Combined Exogenous Activation of Bovine Oocytes: Effects on Maturation-Promoting Factor, Mitogen-Activated Protein Kinases, and Embryonic Competence.

Oocyte activation via dual inhibition of protein synthesis and phosphorylation has improved in vitro embryo production in different mammalian species. In this study, we evaluated the effects of the combination of cycloheximide (CHX), dimethyl amino purine (DMAP), and anisomycin (ANY) on the activation of bovine oocytes, particularly on dynamics of MPF and MAPKs, embryonic developmental potential, and quality. The results showed that the cleavage and blastocyst rates, as well as levels of CCNB1, CDK1, p-CDK1Thr161, and p-CDK1Thr14-Tyr15, were similar among groups; ANY and ANY + CHX reduced the expression of ERK1/2 compared to DMAP-combinations (p < 0.05), whereas ANY + DMAP, CHX + DMAP, and ANY + CHX + DMAP reduced p-ERK1/2 compared to ANY and ANY + CHX treatments (p < 0.05). The quality of blastocysts in terms of cell counts, their allocation, and the numbers of TUNEL-positive cells did not differ among groups. However, transcript levels of POU5F1 were higher in embryos derived from ANY + CHX + DMAP treatment compared to other groups, while expression levels of CDX2 did not show differences. In addition, the BCL2A1/BAX ratio of the ANY + CHX + DMAP treatment was significantly low compared to the ANY treatment (p < 0.05) and did not differ significantly from the other treatments. In conclusion, oocyte activation by dual inhibition of protein synthesis and phosphorylation induces MPF inactivation without degradation of CCNB1, while MAPK inactivation occurs differentially between these inhibitors. Thus, although the combined use of these inhibitors does not affect early developmental competence in vitro, it positively impacts the expression of transcripts associated with embryonic quality.

Cilostamide, a phosphodiesterase 3A inhibitor, sustains meiotic arrest of rat oocytes by modulating cyclic adenosine monophosphate level and the key regulators of maturation promoting factor.

Cilostamide, a phosphodiesterase 3A (Pde3A) inhibitor, is known to increase intraoocyte cyclic adenosine monophosphate (cAMP) level which is involved in sustaining meiotic arrest of the oocytes. To explore the mechanisms involved in the cilostamide-mediated meiotic arrest of the oocytes, the present study describes the effects of cilostamide on cAMP level and related factors involved in maturation of the oocytes at its different meiotic stages; diplotene, metaphase I (MI) and metaphase II (MII). The oocytes from these three stages were collected from rat ovary and incubated with 10 µM cilostamide for 3 h in CO2 incubator. The levels of cAMP, cyclic guanosine monophosphate (cGMP) and the key players of maintaining meiotic arrest during oocyte maturation; Emi2, Apc, Cyclin B1, and Cdk1, were analyzed in diplotene, MI and MII stages. Pde3A was found to be expressed at all three stages but with the lowest level in MI oocyte. As compared to the control sets, the cAMP concentration was found to be highest in MII whereas cGMP was highest in the diplotene stage of cilostamide-treated group. The treated group showed declined reactive oxygen species level as compared with the control counterparts. Relatively increased levels of the Emi2, Cyclin B1, and phosphorylated thr161 of Cdk1 versus declined levels of phosphorylated thr14/tyr15 of Cdk1 in diplotene and MII stage oocytes are known to be involved in maintaining meiotic arrest and all these factors were found to undergo similar pattern of change due to the treatment with cilostamide. The findings thus suggest that cilostamide treatment promotes meiotic arrest by Pde3A inhibition led increase of both cAMP and cGMP level vis-a-vis modulation of the related regulatory factors such as Emi2, CyclinB1, and phosphorylated status of Cdk1 in diplotene and MII stage oocytes. Such a mechanism of meiotic arrest could allow the oocyte to prepare itself for meiotic maturation and thereby to improve oocyte quality.

TOP-2 is differentially required for the proper maintenance of the cohesin subunit REC-8 on meiotic chromosomes in Caenorhabditis elegans spermatogenesis and oogenesis.

