Concentrations Of 17beta-estradiol Research Articles

Abstract 4251: Establishment of hormone-dependent endometrial tumoroids in a conditioned-medium free, serum-free medium

Abstract Tumoroids, also known as cancer organoids, are self-assembled three-dimensional cultures of patient-derived tumor cells. Compared to traditional 2D cancer cell lines, tumoroids better maintain key characteristics of the original tumor including the genotype and transcriptome, which lead to better in vitro modeling of clinically relevant functional responses such as drug sensitivity. An under-studied aspect of tumoroid cultures has been their ability to recapitulate dependence on hormone signaling in vitro, with some reports indicating hormone dependence is lost over time in culture. To investigate this, we utilized Gibco™ OncoPro™ Tumoroid Culture Medium and defined tissue-specific supplements, FGF10 and beta-estradiol, that when added to the medium enabled the derivation of endometrial tumoroids from tumor resections. Derivation of tumoroid lines compatible with long-term growth depended on a variety of factors, including tissue quality and time from resection to culture initiation. Each of the 15 samples processed formed tumoroids when initially seeded into culture. 11/15 samples reformed tumoroids through multiple passages (typically 1-2 weeks per passage) over 1-3 months in culture. 7/15 samples met our high criteria for stable tumoroid line growth of >5 passages and reaching >5 cumulative population doublings (from initial number of cells seeded post-resection) in culture. Next-generation sequencing of the established tumoroids and the uncultured tumor material demonstrated a high degree of correlation (pearson’s r>0.9) of genomic mutations in a targeted panel; few differentially expressed genes were detected beyond down-regulation of immune-related genes, presumably because the medium is optimized for outgrowth of cancer epithelial cells, and immune cells do not persist during long-term culture. Endometrial tumoroids were capable of growing embedded in basement membrane extract (BME) or in suspension with dilute BME added and could be cryopreserved and recovered. Endometrial tumoroids retained gene expression levels (both presence and absence) of estrogen receptor and progesterone receptor for up to the 20 passages tested in vitro. A subset of the models was tested and showed increased growth rates in response to increased concentrations of beta-estradiol. Of those tested, 2/3 tumoroid cultures stopped growing within 1-2 weeks after removal of beta-estradiol from the medium, demonstrating dependence on this hormone for growth. Maintenance of hormone receptor expression and dependency should enable in vitro studies to understand the signaling mechanisms and potential ways to modulate them in the context of cancer. Overall, this method provides a toolset for studying endometrial cancer, particularly hormone dependency, and may be more broadly applicable to other gynecological or hormone dependent cancers. Citation Format: Chris Yankaskas, Brittany Balhouse, Colin Paul, Pradip Shahi Thakuri, Shyanne Salen, Mark Kennedy, Matt Dallas, David Kuninger. Establishment of hormone-dependent endometrial tumoroids in a conditioned-medium free, serum-free medium [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 4251.

Expression of Apoptosis-Related Genes in Cultured Ovarian Tissue of Mouse in Presence of Coenzyme Q10.

Oxidative stress affects the development of ovarian follicles during in vitro culture thus applying an antioxidant is necessary. The aim of this study was to investigate the effect of coenzyme Q10 (CoQ10) on the expression of apoptosis-related genes of mouse ovaries during in vitro culture. The immature mouse ovaries were cultured in the presence of 50 μM CoQ10 for 7 days. Histological examinations were performed and the 17 beta-estradiol concentration was measured on the seventh day of culture. The relative expression of Caspase 3, Bax, Bad, and Bcl 2 genes were investigated by real-time RT-PCR. The rates of normal follicles in the presence of CoQ10 was significantly increased compared to the control group. Also, in CoQ10-trated group a significant increase in the level of 17 beta-estradiol was seen compared to the control group. The mRNA expression of anti-apoptotic gene Bcl2 was significantly increased while the expression of pro-apoptotic genes (Caspase3, Bax and Bad) significantly declined in CoQ10 treated group compared to those of control group. The supplementation of the ovarian culture media with CoQ10 improved the follicular development through alteration in expression of apoptosis-related genes and stimulated the production of estradiol.

Estradiol provokes hypercoagulability and affects fibrin biology: A mechanistic exploration of sex dimorphisms in coagulation.

