Comparison of gonadotropin-releasing hormone and estradiol benzoate plus gonadotropin-releasing hormone to initiate a progesterone-based timed artificial insemination resynchronization protocol in lactating dairy cows.

The present study compared 2 strategies to initiate a progesterone (P4)-based timed artificial insemination (TAI) protocol for lactating dairy cows: only GnRH or estradiol benzoate (EB) plus GnRH (EB+GnRH). Lactating Holstein cows (n = 487; 184 primiparous and 303 multiparous) from 2 commercial dairy herds were used for their second or greater services postpartum. Each week, cows that were nonpregnant at the pregnancy diagnosis 32 d after a previous AI were randomly assigned to 1 of 2 experimental groups that differed only in the strategy to initiate (d 0) the TAI protocol. On d 0, every cow received a 2.0-g P4 implant; in the EB+GnRH group, cows were treated with 2.0 mg i.m. of EB and 16.8 µg i.m. of the GnRH analog buserelin acetate, whereas in the GnRH group, cows received only 16.8 µg i.m. of GnRH. On d 7 after the initial treatment, 0.530 mg i.m. of cloprostenol sodium (PGF) was administered in all cows, followed by a second dose on d 8, concomitant with 1.0 mg i.m. of estradiol cypionate and P4 implant withdrawal. The TAI was performed on d 10 (48 h after P4 device withdrawal) in both experimental groups. Only conventional Holstein semen was used throughout the study. The percentage of cows with corpus luteum (CL) on d 0 (73%) and overall ovulation rate after d 0 (54%) did not differ between groups. The CL regression between d 0 and the first PGF treatment was greater in the EB+GnRH group than the GnRH group (42% vs. 31%). Consequently, the proportion of cows with CL at PGF was greater when only GnRH was used on d 0 compared with EB+GnRH (86% vs. 82%), and the mean number of CL at PGF was greater (1.23 vs. 1.11). The expression of estrus near TAI was greater in GnRH group (84% vs. 77%), and cows showing estrus had greater (44% vs. 10%) pregnancy per AI (P/AI) on d 32 for both treatments. We found no effect of the presence of CL on d 0 or at PGF, nor of ovulation after d 0 or CL regression between d 0 and d 7 on fertility. However, fertility was critically impaired when cows did not have CL at both times, d 0 and at PGF treatment. We did not observe any interaction between treatment and other variables, and the P/AI was similar in cows receiving EB+GnRH or only GnRH on d 0 (37.8% vs. 36.6%). In summary, although there was no detectable difference in P/AI between treatments, this study demonstrated potential negative physiological outcomes caused by EB treatment on d 0 (greater incidence of luteolysis after d 0 and fewer cows with CL at PGF treatment). Overall, we found no benefit of adding EB at the initiation of a P4-based TAI protocol on fertility compared with using GnRH alone, despite differences in ovarian dynamics and expression of estrus.

Identification of Estradiol Benzoate as an Inhibitor of HBx Using Inducible Stably Transfected HepG2 Cells Expressing HiBiT Tagged HBx.

HBx plays a significant role in the cccDNA epigenetic modification regulating the hepatitis B virus (HBV) life cycle and in hepatocyte proliferation and carcinogenesis. By using the sleeping-beauty transposon system, we constructed a tetracycline-induced HBx-expressing stable cell line, SBHX21. HBx with a HiBiT tag can be quickly detected utilizing a NanoLuc-based HiBiT detection system. By screening a drug library using SBHX21 cells, we identified estradiol benzoate as a novel anti-HBx agent. Estradiol benzoate also markedly reduced the production of HBeAg, HBsAg, HBV pgRNA, and HBV DNA in a dose-dependent manner, suggesting that estradiol benzoate could be an anti-HBV agent. Docking model results revealed that estradiol benzoate binds to HBx at TRP87 and TRP107. Collectively, our results suggest that estradiol benzoate inhibits the HBx protein and HBV transcription and replication, which may serve as a novel anti-HBV molecular compound for investigating new treatment strategies for HBV infection.

Intravaginal administration of estradiol benzoate capsule for estrus synchronization in goats.

