Inhibition Of Meiosis Research Articles

Cyclin B Export to the Cytoplasm via the Nup62 Subcomplex and Subsequent Rapid Nuclear Import Are Required for the Initiation of Drosophila Male Meiosis.

The cyclin-dependent kinase 1 (Cdk1)-cyclin B (CycB) complex plays critical roles in cell-cycle regulation. Before Drosophila male meiosis, CycB is exported from the nucleus to the cytoplasm via the nuclear porin 62kD (Nup62) subcomplex of the nuclear pore complex. When this export is inhibited, Cdk1 is not activated, and meiosis does not initiate. We investigated the mechanism that controls the cellular localization and activation of Cdk1. Cdk1-CycB continuously shuttled into and out of the nucleus before meiosis. Overexpression of CycB, but not that of CycB with nuclear localization signal sequences, rescued reduced cytoplasmic CycB and inhibition of meiosis in Nup62-silenced cells. Full-scale Cdk1 activation occurred in the nucleus shortly after its rapid nuclear entry. Cdk1-dependent centrosome separation did not occur in Nup62-silenced cells, whereas Cdk1 interacted with Cdk-activating kinase and Twine/Cdc25C in the nuclei of Nup62-silenced cells, suggesting the involvement of another suppression mechanism. Silencing of roughex rescued Cdk1 inhibition and initiated meiosis. Nuclear export of Cdk1 ensured its escape from inhibition by a cyclin-dependent kinase inhibitor. The complex re-entered the nucleus via importin β at the onset of meiosis. We propose a model regarding the dynamics and activation mechanism of Cdk1-CycB to initiate male meiosis.

Study on the Effect of Topical Pre-operative Nonsteroidal Anti-inflammatory Drugs in Maintaining Mydriasis during Small Incision Cataract Surgery

A prospective, randomized, double masked, single centered comparative study done in the Bangladesh Naval Ship Patenga, Chattogram, Bangladesh, from July 2021 to June 2022, to assess the clinical benefits and effectiveness of prophylactic pre-operative topical administration of NSAIDs in maintaining mydriasis during small incision cataract surgery. Thirty patients underwent small incision cataract surgery with intra ocular lens implantation were randomized to compare the efficacy of topical NSAIDs drops (Group-A) and placebo drops (Group-B) in maintaining peroperative mydriasis. Balanced salt solution containing non preservative adrenaline was used in all patients. Pupil size was measured prior to the corneal incision and after the completion of the operation. The two groups were analyzed with respect to the changes in pupillary diameter at the beginning and at the end of the operation. The change in pupil size was significantly different among the two groups at the end of the surgery. The mean vertical and horizontal diameter of the two groups were near about similar at the beginning of the surgery. Significant differences were seen after IOL implantation with the NSAIDs group having the larger mean diameter in both vertical (P=0.003) and horizontal (P=0.008) pupillary measurements at the conclusion of surgery. Topical NSAIDs has been shown to be a more effective inhibitor of meiosis during per operative uncomplicated small incision cataract surgery and provides a more stable mydriatic effect during surgery compared to the placebo group.
 CBMJ 2023 January: Vol. 12 No. 01 P: 22-26

Bi-allelic MEI1 variants cause meiosis arrest and non-obstructive azoospermia.

Non-obstructive azoospermia (NOA) is characterized by the failure of sperm production due to testicular disorders and represents the most severe form of male infertility. Growing evidences have indicated that gene defects could be the potential cause of NOA via genome-wide sequencing approaches. Here, bi-allelic deleterious variants in meiosis inhibitor protein 1 (MEI1) were identified by whole-exome sequencing in four Chinese patients with NOA. Testicular pathologic analysis and immunohistochemical staining revealed that spermatogenesis is arrested at spermatocyte stage, with defective programmed DNA double-strand breaks (DSBs) homoeostasis and meiotic chromosome synapsis in patients carrying the variants. In addition, our results showed that one missense variant (c.G186C) reduced the expression of MEI1 and one frameshift variant (c.251delT) led to truncated proteins of MEI1 in in vitro. Furthermore, the missense variant (c.T1585A) was assumed to affect the interaction between MEI1 and its partners via bioinformatic analysis. Collectively, our findings provide direct genetic and functional evidences that bi-allelic variants in MEI1 could cause defective DSBs homoeostasis and meiotic chromosome synapsis, which subsequently lead to meiosis arrest and male infertility. Thus, our study deepens our knowledge of the role of MEI1 in male fertility and provides a novel insight to understand the genetic aetiology of NOA.

