Estradiol-17β Concentrations Research Articles

3D Artificial Skin Platform for Investigating Pregnancy-Related Skin Pigmentation.

In this study, we created a 3D Artificial Skin Platform that can be used for the treatment of pigmentation by artificially realizing the skin of pregnant women. For the stable realization of 3D artificial skin, a bilayer hydrogel composed of collagen type I and fibrin was designed and applied to the study to reduce the tension-induced contraction of collagen type I, the extracellular matrix (ECM) of artificial skin, by dynamic culture. Oxygen concentration and 17β-Estradiol (E2) concentration, which are highly related to melanin production, were selected as parameters of the pregnancy environment and applied to cell culture. Oxygen concentration, which is locally reduced in the first trimester (2.5-3%), and E2, which is upregulated in the third trimester, were applied to the cell culture process. We analyzed whether the 3D artificial skin implemented in the 3D Artificial Skin Platform could better represent the tendency of melanin expression in pregnant women than cells cultured under the same conditions in 2D. The expression levels of melanin and melanin-related genes in the 2D cell culture did not show a significant trend that was similar to the melanin expression trend in pregnant women. However, the 3D artificial skin platform showed a significant trend towards a 2-6-fold increase in melanin expression in response to low oxygen concentrations (2.5%) and E2 concentrations (17 ng/mL), which was similar to the trend in pregnant women in vivo. These results suggest that 3D artificial skin cultured on the Artificial Skin Platform has the potential to be used as a substitute for human pregnant skin in various research fields related to the treatment of pigmentation.

Influence of dietary chitosan-oligosaccharides supplementation on productive and reproductive performance of laying hens

Abstract This experiment aimed to ascertain whether adding chitosan-oligosaccharides (COS) to the diet will affect Mandarah laying hens’ productivity and reproductive efficiency. At the age of 34 weeks, 120 laying hens and 12 cocks of the Mandarah chicken strain were separated into four groups, each consisting of 30 hens and three cocks. The 1st group served as a control group, receiving only a basic diet. The 2nd, 3rd, and 4th experimental groups received 0.1, 0.2, and 0.5 g/kg of chitosan-oligosaccharides as an addition to the base diet, respectively. According to the findings of this study, the majority of the analyzed attributes differed significantly between treatment groups. Comparing the COS-treated birds to the control birds, the COS-treated birds significantly increased egg production, improved feed conversion, Haugh unit score, shell thickness, fertility, hatchability, ovary weight, and the number of yellow ovarian follicles as well as blood estradiol-17β (E2) and testosterone concentrations. Furthermore, compared to the other treated groups, introducing COS at a level of 0.1 g/kg diet resulted in the best laying rate, egg mass, and feed conversion. The highest percentage of fertility, hatchability of hatching eggs, ovary weight, number of yellow ovarian follicles, and serum E2 concentration was also found in birds fed COS at a dose of 0.2 g/kg feed. In addition, compared to the control diet, COS treatment at 0.1, 0.2, and 0.5 g/kg enhanced relative economic efficiency by 464.43, 457.41, and 352.75%, respectively. It is concluded that chitosan improves economic efficiency, reproductive performance, productivity in laying hens, and overall health when used at 0.1 g/kg diet.

Abstract 568: Modulation of HER4 isoforms expression in estrogen-stimulated in vitro models

