Higher Percentage Of Oocytes Research Articles

Effect of FSH and eCG on Alpaca (Vicugna pacos) Oocyte Maturation in vitro

This study evaluated alpaca oocytes that developed to metaphase II (MII), using different concentrations of follicle stimulating hormone (FSH) with and without equine chorionic gonadotropin (eCG) during in vitro maturation. Oocytes were obtained from ovaries of slaughtered alpacas. Oocytes were matured in vitro for 36h in TCM-99 and supplemented in groups with different doses of FSH: 0.5, 0.25, 0μg.mL-1 and eCG: 15, 5, 0IU.mL-1. Oocytes were stained with 2% lacmoid, and examined for their nuclear status. The parameter for comparisons between groups was the percentage of oocytes in MII. There was an interaction between FSH and eCG (P<0.05). A higher percentage of oocytes in MII were obtained when using 0.5µg.mL-1 of FSH with any concentration of 15, 5 or 0IU.mL-1 of eCG [58.4±1.94% (n=78); 59.5±1.94% (n=85); 54.3±0.56% (n=81); P<0.05]. A similar percentage was also found using 0.25µg.mL-1 of FSH plus 15IU.mL-1 of eCG [52.3±1.66% (n=86); P>0.05] followed by 0.25µg.mL-1 of FSH with 5 or 0IU.mL-1 of eCG [47.3±1.94% (n=82); 45.3±1.94% (n=86); P<0.05]. The lowest maturation percentages were found using any concentration of eCG without FSH [8.2±0.73% (n=84); 8.0±0.66% (n=74); 11.4±1.23% (n=71); P<0.05]. In conclusion, the addition of eCG to the maturation medium would reduce the amount of FSH required in the in vitro maturation of alpaca oocytes.

132 Influence of oocyte retrieval methods and maturation media on invitro development of polar body extrusion in pig oocytes

Oocyte recovery is a reproductive technology that can be done by using two techniques, aspiration and slicing. Invitro maturation (IVM) is an additional reproductive technology used to advance an oocyte to a maturation stage; thereafter, it may be used during IVF. The objectives of the present study were (1) to compare two different oocyte retrieval methods (aspiration and slicing) from pig ovaries on oocyte quality and quantity, and (2) to compare three different IVM media [NCSU 37, TCM-199, and modified porcine follicular fluid (mpFF=porcine follicular fluid+FSH+LH] on oocytes’ polar body extrusion. During aspiration, an 18G needle was attached to a 10-mL syringe and all visible follicles were aspirated. During slicing, a surgical blade was used to slice the ovaries held in mDPBS. Follicular fluid collected from both methods was assessed for the presence of oocytes with the aid of a microscope. The collected oocytes were then categorized as Grade A, B, or C: Grade A=oocytes with compacted, multilayered cumulus cells and a homogeneous ooplasm; Grade B=oocytes with a compact cumulus cell layer with homogeneous ooplasm; Grade C=oocytes with a less compact cumulus cell layer with irregular ooplasm containing dark granules. The IVM media were placed in a four-well multidish; thereafter Grades A and B oocytes were allocated per treatment groups and matured for 44h. The treatment means were compared using the Fisher’s protected t-test least significant difference. The results showed significant differences between the grades of oocytes (P<0.05) with Grade A and B oocytes accounting for 50.8% of total oocytes (193.8) for aspiration and 58.7% of total oocytes (488.6) for slicing. The oocytes polar body extrusion was recorded as 25.3, 84.2, and 73.8% for NCSU 37 (P<0.05) and TCM-199 and mpFF respectively (P>0.05). In conclusion, the slicing method proved to be better than aspiration with regards to the retrieval of Grades A and B oocytes as well as the total number of oocytes retrieved. The TCM-199 and mpFF media had a higher percentage of oocytes with polar body extrusion than NCSU 37.