OsPRD2 is essential for double-strand break formation, but not spindle assembly during rice meiosis.

Meiotic recombination starts with the programmed formation of double-strand breaks (DSB) in DNA, which are catalyzed by SPO11, a type II topoisomerase that is evolutionarily conserved, and several other accessary proteins. Homologs of MEIOSIS INHIBITOR 4 (MEI4/REC24/PRD2) are proteins that are also essential for the generation of meiotic DSBs in budding yeast, mice and Arabidopsis thaliana. In Arabidopsis, the protein ARABIDOPSIS THALIANA PUTATIVE RECOMBINATION INITIATION DEFECTS 2/MULTIPOLAR SPINDLE 1 (AtPRD2/MPS1) has been shown to have additional roles in spindle assembly, indicating a functional diversification. Here we characterize the role of the rice MEI4/PRD2 homolog in meiosis. The osprd2 mutant was completely male and female sterile. In male meiocytes of osprd2, no γH2AX foci were detected and twenty-four univalents were produced at diakinesis, suggesting that OsPRD2 is essential for DSB generation. OsPRD2 showed a dynamic localization during meiosis. For instance, OsPRD2 foci first appeared as discrete signals across chromosome at leptotene, and then became confined to the centromeres during zygotene, suggesting that they might be involved in assembly of the spindle. However we did not observe any obvious aberrant morphologies in neither the organization of the bipolar spindle nor in the orientation of the kinetochore in the mutant. These findings suggest that in rice PRD2 might not be required for spindle assembly and organization, as it does in Arabidopsis. Taken together our results indicate that plant MEI4/PRD2 homologs do play a conserved role in the formation of meiotic DSBs in DNA, but that their involvement in bipolar spindle assembly is rather species-specific.

Single-cell RNA sequencing of mitotic-arrested prospermatogonia with DAZL::GFP chickens and revealing unique epigenetic reprogramming of chickens

BackgroundGerm cell mitotic arrest is conserved in many vertebrates, including birds, although the time of entry or exit into quiescence phase differs. Mitotic arrest is essential for the normal differentiation of male germ cells into spermatogonia and accompanies epigenetic reprogramming and meiosis inhibition from embryonic development to post-hatch. However, mitotic arrest was not well studied in chickens because of the difficulty in obtaining pure germ cells from relevant developmental stage.ResultsWe performed single-cell RNA sequencing to investigate transcriptional dynamics of male germ cells during mitotic arrest in DAZL::GFP chickens. Using differentially expressed gene analysis and K-means clustering to analyze cells at different developmental stages (E12, E16, and hatch), we found that metabolic and signaling pathways were regulated, and that the epigenome was reprogrammed during mitotic arrest. In particular, we found that histone H3K9 and H3K14 acetylation (by HDAC2) and DNA demethylation (by DNMT3B and HELLS) led to a transcriptionally permissive chromatin state. Furthermore, we found that global DNA demethylation occurred gradually after the onset of mitotic arrest, indicating that the epigenetic-reprogramming schedule of the chicken genome differs from that of the mammalian genome. DNA hypomethylation persisted after hatching, and methylation was slowly re-established 3 weeks later.ConclusionsWe found a unique epigenetic-reprogramming schedule of mitotic-arrested chicken prospermatogonia and prolonged hypomethylation after hatching. This will provide a foundation for understanding the process of germ-cell epigenetic regulation in several species for which this process is not clearly described. Our findings on the biological processes related to sex-specific differentiation of prospermatogonia could help studying germline development in vitro more elaborately.

Programmed self-assembly of enzyme activity-inhibited nanomedicine for augmenting chemodynamic tumor nanotherapy.

