Degradation meets development: Implications in ß-cell development and diabetes.

Pancreatic development is orchestrated by timely synthesis and degradation of stage-specific transcription factors (TFs). The transition from one stage to another stage is dependent on the precise expression of the developmentally relevant TFs. Persistent expression of particular TF would impede the exit from the progenitor stage to the matured cell type. Intracellular protein degradation-mediated protein turnover contributes to a major extent to the turnover of these TFs and thereby dictates the development of different tissues. Since even subtle changes in the crucial cellular pathways would dramatically impact pancreatic ß-cell performance, it is generally acknowledged that the biological activity of these pathways is tightly regulated by protein synthesis and degradation process. Intracellular protein degradation is executed majorly by the ubiquitin proteasome system (UPS) and Lysosomal degradation pathway. As more than 90% of the TFs are targeted to proteasomal degradation, this review aims to examine the crucial role of UPS in normal pancreatic ß-cell development and how dysfunction of these pathways manifests in metabolic syndromes such as diabetes. Such understanding would facilitate designing a faithful approach to obtain a therapeutic quality of ß-cells from stem cells.

Use of sensitivity-enhanced nuclear magnetic resonance spectroscopy equipped with a 1.7-mm cryogenically cooled micro-coil probe in identifying human sperm intracellular metabolites.

CONTEXT: The clinical value of human sperm metabolites has not been established due to the technical complexity in detecting these metabolites when sperm numbers are low. AIMS: To detect endogenous intracellular metabolites in fresh and post-thaw human spermatozoa using 800MHz nuclear magnetic resonance (NMR) spectroscopy equipped with a 1.7-mm cryo-probe. METHODS: Processed spermatozoa from 25 normozoospermic ejaculates were subjected to extraction of intracellular metabolites and then profiled by sensitivity-enhanced NMR spectroscopy equipped with a 1.7-mm cryogenically cooled micro-coil probe. In parallel, some of the processed sperm fractions were subjected to freeze-thawing and were then analysed for intracellular metabolites. KEY RESULTS: Twenty-three metabolites were profiled from only 1.25million sperm cells. Comparison of the metabolomic signature of pre-freeze and post-thaw sperm cells did not show significant changes in the levels of metabolites. CONCLUSIONS: Sensitivity-enhanced NMR spectroscopy equipped with a 1.7-mm cryogenically cooled micro-coil probe is a potential tool for identifying intracellular metabolites when sperm number is low. IMPLICATIONS: Use of sensitivity-enhanced NMR spectroscopy opens up the opportunity to test for endogenous metabolites in samples with a limited number of spermatozoa, to understand the patho-physiology of infertility.

Anti-tuberculosis drugs used in a directly observed treatment short course (DOTS) schedule alter endocrine patterns and reduce the ovarian reserve and oocyte quality in the mouse.

CONTEXT: Tuberculosis is one of the major infectious diseases, with people of reproductive age group having a high risk of infection. AIMS: The present study was designed to understand the consequences of anti-tuberculosis drugs (ATDs) used in DOTS (directly observed treatment short course) schedule on ovarian function. METHODS: Adult female Swiss albino mice were orally administered with combinations of ATDs used in the DOTS schedule every day for 4weeks. At 2weeks after the cessation of ATDs administration, the endocrine changes and ovarian function were assessed in mice. KEY RESULTS: Administration of ATDs to mice resulted in a prolonged estrous cycle, reduced ovarian follicle reserve, alteration in FSH, LH, and progesterone level, and decreased the number of ovulated oocytes. Further, the degree of fragmentation, degeneration, abnormal distribution of cytoplasmic organelles, abnormal spindle organisation, and chromosomal misalignment were higher in oocytes that were ovulated following superovulation. Blastocysts derived from ATDs treated mice had significantly lower total cell numbers and greater DNA damage. A marginal increase in the number of resorbed fetuses was observed in all the ATDs treated groups except in the multidrug resistance treatment group. Male progeny of ATDs treated mice had decreased sperm count and lower progressive motility, while female progeny exhibited a non-significant reduction in the number of oocytes ovulated. CONCLUSIONS: Theresults of this study suggest that ATDs can have significant adverse effects on the ovarian reserve, cytoplasmic organisation of oocytes, and can potentially cause transgenerational changes. IMPLICATIONS: The findings of the present study indicate ovarian toxicity of ATDs and warrant further research in the direction of identifying alternate drugs with minimal toxicity, and strategies to mitigate the ovarian toxicity induced by these drugs.