129 Polar body extrusion following exposure of pig oocytes to different invitro maturation media

The invitro maturation (IVM) rate of pig oocytes is a critical step for subsequent invitro embryo production. The objective of the present study was to compare different IVM media (NCSU 23, NCSU 37, TCM-199, pFF, BO-IVM commercial, and Bovine IVM) on polar body extrusion in pig oocytes following 44h of maturation duration. Ovaries from sows were collected from the local slaughterhouse and transported in warmed (38°C) saline to the laboratory, within 2h following slaughter. The slicing method was used to retrieve oocytes from the ovaries. Collected oocytes (n=353) were selected and washed three times in TCM-199+10% fetal bovine serum. The oocytes were then subjected to IVM as per treatment groups for 44h. Following maturation period, oocytes were removed from each maturation medium and denuded of granulosa cells by vortexing. The oocytes polar body extrusion was evaluated with the aid of OosightTM Imaging Sytem connected to an inverted research microscope. Treatment means were compared using Fisher’s protected least significant difference t-test. Polar body extrusion was 30.0, 18.5, 32.8, 22.1, 32.1, and 27.0% for BO IVM commercial, Bovine IVM, NCSU 23, NCSU 37, TCM-199, and pFF media, respectively (P>0.05). The NCSU 23 and TCM-199 had numerically higher percentages of oocytes with extruded polar body compared with other treatments groups (P>0.05). The lowest (18.5%) polar body extrusion was recorded in Bovine IVM medium. In conclusion, all the IVM media tested can be used during IVM of pig oocytes, with NCSU 23 and TCM-199 having numerically higher polar body extrusion percentages.

Antral follicular count and its relationship with ovarian volume, preantral follicle population and survival, oocyte meiotic progression and ultrastructure of in vitro matured bovine cumulus-oocyte complexes.

This study aimed to evaluate the relationship between antral follicular count (AFC) and ovarian volume (OV), preantral follicular population and survival, meiotic progression and ultrastructure of cumulus-oocyte complexes (COCs) after in vitro maturation. In experiment 1, the relationship between AFC and preantral follicle population and survival was evaluated by classical histology. In experiment 2, the relationship among AFC, OV, ability of oocytes to resume meiosis and ultrastructure of in vitro matured bovine COCs was studied. A positive correlation (P < 0.05) between AFC and the numbers of healthy primordial, degenerate and total follicles was observed, as well as with healthy secondary follicles and total follicles. The numbers of grades I and II oocytes in ovaries of high AFC class were higher compared with those with intermediate or lower AFC. After in vitro maturation, COCs from ovaries of high AFC had a higher percentage of oocytes in metaphase II compared with those of intermediate and low AFC (P < 0.0001). Ovaries of intermediate AFC had a higher percentage of oocytes in metaphase II compared with ovaries with low AFC (P < 0.0001). The proportion of oocytes in metaphase I, telophase I and anaphase I in COCs from ovaries of intermediate AFC (26.04%) was higher (P < 0.05) compared with that seen in COCs of ovaries with high (8.55%) and low (14.15%) AFC. No differences in the ultrastructure of oocytes were seen. In conclusion, after in vitro maturation, cow ovaries with high AFC have higher numbers of oocytes that reach in metaphase II (MII), but they also have higher numbers of degenerated primordial and primary follicles.

183 Effects of epidermal growth factor and progesterone on invitro oocyte growth, meiotic resumption, and expression of maturation-related transcripts in oocytes from bovine small antral follicles