Estrus synchronization requires multiple treatments of hormonal drugs, requiring considerable time and cost. The aim of the present study was to develop an estrus synchronization protocol using intravaginal administration of estradiol benzoate (EB) capsules in goats. Two types of capsules were prepared: an EB capsule that melted immediately after administration and a sustained-release (SR) EB capsule that dissolved slowly and reached a peak after 24 h. Goats with functional corpus lutea were intramuscularly treated with prostaglandin F2α (PG). At 24 h after PG administration, goats were administered 1 mg of EB solution intramuscularly (PG + 24IM; n = 6) or 1 mg of EB capsule intravaginally (PG + 24EB; n = 6). The SR EB capsule was administered intravaginally at the time of PG administration (PG + SR; n = 6). The control group (n = 6) received only PG. All groups showed estrus within 72 h after PG administration. The onset of estrus did not differ significantly between the PG + 24IM and PG + SR groups but was earlier than in the control group. Estradiol concentration in the PG + SR group peaked at 11.5 ± 6.1 h after EB and PG administration. Peak estradiol concentrations were not significantly different between the PG + 24IM and PG + SR groups (78.0 ± 25.8 and 64.0 ± 38.1 pg/ml, respectively), and were higher than the PG + 24EB and control groups (27.3 ± 8.8 and 14.6 ± 6.1 pg/ml, respectively). These results suggest that intravaginal administration of an EB capsule with a sustained-drug release base is applicable for estrus synchronization, as an alternative to intramuscular administration.

Methyl palmitate reversed estradiol benzoate-induced endometrial hyperplasia in female rats.

Early detection and treatment of endometrial hyperplasia (EH) is mandatory for endometrial cancer prevention. Several bioactive agents of plant origin have been shown to elicit their chemotherapeutic effect against tumors and cancer via induction of mitochondrial permeability transition(mPT) pore opening. This research was therefore aimed at evaluating the potential chemopreventive effect of methyl palmitate (MP), on estradiol benzoate(EB)-induced EH, looking at the mitochondrial-mediated pathway and other possible mechanisms of action. Mitochondria were isolated using differential centrifugation. The mPT pore, mitochondrial ATPase (mATPase) activity, lipid peroxidation and cytochrome c release were determined by standard methods using spectrophotometer. Uterine interleukin 1b, MDA levels and SOD, GSH activities, were determined using commercially available kits. The uterine histological and immunohistochemical assessment of estrogen receptor (ERα), IL-1b and caspas-3 were carried out. The fibroblast cell count density was determined using histomorphometry. At all the concentrations of MP used, there was no significant induction of mPT pore opening, neither any enhancement of mATPase activity nor release of cytochrome c when compared to the control. Similar pattern of results were recorded for the in vivo study. However, there was marked increase in the uterine MDA and interleukin 1b levels, with concurrent decrease in SOD and GSH activities, in the EB-treated group, which was significantly reversed by MP co-administration. Endometrial Hyperplasia observed in the EB-treated group was ameliorated by MP co-administration. The immunoexpression of ERα and IL-1b in the EB-treated group was reversed by MP co-administration. This study suggests anti-inflammatory, antioxidant and anti-proliferative potential of MP against EB-induced EH.

Inhibition of expression of glucocorticoids receptors may contribute to postpartum depression.

Although postpartum depression (PPD) is the leading cause of disability worldwide, its molecular mechanisms are poorly understood. Recent evidence has suggested that impaired glucocorticoid receptor (GR), the signaling of key molecules of the HPA axis, plays a key role in the behavioral and neuroendorcrine alterations of major depression. However, the role of GR in postpartum period, which following with the abrupt withdrawal of placental corticotropin releasing hormone (CRH) and resulting in a re-equilibration of the maternal HPA axis in the days of post-delivery, is still not entirely clear. Previously, a hormone-simulated pregnancy (HSP), and the subsequent 'postpartum' withdrawal in estrogen has been employed to mimic the fluctuations in estradiol associated with pregnancy and postpartum. Using the HSP model, we investigated here the effect of 'postpartum' withdrawal in estrogen as well as depression- and anxiety-like behavior by intra-hippocampal infusion with GR inhibitor-RU486. Following the successful acquisition of PPD model by withdrawal in estrogen, reduced GR expression was observed in hippocampus. Further, HSP-rats suffered intra-hippocampal RU486 infusion presented depression- and anxiety-like behavior as postpartum depression. Together, these results suggest an important, though complex, role for GR in the behavioral regulation of postpartum depression.

Comparative study of stress response, growth and development of uteri in post-weaning gilts challenged with zearalenone and estradiol benzoate.