Abstract Introduction HER4, a tyrosine kinase receptor belonging to the Human Epidermal Growth Factor/HER/ErbB-family, has a complex biology that includes four isoforms (JMa-CYT1/2, and JMb-CYT1/2) and tissue specific oncogenic or tumour suppressor roles. In estrogen receptor positive (ER+) breast cancer (BC), HER4 isoforms can either act as a co-regulator of ESR1-mediated gene expression by localising to the nucleus, or activate apoptosis by localising to mitochondria. There is very little known about HER4 function in cutaneous melanoma, despite cutaneous melanoma showing the highest HER4 genomic alteration frequency (17.34%) across different cancers (cBioPortal). Due to the interplay between estrogen signalling and HER4 isoforms in ER+BC, this study investigates the RNA expression of HER4 isoforms along with hormone and nuclear receptors associated with 17β-estradiol stimulation in ER+BC and cutaneous melanoma in vitro models. Material and Methods CAMA1 (ER+BC) and SKMEL24 (cutaneous melanoma) cell lines were serum starved for 4h prior to stimulation with 17β-estradiol (0.5nM, 1nM, 5nM and 10nM). RNA was extracted from cells after 24h using TRIzol, followed by qRT-PCR to assess the expression of HER4 isoforms, the hormone receptors estrogen receptor 1 (ESR1/ER-alpha), estrogen receptor 2 (ESR2/ER-beta), progesterone receptor (PGR/PR), G protein-coupled estrogen receptor 1 (GPER1) and the nuclear receptors retinoic acid receptor alpha (RARA) and retinoid X receptor alpha (RXRA). Cell proliferation and caspase3/7 apoptosis analysis were performed at 24/48/72h in the 17β-estradiol stimulated cells by using the IncuCyte Live Cell Analysis Instrument (Sartorius). SN-38 (150nM) was used as positive control for apoptosis induction. Two-way ANOVA was used for the statistical analysis, p<0.05 was significant. Results The qRT-PCR analysis showed that upon stimulation of CAMA1 with 1nM of 17β-estradiol there is a numerical shift in the CYT-1/CYT-2 ratio towards CYT-2 (not statistically significant, p>0.05) and a significant increase in PGR (p= 0.0027) and RAR-A (p= 0.0248) expression compared to control. CAMA1 stimulated with any concentration of 17β-estradiol showed an increase in proliferation compared to the control. The stimulation of SKMEL24 with 5nM of 17β-estradiol showed an increase in JM-a (p=0.0075) and CYT-1 (p=0.0271) HER4 isoforms, GPER1 (p= 0.0286), and a dose-dependent increase in RARA (p=0.0011) compared to control, even in the absence of ESR1, ESR2 and PGR. SKMEL24 stimulated with 17β-estradiol showed a decrease in proliferation compared to control, but no apoptosis induction was found. Conclusion These findings suggest potential interplay between 17β-estradiol and HER4 isoforms leading to the activation of intracellular pathways that could influence tumour progression/suppression in melanoma. Further studies are needed to clarify the specific receptor interactions and pathways involved. Citation Format: Marta Valenti, Georg F. Bischof, John P. Crown, Denis M. Collins. Modulation of HER4 isoforms expression in estrogen-stimulated in vitro models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 568.

Plasma relaxin, estrogens and progestogens concentration in coldblooded and hot-blood mares in the second half of gestation

Monitoring estrogens, progestogens, and relaxin plasma concentration can be important for determining pregnancies at risk of failure in mares. However, significant breed-related differences in values of reproductive hormones are known, and data concerning coldblooded (CB) mares are missing. The aim of the study was to determine and compare serum relaxin, estrone sulphate (E1S), estradiol-17β (E2), and progestogens concentration in pregnant CB and hot-blood (HB) mares during the second half of gestation. Blood samples obtained from 94 healthy mares in the second half of gestation with known mating dates were analyzed. Samples were collected from 36 CB mares aged from 3 to 16 years and 58 HB mares aged from 4 to 17 years. The hormone concentrations were measured using immunoenzymatic equine species-specific commercial kits. The serum relaxin concentration was almost three times lower in CB, whereas E1S and E2 almost twice as low, as compared to HB mares (P < 0.05). In conclusion, the extremely low concentration of relaxin in CB mares could be one reason for the increased amounts of difficult parturition requiring veterinary intervention noted in these breeds.

17β-Estradiol Effects in Skeletal Muscle: A 31P MR Spectroscopic Imaging (MRSI) Study of Young Females during Early Follicular (EF) and Peri-Ovulation (PO) Phases

The natural variation in estrogen secretion throughout the female menstrual cycle impacts various organs, including estrogen receptor (ER)-expressed skeletal muscle. Many women commonly experience increased fatigue or reduced energy levels in the days leading up to and during menstruation, when blood estrogen levels decline. Yet, it remains unclear whether endogenous 17β-estradiol, a major estrogen component, directly affects the energy metabolism in skeletal muscle due to the intricate and fluctuating nature of female hormones. In this study, we employed 2D 31P FID-MRSI at 7T to investigate phosphoryl metabolites in the soleus muscle of a cohort of young females (average age: 28 ± 6 years, n = 7) during the early follicular (EF) and peri-ovulation (PO) phases, when their blood 17β-estradiol levels differ significantly (EF: 28 ± 18 pg/mL vs. PO: 71 ± 30 pg/mL, p &lt; 0.05), while the levels of other potentially interfering hormones remain relatively invariant. Our findings reveal a reduction in ATP-referenced phosphocreatine (PCr) levels in the EF phase compared to the PO phase for all participants (5.4 ± 4.3%). Furthermore, we observe a linear correlation between muscle PCr levels and blood 17β-estradiol concentrations (r = 0.64, p = 0.014). Conversely, inorganic phosphate Pi and phospholipid metabolite GPC levels remain independent of 17β-estradiol but display a high correlation between the EF and PO phases (p = 0.015 for Pi and p = 0.0008 for GPC). The robust association we have identified between ATP-referenced PCr and 17β-estradiol suggests that 17β-estradiol plays a modulatory role in the energy metabolism of skeletal muscle.

