Immature Female Sprague-Dawley Rats Research Articles

Effect of kisspeptin-54 on ovarian levels of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in an experimental model of ovarian hyperstimulation syndrome (OHSS).

The purpose of this study was to investigate the effect of kisspeptin-54 on ovarian morphology and vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), protein kinase A (PKA) and protein kinase C (PKC) levels in an ovarian hyperstimulation syndrome (OHSS) rat model, which is a possible complication of controlled ovarian hyperstimulation. For this purpose, immature female Sprague-Dawley rats (25days old, 30-40g) were randomly divided into five groups (control, sham, OHSS model, short-term kisspeptin-54 administered OHSS model and long-term kisspeptin-54 administered OHSS model). Serum LH and FSH levels were determined by enzyme-linked immunosorbent assay. Immunohistochemistry and quantitative RT-PCR were performed for VEGF, PEDF, PKA and PKC in ovaries and granulosa cells, respectively. It was observed that there was dilatation in fallopian tubes and an abnormal increase in ovarian weight and volume in the OHSS group, and these morphologies decreased with kisspeptin-54 treatment. After the administration of kisspeptin-54 in the OHSS group, VEGF, PKA and PKC levels reduced and PEDF levels increased in both mRNA and protein levels. It was determined that in the OHSS model, VEGF increased as PEDF decreased, and kisspeptin-54 reduced the effects of OHSS. It was determined that long-term kisspeptin-54 treatment was more effective than short-term administration. It is considered that kisspeptin-54 is an agent that protects ovarian reserve and oocyte maturation in women at risk of OHSS.

Novel molecular mechanisms underlying the ameliorative effect of N-acetyl-L-cysteine against ϒ-radiation-induced premature ovarian failure in rats

Radiotherapy represents a critical component in cancer treatment. However, premature ovarian failure (POF) is a major hurdle of deleterious off-target effects in young females, which, therefore, call for an effective radioprotective agent. The present study aimed to explore the molecular mechanism underlying the protective effects of N-acetyl-L-cysteine (NAC) against γ-radiation-provoked POF. Immature female Sprague-Dawley rats were orally-administered NAC (50 mg/kg) and were exposed to a single whole-body dose of 3.2 Gy ϒ-radiation. NAC administration remarkably reversed abnormal serum estradiol and anti-Müllerian hormone levels by 73% and 40%, respectively while ameliorating the histopathological and ultrastructural alterations-triggered by γ-radiation. Mechanistically, NAC alleviated radiation-induced oxidative damage through significantly increased glutathione peroxidase activity by 102% alongside with decreasing NADPH oxidase subunits (p22 and NOX4) gene expressions by 48% and 38%, respectively compared to the irradiated untreated group. Moreover, NAC administration achieved its therapeutic effect by inhibiting ovarian apoptosis-induced by radiation through downregulating p53 and Bax levels by 33% and 16%, respectively while increasing the Bcl-2 mRNA expression by 135%. Hence, the Bax/Bcl2 ratio and cytochrome c expression were subsequently reduced leading to decreased caspase 3 activity by 43%. Importantly, the anti-apoptotic property of NAC could be attributed to inactivation of MAPK signaling molecules; p38 and JNK, and enhancement of the ovarian vascular endothelial growth factor (VEGF) expression. Taken together, our results suggest that NAC can inhibit radiotherapy-induced POF while preserving ovarian function and structure through upregulating VEGF expression and suppressing NOX4/MAPK/p53 apoptotic signaling.

Mechanistic approach of the inhibitory effect of chrysin on inflammatory and apoptotic events implicated in radiation-induced premature ovarian failure: Emphasis on TGF-β/MAPKs signaling pathway

