Ml Of Sesame Oil Research Articles

Effects of 17β-estradiol on the uterine luteolytic cascade in bovine females at the end of diestrus

In cattle, 17β-estradiol (E2) is essential for triggering luteolysis via the synthesis of prostaglandin F2α (PGF2α). We aimed to evaluate the effects of E2-treatment on day 15 of the estrous cycle on the transcript abundance of genes involved in the PGF2α synthetic cascade. Nelore heifers (N = 50) were subjected to a hormonal protocol for the synchronization of ovulation. Between days 14 and 23 after estrus, the area (cm2) and blood perfusion (%) of the corpus luteum (CL) and progesterone (P4) plasma concentrations were evaluated daily. On day 15, the heifers were assigned to the Control (2 mL of pure sesame oil, N = 21) or Estradiol group (1 mg of E2 diluted in 2 mL of sesame oil, N = 23). After the treatments at 0 h, uterine biopsies were collected at times 1.5 h (C1.5h, N = 8 and E1.5h, N = 10) or 3 h (C3h, N = 8 and E3h, N = 11); and blood samples were obtained from 0, 3, 4, 6 and 7 h for the measurement of 13,14-dihydro-15-keto-PGF2α (PGFM) concentrations by ELISA. Transcript abundance was determined by RT-qPCR and protein abundance of ESRβ and OXTR was determined by Western Blotting. The Estradiol group showed greater (P < 0.05) concentrations of PGFM at 6 and 7 h compared to the Control group. A progressive decrease in plasma P4 concentrations characterized a hastened functional luteolysis, followed by structural luteolysis in the Estradiol group (P < 0.05). Among the treatment groups, no significant difference was detected for the abundance of PRKCα, PRKCβ, AKR1B1, PTGS2 and ESRα transcripts (P > 0.05). Estradiol treatment decreased the abundance of PLA2G4A, AKR1C4, and ESRβ both 1.5h and 3h after treatment (P < 0.05). The relative expression of PGR and OXTR was greater in E3h compared to the C3h (P > 0.05). Protein abundance did not differ between treatment groups at either experimental times (P > 0.05). Overall, E2 promoted an increase in PGFM concentrations and the hastening of functional and structural luteolysis in Nelore heifers through the upregulation of PGR and OXTR, demonstrating for the first time that the expression of these receptors within 3 h after E2 stimulus was associated with triggering luteolysis in cattle.

P-343 The role of Yes-Associated protein (YAP) activity in uterus adenogenesis process

Abstract Study question Could Yes-Associated protein(YAP) be effective in the adenogenesis process that occurs in the mouse uterus in the postnatal period? Summary answer It was observed that the YAP and phospho-YAP levels decreased in mice with inhibited gland development during adenogenesis process when compared to the control groups. What is known already The mouse uterus is composed of undifferentiated mesenchyme surrounded by a single layer of epithelium at birth. On the 5th Postnatal Day (PD), the luminal epithelium begins to bud, and the process called adenogenesis, which is termed as gland development in the uterus, begins. The gland development is evident on the PD 10th day. The uterus shows adult tissue characteristics by PD 15th-20th days. Although it has been stated that various signaling pathways are active in this process, Hippo signaling pathway role in the adenogenesis process have not been clarified yet. Study design, size, duration The uterus with normal gland development and gland development inhibited by Progesterone were evaluated for YAP and phospho-YAP, which is one of the main components of Hippo signaling pathway, by immunohistochemistry, western blotting, and q-RT-PCR methods. For experimental groups, 50 µg/g Progesterone was dissolved in 0.1 mL Sesame oil and for control groups, only 0.1 mL of Sesame oil given subcutaneously on PN 2nd-10th days. Animals were sacrificed on the PN 5th-10th and 15th days. Participants/materials, setting, methods In total of 30 newborn Balb/c female mice were used. They were divided as control (n = 15) and experimental(n = 15) groups. After the injections were finished, mice from both groups at PN5th(n = 5), PN10th(n = 5), and PN15th(n = 5) days were cervical dissociated, and uteri were collected. For immunohistochemical analyses, tissues were taken into buffered formalin, paraffin blocks were made, and the sections were taken. Protein and RNA isolations were performed from uteri for western blotting and q-RT-PCR analyses, respectively. Main results and the role of chance When YAP expression was evaluated by immunohistochemistry, western blotting and q-RT-PCR methods, there was no difference between the groups on the PN 5th day. But there was an active signaling was observed in the control groups on the PN10th day, which is very important for gland formation, and on the PN 15th day, that is effective in the maturation process. On the PN10th and 15th days, it was determined that YAP and phospho-YAP signal expression were decreased significantly in the experimental groups when compared with the control groups in immunohistochemically and protein levels. In addition, these findings were supported by mRNA analysis with YAP. Limitations, reasons for caution The study was performed in vivo mouse model. Further studies can be done with a transgenic animal model. Wider implications of the findings Considering the contribution of uterine gland development to the implantation process, errors in the development of these glands may cause infertility. This study can bring a new perspective to researching the uterus adenogenesis period and causes of infertility and treatment approaches. Trial registration number not applicable