Ionic Liquids Assisted Topical Drug Delivery for Permeation Enhancement: Formulation Strategies, Biomedical Applications, and Toxicological Perspective.

Topical drug delivery provides several benefits over other conventional routes by providing localizing therapeutic effects and also avoids the gastrointestinal tract circumventing the first-pass metabolism and enzymatic drug degradation. Being painless, the topical route also prevents the difficulties linked with the parenteral route. However, there are limitations to the current topical systems which necessitate the need for further research to find functional excipients to overcome these limitations. This review deals in depth with the ionic liquids concerning their physicochemical properties and applicability as well as their role in the arena of topical drug delivery in permeation enhancement, bioavailability enhancement of the drugs by solvation, and drug moiety modification. The review gives a detailed insight into the recent literature on ionic liquid-based topical formulations like ionic liquid-based emulsions, active pharmaceutical ingredient-ionic liquids, ionic liquid-based bacterial cellulose membranes, topical small interfering RNA (siRNA) delivery, and ionogels as a possible solutions for overcoming the challenges associated with the topical route. This review also takes into account the toxicological aspects and biomedical applications of ionic liquids.

Curcumin nanocrystals attenuate cyclophosphamide-induced testicular toxicity in mice.

The cancer therapy using cyclophosphamide (CP) has been associated with adverse effects on the testicular function that raises concerns about the future fertility potential among cancer survivors. Curcumin, a polyphenol, has shown to possess a plethora of biological functions including tissue protective effects. In the present study, we investigated the protective effects of curcumin nanocrystals (NC) in mitigation of CP-induced testicular toxicity. Healthy adult (8-10 week) and prepubertal (2 week) male Swiss albino mice were injected with a single dose of CP (200 mg/kg) intraperitoneally (i.p). NC (4 mg/kg, i.p.) was administered every alternate day, for 35 days in adult mice while, a single dose of NC was injected intraperitoneally to prepubertal mice 1 h prior to CP. Administration of multiple doses of NC ameliorated CP-induced testicular toxicity in adult mice, which was evident from the improved sperm functional competence, sperm chromatin condensation, seminiferous tubule architecture and decreased apoptosis in testicular cells. Further, administration of NC 1 h prior to CP in prepubertal mice modulated the expression of genes pertaining to proliferation, pluripotency, DNA damage and DNA repair in spermatogonial cells at 24 h after the treatment. Overall, these results suggest that NC could be a promising chemoprotective agent, which can have potential application in male fertility preservation.

Nanoconstructs as a versatile tool for detection and diagnosis of Alzheimer biomarkers.

The current review focuses towards the advancements made in the past decade in the field of nanotechnology for the early Alzheimer's disease (AD) diagnosis. This review includes the application of nanomaterials and nanosensors for the early detection of the main AD biomarkers (amyloid beta, phosphorylated tau, apolipoprotein E4 allele or APOE4, microRNAs, cholesterol, hydrogen peroxide etc) in biological fluids, to detect the biomarkers at a very low concentration ranging in pico, femto and even atto molar concentrations. The field of drug development has always aimed and is constantly working on developing disease modifying drugs, but these drugs will only succeed when given in the early disease stages. Thus, developing efficient diagnostic tools is of vital importance. Various nanomaterials such as liposomes; dendrimers; polymeric nanoparticles; coordination polymers; inorganic nanoparticles such as silica, manganese oxide, zinc oxide, iron oxide, super paramagnetic iron oxides; quantum dots, silver nanoparticles, gold nanoparticles, and carbon based nanostructures (carbon nanotubes, graphene oxide, nanofibres, nanodiamonds, carbon dots); Up-conversion nanoparticles; 2D nanomaterials; and radioactive nanoprobes have been used in constructing and improving efficiency of nano-sensors for AD biosensing at an early stage of diagnosis.

