Ameliorating Effect of Sodium Selenite on Developmental and Molecular Response of Bovine Cumulus-Oocyte Complexes Matured in Vitro Under Heat Stress Condition.

Selenium (Se), an essential trace element, plays an important role in the antioxidative defense mechanism, and it has been proven to improve fertility and reproductive efficiency in dairy cattle. The present study evaluated the potential protective action of Se supplement of in vitro maturation (IVM) media on the maturation and subsequent development of bovine cumulus-oocyte complexes (COCs) exposed to heat stress (HS). The treatment with Se improved the viability of cumulus cells (CCs) and oocytes (P < 0.05). The proportion of oocytes reached metaphase II (MII) and those arrested at metaphase I (MI) was greater and lower in treatment than control respectively (P < 0.05). Supplementation with Se increased the percentage of cleaved embryos, total blastocysts, and blastocyst/cleavage ratio (P < 0.05). Moreover, the upregulation of CCND1, SEPP1, GPX-4, SOD, CAT, and downregulation of GRP78, CHOP, and BAX in both Se-treated CCs and oocytes were recorded. The upregulation of NRF2 was detected in Se-treated CCs other than in oocytes, which showed upregulation of IGF2R and SOX-2 as the markers of quality as well. Se supplement in IVM media improved the viability, maturation, and the level of transcripts related to antioxidant defense and quality of heat-treated oocytes, which coincided with greater subsequent development outcomes. Se ameliorated the viability of CCs along with upregulation of antioxidative candidate gene expression and downregulation of apoptosis-related ones to support their protective role on restoring the quality of oocytes against compromising effects of HS.

Supplementation of media with gamma-oryzanol as a novel antioxidant to overcome redox imbalance during bovine oocyte maturation in vitro.

This study evaluated the effect of supplementing IVM media with γ-oryzanol (ORY), a nutraceutical derived from rice bran oil, on the development of bovine oocytes and hindering the compromising effect of redox imbalance. An in vitro model of the bovine cumulus-oocyte complex was used for the evaluation of nuclear maturation and development. Antioxidant activity was investigated by assessing the level of ROS (Reactive Oxygen Species) and GSH (glutathione) in oocytes and quantitative changes in gene expression in matured oocytes and their respective cumulus cells. ORY supplementation increased the proportion of MII oocytes, cleaved embryos, and total blastocysts (p < .05) and was linked to higher and lower levels of intracellular GSH and ROS, respectively (p < .05). The treated oocytes and their respective cumulus-granulosa cells showed a modulation in the expression of genes related to apoptosis (downregulation of BAX and CHOP) and oxidative stress (upregulation of NRF2, CAT, and SOD). Also, relative upregulation of OCT-4 and IGF2R in treated oocytes was concomitant with higher subsequent development in terms of cleavage and total blastocyst rates (p < .05). Based on our findings, it appears that ORY supplementation can improve the nuclear maturation and development of bovine oocytes into blastocysts and augment their enzymatic and non-enzymatic antioxidant systems, maintaining the Redox balance and high enzymatic activity against ROS generation.

Development and evaluation of an injectable slow-release progesterone formulation for estrus synchronization in ewes out of the breeding season.

This study was aimed at developing a type of slow-release progesterone micro-particles useable in a single intramuscular injection for estrus synchronization in non-breeding season ewes. A total of 66 ewes were randomly assigned into four groups: CIDR (n = 16): exposed to intravaginal CIDR for 12 days, and three experimental groups, i.e., T100 (n = 16), T150 (n = 17) and T200 (n = 17), receiving a single intramuscular injection of 100, 150 and 200 mg slow-release progesterone, respectively. Blood sampling was performed on all ewes at five different times, and the ELISA method measured progesterone levels. No significant differences were observed in progesterone levels among the groups in each sampling time. More than 90% of ewes in the CIDR, T100 and T150 groups and all those in T200 showed estrus behaviour, and the rate was not significantly different between groups. The difference in the mean interval from progesterone treatment to estrus was also insignificant. The parturition rate declined by increasing the dose of injected progesterone; although it was similar in CIDR and T100 groups, it decreased significantly in T150 and T200 . Since our injectable progesterone formulation was successful in the induction and synchronization of estrus in ewes out of the breeding season, it can be applied as an alternative to the conventional progesterone containing intravaginal devices.