Radiotherapy is one of the most relevant treatment modalities for various types of malignancies. However, it causes premature ovarian failure (POF) and subsequent infertility in women of reproductive age; hence urging the development of effective radioprotective agents. Chrysin, a natural flavone, possesses several pharmacological activities owing to its antioxidant, anti-inflammatory and anti-apoptotic properties. Therefore, the aim of this study was to investigate the efficacy of chrysin in limiting γ-radiation-mediated POF and to elucidate the underlying molecular mechanisms. Immature female Sprague-Dawley rats were subjected to a single dose of γ-radiation (3.2 Gy) and/or treated with chrysin (50 mg/kg) once daily for two weeks before and three days post-irradiation. Chrysin prevented the radiation-induced ovarian dysfunction by restoring estradiol levels, preserving the normal ovarian histoarchitecture and combating the follicular loss. Eelectron microscopic analysis showed that the disruption of ultrastructure components due to radiation exposure was hampered by chrysin administration. Mechanistically, chrsyin was able to reduce the levels of the inflammatory markers NF-κB, TNF-α, iNOS and COX-2 in radiation-induced ovarian damage. Chrysin also exhibited potent anti-apoptotic effects against radiation-induced cell death by downregulating the expression of cytochrome c and caspase 3. Radiation obviously induced upregulation of TGF-β protein with subsequent phospholyration and hence activation of downstream mitogen-activated protein kinases (MAPKs); p38 and JNK. Notably, administration of chrysin successfully counteracted these effects. These findings revealed that chrysin may be beneficial in ameliorating radiation-induced POF, predominantly via downregulating TGF-β/MAPK signaling pathways leading subsequently to hindering inflammatory and apoptotic signal transduction pathways implicated in POF.

MIF May Participate in Pathogenesis of Polycystic Ovary Syndrome in Rats through MAPK Signalling Pathway.

The polycystic ovary syndrome (PCOS) model was established in rats and correlation between the expression of macrophage migration inhibitory factor (MIF) and cytokinesis with the MAPK signalling pathway in the rat ovary was measured. The PCOS model in rats was established by dehydroepiandrosterone (DHEA). Thirty sexually immature female Sprague-Dawley rats were randomly and equally assigned to three groups: control group, PCOS group, and PCOS with high-fat diet (HFD) group. Serum hormones were assayed by radioimmunoassay (RIA). The ovaries were immunohistochemically stained with MIF, and the expression of MIF, p-JNK and p-p38 was detected by Western blotting in ovaries. The serum testosterone level, LH concentration, LH/FSH ratio, fasting insulin level and HOMA IR index in the PCOS group (6.077±0.478, 13.809±1.701, 1.820±0.404, 10.83±1.123 and 1.8692±0.1096) and PCOS with HFD group (6.075±0.439, 14.075±1.927, 1.779±0.277, 10.20±1.377 and 1.7736±0.6851) were significantly higher than those in the control group (4.949±0.337, 2.458±0.509, 1.239±0.038, 9.53±0.548 and 1.5329±0.7363), but there was no significant difference between the PCOS group and PCOS with HFD group. The expression levels of MIF, p-JNK, and p-p38 in the PCOS group (0.4048±0.013, 0.6233±0.093 and 0.7987±0.061) and PCOS with HFD group (0.1929±0.012, 0.3346±0.103 and 0.3468±0.031) were obviously higher than those in control group (0.2492±0.013, 0.3271±0.093 and 0.3393±0.061), but no significant difference was observed between PCOS group and PCOS with HFD group. It was suggested that MIF may participate in the pathogenesis of PCOS through the MAPK signalling pathway in PCOS rats induced by DHEA.

Pulmonary apoptotic and oxidative damaging effects of Triclosan alone or in combination with Fluoride in Sprague Dawley rats

This study aimed to assess the potential apoptotic and oxidative damaging impacts of Triclosan (TCS) and Sodium Fluoride (NaF) administered separately or in combination, in rats for thirty days. For this purpose, forty immature female Sprague-Dawley rats were equally allocated into five groups. TCS group administered 185mg TCS/kg Bw dissolved in distilled water (DW), while its control group received only DW. NaF group was given 50mg/kg NaF in corn oil BW and its respective control had corn oil alone. The co-treated group was administered TCS and NaF. The oxidative stress biomarkers were evaluated in lung tissue homogenate and apoptotic proteins (BcL-2 and Caspase-3) expression were quantified in lung tissues. The results of TCS or NaF treated groups revealed a prominent depletion of super oxide dismutase (SOD), Catalase (CAT) and Glutathione (GSH) in lung tissue homogenate. On contrary, a marked increase in the tissue levels of Malondialdehyde (MDA) and lactate dehydrogenase (LDH) enzymatic assay levels. The co-exposed group evoked less severity in the oxidative stress biomarkers concentration than individually exposed groups. The apoptotic genes protein expression was significantly higher in TCS or NaF treated rats when compared to the control with intense to moderate immunolabeling of the bronchiolar lining epithelium and surrounding mononuclear inflammatory cells. On the contrary, no significant differences were detected in the expression of the investigated apoptotic biomarkers between the control and the combined exposed group. We concluded that the exposure to either TCS or NaF resulted in significant perturbations in lung tissue after short term oral administration at variable instances but the co-exposure resulted in less severe toxicological consequences.

