Meiosis Block Research Articles

299 DEVELOPMENTAL COMPETENCE OF TRANSGENIC BOVINE EMBRYOS RECONSTRUCTED BY NUCLEAR TRANSFER USING MEIOSIS-BLOCKED OOCYTES

Recent progress in animal cloning by nuclear transfer (NT) has made it feasible to produce transgenic animals using genetically modified cell lines. Healthy cells and competent oocytes are needed to maximize the number of transgenic calves produced. Oocyte maturation plays a central role in oocyte competence. Prematuration inducing meiosis block is, therefore, a possible tool in transgenesis, because it allows further optimization of oocyte maturation protocols. In field conditions, it is not always possible to precisely control timing between oocyte collection and NT procedures. Therefore, temporarily and reversibly blocking maturation may also be used as a strategy to optimize cloning protocols. The aim of this study was to analyze the developmental competence of embryos reconstructed by NT using cells modified genetically as nuclei donors and oocytes submitted, or not, to meiosis block as cytoplasts. The hypothesis was that blocking meiosis does not alter the embryonic developmental physiology nor production of transgenic cloned bovine blastocysts. Ovaries were collected at a slaughterhouse and follicles between 3 and 6 mm were aspirated. Oocytes were divided into 2 groups. The first group (CTR, n = 145) was matured in vitro (IVM) with TCM-199 supplemented with 10% fetal calf serum, 5.0 µg mL–1 of LH, 0.5 µg mL–1 of FSH, 0.2 mm pyruvate, and 10 µg mL–1 of gentamicin for 18 h at 38.5�C under 5% CO2 in a humidified atmosphere. The second group (BL, n = 153) had meiosis blocked by in vitro culture with TCM-199 supplemented with 0.2 mm pyruvate, 10 µg mL–1 of gentamicin, and 10 µm butyrolactone I for 24 h, and then matured in vitro for 18 h. Parthenogenetic embryos resulted from meiosis-blocked and non-blocked oocytes were used as controls for the respective groups of NT embryos. After IVM, oocytes from both groups were reconstructed using bovine fetal fibroblasts transduced previously with a lentivirus and expressing the green fluorescent protein gene. The effect of treatment on fusion rates in cloned embryos, cleavage rates on Day 2, and blastocyst rates on Day 7 of in vitro culture of cloned and parthenogenetic embryos from CTR and BL groups were analyzed by chi-square test at 5% significance. Meiosis block did not affect fusion rates (n = 68, 46.90% and n = 86, 56.21% for CTR and BL cloned groups, respectively). Cleavage rates did not differ between cloned groups (n = 43, 63.24% and n = 49, 56.98% for CTR and BL groups) or between parthenogenetic groups (n = 15, 50% and n = 21, 70% for CTR and BL groups). Also, no difference was observed in blastocyst rates between cloned groups (n = 5, 7.35% and n = 6, 6.98% for CTR and BL groups) and between parthenogenetic groups (n = 11, 36.67% and n = 9, 30% for CTR and BL groups). It was concluded that meiosis block does not affect embryo development to the blastocyst stage. It is suggested that temporarily blocking meiosis may be a useful strategy to optimize NT protocols. FAPESP, Brazil.

The Aspergillus nidulans F‐box protein GrrA links SCF activity to meiosis

Cellular differentiation relies on precise and controlled means of gene expression that act on several levels to ensure a flexible and defined spatio-temporal expression of a given gene product. In our aim to identify transcripts enriched during fruiting body formation of the homothallic ascomycete Aspergillus (Emericella) nidulans, the grrA gene could be identified in a negative subtraction hybridization screening procedure. It encodes a protein similar to fungal F-box proteins, which function as substrate receptors for ubiquitin ligases, and that is highly related to the Saccharomyces cerevisiae regulatory protein Grr1p. Expression studies confirmed induction of grrA transcription and expression of its gene product during cleistothecial development of A. nidulans. Functional complementation of a yeast grr1Delta mutant was achieved by overexpression of the grrA coding sequence. A grrADelta deletion mutant resembles the wild-type in hyphal growth, asexual sporulation, Hülle cell formation or development of asci-containing cleistothecia, but is unable to produce mature ascospores due to a block in meiosis as demonstrated by cytological staining of cleistothecial contents. Our results specify a particular involvement of the E3 ubiquitin ligase SCFGrrA in meiosis and sexual spore formation of an ascomyceteous fungus and shed light on the diverse functions of ubiquitin-proteasome-mediated protein degradation in eukaryotic development.