PCOS model: Apoptotic changes and role of vitamin D

Aim: This research was aimed to observe apoptotic marker (caspase 3) levels in different groups after treatment with vitamin D in an animal model after inducing polycystic ovary syndrome (PCOS) with dehydroepiandrosterone (DHEA). Materials and Methods: 30 pre-pubertal female sprague dawley dams were recruited. The animals were distributed 10 each in control, PCOS and vitamin D treated groups. In control group, 0.2 ml of sesame oil was given. PCOS group was administered DHEA by the daily dose of 6 mg/kg for 30 days. In vitamin D treated group, animals were injected 6 mg/ kg/day DHEA daily and 120 ng 1, 25(OH) 2D3/100 g subcutaneously once a week. The occurrence of programmed cell death in PCOS by apoptotic marker (caspase 3) levels. Results: The results of this study showed significant weight gain, obesity, changes in caspase 3 levels in PCOS group as compared to control and vitamin D treated group. Conclusion: Administration of vitamin D (120 ng 1, 25(OH) 2D3/100) reduced body weight as well as improved the caspase 3 levels in PCOS induced animals. The results support the effect of vitamin D treatment for metabolic and reproductive characteristic features in PCOS females.

Morphological changes in the experimental model of polycystic ovary syndrome and effects of vitamin D treatment.

This research was designed to develop an animal model by inducing polycystic ovary syndrome (PCOS) with dehydroepiandrosterone (DHEA) and observe reproductive and morphological changes after treatment with vitamin D. Thirty pre-pubertal female Sprague-Dawley (SD) dams were recruited. The animals were distributed 10 each in control, PCOS and vitamin D-treated groups. In control group 0.2 ml of sesame oil was given. PCOS group was administered DHEA by the daily dose of 6 mg/kg for 30 days. In vitamin D-treated group, animals were injected 6 mg/kg/day DHEA daily and 120 ng 1, 25(OH) 2D3/100 g subcutaneously once a week. The occurrence of reproductive phenotypic PCOS was evaluated by estrous cycle, morphology and histological changes of ovary, uterus on light microscope. The results of this study showed significant weight gain, obesity, and estrous irregularity in PCOs group as compared to control and vitamin D-treated group. Administration of vitamin D (120 ng 1, 25(OH) 2D3/100) improved the cycle characteristics, reduced body weight and morphological features in PCOS induced animals. The results support the effect of vitamin D treatment for metabolic and reproductive characteristic features in PCOS females.

Therapeutic effects of isoflavone-aglycone fraction from soybean (Glycine max L. Merrill) in rats with estradiol valerate-induced polycystic ovary syndrome as an inflammatory state

Polycystic ovary syndrome (PCOS) is a proinflammatory/oxidative state resulting in metabolic dysregulation and ovarian dysfunction. Isoflavones in soybean seed possess anti-inflammatory/antioxidant properties. So, in this study, the effects of soybean isoflavone-aglycones on tissue inflammation, oxidative status, and ovarian histology in an animal model of PCOS were considered to achieve a novel therapeutic agent. Thirty-two female Wistar rats were divided into four groups (n = 8): a control group receiving the vehicle (CON−); a group with PCOS receiving no treatment (PCOS); and two groups of PCOS rats treated with soybean isoflavone-aglycone fraction (SISAF) at 50 and 100 mg/kg (SISAF50 and SISAF100). PCOS was induced by injecting a single dose of estradiol valerate (4 mg/kg, IM) dissolved in 0.2 ml of sesame oil following 60 days for the full development of polycystic ovaries. The SISAF treatments were administered orally once a day for 21 days. Then, the ovaries were harvested for the assessment of oxidative/antioxidative parameters, interleukin (IL)-6, and tumor necrosis factor (TNF)-α levels concomitant with histological studies. Treatment with SISAF reduced the number of cystic follicles and thickness of the theca layer, as well as increasing the number of corpora lutea and granulosa cells in PCOS rats. Also, SISAF treatment significantly decreased ovarian tissue IL-6 and TNF-α levels, and improved total oxidative/antioxidative status compared to the PCOS group. Isoflavones may provide therapeutic effects in PCOS owing to the antioxidant and anti-inflammatory properties.