Resveratrol inhibits inflammatory signaling implicated in ionizing radiation-induced premature ovarian failure through antagonistic crosstalk between silencing information regulator 1 (SIRT1) and poly(ADP-ribose) polymerase 1 (PARP-1)

This study hypothesized that resveratrol, a silencing information regulator 1 (SIRT1) activator, would counteract the inflammatory signaling associated with radiotherapy-induced premature ovarian failure (POF). Immature female Sprague–Dawley rats were subjected to a single dose of γ-radiation to induce POF and treated with resveratrol (25mg/kg) once daily for two weeks before and three days post irradiation. Resveratrol preserves the entire ovarian follicle pool manifested by increasing serum anti-Müllerian hormone (AMH) levels. Radiation triggered inflammatory process in the ovary through enhanced NF-κB and poly(ADP-ribose) polymerase (PARP)-1 expression which convinced the expression of inflammatory markers including IL-6, IL-8, and visfatin mRNA levels, as well as inducible nitric oxide synthase and cyclooxygenase-2 protein expression with a concomitant reduction in IL-10 mRNA levels. Resveratrol significantly counteracted the effect of radiation and upregulated the gene expression of peroxisome proliferator-activated receptor γ (PPAR-γ) and SIRT1. Resveratrol-activated SIRT1 expression was associated with inhibition of PARP-1 and NF-κB expression-mediated inflammatory cytokines. Our findings suggest that resveratrol restored ovarian function through increasing AMH levels, and diminishing ovarian inflammation, predominantly via upregulation of PPAR-γ and SIRT1 expression leading to inhibition of NF-κB provoked inflammatory cytokines.

Growth Hormone Ameliorates the Radiotherapy-Induced Ovarian Follicular Loss in Rats: Impact on Oxidative Stress, Apoptosis and IGF-1/IGF-1R Axis.

Radiotherapy is one of the standard cytotoxic therapies for cancer. However, it has a profound impact on ovarian function leading to premature ovarian failure and infertility. Since none of the currently available methods for fertility preservation guarantees future fertility, the need for an effective radioprotective agent is highly intensified. The present study investigated the mechanisms of the potential radioprotective effect of growth hormone (GH) on γ irradiation-induced ovarian failure and the impact of the insulin like growth factor 1 (IGF-1) in the underlying protection. Immature female Sprague-Dawley rats were either exposed to single whole body irradiation (3.2 Gy) and/or treated with GH (1 mg/kg s.c). Experimental γ-irradiation produced an array of ovarian dysfunction that was evident by assessment of hormonal changes, follicular development, proliferation marker (PCNA), oxidative stress as well as apoptotic markers. In addition, IGF-1/IGF-1R axis expression was assessed using real-time PCR and immunolocalization techniques. Furthermore, after full maturity, fertility assessment was performed. GH significantly enhanced follicular development and restored anti-Mullerian hormone serum level as compared with the irradiated group. In addition, GH significantly ameliorated the deleterious effects of irradiation on oxidative status, PCNA and apoptosis. Interestingly, GH was shown to enhance the ovarian IGF-1 at transcription and translation levels, a property that contributes significantly to its radioprotective effect. Finally, GH regained the fertility that was lost following irradiation. In conclusion, GH showed a radioprotective effect and rescued the ovarian reserve through increasing local IGF-1 level and counteracting the oxidative stress-mediated apoptosis.

Effects of thyroid hormones on the antioxidative status in the uterus of young adult rats.

