4-vinylcyclohexene Diepoxide Research Articles

Cardiac changes during the peri-menopausal period in a VCD-induced murine model of ovarian failure.

AimCardiovascular disease (CVD) risk is lower in pre‐menopausal females vs age matched males. After menopause risk equals or exceeds that of males. CVD protection of pre‐menopausal females is ascribed to high circulating oestrogen levels. Despite experimental evidence that oestrogen are cardioprotective, oestrogen replacement therapy trials have not shown clear benefits. One hypothesis to explain the discrepancy proposed hearts remodel during peri‐menopause. Peri‐menopasual myocardial changes have never been investigated, nor has the ability of oestrogen to regulate heart function during peri‐menopause.MethodsWe injected female mice with 4‐vinylcyclohexene diepoxide (VCD, 160 mg/kg/d IP) to cause gradual ovarian failure over 120d and act as a peri‐menopausal modelResultsLeft ventricular function assessed by Langendorff perfusion found no changes in VCD‐injected mice at 60 or 120 days compared to intact mice. Cardiac myofilament activity was altered at 60 and 120 days indicating a molecular remodelling in peri‐menopause. Myocardial TGF‐β1 increased at 60 days post‐VCD treatment along with reduced Akt phosphorylation. Acute activation of oestrogen receptor‐α (ERα) or ‐β (ERβ) depressed left ventricular contractility in hearts from intact mice. ER‐regulation of myocardial and myofilament function, and myofilament phosphorylation, were disrupted in the peri‐menopausal model. Disruption occurred without alterations in total ERα or ERβ expression.ConclusionsThis is the first study to demonstrate remodelling of the heart in a model of peri‐menopause, along with a disruption in ER‐dependent regulation of the heart. These data indicate that oestrogen replacement therapy initiated after menopause affects a heart that is profoundly different from that found in reproductively intact animals.

Huyang yangkun formula protects against 4-Vinylcyclohexene diepoxide-induced premature ovarian insufficiency in rats via the Hippo–JAK2/STAT3 signaling pathway

Ethnopharmacological relevanceHuyang Yangkun Formula (HYF) has been prescribed for premature ovarian insufficiency (POI) for decades in the clinical setting. Little is known regarding its underlying molecular mechanism. This study was conducted to elucidate the possible mechanism of the protective potential of HYF against POI induced by the industrial chemical 4-vinylcyclohexene diepoxide (VCD) in rats. Aim of the studyQuality control of HYF was conducted via HPLC and UPLC-MS. Female rats were injected with VCD (160 mg/kg) daily for 15 days. Then, 1.35 g/kg (low dose) or 0.235 g/kg (high dose) HYF was administered once/day for 25 days. Serum AMH, FSH, E2, ALT, AST, BUN and Cr levels were detected through ELISA and HE-stained follicles were counted in ovarian sections. Additionally, RNA-seq profiling analysis and functional assays were used to screen for differentially expressed genes and key regulators with potentially important roles associated with HYF. ResultsThe ovaries of POI rats contained fewer antral and maturing follicles (p < 0.05) than those of control rats, whereas atretic follicles were increased significantly (p < 0.05), and AMH levels were significantly lower in the VCD group than in the control group (p < 0.05). These conditions showed some improvement after low- and high-dose HYF treatment. Low- and high-dose HYF increased AMH levels by 42.4% and 25.9% and decreased FSH levels by 17.5% and 24.1%, respectively, in comparison to the VCD group. The two HYF dosage groups showed significantly increased numbers of antral and maturing follicles but a reduced number of atretic follicles (p < 0.05). HYF down-regulation of JAK, Lats2 and YAP mRNA expression gene expression (p < 0.05) compared with the VCD group. HYF resulted in a strongly attenuated VCD-induced phosphorylation of JAK2 and STAT3 (p < 0.01) and YAP (p < 0.001), but induced an increase in protein levels of LATS2 (p < 0.05). ConclusionOur findings demonstrated the treatment efficacy of HYF in POI rats and showed that HYF repairs the dysfunction and enhances the ovarian function of POI rats through the Hippo–JAK2/STAT3 signaling pathway.

Menopause and FOXP3+ Treg cell depletion eliminate female protection against T cell-mediated angiotensin II hypertension.

