Magnoliae Flos (MF) is a medicinal herb widely employed in traditional medicine for relieving sinusitis, allergic rhinitis, headaches, and toothaches. Here, we investigated the potential preventive effects of MF extract (MFE) against 4-vinylcyclohexene diepoxide (VCD)-induced ovotoxicity in ovarian cells and a mouse model of premature ovarian insufficiency (POI). The cytoprotective effects of MFE were assessed using CHO-K1 or COV434 cells. In vivo, B6C3F1 female mice were intraperitoneally injected with VCD for two weeks to induce POI, while MFE was orally administered for four weeks, beginning one week before VCD administration. VCD led to a significant decline in the viabilities of CHO-K1 and COV434 cells and triggered excessive reactive oxygen species (ROS) production and apoptosis specifically in CHO-K1 cells. However, pretreatment with MFE effectively prevented VCD-induced cell death and ROS generation, while also activating the Akt signaling pathway. In vivo, MFE increased relative ovary weights, follicle numbers, and serum estradiol and anti-Müllerian hormone levels versus controls under conditions of ovary failure. Collectively, our results demonstrate that MFE has a preventive effect on VCD-induced ovotoxicity through Akt activation. These results suggest that MFE may have the potential to prevent and manage conditions such as POI and diminished ovarian reserve.

Some rodent species cause significant damage to agriculture and forestry, and some can transmit pathogens to humans and livestock. The common vole (Microtus arvalis) is widespread in Europe, and its population outbreaks have resulted in massive crop loss. Bait-based fertility control could contribute to rodent pest management. Bait containing 4-vinylcyclohexene diepoxide (VCD) and triptolide (TP), registered as ContraPest®, was delivered to male common voles for 14 or 28 consecutive days. The effects on reproductive structures and residues in the liver and testes were assessed. There was no effect on testis weight, sperm viability, sperm motility and oxidative stress in sperm cells. Results regarding the mitochondrial membrane potential of sperm, DNA fragmentation and progressively motile sperm cells were inconclusive. However, there was an increase in morphological sperm defects in voles treated for 14/28 days and fewer normal sperm cells in voles treated for 28 days. There were no TP residues in the testes, few and low TP residues and no VCD residues in liver tissues, making considerable secondary exposure to non-target species unlikely. Treatments with VCD + TP seemed to have minor effects on the reproductive organs of males. Further studies should evaluate the effect of VCD + TP on females and on the reproductive success of common voles and other pest rodent species.

The occupational chemical 4-Vinylcyclohexene diepoxide (VCD) is a reproductively toxic environmental pollutant that causes follicular failure, leading to premature ovarian insufficiency (POI), which significantly impacts a woman\'s physical health and fertility. Investigating VCD\'s pathogenic mechanisms can offer insights for the prevention of ovarian impairment and the treatment of POI. This study established a mouse model of POI through intraperitoneal injection of VCD into female C57BL/6 mice for 15 days. The results were then compared with those of the control group, including a comparison of phenotypic characteristics and transcriptome differences, at two time points: day 15 and day 30. Through a comprehensive analysis of differentially expressed genes (DEGs), key genes were identified and validated some using RT-PCR. The results revealed significant impacts on sex hormone levels, follicle number, and the estrous cycle in VCD-induced POI mice on both day 15 and day 30. The DEGs and enrichment results obtained on day 15 were not as significant as those obtained on day 30. The results of this study provide a preliminary indication that steroid hormone synthesis, DNA damage repair, and impaired oocyte mitosis are pivotal in VCD-mediated ovarian dysfunction. This dysfunction may have been caused by VCD damage to the primordial follicular pool, impairing follicular development and aggravating ovarian damage over time, making it gradually difficult for the ovaries to perform their normal functions.