Impact of 17β-Estradiol on the Shape, Survival, Osteogenic Transformation, and mRNA Expression of Gingiva-Derived Stem Cell Spheroids.

Background and Objectives: Mesenchymal stem cells hold promise for tissue regeneration, given their robust growth and versatile differentiation capabilities. An analysis of bone marrow-sourced mesenchymal stem cell proliferation showed that 17β-estradiol could enhance their growth. This study aims to investigate the influence of 17β-estradiol on the shape, survival, osteogenic differentiation, and mineralization of human mesenchymal stem cells. Materials and Methods: Spheroids made from human gingiva-derived stem cells were cultivated with varying concentrations of 17β-estradiol: 0, 0.01, 0.1, 1, and 10 nM. Morphology was assessed on days 1, 3, and 5. The live/dead kit assay was employed on day 3 for qualitative cell viability, while cell counting kit-8 was used for quantitative viability assessments on days 1, 3, and 5. To evaluate the osteogenic differentiation of the spheroids, a real-time polymerase chain reaction assessed the expressions of RUNX2 and COL1A1 on day 7. Results: The stem cells formed cohesive spheroids, and the inclusion of 17β-estradiol did not noticeably alter their shape. The spheroid diameter remained consistent across concentrations of 0, 0.01, 0.1, 1, and 10 nM of 17β-estradiol. However, cellular viability was boosted with the addition of 1 and 10 nM of 17β-estradiol. The highest expression levels for RUNX2 and COL1A1 were observed with the introduction of 17β-estradiol at 0.1 nM. Conclusions: In conclusion, from the results obtained, it can be inferred that 17β-estradiol can be utilized for differentiating stem cell spheroids. Furthermore, the localized and controlled use, potentially through localized delivery systems or biomaterials, can be an area of active research. While 17β-estradiol holds promise for enhancing stem cell applications, any clinical use requires a thorough understanding of its mechanisms, careful control of its dosage and delivery, and extensive testing to ensure safety and efficacy.

Severe body condition loss lowers hepatic output of IGF1 with adverse effects on the dominant follicle in dairy cows

The severe loss of body condition score (BCS) during the early lactation period has been associated with infertility in cows. However, the mechanisms are not fully understood. The aim of this study was to examine the effect of BCS loss on liver health, and ovarian functions in cows during early lactation. Retrospectively multiparous cows from two farms were categorized based on units of BCS (1–5 scale) loss as Moderate (MOD, <0.75 units; n = 11) or Severe (SEV, ≥0.75 units; n = 9) loss groups. From Weeks −3 to 7, relative to calving, MOD and SEV cows lost on average 0.4 and 1.0-unit BCS, respectively. All data except hepatic transcriptomes were analyzed with PROC MIXED procedure of SAS. The plasma concentration of non-esterified fatty acids at Week 0 and 1, ß-hydroxy butyrate at Week 1, and γ-glutamyl transferase at Weeks 1 and 7 relative to calving were higher in SEV cows. Hepatic transcriptome analysis showed that 1 186 genes were differentially expressed in SEV (n = 3) compared to MOD (n = 3) cows at Week 7 after calving. Pathway analysis revealed that significant DEGs in SEV cows enriched in lipid metabolisms including, lipid metabolic process, ether lipid metabolism, fatty acid beta-oxidation, fatty acid biosynthetic process, fatty acid metabolic process, fat digestion and absorption, linoleic acid metabolism, alpha-linolenic acid metabolism. The impaired liver function in SEV cows was associated with 1.5-fold reduction of hepatic IGF1 gene expression and lower serum IGF1 concentrations. At the ovarian level, SEV cows had lower IGF1 concentration in the follicular fluid of the dominant follicle of the synchronized follicular wave compared to that of MOD cows at 7 weeks after calving. Further, the follicular fluid concentration of estradiol-17β was lower in SEV cows along with lower transcript abundance of genes from granulosa cells associated with dominant follicle competence, including CYP19A1, NR5A2, IGF1, and LHCGR. These data show that SEV loss of BCS during early lactation leading up to the planned start of breeding is associated with liver dysfunction, including lower IGF1 secretion, and impaired function of the dominant follicle in the ovary.