Although it is known that the prevalence and severity of hypertension increases in women after menopause, the contribution of T cells to this process has not been explored. Although the immune system is both necessary and required for the development of angiotensin II (ANG II) hypertension in men, we have demonstrated that premenopausal women are protected from T cell-mediated hypertension. The goal of the current study was to test the hypotheses that 1) female protection against T cell-mediated ANG II hypertension is eliminated following progression into menopause and 2) T regulatory cells (Tregs) provide premenopausal protection against ANG II-induced hypertension. Menopause was induced in Rag-1-/- mice (via 4-vinylcyclohexene diepoxide), and all mice received a 14-day ANG II infusion. Donor CD3+ T cells were adoptively transferred 3 wk before ANG II infusion. In the absence of T cells, systolic blood pressure responses to ANG II were similar to those seen in premenopausal mice (Δ12 mmHg). After adoptive transfer of T cells, ANG II significantly increased systolic blood pressure in postmenopausal females (Δ28 mmHg). A significant increase in F4/80 positive renal macrophages, an increase in renal inflammatory gene expression, along with a reduction in renal expression of mannose receptor C-type 1, a marker for M2 macrophages, accompanied the increase in systolic blood pressure (SBP). Flow cytometric analysis identified that Tregs were significantly decreased in the spleen and kidneys of Rag-1-/- menopausal mice versus premenopausal females, following ANG II infusion. In a validation study, an anti-CD25 antibody was used to deplete Tregs in premenopausal mice, which induced a significant increase in SBP. These results demonstrate that premenopausal protection against T cell-mediated ANG II hypertension is eliminated once females enter menopause, suggesting that a change in hormonal status upregulates macrophage-induced proinflammatory and T cell-dependent responses. Furthermore, we are the first to report that the presence of Tregs are required to suppress ANG II hypertension in premenopausal females.NEW & NOTEWORTHY Whether progression into menopause eliminated female protection against T cell-mediated hypertension was examined. Menopausal mice without T cells remained protected against angiotensin II (ANG II) hypertension; however, in the presence of T cells, blood pressure responses to ANG II increased significantly in menopause. Underlying mechanisms examined were anti-inflammatory protection provided by T regulatory cells in premenopausal females and renal inflammatory processes involving macrophage infiltration and cytokine activation.

Ovarian failure induced by 4-vinylcyclohexene diepoxide worsens the autonomic cardiovascular response to chronic unpredictable stress in rats

AimsAfter menopause, women are more responsive to stress and more prone to exhibit hypertension, which elevates the risk of cardiac diseases. This vulnerability is due, in part, to the decline of ovarian steroids plasma levels. The 4-vinylciclohexane diepoxide (VCD) causes a gradual depletion of ovarian follicles causing loss of the normal ovarian function and a hormonal profile comparable to menopause in humans. We aimed to verify whether the ovarian failure (OF) worsens the cardiovascular autonomic response to stress. Main methodsRats were treated with VCD (160 mg/kg) or oil for 15 days, exposed to chronic unpredictable stress (CUS) for 10 days and studied 80 and 180 days after VCD treatment. Key findings80 days after VCD-treatment, stressed rats showed increased sympathetic nerve activity, reduced parasympathetic activity and an increase in the overall spontaneous baroreflex sensitivity (BRS). 180 days after VCD treatment, BRS was impaired and the vascular sympathetic activity was increased, independently of stress exposure. SignificanceNeither 80 nor 180 days after the onset of VCD-treatment the hypertensive effects of stress were enhanced in rats. However, OF led to a worsening on different aspects of the cardiovascular response to stress, which can cause cardiovascular complications when associated with ovarian aging.

L-arginine supplementation reduces blood pressure and plasma lipid levels in an animal model of perimenopause induced by 4-Vinylcyclohexene diepoxide

The incidence of women developing high blood pressure during perimenopause has been documented and is sustained till menopause. However, no study till date on beneficial effects of L-arginine supplementation on BP during perimenopause in humans and animal models of perimenopause.Female rats 28 days old were divided into 3 groups; Control group was injected (SC) daily with Corn oil (2.5 μl/g BW) for 15 days, allowed to grow till 12th week; VCD group was injected (SC) daily with 4-vinylcyclohexene diepoxide (160 mg/kg BW) diluted in Corn oil (2.5 μl/g BW) for 15 days, allowed to grow till 12th week; VCD + L-ARG group was injected as VCD group, allowed to grow till 8th week, then administered oral 100mg/kg L-arginine daily for additional 4 weeks. Caudal BP was measured with tail-cuff apparatus (Kent Scientific CODA system) at weeks eight, ten, and twelve. Terminal BP was also measured with a power-lab apparatus and blood samples were subsequently collected for measurement of plasma lipid profile.L-arginine supplementation significantly reduced systolic, diastolic and mean arterial BP parameters in the VCD + L-ARG group compared to the Control and VCD groups (P < 0.05). It also significantly reduced total cholesterol and LDL concentrations in the VCD + L-ARG group compared to the Control and VCD groups (P < 0.05). HDL concentration was significantly higher in the VCD and VCD + L-ARG groups compared to the Control group (P < 0.05). These results show that L-arginine supplementation ameliorates some cardiovascular risk factors during perimenopausal transitory period.