The protective effects of astaxanthin on pre-antral follicle degeneration in ovine vitrified/warmed ovarian tissue.

This study assesses the protective effects of astaxanthin (AST) against vitrification/warming-induced cryoinjuries of ovarian tissue slices in sheep. Cortical slices of slaughterhouse acquired-ovine ovaries were randomly distributed in different groups: fresh, toxicity, and five vitrification groups including vitrification in presence of 0 (control group), 1, 10 and 100 µM astaxanthin or 100 µM vitamin E. After vitrification/warming and 24 h culturing, the samples were subjected to histological studies, antioxidant evaluation by TAC and TBAR assays, and assessment of relative expression of BMP4, BMP15, GDF9 and KITLG genes related to folliculogenesis and follicular growth regulation. According to the results, vitrification reduced the percentage of morphologically intact follicles compared to the fresh and toxicity groups (p < 0.05). In vitrification groups, vitamin E and all three concentrations of AST increased the percentage of intact pre-antral follicles and antioxidant activity relative to the vitrified control (p < 0.05). This enhancement significantly occurred in 10 µM AST group more than vitamin E (p < 0.05). Also, 10 µM concentration of AST enhanced the expression of all the examined genes compared to the control (p < 0.05), while the expression of BMP4, BMP15 and KITLG was higher in the AST than vitamin E (p < 0.05). The latter could increase only the expression of GDF9 compared to the control group (p = 0.011). In conclusion, AST is a highly effective antioxidant for maintaining the survival of pre-antral follicles, retaining cell density, increasing total antioxidant capacity, and increasing the expression of some genes related to follicular development after short-term culture of vitrified/warmed ovarian tissue slices.

The effect of quercetin in the maturation media on cumulus-granulosa cells and the developmental competence of bovine oocytes.

The present study was designed to investigate the effects of Quercetin on the developmental competence of bovine oocytes and cumulus-granulosa cells (CGs). Two groups of immature cumulus-oocyte complexes (COCs) were subjected to IVM with or without Quercetin. The viability, nuclear status, early and late apoptosis of oocytes and CGs were evaluated using gene expression analysis and staining methods. Embryonic development was assessed morphologically by recording Post-IVF survival, cleavage, and blastocyst rates. The proportion of oocytes reaching the MII stage was greater and the number of early-apoptotic oocytes was lower in the group matured with Quercetin compared to the Control (p < 0.05). Relative upregulation of OCT-4, IGF2R and Bcl-2, and downregulation of CHOP was seen in treated oocytes. Also, downregulation of Bax and upregulation of Glut-4 was detected in treated CGs. The treated oocytes experienced higher post-IVF survival and cleavage rates compared to the untreated group (p < 0.05); more cleaved embryos reached ≥16-cell stage and blastocyst at days 4th and 7th respectively. In addition, total blastocyst rate was significantly improved. It is concluded that supplementing maturation media with Quercetin can enhance the quality of bovine oocytes and endow them with protective potential against early apoptotic damage possibly through CHOP regulation of BCL2 gene family, triggering expression of a gene in CGs to maintain the intactness of oocyte against apoptotic signals and providing oocytes with more energy substrates. It also boosts the subsequent development of oocyte to blastocyst and improves the efficacy of bovine embryo production in vitro.

Ram epididymal sperm frozen in an extender containing ethylene glycol have higher post-thaw longevity and in vitro fertility.