The objective of this study was to evaluate the effects of zearalenone (ZEA) and estradiol benzoate (EB) on stress injury and uterine development in post-weaning gilts. Thirty healthy post-weaning female gilts (Duroc × Landrace × Large White) aged 28-32 days were randomly allocated to three treatments as follows: (a) basal diet (Control), (b) basal diet plus 1.0 mg/kg purified ZEA (ZEA) and (c) basal diet plus 0.75 ml (1.5 mg) EB per pig at 3-days intervals by intramuscular injection (EB). The serum estradiol (E2 ), the final and the increased vulvar area, uterine index, thickness of the myometrium and endometrium, and protein expression of heat shock protein 70 (HSP70) in ZEA group were higher than those in the control group (p < .05), but lower than those in the EB group (p < .05). The serum luteinizing hormone in ZEA group was lower than that of the control group (p < .05), but higher than that in the EB group (p < .05). Higher serum follicle-stimulating hormone and progesterone were observed in the ZEA and control groups than those in the EB group (p < .05). The serum glutathione peroxidase activity in the ZEA group was lower than that in the control and EB groups (p < .001), and the malondialdehyde in the ZEA group was higher than that in the control and EB groups (p < .001). Moreover, the relative mRNA and protein expression of growth hormone receptor (GHR) and relative mRNA expression of HSP70 in the ZEA and EB groups were higher than those in the control group (p < .05). In conclusion, both ZEA (1.0 mg/kg) and EB (1.5 mg at 3 days intervals by intramuscular injection) stimulated vulvar swelling and uterine hypertrophy by disordering serum hormones and up-regulating GHR expression, and induced stress by different mechanisms in this study. Furthermore, the observed up-regulating HSP70 expression challenged by ZEA or EB may be part of the mechanism to resist stress injury.

[Estradiol benzoate induces abnormal proliferation of spermatogenic cells in the testis of infertile male mice].

OBJECTIVE: To explore the change in the proliferation of spermatogenic cells in the male mouse with infertility induced by exogenous estradiol benzoate (EB). METHODS: Sixty male mice aged 4 weeks were randomly divided into a control, a low-dose EB, and a high-dose EB group to be injected intramuscularly with corn oil at 150 µl or EB at 5 or 10 mg/kg, respectively, every other day for 4 weeks. Then, we obtained the weight and indexes of the testis, performed HE staining of the paraffin sections of the testis tissue and epididymal cauda, counted the spermatozoa in the epididymal sperm suspension, and determined the expression of the proliferating cell nuclear antigen (PCNA), the mRNA expressions of CyclinA1, CyclinB1, VASA and p53, and the protein expressions of p53 and phosphorylated p53 in the testis by immunohistochemistry, qRT-PCR and Western blot, respectively. RESULTS: In comparison with the controls, the mice treated with EB showed significantly decreased testicular indexes (P <0.05), no sperm in the sperm suspension or epididymal tubes, remarkably reduced numbers of spermatogonia, primary spermatocytes and Sertoli cells (P <0.05), down-regulated expression of PCNA (P <0.05) and mRNA expressions of CyclinA1, CyclinB1, PCNA and VASA in the seminiferous tubules (P <0.05), but a dose-dependent increase of the p53 level (P <0.05). Western blot revealed markedly higher levels of p53 protein expression and phosphorylation in the EB than in the control group (P <0.05) and even higher in the 10 mg/kg than in the 5 mg/kg EB group (P <0.05). CONCLUSIONS: EB inhibited the proliferation of spermatogenic cells by down-regulating the expressions of cell cycle-related factors in a dose-dependent manner, which might contribute to abnormal proliferation of spermatogenic cells in the testis of infertile male mice.

Incorporation of estradiol benzoate to CIDR protocol improves the reproductive responses in crossbred dairy heifers.

The present study was designed to determine the effect of estradiol benzoate (EB) on reproductive response following a controlled internal drug release (CIDR) protocol in crossbred (Sahiwal × Friesian) dairy heifers. In the first trial, a total of 100 crossbred dairy heifers were treated with CIDR protocol for 7 days and injected with the PGF2α on day 6. After 24 h of CIDR removal, one group (EB = 50) was injected with estradiol benzoate whereas the other (control = 50) remained untreated. Estrus intensity and response were recorded visually and ovulation rate was recorded by ultrasonography. All heifers were artificially inseminated at 48 and 60 h following CIDR removal. Heifers were scanned for pregnancy within days 30-40 of artificial insemination (AI). In the second trial, two subgroups of heifers were included to observe the estrus and ovulatory events. The results of the first trial revealed that estrus response was achieved 100% in both the treatment groups. Estrus intensity (2.9 ± 0.1 vs. 2.0 ± 0.7) and ovulation rate (100 vs. 88%) differed significantly (P < 0.05) between the EB and control groups. However, a tendency for higher pregnancy per AI was observed (54 vs. 36%; P = 0.07) in EB than that in control groups. The results of the second trial revealed that a significantly (P < 0.05) shorter estrus and earlier ovulatory events were observed in EB-treated heifers. It is concluded that the incorporation of estradiol benzoate to the CIDR protocol is helpful to improve the estrus signs and enhance the ovulation and the pregnancy per AI in crossbred dairy heifers.

Effect of arousing stimuli on circulating corticosterone and the circadian rhythms of luteinizing hormone (LH) surges and locomotor activity in estradiol-treated ovariectomized (ovx+EB) Syrian hamsters.