During meiotic prophase I, accurate segregation of homologous chromosomes requires the establishment of chromosomes with a meiosis-specific architecture. The sister chromatid cohesin complex and the enzyme Topoisomerase II (TOP-2) are important components of meiotic chromosome architecture, but the relationship of these proteins in the context of meiotic chromosome segregation is poorly defined. Here, we analyzed the role of TOP-2 in the timely release of the sister chromatid cohesin subunit REC-8 during spermatogenesis and oogenesis of Caenorhabditis elegans. We show that there is a different requirement for TOP-2 in meiosis of spermatogenesis and oogenesis. The loss-of-function mutation top-2(it7) results in premature REC-8 removal in spermatogenesis, but not oogenesis. This correlates with a failure to maintain the HORMA-domain proteins HTP-1 and HTP-2 (HTP-1/2) on chromosome axes at diakinesis and mislocalization of the downstream components that control REC-8 release including Aurora B kinase. In oogenesis, top-2(it7) causes a delay in the localization of Aurora B to oocyte chromosomes but can be rescued through premature activation of the maturation promoting factor via knockdown of the inhibitor kinase WEE-1.3. The delay in Aurora B localization is associated with an increase in the length of diakinesis bivalents and wee-1.3 RNAi mediated rescue of Aurora B localization in top-2(it7) is associated with a decrease in diakinesis bivalent length. Our results imply that the sex-specific effects of TOP-2 on REC-8 release are due to differences in the temporal regulation of meiosis and chromosome structure in late prophase I in spermatogenesis and oogenesis.

SGK1 is essential for meiotic resumption in mammalian oocytes.

In mammalian females, oocytes are stored in the ovary and meiosis is arrested at the diplotene stage of prophase I. When females reach puberty oocytes are selectively recruited in cycles to grow, overcome the meiotic arrest, complete the first meiotic division and become mature (ready for fertilization). At a molecular level, the master regulator of prophase I arrest and meiotic resumption is the maturation-promoting factor (MPF) complex, formed by the active form of cyclin dependent kinase 1 (CDK1) and Cyclin B1. However, we still do not have complete information regarding the factors implicated in MPF activation. In this study we document that out of three mammalian serum-glucocorticoid kinase proteins (SGK1, SGK2, SGK3), mouse oocytes express only SGK1 with a phosphorylated (active) form dominantly localized in the nucleoplasm. Further, suppression of SGK1 activity in oocytes results in decreased CDK1 activation via the phosphatase cell division cycle 25B (CDC25B), consequently delaying or inhibiting nuclear envelope breakdown. Expression of exogenous constitutively active CDK1 can rescue the phenotype induced by SGK1 inhibition. These findings bring new insights into the molecular pathways acting upstream of MPF and a better understanding of meiotic resumption control by presenting a new key player SGK1 in mammalian oocytes.

Topoisomerase II dysfunction causes metaphase I arrest by activating Aurora B, SAC and MPF and prevents PB1 abscission in mouse oocytes†.

Oocyte aneuploidy is caused mainly by chromosome nondisjunction and/or unbalanced sister chromatid pre-division. Although studies in somatic cells have shown that topoisomerase II (TOP2) plays important roles in chromosome condensation and timely separation of centromeres, little is known about its role during oocyte meiosis. Furthermore, because VP-16, which is a TOP2 inhibitor and induces DNA double strand breaks, is often used for ovarian cancer chemotherapy, its effects on oocytes must be studied for ovarian cancer patients to recover ovarian function following chemotherapy. This study showed that inhibiting TOP2 with either ICRF-193 or VP-16 during meiosis I impaired chromatin condensation, chromosome alignment, TOP2α localization, and caused metaphase I (MI) arrest and first polar body (PB1) abscission failure. Inhibiting or neutralizing either spindle assembly checkpoint (SAC), Aurora B or maturation-promoting factor (MPF) significantly abolished the effect of ICRF-193 or VP-16 on MI arrest. Treatment with ICRF-193 or VP-16 significantly activated MPF and SAC but the effect disappeared when Aurora B was inhibited. Most of the oocytes matured in the presence of ICRF-193 or VP-16 were arrested at MI, and only 11-27% showed PB1 protrusion. Furthermore, most of the PB1 protrusions formed in the presence of ICRF-193 or VP-16 were retracted after further culture for 7 h. In conclusion, TOP2 dysfunction causes MI arrest by activating Aurora B, SAC, and MPF, and it prevents PB1 abscission by promoting chromatin bridges.

