to the first polar body (angle between the spindle and the first polar body varying from 0 to 60°) in both groups (117/125, 93.6% and 212/227, 93.4%, respectively for endometriosis and controls). CONCLUSIONS: Non invasive analysis of the presence and position of spindles of in vivo matured oocytes from stimulated cycles demonstrated similar results between endometriosis and controls. However, data regarding the proportion of meiotic abnormalities in live oocytes with spindle visible are not available.

Evaluation of meiotic spindle and chromosomal configurations of in vitro matured oocytes obtained from stimulated cycles of endometriotic patients: preliminary results

Evaluation of meiotic spindle and chromosomal configurations of in vitro matured oocytes obtained from stimulated cycles of endometriotic patients: preliminary results

Stimulation of in vitro ovine oocyte maturation with a novel peptide isolated from follicular fluid of the buffalo ( Bubalus bubalis)

In vitro oocyte maturation is an important step in the laboratory production of embryos. The technique of in vitro maturation (IVM) has generally been standardized in sheep, but now the efforts are aimed at reducing the cost of the technology by replacing expensive inputs of the IVM medium with less expensive and chemically defined inputs. In this study, a novel peptide (Mr 26.6 kDa) isolated and partially purified from buffalo ovarian follicular fluid was incorporated at various dose levels to study its effect on the IVM of ovine oocytes. Oocytes were retrieved from sheep ovaries via a slicing technique. The basic culture medium (control) was comprised of the tissue culture medium (TCM-199) + BSA (3 mg ml −1) with gentamicin (50 ng ml −1). The isolated peptide was added to the basic culture medium at dose levels of 10, 15, 20, 25, 50, 100, 1000, 2000, 5000 ng ml −1 of culture medium. An additional treatment group was incorporated comprised of 2000 ng peptide per milliliter plus epidermal growth factor (EGF: 20 ng ml −1). A positive control group comprising of basic culture medium along with steer serum (20%, v/v) and EGF (20 ng ml −1) was also tested. The oocytes in the culture medium were placed in Petri dishes with overlaying mineral oil and incubated for 24 h at 38.5 °C, 95% relative humidity and 5% CO 2/air. The biological effect of the isolated peptide was evaluated by a cumulus expansion score and the in vitro maturation rates, which were calculated based on the cumulus expansion and extrusion of first polar body. There was a dose-dependent increment in the cumulus expansion as well as in vitro maturation rates of oocytes when the basic medium was supplemented with the isolated peptide. The in vitro maturation rates obtained with different doses of the peptide, i.e. 0, 10, 15, 20, 25, 50, 100, 1000, 2000, 5000, 2000 ng + EGF and positive control group were 18.9, 37.5, 50.7, 63.0, 64.5, 67.0, 76.5, 78.2, 81.5, 83.1, 90.4 and 88.1%, respectively. Even at a low dose level of 20 ng ml −1, the isolated peptide could stimulate the IVM rate and at 100 ng ml −1 dose, it yielded a high maturation rate (>75%). In view of the observations made in this study, the novel peptide can be incorporated at 100 ng dose in IVM medium of ovine oocytes for augmenting the efficiency of in vitro embryo production technology (IVEP) in sheep.

Timing of sequential changes in cumulus cells and first polar body extrusion during in vitro maturation of buffalo oocytes