BACKGROUND: Establishing an efficient, simple and inexpensive method for freezing ram epididymal sperm so that the quality and fertility of spermatozoa could be maintained for a longer period after thawing is of great practical value. OBJECTIVES: To optimize freezing and thawing protocol for ram epididymal sperm using either ethylene glycol (EG) or glycerol (GLY) as cryoprotectants (CPAs). Then, to evaluate the post-thaw longevity and in vitro fertility of spermatozoa that were frozen and thawed according to the optimized protocol. MATERIALS AND METHODS: At first, an optimum protocol for freezing and thawing sperm using EG or GLY were investigated, and the next experiments were performed using the spermatozoa that had been frozen and thawed according to the optimized protocol for each CPA. In the next experiments, frozen-thawed and fresh sperm were diluted in an isotonic culture medium and subsequently incubated at 39°C for 4 h. The motility characteristics and functional membrane integrity (FMI) of spermatozoa were evaluated after thawing, after dilution (t0 ), and after incubation (t4 ). The in vitro fertility of the spermatozoa was assessed at t0 and t4 . RESULTS: For both CPAs, the highest motility parameters and FMI was found for spermatozoa frozen at 3 cm above LN2 and thawed at 50 and 65°C (P < 0.05). In comparison to the spermatozoa of GLY group, the spermatozoa of the EG group had higher total and progressive motility at t0 , as well as higher FMI, total and progressive motility, and linearity at t4 (P < 0.05). Fertility of frozen-thawed sperm was higher than that of fresh sperm at t0 (P < 0.05). Incubation treatment increased the fertility of fresh sperm while decreased the fertility of frozen-thawed sperm, and this decline was more severe in GLY than in the EG group. CONCLUSION: Based on the findings, EG can be a more suitable CPA for freezing ram epididymal sperm.

Quercetin effect on the efficiency of ovine oocyte vitrification at GV stage.

The widely adopted method of vitrification is known to induce some negative effects on oocytes. In order to enhance the efficiency of this process performed on ovine oocytes at germinal vesicle stage, vitrification and warming (VW) solutions, and maturation media were supplemented with 5 µM Quercetin (Q). Four groups of vitrified and fresh immature oocytes were subjected to IVM, IVF and IVC, and their survival rate, apoptosis, nuclear status and developmental competence were assessed. Non-vitrified oocytes treated with Quercetin experienced higher cleavage rate relative to those matured without Quercetin (p < 0.05). Supplementation of VW and maturation media with Quercetin resulted in increased survival, cleavage and total blastocyst rates relative to the untreated oocytes. The post-IVM survival rate of non-vitrified oocytes showed no difference among those matured with and without Quercetin, but was higher for oocytes vitrified, warmed and matured with Quercetin relative to VW group lacking Quercetin. The proportion of early-apoptotic (AV+) oocytes was affected by Quercetin supplementation in both control and VW groups (p < 0.05). The number of AV positive oocytes was lower and the proportion of oocytes reaching MII stage was greater in non-vitrified and VW groups matured with Quercetin, in comparison with their untreated respective controls (p < 0.05). There was no difference in the number of late-stage apoptotic oocytes among different groups. It is concluded that supplementing vitrification and warming solutions with Quercetin endows vitrified ovine oocytes with protective potential against early apoptotic damage, and improves viability, maturation rate and developmental competence at GV stage.

Evaluation of the efficiency of TaqMan duplex real-time PCR assay for non-invasive pre-natal assessment of foetal sex in equine.

Accurate diagnosis of foetal sex in pregnant mare is helpful for many breeders, both for private or commercial purposes. In this study, in order to pre-natal foetal sexing in equine, we used TaqMan duplex real-time PCR to detect the specific regions of SRY and TSPY genes on extracted cell-free foetal DNA from maternal blood. Peripheral blood samples from 50 pregnant Arabian mares with singleton foetuses were collected. Cell-free foetal DNA was extracted from maternal plasma, and duplex real-time PCR assays were performed with TaqMan probes and primers. Amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as control of DNA extraction procedure. From the 50 sampled mares, 28 cases had female and 22 mares had male foetuses. The final results for 46 samples were conclusive, and from them, 43 cases were predicted correctly. Sensitivity, specificity and accuracy of the test were 90.48%, 96% and 93.48%, respectively. In conclusion, a TaqMan duplex real-time PCR was set up to pre-natal detection of foetal sex in equine. The method was fast and decreased the false-positive and false-negative results. The technique can be used as a routine procedure in farms by collecting only a blood sample.