Hypothalamic-pituitary-gonadal axis response to photoperiod changes in female guanacos (Lama guanicoe)

The guanaco, a wild South American camelid, is renowned for its remarkable resilience to extreme conditions. Despite this, little is known about how reproductive hormones in female camelids are influenced during their seasonal breeding period, which occurs during long photoperiod. To explore this, the study investigated the response of the hypothalamic-pituitary–gonadal axis in female guanacos during short days (10L:14D; July) and long days (16L:8D; December) in the Mediterranean ecosystem (33°38′28″S, 70°34′27″W). Blood samples from 14 adult animals were collected, and measurements of melatonin, 17β-estradiol, FSH, and LH concentrations were taken. The results showed that melatonin concentration was lower (P < 0.05) during long days than short days, whereas 17β-estradiol, FSH, and LH concentrations were higher (P < 0.05) during long days compared to short days. Furthermore, the study detected the expression of the melatonin receptor 1A and kisspeptin in the hypothalamus and pituitary, suggesting that the pineal gland of female guanacos is sensitive to seasonal changes in day length. These findings also indicate a seasonal variation in the concentration of reproductive hormones, likely linked to the distinct modulation of the hypothalamic-pituitary-gonadal axis of female guanacos during short and long days.

Reduced period from follicular wave emergence to luteolysis generated greater steroidogenic follicles and estrus intensity in dairy cows

The onset of productive life in dairy cattle, concomitant to parturition, is accompanied by a substantial decrease in fertility in comparison with non-lactating, nulliparous heifers. Follicular growth patterns differ between parous and nulliparous dairy cattle. Nulliparous heifers ovulate follicles with reduced antral age (RAA). This study aimed to exogenously reduce ovulatory follicle age in lactating dairy cows from 7 to 5 days old. Cows (n = 80) had their estrous cycles synchronized with the Double-Ovsynch program. At the final portion of this program, luteolysis was induced at either 5 (RAA) or 7 (Control) days following follicular wave emergence. RAA outcomes were estimated in comparison with Controls. RAA resulted in smaller follicles 2 days post-treatment. Despite lower serum concentrations of 17β-estradiol before treatment compared with Controls, the rate of increase in this hormone was greater for the RAA treatment. There was no difference in luteolysis rates between treatments. Proestrus (luteolysis onset to estrus onset) was prolonged in RAA cows. Cows with RAA had more intense estruses. Collectively, these results indicate that decreasing the age of the ovulatory follicle may improve the steroidogenic capacity of the dominant follicle and estrus expression intensity in lactating dairy cows.

Embryonic Exposure to the Benzotriazole Ultraviolet Stabilizer 2-(2H-benzotriazol-2-yl)-4-methylphenol Decreases Fertility of Adult Zebrafish (Danio rerio).

Benzotriazole ultraviolet stabilizers (BUVSs) are emerging contaminants of concern. They are added to a variety of products, including building materials, personal care products, paints, and plastics, to prevent degradation caused by ultraviolet (UV) light. Despite widespread occurrence in aquatic environments, little is known regarding the effects of BUVSs on aquatic organisms. The aim of the present study was to characterize the effects of exposure to 2-(2H-benzotriazol-2-yl)-4-methylphenol (UV-P) on the reproductive success of zebrafish (Danio rerio) following embryonic exposure. Embryos were exposed, by use of microinjection, to UV-P at <1.5 (control), 2.77, and 24.25 ng/g egg, and reared until sexual maturity, when reproductive performance was assessed, following which molecular and biochemical endpoints were analyzed. Exposure to UV-P did not have a significant effect on fecundity. However, there was a significant effect on fertilization success. Using UV-P-exposed males and females, fertility was decreased by 8.75% in the low treatment group and by 15.02% in the high treatment group relative to control. In a reproduction assay with UV-P-exposed males and control females, fertility was decreased by 11.47% in the high treatment group relative to the control. Embryonic exposure to UV-P might have perturbed male sex steroid synthesis as indicated by small changes in blood plasma concentrations of 17β-estradiol and 11-ketotestosterone, and small statistically nonsignificant decreases in mRNA abundances of cyp19a1a, cyp11c1, and hsd17b3. In addition, decreased transcript abundances of genes involved in spermatogenesis, such as nanos2 and dazl, were observed. Decreases in later stages of sperm development were observed, suggesting that embryonic exposure to UV-P impaired spematogenesis, resulting in decreased sperm quantity. The present study is the first to demonstrate latent effects of BUVSs, specifically on fish reproduction. Environ Toxicol Chem 2024;43:385-397. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Removal of Soluble ACE2 in VeroE6 Cells by 17β-Estradiol Reduces SARS-CoV-2 Infectivity.