Evaluation of the Rho A/Rho-kinase pathway in the uterus of the rat model of polycystic ovary syndrome

The aim of this study was to investigate the expression of RhoA/Rho-kinase in the uterus and the effect of Rho-kinase inhibitors on uterine contractions of dehydroepiandrosterone (DHEA) induced polycystic ovary syndrome (PCOS) rats. Forty-four female Sprague-Dawley (21 days old) rats divided into three groups: The control group (n = 14, any procedure was not performed), vehicle group (n = 14, 0.2 ml of sesame oil, subcutaneous injection, 20 days) and PCOS group (n = 16, DHEA 6 mg/100 g in 0.2 ml of sesame oil, subcutaneous injection, 20 days). The myometrium thickness and uterine wet weight were assessed. The mRNA and protein expressions of Rho A, the effect of Rho-kinase inhibitors (fasudil and Y-27632) on KCl, carbachol, and PGF2α induced contractions were evaluated in the uterus. In the PCOS group, the myometrium thickness and uterine wet weight significantly increased compared to the control group and vehicle group. The mRNA expression level and the immunoreactive score of Rho A, ROCK 1, ROCK 2 were similar in all groups. In the PCOS group, KCl, carbachol, and PGF2α induced uterine contractions significantly increased compared to the control group and vehicle group. Fasudil and Y-27632 significantly inhibited KCl, carbachol, and PGF2α induced uterine contractions in all groups. In conclusion, the expression of Rho A, ROCK 1, ROCK 2 not changed although myometrium thickness, uterine wet weight and the contractile responses of uterus increased in the PCOS group. The results suggest that the Rho-kinase inhibitors effectively suppressed increased contractions in the PCOS group they might be potential therapeutic agents.

GDF9 and BMP15 Expressions and Fine Structure Changes During Folliculogenesis in Polycystic Ovary Syndrome.

Background:Polycystic ovary syndrome is the most frequently seen endocrine disorder in women of reproductive age with a prevalence of about 10%.Aims:To investigate the efficiency of growth differentiation factor 9 and bone morphogenetic protein 15 during folliculogenesis in a dehydroepiandrosterone-induced mouse Polycystic ovary syndrome model.Study Design:Animal experimentation.Methods:Mice were divided into 3 groups: control, vehicle and Polycystic ovary syndrome. Polycystic ovary syndrome model mice were developed by the injection of dehydroepiandrosterone dissolved in 0.1 mL of sesame oil. Ovarian tissues were examined for growth differentiation factor 9 and bone morphogenetic protein 15 using immunofluorescent labelling and electron microscopic examinations.Results:The immunoreactivity of growth differentiation factor 9 and bone morphogenetic protein 15 proteins decreased (p<0.05) in the Polycystic ovary syndrome group (27.73±8.43 and 24.85±7.03, respectively) compared with the control group (33.72±11.22 and 31.12±11.05, respectively) and vehicle group (33.95±10.75 and 29.99±10.72, respectively). Apoptotic changes were observed in granulosa cells, lipid vacuoles increased in Theca cells and thickening and irregularities were noted in the basal lamina of granulosa cells. An increased electron density in the zona pellucida in some of the multilaminar primary and secondary follicles in the Polycystic ovary syndrome model was also observed at the ultrastructural level.Conclusion:These results suggest that the decrease in the growth differentiation factor 9 and bone morphogenetic protein 15 expression initiated at the primary follicle stage effect the follicle development and zona pellucida structure and may cause subfertility or infertility in Polycystic ovary syndrome.

Effect of altering the intervals between consecutive superovulatory doses of porcine follicle-stimulating hormone on ovarian responses and embryo yields in anestrous ewes