Mouse SYCP2 is required for synaptonemal complex assembly and chromosomal synapsis during male meiosis

During meiosis, the arrangement of homologous chromosomes is tightly regulated by the synaptonemal complex (SC). Each SC consists of two axial/lateral elements (AEs/LEs), and numerous transverse filaments. SC protein 2 (SYCP2) and SYCP3 are integral components of AEs/LEs in mammals. We find that SYCP2 forms heterodimers with SYCP3 both in vitro and in vivo. An evolutionarily conserved coiled coil domain in SYCP2 is required for binding to SYCP3. We generated a mutant Sycp2 allele in mice that lacks the coiled coil domain. The fertility of homozygous Sycp2 mutant mice is sexually dimorphic; males are sterile because of a block in meiosis, whereas females are subfertile with sharply reduced litter size. Sycp2 mutant spermatocytes exhibit failure in the formation of AEs and chromosomal synapsis. Strikingly, the mutant SYCP2 protein localizes to axial chromosomal cores in both spermatocytes and fetal oocytes, but SYCP3 does not, demonstrating that SYCP2 is a primary determinant of AEs/LEs and, thus, is required for the incorporation of SYCP3 into SCs.

337 GENE EXPRESSION IN BOVINE OOCYTES AFTER MEIOSIS BLOCK WITH BUTYROLACTONE I

The present study aimed to assess the effect of meiosis block on the expression of genes involved in apoptosis. Slaughterhouse bovine ovaries were collected soon after slaughter and transported to the laboratory in saline. Follicles with a diameter of 2–6 mm were aspirated, and oocytes were selected and distributed to the different treatments. For meiosis block (MB), oocytes were cultured for 24 h in TCM-199 supplemented with 100 �M butyrolactone I (BLI) and 3 mg mL-1 BSA (B100) or with 10 �M BLI, but without BSA (B10). After the 24-h MB culture, the oocytes were subjected to IVM in TCM-199 supplemented with 10% fetal calf serum (FCS), 5.0 �g mL-1 LH, 0.5 �g mL-1 FSH, 0.2 mM pyruvate, and 10 �g mL-1 gentamicin for 22 h. All cultures were at 38.5�C under an atmosphere of 5% CO2 in air. As controls (C), a group of oocytes was collected immediately after aspiration (immature oocytes) and after IVM without prior meiosis block (mature oocytes). In the treated groups, oocytes were collected at the end of MB culture (immature oocytes) and at the end of IVM post-MB (mature oocytes). After removal of cumulus cells and zonae pelucidae, oocytes were frozen at -80�C in PBS with 0.1% PVA and 1 IU �L-1 RNase inhibitor. Reverse transcription was performed using SuperScript II Reverse Transcriptase (Invitrogen Brasil, Ltda., Sao Paulo, Brazil) according to the manufacturer's recommendations in an Applied Biosystems 7500 fast real-time PCR system (Applied Biosystems, Sao Paulo, Brazil). Efficiency of the reactions was assessed by the software LinRegPCR (Ramakers et al. 2003 Neurosci. Lett. 339, 62–66) and submitted to REST Beta 2005 V1.9.9 analysis (Relative Expression Software Tool). A level of significance of 5% was used. Relative expression of BAX and BCL2 genes did not differ (P > 0.05) when the expression of the constitutive gene GAPDH was used to normalize the data. However, without normalization, a significant reduction in expression was observed after IVM (P < 0.05). For immature oocytes, GAPDH, BAX, and BCL2 expression was 1.0% (for the three genes) for control; 0.26, 0.36, and 0.56% for B100; and 0.6, 0.44, and 0.26% for B10, respectively. After IVM, expressions were 0.06, 0.33, and 0.26% for control; 0.06, 0.19, and 0.25% for B100; and 0.02, 0.15, and 0.3% for B10, respectively. The results show a reduction in transcripts (GAPDH, BAX, and BCL2) after meiosis block, and this reduction was more pronounced in matured oocytes, irrespective of treatment. The reduction in transcripts during MB could influence oocyte competence, but the reduction of these transcripts during maturation seems to be inherent to the oocytes. This work was supported by Fapesp, Brazil, grant # 03/01479-6.