Thyroid hormones and oxidative stress play significant roles in the normal functioning of the female reproductive system. Nitric oxide (NO), a free radical synthesized by nitric oxide synthases (NOS), participates in the regulation of thyroid function and is also a good biomarker for assessment of the oxidative stress status. Therefore, the purpose of this study was to investigate effects of thyroid hormones on uterine antioxidative status in young adult rats. Thirty immature female Sprague-Dawley rats were randomly divided into three groups: control, hypothyroid (hypo-T) and hyperthyroid (hyper-T). The results showed the body weights decreased significantly in both the hypo-T and hyper-T groups and that uterine weights were decreased significantly in the hypo-T group. The serum concentrations of total triiodothyronine (T3) and thyroxine (T4), as well as estradiol (E2), were significantly decreased in the hypo-T group, but increased in the hyper-T group. The progesterone (P4) concentrations in the hypo- and hyperthyroid rats markedly decreased. Immunohistochemistry results provided evidence that thyroid hormone nuclear receptor α/β (TRα/β) and three NOS isoforms were located in different cell types of rat uteri. The NO content and total NOS and inducible NOS (iNOS) activities were markedly diminished in the hypo-T group but increased in the hyper-T group. Moreover, the activities of both glutathione peroxidase (GSH-Px) and catalase (CAT) exhibited significant decreases and increases in the hypo-T and hyper-T groups, respectively. The malondialdehyde (MDA) contents in both the hypo-T and hyper-T groups showed a significant increase. Total superoxide dismutase (T-SOD) activity in the hypo- and hyper-T rats markedly decreased. In conclusion, these results indicated that thyroid hormones have an important influence on the modulation of uterine antioxidative status.

Neurotrophic effects of perfluorocarbon emulsion gel: a pilot study

BackgroundPositive neurotrophic effects of hyperbaric oxygen treatment may be more easily achieved by applying a Perflourocarbon (PFC) emulsion gel to the repair site. PFCs are halogen substituted carbon oils with unique oxygen transport potentials that are capable of increasing oxygen availability in local tissues. The purpose of this study was to determine if the application of a PFC emulsion to a repaired nerve would improve recovery.Materials and methodsThe left tibial nerve of 21 immature female Sprague-Dawley rats was transected, immediately repaired, and then circumferentially coated with PFC gel (Group A, n = 7), PFC-less gel (Group B, n = 7), or nothing (suture only, Group C, n = 7). At eight weeks post surgery, electrophysiological testing and histological and morphological analysis was performed.ResultsNo statistically significant differences between experimental groups were found for muscle size and weight, axon counts, or nerve conduction velocity. Group A had a significantly smaller G-ratio than Groups B and C (p < .0001).ConclusionOverall results do not indicate a functional benefit associated with application of a PFC emulsion gel to rodent tibial nerve repairs. A positive effect on myelination was seen.

Effect of calcium treatment on blood parameters, gonadal development and the structure of bone in immature female rats

Calcium is an essential nutrient required for critical biological functions. Calcium supplementation is to be evaluated using immature female rats. The present study focused on some blood parameters, gonadal development and bone structure. Forty immature female Sprague-Dawley rats were randomly divided into four equal-sized groups (80 g average body weight) to receive calcium chloride dihydrate (group I: control; groups II, III and IV: received 20 mg, 40 mg and 60 mg per kg body weight, respectively) for 5 weeks. Rats were decapitated, and their trunk blood was sampled for biochemical assays. Cholesterol, triglycerides, glucose and calcium were measured. Gonadal and bone structure were histologically evaluated. Results revealed that treatment of developing female rats with three calcium doses used have no marked effect on the serum calcium and cholesterol levels. However, serum triglyceride level and body weight gain are significantly decreased in rats treated with all of the three calcium doses. Serum glucose level showed a marked increase in animals treated with the higher calcium doses. Moreover, observable histological alterations are recognized in the ovaries. Bones of the experimental animals also showed morphological alterations. These results suggest that increasing calcium supplementation decreases triglycerides and percentage body weight gain and positively affects the bone and gonadal development.

Administration of a gonadotropin-releasing hormone agonist affects corpus luteum vascular stability and development and induces luteal apoptosis in a rat model of ovarian hyperstimulation syndrome