In most proestrous hamsters, novel wheel exposure phase advances activity rhythms and blocks the preovulatory LH surge, which occurs 2h earlier the next day. Because wheel immobilization does not prevent these effects we hypothesized that arousal alone blocks and phase advances the LH surge. Ovariectomized (ovx) hamsters received a jugular vein cannula and estradiol benzoate (EB) or vehicle was injected sc. The next day (Day 1), at zeitgeber time (ZT) 4-5 (ZT 12 = lights off), after obtaining a blood sample, each hamster was exposed to constant darkness (DD), and either remained in her home cage or was transferred to a new cage and exposed to a running wheel or a 2-hour arousal paradigm. Blood samples were obtained in dim red light and activity was recorded hourly until ~ZT 10-11 on Days 1 and 2. For the next 1-2 weeks, activity was monitored in DD. Plasma LH and corticosterone were assessed by RIA. Novel wheel exposure or arousal at ZT 4 greatly attenuated the Day 1 LH surge in ovx+EB hamsters, and phase advanced the Day 2 LH surge by about 2h. In proestrous hamsters, novel wheel exposure led to a prolonged (>2h) increase in corticosterone levels only when LH surges were blocked. Phase advances in activity rhythms were enhanced by estradiol and arousal. The results suggest that estradiol modulates the effectiveness of non-photic stimuli. The role of the increased activity of the hypothalamic-pituitary-adrenal axis associated with novel wheel-induced attenuation of LH surges in ovx+EB hamsters remains to be determined.

Increasing estradiol benzoate, pretreatment with gonadotropin-releasing hormone, and impediments for successful estradiol-based fixed-time artificial insemination protocols in dairy cattle.

With the objective to optimize fixed-time artificial insemination (FTAI) protocols based on estradiol benzoate (EB) and progesterone (P4), we performed 2 experiments (Exp.) in dairy cows. In Exp. 1 (n=44), we hypothesized that increased EB (EB3=3 mg vs. EB2=2 mg) on d 0 would improve synchronization of ovarian follicle wave emergence. Likewise, in Exp. 2 (n=82), we hypothesized that a GnRH treatment on d -3 (early in a follicular wave on d 0) versus d -7 (presence of a dominant follicle on d 0) would better synchronize wave emergence. Moreover, results from both experiments were combined to identify reasons for the lack of synchronization. All cows were treated with EB at the time of introduction of a P4 implant (d 0). On d 7, cows were given 25 mg of prostaglandin F2α; on d 8, the implant was removed and cows were given 1mg of estradiol cypionate. All cows received FTAI on d 10. In both experiments, daily ultrasound evaluations were performed and, in Exp. 2, circulating P4 was evaluated during the protocol. Pregnancy per artificial insemination (P/AI) was determined on d 31 and 59 after FTAI. In Exp. 1, EB dose did not change time to wave emergence, but EB3 compared with EB2 decreased the percentage of cows with a corpus luteum on d 7 (19.8 vs. 55.3%) and time to ovulation (10.4 vs. 10.9 d). In Exp. 2, although we detected a tendency for delayed follicle wave emergence after the start of the FTAI protocol in cows ovulating to GnRH given on d -7, there was no difference in percentage of cows with a synchronized wave emergence (~80%). Regardless of treatment, more cows with P4<0.1 ng/mL, compared with P4≥0.1 and <0.22 ng/mL at the time of AI, ovulated to the protocol (81.2 vs. 58.0%) and had increased P/AI (47.4 vs. 21.4%). An analysis of data from both experiments showed that only 73.8% (93/126) of cows had synchronized wave emergence, and only 77.8% (98/126) of cows ovulated at the end of the protocol. Fertility was much greater in cows that had emergence of a new wave synchronized and ovulated to end of the protocol [P/AI 61.3% (46/75)] compared with cows that failed to present one or both of the outcomes above [15.7% (8/51)]. Thus, although current FTAI protocols using EB and P4 produce P/AI between 30 and 40% for lactating dairy cows, there remains room for improvement because less than 60% (75/126) of the cows were correctly synchronized. Starting the FTAI protocol without the dominant follicle or increasing the dose of EB to 3mg was not effective in increasing synchronization rate.

Effect of Yin-Zhi-Huang on up-regulation of Oatp2, Ntcp, and Mrp2 proteins in estrogen-induced rat cholestasis.