The Impact of Educational Intervention on the Anxiety of Family Caregivers of the Elderly with Dementia: A Randomized Controlled Trial.

BACKGROUND: Due to the increasing population of elderly and the consequent increase in the number of chronic diseases such as dementia, the psychological complications such as anxiety in the family caregivers increase. The aim of this study was to determine the effect of educational intervention on the anxiety of family caregivers of the elderly people with dementia. METHODS: This randomized controlled trial was performed in the elderly Neurology Clinics in Shiraz from May to August 2017. This study was conducted on 70 families with elderly people with dementia who were randomly divided into an intervention (receiving in groups for seven sessions of educational intervention) and a control group (Conventional care). Data collection tool was Spielberger's Anxiety Inventory (40-items, score=20-80). Data were analyzed by SPSS version 21 using ANOVA test with repeated measures and independent t-test. RESULTS: One and three months after the interventions, the mean scores of anxiety in the intervention group were 70.51±3.78 and 70.31±3.43 and in the control groups they were 76.45±3.45 and 76.22±5.08 respectively. The results showed significant differences between the two groups regarding anxiety after the intervention (P<0.001). CONCLUSION: Educational programs held to promote and maintain the physical and mental health of caregivers could reduce the anxiety of family caregivers of the elderly with dementia.Trial Registration Number: IRCT2017080915426N4.

Effects of cryopreservation on stallion sperm protamine messenger RNAs.

Protamines substitute DNA-binding histones during late spermatogenesis in sperm nucleus. Stallion sperm contains all three variants of these arginine-rich and positively charged nuclear proteins (P1, P2 and P3). Two variants of protamine-2, that is P2 and P3, constitute approximately 15% of the entire protamine content. Also, the ratio of protamine-1 to protamine-2 varies among different mammalian species, and abnormal protamine ratios and protamine content are correlated with male infertility. In this study, changes in protamine mRNA abundance for all three protamines were investigated in stallion sperm during cryopreservation. Twelve ejaculates were collected from six sexually mature stallions. Sperm samples were divided into two parts for total mRNA extraction: one as fresh and the other as cryopreserved sample. Levels of three protamine transcripts were determined by real-time reverse transcriptase polymerase chain reaction. Results of relative expression showed that cryopreservation can significantly alter protamine transcripts: protamine 2 was downregulated, while protamine 3 was upregulated in cryopreserved samples relative to the control. Changes in protamine 1 were not significant after cryopreservation. This study is the first to evaluate changes in mRNA abundance of protamine genes in stallion sperm following cryopreservation. Such evaluations are important in finding transcriptomic markers for success in fertilization and assisted reproductive techniques.

Sericin Ameliorates the Capacitation State and Chromatin Integrity of Frozen-Thawed Stallion Spermatozoa by Reducing Oxidative Stress.

BACKGROUND: In the process of sperm cryopreservation, apart from cryoinjury, the production of Reactive Oxygen Species (ROS) can adversely affect the integrity of chromatin and cellular membranes. Addition of natural antioxidants to freezing medium is an approach to reduce the destructive effects of ROS on sperm. METHODS: In this study, during 60 min of cooling process, the ejaculates of five stallions were diluted in the following media: INRA 82 medium as Control (C), INRA 82 medium supplemented with 0.25% Sericin (S), INRA 82 medium supplemented with 1.5 mM Glutathione (G), and INRA 82 medium supplemented with 0.25% Sericin+1.5 mM Glutathione (S+G). RESULTS: In the frozen/thawed sericin supplemented group, while the integrity of DNA and the activity of catalase and Glutathione Peroxidase (GPx) were increased, the lipid peroxidation and midpieceab normality decreased, compared with other groups (p<0.05). The proportions of sperms with abnormal head in group S and the sperm with distal droplet in G and S+G groups decreased, compared with group C (p<0.05). In CTC assay, the percentage of capacitated spermatozoa in treatment groups was lower than control (p<0.01). CONCLUSION: In conclusion, the presence of sericin in freezing medium of stallion semen could improve sperm DNA integrity and its resistance to ROS and lipid peroxidation.

