Pranab Lal Pakrasi

Cyclooxygenase-2-derived endogenous prostacyclin reduces apoptosis and enhances embryo viability in mouse

The role of prostaglandins (PGs) in apoptosis in preimplantation mice embryo development is reported in this study. It is known that apoptosis plays a very important role in normal mice embryo development. Very few reports are available on this subject. Embryos (6-8 cells) were cultured in the presence of a selective cyclooxygenase (COX)1 inhibitor (SC560), a selective COX2 inhibitor (NS398) and a selective prostacyclin synthase (PGIS) inhibitor (U51605) in a 48-h culture. In another experiment, culture media were supplemented with prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2 or prostacyclin) analogues. The apoptosis was evaluated by detection of active caspase-3. It was strongly detected in the presence of selective COX-2 and PGIS inhibitors, which can be decreased by a PGI2 analogue. In our embryo transfer experiment, the implantation rate decreased with exposure to either the COX2 or the PGIS inhibitor which is increased further after PGI2 supplementation. The level of PGI2 is also higher at the 8-16-cell stage, compaction and blastocyst stage than PGE2. All these results indicate that COX2-derived PGI2 plays an important role in preimplantation embryo development and acts as an antiapopetic factor in in vitro culture.

Estrogenic and antiestrogenic properties of clomiphene citrate in laboratory mice

The estrogen agonistic and antagonistic properties of clomiphene citrate were investigated in the mice. Clomiphene citrate was tested at various doses of 0.1, 1.0, 10 and 100 μg for three consecutive days in immature and mature bilaterally ovariectomized mice. Clomiphene citrate showed uterotrophic activity in both immature and ovariectomized conditions. The lower doses of 0.1 and 1.0 μg were ineffective to show any uterotrophic stimulation. Clomiphene citrate at 10 μg dose produced 305.56% increase in uterine weight i.e., 27.70 ± 0.24 vs 6.83 ± 0.06 in immature and 182.27% i.e., 42.68 ± 1.12 vs 15.12 ± 0.57 in ovariectomized mice. Clomiphene citrate at 100 μg dose showed significant uterotrophic effect e.g., 435.57% i.e., 36.58 ±0.34 vs 6.83 ± 0.06 in immature and 586% i.e., 103.80 ± 0.60 in ovariectomized mice. When clomiphene citrate was administered in combination with 0.32 μg of estradiol 17-β it caused significant antagonistic effect (decrease in uterine weight) at 10 and 100 μg respectively. Clomiphene citrate at 10 μg dose produced 32% i.e., 28.93 ± 0.43 vs 38.04 ± 2.68 in immature and 35% i.e., 59.64±1.44 vs 83.34 ±0.25 in ovariectomized mice respectively. Histological observation clearly showed that clomiphene citrate at 10 and 100 μg doses did not cause any differential hypertrophy of the epithelial layer. Similar doses in combination with estradiol produced significant antagonistic effect on uterine weight and luminal epithelial cell height.

PROSTAGLANDINS AND OVUM IMPLANTATION IN MICE

The possible involvement of prostaglandins (PGs) in events of ovum implantation is investigated. The levels of PGF and PGE-A were measured by radioimmunoassay in the implantation and interimplantation sites on days 4, 5, 6, and 7 of pregnancy. The concentration of prostaglandins was determined in morulae and blastocysts also. The levels of PGE-A were higher in implantation sites in comparison to PGF. PGE-A level showed a peak on d5 and remained significantly higher on d6 and d7, in comparison to d4. The concentration of PGF was found to be very low in both interimplantation sites and implantation sites before implantation. The concentration of PGF showed a significant increase on d6 in the interimplantation sites which peaked up to 32.61 +/- 2.01 ng on d7. The embryos showed an increase in PGE-A concentration along with development. However, PGF could not be found in the embryos. The present result shows that in mice PGE is the main prostaglandin involved in ovum implantation and PGF is associated with maintaining the embryos in the uterus. It is also predicted that in mice, blastocysts differentially stimulate PG synthesis in the uterus between implantation sites and interimplantation sites.