Organophosphorus pesticide quinalphos (Ekalux 25 E.C.) reduces sperm functional competence and decreases the fertilisation potential in Swiss albino mice.

Quinalphos (QP) is one of the most commonly used organophosphate pesticide for agriculture. In this study, adult Swiss albino male mice were orally administered with 0.25, 0.5 and 1.0 mg/kg of QP (Ekalux 25 E.C.) for ten consecutive days and the reproductive function was assessed at 35 and 70 days after QP treatment. At highest dose (1.0 mg/kg), QP exposure resulted in significant decrease in motility and increase in sperm head defects and DNA damage. Pharmacokinetic data showed a threefold increase in concentration of QP in the testis as compared to serum. QP was detectable in testes even after 24 hr of administration indicating slow clearance from tissue. In addition, high oestradiol, low testosterone level with a parallel increase in aromatase and cytochrome P450 transcript levels was observed. Significant decrease in fertilisation, lower blastocyst rate and poor blastocyst quality was observed when spermatozoa collected from QP exposed mice were subjected to in vitro fertilisation. In conclusion, exposure of QP to male mice decreases the sperm functional competence and fertilising ability, which appears to be mediated through elevated oxidative stress and altered steroidogenesis in testes.

Fertility preservation during the COVID-19 pandemic: mitigating the viral contamination risk to reproductive cells in cryostorage.

Reopening fertility care services across the world in the midst of a pandemic brings with it numerous concerns that need immediate addressing, such as the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on the male and female reproductive cells and the plausible risk of cross-contamination and transmission. Due to the novelty of the disease the literature contains few reports confirming an association of SARS-CoV-2 with reproductive tissues, gametes and embryos. Cryobanking, an essential service in fertility preservation, carries the risk of cross-contamination through cryogenic medium and thus calls for risk-mitigation strategies. This review aims to address the available literature on the presence of SARS-CoV-2 on tissues, gametes and embryos, with special reference to the possible sources of cross-contamination through liquid nitrogen. Strategies for risk mitigation have been extrapolated from reports dealing with other viruses to the current global crisis, for safety in fertility treatment services in general, and specifically for oncofertility.

High-fat diet leads to elevated lipid accumulation and endoplasmic reticulum stress in oocytes, causing poor embryo development.

The present study was designed to investigate the effect of diet-induced obesity on endoplasmic reticulum (ER) stress in oocytes. Swiss albino mice (3 weeks old) were fed with a high-fat diet (HFD) for 8 weeks. Oocytes were assessed for lipid droplet accumulation, oxidative stress, ER stress and their developmental potential invitro. High lipid accumulation (P<0.01) and elevated intracellular levels of reactive oxygen species were observed in both germinal vesicle and MII oocytes of HFD-fed mice (P<0.05 and P<0.01 respectively compared with control). Further, expression of the ER stress markers X-box binding protein 1 (XBP1), glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF4) and activating transcription factor 6 (ATF6) was significantly (P<0.001) higher in oocytes of the HFD than control group. Oocytes from HFD-fed mice exhibited poor fertilisation and blastocyst rates, a decrease in total cell number and high levels of DNA damage (P<0.01) compared with controls. In conclusion, diet-induced obesity resulted in elevated lipid levels and higher oxidative and ER stress in oocytes, which contributed to the compromised developmental potential of embryos.

Germinal stage vitrification is superior to MII stage vitrification in prepubertal mouse oocytes.