The effects of 4-vinylcyclohexene diepoxide on the testes of dogs

The effects of 4-vinylcyclohexene diepoxide on the testes of dogs

119 Combined exposure to phthalates and 4-vinylcyclohexene diepoxide accelerate the loss of ovarian follicles, leading to premature ovarian failure in rat model

Premature ovarian failure (POF) is observed in women under 40 years with primary or secondary amenorrhea. The causes for POF are idiopathic, genetic, iatrogenic incidence, autoimmunity, and adverse effects from toxic chemical exposures. Several classes of chemicals have been shown to alter follicle development and reduce fertility, leading to POF in mammals. We investigated the synergistic effects of 4-vinylcyclohexene diepoxide (VCD) and phthalates, including di(2-ethylhexyl) phthalate (DEHP), butyl benzyl phthalate (BBP), and di-n-butyl phthalate (DBP), on POF. Both VCD and these phthalates have been reported to disrupt the folliculogenesis and steroidogenesis in many species. In the vehicle group, female Sprague-Dawley rats (8 weeks of age) received an IP injection of corn oil daily for 2 weeks. The 7 other groups received VCD+corn oil, DEHP, DEHP+VCD, BBP, BBP+VCD, DBP, or DBP+VCD. After receiving the VCD or vehicle injection IP for 2 weeks, corn oil or a phthalate were orally administered once a day for the entire 6-week study period. The mRNA expressions of Amh and Sohlh2 were significantly decreased in the combination groups compared to the control and individual groups. Serum Amh levels were significantly lower in the combination groups. Additionally, serum levels of FSH were markedly higher in combination groups. Our findings suggest that the combination of VCD and phthalates can accelerate the POF by disturbance in folliculogenesis and hormone regulation.

Thyroid hormone measurement in developmental toxicity studies – technical and interpretation issues

Humans are at daily risk by simultaneous exposures to a broad spectrum of man-made chemicals in the commercial products. Several classes of chemicals have been shown to alter follicle development and reduce fertility, leading to premature ovarian failure (POF) in mammals. We investigate the synergistic effects of 4-vinylcyclohexene diepoxide (VCD) and phthalate, including di(2-ethylhexyl) phthalate (DEHP), butyl benzyl phthalate (BBP) and di-n-butyl phthalate (DBP) on POF. Combination exposure with VCD and phthalate significantly reduced the numbers of primary follicles. The expressions of Amh and Sohlh2 were significantly decreased in the combination groups. Serum Amh levels were significantly lower in the combination groups. Additionally, serum levels of follicle-stimulating hormone were significantly increased in combination groups. Taken together, exposure to phthalates promotes the depletion of follicular follicles and consequently increases the risk of premature menopause, and combined exposure of phthalates and VCD to early menopausal women is likely to aggravate the POF syndrome.

The microbiome and brain development

Humans are at daily risk by simultaneous exposures to a broad spectrum of man-made chemicals in the commercial products. Several classes of chemicals have been shown to alter follicle development and reduce fertility, leading to premature ovarian failure (POF) in mammals. We investigate the synergistic effects of 4-vinylcyclohexene diepoxide (VCD) and phthalate, including di(2-ethylhexyl) phthalate (DEHP), butyl benzyl phthalate (BBP) and di-n-butyl phthalate (DBP) on POF. Combination exposure with VCD and phthalate significantly reduced the numbers of primary follicles. The expressions of Amh and Sohlh2 were significantly decreased in the combination groups. Serum Amh levels were significantly lower in the combination groups. Additionally, serum levels of follicle-stimulating hormone were significantly increased in combination groups. Taken together, exposure to phthalates promotes the depletion of follicular follicles and consequently increases the risk of premature menopause, and combined exposure of phthalates and VCD to early menopausal women is likely to aggravate the POF syndrome.