The satisfactory therapeutic effects of chemodynamic therapy (CDT) dependent solely on endogenous hydrogen peroxide (H2O2) from tumor cells are difficult to achieve. This is closely attributed to the high metabolic activity of malignant cancer cells, prompting the rapid self-protection and proliferation. Here, we report a programmed self-assembly multilayered nanostructure, thioglycolic acid (TGA)-Cu coordination nanoparticles with rapid GSH-response characteristics, for intensifying the CDT efficiency and comprehensively inhibiting the tumor metabolic activity via exchanging the TGA ligand with glutathione (GSH) in the tumor cell. In the formulation, TGA, a small toxic molecule, was combined with Cu ions and securely delivered to the destination for inactivating the functional protein by depriving their spatial structure, then inducing the inhibition of metabolism and meiosis. Simultaneously, the oxidative stress that originated from the oxidized glutathione (GSSG)-Cu complex triggering H2O2 compels the cancer cells to perform active and passive death processes in concert with the inhibition of intracellular enzyme activities. Thus, this work is not only expected to be a heuristic strategy for amplifying the therapeutic effect of CDT together with the inhibition of enzyme activity, but also may advance the construction of stimulus-response bio-functional materials.

Nanostructured Titanium Dioxide (TiO₂) Reduces Sperm Concentration Involving Disorder of Meiosis and Signal Pathway.

Nano-titanium dioxide (nano-TiO₂) has been widely used in food and cosmetic industries, and the medical sector. However, nano-TiO₂ is potentially toxic to the reproductive system. Previous research has shown that nano-TiO₂ can reduce sperm concentration but do not yet known whether this effect occurs because of dysfunctional meiosis in spermatogenic cells. In the present paper, we demonstrate that Nano-TiO₂ can penetrate through the blood-testis barrier of a mouse model and enter the testicular tissue, thus causing damage to the testis and epididymis. This reduced the number of developing sperm; we demonstrated that the mechanism underlying this effect was the inhibition or destruction of meiosis in spermatogenic cells, particularly during meiosis I. We also found that the inhibition of meiosis I caused by nano-TiO₂ exposure was related to dysfunctional meiosis and that the abnormal expression of meiosis-related factors. Therefore, our data demonstrate that nano-TiO₂ reduces sperm concentration by disrupting meiosis and related signaling pathways.

Identification and Screening of Selective WEE2 Inhibitors to Develop Non-Hormonal Contraceptives that Specifically Target Meiosis.

We used a progressive elimination strategy to identify oocyte-specific WEE2 kinase inhibitors for potential non-hormonal contraceptives that target meiosis. Beginning with an in-house library of over 300,000 compounds, virtual high throughput screening identified 57 WEE2 inhibitors with preferential predicted binding over the somatic variant WEE1. Seven compounds were further evaluated in vitro by enzyme-linked immunosorbent assay to measure biochemical inhibition on WEE1 and WEE2 phosphorylation of CDK1. To assess specificity, we evaluated WEE2-mediated inhibition of meiosis using in vitro oocyte fertilization, and WEE1-mediated inhibition of mitosis using a somatic cell proliferation assay. Our results from these assays identified three candidates for further development: 6-(2,6-dichlorophenyl)-2-((4-(2-(diethylamino)ethoxy) phenyl)amino)-8-methylpyrido[2,3-d]pyrimidin-7(8H)-one (2), 6-(2,6-dichlorophenyl)-8-methyl-2-((4-morpholinophenyl) amino)pyrido[2,3-d]pyrimidin-7(8H)-one (12), and 3-((6-(2,6-dichlorophenyl)-8-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)benzoic acid (16).

Proteomic and N-Terminomic TAILS Analyses of Human Alveolar Bone Proteins: Improved Protein Extraction Methodology and LysargiNase Digestion Strategies Increase Proteome Coverage and Missing Protein Identification.