This study investigated if in vitro maturation (IVM) before or after vitrification would be more successful for prepubertal oocytes. To mimic prepubertal conditions in an experimental setup, oocytes were collected from healthy 14, 21 and 28day old Swiss albino mice. The germinal vesicle (GV) stage oocytes and in vitro matured MII oocytes were subjected to vitrification-warming. Both structural (meiotic spindle morphology, mitochondrial integrity, cortical granules) and functional (sperm zona binding, fertilization) characteristics were assessed in oocytes after warming. This study demonstrated that IVM was more detrimental to prepubertal oocytes than to young adults. Further, vitrification of the IVM oocytes resulted in an increase in the number of abnormal meiotic spindles, a change in the cortical distribution pattern, a reduction in sperm zona binding and the fertilization rate. Importantly, oocyte integrity was better when prepubertal oocytes were vitrified before, rather than after, IVM. The above observations support GV stage vitrification for prepubertal oocytes requiring fertility preservation. Understanding the mechanisms behind the differing outcomes for oocytes from immature females will help in refining current protocol, thereby retaining the oocytes' maximum structural and functional integrity Further investigation is necessary to determine whether human prepubertal oocytes also behave in a similar way. It is to be noted here, with great emphasis, that a major limitation of this study is that the oocytes' abilities were tested only until fertilisation, as a consequence of which the study cannot reveal the developmental potentials of the embryos beyond fertilisation.

Antidiabetic drug metformin affects the developmental competence of cleavage-stage embryos.

PURPOSE: Metformin is the most commonly prescribed drug in the management of metabolic disorders such as polycystic ovarian syndrome (PCOS) and gestational diabetes in women of reproductive age. Insulin-sensitizing effect of metformin helps in improving from PCOS features such as hyperandrogenism, anovulation, and infertility. However, its ability to cross placental barrier raises concern about safety of the drug on early embryonic development. In this study, we evaluated the effect of metformin on the ovarian function and embryo development. METHODS: Adult Swiss albino female mice were administered with metformin (0, 50, 100, and 200 mg/kg body weight) for 4 weeks and assessed for reproductive function and preimplantation embryo development. Further, effect of metformin (0, 10, 25, 50, 100, 250, and 500 µg/mL) exposure to 2-cell-stage embryos was tested under in vitro conditions. RESULTS: Metformin did not alter the body weight, blood glucose, ovarian weight, and follicular reserve. However, the early embryo development was significantly affected in mice treated with metformin in vivo at highest dose. Moreover, embryos which were exposed to metformin in vitro showed dose-dependent decline in blastocyst rate and hatching rate. Furthermore, at highest concentration of metformin (500 µg/mL), all the embryos were arrested at compaction stage. CONCLUSION: The study revealed that metformin affects the early embryonic development and raises concern about its use during conception.

Epigallocatechin-3-gallate (EGCG) protects the oocytes from methyl parathion-induced cytoplasmic deformities by suppressing oxidative and endoplasmic reticulum stress.

Methyl parathion (MP) is a commonly used organophosphorus insecticide in commercial farming. It is well known that MP exposure can affect the function of nervous, respiratory, cardiovascular and reproductive systems. In our previous report we have demonstrated that MP exposure results in poor oocyte maturation and defective embryo development which is mainly mediated through oxidative stress. The present investigation was designed to explore whether using a potent free radical scavenger like Epigallocatechin-3-gallate (EGCG) can help in reducing the detrimental effects of MP on the oocytes. For the study, germinal vesicle (GV) stage oocytes collected from the ovaries of adult Swiss albino mice were subjected to in vitro maturation (IVM) in the presence or absence of MP (100 µg/mL) and/or EGCG (0.25 µM). MP significantly reduced the nuclear maturation rate, and resulted in poor cytoplasmic organization which was evident from the altered distribution pattern of mitochondria, endoplasmic reticulum and abnormal spindle organization. These changes were associated with significant elevation in oxidative stress and expression of ER stress markers such as 78 kDa Glucose regulated protein (GRP78) as well as X-box binding protein-1 (XBP1) in the oocytes. Further, the oocytes exposed to MP had lower activation rate and developmental potential. Supplementation of EGCG during IVM not only improved the nuclear maturation rate but also reduced the cytoplasmic abnormalities. These beneficial effects appear to be due to mitigation of oxidative and ER stress in oocytes. In conclusion, results of our study indicate that EGCG can help in alleviating MP-induced oocyte abnormalities.