Dopamine and prolactin interaction in rat developmental and reproductive toxicity studies

Humans are at daily risk by simultaneous exposures to a broad spectrum of man-made chemicals in the commercial products. Several classes of chemicals have been shown to alter follicle development and reduce fertility, leading to premature ovarian failure (POF) in mammals. We investigate the synergistic effects of 4-vinylcyclohexene diepoxide (VCD) and phthalate, including di(2-ethylhexyl) phthalate (DEHP), butyl benzyl phthalate (BBP) and di-n-butyl phthalate (DBP) on POF. Combination exposure with VCD and phthalate significantly reduced the numbers of primary follicles. The expressions of Amh and Sohlh2 were significantly decreased in the combination groups. Serum Amh levels were significantly lower in the combination groups. Additionally, serum levels of follicle-stimulating hormone were significantly increased in combination groups. Taken together, exposure to phthalates promotes the depletion of follicular follicles and consequently increases the risk of premature menopause, and combined exposure of phthalates and VCD to early menopausal women is likely to aggravate the POF syndrome.

Mullerian-inhibiting substance augments ovarian response to superovulation in mice with diminished ovarian reserve

To determine whether pretreatment with Mullerian-inhibiting substance (MIS) affects the oocyte yield of superovulation in a mouse model of diminished ovarian reserve (DOR). Preclinical placebo-controlled animal study. The DOR mouse model was created by giving intraperitoneal (IP) injections of 4-vinylcyclohexene diepoxide (VCD) to 6-week old nu/nu mice (n=3) at 160 mg/kg daily for 5 days. Control mice (n=3) were injected IP with vehicle control (corn oil). Mice were euthanized 14 days later. Dissected ovaries were fixed in 4% paraformaldehyde, embedded in paraffin blocks in an automated tissue processor, cut into 8-um sections, and mounted onto glass slides, which were stained with hematoxylin and eosin. Under light microscopy, ovarian follicles were classified into five groups - primordial, primary, secondary, antral, and atretic - and counted. A cohort of DOR mice was subsequently treated with subcutaneous recombinant human MIS protein (750 ug/kg, n=3) or saline (n=4) twice daily for 40 days. After 30 days of observation, superovulation was performed in standard fashion with pregnant mare serum gonadotropin 7.5 IU IP, followed by human chorionic gonadotropin (hCG) 7.5 IU IP 49 hours later to trigger ovulation. 16 hours after hCG, oocytes were dissected from the oviducts of euthanized mice and were counted. Morphologic assessment of collected oocytes was performed under a stereo microscope, and the percentage of degenerated oocytes collected from each mouse was noted. Oocyte yields and percentage of degenerated oocytes of MIS-treated and saline control mice were compared using student’s t-test. A p-value of < 0.05 was considered significant. VCD administration as described above creates a DOR phenotype in mice, by depleting primordial follicle reserve by 73%, while maintaining ovulatory function. DOR mice treated with MIS produced, on average, 29 oocytes through superovulation compared to saline control mice who produced only 11 oocytes (p = 0.038). Furthermore, more oocytes were morphologically abnormal in controls compared to MIS-treated mice (52.2% versus 30.1%, p = 0.026). In the setting of DOR, treatment with MIS protein significantly enhances oocyte yield following superovulation and leads to a lower proportion of degenerated oocytes. These findings suggest a future clinical application of MIS in IVF protocols, particularly for patients with DOR.

Abstract 559: AMP-Activated Protein Kinase And Estrogen-Dependent Mechanisms Underlying Increased Susceptibility To Cardiovascular Disease During Menopause

According to the CDC (2017), more women than men have died from heart disease over the last 20-25 years. Yet, premenopausal women are protected against heart and cardiovascular disease (CVD) compared to age-matched men. The cellular and molecular mechanisms underlying the transition from premenopause/perimenopause (CVD-resistance) to postmenopause (CVD-susceptible) in women is unknown and is the focus of this project. The critical barrier impeding translational progress is the lack of appropriate models to study menopause. Most studies have used surgical removal of ovaries (ovariectomy) as a model of menopause; yet this technique poorly recapitulates the natural, physiological transition to menopause that 90% of women experience. We overcome this barrier with administration of the chemical, 4-vinylcyclohexene diepoxide (VCD), where we can mirror progressive ovarian failure and preserve the critical “perimenopause” transitional period, identical to humans. Using this model, we demonstrate that perimenopausal , like cycling (premenopausal) females, are protected from pathological angiotensin II (Ang II)-induced cardiac remodeling, while menopausal females are not. Our novel finding that perimenopausal females remain protected, despite irregular cycling (prior to complete loss of estrogen), underscores the importance of studying the role of estrogen in CVD, across the transition from perimenopause to menopause. Multiple molecular, genetic and cellular mechanisms have been suggested to underlie protection against CVD in non-cycling females, many of which put estrogen as the key mediator. We discovered that one of these pathways, the adenosine monophosphate-activated kinase (AMPK) signaling axis, is activated by estrogen through direct binding of estrogen receptors to the α-catalytic subunit of AMPK. Taken together with our published and preliminary data, we hypothesis that loss of AMPK signaling is responsible for the exacerbated pathological cardiac remodeling in menopause (see Fig.). To date, there are no studies that have tested the ability of AMPK activation to mitigate menopausal susceptibility to CVD. Aim: Determine if AMPK is necessary and/or sufficient to impart protection against pathological cardiac remodeling in menopausal females. Rationale: We discovered that AMPK signaling is severely disabled during menopause where females are more susceptible to CVD. Using the VCD mouse model of menopause, we will determine if protection against Ang II-induced cardiac remodeling during menopause is AMPK-dependent. Summary: We plan to determine the cellular and molecular mechanisms in premenopausal and perimenopausal females that prevent associated cardiovascular morbidities; and determine the underlying shift that gives rise to increased susceptibility to CVD in menopausal females.