Ovarian hyperstimulation syndrome (OHSS) is a complication of ovarian stimulation with gonadotropins followed by the administration of human chorionic gonadotropin (hCG) to trigger the final steps of oocyte maturation. Gonadotropin-releasing hormone (GnRH) analogs are thought to be effective in preventing this complication and a clinical trial has found a lower incidence of OHSS in patients treated with these molecules. Our aim was to analyze the in vivo effect of a GnRH-I agonist on corpus luteum development and regression, ANGPT-1, ANGPT-2 and Tie-2 protein expression and luteal blood vessel stabilization, the expression of the steroidogenic acute regulatory protein (StAR) and the cytochrome P450 side-chain cleavage enzyme (P450scc) and cell proliferation, in ovaries from an OHSS rat model. To this end immature female Sprague-Dawley rats were hyperstimulated and treated with a GnRH-I agonist from the start of pregnant mare serum gonadotropin (PMSG) administration until the day of hCG injection for 5 consecutive days. Blood and tissue samples were collected 48h after hCG injection. Vascular endothelial growth factor VEGF levels were evaluated in the peritoneal fluid by ELISA. Serum progesterone and estradiol were measured by RIA. Histological features of sectioned ovaries were assessed in hematoxylin and eosin (H&E) stained slides. Luteal blood vessel stability, cell proliferation and apoptosis were assessed by immunohistochemistry for SMCA, PCNA, and TUNEL, respectively. P450scc, StAR, FLK-1, ANGPT-1, ANGPT-2, Tie-2 and PCNA protein levels were evaluated by Western blot from dissected corpora lutea (CL). The treatment with the GnRH-I agonist significantly decreased serum progesterone and estradiol levels as well as P450scc and StAR protein expression in the untreated OHSS group. In addition, the agonist significantly decreased the number of CL in the OHSS group, as compared with the untreated OHSS group. In the OHSS group, the area of periendothelial cells in the CL was larger than that of the control group. However, the treatment with the GnRH-I agonist significantly reduced the area of periendothelial cells in the CL in the OHSS group. The luteal levels of ANGPT-1 and its receptor Tie-2 significantly increased in the OHSS group when compared with the control group. Conversely, the administration of the GnRH-I agonist significantly decreased the levels of these factors in the CL from the OHSS group, as compared with the untreated OHSS group. In addition, the treatment with the GnRH-I agonist reduced the diameter of CL and decreased CL cell proliferation as compared with that observed in the untreated OHSS group. Finally, the GnRH-I agonist increased apoptosis in the CL from the OHSS group. In conclusion, these results show that GnRH-I agonist exerts diverse actions on the CL from a rat OHSS model. The decrease in P450scc, StAR, ANGPT-1 and Tie-2 expression, blood vessel stability and luteal proliferation leads to CL regression in the ovaries from OHSS rats. Moreover, our results suggest that the downregulation of ANGPT-1 and its receptor is a possible mechanism whereby GnRH-I agonists could prevent early OHSS.

Potential Estrogenic Effect(s) of Parabens at the Neonatal Stage of an Immature Female Rat Mode.

This study was performed to examine the estrogenic effects of parabens on hormonal responsiveness and on the morphology of reproductive tissues during a critical developmental stage of female rats. Two hundred immature female Sprague-Dawley rats (n = 10/group) were orally treated with methyl-, ethyl-, propyl-, isopropyl-, butyl-, and isobutylparaben from postnatal day 21 to 40 in a dose-dependent manner based on our previous study [62.5, 250, and 1000 mg/kg of body weight (BW) per day]. A high dose of methyl- and isopropyl paraben (1000 mg/kg of BW per day) resulted in a significant delay in the date of vaginal opening and a decrease in length of the estrous cycle. In measurements of organ weight and body weight, we observed significant weight changes (i.e., decresed in ovaries, kidneys and increased in adrenal glands, thyroid glands, liver) conversely, body weight was not altered following paraben treatment. In all groups exposed to paraben treatment, histological analysis of the ovaries from the immature rats revealed interstitial cell disorders, decreased corpora lutea, an increase in the number of cystic follicles, and thinning of the follicular epithelium, which occurred in a dose-dependent manner. In addition, morphological study of the uterus revealed the myometrial dysplasia such as myometrial hyperplasia in the highest dose of propyl- and isopropyl paraben and in all dose of butyl- and isobutyl parabens. We also observed a significant decrease in serum estradiol and T4 concentrations in methyl-, ethyl-, propyl-, isopropyl-, and isobutylparaben-treated groups (P < 0.01 and 0.05). Taken together, long-term exposure to parabens can produce suppressive effects on hormonal responsiveness and can disrupt the morphology of reproductive target tissues during this critical stage of development in immature female rats. (poster)

Comparison of the Performance of Chronically Versus Freshly Denervated Autograft in Nerve Repair