Dehydroepiandrosterone (DHEA) is frequently integrated as an adjuvant in over a quarter of controlled ovarian hyperstimulation (COH) protocols, despite the ongoing debate regarding its impact. This study aimed to evaluate the efficacy and mechanism of action of DHEA on ovarian follicular development and ovarian response in rats with varying ovarian reserves. The study involved 75 rats categorized into 15 distinct groups. The ovarian tissues of rats in both the normal ovarian reserve group and the premature ovarian insufficiency (POI) group, induced by 4-vinylcyclohexene diepoxide (VCD) injection, were subjected to histomorphological and biochemical analyses following the administration of DHEA, either alone or in combination with COH. Follicle counting was performed on histological sections obtained from various tissues. Serum concentrations of anti-Müllerian hormone (AMH) and the quantification of specific proteins in ovarian tissue, including phosphatase and tensin homolog of chromosome 10 (PTEN), phosphoinositide 3-kinase (PI3K), phosphorylated protein kinase B (pAKT), cyclooxygenase 2 (COX-2), caspase-3, as well as assessments of total antioxidant status and total oxidant status, were conducted employing the ELISA method. The impact of DHEA exhibited variability based on ovarian reserve. In the POI model, DHEA augmented follicular development and ovarian response to the COH protocol by upregulating the PTEN/PI3K/AKT signaling pathway, mitigating apoptosis, inflammation, and oxidative stress, contrary to its effects in the normal ovarian reserve group. In conclusion, it has been determined that DHEA may exert beneficial effects on ovarian stimulation response by enhancing the initiation of primordial follicles and supporting antral follicle populations.

4-Vinylcyclohexene diepoxide (VCD) is a potentially hazardous industrial chemical that may enter a goat\'s body in various ways during industrial breeding. Ovarian granulosa cells (GCs) play a critical role in supporting follicle development and hormone synthesis. However, there are few studies on the effect of VCD on goat ovarian GCs. In this study, goat ovarian GCs were isolated and treated with VCD. The results showed that treatment with VCD increased the proportion of S phase and G2/M cells, but decreased the proportion of G1 phase. VCD treatment significantly inhibited the expression of cyclin A and cyclin-dependent kinase 2 (CDK2). But the expression levels of p21 and p27 were increased. VCD could induce an apparent increase in the proportion of apoptosis and the level of cleaved caspase 3. Treatment with VCD significantly reduced the progesterone and estrogen concentration in the medium in which goat ovarian GCs were cultured. Correspondingly, the expression level of steroidogenic acute regulatory protein (STAR) was significantly downregulated. Treatment with 0.25 and 0.5 mM VCD, the protein expression level of insulin-like growth factor 1 receptor (IGF1R) and Akt were significantly decreased. Moreover, treatment with 0.25 mM VCD significantly inhibited the phosphorylation of Akt. In conclusion, VCD exposure had cytotoxic effects such as decreased cell viability, disordered cell cycle, increased apoptosis, and interference with steroid hormone synthesis on goat GCs. These cytotoxic effects of VCD on goat GCs may be due to the downregulation of IGF1R and the inhibition of IGF1R/Akt signaling pathway.

The transition to menopause is characterized by mood, behavioral and metabolic changes. However, little is known about the changes in adrenal response to stress.
The aim of the study was to evaluate, in an animal model of perimenopause induced by 4-vinylcyclohexene diepoxide (VCD), (1) the endocrine and neuronal stress system activity in response to acute restraint stress and (2) the effect of hormonal therapy in this response.
Prepubertal female Wistar rats received daily injections (s.c) of oil or VCD (160 mg/kg) for 15 days. On 56th-66th days after treatment onset, the groups to be stressed received s.c implants containing placebo (PL), 17β-estradiol (E2), progesterone (P4), or E2P4. At 80 ± 5 days after VCD/oil injections, stress was applied for 30 min. Blood samples were collected immediately after and 60 min after the end of stress session from the tail tip followed by transcardial perfusion with PFA 4% for the assessment of c-Fos expression in the medial and posterior parvocellular (PaMP and PaPo) subdivisions of the paraventricular nucleus (PVN) and c-Fos/tyrosine hydroxylase in the locus coeruleus (LC) using immunohistochemistry. Control groups were not stressed nor received hormone therapy.
While basal corticosterone levels were similar between VCD-periestropausal and control rats, the secretion in response to stress in the VCD group was lower. This effect was prevented by P4 therapy. Inversely, basal levels of P4 were lower in VCD-periestropausal rats than in the controls, and no differences were found in response to stress between the groups. As expected, 30-min restraint stress increased c-Fos immunoreactivity in all brain areas studied in both control and VCD-periestropausal rats. However, the c-Fos increase in the PaMP region was attenuated. In all areas examined, there were no significant differences in the number of c-Fos-positive neurons across hormonal therapies.
This is the first study to demonstrate in a perimenopausal rat model that reproductive aging is accompanied by inadequate secretion of corticosterone in response to acute stress in association with the hypoactivation of the PaMP region of the PVN, while adrenal P4 response is preserved. Moreover, P4 therapy was shown to attenuate the effects of progressive ovarian failure on adrenal functioning during stress.