Oocytes from small antral follicles are not able to undergo nuclear maturation because they still need to accumulate mRNA and proteins. We hypothesised that invitro growth of cumulus-oocyte complexes (COCs) of these follicles in the presence of epidermal growth factor (EGF) and progesterone (P4) increase the levels of transcripts and have a positive impact on maturation. This study aimed to evaluate the effects of EGF and P4 on growth, maturation, and expression of mRNAs for growth and differentiation factor 9 (GDF9), cyclin B1, oocyte-specific linker histone (H1FOO), quinase cMOS, poly(A) ribonuclease (PARN), and initiation factor 4E (eIF4E) after growth and prematuration of COCs. Bovine COCs (n=400) were aspirated from antral follicles (1-3mm) and cultured for 48h in control medium (TCM-199 plus 5.0mgmL−1 LH, 0.5mgmL−1 FSH, and 5% fetal bovine serum) alone or with EGF (10ngmL−1), P4 (100 µM), or both EGF and P4. After growth, the COCs were pre-matured for 20h in TCM-199 containing 5.0mgmL−1 LH, 0.5mgmL−1 FSH, 0.4% bovine serum albumin, and 10μM cilostamide. Then, COCs were matured invitro in the same medium used for prematuration, but without cilostamide. Oocyte diameter and meiotic progression were evaluated after growth, prematuration, and maturation periods. After prematuration, oocytes were stored until RNA extraction. Total RNA was extracted using Trizol according to the manufacturer's instructions (Invitrogen). After reverse transcription, quantification of mRNAs for GDF9, cyclin B1, H1FOO, cMOS, PARN, eIF4E, and GAPDH was performed by real-time PCR. The delta-delta-cycle threshold (ΔΔCT) method was used to normalise the data. The percentages of oocytes in each stage of maturation were compared by Mann-Whitney test. Differences in expression of mRNAs were analysed by Kruskal-Wallis test (P&amp;lt;0.05). The results showed that, compared with time 0, an increase in oocyte diameter was observed after 48h of culture for COCs in all groups (P&amp;lt;0.05), except for those cultured in control medium alone or with only P4. However, when compared with control medium, no effects of EGF, P4, or both were seen after the growth and prematuration periods. After prematuration, a higher percentages of oocytes at the germinal vesicle stage was observed for COCs cultured with P4 compared with those cultured in TCM-199 (P&amp;lt;0.05). After IVM, the rate of meiosis resumption was significantly reduced in oocytes cultured with P4 (P&amp;lt;0.05). The levels of mRNA for cMOS, H1FOO, and cyclin B1 in oocytes cultured with P4 were higher than those cultured in the control medium (P&amp;lt;0.05). The mRNA levels for eIF4E in oocytes cultured with both EGF and P4 were increased (P&amp;lt;0.05) compared with those cultured with EGF. The levels of transcripts for PARN in oocytes cultured with both EGF and P4 were higher than those in COCs cultured with only P4. In contrast, mRNA levels of GDF9 did not show differences between treatments. In conclusion, P4 inhibited oocyte meiotic resumption and increased mRNA levels for cMOS, H1FOO, and cyclin B1 in COCs after culture and prematuration invitro.

FERTILITAS DAN PERSENTASE EMBRIO KERBAU SAMPAI MORULA YANG DIKULTUR DENGAN PENAMBAHAN GLUTATHIONE SECARA IN VITRO

The concentration of high fat content in buffalo embryo culture media is very influential on the increase in oxidative stress that occurs in conditions of in vitro. The occurrence of increased oxidative stress in the process of embryo culture in vitro, would result in lower percentage of embryo culture until the morula stage (32) cells. The purpose of this study was to determine the percentage of fertilized oocytes supplemented with glutathione. To know the development of the embryo until the morula stage (32) cells were cultured in the culture medium supplemented with glutathione (GSH). To determine the viability of morula stage embryo or 32 cells. The materials used in this study is ovarian buffaloes and methods used in this study is an experimental method in the laboratory. Based on these results it can be concluded that the addition of glutathione 1.5 mM in media oocyte maturation buffalo in vitro provide a significant influence on the percentage of maturation ie (P &lt;0:01) or 62.5% and in line with the high percentage of oocytes matured in treatment increase glutathione 1.5 mM, then the percentage of oocytes were successfully fertilized also higher, namely 88.98%. Furthermore, the addition of 3 mM glutathione in embryo culture media in vitro buffalo give a significant influence on the percentage of embryo development to the morula stage or cell 32, namely (P &lt;0.01) in, or 40.73%.

Influência do horário de aplicação e da variedade genética em fêmeas de tilápias Oreochromis niloticus submetidas à indução hormonal com hCG

Objetivou-se com este trabalho avaliar a influência do horário de aplicação e da variedade genética de fêmeas submetidas à indução com hCG. O experimento foi realizado nos meses de julho e agosto de 2012. Utilizou-se delineamento inteiramente ao acaso, com esquema fatorial 2x4x2 (duas variedades, quatro horários de aplicação, com e sem hCG), sendo quatro repetições para os tratamentos controle e seis para induzidos com hCG. Foram utilizadas 40 fêmeas das variedades GIFT e UFLA, microchipadas, alojadas em um sistema com recirculação de água. Foram utilizados quatro horários de aplicação: seis; 12; 18 e 24 horas. A dosagem de hCG foi de 5UI/grama de peso de peixe, dividida em duas aplicações. A extrusão dos ovócitos foi realizada 720 horas-graus após a última aplicação, sendo observado o número de animais de cada tratamento que apresentaram desova. Os dados obtidos foram submetidos à análise de variância, e as médias comparadas pelo teste SNK a 5% de significância. A indução com hCG proporcionou melhores (P&lt;0,05) resultados paras as variáveis: índice de desova (ID), fecundidade absoluta relativa ao comprimento (FARC) e peso de desova (PD), independentemente da variedade utilizada. A variedade UFLA não foi influenciada pelo horário de aplicação (P&gt;0,05). Já a GIFT apresentou maior ID (P&lt;0,05) quando a indução hormonal foi realizada às 24h em relação à aplicação realizada às 18h. O diâmetro do ovócito da variedade UFLA é maior do que o da GIFT (P&lt;0,05). Ao se observar o grupo controle, verificou-se que a variedade UFLA apresentou maior porcentagem de ovócitos com posição periférica de vesícula germinativa (P&lt;0,05) em relação à GIFT. A indução hormonal com hCG foi influenciada pela variedade e pelo horário de aplicação.