Graft choice is one of the few variables over which the surgeon has control when reconstructing nerve gaps. Because repair of chronically denervated nerves generally yields inferior recovery, we hypothesized that the use of chronically denervated nerve tissue as a graft source may compromise axonal regeneration and clinical results. A total of 45 immature female Sprague-Dawley rats underwent transection of one peroneal nerve before being divided into 3 experimental groups: group A (n = 15) had acutely denervated nerve graft, group B (n = 15) had 2-month denervated nerve graft, and group C (n = 15) had 4-month denervated nerve graft. We included 10 additional rats as a sham group. After 2 months, groups A and B underwent removal of 1 cm of the contralateral peroneal nerve. For group A, this section of nerve was immediately sutured back in place to serve as a model for acute denervation. For group B, the defect was repaired with a 1-cm graft from the distal stump of the previously transected (denervated) peroneal nerve. Group C underwent the same procedure as group B, but after an additional 2 months. After 8 weeks of regeneration time, the 3 experimental groups and the sham group underwent testing. We assessed twitch contraction forces of the reinnervated extensor digitorum longus before we harvested the muscle belly for morphologic measurements. Histological nerve tissue evaluation assessed axonal regeneration. We detected no statistical differences for mean muscle contraction strengths between the experimental groups; nevertheless, the reinnervated extensor digitorum longus muscle bellies from the 4-month denervated nerve graft group were statistically smaller than muscles from the other 2 experimental groups (p < .05). Axon counts decreased, whereas axon diameters increased in direct correlation with the length of time of graft denervation (p < .05). No difference in axon myelination was found between experimental groups. Prolonged denervation of nerve graft material compromised both axon and reinnervated muscle recovery in this rodent model.

259 SODIUM/POTASSIUM/CALCIUM EXCHANGER 3 IS REGULATED BY STEROID HORMONES, ESTROGEN AND PROGESTERONE, IN THE UTERUS OF RAT DURING ESTROUS CYCLE

As a member of the family of potassium-dependent sodium/calcium exchangers, a sodium/calcium/potassium exchanger, NCKX3, plays a critical role in transport of 1 intracellular calcium and potassium ion in exchange for 4 extracellular sodium ions. Transcripts of NCKX3 were most abundant in the brain and smooth muscle, but in the uterus, aorta, and intestine, this gene expressed at lower levels. Its expression and role in the rat uterus during the estrus cycle have not been elucidated yet. In this study, we further examined the uterine expressions of NCKX3 mRNA and protein in stages during the estrous cycle of mature female Sprague-Dawley (SD) rats and in the absence or presence of sex-steroid hormones estrogen (E2) and progesterone (P4) in immature female SD rats. NCKX3 mRNA was verified by RT-PCR. Western blot analysis was applied to detect NCKX3 with an anti-NCKX3 goat polyclonal antibody. During the rat estrous cycle, uterus expressions of NCKX3 mRNA and proteins were highly expressed 2.5-fold at proestrus compared with those at estrus and diestrus. To examine the role(s) of sex steroids on the regulation of NCKX3 in the uterus of the immature female rat, hormones were prepared in ethanol and the rats were treated with E2 [40 (μg/kg of body weight (BW)], P4 (4 mg/kg of BW), or E2 plus P4 for 3 days. The expression of NCKX3 mRNA and protein increased 2-fold with E2, whereas P4 antagonized E2-stimulated NCKX3 expression, as similarly observed in the uterus. In addition, spatial expression of NCKX3 protein was detected by immunohistochemistry. Uterine NCKX3 protein was abundantly localized in the cytoplasm of the luminal and glandular epithelial cells at proestrus. Taken together, these results indicate that uterine NCKX3 is abundantly expressed in the uterus and controlled by steroid hormones E2 and P4, suggesting that uterine expression of NCKX3 might be involved in reproductive function during the cycle in female rat.