Studies were conducted to investigate the degree of the cumulus cell expansion and expulsion of the first polar body in relation to time of incubation in three different culture media during in vitro maturation of buffalo oocytes and to suggest a suitable practical method for assessment of in vitro maturation rate of buffalo oocytes. Buffalo oocytes were aspirated from ovaries collected from a local slaughterhouse. Only oocytes with more than two layers of cumulus cells and homogenous ooplasm were cultured into 50 μl droplets of three different culture systems: (1) TCM-199+steer serum (10%); (2) TCM-199+steer serum (10%)+PMSG (40 IU/ml); and (3) TCM-199+steer serum (10%)+PMSG (40 IU/ ml)+ estradiol 17β (1 μg/ml) in a 35 mm Petri dish. The droplets were covered with warm (39 °C) mineral oil and incubated in a CO 2 incubator (39 °C, 5% CO 2 in air, 90–95% relative humidity) for 16–18, 20, 22, and 24 h. The maturation rate was assessed by evaluation of degree of cumulus cells expansion and identifying first polar body extrusion into the perivitelline space under stereo zoom microscope. Matured oocytes were inseminated in vitro with 9–10 million sperm/ml of Brackett and Oliphant (BO) medium. Cleaved embryos were cultured in TCM-199 supplemented with steer serum (10%) for 8 days. Cumulus expansion and extrusion of first polar body commenced at 16 and 17 h, respectively, of buffalo oocyte culture. These events mainly exhibited during 22–24 h of culture. Oocytes with Degrees 1 and 2 cumulus cells expansion and extruded first polar body in degree 0 oocytes may be considered as matured and can be used in IVF studies.

Cytostatic Activity Develops during Meiosis I in Oocytes of LT/Sv Mice

Oocytes of wild-type mice are ovulated as the secondary oocytes arrested at metaphase of the second meiotic division. Their fertilization or parthenogenetic activation triggers the completion of the second meiotic division followed by the first embryonic interphase. Oocytes of the LT/Sv strain of mice are ovulated either at the first meiotic metaphase (M I) as primary oocytes or in the second meiotic metaphase (M II) as secondary oocytes. We show here that duringin vitromaturation a high proportion of LT/Sv oocytes progresses normally only until metaphase I. In these oocytes MAP kinase activates shortly after histone H1 kinase (MPF) activation and germinal vesicle breakdown. However, MAP kinase activation is slightly earlier than in oocytes from wild-type F1 (CBA/H × C57Bl/10) mice. The first meiotic spindle of these oocytes forms similarly to wild-type oocytes. During aging, however, it increases in size and finally degenerates. In those oocytes which do not remain in metaphase I the extrusion of first polar bodies is highly delayed and starts about 15 h after germinal vesicle breakdown. Most of the oocytes enter interphase directly after first polar body extrusion. Fusion between metaphase I LT/Sv oocytes and wild-type mitotic one-cell embryos results in prolonged M-phase arrest of hybrids in a proportion similar to control LT/Sv oocytes and control hybrids made by fusion of two M I LT/Sv oocytes. This indicates that LT/Sv oocytes develop cytostatic factor during metaphase I. Eventually, anaphase occurs spontaneously and the hybrids extrude the polar body and form pronuclei in a proportion similar as in controls. In hybrids between LT/Sv metaphase I oocytes and wild-type metaphase II oocytes (which contain cytostatic factor) anaphase I proceeds at the time observed in control LT/Sv oocytes and hybrids between two M I LT/Sv oocytes, and is followed by the parthenogenetic activation and formation of interphase nuclei. Also the great majority of hybrids between M I and M II wild-type oocytes undergoes the anaphase but further arrests in a subsequent M-phase. These observations suggest that an internally triggered anaphase I occurs despite the presence of the cytostatic activity both in LT/Sv and wild-type M I oocytes. Anaphase I triggering mechanism must therefore either inactivate or override the CSF activity. The comparison between spontaneous and induced activation of metaphase I LT/Sv oocytes shows that mechanisms involved in anaphase I triggering are altered in these oocytes. Thus, the prolongation of metaphase I in LT/Sv oocytes seems to be determined by delayed anaphase I triggering and not provoked directly by the cytostatic activity.