Effect of Oocyte Recovery Techniques on &amp;lt;i&amp;gt;in Vitro&amp;lt;/i&amp;gt; Production of Swine Embryos

In Vitro production of swine embryos is a valuable tool to generate clones and genetically modified pigs during a short period of time. However, the efficiency of the existing methods is extremely low and the oocyte quality and quantity represent important obstacles on the success of in Vitro production of embryos. Therefore, the aim of this study was to compare the in Vitro maturation, fertilization and subsequent embryo development rates of oocytes recovered by ovary slicing or follicular aspiration. The oocyte recovery rate (grade 1 COC/ovary) was higher (p = 0.0083) in the slicing group when compared to the aspiration group. No differences were observed between groups regarding in Vitro maturation and early cleavage rates. A higher percentage of oocytes recovered by follicular aspiration reached the blastocyst stage after IVF when compared to the ovary slicing method (p = 0.0395). However, no difference on blastocyst cell number was observed. Although the recovery of oocytes using the slicing technique yielded more grade 1 oocytes per ovary than the aspiration method, the number of oocytes that reached the blastocyst stage after IVF by the slicing method was lower when compared with oocytes obtained by aspiration, as observed by lower blastocyst rates. In conclusion, the follicular aspiration is the method of choice for porcine in Vitro embryo production.

289 THE EFFECTS OF MEIOSIS BLOCKING BY CDK INHIBITORS ON THE ACTIVITY OF MATURATION PROMOTING FACTOR, ERK1 AND 2, AND THE DISTRIBUTION OF CYTOPLASMIC ORGANELLES IN IN VITRO-MATURED BOVINE OOCYTES

Studies have suggested that the prematuration with meiotic blockers can improve oocyte quality promoting embryonic development; however, its exact effects on cytoplasmic characteristics remain unclear. Thus, this study aimed to evaluate the effects of meiosis block of bovine oocytes with the CDK inhibitors roscovitine (ROS) and butyrolactone (BL-I) on nuclear maturation, expression, and localization of ERK 1 and 2, cyclin B1, and p34cdc2 proteins and the ultrastructure of oocytes. Immature oocytes from the slaughterhouse were divided into the following groups: (1) control, in vitro maturated (IVM) in TCM 199 for 24 h; (2) ROS 12.5 µM; (3) BL-I 50 µM; and (4) ROS (6.25 µM) + BL-I (25 µM) treatment for 6 h followed by IVM in CDK inhibitor-free medium for 18 h. Immature oocytes and IVM oocytes in each group were then fixed stained by immunofluorescence for nuclear visualisation (n = 600), localization, and expression of ERK1 and 2 proteins, cyclin B1 and p34cdc2 protein (n = 350), and prepared for evaluation of the ultrastructure by electron microscopy (n = 100). Data were analysed using the ANOVA test (nuclear visualisation), Student-Newman-Keuls test (expression of ERK1 and 2 proteins, cyclin B1 and p43cdc2) by the PROC GLM procedure of SAS (SAS Inst. Inc., Cary, NC, USA). At 6 h of IVM, a lower (P &lt; 0.05) percentage of oocytes were at the metaphase I (MI) stage in the control group (C = 18.2 ± 5.4%) compared with other groups and a higher percentage of oocytes were degenerated in the ROS group (16.3 ± 5.6%) compared with other groups (C = 13.6 ± 4.6%, BL-I = 10.0 ± 4.5%, BL-I+ROS = 8.0 ± 5.6%). At 24 h of IVM, higher (P &lt; 0.05) percentages of oocytes were at the MI stage in the control and ROS group (8.3 ± 5.9% and 6.8 ± 6.4%, respectively). There was no difference (P &gt; 0.05) in percentage of metaphase II (MII) oocytes among the groups. Only the ROS group showed lower fluorescence intensity of ERK1 and 2 proteins in the ooplasma (P &lt; 0.05). Immature oocytes showed higher expression of cyclin B1 and p34cdc2 (P &lt; 0.05). There was no difference in the localization of these proteins in the ooplasm and there was no difference in the oocyte ultrastructure (mitochondria, cortical granules, endoplasmic reticulum, microvilli, zona pellucida, lipid granules) among treatments (P &gt; 0.05). The results suggest that a temporary blocking of oocyte maturation by CDK inhibitors affect neither the expression and distribution of MPF components (cyclin B1/cdc2) nor the distribution of cytoplasmic organelles in IVM oocytes. However, the expression of ERK 1 and 2 in mature oocytes may be reduced by pre-IVM with ROS.