Women are less susceptible than men to coronavirus disease 2019 (COVID-19), which might be due to the female steroid hormone 17β-estradiol. We hypothesized that 17β-estradiol removes the soluble portion of angiotensin-converting enzyme 2 (sACE2) to which severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds in host cells and that sACE2 then binds to the virus, thereby reducing the infectivity. In the present study, VeroE6/serine protease 2 transmembrane protein (TMPRSS2) cells were infected with pseudo SARS-CoV-2 viruses and used as our model system. This infectivity was reduced by the application of 17β-estradiol. After applying 17β-estradiol to the VeroE6/TMPRSS2 cells, we used a sandwich enzyme-linked immunosorbent assay (ELISA) to measure the sACE2 concentration in the culture medium. Our findings revealed that 17β-estradiol removes sACE2 from VeroE6/TMPRSS2 cells. Furthermore, the pseudo SARS-CoV-2 viruses were incubated in culture medium with ACE2 collected from 17β-estradiol-treated VeroE6/TMPRSS2 cells, and the viruses were measured with an ultrasensitive ELISA using anti-spike protein antibodies. The amount of spike proteins decreased according to the concentration of 17β-estradiol applied. These results clearly demonstrated that the soluble portion of ACE2, which was removed from 17β-estradiol-treated VeroE6/TMPRSS2 cells, bound to the spike proteins of SARS-CoV-2, thereby reducing COVID-19 infectivity.

Urine levels of estrogen and its metabolites in premenopausal middle-aged women with different degrees of obesity and their correlation with glucose and lipid metabolism

ObjectivesTo determine the levels of estrogen and estrogen metabolites in the urine of premenopausal women with obesity and their correlation with glucose and lipid metabolism. Methords135 premenopausal women were selected from the same area. According to the body mass index (BMI), they were divided into four different groups. High performance liquid chromatography-mass spectrometry (HPLC/MS) was adopted to detect the concentrations of estrogen and estrogen metabolites in the urine. The influencing factors of BMI were analyzed, the correlation between the urinary degrees of estrogen and estrogen metabolites and glucose and lipid metabolism levels was assessed. Results(1) The concentrations of 17β-estradiol (E2), estrone (E1), 16α-hydroxyestrone (16α-OHE1) and 2-hydroxyestrone (2-OHE1) gradually increased with increasing BMI (p < 0.05). (2) Stepwise regression analysis displayed that the concentrations of E2, 16α-OHE1 and 2-OHE1 in urine were significantly positively correlated with BMI (p < 0.05). (3) The concentrations of E2, E1, 16α-OHE1, 2-OHE1 and 16α-OHE1/2-OHE1 in urine were greatly positively related to fasting insulin (FIN), Triglyceride (TG), Total Cholesterol (TC) and Low-density lipoprotein (LDL) (p < 0.05). And they were greatly negatively related to High-density lipoprotein (HDL) (p < 0.05). ConclusionsEarly screening can reflect the degree of obesity and glucose and lipid metabolism disorders in premenopausal middle-aged women, thereby providing guidance for improving the prognosis of obese women.

Comparison of 17β-estradiol, bisphenol-A and caffeine concentration levels before and after the water treatment plant.

This article compares the concentration levels of 17β-estradiol (E2), bisphenol-A (BPA) and caffeine (CAF) in the Sinos River, Brazil, which is a source of drinking water and the presence of contaminants after the conventional treatment in a municipal water treatment plant (WTP). A total of nine sampling campaigns were carried out, with sample collection in the Sinos River, upstream and downstream of the WTP, in addition to a drinking water sample (DW). The samples were extracted with solid phase extraction (SPE) and the concentration by liquid chromatography coupled to mass spectrometry (LC-MS). The maximum concentration in the Sinos River was 6,127.99 ng·L-1 for E2, 3,294.63 ng·L-1 for BPA and 1,221.95 ng·L-1 for CAF. In drinking water, the concentration range of E2, BPA and CAF was from less than the Detection Limit (DL) up to 437.50 ng·L-1, <DL up to 2,573.34 ng·L-1 and <DL up to 832.30 ng·L-1, respectively. In conclusion, the concentrations of these pollutants present in the Sinos River are high, which may represent a negative environmental impact on this water source. Drinking water indicates the need for a new treatment process that could promote the removal of these compounds.