293 TWO-STEP MATURATION OF BOVINE OOCYTES WITHOUT CDK INHIBITORS: AN ALTERNATIVE TO AFFECT THEIR SUBSEQUENT DEVELOPMENTAL COMPETENCE

The acquisition of meiotic and developmental competence seems to correlate not only with the size of follicles and oocytes but also with the morphology and transcriptional activity of the oocyte nuclei and nucleoli. To secure or increase the fertilization and the developmental competence of bovine oocytes, we have developed a two-step culture system using the specific cyclin dependent kinase inhibitors (Butyrolactone I, Bohemine). However, these drugs have several side effects during the prolonged time of culture. To avoid this disadvantage, we have used in the present experiments modified culture conditions simulating the intrafollicular block of meiosis. In the first step of culture, bovine oocytes isolated from small, medium, and large follicles (2–3, 3–4, and 4–6 mm in diameter, respectively) were kept under conditions that secured for at least 48 h the intact germinal vesicle stage (GV) in more than 90% of oocytes. The second step represented the subsequent 20–22 h in conditions stimulating resumption of meiosis. The effectiveness of this model depended mainly on medium composition: reduced NaHCO3, substitution of serum with serum albumin, addition of antioxidants (curcumin), increased viscosity of a medium by agar (0.3%), and reduction of oxygen concentration (within 6–9%). The reduction of the proportion between the number of cumulus-oocyte complexes (COC) and the amount of medium (within 6–7 mL per COC) should amplify the GVBD-inhibiting effect of oocyte-surrounding granulosa cells. The COC were situated in clots of 6–7 COC per clot. The effectiveness and reversibility of GVBD inhibition depends also on the duration of COC isolation. The full reversibility of GVBD inhibition was controlled morphologically and also by measuring histone H1 and MAP kinase activities. The two-step versus one-step (24 h) maturation technique was evaluated by the percentage of total and hatched Day 9 blastocysts. When compared with one-step maturation, the two-step culture showed a slightly increased proportion of total and hatched blastocysts developed from the smallest follicular category (13.9 vs. 7.1% and 9.2 vs. 3.3% for total and hatched blastocysts, respectively). No significant difference was noticed between between one- and two-step culture when oocytes from large healthy follicles were used. However, the two-step maturation of oocytes from regressing follicles substantially reduced the blastocyst yield (9.7 vs. 39.1% and 4.9 vs. 26.7% for total and hatched blastocysts, respectively). This study was supported by grant of GA CR No. 524/02/0674.

274 NUCLEAR MATURATION KINETICS AND IN VITRO EMBRYO DEVELOPMENT OF BOVINE OOCYTES TREATED WITH BUTYROLACTONE I COMBINED OR NOT WITH ROSCOVITINE