With 2129 proteins still classified by the Human Proteome Organisation Human Proteome Project (HPP) as "missing" without compelling evidence of protein existence (PE) in humans, we hypothesized that in-depth proteomic characterization of tissues that are technically challenging to access and extract would yield evidence for tissue-specific missing proteins. Paradoxically, although the skeleton is the most massive tissue system in humans, as one of the poorest characterized by proteomics, bone falls under the HPP umbrella term as a "rare tissue". Therefore, we aimed to optimize mineralized tissue protein extraction methodology and workflows for proteomic and data analyses of small quantities of healthy young adult human alveolar bone. Osteoid was solubilized by GuHCl extraction, with hydroxyapatite-bound proteins then released by ethylenediaminetetraacetic acid demineralization. A subsequent GuHCl solubilization extraction was followed by solid-phase digestion of the remaining insoluble cross-linked protein using trypsin and then 6 M urea dissolution incorporating LysC digestion. Bone extracts were digested in parallel using trypsin, LysargiNase, AspN, or GluC prior to liquid chromatography-mass spectrometry analysis. Terminal Amine Isotopic Labeling of Substrates was used to purify semitryptic peptides, identifying natural and proteolytic-cleaved neo N-termini of bone proteins. Our strategy enabled complete solubilization of the organic bone matrix leading to extensive categorization of bone proteins in different bone matrix extracts, and hence matrix compartments, for the first time. Moreover, this led to the high confidence identification of pannexin-3, a "missing protein", found only in the insoluble collagenous matrix and revealed for the first time by trypsin solid-phase digestion. We also found a singleton proteotypic peptide of another missing protein, meiosis inhibitor protein 1. We also identified 17 proteins classified in neXtprot as PE1 based on evidence other than from MS, termed non-MS PE1 proteins, including ≥9-mer proteotypic peptides of four proteins.

Treatment with roscovitine and butyrolactone I prior to in vitro maturation alters blastocyst production.

This study evaluated the effects of oocyte meiosis inhibitors roscovitine (ROS) and butyrolactone I (BL-I) on in vitro production of bovine embryos. Bovine oocytes were maintained in pre in vitro maturation (pre-IVM) with 25 µM ROS or 100 µM BL-I for 24 h to delay meiosis and for 24 h in in vitro maturation (IVM). Following this treatment, the nuclear maturation index was evaluated. All embryos degenerated following this procedure. In the second set of experiments, oocytes were maintained for 6 or 12 h in pre-IVM with the following three treatments: ROS (25 µM or 12.5 µM), BL-I (100 µM or 50 µM) or a combination of both drugs (6.25 µM ROS and 12.5 µM BL-I). Oocytes were cultivated for 18 or 12 h in IVM. When a meiosis-inducing agent was used during pre-IVM for 24 h, more degenerated oocytes were observed at the end of the IVM period. This effect decreased when the meiotic blocking period was reduced to 6 or 12 h. No significant differences were observed in the blastocyst production rate of oocytes in pre-IVM for 6 h with ROS, BL-I, or ROS + BL-I compared with that of the control group (P > 0.05). However, inhibition of oocytes for 12 h resulted in decreased embryo production compared with that in the controls (P < 0.05). There was no difference in the post-vitrification embryo re-expansion rate between the study groups, showing that the meiotic inhibition for 6 or 12 h did not alter the embryo cryopreservation process.

Gene Expression in the Supporting Cells at the Onset of Meiosis in Rabbit Gonads

Subsequent to somatic gonadal sexual differentiation, germ cells enter meiosis or mitotic arrest in the ovary or testis, respectively. Among mice, these processes occur almost synchronically in fetal gonads and depend, among other factors, on the levels of retinoic acid (RA). In contrast to those in mice, rabbit germ cells enter meiosis or mitotic arrest after birth and coexist with proliferating germ cells. Here, we studied the somatic cell context in which germ cells enter meiosis or mitotic arrest in the rabbit. Using confocal immunofluorescence and real-time PCR, we studied the expression profiles of ALDH1A1 and ALDH1A2 and, comprising 2 genes required for RA synthesis, 2 meiosis markers STRA8 and SYCP3 as well as 2 genes involved in meiosis inhibition, CYP26B1 and NANOS2. We found that although both meiosis and mitotic arrest initiate after birth, these 2 processes are regulated in a way similar to the human fetal gonad. Current results reinforce the value of the neonatal rabbit gonad as an alternative experimental model for analyzing the direct effect of environmental factors during critical stages of germ cell establishment.