[Corrigendum] Antiproliferative effects of imatinib mesylate on ZR­75­1 and MDA­MB­231 cell lines via PDGFR­ß, PDGF­BB, c­Kit and SCF expression.

An interested reader drew to our attention that the above study appeared to contain a high level of overlap with an article by the same authors published in the journal Drug Design, Development and Therapy [Kadivar A, Kamalidehghan B, Akbari Javar H, Karimi B, Sedghi R and Noordin MI: Antiproliferation effect of imatinib mesylate on MCF7, T­47D tumorigenic and MCF 10A nontumorigenic breast cell lines via PDGFR­ß, PDGF­BB, c­Kit and SCF genes. Drug Des Devel Ther 11: 469­481, 2017]. Following an internal investigation and also in liaison with the authors, it was established that, although the studies were conducted along broadly similar lines, the papers contained entirely different data involving two different subsets of cell lines; the submission to Drug Des Devel Ther aimed to explore the effects of imatinib mesylate on three different groups, with each group being represented by a cell line, whereas the submission to Int J Mol Med explored the effectiveness of imatinib mesylate in breast cancer cell lines. In spite of this, considering the relatedness of the articles and the fact that the paper to Drug Des Devel Ther was submitted first and published while the Int J Mol Med paper was passing through the peer­review process, the authors concede that they should have properly referenced their paper submitted to Drug Des Devel Ther in the Int J Mol Med paper. Note that the publishers of Drug Des Devel Ther, with whom we were liaising, agreed with the decision to issue a Corrigendum for this paper that acknowledges the article published in Drug Des Devel Ther. The authors regret their failure to acknowledge the related paper in this instance, and apologize to the readership for this oversight. [the original article was published in International Journal of Molecular Medicine 14: 414­424, 2018; DOI: 10.3892/ijmm.2018.3590].

Antiproliferative effects of imatinib mesylate on ZR­75­1 and MDA­MB­231 cell lines via PDGFR­ß, PDGF­BB, c­Kit and SCF expression.

Imatinib mesylate is an anti­neoplastic targeted chemotherapeutic agent, which can inhibit tyrosine kinase receptors, including BCR­ABL, platelet­derived growth factor receptors (PDGFRs) and c­Kit. Cellular processes, including differentiation, proliferation and survival are regulated by these receptors. The present study aimed to evaluate the antiproliferative effects of imatinib mesylate, and its effects on apoptotic induction and cell cycle arrest in breast cancer cell lines. In addition, the study aimed to determine whether the effects of this drug were associated with the mRNA and protein expression levels of PDGFR­ß, c­Kit, and their corresponding ligands PDGF­BB and stem cell factor (SCF), which may potentially modulate cell survival and proliferation. To assess the antiproliferative effects of imatinib mesylate, an MTS assay was conducted following treatment of cells with 2­10 µM imatinib mesylate for 96, 120 and 144 h; accordingly the half maximal inhibitory concentration of imatinib mesylate was calculated for each cell line. In addition, the proapoptotic effects and cytostatic activity of imatinib mesylate were investigated. To evaluate the expression of imatinib­targeted genes, PDGFR­ß, c­Kit, PDGF­BB and SCF, under imatinib mesylate treatment, mRNA expression was detected using semi­quantitative polymerase chain reaction and protein expression was detected by western blot analysis in ZR­75­1 and MDA­MB­231 breast carcinoma cell lines. Treatment with imatinib mesylate suppressed cell proliferation, which was accompanied by apoptotic induction and cell cycle arrest in the investigated cell lines. In addition, PDGFR­ß, PDGF­BB, c­Kit and SCF were expressed in both breast carcinoma cell lines; PDGFR­ß and c­Kit, as imatinib targets, were downregulated in response to imatinib mesylate treatment. The present results revealed that at least two potential targets of imatinib mesylate were expressed in the two breast carcinoma cell lines studied. In conclusion, the antiproliferative, cytostatic and proapoptotic effects of imatinib mesylate may be the result of a reduction in the expression of c­Kit and PDGFR tyrosine kinase receptors, thus resulting in suppression of the corresponding ligand PDGF­BB. Therefore, imatinib mesylate may be considered a promising target therapy for the future treatment of breast cancer.