Cyclin-dependent kinase inhibitors (CDKIs) have been used for prematuration culture the aim at improving oocyte competence. However, CDKIs seem to accelerate nuclear maturation (Hashimoto et al., 2002 Biol. Reprod. 66, 1696–1701). The aim of the present work was to compare the effect of butyrolactone I (BLI) alone or combined with roscovitine (ROS) at low dose (Ponderato et al, 2001 Mol. Reprod. Dev. 60, 579–585) on nuclear maturation kinetics and embryo development. To assess maturation kinetics (Experiment 1), oocytes were cultured in 100 μM BLI (B) or 6.25 μM BLI + 12.5 μM ROS (BR) in TCM-199 for 24 h. After prematuration, oocytes were submitted to in vitro maturation (IVM in TCM-199 + 0.5 μg mL−1 FSH, 50 μg mL−1 LH, 10% FCS) for another 24 h. Oocytes were fixed every 3 h (40–50 oocytes/time point/group in 4 replicates) to assess nuclear status. In Experiment 2, oocytes were submitted to prematuration, but the inhibitors were diluted in TCM-199 or DMEM. IVM lasted 21 h in DMEM (same hormone supplementation as in TCM-199 + 5% FCS and 50 ng mL−1 EGF). After IVM, all groups (140–150 oocytes/group in 7 replicates) were in vitro fertilized. Oocytes and sperm (2 × 106 sperm cells mL−1) were co-cultured for 18 h. Embryos were cultured in CR2aa in co-culture with granulosa cells for 8 days. All cultures were in microdrops under oil, at 38.5°C under 5% CO2 in air. In both experiments, control oocytes (C) were submitted only to IVM. Data were analyzed by GLM and GENMOD procedures (SAS program; SAS Institute, Inc., Cary, NC, USA), for Experiments 1 (4 replicates) and 2 (7 replicates), respectively. Cell numbers were analyzed by ANOVA and Tukey test. In Experiment 1, at 0 h, C and B oocytes were all (100%) at germinal vesicle stage (GV). BR had less GV oocytes (89 ± 1%, P < 0.05), indicating that BR was less effective in maintaining meiotic block for 24 h. After 3 h IVM, B and BR had less oocytes in GV (85 ± 2 and 80 ± 1%, respectively; P > 0.05) than C (100%, P < 0.05), suggesting an acceleration of oocyte maturation. At 12 h, however, most oocytes were at intermediate stages (metaphase I to telophase I) in all groups (78 ± 1–83 ± 2%, P > 0.05). After 21 and 24 h, all groups had similar metaphase II (MII) rates (77 ± 1–89 ± 1 for 21 h and 85 ± 2–96 ± 8 for 24 h P > 0.05). These results suggest that after 12 h, meiosis acceleration was less evident and oocytes proceeded nuclear maturation at similar rates. In Experiment 2, cleavage (79 ± 3–84 ± 3%, P > 0.05) and Day 7 blastocyst rates (26 ± 4–37 ± 4%, P > 0.05) were similar for all groups. After 8 days in culture, all groups presented similar blastocyst rates (35 ± 4–40 ± 4%, P > 0.05), except for the group prematured with BR in DMEM, which presented lower blastocyst rates (32.3 ± 4%) only when compared with C (40 ± 4%, P < 0.05). Hatching rates were similar (10 ± 3–16 ± %3, P > 0.05) as were total cell numbers (141 ± 5–170 ± 10). In conclusion: (a) BR is less effective in maintaining meiosis block; (b) B and BR accelerate the first half of meiosis progression in about 3 h; and (c) BR used in DMEM during prematuration may negatively affect developmental rates. Financial support was provided by Fapesp, Brazil.

Effect of phosphodiesterase type 3 inhibitor on developmental competence of immature mouse oocytes in vitro.

In vitro use of arresters of meiosis could improve cytoplasmic maturation of immature oocytes by controlling the period of prophase I. Phosphodiesterases (PDE) are responsible for the breakdown and concomitant inactivation of the cyclic nucleotides cAMP and cGMP and are implicated in the regulation of oocyte meiotic maturation. Selective inhibitors of phosphodiesterase type 3 (PDE3) prevent meiotic resumption of mammalian oocytes. This study evaluated the impact of meiosis arrest by PDE3 inhibitor, Org 9935, on developmental competence of geminal vesicle (GV)-stage oocytes from small antral follicles. Cumulus-oocyte complexes (COC), retrieved from antral follicles 24 h after eCG exposure and cultured in the presence of PDE3 inhibitor (10 microM) for an additional 24 h, remained arrested in the meiotic prophase. The GV configuration of oocytes before and after the arrest by PDE3 inhibitor was examined. After the period of meiosis arrest, a significantly increased proportion of oocytes had acquired a nucleolus surrounded by a condensed chromatin rim at the GV, which is a morphological correlate of transcriptional repression. Removal of inhibitor resulted in 90.6% +/- 8.3% of oocytes with the first polar body extruded. Fertilization was significantly improved in oocytes that had been arrested compared with oocytes collected 24 h after eCG and undergoing in vitro maturation immediately. Embryonic preimplantation and live offspring rates of arrested oocytes were higher, although not significantly, than those of nonarrested oocytes. These results suggest that a temporal block of meiosis by PDE3 inhibitor promotes developmental competence of mice oocytes retrieved from small antral follicles.