Abstract 377: Time-restricted feeding: A dietary intervention to treat breast cancer in postmenopausal obese mice

Abstract Background: Abundant evidence indicates that obesity increases risk for breast cancer and the incidence further increases by 40% in obese postmenopausal women. Therefore it is envisioned by diverse research groups that the risk of breast cancer in obese postmenopausal women is an ever-increasing menace that needs to be curbed soon with effective practical strategies that could have a far-reaching impact. In this setting, time-restricted feeding (TRF), the practice of consuming ad libitum energy during the normal active phase, has been demonstrated to reinforce normal metabolic regulation, thereby attenuating obesity-driven metabolic deregulation. Although studies have assessed the effect of TRF on metabolism, no investigations have been carried out in human or mouse exploring TRF in cancer remission. Therefore, the present work is to understand the efficacy of TRF for improved breast cancer remission in postmenopausal obese female mice. Further, the work explores the mechanistic link of TRF for reduced tumor growth. Methods: Ovariectomized and 4-vinylcyclohexene diepoxide (VCD) treated mice were used as a postmenopausal model. Both ovariectomized and VCD mice were made obese by feeding them with 60% high-fat diet (HFD) and then grouped into ad libitum group (24 h access to food) and TRF group (8 hr access to food at night) to assess metabolic and tumor growth effect of TRF. To develop breast tumor, mice were injected with py230 breast cancer cell line into the mammary fat pad. The metabolic effect of TRF was assessed by glucose tolerance test, insulin tolerance test, body weight measurement, food intake, lipid accumulation in liver by HE staining and measurement of different tissue weight. Measuring the tumor volume over time and tumor weight assessed TRF effect on tumor growth. Performing a tumor growth study in mice fed with HFD and HFD containing diazoxide assessed insulin dependency of tumor growth in ad libitum group. Insulin-dependent tumor growth was validated by tumor growth study in normal chow-fed mice implanted with insulin pump or without pump (control). Results: The preliminary studies in ovariectomized and VCD-treated postmenopausal mice suggest that restricting access to Western-style HFD in active night phase improves insulin resistance, glucose tolerance, and hepatic steatosis. Further, TRF exhibited reduced tumor growth compared to ad libitum group. More importantly, the results from tumor growth study in mice fed with HFD with/without diazoxide or normal chow mice with/without insulin pump, suggest that the tumor growth is insulin dependent and TRF may be acting through attenuating insulin signaling. Conclusion: Experimental and animal model data corroborate that TRF improves metabolic deregulation and reduces breast tumor growth in HFD-fed obese mice. The results suggest putative application of such therapeutic intervention for breast cancer therapy. Citation Format: Manasi Das, Emilie Gross, Deepak Kumar, Consuelo Sauceda, Hyun-Tae Park, Dorothy D. Sears, Lesley Ellies, Nicholas Webster. Time-restricted feeding: A dietary intervention to treat breast cancer in postmenopausal obese mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 377.

Cardiovascular Autonomic Responses in the VCD Rat Model of Menopause: Effects of Short- and Long-Term Ovarian Failure.

After menopause, hypertension elevates the risk of cardiac diseases, one of the major causes of women's morbidity. The gradual depletion of ovarian follicles in rats, induced by 4-vinylcyclohexene diepoxide (VCD), is a model for studying the physiology of menopause. 4-Vinylcyclohexene diepoxide treatment leads to early ovarian failure (OF) and a hormonal profile comparable to menopause in humans. We have hypothesized that OF can compromise the balance between sympathetic and parasympathetic tones of the cardiovascular system, shifting toward dominance of the former. We aimed to study the autonomic modulation of heart and blood vessels and the cardiovascular reflexes in rats presenting short-term (80 days) or long-term (180 days) OF induced by VCD. Twenty-eight-day-old Wistar rats were submitted to VCD treatment (160 mg/kg, intraperitoneally) or vehicle (control) for 15 consecutive days and experiments were conducted at 80 or 180 days after the onset of treatment. Long-term OF led to an increase in the sympathetic activity to blood vessels and an impairment in the baroreflex control of the heart, evoked by physiological changes in arterial pressure. Despite that, long-term OF did not cause hypertension during the 180 days of exposure. Short-term OF did not cause any deleterious effect on the cardiovascular parameters analyzed. These data indicate that long-term OF does not disrupt the maintenance of arterial pressure homeostasis in rats but worsens the autonomic cardiovascular control. In turn, this can lead to cardiovascular complications, especially when associated with the aging process seen during human menopause.