Effects of heat shock during the early stage of oocyte maturation on the meiotic progression, subsequent embryonic development and gene expression in ovine.

Heat shock may affect different aspects of oocyte maturation and its subsequent development to the blastocyst stage. A series of in vitro experiments was performed to determine whether physiologically heat shock (41°C) disrupts the progression of the ovine oocytes through meiosis, activation and blastocyst formation. Cumulus-oocyte complexes (COCs) were aspirated from 2-6-mm follicles and cultured at 38.5°C (control) or 41°C (heat shock) for the first 12 h of maturation. The oocytes were incubated at 38.5°C during the last 10 h of maturation and 8 days after activation. Results showed that most of the oocytes matured under heat-shock conditions remained at the germinal vesicle breakdown (GVBD) stage and they showed an aberrant chromatin configuration. After heat shock, oocyte diameter and time spent for zona pellucida dissolution increased (P < 0.05). The heat-shocked group had a higher percentage of oocytes with incomplete migration of cortical granules (P < 0.05). The heat-shock condition decreased (P < 0.05) cleavage rates (56.19 versus 89.28%) and morula formation (26.85 versus 37.81%). However, there was no significant difference in blastocyst formation and percentage of hatched blastocysts. At 12 h, heat shock had an adverse effect on embryo quality and reduced inner cell mass number (P < 0.05). Quantitative gene expression analysis showed greater transcripts (P < 0.05) for Na/K-ATPase mRNA in heat-shocked oocytes. To sum up, heat shock has disruptive effects on ovine oocyte maturation and can impair cellular and molecular factors that are important for embryo development.

Chromatin Patterns of Immature Canine Oocytes after In Vitro Maturation

In canine species, in vitro maturation (IVM) rates of oocytes collected from anoestrous ovaries are low (<20%). Several IVM media have been tested without significant improvements. A critical step in the evaluation of culture conditions is the observation of the meiotic stage reached by the oocytes. The present study was designed to investigate the chromatin patterns of in vitro matured oocytes by visualizing Germinal Vesicle (GV) and Germinal Vesicle Breakdown (GVBD) structures at 72 h of IVM. Nuclear stages of 1678 oocytes were evaluated by confocal microscopy after IVM. 1204 oocytes were non-degenerated, and 94.4% were still immature and at GV stage. Five different patterns of chromatin configuration were observed. Higher percentages of oocytes with unmodified GV and with diffuse (58%; Type A) and filamentous chromatin (19%; Type B) were observed in comparison with those with modifications in the GV such as patched chromatin (12.5%; Type C), surrounded-nucleolus (3%; Type D) and in vivo type chromatin/fully grouped chromatin (2.5%; Type E). These results indicate that GVBD (absence of nucleolus, nucleus breakdown) is rarely observed in vitro. The percentage of type C-D-E GVs and MI (meiotic resumption) and of MII (completion of meiosis) can be used to evaluate meiotic resumption after IVM. Our results indicate that although a low number of in vitro matured oocytes exhibit the chromatin configurations observed in in vivo collected oocytes, chromatin changes in the GV can be induced during IVM.