CONTEXT: Yin-Zhi-Huang (YZH), a prescription of traditional Chinese medicine, is widely used to treat neonatal jaundice or cholestasis. OBJECTIVE: This study investigates the regulatory effect of YZH on the localization and expression of organic anion transporting polypeptides 2 (Oatp2), Na(+)-taurocholate co-transporting polypeptide (Ntcp), multidrug-resistance-associated protein 2 (Mrp2), and bile salt export pump (Bsep) in estrogen-induced cholestasis rats. MATERIAL AND METHODS: Cholestasis model rats were induced via subcutaneous injection of estradiol benzoate (EB, 5 mg/kg/d) for 5 d. Other EB-induced rats were treated with saline (2 ml) or YZH (1.5 g/kg, two times a day) for 7, 14, and 21 d. The biochemical and pathologic examinations were performed. Moreover, the localization and expression of Oatp2, Ntcp, Mrp2, and Bsep were determined by immunohistochemisty and Western blotting, respectively. RESULTS: YZH treatment could significantly decrease the serum total bile acids (TBA) (4.9 ± 0.6-2.8 ± 0.8) and direct bilirubin (DBIL) (2.6 ± 0.7-1.0 ± 0.1) levels, improve the histological disorganization, and, respectively, increase the expression of Oatp2 and Ntcp by 46% and 28% compared with saline-treated (p < 0.05) rats at 14 d. The expression of Mrp2 increased by 45% was observed in YZH treated compared with saline-treated (p < 0.05) rats at 7 d. However, there was a little change in the expression of Bsep (p > 0.05) after YZH treatment for 7, 14, and 21 d. DISCUSSION AND CONCLUSION: In conclusion, the therapeutic effect of YZH to cholestasis could be attributed to the regulation of Oatp2, Ntcp, Mrp2, and Bsep.

The effectiveness of different hormone protocols for improving ovarian function before ovum pick-up in crossbred Japanese black cattle.

Background and Aim: Producing and transferring embryos in vitro are profitable for enhancing premium beef genetics. Reducing costs and enhancing the effectiveness of hormone protocols before ovum pick-up (OPU) yield advantages. This study aimed to confirm that estradiol benzoate (EB) treatment resulted in more medium- and large-sized follicles before OPU and of higher oocyte quality after OPU than non-hormonal treatments, comparable to those undergoing gonadotropin-releasing hormone (GnRH) with follicle-stimulating hormone (FSH) plus prostaglandin F2α (PGF2α) and progesterone-releasing controlled internal drug release (CIDR) or EB with progesterone-releasing CIDR hormonal treatments. Materials and Methods: 30 crossbred Japanese black cows were divided into five equal groups, which were either untreated or treated with different hormone protocols before OPU. Group 1 (cows in estrus) and group 2 (cows in diestrus) were the untreated controls. Cows in group 3 were treated with GnRH + FSH + CIDR + PGF2α. Cows in group 4 received EB, and those in group 5 received EB + CIDR + PGF2α. After administering the protocols, all cow follicles were examined through ultrasonography and categorized by size. Subsequently, all cows underwent OPU, and the oocytes were collected and graded from A to D according to standard criteria. Results: Group 3 presented the highest large follicle numbers, and groups 3-5 had more medium follicle numbers, not different among groups but they had this parameter more than those of control groups 1 and 2. Moreover, groups 3-5 did not differ in combined grades A + B oocytes (good-quality oocytes). Groups 3 and 4 had more A + B oocytes than control groups 1 and 2, whereas group 5 was not different in this parameter from group 1. Conclusion: Among the three hormone protocols, EB treatment proved the most cost-effective and efficient, yielding more high-quality oocytes compared to the non-treatment protocols. To reduce the limitations of EB use in the future, this study suggests researching natural EB phytoestrogens as alternative treatments for improving ovarian function before OPU in other cattle breeds.

Gut Microbiota and Acylcarnitine Metabolites Connect the Beneficial Association between Estrogen and Lipid Metabolism Disorders in Ovariectomized Mice.

Decreased estrogen level is one of the main causes of lipid metabolism disorders and coronary heart disease in women after menopause. Exogenous estradiol benzoate is effective to some extent in alleviating lipid metabolism disorders caused by estrogen deficiency. However, the role of gut microbes in the regulation process is not yet appreciated. The objective of this study was to investigate the effects of estradiol benzoate supplementation on lipid metabolism, gut microbiota, and metabolites in ovariectomized (OVX) mice and to reveal the importance of gut microbes and metabolites in the regulation of lipid metabolism disorders. This study found that high doses of estradiol benzoate supplementation effectively attenuated fat accumulation in OVX mice. There was a significant increase in the expression of genes enriched in hepatic cholesterol metabolism and a concomitant decrease in the expression of genes enriched in unsaturated fatty acid metabolism pathways. Further screening of the gut for characteristic metabolites associated with improved lipid metabolism revealed that estradiol benzoate supplementation influenced major subsets of acylcarnitine metabolites. Ovariectomy significantly increased the abundance of characteristic microbes that are significantly negatively associated with acylcarnitine synthesis, such as Lactobacillus and Eubacterium ruminantium group bacteria, while estradiol benzoate supplementation significantly increased the abundance of characteristic microbes that are significantly positively associated with acylcarnitine synthesis, such as Ileibacterium and Bifidobacterium spp. The use of pseudosterile mice with gut microbial deficiency greatly facilitated the synthesis of acylcarnitine due to estradiol benzoate supplementation and also alleviated lipid metabolism disorders to a greater extent in OVX mice. IMPORTANCE Our findings establish a role for gut microbes in the progression of estrogen deficiency-induced lipid metabolism disorders and reveal key target bacteria that may have the potential to regulate acylcarnitine synthesis. These findings suggest a possible route for the use of microbes or acylcarnitine to regulate disorders of lipid metabolism induced by estrogen deficiency.