The effect of varying intervals between successive gonadotropin injections on the superovulatory outcomes in anestrous Rideau Arcott ewes superstimulated for ovarian follicular development with multiple doses of porcine FSH (pFSH) was evaluated in a single study. Twenty-five animals received six (1×2.5ml and 5×1.25ml) injections of Folltropin®-V given at 0800 and 1600h or at 0800 and 2000h in Group 1 (n=9) or Group 2 (n=16), respectively. An i.m. injection of 500 IU of equine chorionic gonadotropin (eCG; Folligon®) was given concurrently with the first pFSH dose. Time of estrus was synchronized among ewes with intravaginal sponges containing 60mg of medroxyprogesterone acetate (Veramix®) that were left in place for 14days; sponges were removed at the time of the 5th pFSH injection. Six days after insertion of MAP sponges, all ewes received an i.m. injection of estradiol-17β dissolved in 1ml of sesame oil (350μg/ewe) to synchronize follicular wave emergence. Following the last pFSH dose, all animals were given a single i.m. injection of 50μg of gonadotropin-releasing hormone (GnRH; Cystorelin®) to induce ovulations before placing in a pen with four fertile rams for 36h. The ovarian responses were assessed and embryos recovered surgically 7days after GnRH injections. The mean number of corpora lutea was greater (P<0.05) in Group 1 compared with Group 2 ewes (21.0±2.9 compared with 10.4±1.6, respectively; mean±SEM) but there was no difference (P>0.05) in the number of transferable embryos (5.4±2.4 compared with 5.4±1.3/ewe, respectively), and Group 1 animals had significantly more degenerated embryos than Group 2 ewes (2.6±1.2 compared with 0.6±0.3/ewe, respectively). A superovulatory protocol wherein pFSH injections were given at 0800 and 1600h was more effective in terms of inducing multiple ovulations than the protocol with 12-h intervals between consecutive pFSH doses, but it was not associated with an increased production of transferable quality embryos by anestrous ewes.

The Effect of Nigella Sativa Extract (Thymoquinone) on Glucose Insulin Levels and Body Weight of Induced Diabetic Female Rats

<i>Background</i>: Diabetes is one of the most common chronic diseases in the world. It characterized by hyperglycemia resulting from defects in insulin secretion or action or both. Chronic complications of diabetes such as cardiovascular damage, cataracts and retinopathy, nephropathy and polyneuropathy. Induction of experimental diabetes in rodentsis used by Streptozotocin (STZ). A number of therapeutic effects including the effect on diabetes have been described for the Nigella sativa extract (Thymoquinone). <i>Aim:</i> The goal of this study to investigate the effect of Nigella sativa extract (Thymoquinone) as a herbal medicines on female induced diabetic rat, and hence investigating the comparison of changes in body weight, levels of glucose and insulin in serum, between normal and diabetic rats. <i>Methods:</i> Adult female Wister rats, weighing 200–250 g, were used. Rats were divided into four groups normal control (Group A), experimental control group (Group B) rats were given 0.5 ml of the single sodium-citrate buffer injection and 1ml of Sesame oil orally via gavage. (Group C) treated STZ-diabetic (60 mg/kg B. W., IP), with of low Nigella sativa extract (Thymoquinone) (5 mg/kg B. W, IP), and (Group D) treated STZ-diabetic (60 mg/kg B. W., IP), with of high Nigella sativa extract (Thymoquinone) (10 mg/kg B. W., IP) and until the end of experiment were evaluated to assess its effect on body weight, glucose and insulin levels in different groups. <i>Results:</i> The results indicated that significant reduction in glucose levels of high dose of treated group with Nigella sativa extract (Thymoquinone) (10 mg/kg b. w.) compared to low dose. Both dose of treated group with <i>Nigella sativa</i> extract (Thymoquinone) (5 and 10 mg/kg b. w.) very high significantly (p &lt; 0.001) reduced in body weight and insulin levels in comparison to both group normal control and experimental control groups. Moreover, there was no significant difference observed in body weight between normal control and experimental control groups. The present findings suggest an antidiabetic effect of Nigella sativa extract (Thymoquinone) may attributed through a decrease in hepatic gluconeogenesis. <i>Conclusions:</i> Thymoquinone has the ability to improve oxidative stress in plasma and tissues of STZ induced diabetic rats as evidenced by improved glycemic. Thus, Thymoquinone could be considered as a treatment strategy for diabetic complications.

Long-term treatment with dehydroepiandrosterone may lead to follicular atresia through interaction with anti-Mullerian hormone.