Depletion of follicles accelerated by combined exposure to phthalates and 4-vinylcyclohexene diepoxide, leading to premature ovarian failure in rats

Humans are at daily risk by simultaneous exposures to a broad spectrum of man-made chemicals in the commercial products. Several classes of chemicals have been shown to alter follicle development and reduce fertility, leading to premature ovarian failure (POF) in mammals. We investigate the synergistic effects of 4-vinylcyclohexene diepoxide (VCD) and phthalate, including di(2-ethylhexyl) phthalate (DEHP), butyl benzyl phthalate (BBP) and di-n-butyl phthalate (DBP) on POF. Combination exposure with VCD and phthalate significantly reduced the numbers of primary follicles. The expressions of Amh and Sohlh2 were significantly decreased in the combination groups. Serum Amh levels were significantly lower in the combination groups. Additionally, serum levels of follicle-stimulating hormone were significantly increased in combination groups. Taken together, exposure to phthalates promotes the depletion of follicular follicles and consequently increases the risk of premature menopause, and combined exposure of phthalates and VCD to early menopausal women is likely to aggravate the POF syndrome.

Peroxiredoxin 2 deficiency accelerates age-related ovarian failure through the reactive oxygen species-mediated JNK pathway in mice

Reactive oxygen species (ROS) produced in biological reactions have been shown to contribute to ovarian aging. Peroxiredoxin 2 (Prx2) is an antioxidant enzyme that protects cells by scavenging ROS; however, its effect on age-related, oxidative stress-associated ovarian failure has not been reported. Here, we investigated its role in age-related ovarian dysfunction and 4-vinylcyclohexene diepoxide (VCD)-induced premature ovarian failure using Prx2-deficient mice. Compared to those in wildtype (WT) mice, serum levels of anti-Müllerian hormone, 17β-estradiol, and progesterone and numbers of follicles and corpora lutea were significantly lower in 18-month-old Prx2−/− mice. Moreover, levels of Bax, cytochrome c, cleaved caspase-3, and phosphorylated JNK proteins were higher and numbers of apoptotic (terminal deoxynucleotidyl transferase dUTP nick end labeling-positive) cells were considerably greater in 18-month-old Prx2−/− ovaries than WT ovaries. Furthermore, the effects of the ovarian toxicant VCD in significantly enhancing ROS levels and apoptosis through activation of JNK-mediated apoptotic signaling were more pronounced in Prx2−/− than WT mouse embryonic fibroblasts. Expression of the steroidogenic proteins StAR, CYP11A1, and 3β-HSD and serum levels of 17β-estradiol and progesterone were also reduced to a greater extent in Prx2−/− mice than WT mice after VCD injection. This reduced steroidogenesis was rescued by addition of the Prx mimic ebselen or JNK inhibitor SP600125. This constitutes the first report that Prx2 deficiency leads to acceleration of age-related or VCD-induced ovarian failure by activation of the ROS-induced JNK pathway. These findings suggest that Prx2 plays an important role in preventing accelerated ovarian failure by inhibiting ROS-induced JNK activation.

Effects of electroacupuncture at "Zhongliao" (BL 33) and "Tianshu" (ST 25) on ovarian function in rats with premature ovarian insufficiency