Cheminformatics Strategies Unlock Marburg Virus VP35 Inhibitors from Natural Compound Library.

The Ebola virus and its close relative, the Marburg virus, both belong to the family Filoviridae and are highly hazardous and contagious viruses. With a mortality rate ranging from 23% to 90%, depending on the specific outbreak, the development of effective antiviral interventions is crucial for reducing fatalities and mitigating the impact of Marburg virus outbreaks. In this investigation, a virtual screening approach was employed to evaluate 2042 natural compounds for their potential interactions with the VP35 protein of the Marburg virus. Average and worst binding energies were calculated for all 20 poses, and compounds that exhibited binding energies <-6 kcal/mol in both criteria were selected for further analysis. Based on binding energies, only six compounds (Estradiol benzoate, INVEGA (paliperidone), Isosilybin, Protopanaxadiol, Permethrin, and Bufalin) were selected for subsequent investigations, focusing on interaction analysis. Among these selected compounds, Estradiol benzoate, INVEGA (paliperidone), and Isosilybin showed strong hydrogen bonds, while the others did not. In this study, the compounds Myricetin, Isosilybin, and Estradiol benzoate were subjected to a molecular dynamics (MD) simulation and free binding energy calculation using MM/GBSA analysis. The reference component Myricetin served as a control. Estradiol benzoate exhibited the most stable and consistent root-mean-square deviation (RMSD) values, whereas Isosilybin showed significant fluctuations in RMSD. The compound Estradiol benzoate exhibited the lowest ΔG binding free energy (-22.89 kcal/mol), surpassing the control compound's binding energy (-9.29 kcal/mol). Overall, this investigation suggested that Estradiol benzoate possesses favorable binding free energies, indicating a potential inhibitory mechanism against the VP35 protein of the Marburg virus. The study proposes that these natural compounds could serve as a therapeutic option for preventing Marburg virus infection. However, experimental validation is required to further corroborate these findings.

Chloroform Fraction of Drymaria cordata Linn (CFDC) Suppresses Estradiol Benzoate- Induced Endometrial Hyperplasia.

BACKGROUND: The diagnosis of uterine dysfunction (endometrial hyperplasia) is on the rise. The available treatment is quite expensive and associated with some side effects. The therapeutic potential of natural products is now being explored, as they are easily available with little or no side effects. Drymaraia cordata is folklorically utilized in the treatment of diverse ailments including uterine fibroids. OBJECTIVES: This study aims to investigate the potential therapeutic effect of chloroform fraction of methanol extract of Drymaria cordata (CFDC) in estradiol benzoate (EB)-induced endometrial hyperplasia. METHODS: Thirty-six rats were randomly divided equally into six groups. These included control group, CFDC: (100 mg/kg), CFDC: (200 mg/kg), EB: (2 mg/kg), EB + CFDC (100 mg/kg), and EB + CFDC (200 mg/kg). Endometrial hyperplasia (EH) was induced by intraperitoneal injection of EB. The levels of estrogen (E2), progesterone (PG), Follicle stimulating hormone (FSH), Luteinizing hormone (LH), Malondialdehyde (MDA), Superoxide dismutase (SOD), and Glutathione peroxidase (GSH-Px) activities were determined using ELISA technique. The uterine histological assessment and immunohistochemical expression levels of estrogen receptor, Ki-67, cytochrome c, and caspase 3 were carried out. RESULTS: EH was severely expressed in the uterine section of EB-treated rats. However, CFDC administration improved the pathological features of the animal model. The sex hormones levels were increased in the EB-treated group, which were significantly reduced by CFDC. The antioxidant indices were also restored by CFDC. Immunoexpression levels of ERα and Ki-67 were downregulated while cytochrome c and caspase 3 were upregulated by CFDC. CONCLUSION: This study suggests that CFDC contains phytochemicals that can protect against EB-induced EH via modulation of hormonal signaling, apoptotic machinery, and oxidative indices.