Developmental competence of bovine oocytes is stimulated by low doses of dibutyryl-cAMP during cycloheximide-induced block of meiosis

The time that precedes the resumption of meiosis is crucial for the acquisition of oocyte developmental competence. However, at present, the mechanisms regulating this process are largely unknown though their understanding would enhance our capacity to improve the quality of in vitro produced embryos. This study was designed to assess the role of cAMP in the acquisition of oocyte developmental competence before meiotic resumption. To achieve this, bovine cumulusoocyte complexes (COCs) were reversibly maintained in meiotic arrest for 6 h by a protein synthesis inhibitor, cycloheximide (CHX), and during this period the intracellular cAMP concentration were modulated with different concentrations of a cAMP analog, dibutyryl-cAMP (dbcAMP) before being matured under standard conditions with serum and gonadotropins. COCs were isolated from ovaries recovered at slaughter by aspirating 2-5 mm follicles in TALP Hepes alone or supplemented with 2 gg/ml of CHX. COCs with compact cumulus layers and homogeneous cytoplasm were selected and incubated for 6 h with or without CHX and various concentrations of dbcAMP. At the end of culture COCs were washed and placed in M199 supplemented with 10% FCS, 1 gg/ml FSH and 5 pg/ml LH for 24 h'then fertilized for 16 h and cultured for 5 d in the presence of bovine oviductal epithelial cells. Frequency of cleavage, morula and blastocyst formation were determined 2 and 6 d post IVF (p.i.) respectively.

Genetics: The use of epididymal and testicular spermatozoa for intracytoplasmic sperm injection: the genetic implications for male infertility

The results and rationale of using testicular and epididymal spermatozoa with intracytoplasmic sperm injection (ICSI) for severe cases of male infertility are reviewed. A total of 72 consecutive microsurgical epididymal sperm aspiration (MESA) cases were performed for congenital absence of the vas (CAV) and for irreparable obstructive azoospermia. ICSI was used to obtain normal embryos for transfer and fertilization in 90% of the cases. The overall fertilization rate was 46% with a normal cleavage rate of 68%. The pregnancy and delivery rates per transfer were 58 and 37% respectively. The delivery rate per cycle was 33%. In many cases, no epididymal spermatozoa were available and so testicular sperm extraction (TESE) was used for sperm retrieval. The transfer rate was lower with TESE (84 versus 96%) and the spermatozoa could not be frozen and saved for use in future cycles. However, there was little difference in pregnancy rates using epidiymal or testicular spermatozoa. The results were not affected by whether the obstruction was caused by CAV or failed vasoepididymostomy. Both fresh and frozen spermatozoa gave similar results; the only significant factor appeared to be the age of the female. Because of the consistently good results obtained using epididymal sperm with ICSI when compared with conventional IVF, and the similarly good results with testicular tissue spermatozoa, ICSI is mandatory for all future MESA patients. All CAV patients and their partners should be offered genetic screening for cystic fibrosis; hence pre-implantation embryo diagnosis should be available in any full service MESA programme. It is now clear that even with non-obstructive azoospermia, e.g. Sertoli-cell only, or maturation arrest, there are usually some small foci of spermatogenesis which allow TESE with ICSI to be carried out. This means that even in men with azoospermia due to absence of spermatogenesis or to a block in meiosis, there are usually a few spermatozoa available in the testes that are adequate for successful ICSI. Finally, it is likely that some forms of severe male factor infertility are genetically transmitted and although ICSI offspring have been shown to be completely normal, it is possible that the sons of these infertile couples will also require ICSI when they grow up and wish to have a family.