BackgroundHyperandrogenism is the primary manifestation of polycystic ovary syndrome (PCOS), which appears to be caused by excess exposure to androgen. As such, androgenized animal models have been developed and investigated to study the etiology of PCOS. Anti-Mullerian hormone (AMH) is known to be associated with follicle growth, and its levels are two to three times higher in women with PCOS than in those with normal ovaries. We studied how duration of androgen administration affects folliculogenesis and AMH expression.MethodsWe divided 30 immature (3-week-old) Sprague Dawley rats into six groups. Three groups were injected each evening with dehydroepiandrosterone (DHEA) (6 mg/100 g body weight/0.2 ml sesame oil) for 7, 15 or 30 days, respectively. The three control groups were injected with 0.2 ml of sesame oil for the corresponding lengths of time. Resected ovaries were sectioned and examined to determine follicle numbers at each developmental stage, and immunostained to assess AMH expression.ResultsOn day 7, follicle numbers and AMH expression levels at each developmental stage of follicle growth were similar in the respective control and DHEA groups. On day 15, the total follicle number (P = 0.041), the percentage of primordial follicles (P = 0.039) and AMH expression were significantly greater in the DHEA than the control group. On day 30, the percentages of primordial (P = 0.005), primary (P = 0.0002) and atretic (P = 0.03) follicles were significantly greater in the DHEA group, whereas the percentage of intermediary follicles (early pre-antral, late preantral, and early antral follicles) was significantly lower in the DHEA group (P = <0.0001). AMH expression in DHEA-treated rats on day 30 was seen exclusively in the primordial (P = 0.0413) and late antral follicles (p = 0.028).ConclusionsAndrogen administration increases AMH production in a process that regulates the growth of primordial follicles. That is, androgen-induced AMH expression provides local negative feedback to folliculogenesis augmented by androgen.

Shelf life of Bottled Sea Squirt Halocynthia roretzi Meat Packed in Vegetable Oil (BSMO)

Fresh sea squirt meat requires a modified processing and preservation process because it has a short shelf life due to its high mois ture content and strong proteolytic enzyme activity. In this study, bottled sea squirt meat prepared in vegetable oil (BSMO) to enhance the consumer acceptability was exposed to γ-ray (Co60, 10 kGy/h) irradiation to extend the shelf life without the use of a heating process. Response surface methodology was used to determine the optimal mixing ratio of BSMO containing 5% dehydrated fresh meat. Texture analysis and nutritional evaluation were also performed on a control and BSMO samples. The volatile basic nitrogen (VBN) content and total cell count were measured to determine the shelf life of irradiated BSMO products during chilled storage at 4°C for 60 days. According to a panel of 10 trained tasters (aged 20-29 years), the optimal mixing formulation was 80 g meat in 60 mL of mixed vegetable oil (30 mL of olive oil and 30 mL of sesame oil). The highest rated formulation, according to a panel of nine trained tasters (aged ≥30 years), was 80 g meat in 60 mL of mixed vegetable oil (42 mL of olive oil and 18 mL of sesame oil). Moisture, ash, and protein contents in BSMO did not change significantly ( P < 0.05) compared with the control. A higher lipid content (0.84 ± 0.23 to 2.13 ± 0.61; P < 0.05) was observed due to the presence of vegetable oil on the surface of BSMO. The vegetable oil raised the hardness, springiness, cohesiveness, gumminess, chewiness, and resilience of BSMO. BSMO products remained edible after 50 days of storage at 4°C based on the VBN content (BSMO 1: 27.92 ± 0.96 mg/100 g, BSMO 2: 24.84 ± 1.95 mg/100 g) and total cell count (BSMO 1: 4.60 ± 0.80 log CFU/mL, BSMO 2: 3.65 ± 0.20 log CFU/mL) when compared with standard levels of VBN (25.00 mg/100 g) and total cell count (5 log CFU/mL), respectively. The results showed that irradiated BSMO products could help to expand the processed seafood market and increase the popularity of seafood among the younger generations.

Effect of the aqueous extract of Foeniculum vulgare (fennel) on the kidney in experimental PCOS female rats.

Foeniculum vulgare seed (F. vulgare) is an herbal plant which is used with phytoestrogene compounds for polycystic ovary syndrome (PCOS) treatment. In this research, renoprotective effect of the aqueous extract of Foeniculum vulgare (AEF) in experimental PCOS female rats is studied. Forty female rats were randomly divided into five groups. The first group served as control, was injected with an equivalent volume (0.2 ml) of normal saline, and received normal diet. Animals in the second group were non poly cystic ovary syndrome (PCOS) rats which were treated with intragastric administration of aqueous extract of F. vulgare (150 mg/kg b.w.). In the third group, the rats were treated with intraperitoneal injection of estradiolvalerate (EV) (4 mg in 0.2 ml of sesame oil). The fourth groups were treated with EV and AEF (150mg/kg bw) with the same route. The fifth groups were treated with EV and AEF (100mg/kg bw). After 4 weeks of study, all of the rats were sacrificed, their kidneys tissues were processed for light microscopy, and some biochemical parameters of serum were measured. The mean values of blood urea nitrogen in PCOS rats treated with low dose of AEF and EV and non-treated, was significantly (p<0.05) increased compared with non-PCOS and PCOS rats treated with high dose of AEF. Moreover, histopathological changes of kidney samples were comparable in PCOS rats with respect to treated groups with AEF. Aqueous extract of fennel seed showed the beneficial effect (especially at dose of 150 mg/kg b.w.) on renal function in PCOS rats.