The Effect of Temperature on Oogenesis and Brain Gene Expression of Hormones Involved in Reproduction and Growth in the Female Blue Gourami ( Trichogaster trichopterus )

Introduction: The blue gourami (Trichogaster trichopterus) female has an asynchronic ovary development and the FOM will occur only in the presence of the male. Thus, each stage of its gonadal development can be controlled and examined separately in the laboratory [4]. Previous studies in our laboratory have shown variations in hypothalamic and pituitary hormonal gene expression during reproduction stages. Gonadotropin releasing hormone 3 (GnRH3), and pituitary adenylate cyclase activating polypeptide (PACAP long and short form [PRP]) gene expression increased in reproductively active females [5,6], and beta-follicle stimulating hormone (beta FSH) and beta-luteinizing hormone (beta LH) gene expression were increased in females in the vitellogenic stage [4]. Accumulated data indicate an involvement of growth factors in the regulation of reproduction, e.g., growth hormone (GH) and prolactin (PRL) [2, 3]. The present study examined the effect of temperature on reproduction and growth-related factors in blue gourami (Trichogaster trichopterus) females under non-reproductive (NRC) and reproductive conditions. Methods: The females, maintained in three different containers at the temperatures as described in prevue study [1], gonads were sampled and mRNA levels of GnRH3, PACAP, PRP, IGF1, GH, beta-LH, beta-FSH and PRL were mustered[1]. Results: A higher percentage of oocytes in the advanced vitellogenic stage was found in FNRC kept at 27 °C than in those kept at 23 °C or 31 °C (P<0.05,Fig. 1a). In contrast, in FRM and FNRM, a higher percentage of oocytes at the FOM stage were observed at 23 °C, than at 27 °C (Fig. 1b). In FNRC kept at 23 oC and 27 oC, as compared to the group at 31 oC, significantly higher mRNA levels of brain GnRH3 were detected, whereas no significant differences in the mRNA levels of brain IGF-1, PACAP and PRP were observed in these females at 23 oC, 27 oC or 31 oC. In FRM, mRNA levels of brain GnRH3, IGF1, PACAP and PRP were higher when kept at 27 °C than at 23 °C (Figs. 2A-D). PRP mRNA levels were also higher at 27 °C than at 31 °C in these fish. On the other hand, mRNA levels of PACAP were greater in FNRM kept at 27 °C than in those fish maintained at 23 °C and 31°C (Fig. 2B); and mRNA levels of IGF-1 were higher in the brains of FNRM kept at 23 °C than in those kept at 31 °C (Fig. 2C).

Oocyte morphological abnormalities in overweight women undergoing in vitro fertilization cycles

The effect of elevated body mass index (BMI) on the oocyte quality was investigated in women undergoing in vitro fertilization (IVF) cycles. A total of 268 patients classified on the basis of BMI subject to the first reproductive treatment were included in this study: the normal weight (NW) group consisted of 160 patients with BMI 19–24.9 kg/m2 and the overweight (OW) group consisted of 108 patients with BMI ≥25 kg/m2. All women were treated with a standard long luteal protocol. The oocyte features were classified as extracytoplasmic or cytoplasmic abnormalities. Outcomes were oocyte morphology, embryo quality, fertilization and implantation rates, and the ovarian response to stimulation. A higher percentage of oocytes with granular cytoplasm was found in women with BMI ≥25 (p = 0.04). However, percentages of mature, immature oocytes and germinal vesicle were similar in both groups. No differences were found in fertilization and cleavage rates and percentages of embryo quality. The implantation rate (p < 0.001) was significantly lower in the OW group than in the NW group. The amount of gonadotrophins was significantly higher in OW group (p = 0.003). These findings suggest that the poor reproductive outcome of obese women is influenced by the release of ova with reduced fertilization potential.

In vitro production of a caprine embryo from a preantral follicle cultured in media supplemented with growth hormone

The objective was to evaluate the effects of growth hormone (GH) on the survival, growth, maturation, and fertilization of oocytes derived from caprine preantral ovarian follicles cultured in vitro. Preantral follicles were isolated from the cortex of caprine ovaries and individually cultured for 18 d in the absence (control) or presence of bovine GH at concentrations of 10 or 50 ng/mL (GH10 and GH50, respectively). Follicle development was evaluated on the basis of survival, antral cavity formation, diameter increase, and the presence of healthy cumulus-oocyte complexes and mature oocytes. After culture, oocytes were subjected to in vitro maturation (IVM) and in vitro fertilization (IVF). The rate of antrum formation after Day 6 of culture was higher in both GH10 and GH50 than in the control (81.0, 92.7, and 47.6%, respectively, P < 0.05). Percentages of grown oocytes that were acceptable for IVM were also higher (P < 0.05) in GH-treated groups than in the control (54.8, 48.8, and 11.9% for GH10, GH50, and Control). A higher percentage of oocytes in the GH50 treatment underwent meiotic resumption (50.0%), produced mature oocytes, and enabled production of an embryo after IVF than in the control group (0.0%; P < 0.05). In conclusion, GH promoted in vitro growth and maturation of goat preantral follicle oocytes and enabled production of an embryo. Furthermore, this study was apparently the first to produce a caprine embryo by in vitro fertilization of oocytes derived from preantral follicles grown in vitro.