The Drosophila cdc25 homolog twine is required for meiosis.

We have identified a second cdc25 homolog in Drosophila. In contrast to string (the first homolog identified in Drosophila) this second homolog, twine, does not function in the mitotic cell cycle, but is specialized for meiosis. Expression of twine was observed exclusively in male and female gonads. twine transcripts are present in germ cells during meiosis, and appear only late during gametogenesis, well after the end of the mitotic germ cell divisions. The sterile Drosophila mutant, mat(2)synHB5, which had previously been isolated and mapped to the same genomic region as twine (35F), was found to carry a missense mutation in the twine gene. This missense mutation in twine abolished its ability to complement a mutation in Schizosaccharomyces pombe cdc25. Phenotypic analysis of mat(2)synHB5 mutant flies revealed a complete block of meiosis in males and severe meiotic defects in females.

Quantitative Parameters of Seminiferous Epithelium in Secretory and Excretory Oligoazoospermia

Testicular biopsy specimens from infertile men (sperm count, less than 10(6)/ml) were evaluated on 1-micron thick sections, and counts of stem cells and differentiated spermatogonia, primary spermatocytes, early and late spermatids, and Sertoli cells were compared to counts in six fertile men. Biopsy specimens were also compared for the appearance of seminiferous tubule wall, blood vessels, and interstitium. Infertile men were grouped according to the following diagnoses: hypospermatogenesis (n = 5), spermatocyte arrest of spermatogenesis (n = 5), and obstruction of the genital tract (n = 7). A low productivity of spermatogenesis in cases of hypospermatogenesis appeared to be due to an exaggerated degeneration of primary spermatocytes and to a yield of abnormal spermatids. A block of meiosis in spermatocyte arrest was associated with a degeneration of primary spermatocytes and with a reduced number of staminal spermatogonia. Abnormal spermiogenesis was observed in cases of obstruction of the genital tract and was associated with an increase in stem cell spermatogonia. A thickening of seminiferous tubule and blood vessel walls could be responsible for the limited functional capacity of Sertoli cells, causing altered spermiogenesis in cases of excretory azoospermia. A severe primitive failure of Sertoli cells in secretory oligoazoospermia could account for a deranged maturation and degeneration of premeiotic and postmeiotic germ cells.

Recessive mutations from natural populations of Neurospora crassa that are expressed in the sexual diplophase.

Wild-collected isolates of Neurospora crassa Shear and Dodge were systematically examined for recessive mutations affecting the sexual phase of the life cycle, which is essentially diploid. Seventy-four of 99 wild-collected isolates from 26 populations in the United States, India and Pakistan carried one or more recessive mutations that reduced fertility significantly when homozygous; mutations affecting spore morphology were also detected. Limited complementation tests indicate that most of the 106 recovered mutations are unique.--The recessive diplophase (= sexual phase) mutations were uncovered by crossing each wild-collected isolate to a marked two-chromosome double-reciprocal translocation strain as "balancer." Surviving progeny receive approximately 60% of their genome from the wild parent, but receive the mating-type allele from the "balancer" parent. These progeny were backcrossed to the wild parent and were also crossed with a standard laboratory strain (fl). Reduced fertility in the backcross vs. normal fertility in the cross with the laboratory standard signals the presence of a recessive mutation in the wild-collected isolate.--Most of the mutants (95 of 106) fall into two major classes: those producing barren perithecia with no or few viable ascospores (51) and those with spore maturation defects (44). Most of the recessive barrens result either from an early block in meiosis of ascus development (25) or from a late disturbance in postmeiotic ascus behavior (18).--These recessive mutations are formally equivalent to recessive lethals in higher eukaryotes and may be important in determining the breeding structure of natural Neurospora populations.