Morphologic, Steroidogenic, and Transcriptomic Assessment of the Corpus Luteum in Holstein Cows after Spontaneous or Hormone-Induced Ovulation.

There is evidence that replacing the gonadotropin-releasing hormone (GnRH) with porcine luteinizing hormone (pLH) to synchronize ovulation prior to artificial insemination (AI) increased pregnancy per AI in dairy cows without affecting blood progesterone (P4) concentrations. Whether morphologic, steroidogenic, and transcriptomic differences exist among corpora lutea (CL) formed after ovulation induced by GnRH and pLH is unclear. Our main objective, therefore, was to compare CL characteristics between GnRH- and pLH-induced CL. In 24 non-lactating Holstein cows, ovulations were spontaneous (Spont-Ov) or induced with 100 µg GnRH, 25 mg pLH, or 1 mg estradiol benzoate (EB), with CL excised 12 d after ovulation. In pLH- versus GnRH-treated cows, the duration of elevated LH (above baseline) was prolonged (10 versus 6 h, respectively, p < 0.01), but CL dimensions, pixel intensity of CL images, proportions of steroidogenic and non-steroidogenic luteal cells, and mean plasma LH did not significantly differ. Post-ovulation mean plasma P4 (ng/mL) did not differ among Spont-Ov (3.0) pLH (3.1) or GnRH (3.0) cows but were lower in EB cows (2.0). In vitro P4 concentration was greater in luteal explants of pLH-treated cows than in all other groups (combined means, 16.0 vs. 12.3 µg/mL, p < 0.02). Relative abundance of mRNA for oxytocin receptor (OXTR) was 2-fold higher (p < 0.01) in CL of pLH vs. GnRH cows and highest in Spont-Ov CL. In summary, pLH-treated cows had a longer LH peak, and greatest luteal tissue concentrations and in vitro production of P4. We inferred that increased P4 concentrations at the ovarian-uterine level in pLH-treated cows could have promoted embryo development and increased pregnancy per AI.

Administration of Estradiol Benzoate Enhances Ovarian and Uterine Hemodynamics in Postpartum Dairy Buffaloes.

The postpartum (PP) period is a crucial stage for the resumption of reproductive performance and ovarian cyclicity in dairy buffaloes. The present study aimed, for the first time, to assess the effect of the administration of estradiol benzoate (EB) on ovarian and uterine hemodynamics in PP dairy buffaloes. Eight pluriparous acyclic domestic buffaloes were enrolled in the present experiment and received a dose of 10 mg of estradiol benzoate (EB) intramuscularly 4 weeks after parturition. All animals were examined two times before EB administration (days -3, and -1) and on the day of EB administration (day 0), followed by examinations on days 1, 3, 5, 7, and 9 post-EB administration. The middle uterine artery (MUA) and ovarian artery (OA) blood flow patterns were assessed using a color Doppler ultrasound device. The reproductive parameters were (1) the cross-sectional diameters (cm) of the OA and MUA, (2) cranial uterine horn thickness (UHT; cm), and (3) hemodynamic changes within the MUA on both the ipsi- and contra-lateral sides of the previous pregnant horn and within the OA corresponding to the ovarian tissues. The examined blood flow parameters were the pulsatility index (PI), resistance index (RI), peak systolic/end-diastolic ratio (S/D), time-averaged maximum velocity (TAV; cm/s), uterine blood flow rate (BFR; bpm), and uterine blood flow volume (BFV; mL/min). Concomitantly, blood samples were collected from the coccygeal vein, and the sera were stored at -18 °C for use in estradiol (E2-17ß) and nitric oxide (NO) assays. The results revealed increases in both OA and MUA cross-sectional diameter (cm) on the ipsi-lateral and contra-lateral (p < 0.05) sides within 24 h until day 9 post-treatment. The values of the RI and PI of blood flow within the OA and MUA on the ipsi-lateral and contra-lateral sides of the previous pregnancy were obviously lower (p < 0.05) at 24 h after the administration of EB, and then, started to gradually elevate, reaching the pre-treatment values on day 9 after EB administration. Both the BFR and BFV in the OA and MUA significantly increased from 24 h to 72 h after EB administration on both the ipsi-lateral and contra-lateral sides (p < 0.05); then, their values started to decrease to reach the pretreatment value on day 9 after EB administration. Both E2 and NO concentrations significantly increased (p < 0.05) from 24 h until day 3 after EB injection and then started to decline after that, reaching the pre-treatment value on day 9. In conclusion, the administration of EB enhances the ovarian and uterine blood flow concomitantly with increased levels of NO in PP dairy buffaloes.

Nutritional and Hormonal Induction of Fatty Liver Syndrome and Effects of Dietary Lipotropic Factors in Egg-type Male Chicks.