N-octanoylated ghrelin peptide inhibits bovine oocyte meiotic resumption

BackgroundStudies have shown that ghrelin plays an important role in the mammalian reproductive system, including the central, gonadal levels, and also during in vitro maturation of oocytes; however, the functions of ghrelin in bovine oocyte meiosis require further investigation. ObjectiveWe aimed to evaluate the effects of an n-octanoylated ghrelin peptide on oocyte meiotic resumption and the developmental competence of mature oocytes in vitro. Experimentaldesign: The expression of GHRL (encoding ghrelin) mRNA and its receptor (the growth hormone secretagogue receptor, GHSR) in the cumulus-oocyte complex (COCs), denuded oocytes (DOs), and cumulus cells (CCs) was assessed using quantitative real-time reverse transcription PCR (qRT-PCR), and the effects of the n-octanoylated ghrelin peptide on meiotic resumption were studied at four different doses (0, 10, 50, and 100 ng/mL) in a 6 h culture system. ResultsqRT-PCR analysis showed that GHRL and GHSR mRNAs were expressed in all tested samples; however, GHRL was predominantly expressed in DOs, and GHSR was predominantly expressed in CCs. Germinal vesicle breakdown was inhibited significantly by 50 ng/mL ghrelin compared with that in the negative control (P < 0.05). Further studies showed that n-octanoylated ghrelin increased the levels of cAMP and cGMP in the CCs and DOs, which inhibited the meiotic resumption of bovine oocytes. And the inhibitory role in the developmental competence of mature oocytes were also included, ghrelin could significantly improve the cleavage rate (P < 0.05) and blastocyst rate (P < 0.05). ConclusionN-octanoylated ghrelin maintained bovine oocytes meiotic arrest and further improved their developmental competence; therefore, n-octanoylated ghrelin could be considered as a potential pharmaceutical inhibitor of meiosis for the in vitro maturation of bovine oocytes.

Retinoic acid enhances germ cell differentiation of mouse skin-derived stem cells

BackgroundRetinoic acid (RA) signaling has been identified as a key driver in male and female gamete development. The presence of RA is a critical step in the initiation of meiosis and is required for the production of competent oocytes from primordial germ cells. Meiosis has been identified as a difficult biological process to recapitulate in vitro, when differentiating stem cells to germ cells. We have previously shown that primordial germ cell-like cells, and more advanced oocyte-like cells (OLCs), can be formed by differentiating mouse skin-derived stem cells. However, the OLCs remain unable to function due to what appears to be failure of meiotic initiation. The aim of this study was to determine the effect of RA treatment, during stem cell differentiation to germ cells, particularly on the initiation of meiosis.ResultsUsing qPCR we found significant increases in the meiosis markers Stra8 and Sycp3 and a significant reduction in the meiosis inhibitor Nanos2, in the differentiating populations. Furthermore, OLCs from the RA treated group, expressed significantly more of the meiosis regulatory gene Marf1 and the oocyte marker Oct4. At the protein level RA treatment was found to increase the expression of the gap junction protein CX43 and the pluripotency marker OCT4. Moreover, the expression of SYCP3 was significantly upregulated and the localization pattern better matched that of control fetal ovarian cells. RA treatment also improved the structural integrity of the OLCs produced by initiating the expression of all three zona pellucida transcripts (Zp1–3) and improving ZP3 expression levels and localization. Finally, the addition of RA during differentiation led to an almost two-fold increase in the number of OLCs recovered and increased their in vitro growth.ConclusionRA is a key driver in the formation of functioning gametes and its addition during stem cell to germ cell differentiation improves OLCs entry into meiosis.

Delaying meiotic resumption during transportation of bovine cumulus-oocyte complexes: effects on development, apoptosis and caspases activity of in vitro-produced embryos.