ELAPOR1 is a secretory granule maturation-promoting factor that is lost during paligenosis.

A single transcription factor, MIST1 (BHLHA15), maximizes secretory function in diverse secretory cells (like pancreatic acinar cells) by transcriptionally upregulating genes that elaborate secretory architecture. Here, we show that the scantly studied MIST1 target, ELAPOR1 (endosome/lysosome-associated apoptosis and autophagy regulator 1), is an evolutionarily conserved, novel mannose-6-phosphate receptor (M6PR) domain-containing protein. ELAPOR1 expression was specific to zymogenic cells (ZCs, the MIST1-expressing population in the stomach). ELAPOR1 expression was lost as tissue injury caused ZCs to undergo paligenosis (i.e., to become metaplastic and reenter the cell cycle). In cultured cells, ELAPOR1 trafficked with cis-Golgi resident proteins and with the trans-Golgi and late endosome protein: cation-independent M6PR. Secretory vesicle trafficking was disrupted by expression of ELAPOR1 truncation mutants. Mass spectrometric analysis of co-immunoprecipitated proteins showed ELAPOR1 and CI-M6PR shared many binding partners. However, CI-M6PR and ELAPOR1 must function differently, as CI-M6PR co-immunoprecipitated more lysosomal proteins and was not decreased during paligenosis in vivo. We generated Elapor1-/- mice to determine ELAPOR1 function in vivo. Consistent with in vitro findings, secretory granule maturation was defective in Elapor1-/- ZCs. Our results identify a role for ELAPOR1 in secretory granule maturation and help clarify how a single transcription factor maintains mature exocrine cell architecture in homeostasis and helps dismantle it during paligenosis.NEW & NOTEWORTHY Here, we find the MIST1 (BHLHA15) transcriptional target ELAPOR1 is an evolutionarily conserved, trans-Golgi/late endosome M6PR domain-containing protein that is specific to gastric zymogenic cells and required for normal secretory granule maturation in human cell lines and in mouse stomach.

Perfluorodecanoic acid induces meiotic defects and deterioration of mice oocytes in vitro.

Perfluorodecanoic acid (PFDA) is a member of the perfluoroalkyl substances, which are toxic to organic functions. Recently, it has been found in follicular fluid, seriously interfering with reproduction. Follicular fluid provides the oocyte with necessary resources during the process of oocytes maturation. However, the effects of PFDA on the oocyte need investigation. Our study evaluated the impacts of PFDA on the meiosis and development potential of mouse oocytes by exposing oocytes to PFDA in vitro at 350, 400, and 450 µM concentrations. The results showed that exposure to PFDA resulted in the first meiotic prophase arrest by obstructing the function of the maturation-promoting factor. It also induced the dysfunction of the spindle assembly checkpoint, expedited the progression of the first meiotic process, and increased the risk of aneuploidy. The oocytes treated with PFDA had a broken cytoskeleton which also contributed to meiotic maturation failure. Besides, PFDA exposure caused mitochondria defections, increased the reactive oxygen species level in oocytes, and consequently induced oocyte apoptosis. Moreover, PFDA produced epigenetic modifications in oocytes and increased the frequency of mature oocytes with declined development potential. In summary, our data indicated that PFDA disturbs the meiotic process and induces oocyte quality deterioration.

Meiotic Instability Generates a Pathological Condition in Mammalian Ovum.