A Progestin and an Estrogen Regulate Early Stages of Oogenesis in Fish1

Using two species of teleost fish, Japanese huchen (Hucho perryi) and common carp (Cyprinus carpio), we investigated whether sex steroids are involved in early oogenesis in vitro. Ovarian fragments were cultured to examine the effects of a progestin, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP), and an estrogen, estradiol-17 beta (E2). DHP and E2 significantly promoted DNA synthesis in ovarian germ cells, as judged by 5-bromo-2-deoxyuridine (BrdU) incorporation into these cells. Furthermore, to detect the initiation of the first meiotic division of early oogenesis, we assessed ultrastructurally the occurrence of synaptonemal complexes (SCs) and analyzed by immunohistochemistry the expression of a meiosis-specific marker, Spo11. In huchen, a higher percentage of oocytes with SC was seen in DHP-treated ovarian fragments than in control or E2-treated ovarian fragments. Spo11 was expressed in germ cells after DHP treatment of carp ovarian explants. These data suggest that the progression of germ cells through early oogenesis involves two sex steroids: E2, which acts directly on oogonial proliferation, and DHP, which acts directly on the initiation of the first meiotic division of oogenesis.

The Relationship Between Oocyte Morphology and Ovarian Status in Cattle

We investigated the relationships between oocyte morphology, follicular size and follicular waves using bovine ovaries derived from local abattoirs. Ovaries at the recruitment and selection phases contained larger numbers of oocytes with good developmental ability, although ovaries at the recruitment phase contained the largest numbers of follicles compared with ovaries at the selection and dominant phases. Dominant phase ovaries contained a high percentage of oocytes with as good developmental ability as selection phase ovaries; however, they contained the lowest total number of oocytes with good developmental ability. Small follicles under 3.0 mm in diameter contained large numbers of small and degenerating oocytes. In contrast, follicles more than 3.0 mm in diameter contained a higher percentage of oocytes with good developmental ability.

Bub1 Prevents Chromosome Misalignment and Precocious Anaphase during Mouse Oocyte Meiosis

In mitosis the checkpoint proteins ensure faithful chromosome segregation by delaying onset of anaphase until all sister chromatids align at the metaphase plate of the bipolar spindle correctly. In the present study we blocked the function of Bub1 during meiosis by microinjecting anti-Bub1 specific antibody into cytoplasm of mouse oocytes, and found that depletion of Bub1 induced evident cyclin B degradation and precocious anaphase onset. Bub1 suppression also overrode the checkpoint-dependent cell cycle arrest provoked by a low dosage of nocodazole. Furthermore, Bub1 depletion induced a significantly higher percentage of oocytes with misaligned chromosomes. In addition, we depicted the localization dynamics of Bub1 in response to spindle damage and its relationship with microtubules and chromosomes, providing further evidence for Bub1’s role as a spindle checkpoint protein. Our data suggest that Bub1 is a critical spindle checkpoint protein that regulates accurate chromosome alignment and homolog disjunction in mammalian oocyte meiosis.

Neonatal Genistein Treatment Alters Ovarian Differentiation in the Mouse: Inhibition of Oocyte Nest Breakdown and Increased Oocyte Survival1