Evaluation of the antidiabetic properties of S-1708 mulberry variety

Background: Diabetes is a metabolic disease prevalent worldwide in all age group of people. The source of diabetes is due to an oxidation process that can produce free radicals. An increase in oxidative free radicals in the body is reported to be one of the several causes of diabetes. The best remedy to combat oxidative stress is the use of antioxidants, which inhibit and scavenge free radicals. Aim: This study has been undertaken to evaluate the antioxidant activity and antidiabetic effect of mulberry leaf extract in diabetic mice. Materials and Methods: Antioxidant activity of mulberry leaves was determined by 2,2-diphenyl-1-picryl-hydrazyl (DPPH) and ferric reducing/antioxidant power (FRAP) assay. Antidiabetic assay of mulberry leaf extract was analyzed by oral administration of leaf extract up to 3 weeks in diabetic mice induced by streptozotocin. Results: In vitro antioxidant activity in both DPPH and FRAP assays showed significantly (P < 0.05) higher inhibition of free radicals than that with ascorbic acid. Diabetic mice fed with mulberry leaf extract showed increment (+25.88%) in body weight and a significant reduction in blood glucose concentration (−71.58%). Further, glucose-6-phosphate dehydrogenase enzyme activity was significantly (P < 0.05) increased, whereas activities of other enzymes particularly catalase, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase were decreased in diabetic mice after oral administration of mulberry leaf extracts. Histology of liver revealed regeneration of hepatocytes, central vein, and nucleus. Conclusion: This study demonstrated that S-1708 mulberry variety has a potential therapeutic value in diabetes and related complications. Abbreviations used: S-1708, DPPH, FRAP.

Expression of ER-α and ER-β during peri-implantation period in uterus is essential for implantation and decidualization in golden hamster

Aims The role of estrogen in embryo implantation in golden hamster (Mesocricetus auratus) is still ambiguous. In order to clarify it, we investigated the spatial distribution and expression of estrogen receptors, ER‐&agr; and ER‐&bgr; in the uterus of pregnant hamster during peri‐implantation period and identified the effect of estrogen receptor antagonist ICI‐182,780 on the embryo implantation. Main methods We performed in vivo experiments on early pregnant hamsters involving treatment with ICI‐182,780, an estrogen receptor antagonist. Immunohistochemistry, western blot analysis and quantitative PCR were employed to evaluate the spatio‐temporal distribution and expression of ER‐&agr; and ER‐&bgr; in the uterus of normal early pregnant and treated hamsters. Key findings Results showed that embryo implantation was completely absent in ICI‐182,780 treated uterine horn while, normal implantation occurred in control and vehicle treated horns. Both the receptors were differentially expressed in the uterus of hamster from day 1 (D1) to day7 (D7). In contrast, treated horns without any implantation site showed no trace of any receptors. Protein and mRNA expression of both the receptors were high around the day of implantation while, ER‐&bgr; expression was up‐regulated on D7 of embryo implantation. P value < 0.05 is considered significant. Significance Spatio‐temporal expression of ERs in the uterus during peri‐implantation period have crucial role for endometrium receptivity and implantation in hamster. Recurrent implantation failure is the devastating problem among the desirable couple and is mainly due to defect in endometrium receptivity. This study may provide a new insight to manage the problem of idiopathic infertility.

Induction of ovulation by clomiphene citrate in the Indian vespertilionid bat, Scotophilus heathi

The ovulation induction property of clomiphene citrate (CC) and human chorionic gonadotropin (hCG) was studied in Scotophilius heathi, an Indian tropical vespertilionid bat, during the period of delayed ovulation between December to early January. The results of the study showed that 10 microg of CC alone was ineffective to induce ovulation, whereas 100 microg CC and 10 IU hCG alone induced ovulation. A significant (P < 0.01) increase in the ovulation rate was observed when 10 microg CC followed by 10 IU hCG, compared to 10 IU hCG and 100 microg CC alone groups. Finally, CC at a 100 microg dose, followed by 10 IU hCG, produced superovulation (14.00 +/- 0. 70), which is significantly different in comparison to all other groups. This is the first report of ovulation induced by CC in the Indian tropical bat as well as in any animal model that exhibits temporary anovulation similar to polycystic ovary syndrome (PCOD) during the normal physiology of reproduction.

Evaluation of the role of leukotrienes on ovum implantation in mice

Prostaglandins are considered to be one of the important mediators of ovum implantation. Various lipoxygenase products also have been implicated along with PGs for this process. A specific rather than preferential inhibitor of 5-lipoxygenase is used to investigate the role of leukotrienes in the event of implantation and decidualization process in mice. AA861, a selective inhibitor of 5-lipoxygenase is used in different dose levels like 50,100 and 500 μg in (A) intact pregnant mice (on D1-D4 and D2-D4 and D4); (B) delayed mice on (D3-D8); (C) pseudopregnant traumatized mice (on D1-D4). All the experimental animals of group A were killed on D6. Estrogen injected delayed animals of group  were killed 48 h after the induction of implantation. Implantation sites were counted as blue spot and compared with those of control animals. Traumatized animals of group C were killed 24 h after the mechanical traumatization and uterine weights were compared with those of vehicle treated controls. Results show that AA861 could not inhibit ovum implantation in either intact or ovariectomized delayed animals. It also did not show any adverse effect on tubal transport or development of embryos. AA861 did not have any inhibitory role on decidualization of pseudopregnant uteri also.This experiment shows that a selective inhibitor of 5-lipoxygenase enzyme may not impair the implantation in mice indicating a doubt about the involvement of 5-lipoxygenase products in implantation.