Antiproliferation effect of imatinib mesylate on MCF7, T-47D tumorigenic and MCF 10A nontumorigenic breast cell lines via PDGFR-ß, PDGF-BB, c-Kit and SCF genes.

Recent cancer molecular therapies are targeting main functional molecules to control applicable process of cancer cells. Attractive targets are established by receptor tyrosine kinases, such as platelet-derived growth factor receptors (PDGFRs) and c-Kit as mostly irregular signaling, which is due to either over expression or mutation that is associated with tumorigenesis and cell proliferation. Imatinib mesylate is a selective inhibitor of receptor tyrosine kinase, including PDGFR-ß and c-Kit. In this research, we studied how imatinib mesylate would exert effect on MCF7 and T-47D breast cancer and MCF 10A epithelial cell lines, the gene and protein expression of PDGFR-ß, c-Kit and their relevant ligands platelet-derived growth factor (PDGF)-BB and stem cell factor (SCF). The MTS assay was conducted in therapeutic relevant concentration of 2-10 µM for 96, 120 and 144 h treatment. In addition, apoptosis induction and cytostatic activity of imatinib mesylate were investigated with the terminal deoxynucleotidyl transferase dUTP nick end labeling TUNEL and cell cycle assays, respectively, in a time-dependent manner. Comparative real-time PCR and Western blot analysis were conducted to evaluate the expression and regulation of imatinib target genes and proteins. Our finding revealed that imatinib mesylate antiproliferation effect, apoptosis induction and cytostatic activity were significantly higher in breast cancer cell lines compared to MCF 10A. This effect might be due to the expression of PDGFR-ß, PDGF-BB, c-Kit and SCF, which was expressed by all examined cell lines, except the T-47D cell line which was not expressed c-Kit. However, examined gene and proteins expressed more in cancer cell lines. Therefore, imatinib mesylate was more effective on them. It is concluded that imatinib has at least two potential targets in both examined breast cancer cell lines and can be a promising drug for targeted therapy to treat breast cancer.

Peroxisome proliferator-activated receptors (PPARα, PPARγ and PPARß/δ) gene expression profile on ram spermatozoa and their relation to the sperm motility.

Peroxisome proliferator-activated receptors (PPARs) are a member of nuclear receptors superfamily, which mainly regulate the expression of target genes involved in lipid and energy metabolism. These receptors are divided to three isotypes: PPARα, PPARγ and PPARß/δ. Each isotype has a distinct tissue distribution relating to the distinct functions. In this study, the mRNA abundance for PPARα, PPARγ and PPARß/δ was evaluated and compared with high and low motile ram spermatozoa. Semen samples from 6 adult rams were fractionated on a two layer discontinuous Percoll gradient to high and low motile sperm and quantitative parameters of sperm motility were determined by CASA. Total RNA was extracted and the mRNA abundance for each gene was measured by relative quantification technique with Real time PCR. The levels of three isotypes of PPAR transcripts were significantly higher in high motile semen samples using quantitative RT-PCR. Some of sperm motility indices were also significantly correlated with PPARα and PPARγ relative expression. This study revealed the novel association of PPAR gene isotypes with sperm motility. Data from our study suggested PPARs are one of the possible factors that can be studied in male infertility.

Correlation of Adiponectin mRNA Abundance and Its Receptors with Quantitative Parameters of Sperm Motility in Rams.