Prenatal hyperandrogenism and lipid profile during different age stages: an experimental study

The present study investigates the effect of prenatal hyperandrogenization on lipid metabolism and oxidant/antioxidant balance. Experimental study. Research institute. Pregnant Sprague Dawley rats were subcutaneously injected with 2 mg free T between days 16 and 19 of pregnancy, and controls (C) received vehicle (0.1 mL of sesame oil). Prenatally hyperandrogenized female offspring (T2) had a condition that resembles polycystic ovary (PCO). Animals were weighed and killed at 21 and 60 days of age (N = 15 rats/group). Ovarian tissue and truncal blood were obtained from the C and T2 groups. Circulating lipid profile (total cholesterol, high-density lipoprotein [HDL], low-density lipoprotein [LDL] cholesterol, and triglycerides) was quantified by colorimetric-enzymatic methods. Ovarian oxidative stress was evaluated by quantifying lipid peroxidation and glutathione content by spectofotometric assays. Ovarian fat content was evaluated by Red Oil staining and ovarian messenger RNA (mRNA) expression of peroxisome proliferator-activated receptor gamma (PPAR-γ) by real-time polymerase chain reaction (PCR). At 60 days of age, 100% of group C rats and 20% of group T2 rats ovulated. At 21 days of age the T2 rats displayed lower body weight than C rats; however, at 60 days of age T2 and C rats showed similar body weights. The lipid profile (total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides) was altered in the anovulatory and ovulatory phenotype of the T2 group, but the levels were higher in the anovulatory phenotype. Lipid peroxidation of rats at 21 and 60 days of age from T2 was similar to C but the antioxidant glutathione level was decreased in 21-day-old rats compared with C rats. The lipid content of ovarian tissue, determined by Red Oil staining, was higher in the T2 than in the C group. The mRNA expression of ovarian PPAR-γ, quantified by real time PCR, decreased in anovulatory rats at 60 days of age from T2 compared to C rats. Our findings reveal the importance of evaluating the complete lipid profile, especially at early stages of life after the prenatal hyperandrogenism condition. In addition, we demonstrated that the antioxidant-reduced glutathione would represent a good marker of oxidative stress as it is altered before lipid peroxidation. Prenatal hyperandrogenization also alters the gene expression of PPAR-γ in rats. Here we demonstrated for the first time that abnormalities in PPAR-γ and lipid profile were higher in rats showing an anovulatory phenotype than those displaying an ovulatory phenotype.

The mechanism of mTOR (mammalian target of rapamycin) in a mouse model of polycystic ovary syndrome (PCOS)

Polycystic ovary syndrome (PCOS) is a common and complex endocrine disorder affecting 5-10% of women in reproductive age that is characterized by hyperandrogenism, oligo- or anovulation and infertility. However the pathophysiology of PCOS still remains unknown. The mammalian target of rapamycin (mTOR) is a central component that regulates various processes including cell growth, proliferation, metabolism, and angiogenesis. mTOR signaling cascade has recently been examined in ovarian follicles where it regulates granulosa cell proliferation and differentiation. mTOR functions as two complexes, mTOR complex 1 and 2. Therefore, we hypothesized that mTORC1 and/or 2 may have important role in proliferation of theca and granulosa cells in PCOS. In the present study, we sought to determine the mTOR signaling pathway in PCOS mouse ovary. We designed 3 groups: Control (C, no treatment), PCOS (P, The injection of DHEA (6 mg/100 g BW in 0.1 ml of sesame oil) (s.c) for 20 consecutive days), Vehicle (V, daily (s.c) sesame oil alone injection). Our results showed that mTORC1 and mTORC2-mediated signaling may play a role in PCOS mouse ovary. These findings provide evidence that mTORC1 and mTORC2 may have responsibility in increased ovarian follicular cell proliferation and growth in PCOS. Consequently, these results suggest that the mTOR signaling pathways (mTORC1 and mTORC 2) may create new clinical strategies to optimize developmental competence of PCOS should target correction of the entire follicle growth, oocyte development process and anovulatory infertility in PCOS.