Maintenance of metaphase-II (M-II) arrest in ovum is required to present itself as a right gamete for successful fertilization in mammals. Surprisingly, instability of meiotic cell cycle results in spontaneous exit from M-II arrest, chromosomal scattering and incomplete extrusion of second polar body (PB-II) without forming pronuclei so called abortive spontaneous ovum activation (SOA). It remains unclear what causes meiotic instability in freshly ovulated ovum that results in abortive SOA. We propose the involvement of various signal molecules such as reactive oxygen species (ROS), cyclic 3',5' adenosine monophosphate (cAMP) and calcium (Ca2+) in the induction of meiotic instability and thereby abortive SOA. These signal molecules through their downstream pathways modulate phosphorylation status and activity of cyclin dependent kinase (cdk1) as well as cyclin B1 level. Changes in phosphorylation status of cdk1 and its activity, dissociation and degradation of cyclin B1 destabilize maturation promoting factor (MPF). The premature MPF destabilization and defects in other cell cycle regulators possibly cause meiotic instability in ovum soon after ovulation. The meiotic instability results in a pathological condition of abortive SOA and deteriorates ovum quality. These ova are unfit for fertilization and limit reproductive outcome in several mammalian species including human. Therefore, global attention is required to identify the underlying causes in greater details in order to address the problem of meiotic instability in ova of several mammalian species icluding human. Moreover, these activated ova may be used to create parthenogenetic embryonic stem cell lines in vitro for the use in regenerative medicine.Graphical abstract.

SMALL ORGAN4 Is a Ribosome Biogenesis Factor Involved in 5.8S Ribosomal RNA Maturation.

Ribosome biogenesis is crucial for cellular metabolism and has important implications for disease and aging. Human (Homo sapiens) glioma tumor-suppressor candidate region gene2 (GLTSCR2) and yeast (Saccharomyces cerevisiae) Nucleolar protein53 (Nop53) are orthologous proteins with demonstrated roles as ribosome biogenesis factors; knockdown of GLTSCR2 impairs maturation of 18S and 5.8S ribosomal RNAs (rRNAs), and Nop53 is required for maturation of 5.8S and 25S rRNAs. Here, we characterized SMALL ORGAN4 (SMO4), the most likely ortholog of human GLTSCR2 and yeast Nop53 in Arabidopsis (Arabidopsis thaliana). Loss of function of SMO4 results in a mild morphological phenotype; however, we found that smo4 mutants exhibit strong cytological and molecular phenotypes: nucleolar hypertrophy and disorganization, overaccumulation of 5.8S and 18S rRNA precursors, and an imbalanced 40S:60S ribosome subunit ratio. Like yeast Nop53 and human GLTSCR2, Arabidopsis SMO4 participates in 5.8S rRNA maturation. In yeast, Nop53 cooperates with mRNA transport4 (Mtr4) for 5.8S rRNA maturation. In Arabidopsis, we found that SMO4 plays similar roles in the 5.8S rRNA maturation pathway than those described for MTR4. However, SMO4 seems not to participate in the degradation of by-products derived from the 5'-external transcribed spacer (ETS) of 45S pre-rRNA, as MTR4 does.

Chronic cadmium exposure causes oocyte meiotic arrest by disrupting spindle assembly checkpoint and maturation promoting factor.

Cadmium (Cd) is a bioaccumulative heavy metal element with potential toxicity on the female reproductive system, but the exact molecular mechanisms have not yet been clearly defined. In this study, female mice were exposed to 0.5 mg/kg/day of CdCl2 for 60 consecutive days. We found that chronic Cd exposure significantly decreased the fecundity of female mice by affecting oocyte meiotic progression as indicated by disrupted spindle assembly, chromosome alignment and kinetochore-microtubule attachments, consequently resulting in aneuploid oocytes. Further studies showed that the periodic fluctuations of MPF activity and cyclin B1 expression were disturbed in Cd-exposed oocytes probably by affecting the spindle assembly checkpoint protein Bub3. In addition, Cd exposure induced oxidative stress as indicated by an increased level of reactive oxygen species and apoptosis in oocytes, leading to oocyte quality deterioration. Taken together, these data suggest that Cd exposure causes disrupted molecular events of meiotic progression and deterioration of oocyte quality via oxidative stress, leading to decrease of female fertility.

Cell cycle period control through modulation of clock inputs.