BACKGROUND: Adiponectin and its receptors (AdipoR1 and AdipoR2), known as adiponectin system, have some proven roles in the fat and glucose metabolisms. Several studies have shown that adiponectin can be considered as a candidate in linking metabolism to testicular function. In this regard, we evaluated the correlation between sperm mRNA abundance of adiponectin and its receptors, with sperm motility indices in the present study. MATERIALS AND METHODS: In this completely randomized design study, semen samples from 6 adult rams were fractionated on a two layer discontinuous percoll gradient into high and low motile sperm cells, then quantitative parameters of sperm motility were determined by computer-assisted sperm analyzer (CASA). The mRNA abundance levels of Adiponectin, AdipoR1 and AdipoR2 were measured quantitatively using real-time reverse transcriptase polymerase chain reaction (qRT-PCR) in the high and low motile groups. RESULTS: Firstly, we showed that adiponectin and its receptors (AdipoR1 and AdipoR2) were transcriptionally expressed in the ram sperm cells. Using Pfaff based method qRT- PCR, these levels of transcription were significantly higher in the high motile rather than low motile samples. This increase was 3.5, 3.6 and 2.5 fold change rate for Adiponectin, AdipoR1 and AdipoR2, respectively. Some of sperm motility indices [curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), wobble (WOB) and straightness (STR)] were also significantly correlated with Adiponectin and AdipoR1 relative expression. The correlation of AdipoR2 was also significant with the mentioned parameters, although this correlation was not comparable with adiponectin and AdipoR1. CONCLUSION: This study revealed the novel association of adiponectin system with sperm motility. The results of our study suggested that adiponectin is one of the possible factors which can be evaluated and studied in male infertility disorders.

Connexin-43: A possible mediator of heat stress effects on ram Sertoli cells.

Sertoli cells are an essential group of cells in seminiferous epithelium which provide nutritional and structural supports for spermatogenic cells via cell junctions. In this study, the gene expression of connexin-43, the most abundantly distributed gap junction protein of cells, was investigated in ram Sertoli cells under mild and severe heat stresses with real-time quantitative PCR. Sertoli cells were isolated from testes of 10 lambs. After culture and 3 passages, they were treated with mild (39 ˚C) and severe (42 ˚C) heat stress for 6 hr. The results showed a significant reduction in the percentage of live cells under severe heat stress compared to the control group (32 ˚C), (p <0.05). Relative quantification analysis revealed significantly higher (3.80 fold increase) values of connexin-43 transcripts in severely heat stressed group than control group (p <0.05). It is concluded that challenging Sertoli cells with 42 ˚C heat could threaten their survival, and overexpression of connexin-43 may cause dysfunction of Sertoli cells due to heat stress. These findings can be useful to identify the molecular mechanisms involved in adverse effects of heat stress on male reproduction and enhance our understanding of its pathogenesis.

Formulation and in vitro, in vivo evaluation of effervescent floating sustained-release imatinib mesylate tablet.

INTRODUCTION: Imatinib mesylate is an antineoplastic agent which has high absorption in the upper part of the gastrointestinal tract (GIT). Conventional imatinib mesylate (Gleevec) tablets produce rapid and relatively high peak blood levels and requires frequent administration to keep the plasma drug level at an effective range. This might cause side effects, reduced effectiveness and poor therapeutic management. Therefore, floating sustained-release Imatinib tablets were developed to allow the tablets to be released in the upper part of the GIT and overcome the inadequacy of conventional tablets. METHODOLOGY: Floating sustained-release Imatinib mesylate tablets were prepared using the wet granulation method. Tablets were formulated using Hydroxypropyl Methylcellulose (HPMC K4M), with Sodium alginate (SA) and Carbomer 934P (CP) as release-retarding polymers, sodium bicarbonate (NaHCO3) as the effervescent agent and lactose as a filler. Floating behavior, in vitro drug release, and swelling index studies were conducted. Initial and total drug release duration was compared with a commercial tablet (Gleevec) in 0.1 N HCl (pH 1.2) at 37 ± 0.5°C for 24 hours. Tablets were then evaluated for various physical parameters, including weight variation, thickness, hardness, friability, and drug content. Consequently, 6 months of physical stability studies and in vitro gastro-retentive studies were conducted. RESULTS AND DISCUSSION: Statistical data analysis revealed that tablets containing a composition of 14.67% w/w HPMC K4M, 10.67%, w/w Na alginate, 1.33%, w/w Carbomer 934P and 9.33%, w/w NaHCO3 produced the most favorable formulation to develop 24-hour sustained-release tablets with optimum floating behavior and satisfactory physicochemical characteristics. Furthermore, in vitro release study revealed that the formulated SR tablet had significantly lower Cmax and higher Tmax compared to the conventional tablet (Gleevec). Thus, formulated SR tablets preserved persistent concentration of plasma up to 24 hours. CONCLUSION: In conclusion, in order to suggest a better drug delivery system with constant favorable release, resulting in optimized absorption and less side effects, formulated CP-HPMC-SA based imatinib mesylate floating sustained-release tablets can be a promising candidate for cancer chemotherapy.