To explore the effects of electroacupuncture (EA) at "Zhongliao" (BL 33) and "Tianshu" (ST 25) on ovarian function in rats with premature ovarian insufficiency (POI). A total of 48 SD female rats with regular estrus were divided into a blank group (n=8), a model group (n=10), an EA group (n=10), a binding group (n=10) and a tamoxifen (TAM) group (n=10). The rats in the model group, EA group, binding group and TAM group were all treated with intraperitoneal injection of 4-vinylcyclohexene diepoxide (VCD, 160 mg/kg) for 15 consecutive days to establish the model of POI; the rats in the blank group were treated with normal diet. After the model was established successfully, the rats in the EA group were treated with EA at "Zhongliao" (BL 33) and "Tianshu" (ST 25) with continuous wave (1 to 3 Hz, 0.1 to 1 mA) for 20 minutes, once a day (five times a week) for the first two weeks and once every other day (three times a week) for the following two weeks. The rats in the TAM group were treated with subcutaneous injection of tamoxifen (1mg/kg), once a day (five times a week) for the first two weeks and once every other day (three times a week) for the following two weeks. The rats in the binding group were bound by a small sack as the EA group. The rats in the blank group and the model group were treated with normal diet. After four weeks, the sexual gland weight and index were tested in each group; the ELISA method was applied to test the level of anti-mllerian hormone (AMH) and inhibin B; the morphology of ovary was observed; the number of primordial follicles, primary follicle, antral follicle and atretic follicle was counted; the expression of insulin-like growth factor-1 (IGF-1) and insulin-like growth factor-1 receptor (IGF-1R) were measured. (1) Compared with the blank group, the ovary weight, ovary index, uterus weight and uterus index were significantly decreased after treatment in the model group, EA group, binding group and TAM group (all P<0.01); but the differences between the model group and the EA group, binding group, TAM group were not significant (all P>0.05). (2) Compared with the blank group, the levels of serum AMH, inhibin B and E2 were significantly reduced; the levels of FSH and LH were significantly increased in the model group; EA group, binding group and TAM group (all P<0.01). Compared with the model group, the levels of serum AMH, inhibin B and E2 were significantly increased, the level of FSH and LH were significantly reduced in the EA group and TAM group (all P<0.01). (3) Compared with the blank group, in the model group, EA group, binding group and TAM group the ovary was dark red and pale, surrounded by particle or not; the morphology was small and atrophic; the primordial follicles was reduced even vanished; the structure of primary follicle was damaged and loosely arranged; the mature follicle was few; the atretic follicle and interstitial gland were increased. (4) Compared with the blank group, the expressions of IGF-1 mRNA and IGF-1R mRNA were increased in the model group (all P<0.01); compared with the blank group, the expression of IGF-1 mRNA was increased in the binding group (P<0.05), but that of IGF-1R mRNA was not significantly different (P>0.05); compared with the model group, the expression of IGF-1 mRNA was not significantly different in the EA group, binding group and TAM group (all P>0.05), but that of IGF-1R mRNA was reduced (P<0.05, P<0.01). EA at "Zhongliao" (BL 33) and "Tianshu" (ST 25) has improvement effect on ovarian function in rats with VCD-induced POI, which is likely to be related to regulating the IGF-1R mRNA expression to improve the IGF-1/ IGF-1R axis.

Protective effects of electroacupuncture pretreatment on ovarian in rats with premature ovarian insufficiency

To explore the protective effect and possible mechanism of electroacupuncture (EA) pretreatment at "Zhongliao" (BL 33) and "Tianshu" (ST 25) on ovarian in rats with premature ovarian insufficiency (POI). A total of 32 SD female rats with regular estrus were randomly divided into a blank group, a model group, an EA group and an immobilization group, 8 rats in each one. The rats in the model group, EA group and immobilization group were treated with intraperitoneal injection of 4-vinylcyclohexene diepoxide (160 mg/kg) for 15 days to establish the model. From the beginning of model establishment, the rats in the EA group were treated with EA pretreatment at "Zhongliao" (BL 33) and "Tianshu" (ST 25), once a day for the first two weeks (5 treatments per week) and once every two days for the last two weeks (3 treatments per week). EA was given 20 min per treatment for four weeks. The rats in the immobilization group were treated with immobilization with identical duration, frequency and total amount as the EA group. No treatment was given in the blank group and model group. When model was successfully established in the model group, rats were sacrificed to test the weight of sexual gland and the sexual gland index; the ELISA method was applied to test the level of anti-müllerian hormone (AMH) and inhibin B; the radioimmunoassay method was applied to test the level of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), progesterone (P) and testosterone (T). In addition, the morphology and the number of ovarian follicles were observed; and western blot method was applied to test the expression of B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax) in ovarian tissue. Compared with the blank group, the bilateral ovary weight, ovary index, uterus weight and uterus index decreased (P<0.05, P<0.01); serum AMH, inhibin B, E2 and P reduced, while serum FSH, LH and T increased in the model group (all P<0.01); primordial follicles and primary follicles decreased, while atretic follicles increased in the model group (all P<0.01); the expression of Bax protein increased, while the expression of Bcl-2 protein decreased in the model group (both P<0.01). Compared with the model group, the bilateral ovary weight, ovary index and uterus weight increased (P<0.05, P<0.01); serum AMH, inhibin B, E2 and P increased, while serum FSH and LH reduced (P<0.05, P<0.01); primordial follicles and primary follicles increased, while atretic follicles reduced (P<0.05, P<0.01); the expression of Bax protein reduced, while the expression of Bcl-2 protein increased in the EA group (P<0.05, P<0.01). The differences of each outcome indexes were not significant between the model group and immobilization group (all P>0.05). EA pretreatment at "Zhongliao" (BL 33) and Tianshu (ST 25) for 4 weeks has certain protective effect on ovarian in POI rats, which is likely to be related with up-regulating ovarian granular cells, inhabiting the expression of Bcl-2 protein and down-regulating the expression of Bax protein.