The bimodular development of ovarian follicles in cyclic guinea pigs and differences between antral follicles developed at different phases of the cycle

The results of a histological, [3H]thymidine uptake, and autoradiographic study of the cyclic guinea pig ovary suggest that there are two consecutive waves of antral follicle development within each estrous cycle, with the first wave extending from days 2 to 10 and the second wave from days 7 to 16 of the 16-day cycle. The bimodular development is apparently made possible by the continuous daily recruitment of growing follicles from the pool of dormant primordial follicles and by the occurrence of two occasions (days 1–3 and days 6–9) when gonadotropin secretory activity allows some of these follicles to progress into the antral phase of development. The antral follicles of the first wave attain their maximum size (0.5 mm3) on day 9. However, they have disappeared by day 12 and in their place, large atretic follicles and the smaller healthy antral follicles of the second wave have become the most prominent follicular features. On days 14 and 15, the antral follicles of the second wave have grown to 0.5 mm3 themselves, and within the next 24 h their volume increases rapidly by 150% to 1.23 mm3. Extrusion of first polar bodies, vaginal estrus, and ovulation then follow in quick succession. The results indicate that the follicles of the first wave become atretic before the final rapid growth phase and resumption of meiotic division. The absence of the rapid growth phase probably indicates that they have never reached final maturity, nor have they achieved the competence to ovulate in response to gonadotropin stimulation.

Chemicals Influencing Maturation, Activation, and Degeneration of Bovine Oocytes in Culture

The effects of the dibutyryl cyclic adenosine 3′,5′-monophosphate, calmodulin antagonist W7, and ovarian glycosaminoglycans on polar body extrusion, “nucleus” formation, and segmentation of bovine oocytes were examined in vitro. Dibutyryl cyclic adenosine 3′,5′-monophosphate (100 μM) accelerated the extrusion of first polar body; however, W7 (20 μM) suppressed the formation of the first polar body. Spontaneous “nucleus” formation resembling pronucleus formation in oocytes was accelerated in the presence of dibutyryl cyclic adenosine 3′,5′-monophosphate at concentrations of greater than 20 μM. The spontaneous segmentation, which is a degenerative change, was suppressed in the presence of bovine follicular fluid-glycosaminoglycans at concentrations of 250μg/ml or greater. These results 5indicate that maturation, activation, and degeneration of bovine oocytes in culture are modulated by dibutyryl cyclic adenosine 3′,5′-monophosphate, W7, and ovarian glycosaminoglycans.

Effects of various concentrations of gonadotropins and 17β estradiol on the in vitro maturation of cattle oocytes selected using brilliant cresyl blue staining

The objective of this study was to determine the in vitro maturation rate of cattle oocytes selected with brilliant cresyl blue (BCB) stain, in tissue culture medium 199 (TCM 199) supplemented with various concentrations of hormones. Oocytes were retrieved from abattoir-derived ovaries by aspiration. Oocytes were then exposed to 26 μM BCB stain, and classified according to the colour of their cytoplasm: BCB+ (oocytes with blue cytoplasm) and BCB- (unstained oocytes). The BCB selected and the non-selected immature oocytes were randomly allocated into TCM 199 + 10% foetal bovine serum (FBS) maturation media supplemented with three concentrations of hormones as treatments (T). The T1 group was matured in the presence of 0.5 μg follicle stimulating hormone (FSH)/mL, 5 mg luteinising hormone (LH)/mL and 2 μg estradiol (E2)/mL. The T2 group was matured in 1 μg FSH, 6 mg LH and 2.5 μg E2/mL. The T3 group was matured in 1.5 μg FSH, 7 mg LH and 4.5 μg E2/mL. The maturation rate of oocytes was determined by the protrusion of the first polar bodies 24 h after maturation. Data were analysed by ANOVA using SAS. Treatment 2 yielded higher maturation rates for with BCB+ (30.5%) and without BCB (35%) oocytes, with T1 giving a lower maturation rate for BCB+ (10.7%) and without BCB (9.7%) oocytes. However, BCB- oocytes had lower polar body extrusion (0.7%, 1% and 2.7%) for T1, T2 and T3, respectively. In conclusion, immature oocytes that were exposed to BCB+ and cultured in TCM 199 supplemented with 10% FBS, 1 μg FSH, 6 mg LH and 2.5 μg E2/mL had a higher number of matured oocytes (extrusion of first polar body), similar to those that were not exposed to BCB (no BCB). Oocyte selection with BCB staining was a useful test for classifying good-quality cattle oocytes.