Growth, reproduction, fatty acid profiles and offspring performance of broodstock yellow catfish Pelteobagrus fulvidraco fed diets with different lipid levels

Parental nutrition plays important roles in reproductive performance and offspring health of broodstock yellow catfish. However, there is a lack of relevant knowledge concerning the broodstocks of many fish species. This study investigated the influence of dietary lipid levels on growth, spawning performance, tissue fatty acid profiles, sex hormone secretion, and offspring health status of yellow catfish (Pelteobagrus fulvidraco). Five experimental diets were designed containing a 7:3 ratio of fish oil and corn oil blends with graded lipid levels (6.3%, 8.2%, 10.8%, 12.2%, and 14.4% by dry weight) and were fed to female broodstock yellow catfish for 8 weeks. The results showed that weight gain, egg diameter, fertilization ratio, and hatching ratio increased initially and then decreased with increasing dietary lipid levels, reaching optimal peaks in the L10.8 group. Broodstock fed a diet with 10.8% lipid obtained the highest 17-β estradiol concentration. The deposition of highly unsaturated fatty acids in tissues increased initially and then decreased with the increase in dietary lipids, reaching a peak in the 10.8% group. Plasma superoxide dismutase activity and total glutathione content were lowest in fish fed a diet with a 6.3% lipid level, while the contents of malondialdehyde and cortisol in larvae 3 days post-hatching showed an opposite trend, indicating that a low-fat diet reduced the antioxidant capacity of broodstock and induced a stress response that had negative effects on offspring health. Broodstock fed a diet with an appropriate lipid level could increase the expression of antioxidant enzymes by activating the nrf2 signaling pathway. Based on the regression of dietary lipid level and weight gain, reproductive performance, and egg quality of fish, the optimal dietary lipid level was 10.1–11.7% for broodstock yellow catfish.

Male Cope’s gray treefrogs (Hyla chrysoscelis) in amplexus have elevated and correlated steroid hormones compared to solitary males

Gonadal steroid hormones are typically elevated during the breeding season—a finding known as an associated reproductive pattern. Though less studied, there is also evidence, in both sexes, for elevated adrenal/interrenal steroids, including acute elevations on the day of mating. I investigated gonadal and interrenal steroids in wild male Cope’s gray treefrogs at breeding aggregations. I collected blood from males found in amplexus with female mates (amplexed males) and males sampled at the same time and location that were actively advertising vocally and without a mate (solo males). Concentrations of plasma corticosterone, testosterone, and 17β-estradiol (CORT, T and E2, respectively) were validated and measured. These two categories of males differed in four ways: (1) amplexed males exhibited significantly elevated concentrations of all three steroids compared to solo males (CORT: +347 %; T: +60 %; and E2: +43 %); (2) these hormone profiles alone accurately predicted male mating category with ca. 83 % accuracy using a discriminant function analysis; (3) amplexed males exhibited significant between-hormone correlations (T and E2 were positively correlated and CORT and E2 were negatively correlated) whereas no correlations were found in solo males; (4) amplexed males showed a negative correlation with CORT concentration and the time of night, whereas no such pattern was present in solo males. These findings suggest an acute and strong coactivation of the interrenal and gonadal axes that could drive phenotypic integration during this fitness-determining moment. I discuss these findings and suggest experiments to determine causation, including the role of motor behavior driving endocrine states and the role of female selection on endocrine profiles.

The concentration of kisspeptin, 17β-estradiol, progesterone and cortisol in the first trimester of pregnancy in cows

In clinically healthy Holstein cows (n=25), the dynamics of hormonal metabolism in the first trimester of cow pregnancy was studied. For this purpose, the concentration of kisspeptin, 17β-estradiol, progesterone and cortisol was determined in the blood serum of cows. The animals were regularly monitored with clinical and obstetricgynecological studies. The level of kisspeptin in the first trimester of pregnancy increased almost throughout the entire period under consideration with a decrease in concentration to 194.23±23.61 pg/ml on day 98. The level of estradiol during the first trimester increased from 49.66±9.68 to 76.20±17.84 pg/ml, respectively, which is also explained by the inclusion of the fetoplacental complex in its synthesis and secretion. Progesterone and cortisol levels were on average at the same level. During the experiment, out of 25 animals, embryonic death was recorded in 14 on the 30th day after insemination. As a result, two groups were formed: the 1st group - animals with a normal course of pregnancy and the 2nd group with embryonic death. Analyzing the data of enzyme immunoassay in animals of the 1st group, one can note a decrease in the level of kisspeptin by 11% on the 28th day compared to the 14th day after insemination. At the same time, the concentration of estradiol, on the contrary, increased by 29%. In the second group, on the contrary, the level of kisspeptin and estradiol did not change and, on average, remained almost at the same level. On the 28th day after insemination in the first group, the cortisol concentration was significantly higher compared to the second group - 15.32±3.23 and 9.00±0.89 nmol/l, respectively. Data for kisspeptin and estradiol showed no significant differences. However, it is of interest that in the first group, with an increase in the level of estradiol, a decrease in the level of kisspeptin occurs. In particular, this is consistent with previous studies, according to which such an interaction of these hormones can be regulated by a high concentration of progesterone.