Evaluation of Molecular Markers in a Rat Model of Mammary Carcinogenesis

We sought to evaluate the molecular markers involved in breast tumorigenesis in a rat model that mimics many essential elements of human breast cancer. Female Sprague-Dawley rats were divided into two groups. Animals in group 1 were given a single dose of 7,12-dimethylbenz[a]anthracene (DMBA) (20 mg/rat) dissolved in 1 ml of sesame oil by intragastric intubation. Group 2 animals received basal diet and served as control. We analyzed DMBA-induced changes in the expression of CYP isoforms (CYP1A1 and 1B1) involved in DMBA metabolism, markers of oxidative stress (4HNE, HEL, and 8-OHdG), cell survival and proliferation (PCNA, NF-kappaB-p50, NF-kappaB-p65, GST-P, and p53), apoptosis (Bcl-2, Bax, caspases, Apaf-1, cytochrome C, and Fas), invasion (uPA, MMP-2, MMP-9, TIMP-2, and RECK), and angiogenesis (VEGF, VEGF-R1, HIF-1alpha, and PLGF) by immunohistochemical localization, Western blot, and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The present study demonstrates increased carcinogen metabolism, oxidative stress, cell proliferation, together with apoptosis evasion, invasion, metastasis, and neovascularization that may confer a selective growth advantage to DMBA-induced mammary tumors. Aberrant expression of multiple molecules in key signaling pathways in Sprague-Dawley rat mammary tumors renders this model as an important tool for monitoring carcinogenic progression and chemointervention.

Long-term administration of estrogen or tamoxifen to ovariectomized rats affords neuroprotection to hippocampal neurons by modulating the expression of Bcl-2 and Bax

Recently we reported that chronic treatment with 17β-estradiol (E2) or tamoxifen (TAM) regulates the ovariectomy-induced downregulation of the key molecules linked to hippocampal synaptic plasticity and signal transduction pathway. We now report modulation of the antiapoptotic (Bcl-2) and proapoptotic (Bax) proteins in the hippocampus of both the ovariectomized (OVX) rats as well as those given E2 or TAM subcutaneously as a daily dose for four weeks post-ovariectomy. Forty bilaterally OVX animals were divided into four groups of 10 each, namely i) OVX + E2 (0.1 mg/kg body weight), ii) OVX + TAM (0.05 mg/kg body weight), iii) OVX + vehicle (0.1 ml of sesame oil) and iv) OVX controls. An additional group of 10 animals constituted the ovary intact controls. Following culmination of treatment regimen, brain tissues of five animals from each group were processed for immunohistochemical staining of Bcl-2 and Bax on perfusion fixed cryo-sections. The remaining animals in each group were utilized for protein and Western blot analyses using unfixed hippocampal tissue. The results revealed that chronic administration of both E2 and TAM prevented the ovariectomy-induced downregulation of Bcl-2 and upregulation of Bax expression while restoring the Bcl-2/Bax ratio as observed in the ovary intact rats. Furthermore, TUNEL assay demonstrated a decline in the percentage of TUNEL positive cells in E2 or TAM treated groups. Confocal microscope studies of ERα and the apoptotic markers revealed that these two proteins co-reside in the same ERα positive hippocampal neurons. Thus, long-term E2 or TAM therapy modulates the apoptotic proteins and affords neuroprotection to the hippocampal neurons. Furthermore the estrogen-like effects of TAM point towards its potential as a beneficial therapeutic agent for neurodegenerative disorders, particularly in the postmenopausal women.

Mammary Epithelial-specific Deletion of the Focal Adhesion Kinase Gene Leads to Severe Lobulo-Alveolar Hypoplasia and Secretory Immaturity of the Murine Mammary Gland

Integrin-mediated cell adhesion and signaling is required for mammary gland development and functions. As a major mediator of integrin signaling, focal adhesion kinase (FAK) has been implicated to play a role in the survival, proliferation, and differentiation of mammary epithelial cells in previously studies in vitro. To assess the role of FAK in vivo, we created mice in which FAK is selectively deleted in mammary epithelial cells. The mammary gland FAK conditional knock-out (MFCKO) mice are viable, fertile, and macroscopically indistinguishable from the control littermates. In virgin MFCKO mice, mammary ductal elongation is retarded at 5 weeks of age but reaches the full extent by 8 weeks of age compared with the control mice. However, the MFCKO females are unable to nurse their pups due to severe lobulo-alveolar hypoplasia and secretory immaturity during pregnancy and lactation. Analysis of the mammary epithelial cells in MFCKO mice showed reduced Erk phosphorylation, expression of cyclin D1, and a corresponding decrease in proliferative capability compared with the littermate controls. In addition, phosphorylation of STAT5 and expression of whey acidic protein are significantly reduced in the mammary glands of MFCKO mice, suggesting defective secretory maturation in these mice. Therefore, the combination of the severe lobulo-alveolar hypoplasia and defective secretory differentiation is responsible for the inability of the MFCKO females to nurse their pups. Together, these results provide strong support for a role of FAK in the mammary gland development and function in vivo.