Sex dimorphisms in coagulation are well established, with female-specific hypercoagulability conferring a survival benefit in the setting of trauma-induced coagulopathy (TIC). The mechanism behind these phenomena remains to be elucidated. We hypothesize that estradiol provokes a hypercoagulable profile and alters clot proteomics and fibrin crosslinking. Whole blood was collected from healthy adult volunteers (n = 30). A battery of thrombelastography (TEG) assays (native, kaolin, platelet-mapping, functional fibrinogen), whole blood thrombin generation, proteomics, and clot structure architecture (via analysis of fibrin crosslinks and fluorescent fibrinogen-visualized clots) were performed after pre-treatment of the blood with physiologic concentrations of beta-estradiol. In addition, a prospective study of coagulation through the menstrual cycle was conducted by collecting blood from women on peak and nadir estrogen days in the standard 28-day menstrual cycle. On TEG, in females, estradiol provoked a hypercoagulable phenotype, specifically a shorter time to clot formation and greater thrombin generation, greater rate of clot propagation and functional fibrinogen, higher clot strength, and diminished clot fibrinolysis. In both males and females, estradiol increased platelet hyperactivity. Similar changes were seen in time to clot formation and clot strength in vivo during peak estrus of the menstrual cycle. On proteomic analysis, in both males and females, estradiol was associated with increases in abundance of several procoagulant and antifibrinolytic proteins. Crosslinking mass spectrometry analysis showed addition of estradiol increased the abundance of several FXIII crosslinks within the FIBA alpha chain in both sexes. Fluorescent fibrinogen analysis revealed a trend toward increased fiber resolvability index after addition of estradiol. Estradiol provokes a hypercoagulable phenotype, affecting time to clot formation, clot propagation, clot strength, clot fibrinolysis, and clot structure. In sum, these data highlight the role of estradiol is driving female-specific hypercoagulability and highlights its potential role as a therapeutic adjunct in resuscitation of TIC.

325 Blood pressure and the kidney cortex transcriptome response to high sodium diet challenge in female nonhuman primates

OBJECTIVES/GOALS: The goal of this study was to understand the impact of a high sodium diet on gene networks in the kidney that correlate with blood pressure in female primates, and translating findings to women. METHODS/STUDY POPULATION: Sodium-naïve female baboons (n=7) were fed a low-sodium (LS) diet for 6 weeks followed by a high sodium (HS) diet for 6 weeks. Sodium intake, serum 17 beta-estradiol, and ultrasound-guided kidney biopsies for RNA-Seq were collected at the end of each diet. Blood pressure was continuously measured for 64-hour periods throughout the study by implantable telemetry devices. Weighted gene coexpression network analysis was performed on RNA-Seq data to identify transcripts correlated with blood pressure on each diet. Network analysis was performed on transcripts highly correlated with BP, and in silico findings were validated by immunohistochemistry of kidney tissues. RESULTS/ANTICIPATED RESULTS: On the LS diet, Na+ intake and serum 17 beta-estradiol concentration correlated with BP. Cell type composition of renal biopsies was consistent among all animals for both diets. Kidney transcriptomes differed by diet; analysis by unbiased weighted gene co-expression network analysis revealed modules of genes correlated with BP on the HS diet. Network analysis of module genes showed causal networks linking hormone receptors, proliferation and differentiation, methylation, hypoxia, insulin and lipid regulation, and inflammation as regulators underlying variation in BP on the HS diet. Our results show variation in BP correlated with novel kidney gene networks with master regulators PPARG and MYC in female baboons on a HS diet. DISCUSSION/SIGNIFICANCE: Previous studies in primates to identify molecular networks dysregulated by HS diet focused on males. Current clinical guidelines do not offer sex-specific treatment plans for sodium sensitive hypertension. This study leveraged variation in BP as a first step to identify correlated kidney regulatory gene networks in female primates after a HS diet.

The Effectiveness of Pharmacological Synchronization of the Estrous Cycle in Hinds (Cervus elaphus L.): A Pilot Field Trial.