The use of Hibiscus esculentus (Okra) gum in sustaining the release of propranolol hydrochloride in a solid oral dosage form.

The effectiveness of Okra gum in sustaining the release of propranolol hydrochloride in a tablet was studied. Okra gum was extracted from the pods of Hibiscus esculentus using acetone as a drying agent. Dried Okra gum was made into powder form and its physical and chemical characteristics such as solubility, pH, moisture content, viscosity, morphology study using SEM, infrared study using FTIR, crystallinity study using XRD, and thermal study using DSC and TGA were carried out. The powder was used in the preparation of tablet using granulation and compression methods. Propranolol hydrochloride was used as a model drug and the activity of Okra gum as a binder was compared by preparing tablets using a synthetic and a semisynthetic binder which are hydroxylmethylpropyl cellulose (HPMC) and sodium alginate, respectively. Evaluation of drug release kinetics that was attained from dissolution studies showed that Okra gum retarded the release up to 24 hours and exhibited the longest release as compared to HPMC and sodium alginate. The tensile and crushing strength of tablets was also evaluated by conducting hardness and friability tests. Okra gum was observed to produce tablets with the highest hardness value and lowest friability. Hence, Okra gum was testified as an effective adjuvant to produce favourable sustained release tablets with strong tensile and crushing strength.

Fine Structures of the Oocyte in Relation to Serum, Follicular Fluid Steroid Hormones and IGF-I in the Ovulatory-Sized Follicles in One-Humped Camel (Camelus dromedarius).

BACKGROUND: The following study was carried out to determine the ultrastructural features of the oocyte of the ovulatory-sized follicles in relation to concentrations of steroids and IGF-I in the Follicular Fluid (FF) and serum in the dromedary camel. METHODS: CAMEL FOLLICLES WITH A CLEAR AND HEALTHY APPEARANCE WERE CATEGORIZED INTO THREE CLASSES: follicles 10 to 13.9, 14-17.9 and 18-30 mm diameter. The FF and serum samples were assayed for estradiol-17ß, progesterone and IGF-I. Recovered Cumulus-Oocyte Complexes (COCs) were prepared for transmission electron microscopy. RESULTS: The mean (±SD) FF concentrations of progesterone and IGF-I was significantly (p < 0.05) higher in follicles 18 to 30 mm diameter compared to other groups of follicles. There was no difference in the mean (±SD) serum estradiol-17ß, progesterone and IGF-I concentrations between camels with different ovulatory-sized follicles (p > 0.05). Oocytes from follicles 18 to 30 mm diameter (group 3) showed more advanced signs of maturation including the disappearance of the nuclear envelope, increased number of microvilli in erect position, the increase in number and size of vesicles and more even distribution of the mitochondria throughout the ooplasm. CONCLUSION: The final stages of oocyte maturation in dromedary camel is associated with increasing progesterone and IGF-I concentrations and constant high estradiol concentration in the follicular fluid which are paralleled with well-defined ultrastructural changes in oocytes.