Sperm-mediated DNA lesions alter metabolite levels in spent embryo culture medium.

Paternal genetic alterations may affect embryo viability and reproductive outcomes. Currently it is unknown whether embryo metabolism is affected by sperm-mediated abnormalities. Hence, using a mouse model, this study investigated the response to paternally transmitted DNA lesions on genetic integrity and metabolism in preimplantation embryos. Spent embryo culture media were analysed for metabolites by nuclear magnetic resonance spectroscopy and embryonic genetic integrity was determined by terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay on embryonic Day 4.5 (E4.5). Metabolic signatures were compared between normally derived embryos (control) and embryos derived from spermatozoa carrying induced DNA lesions (SDL). SDL embryos showed a significant reduction in blastocyst formation on E3.5 and E4.5 (P<0.0001) and had an approximately 2-fold increase in TUNEL-positive cells (P<0.01). A cohort of SDL embryos showing delayed development on E4.5 had increased uptake of pyruvate (P<0.05) and released significantly less alanine (P<0.05) to the medium compared with the corresponding control embryos. On the other hand, normally developed SDL embryos had a reduced (P<0.001) pyruvate-to-alanine ratio compared with normally developed embryos from the control group. Hence, the difference in the metabolic behaviour of SDL embryos may be attributed to paternally transmitted DNA lesions in SDL embryos.

Supplementation of biotin to sperm preparation medium enhances fertilizing ability of spermatozoa and improves preimplantation embryo development.

PURPOSE: Motility of spermatozoa helps not only in planning the type of infertility treatment but also directly reflects the success rate in assisted reproductive technology (ART). Previously, biotin, a water-soluble vitamin, has been shown to increase the motility and longevity of cryopreserved human spermatozoa. The present study was designed to understand the molecular basis of the beneficial effects of presence of biotin in sperm wash medium on early embryo development. METHODS: The effect biotin supplementation to sperm wash medium on the sperm parameters were assessed in swim-up fraction of normozoospermic and asthenozoospermic ejaculates collected from infertile men. Fertilization and early embryo development was studied using Swiss albino mice. RESULTS: Even though both biotin and pentoxifylline (PTX) enhanced the motility of spermatozoa from normozoospermic and asthenozoospermic samples, biotin group exhibited higher in vitro survival. Using mouse model, we observed that presence of biotin or PTX in sperm wash medium improved the fertilization rate and blastocyst rate compared to control. Blastocysts from these groups had significantly higher total cell number (P < 0.01) and lower apoptotic index. In silico target prediction revealed that GTPase HRas (HRas), tyrosine-protein phosphatase nonreceptor type 1 (PTP1B), and glucokinase are the probable targets for biotin. Solution-state Nuclear Magnetic Resonance (NMR) studies confirmed that biotin interacts both with human HRas and PTP1B. CONCLUSION: Our results indicate that presence of biotin in sperm wash medium can improve the fertilization potential and preimplantation embryo development and can be considered as a safe alternate to PTX.

Sunscreen creams containing naringenin nanoparticles: Formulation development and in vitro and in vivo evaluations.