A novel fluorescent sensor based on aptamer recognition and DNA walker amplification strategy and its determination of 17β-estradiol

17β-Estradiol (E2) is a common environmental endocrine and excessive estrogen will disturb the endocrine balance of human body and lead to diseases because of its accumulation in human body. The main aim of this study is to determine the trace amount of E2 in various foods. Based on aptamer specific recognition, DNA walker molecular machine and Exonuclease III-assisted dual cycle signal amplification technology, a fluorescent aptamer sensor was constructed and applied to the detection of 17β-estradiol. Atomic force microscopy (AFM) and polyacrylamide gel electrophoresis (PAGE) were used to verify the feasibility of DNA walker participating in the reaction. Under the optimal conditions, 17β-estradiol concentration showed a good linear relationship with fluorescence in the range of 5.0 × 10−10–1.8 × 10−8 M, and the detection limit was 5.6 × 10−11 M. The fluorescent aptamer sensor established in this study has good selectivity, simple operation and good specificity, and the results of its application in the determination of milk samples are satisfactory. Research findings revealed that the E2 concentration in water and milk is less than the safe limits, indicating the foods in the market are safe and can be consumed. Though the foods are safe in present content, the health risk still exists because of continuous intake of trace E2.

FRI273 Generation And In Vitro Characterization Of A Novel Immortalized Ovine Neuronal KNDy Cell Line

Abstract Disclosure: R. Toyoizumi: None. C.Y. Markgraf: None. A. Starman: None. E. Rodriguez: None. B.L. Lee: None. R. Bahn-Bales: None. O. Stricklin: None. C. Estill: None. C.E. Roselli: None. P.E. Chappell: None. The multiphenotypic hypothalamic KNDy neurons play a critical role in the neuroendocrine regulation of reproduction, and via their stimulation of GnRH release, are requisite for fertility and pubertal progression. In vivo evidence has accumulated identifying this more caudal population of Kisspeptin neurons in mediating predominantly negative feedback effects of gonadal steroids, and their ability to synthesize and secrete kisspeptin (KP), neurokinin B (NKB/Tac3), and dynorphin (Dyn) implicates them as crucial regulators of GnRH pulse generation. We have previously generated murine immortalized arcuate KNDy neuronal cell lines to explore molecular mechanisms underlying the steroid hormone regulation of these cells, and in the current study, we characterize a novel immortalized ovine neuronal cell line model of KNDy (oKNDy) cells derived from fetal female sheep brain (∼133 days of fetal development). Clonal lines were derived from isolated mediobasal hypothalamic explants using lentiviral infection of neurons with plasmids encoding SV40 large T-antigen. These neurons express ovine kiss1, tac3, and pdyn, as well as steroid receptors esr1, esr2, and pgr. Similar to KNDy neurons in vivo, receptors for Tac3 (tacr3) and Dyn (oprk1) were also found to be expressed in our oKNDy cells. Further, low physiological concentrations (5-50pM) of 17β-estradiol (E2) were found to significantly repress kiss1 and tac3 expression, while having no effect on tacr3. Concentrations of E2 and progesterone (P4) approximating proestrous levels maximally repressed kiss1 and tac3 after 24h, with elevations in pdyn noted under the same conditions. While cells exposed to higher E2 levels exhibited greater kiss1/tac3 repression, P4 exposure alone revealed stimulation of expression with increasing P4. Since these neurons were derived from the short-day seasonally breeding sheep, they can also be used to explore how circannual timing cues derived from the pituitary pars tuberalis may exert gating signals on the reproductive axis as previously modeled in vivo. Consistent with this, we have found high levels of thyroid hormone receptor α (thra) expression in our oKNDy neurons, suggesting they can respond to locally-synthesized T3. We are continuing to characterize these oKNDy neuronal cells in culture in order to provide insight into GnRH pulse generation and construct molecular models of neuronal responsiveness to steroids and other regulatory inputs. Presentation: Friday, June 16, 2023

Effect of Sex and Cross-Sex Hormone Treatment on Renal Monocarboxylate-Transporter Expression in Rats.