This experiment was conducted with male chicks to investigate the influence of hormones and nutrients on the development of fatty liver syndrome (FLS) as well as the effects of dietary lipotropic factors on hepatic fat accumulation and lipogenic enzyme gene expression. A total of two-hundred sixteen 4-wk-old Hy-Line male chicks were divided into six groups and fed an experimental diet (T1, low-energy diet with low levels of lipotropic factors; T2, high-energy diet with low levels of lipotropic factors; T3 and T5, low-energy diet with high levels of lipotropic factors; T4 and T6, high-energy diet with high levels of lipotropic factors) for six weeks. The chicks in T5 and T6 groups were treated with intramuscular injections of estradiol benzoate for three days prior to biopsy and clinical analysis of FLS. Chicks treated with estrogen had significantly greater liver weights than untreated chicks. The abdominal fat contents were increased in chicks consuming high-energy diets as compared to those consuming low-energy diets. Treatment with estrogen significantly increased the concentrations of serum cholesterol, triacylglycerol and phospholipid (p<0.05). The hepatic triacylglycerol levels were tenfold higher in the estrogen treated chicks than in the untreated chicks. There were no significant differences in malondialdehyde levels between the treatment groups. Estrogen treatment dramatically increased the levels of fatty acid synthetase, acetyl-CoA carboxylase and ApoB mRNA. The results indicated that treatment with exogenous estrogen in growing male chicks induced hepatic fat accumulation, which might be partially due to increased lipogenic enzyme gene expression.

Piceatannol SNEDDS Attenuates Estradiol-Induced Endometrial Hyperplasia in Rats by Modulation of NF-κB and Nrf2/HO-1 Axes.

Endometrial hyperplasia (EH) is the most common risk factor for endometrial malignancy in females. The pathogenesis of EH has been directly linked to uterine inflammation, which can result in abnormal cell division and decreased apoptosis. Piceatannol (PIC), a natural polyphenolic stilbene, is known to exert anti-inflammatory, antioxidant and anti-proliferative activities. The aim of the present study was to examine the potential preventive role of PIC in estradiol benzoate (EB)-induced EH in rats. A self-nanoemulsifying drug delivery system (SNEDDS) was prepared to improve the solubility of the PIC. Therefore, thirty female Wistar rats were divided into five groups: (1) control, (2) PIC SNEDDS (10 mg/kg), (3) EB (0.6 mg/kg), (4) EB + PIC SNEDDS (5 mg/kg) and (5) EB + PIC SNEDDS (10 mg/kg). The administration of PIC SNEDDS prevented EB-induced increases in uterine weights and histopathological changes. Additionally, it displayed pro-apoptotic and antioxidant activity in the endometrium. Immunohistochemical staining of uterine sections co-treated with PIC SNEDDS showed significantly decreased expression of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and nuclear transcription factor-kappa B (NF-κB). This anti-inflammatory effect was further confirmed by a significant increase in Nrf2 and heme oxygenase-1 (HO-1) expression. These results indicate that SNEDDS nanoformulation of PIC possesses protective effects against experimentally induced EH.

Investigation of Antiparasitic Activity of Two Marine Natural Products, Estradiol Benzoate, and Octyl Gallate, on Toxoplasma gondii In Vitro.

Toxoplasmosis, caused by Toxoplasma gondii, is a common disease worldwide and could be severe and even fatal in immunocompromised individuals and fetuses. Limitation in current available treatment options drives the need to develop novel therapeutics. This study assessed the anti-T. gondii potential of 103 marine natural products. A luminescence-based ß-galactosidase activity assay was used to screen the marine natural products library. Afterward, those compounds that displayed over 70% parasite inhibition ratio were further chosen to assess their cytotoxicity. Compounds exhibiting low cytotoxicity (≥80% cell viability) were applied to evaluate the inhibition efficacy on discrete steps of the T. gondii lytic cycle, including invasion, intracellular growth, and egress abilities as well as the cell cycle. We found that both estradiol benzoate and octyl gallate caused >70% inhibition of tachyzoite growth with IC50 values of 4.41 ± 0.94 and 5.66 ± 0.35 µM, respectively, and displayed low cytotoxicity with TD50 values of 34.11 ± 2.86 and 26.4 ± 0.98 µM, respectively. Despite their defects in inhibition of invasion and egress of tachyzoite, the two compounds markedly inhibited the tachyzoite intracellular replication. Flow cytometric analyses further suggested that the anti-T. gondii activity of estradiol benzoate, rather than octyl gallate, may be linked to halting cell cycle progression of tachyzoite from G1 to S phase. Taken together, these findings suggest that both estradiol benzoate and octyl gallate are potential inhibitors for anti-T. gondii infection and support the further exploration of marine natural products as a thinkable source of alternative and active agents against T. gondii.