There is a sharp rise in cardiovascular disease (CVD) risk and progression with the onset of menopause. The 4-vinylcyclohexene diepoxide (VCD) model of menopause recapitulates the natural, physiological transition through perimenopause to menopause. We hypothesized that menopausal female mice were more susceptible to CVD than pre- or perimenopausal females. Female mice were treated with VCD or vehicle for 20 consecutive days. Premenopausal, perimenopausal, and menopausal mice were administered angiotensin II (ANG II) or subjected to ischemia-reperfusion (I/R). Menopausal females were more susceptible to pathological ANG II-induced cardiac remodeling and cardiac injury from a myocardial infarction (MI), while perimenopausal, like premenopausal, females remained protected. Specifically, ANG II significantly elevated diastolic (130.9 ± 6.0 vs. 114.7 ± 6.2 mmHg) and systolic (156.9 ± 4.8 vs. 141.7 ± 5.0 mmHg) blood pressure and normalized cardiac mass (15.9 ± 1.0 vs. 7.7 ± 1.5%) to a greater extent in menopausal females compared with controls, whereas perimenopausal females demonstrated a similar elevation of diastolic (93.7 ± 2.9 vs. 100.5 ± 4.1 mmHg) and systolic (155.9 ± 7.3 vs. 152.3 ± 6.5 mmHg) blood pressure and normalized cardiac mass (8.3 ± 2.1 vs. 7.5 ± 1.4%) compared with controls. Similarly, menopausal females demonstrated a threefold increase in fibrosis measured by Picrosirus red staining. Finally, hearts of menopausal females (41 ± 5%) showed larger infarct sizes following I/R injury than perimenopausal (18.0 ± 5.6%) and premenopausal (16.2 ± 3.3, 20.1 ± 4.8%) groups. Using the VCD model of menopause, we provide evidence that menopausal females were more susceptible to pathological cardiac remodeling. We suggest that the VCD model of menopause may be critical to better elucidate cellular and molecular mechanisms underlying the transition to CVD susceptibility in menopausal women.NEW & NOTEWORTHY Before menopause, women are protected against cardiovascular disease (CVD) compared with age-matched men; this protection is gradually lost after menopause. We present the first evidence that demonstrates menopausal females are more susceptible to pathological cardiac remodeling while perimenopausal and cycling females are not. The VCD model permits appropriate examination of how increased susceptibility to the pathological process of cardiac remodeling accelerates from pre- to perimenopause to menopause.

Background The ovotoxicity of 4-vinylcyclohexene diepoxide (VCD) has been established in several experimental models. Hesperidin (HSD) is a bi-flavonoid found in citrus fruits and has been reported to be a potent antioxidant and anti-inflammatory agent. Here, we have evaluated the rescue role of hesperidin on VCD-induced toxicity in the brain, ovary, and uterus of rats. Methods Six groups of rats containing ten rats in each group were orally given corn oil (control), hesperidin (100 mg/kg), hesperidin (200 mg/kg), VCD (250 mg/kg), VCD [(250 mg/kg)+hesperidin (100 mg/kg)] and VCD [(250 mg/kg)+hesperidin (200 mg/kg)] once a day for 30 days, respectively. Thereafter, we determined the selected biomarkers of oxidative damage, inflammation, endocrine balance, and histology of the reproductive organs. Results The data showed that hesperidin rescued VCD-induced increase in oxidative stress (hydrogen peroxide and malondialdehyde) and inflammatory (nitric oxide) biomarkers. In addition, hesperidin restored the reduction in antioxidant enzymes (catalase, glutathione S-transferase, glutathione peroxidase) activities and glutathione level in the brain, ovary, and uterus of rats (p<0.05). Lastly, hesperidin preserved the histological structure of the ovary and uterus of rats exposed to VCD. Conclusions Overall, the rescue role of hesperidin on VCD-induced toxicity in the brain and reproductive organs of female rats may be due to its antioxidative and anti-inflammatory properties.

OBJECTIVE: After 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.
METHODS: Rats 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.
RESULTS: 80 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.
CONCLUSIONS: Neither 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.

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.