In this work, we study period control of the mammalian cell cycle via coupling with the cellular clock. For this, we make use of the oscillators' synchronization dynamics and investigate methods of slowing down the cell cycle with the use of clock inputs. Clock control of the cell cycle is well established via identified molecular mechanisms, such as the CLOCK:BMAL1-mediated induction of the wee1 gene, resulting in the WEE1 kinase that represses the active form of mitosis promoting factor (MPF), the essential cell cycle component. To investigate the coupling dynamics of these systems, we use previously developed models of the clock and cell cycle oscillators and center our studies on unidirectional clock [Formula: see text] cell cycle coupling. Moreover, we propose an hypothesis of a Growth Factor (GF)-responsive clock, involving a pathway of the non-essential cell cycle complex cyclin D/CDK4. We observe a variety of rational ratios of clock to cell cycle period, such as: 1:1, 3:2, 4:3, and 5:4. Finally, our protocols of period control are successful in effectively slowing down the cell cycle by the use of clock modulating inputs, some of which correspond to existing drugs.

Hydrogen Sulfide Impairs Meiosis Resumption in Xenopuslaevis Oocytes.

The role of hydrogen sulfide (H2S) is addressed in Xenopuslaevis oocytes. Three enzymes involved in H2S metabolism, cystathionine ß-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase, were detected in prophase I and metaphase II-arrested oocytes and drove an acceleration of oocyte meiosis resumption when inhibited. Moreover, meiosis resumption is associated with a significant decrease in endogenous H2S. On another hand, a dose-dependent inhibition was obtained using the H2S donor, NaHS (1 and 5 mM). NaHS impaired translation. NaHS did not induce the dissociation of the components of the M-phase promoting factor (MPF), cyclin B and Cdk1, nor directly impacted the MPF activity. However, the M-phase entry induced by microinjection of metaphase II MPF-containing cytoplasm was diminished, suggesting upstream components of the MPF auto-amplification loop were sensitive to H2S. Superoxide dismutase and catalase hindered the effects of NaHS, and this sensitivity was partially dependent on the production of reactive oxygen species (ROS). In contrast to other species, no apoptosis was promoted. These results suggest a contribution of H2S signaling in the timing of amphibian oocytes meiosis resumption.

The chromatin remodeler Snf2h is essential for oocyte meiotic cell cycle progression.

Oocytes are indispensable for mammalian life. Thus, it is important to understand how mature oocytes are generated. As a critical stage of oocytes development, meiosis has been extensively studied, yet how chromatin remodeling contributes to this process is largely unknown. Here, we demonstrate that the ATP-dependent chromatin remodeling factor Snf2h (also known as Smarca5) plays a critical role in regulating meiotic cell cycle progression. Females with oocyte-specific depletion of Snf2h are infertile and oocytes lacking Snf2h fail to undergo meiotic resumption. Mechanistically, depletion of Snf2h results in dysregulation of meiosis-related genes, which causes failure of maturation-promoting factor (MPF) activation. ATAC-seq analysis in oocytes revealed that Snf2h regulates transcription of key meiotic genes, such as Prkar2b, by increasing its promoter chromatin accessibility. Thus, our studies not only demonstrate the importance of Snf2h in oocyte meiotic resumption, but also reveal the mechanism underlying how a chromatin remodeling factor can regulate oocyte meiosis.

Effect of single and combined treatments with MPF or MAPK inhibitors on parthenogenetic haploid activation of bovine oocytes.

In bovine, correct oocyte artificial activation is a key step in ICSI and other reproductive biotechnologies, and still needs to be improved. The current study was designed to compare the activating efficiency of ionomycin (Io) followed by: a 4 h time window and ethanol (4h-Et), roscovitine (Rosc), dehydroleucodine (DhL), cycloheximide (CHX) or PD0325901 (PD), each as a single treatment, and then combine them in novel protocols. Parthenogenetic haploid activation was evaluated in terms of pronuclear (PN) formation, second polar body (2PB) extrusion, ploidy of day 2 embryos and in vitro development. Combined treatments with Io-4h-Et-Rosc and Io-Rosc/CHX increased PN formation (92.2% and 96%, respectively) compared with Io-Rosc, Io-CHX or Io-4h-Et, which were equally efficient at inducing PN formation (82-84%) and 2PB extrusion (62.1-70.5%). Oocyte activation with Io-DhL and Io-Rosc/DhL resulted in higher 2PB extrusion rates (90% and 95.9%, respectively) but lower PN formation (49.4-58.8%) and cleavage rates (36-57.9%), as occurred with Io-CHX/DhL (76.4% and 70.4%, respectively). For the first time, results show that Io followed by the MAPK inhibitor PD induces PN formation and 2PB extrusion, but PD combined with Rosc or CHX resulted in low rates of haploid day 2 embryos. In conclusion, DhL strongly induces 2PB extrusion but leads to poor PN formation and embryo development. PD induces bovine oocyte activation but results in low rates of haploid embryos. In contrast, the improved PN formation rates after treatment with combined Io-4h-Et-Rosc and Io-Rosc/CHX suggest they should be further evaluated in ART, aiming to increase success rates in bovine.