BACKGROUND: The aim of this study was to develop sunscreen creams containing polymeric nanoparticles (NPs) of naringenin for photoprotective and antioxidant effects. METHODS: Polymeric NPs of naringenin were prepared and optimized. The NPs were incorporated into sunscreen creams and evaluated for in vitro and in vivo skin retention. RESULTS: The optimized naringenin NPs showed a size of 131.2 nm, zeta potential -25.4 mV, and entrapment efficiency 32.45%. The absence of drug-excipient interaction was confirmed by Fourier transform infrared spectroscopy and differential scanning calorimetry. X-Ray diffraction analysis demonstrated the amorphization of naringenin in nanoparticles. Transmission electron microscopy showed the sphericity of the NPs with the size of <200 nm. Cytotoxicity assessment in HaCaT cells indicated non-toxic nature of naringenin NPs. In vitro skin permeation studies demonstrated that higher amount of naringenin permeated at the end of 12 hours (Q12 hours  = 184.03 ± 3.37 µg/cm2 ) and deposited in the skin (10.38 ± 0.48 µg/cm2 ) from NPs as compared to plain naringenin. Sunscreen creams (SC1-SC5) containing plain naringenin or NPs with/without nano-zinc oxide and nano-titanium dioxide were prepared and evaluated. Optimized cream (SC5) containing naringenin NPs showed highest SPF value and enhanced skin retention of naringenin in comparison with NPs in suspension form and other cream formulations. CONCLUSION: Optimized nanoparticulate sunscreen cream exhibited highest skin retention and negligible skin permeation of naringenin besides showing excellent SPF value.

Haploid parthenotes express differential response to in vitro exposure of ammonia compared to normally fertilized embryos.

In the present study, we assessed whether absence of paternal genome imparts any differential response in embryos to chemical stress such as ammonia. Parthenogenesis was induced in MII stage oocytes using 10 mM SrCl2 in M16 medium. Parthenotes and normally fertilized embryos at 2 cell stage were exposed to different concentrations of ammonia and cultured till blastocyst. Exposure of ammonia to normally fertilized embryos resulted in significant decrease in the developmental potential (p < 0.0001) and blastocyst quality (p < 0.001). Whereas, in parthenotes, even though lower concentrations of ammonia did not have any effect, at 200 µM concentration the blastocyst rate was two times higher than control. The baseline apoptotic index was higher in parthenotes compared to normally fertilized embryos, which further increased after ammonium exposure (p < 0.001). Unlike in normally fertilized embryos ammonia exposure altered the mitochondrial distribution pattern and lead to increased expression of Oct4, Nanog and Na+/K+ ion exchange channel, while the cytochrome C expression was downregulated. This indicates that haploidy and/or absence of paternal factors in the embryo results in differential tolerance to stress induced by ammonia.

Supplementing zinc oxide nanoparticles to cryopreservation medium minimizes the freeze-thaw-induced damage to spermatozoa.

The sperm DNA integrity post cryopreservation of human semen samples is one of the serious concerns in human infertility treatment. In the present study, the beneficial effects of zinc oxide nanoparticles in preserving the functional ability of spermatozoa was explored. Ejaculates of normozoospermic men cryopreserved along with Zinc oxide nanoparticles (ZnONPs) exhibited non-significantly higher percentage of total and progressive motility in frozen-thawed samples compared to control. The sperm chromatin damage and malondialdehyde (MDA) level was significantly lower in ZnONPs group (P < 0.01 and P < 0.05 respectively) and the spermatozoa's ability to undergo acrosome reaction was also unaltered. Fluorescence microscopy and High resolution transmission electron microscopy analysis demonstrated that the ZnONPs do not penetrate the membrane of spermatozoa but stay around the spermatozoa. In conclusion, the presence of ZnONPs during cryopreservation appears to be beneficial to the spermatozoa as they withstand freeze-thaw process competently better than control, without any adverse effect shown.

Sperm-derived factors enhance the in vitro developmental potential of haploid parthenotes.