Effect of Ovarian Steroids on Colonic Epithelial Cell Proliferation and Apoptosis in Rats

The aim of this study was to investigate the effects of steroid hormones on proliferation and apoptosis in the colon crypt epithelium. The research was conducted on adult ovariectomized (Ovx) rats (Sprague Dawley). Ovx rats were injected for 15 days with 0.2 ml of sesame oil (control; C), or 17β-oestradiol (10 μg/d; E), or progesterone (2 mg/d; P), or E + P. Proliferative activity in the colon was assessed by using proliferating cell nuclear antigen (PCNA) antibody. The proliferation index (PI), the number of PCNA positive cells divided by the total number of cells counted in the crypt column multiplied by 100, was calculated. PI was lower in the hormonetreated groups, especially in group P compared to that in group C. The apoptotic index (AI), the mean number of apoptotic cells, was detected by active caspase 3 immunoreactivity per crypt in the colon. AI was lower in the colon crypt epithelium of group E than that of the other groups. However, AI in the colon crypt epithelium in groups P and E + P was higher than that of both group E and group C. In addition, the colon crypt size (the number of epithelial cells lining one side of 10 well-oriented, longitudinally cut crypts) was considerably lower in group E than that of the other groups. In conclusion, we showed that the decrease of AI in group E was balanced by progesterone; the decrease of PI in group P was also depressed by oestrogen.

Identification of testosterone-regulated genes in testes of hypogonadal mice using oligonucleotide microarray.

FSH and testosterone (T) are required for normal spermatogenesis in mammalian males. These hormones regulate the function of Sertoli cells, which in turn support the differentiation of germ cells in the seminiferous tubules. The molecular targets for these hormones in the testis remain elusive. In this study, we have used hypogonadal (hpg) mice as an in vivo model to examine the actions of T on gene expression in murine testis. This expression pattern was analyzed using Affymetrix Murine GeneChip U74v.2 A, B, C (36,899 transcripts) along with Microarray Suite version 5.0, GeneSpring software, and real-time PCR. hpg mice aged 35-45 d were injected sc with 25 mg testosterone proprionate (TP) in 100 ml of sesame oil, and the animals were killed 4, 8, 12, or 24 h after TP treatments. Untreated hpg mice were used as controls. Gene expression from testes of hpg mice treated with TP was compared with that of testes of untreated hpg mice. At all experimental time points earlier than 24 h, there were more mRNAs with reduced than increased abundance in testes of hpg mice after TP treatment. This study suggests that in murine testis, the primary action of T might be to repress gene expression.

Effect of all-trans and 9-cis retinoic acid on growth and metastasis of xenotransplanted canine osteosarcoma cells in athymic mice.

To determine effects of all-trans and 9-cis retinoic acid (RA) on tumor growth and metastatic ability of canine osteosarcoma cells transplanted into athymic (nude) mice. Forty-five 5-week-old female BALB/c nude mice. 1 X 10(7) POS osteosarcoma cells were transplanted subcutaneously into the intrascapular region of mice. All-trans RA (3 or 30 microg/kg of body weight in 0.1 ml of sesame oil), 9-cis RA (3 or 30 mg/kg in 0.1 ml of sesame oil), or sesame oil (0.1 ml; control treatment) were administered intragastrically 5 d/wk for 4 weeks beginning 3 days after transplantation (n = 4 mice/group) or after formation of a palpable tumor (5 mice/group). Tumor weight was estimated weekly by measuring tumor length and width, and retinoid toxic effects were evaluated daily. Two weeks after the final treatment, mice were euthanatized, and number of mice with pulmonary metastases was determined. Adverse treatment effects were not detected. Tumor weight was less in mice treated with either dose of 9-cis RA than in control mice, although this difference was not significant. Treatment with 30 mg of 9-cis RA/kg initiated after tumor formation significantly reduced the incidence of pulmonary metastasis, compared with the control group. 9-cis RA decreased the incidence of pulmonary metastasis in nude mice transplanted with canine osteosarcoma cells and may be a potential adjunct therapy for treatment of osteosarcoma in dogs.