Microcystin-LR influences the in vitro oocyte maturation of zebrafish by activating the MAPK pathway.

Harmful cyanobacteria and their production of microcystins (MCs) exert significant toxicity on reproduction of fish, especially the process of oogenesis. Our previous studies demonstrated that MCs have negative impacts on the quantity and quality of mature oocytes in female zebrafish. However, the underlying mechanisms of MCs disrupting oocyte maturation (OM) have been rarely reported. In the present study, in vitro oocytes (immature) were separated from zebrafish and treated with 1, 10, 100 µg/L MC-LR. The serine/threonine protein phosphatase 2A (PP2A) activity was downregulated significantly in oocytes exposed to 10 and 100 µg/L MC-LR for both 2 and 4 h. The phosphorylation levels of mitogen-activated protein kinase (MAPK) were detected without noticeable change in all oocytes treated with MC-LR for 2 h, whereas the activated levels of MAPK subtypes (ERK, p38 and JNK) increased remarkably in the 100 µg/L MC-LR treatment of 4 h. In the oocytes exposed to 100 µg/L MC-LR for 4 h, germinal vesicle breakdown (GVBD) rates changed abnormally and maturation-promoting factor (MPF) activity increased significantly, in accordance with the upregulation of Cyclin B protein levels. Moreover, the MAPK inhibitors (10 µM) were applied to explore the role of MAPK subtypes during MC-LR influencing OM and results showed that ERK inhibitor U0126 and p38 inhibitor SB203580 mitigated the effects of 100 µg/L MC-LR-induced MAPK hyper-phosphorylation and elevated GVBD in the oocytes. In conclusion, the present study indicates that microcystins disrupt the meiotic maturation by the pathway of MC-PP2A-MAPK-OM due to the phosphorylation disorder in oocytes.

CKS1 Germ Line Exclusion Is Essential for the Transition from Meiosis to Early Embryonic Development.

Cell division cycle (Cdc) kinase subunit (CKS) proteins bind cyclin-dependent kinases (CDKs) and play important roles in cell division control and development, though their precise molecular functions are not fully understood. Mammals express two closely related paralogs called CKS1 and CKS2, but only CKS2 is expressed in the germ line, indicating that it is solely responsible for regulating CDK functions in meiosis. Using cks2-/- knockout mice, we show that CKS2 is a crucial regulator of maturation-promoting factor (MPF; CDK1-cyclin A/B) activity in meiosis. cks2-/- oocytes display reduced and delayed MPF activity during meiotic progression, leading to defects in germinal vesicle breakdown (GVBD), anaphase-promoting complex/cyclosome (APC/C) activation, and meiotic spindle assembly. cks2-/- germ cells express significantly reduced levels of the MPF components CDK1 and cyclins A1/B1. Additionally, injection of MPF plus CKS2, but not MPF alone, restored normal GVBD in cks2-/- oocytes, demonstrating that GVBD is driven by a CKS2-dependent function of MPF. Moreover, we generated cks2cks1/cks1 knock-in mice and found that CKS1 can compensate for CKS2 in meiosis in vivo, but homozygous embryos arrested development at the 2- to 5-cell stage. Collectively, our results show that CKS2 is a crucial regulator of MPF functions in meiosis and that its paralog, CKS1, must be excluded from the germ line for proper embryonic development.

Phosphoric acid and phosphorylation levels are potential biomarkers indicating developmental competence of matured oocytes.