This study examined the effects of meiosis inhibition during bovine oocyte transportation on developmental competence and quality of produced embryos. The transportation medium was supplemented with: 100 μM butyrolactone I (BL), 500 μM IBMX + 100 μM forskolin (mSPOM), 100 μM milrinone (MR) or follicular fluid (bFF), and was carried out in a portable incubator for 6 h. Next, oocytes were in vitro matured (IVM) for 18 h, without the meiotic inhibitors, with the exception of mSPOM group, in which was added 20 μM cilostamide. The three control groups were IVM with 10% fetal calf serum (FCS) (Control Lab FCS) or 0.6% bovine serum albumin (BSA) (Control Lab BSA) in a CO2 in air incubator or in the portable incubator with 0.6% BSA (Control Transp BSA). Higher cleavage rates (P 0.05) to the other groups (23.6 ± 3.3% to 28.8 ± 2.7%). The total number of blastomeres was higher (P 0.05) from bFF (67.7 ± 4.2). No differences (P > 0.05) were found in apoptosis by the activity of caspases (139.0 ± 3.2 to 152.4 ± 6.5, expressed in fluorescence intensity) as well as the percentage of TUNEL-positive cells (12.3 ± 2.0% to 15.7 ± 1.7%). In conclusion, the transportation of oocytes over 6 h with BL, mSPOM or bFF enabled the acquisition of developmental competence at similar rates to the Control Lab FCS group.

Production of functionally sterile triploid Nibe croaker Nibea mitsukurii induced by cold-shock treatment with special emphasis on triploid aptitude as surrogate broodstock

Among the pelagic egg spawning marine teleosts, spermatogonial transplantation techniques were first developed in the Nibe croaker Nibea mitsukurii; however, production of donor-derived offspring had not yet been achieved. In salmonids, when triploid individuals were used as recipients, the resulting surrogate broodstock produced only donor-derived gametes, as the triploids were incapable of producing their own gametes. Therefore, in the present study, we determined the optimal parameters for cold-shock inducing triploidy through the suppression of meiosis II resulting in retention of the second polar body in fertilized Nibe croaker eggs. It was determined that cold-shock treatment, which was carried out at 10°C for a period of 15min beginning 5min after fertilization, following pre-shock treatment at 22°C achieved a 100% triploidization rate as well as hatching rates of which were high enough for practical application in the pelagic egg spawning marine teleost (16.4±10.4%). Average chromosome numbers of cold-shock treated larvae counted using mitotic figures found on the fin membrane of 1day post-hatch larvae were 67.3±0.7, which was approximately 1.5 times larger than that of control larvae (46.8±1.1), suggesting that triploidy had been induced. No differences in survival rates of hatched larvae during the rotifer-feeding stage existed between treated and non-treated groups. Gonadosomatic index and gametogenesis were impaired in both sexes of triploid Nibe croaker. Although triploids produced small amounts of gametes, no progeny from crosses between triploids and diploids survived later than 24h subsequent to hatching. These results indicated that the triploid Nibe croaker developed in this study could be effectively used as surrogate recipients in spermatogonial transplantation. Statement of relevanceIn intraperitoneal germ-cell transplantation techniques in salmonids, donor-derived diploid germ cells proliferated and differentiated within triploid recipient gonads, and the surrogate recipients produced only donor-derived gametes, which were capable of generating viable offspring when used in fertilization. To date, germ-cell transplantation techniques have been developed for use in perciforms, including sciaenids, however, triploid recipients have not yet been utilized in marine fishes. In this study, triploid Nibe croaker was produced at high triploidization rates (~100%) using cold-shock treatments. Analysis of the gonadal development and gametogenesis of triploid Nibe croaker in two consecutive spawning seasons revealed that the triploid Nibe croaker was functionally sterile in both sexes and that the triploids would be suitable for use as surrogate recipients.

Ameliorative Effect of Grape Seed Proanthocyanidin Extract on Cadmium-Induced Meiosis Inhibition During Oogenesis in Chicken Embryos.