Parthenotes are characterized by poor in vitro developmental potential either due to the ploidy status or the absence of paternal factors. In the present study, we demonstrate the beneficial role of sperm-derived factors (SDF) on the in vitro development of mouse parthenotes. Mature (MII) oocytes collected from superovulated Swiss albino mice were activated using strontium chloride (SrCl2) in the presence or absence of various concentrations of SDF in M16 medium. The presence of SDF in activation medium did not have any significant influence on the activation rate. However, a significant increase in the developmental potential of the embryos and increased blastocyst rate (P < 0.01) was observed at 50 µg/ml concentration. Furthermore, the activated oocytes from this group exhibited early cleavage and cortical distribution of cortical granules that was similar to that of normally fertilized zygotes. Culturing 2-cell stage parthenotes in the presence of SDF significantly improved the developmental potential (P < 0.05) indicating that they also play a significant role in embryo development. In conclusion, artificial activation of oocytes with SDF can improve the developmental potential of parthenotes in vitro.

Epigenetic changes in preimplantation embryos subjected to laser manipulation.

The advantage of using laser for assisted hatching in routine assisted reproductive technology (ART) practice is debatable. Recently, it has been shown that laser-manipulated mouse embryos had compromised genetic integrity. However, the impact of laser-assisted hatching (LAH) on the epigenetic integrity of the preimplantation embryos is not elucidated so far. Since continuous thermal stress on embryos was found to lower mRNA levels of de novo (bovine) methyl transferases in embryos, we hypothesize that thermal energy induced during LAH may alter the epigenetic signature through abnormal de novo methyl transferases (Dnmts) levels. Thus, using mouse model, we made an attempt to look into the expression of Dnmt3a and Dnmt3b in laser-manipulated embryos and their effects on global methylation. This experimental prospective study used mouse embryos from varying developmental stages (2-cell, 6-8-cell, and blastocyst) which were subjected to LAH using a 1480-nm diode laser. Two pulses of 350 µs frequency were applied to breach the zona pellucida, and then, embryos were assessed for the expression of two de novo methyl transferases (Dnmt3a and Dnmt3b) and LINE-1 (long interspersed element-1) methylation when LAH embryos developed to blastocyst stage. Results from this study have shown that blastocysts subjected to LAH at two-cell stage had significantly lower mRNA transcripts of Dnmt3a (P < 0.01) and Dnmt3b (P < 0.05) whereas LAH at six- to eight-cell and blastocyst stages did not affect the mRNA level significantly. On the other hand, LINE-1 methylation did not change significantly between LAH and control group in all the stages studied. These results suggest that two-cell-stage laser manipulation of embryos changes the mRNA level of Dnmts without affecting the global DNA methylation.

Distribution pattern of cytoplasmic organelles, spindle integrity, oxidative stress, octamer-binding transcription factor 4 (Oct4) expression and developmental potential of oocytes following multiple superovulation.

The aim of the present study was to determine the effects of repeated superovulation on oocyte quality and embryo developmental potential. Female Swiss albino mice were injected with 5IU pregnant mare's serum gonadotropin followed 48h by 10IU human chorionic gonadotropin. Mice were superovulated up to four times with a gap of 7 days between each superovulation cycle. Ovarian weight increased significantly with an increasing number of superovulation cycles. Although the first stimulation cycle resulted in a threefold increase in the number of oocytes, the number of oocytes decreased gradually after subsequent stimulations. Increased cytoplasmic fragmentation, abnormal mitochondrial distribution, aggregation of Golgi apparatus, spindle damage, increased intracellular oxidative stress and a decrease in expression of octamer-binding transcription factor 4 (Oct4) expression were observed in these oocytes. Further, embryos derived from mice subjected to multiple stimulation cycles exhibited a low blastocyst rate, decreased hatching rate and increased apoptosis in blastocysts. In conclusion, the present study demonstrates that repeated superovulation adversely affects mouse oocyte quality by altering the distribution of cytoplasmic organelles, increasing oxidative stress and decreasing Oct4 expression, resulting in poor developmental potential of the embryos.