Proton- and sodium-dependent monocarboxylate transporters (MCTs/SMCTs) are determinants of renal clearance through the renal reabsorption of monocarboxylate substrates. Prior studies with intact females and males, ovariectomized females and castrated males have revealed the hormonal regulation of renal monocarboxylate-transporter expression, prompting investigation into the regulatory role of individual hormones. The aim of the present study is to evaluate the effect of exogenous sex and cross-sex hormones on renal MCT1, MCT4, CD147 and SMCT1 mRNA and membrane-bound protein expression. Ovariectomized (OVX) females and castrated (CST) male Sprague Dawley rats received estrogen and/or progesterone, testosterone, or a corresponding placebo treatment for 21 days prior to kidney collection. The quantitative measurement of mRNA and membrane-protein levels were conducted using qPCR and Western blot. Quantitative analysis revealed the combination estrogen/progesterone treatment reduced membrane MCT1 and 4 expression and increased SMCT1 expression, while testosterone administration increased MCT1 membrane-protein expression. Correlation analysis indicated that plasma 17β-estradiol was negatively correlated with MCT1 and MCT4 membrane expression, while testosterone was positively correlated. In contrast, SMCT1 membrane expression was positively correlated with 17β-estradiol and progesterone concentrations. MCT1, MCT4, CD147 and SMCT1 renal expression are significantly altered in response to female and male sex hormones following sex and cross-sex hormone treatment in OVX and CST rats. Further studies are needed to understand the complex role of sex hormones, sex hormone receptors and the impact of puberty on MCT/SMCT regulation.

17β-estradiol inhibits TGF-β-induced collagen gel contraction mediated by human Tenon fibroblasts via Smads and MAPK signaling pathways.

To investigate the impact of 17β-estradiol on the collagen gels contraction (CGC) and inflammation induced by transforming growth factor (TGF)-β in human Tenon fibroblasts (HTFs). HTFs were three-dimensionally cultivated in type I collagen-generated gels with or without TGF-β (5 ng/mL), 17β-estradiol (12.5 to 100 µmol/L), or progesterone (12.5 to 100 µmol/L). Then, the collagen gel diameter was determined to assess the contraction, and the development of stress fibers was analyzed using immunofluorescence staining. Immunoblot and gelatin zymography assays were used to analyze matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) being released into culture supernatants. Enzyme-linked immunosorbent assay (ELISA) and reverse transcription-quantitative polymerase chain reaction (RT-PCR) were used to detect interleukin (IL)-6, monocyte chemoattractant proteins (MCP)-1, and vascular endothelial growth factor (VEGF) in HTFs at the translational and transcriptional levels. The phosphorylation levels of Sma- and Mad-related proteins (Smads), mitogen-activated protein kinases (MAPKs), and protein kinase B (AKT) were measured by immunoblotting. Statistical analysis was performed using either the Tukey-Kramer test or Student's unpaired t-test to compare the various treatments. The CGC caused by TGF-β in HTFs was significantly inhibited by 17β-estradiol (25 to 100 µmol/L), and a statistically significant difference was observed when comparing the normal control group with 17β-estradiol concentrations exceeding 25 µmol/L (P<0.05). The suppressive impact of 17β-estradiol became evident 24h after administration and peaked at 72h (P<0.05), whereas progesterone had no impact. Moreover, 17β-estradiol attenuated the formation of stress fibers, and the production of MMP-3 and MMP-1 in HTFs stimulated by TGF-β. The expression of MCP-1, IL-6, and VEGF mRNA and protein in HTFs were suppressed by 100 µmol/L 17β-estradiol (P<0.01). Additionally, the phosphorylation of Smad2 Smad3, p38, and extracellular signal-regulated kinase (ERK) were downregulated (P <0.01). 17β-estradiol significantly inhibits the CGC and inflammation caused by TGF-β in HTFs. This inhibition is likely related to the suppression of stress fibers, inhibition of MMPs, and attenuation of Smads and MAPK (ERK and p38) signaling. 17β-estradiol may have potential clinical benefits in preventing scar development and inflammation in the conjunctiva.