Early in ovarian differentiation, female mouse germ cells develop in clusters called oocyte nests or germline cysts. After birth, mouse germ cell nests break down into individual oocytes that are surrounded by somatic pregranulosa cells to form primordial follicles. Previously, we have shown that mice treated neonatally with genistein, the primary soy phytoestrogen, have multi-oocyte follicles (MOFs), an effect apparently mediated by estrogen receptor 2 (ESR2, more commonly known as ERbeta). To determine if genistein treatment leads to MOFs by inhibiting breakdown of oocyte nests, mice were treated neonatally with genistein (50 mg/kg per day) on Days 1-5, and the differentiation of the ovary was compared with untreated controls. Mice treated with genistein had fewer single oocytes and a higher percentage of oocytes not enclosed in follicles. Oocytes from genistein-treated mice exhibited intercellular bridges at 4 days of age, long after disappearing in controls by 2 days of age. There was also an increase in the number of oocytes that survived during the nest breakdown period and fewer oocytes undergoing apoptosis on Neonatal Day 3 in genistein-treated mice as determined by poly (ADP-ribose) polymerase (PARP1) and deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end-labeling (TUNEL). These data taken together suggest that genistein exposure during development alters ovarian differentiation by inhibiting oocyte nest breakdown and attenuating oocyte cell death.

Porcine sperm factor supports activation and development of bovine nuclear transfer embryos.

A study was undertaken to determine whether injection of porcine sperm factors (pSF), which trigger oscillations in intracellular calcium concentration ([Ca(2+)](i)) in mammalian oocytes, could be used to activate bovine oocytes during nuclear transfer. To date, only combined treatments that induce a monotonic rise in [Ca(2+)](i) and inhibit either phosphorylation or protein synthesis have been utilized in nuclear transfer. Several doses of pSF were tested. Injection of 5 mg/ml pSF triggered [Ca(2+)](i) oscillations that resembled those associated with fertilization with respect to amplitude and periodicity, and as a result, a high percentage of oocytes underwent activation. Furthermore, this concentration of pSF supported in vitro and in vivo development up to 60-90 days of gestation, comparable to development in control nuclear transfer embryos. Nevertheless, neither activation procedure supported development as well as did fertilization. The effectiveness of pSF as an activating agent in bovine oocytes may have been compromised because pSF was unable to support oscillations past 3-5 h postinjection and a second injection was necessary to extend the [Ca(2+)](i) oscillations. Likewise, a single injection of pSF failed to trigger downregulation of the inositol 1,4,5-trisphosphate receptor 1 subtype, whereas a second injection downregulated the receptor in a manner similar to that seen in fertilized oocytes. These results demonstrate that soluble factor(s) from porcine sperm can support early development in bovine nuclear transfer embryos; however, the efficacy may be limited because of the premature cessation of the induced oscillations.

Intracytoplasmic sperm injection of in vitro matured oocytes of domestic cats with frozen-thawed epididymal spermatozoa

The ability to mature and fertilize oocytes of endangered species may allow us to sustain genetic and global biodiversity. The first objective of this study was to compare the effect of two different culture media and two different incubation times on in vitro maturation (IVM) of domestic cat oocytes. The second objective was to determine the developmental competence of in vitro matured cat oocytes after intracytoplasmic sperm injection (ICSI) with cat spermatozoa. Oocytes recovered from ovaries of ovariectomized cats were cultured either in TCM 199 medium or in synthetic oviductal fluid (SOF), both of which were supplemented with cysteamine, BSA, FSH, LH. Nuclear maturation was assessed after 24 h and 40 h of incubation. Results of IVM showed that the percentage of oocytes reaching MII after 24 h and 40 h of incubation were significantly higher (P<0.001) after culture with SOF (88/110, 80% and 159/192, 82.8%) than TCM 199 (86/129, 66.7% and 58/90, 64.4%). Oocytes (n = 231) matured in vitro in SOF for 24 h were fertilized by ICSI with frozen-thawed epididymal cat spermatozoa. After ICSI, one group of oocytes (n = 129) was activated with ethanol, and a second group (n = 102) was not activated. The developmental competence of all ICSI oocytes was examined after 7 days of in vitro culture. After 28 h of culture, the cleavage frequency of ICSI-activated oocytes was significantly higher (P<0.001) than that of ICSI nonactivated (106/129, 82.2% vs 42/102, 41.2%). After 7 d in vitro culture, the frequency of in vitro development of cleaved embryos to morula stage was higher (P<0.001) in the ICSI activated group (37/106, 34.9%) that the ICSI nonactivated (2/102, 4.8%), while the development to blastocyst was not different (0 vs 7/106, 6.6%; P=0.088). We conclude that a higher percentage of oocytes will undergo meiotic maturation in SOF than in TCM 199. We also conclude that embryos can be produced with ICSI of in vitro matured oocytes using frozen-thawed epididymal cat semen.