Use of the REVERT® total protein stain as a loading control demonstrates significant benefits over the use of housekeeping proteins when analyzing brain homogenates by Western blot: An analysis of samples representing different gonadal hormone states.

Western blot is routinely used to quantify differences in the levels of target proteins in tissues. Standard methods typically use measurements of housekeeping proteins to control for variations in loading and protein transfer. This is problematic, however, when housekeeping proteins also are affected by experimental conditions such as injury, disease, and/or gonadal hormone manipulations. Our goal was to evaluate an alternative and perhaps superior method for conducting Western blot analysis of brain tissue homogenates from rats with distinct physiologically relevant gonadal hormone states. Tissues were collected from the hippocampus, frontal cortex, and striatum of young adult female rats that either were ovariectomized to model surgical menopause, or were treated with the ovatotoxin 4-vinylcyclohexene diepoxide (VCD) to model transitional menopause. Tissues also were collected from rats with a normal estrous cycle killed at proestrus when estradiol levels are high, and at diestrus when estradiol levels are low. Western blot detection of α-tubulin, β-actin, and GAPDH was performed and were compared for sensitivity and reliability with a fluorescent total protein stain (REVERT®). Results show that the total protein stain was much less variable across samples and had a greater linear range than α-tubulin, β-actin, or GAPDH. The stain was stable and easy to use, and did not interfere with the immunodetection or multiplexed detection of the housekeeping proteins. In addition, we show that normalization of our data to total protein, but not to GAPDH, revealed significant differences in α-tubulin expression in the hippocampus as a function of treatment, and that gel-to-gel consistency in measuring differences between paired samples run on multiple gels was significantly better when data were normalized to total protein than when normalized to GAPDH. These results demonstrate that the REVERT® total protein stain can be used in Western blot analysis of brain tissue homogenates to control for variations in loading and protein transfer, and provides significant advantages over the use of housekeeping proteins for quantifying changes in the levels of multiple target proteins.

ContraPest®, a New Tool for Rodent Control

Author(s): Pyzyna, Brandy R.; Trulove, Nicholas F.; Mansfield, Clayton H.; McMillan, Ryan A.; Ray, Courtney N.; Mayer, Loretta P. | Abstract: Lethal rodenticides and other lethal tools of managing commensal rodent populations long-term are not sustainable due to population rebounds and increasing resistance to rodenticides. The use of integrated pest management (IPM) programs are more prevalent due to consumer desire to decrease rodenticide use and utilize environmentally friendly, humane methods. IPM plans often require multiple tools to control an infestation, such as physical, biological and chemical measures. Here, we propose that rodent population management would benefit from a new tool aimed at targeting the biological source of overabundance: reproduction. SenesTech, Inc. (Flagstaff, AZ USA) has developed ContraPest®, a liquid bait that limits the reproductive capacity of both male and female wild Norway and Roof rats. The two active ingredients, 4-vinylcyclohexene diepoxide (VCD) and triptolide, deplete all stages of follicles in the female and disrupt spermatogenesis in the male. Laboratory and field studies reveal that ContraPest® is palatable and repeatedly consumed by rats even when provided with ad libitum food and water. Studies involving laboratory and wild caught rats have demonstrated a 93 - 100% reduction in litter sizes of rats treated with ContraPest® compared to control rats. ContraPest® was tested on free ranging rat populations in agricultural and urban settings. Rat populations on protein production farms decreased by an average of 46% following 100 days of treatment with ContraPest®. In a complex urban environment, where property boundaries limit access to populations and foraging areas, ContraPest® reduced the seasonal population peak by 67% after 133 days of baiting. These studies, combined with all of our field studies and population reduction models, demonstrate that ContraPest® is a highly effective rodent contraceptive bait in a variety of environments. We strongly believe that adding/implementing fertility management via ContraPest® to an IPM program would enhance long-term rodent population control in rural, urban, and agricultural environments.