Cadmium (Cd) is an environmental endocrine disruptor that has toxic effects on the female reproductive system. Here the ameliorative effect of grape seed proanthocyanidin extract (GSPE) on Cd-induced meiosis inhibition during oogenesis was explored. As compared with controls, chicken embryos exposed to Cd (3 µg/egg) displayed a changed oocyte morphology, decreased number of meiotic germ cells, and decreased expression of the meiotic marker protein γH2AX. Real time RT-PCR also revealed a significant down-regulation in the mRNA expressions of various meiosis-specific markers (Stra8, Spo11, Scp3, and Dmc1) together with those of Raldh2, a retinoic acid (RA) synthetase, and of the receptors (RARα and RARβ). In addition, exposure to Cd increased the production of H2 O2 and malondialdehyde in the ovaries and caused a corresponding reduction in glutathione and superoxide dismutase. Simultaneous supplementation of GSPE (150 µg/egg) markedly alleviated the aforementioned Cd-induced embryotoxic effects by upregulating meiosis-related proteins and gene expressions and restoring the antioxidative level. Collectively, the findings provided novel insights into the underlying mechanism of Cd-induced meiosis inhibition and indicated that GSPE might potentially ameliorate related reproductive disorders.

Effect of Temporary Meiotic Attenuation of Oocytes with Butyrolactone I and Roscovitine in Resistance to Bovine Embryos on Vitrification.

This study aimed to produce in vitro bovine embryos by the addition of two drugs, which is responsible for oocyte meiosis inhibition: roscovitine (ROS) and butyrolactone I (BL-I). Oocytes were recovered from slaughtered cows and matured in a commercial medium and maintained in a 5% CO2 atmosphere. Oocytes were maintained for 6 h in an in vitro maturation (IVM) medium containing ROS (12.5 μm), BL-I (50 μm) and association of drugs (ROS 6.25 μm and BL-I 25 μm). Oocytes were cultured for 18 h in an agent-free medium for the resumption of meiosis. After 24 h of maturation, oocytes were inseminated in the commercial in vitro fertilization (IVF) medium. Presumptive zygotes were cultured in SOFaa medium in a 5% CO2 atmosphere. On day 3, rate of cleavage was evaluated and on days 6 and 7, rate of blastocyst formation. BL-I and its association with the ROS increased the rates of cleavage and blastocyst formation (p < 0.05). The ROS alone was inefficient, impairing embryonic development, with low rates of blastocyst formation when compared to the control group and other treatments (p < 0.05). The embryos from BL-I and ROS+BL-I groups presented higher number of cells and lower rates of cellular apoptosis compared to other groups, either for the fresh or for post-thawing embryos. Embryos from ROS+BL-I group showed to be more resistant to the vitrification process, presenting a higher rate of embryonic re-expansion (p < 0.05). In conclusion, block of meiosis using BL-I or its association with ROS increased the rate of blastocyst formation, and the association of ROS+BL-I resulted in a better resistance to the embryo cryopreservation process.

Silica nanoparticles induce start inhibition of meiosis and cell cycle arrest via down-regulating meiotic relevant factors.

Silica nanoparticles have been shown to induce reproductive toxicity, but the mechanism is unknown. To investigate the toxic mechanism of SiNPs, 60 male mice were randomly divided into three groups: a control group, a saline group and a SiNPs group, with two evaluation time points (45 and 75 days after the first dose) per group. Mice in the SiNPs group were treated with SiNPs at a dose of 2.0 mg kg-1 every three days, a total of 15 times in 45 days, mice in the saline group were given the same volume of physiological saline, and the control group was treated with nothing. Then, half of the mice in each group were sacrificed for tissue samples on days 45 and 75. In vitro, GC-2spd cells were exposed to various concentrations of SiNPs for 24 h. The results showed that SiNPs damaged seminiferous epithelium, leading to a decrease in sperm quality and an increase in the sperm abnormality rate. Moreover, expressions of Sohlh1/cyclin A1/cyclin B1/CDK1/CDK2 were greatly down-regulated and the ROS level in the testicular tissue of the mice was significantly increased on day 45. However, these changes were reversed by day 75. In vitro, SiNPs induced G0/G1-phase cell cycle arrest and proliferation inhibition in GC-2spd cells. These results suggested that SiNPs might induce cell cycle arrest and inhibit cell proliferation by down-regulating expressions of meiotic regulators, whereas DNA damage caused by oxidative stress may be associated with meiosis and sperm production. In addition, damage to the male reproductive system caused by SiNPs may be reversible.