Here, we aimed to identify biomarkers for mice oocyte maturation in metaphase II in vivo and in situ using Raman spectroscopy. Principal component analysis of 324 Raman data points of oocytes at Phase I, II, III, and IV showed that the phosphoric acid concentration uniformly increased in oocytes with higher developmental competence than in oocytes at other maturation stages, and proteins were more phosphorylated. The maturation phases were successfully predicted by linear discriminant analysis with high accuracy (90.7%) using phosphoric molecular information mentioned above. Furthermore, detections of higher concentration of unsaturated fatty acids in overmatured oocytes indicated that a decline in metabolic activity due to overmaturation induced a surplus of these lipid components. Upon assessing invasiveness by laser irradiation, about 50% irradiated oocytes progressed to morula and blastocyst stages in good conditions. Thus, Raman spectroscopy holds promise in evaluating oocyte maturation and quality based on molecular information in infertility treatment.

Effect of EGF on expression and localization of maturation-promoting factor, mitogen-activated protein kinase, p34cdc2 and cyclin B during different culture periods on in vitro maturation of canine oocytes.

This study aimed to investigate the localization of MPF, MAPK, p34cdc2 and cyclin B1 proteins, before and after treatment with EGF during different moments of oocyte maturation. The ovaries obtained from 350 domestic dogs were aseptically isolated, immersed in physiological solution and transported at 4°C. In the laboratory, the ovaries were sectioned for the release of cumulus-oocyte complexes. Cumulus-oocyte complexes were selected and divided into treatment groups with and without EGF and cultured for 24, 48 and 72 hr. Immunofluorescence was used for the detection and the localization of MAPK, MPF, p34cdc2 and cyclin B1 proteins. We observed that the expression and localization of MPF, MAPK, p34cdc2 and cyclin B1 proteins are associated with meiosis resumption and cell cycle progression, and that EGF influences cell signalling pathways by promoting alterations in the localization of these proteins, improving the acquisition of oocyte competence. This is the first report of the localization of crucial proteins for meiosis progression in domestic dogs and identification of the expression and localization of proteins for cell cycle progression performed in this study represented a step of great importance to elucidate the mechanisms involved in the meiosis block in domestic dogs, allowing the advance in this research area.

Increased Expression of Maturation Promoting Factor Components Speeds Up Meiosis in Oocytes from Aged Females.

The rate of chromosome segregation errors that emerge during meiosis I in the mammalian female germ line are known to increase with maternal age; however, little is known about the underlying molecular mechanism. The objective of this study was to analyze meiotic progression of mouse oocytes in relation to maternal age. Using the mouse as a model system, we analyzed the timing of nuclear envelope breakdown and the morphology of the nuclear lamina of oocytes obtained from young (2 months old) and aged females (12 months old). Oocytes obtained from older females display a significantly faster progression through meiosis I compared to the ones obtained from younger females. Furthermore, in oocytes from aged females, lamin A/C structures exhibit rapid phosphorylation and dissociation. Additionally, we also found an increased abundance of MPF components and increased translation of factors controlling translational activity in the oocytes of aged females. In conclusion, the elevated MPF activity observed in aged female oocytes affects precocious meiotic processes that can multifactorially contribute to chromosomal errors in meiosis I.

Cyclin B2 can compensate for Cyclin B1 in oocyte meiosis I.

Mammalian oocytes are arrested at the prophase of the first meiotic division for months and even years, depending on species. Meiotic resumption of fully grown oocytes requires activation of M-phase-promoting factor (MPF), which is composed of Cyclin B1 and cyclin-dependent kinase 1 (CDK1). It has long been believed that Cyclin B1 synthesis/accumulation and its interaction with CDK1 is a prerequisite for MPF activation in oocytes. In this study, we revealed that oocyte meiotic resumption occurred in the absence of Cyclin B1. Ccnb1-null oocytes resumed meiosis and extruded the first polar body. Without Cyclin B1, CDK1 could be activated by up-regulated Cyclin B2. Ccnb1 and Ccnb2 double knockout permanently arrested the oocytes at the prophase of the first meiotic division. Oocyte-specific Ccnb1-null female mice were infertile due to failed MPF activity elevation and thus premature interphase-like stage entry in the second meiotic division. These results have revealed a hidden compensatory mechanism between Cyclin B1 and Cyclin B2 in regulating MPF and oocyte meiotic resumption.