242 POTENTIAL ESTROGENIC EFFECT(S) OF PARABENS AT THE NEONATAL STAGE OF AN IMMATURE FEMALE RAT MODEL

In this study, to examine the estrogenic activity effects of parabens on hormonal responsiveness and on change in the morphology of reproductive target tissues during a critical development stage in female rats, analyses for parabens including methyl-, ethyl-, propyl-, isopropyl-, butyl-, and isobutylparaben were performed in an immature female Sprague-Dawley rat model. Two hundred female immature rats (n = 10/group) were orally treated with these parabens from postnatal day 21 to 40 in a dose-dependent manner based on our previous study [62.5, 250, and 1000 mg/kg of body weight (BW) per day]. 17α-ethinylestradiol (EE;1 mg/kg of BWper day) was used as a positive control and corn oil as a vehicle.A high doseofmethyl- and isopropylparaben (1000 mg/kg of BW per day) resulted in a significant delay in the date of vaginal opening and a decrease in length of the estrous cycle (P &lt; 0.05). In measurements of organ weight and body weight, we observed significant weight changes in ovaries, adrenal glands, thyroid glands, liver, and kidneys(P &lt; 0.05); conversely, body weight was not altered following paraben treatment. In all groups exposedto paraben treatment, histological analysis of the ovaries from the immature rats revealed interstitial cell disorders, a lack of corpora lutea, an increase in the number of cystic follicles, and thinning of the follicular epithelium, which occurred in a dose-dependent manner. In addition, morphological studies of the uterus revealed the myometrial dysplasia suchas myometrial hyperplasia inthe high-doseofpropyl- and isopropylparaben (1000 mg/kgof BWper day) group and in all dose of butyl- and isobutylparabens groups. We also observed a significant decrease in serum estradiol and T4 concentrations in methyl-, ethyl-, propyl-, isopropyl-, and isobutylparaben-treated groups (P &lt; 0.01 and 0.05).A receptor-binding assay indicated that the relative binding affini- ties of parabens to estrogen receptors occurred in the order: isobutylparaben &gt; butylparaben &gt; isopropylparaben = propylparaben &gt; ethylparaben. These values were much less than the binding affinity for 17?-estradiol. Taken together, long-term exposure to parabens, which show less estrogenic activity than EEl, can produce suppressive effects on hormonal responsiveness and can disrupt the morphology of reproductive target tissues during this critical stage of development in immature female rats.

In vivo evaluation of substituted 3-phenyl,7-methoxy-benzopyrans as modified estrogens

AbstractSubstituted 3-phenyl,7-methoxy-benzopyran derivatives and vitamin D 3 (cholecalciferol, 1) were evaluated for their estrogen agonistic and antagonistic activities in immature female Sprague-Dawley rat model. The benzopyran derivatives 17 and 18, which were made as hybrids of estrogen and vitamin D 3 (pseudo vitamin D 3 analogs), showed significant estrogen agonistic activity (up to 48%) and weak estrogen antagonistic activity (up to 6%) at 10 mg/kg, whereas vitamin D 3 showed significant estrogen agonistic (82%) and antagonistic activities (39%) at 10 mg/kg.Graphical abstractSubstituted 3-phenyl, 7-methoxy-benzopyran derivavtives and vitamin-D3 (Cholecalciferol, 1) wereevaluated for their estrogen agonistic and antagonistic activities in mature female Sprague-Dawley ratmodel. The benzopyran derivatives, which were made as hybrids of estrogen and vitamin-D3 (pseudovitamin-D3 analogs), showed significant estrogen agonistic activity (upto 48%) and weak estrogenantagonistic activity (upto 6%) at 10 mg kg -1 . Whereas, vitamin-D3 showed significant estrogenagonistic (82%) and antagonistic activities (39%) at 10 mg kg -1 .[IMAGE]

Coexistence of different tissue tumourigenesis in an N-methyl-N-nitrosourea-induced mammary carcinoma model: a histopathological report in Sprague-Dawley rats

N-methyl-N-nitrosourea (MNU), a highly potent carginogen, is widely used to generate mammary tumours in murine species. In a model of MNU-induced mammary carcinogenesis using immature female Sprague-Dawley rats, large mammary tumours (largest dimension > or =0.5 cm) were obtained within a very short period of time. In addition, in the rats bearing MNU-induced mammary carcinomas, there were a number of tumours whose origins were not from mammary tissue but from several different tissues and from mammary non-epithelial tissue. The tumours were of mesenchymal or epithelial origin and they were located in the inguinal region. These tumours were diagnosed as fibroadenoma, combined tubular adenoma and fibroadenoma, hyperkeratotic papilloma, keratinous cyst and malignant peripheral nerve sheath tumour (MPNST) with smooth muscle differentiation. The occurrence of these other tumours in addition to the development of the mammary carcinomas may be attributed to a direct local effect of the intraperitoneal administration of MNU during the sexual development of the immature rats. In the MNU-induced mammary tumour model, coexistence of tumourigenesis in various non-mammary tissues should be considered an important factor that may interfere with experimental procedures and results and also the quality of life of the tumour-bearing animals.

Evaluation of a bromine-76-labeled progestin 16alpha,17alpha-dioxolane for breast tumor imaging and radiotherapy: in vivo biodistribution and metabolic stability studies.