Gene expression in sheep cumulus-oocyte complexes meiotically inhibited with roscovitine

The majority of mammalian oocytes destined for in vitro maturation (IVM) have not undergone all molecular and structural changes necessary for competence acquisition to support the fertilization and early embryogenesis. In this context, different methods able to provide a transient arrest of meiosis resumption have been tested in order to improve the in vitro developmental potential of oocytes. Based on that, our study aimed to evaluate the effect of temporary meiosis inhibition using roscovitine on gene expression in sheep oocytes and cumulus cells. For this, cumulus-oocyte complexes (COCs) were cultured for 6h in modified TCM199 medium with (Rosco) and without (Control) 75μM roscovitine. Subsequently, they were in vitro matured for a further 18h in inhibitor-free TCM199 medium supplemented with gonadotropins. At 0, 6 and 24h of culture, nuclear status of oocytes and expression of selected genes were evaluated by Hoescht staining and qRT-PCR, respectively. The analysis of oocyte chromatin organization revealed that roscovitine efficiently inhibited the meiosis of sheep oocytes for 6h and its action was completely reversed after 18h of in vitro maturation in inhibitor-free medium. Besides, no detrimental effect on cumulus expansion was observed. The expression profile of most investigated genes in cumulus cells (PTX3, GREM1, GLUT1, PTGS2, ALK5, ALK6) and oocytes (ZAR1, NLRP5, SOD1, BMP15, GDF9) was similar between Control and Rosco treatments and the ratio BCL2/BAX was maintained in both cell types even in the presence of roscovitine. These results indicate that reversible meiotic arrest promoted by roscovitine, at the concentration and exposure time studied, neither impaired nor improved the expression of investigated genes in sheep oocytes and cumulus cells. Moreover, the efficiency of temporary meiotic arrest and the absence of deleterious effect on COCs suggest that roscovitine provides a useful method for transportation or manipulation of sheep oocytes at onset of maturation. However, further investigations are necessary to evaluate the benefits of roscovitine treatment on in vitro development of sheep embryos and its effects on cellular ultrastructure.

Endogenously produced hydrogen sulfide is involved in porcine oocyte maturation in vitro

Hydrogen sulfide, one of three known gasotransmitters, is involved in physiological processes, including reproductive functions. Oocyte maturation and surrounding cumulus cell expansion play an essential role in female reproduction and subsequent embryonic development. Although the positive effects of exogenous hydrogen sulfide on maturing oocytes are well known, the role of endogenous hydrogen sulfide, which is physiologically released by enzymes, has not yet been described in oocytes. In this study, we observed the presence of Cystathionine β-Synthase (CBS), Cystathionine γ-Lyase (CTH) and 3-Mercaptopyruvate Sulfurtransferase (3-MPST), hydrogen sulfide-releasing enzymes, in porcine oocytes. Endogenous hydrogen sulfide production was detected in immature and matured oocytes as well as its requirement for meiotic maturation. Individual hydrogen sulfide-releasing enzymes seem to be capable of substituting for each other in hydrogen sulfide production. However, meiosis suppression by inhibition of all hydrogen sulfide-releasing enzymes is not irreversible and this effect is a result of M-Phase/Maturation Promoting Factor (MPF) and Mitogen-Activated Protein Kinase (MAPK) activity inhibition. Futhermore, cumulus expansion expressed by hyaluronic acid (HA) production is affected by the inhibition of hydrogen sulfide production. Moreover, quality changes of the expanded cumuli are indicated. These results demonstrate hydrogen sulfide involvement in oocyte maturation as well as cumulus expansion. As such, hydrogen sulfide appears to be an important cell messenger during mammalian oocyte meiosis and adequate cumulus expansion.