Progesterone receptors (PRs) are present in many breast tumors, and their levels are increased by certain endocrine therapies. They can be used as targets for diagnostic imaging and radiotherapy. 16alpha,17alpha-[(R)-1'-alpha-(5-[(76)Br]Bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione ([(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione (3)), a PR ligand with relative binding affinity (RBA)=65 and log P(o/w)=5.09+/-0.84, was synthesized via a two-step reaction, and its tissue biodistribution and metabolic stability were evaluated in estrogen-primed immature female Sprague-Dawley rats. [(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione 3 was synthesized in 5% overall yield with specific activity being 200-1250 Ci/mmol. [(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione 3 demonstrated high PR-mediated uptake in the target tissue uterus (8.72+/-1.84 %ID/g at 1 h) that was reduced by a blocking dose of unlabeled progestin R5020, but the nonspecific uptake in blood and muscle (2.11+/-0.14 and 0.89+/-0.16 %ID/g at 1 h, respectively) was relatively high. [(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione 3 was stable in whole rat blood in vitro, but it was not stable in vivo due to the fast metabolism that occurred in the liver, resulting in the formation of a more polar radioactive metabolite and free [(76)Br]bromide. The level of free [(76)Br]bromide in blood remained high during the experiment (2.11+/-0.14 %ID/g at 1 h and 1.52+/-0.24 %ID/g at 24 h). The tissue distribution of [(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione 3 at 1 and 3 h was compared with that of the (18)F analogs, [(18)F]FFNP fluoro furanyl norprogesterone (FFNP) 1 and ketal 2. [(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione 3 may have potential for imaging PR-positive breast tumors at early time points, but it is not suitable for imaging at later times or for radiotherapy.

Synthesis and biodistribution studies of two novel radioiodinated areno-annelated estra-1,3,5(10),16-tetraene-3-ols as promising estrogen receptor radioligands

Two novel radioiodinated areno-annelated estra-1,3,5(10),16-tetraenes, [125I]2-iodo-1′-methoxybenzo[4′,3′:16,17]estra-1,3,5(10),16-tetraene-3-ol (2-[125I]-MEBE) and [125I]4-iodo-1′-methoxybenzo[4′,3′:16,17]estra-1,3,5(10),16-tetraene-3-ol, (4-[125I]-MEBE) were synthesized for evaluation as potential ligands for the estrogen receptor. Radioiodination of 1′-methoxybenzo[4′,3′:16,17]estra-1,3,5(10),16-tetraene-3-ol at the A ring was accomplished by electrophilic aromatic substitution using [125I] sodium iodide and chloramine-T as oxidant. After purification by reverse phase HPLC, the two radioisomers (2-[125I]-MEBE and 4-[125I]-MEBE) were obtained in a radiochemical yield of 42 and 48%, respectively, in a radiochemical purity of greater than 95% and a high specific activity. The effect of the site of radioiodination (C2 vs C4) on the biological behaviour of the molecules was evaluated through biodistribution studies in immature female Sprague-Dawley rats. Both 2-[125I]-MEBE and 4-[125I]-MEBE are stable in vivo and are mainly excreted through the hepatobiliary pathway. Both localize in the uterus and ovaries via a receptor-mediated process, where the 2-[125I]-MEBE isomer has the higher specific ER binding and uterus selectivity. The favourable in vitro/in vivo stability and biodistribution profiles suggest that these radioligands are good candidates for further exploration of their potential clinical application. Copyright © 2006 John Wiley & Sons, Ltd.

Effect of Symplocos racemosa Roxb. on gonadotropin release in immature female rats and ovarian histology

In the present study we are reporting in vivo effect of aqueous extracts of Symplocos racemosa Roxb. (Fam. Symplocaceae) on serum FSH and LH levels in immature female Sprague–Dawley rats under basal conditions. Symplocos racemosa is used in Indian System of Medicine (ISM) for various female disorders. Aqueous extract on oral administration significantly stimulated serum FSH level ( P < 0.016) along with the rise in serum LH level ( P < 0.001). Moreover, histopathological studies revealed enhanced folliculogenesis, presence of mature follicles and detached oocytes, which are result of increased FSH and LH levels. Further, an increase in the ovary weight of treated animals was found due to observed FSH surge. These results are in concordance with the traditional use of the drug for female disorders.