Simple SummaryReproductive biotechnics of Cevidae species may increase valuable traits for the agricultural sector, and improve genetic diversity in relatively small farmed populations. The first step of biotechnics in terms of suitability for embryo transfer or insemination is preparation of females for proper phase of the estrous cycle. Based on available synchronization protocols applicated in red deer and domestic ruminant females we estimated the protocol for hinds in view of future routine use. Progestin analogues in the form of an insert (CIDR) or a sponge (Chronogest) and injection of equine chorionic gonadotropin (eCG; Folligon) were used on the appropriate day for inducing the estrus. The effect of synchronization was checked by measurement of progesterone (P4) and 17-beta estradiol (E2) concentration by RIA, pregnancy-associated glycoproteins (PAG)s concentration by EIA and insemination effectiveness—pregnancy rate. The better method of the estrous cycle synchronization found down to be progestin sponge and eCG based protocol in hinds considering hormone responsiveness and pregnancy rate.The aim was to estimate the effective pharmacological method of the estrous cycle synchronization by checking the effects of synchronization by measurement of progesterone (P4) and 17-beta estradiol (E2) concentration by RIA and artificial insemination. The experiment was performed at the red deer farm in Rudzie (North-East Poland; 3 year’s old). The herd (N = 14) was kept away from bulls and was divided in two groups of seven animals. In the Group I, CIDR insert (0.3 g of P4) was applicated intravaginally for 12 days; a second insert replaced the first one for the next 12 days, and next 200 IU of equine chorionic gonadotropin (eCG) was injected intramuscularly (Folligon). Estrus was expected 48 h after eCG injection. In the Group II, Chronogest sponge (20 mg of flugestone acetate) was applicated intravaginally and after 7 days replaced with second chronogest sponge for 7 days. After removing the sponge, on the same day eCG was injected and estrus was expected after 48 h. Artificial insemination was provided with frozen-thawed semen twice: 12 and 24 h after expected estrus. The peripheral blood from the jugular vein was collected each time when the inserts or sponge were applicated and 40 days after insemination. The concentration of P4 and E2 in plasma was measured by RIA. The effectiveness of insemination was monitored by pregnancy-associated glycoproteins determination and observed by the number of calves born. Two pregnancies were confirmed in Group I and five in Group II based on PAG concentration. One newborn was observed in Group I and five in Group II. Both methods of synchronization are effective in hinds based on the received profile of steroids. Although the sponge shape in case of chronogest is better comparing with CIDR, which was not completely deposited in the vagina of hind, potentially leads to bacteria inflammation, and it disturbs the rightful endocrine regulation. Moreover, pregnancy rate and hormone responsiveness were better in Group II.

Association among lipopolysaccharide, the transforming growth factor–beta superfamily, follicular growth, and transcription factors in spontaneous bovine ovarian cysts

The aim of this study was to investigate some of the growth and transcriptional factors originating from oocytes and granulosa cells in follicular fluid and to identify the relationships between the basic blood metabolite-metabolic hormones and intrafollicular lipopolysaccharide (LPS) concentrations. Thirty cows included in the study were allocated into 2 groups comprising 15 cows with healthy preovulatory follicles (cyclic cows) and 15 cows with confirmed cystic follicles. The ovaries and uteri of all cows were assessed by transrectal ultrasonographic examination. Blood serum samples were collected at 15, 25, 35, 45, and 55 d after calving for analysis of nonesterified fatty acids, β-hydroxybutyrate, insulin, glucose, IGF-I, ACTH, and cortisol. Ovaries and uteri were examined using transrectal ultrasound. Vaginal discharge was evaluated on the same days. Follicular fluid was also aspirated on days 35–55 from the healthy preovulatory follicles and cystic follicles using a transvaginal ovum pickup method. The densitometric levels of inhibin-α, growth and differentiation factor (GDF-9), bone morphogenetic protein (BMP-6), and GATA-4 and GATA-6 proteins were analyzed by the Western blotting technique; the concentrations of antimullerian hormone (AMH), IGF-I, estradiol-17 beta (E2), and progesterone (P4) were determined by ELISA; and the concentrations of LPS in the follicular fluid were measured by the Limulus amebocyte lysate test. The serum insulin, ACTH, and cortisol concentrations were higher in cystic cows than cyclic cows, but serum IGF-I concentrations were lower in cystic cows. The IGF-I concentrations of cystic follicular fluids were lower, whereas AMH levels were significantly greater than those of healthy preovulatory follicular fluids. The cystic follicles had significantly lower expression levels of GDF-9, BMP-6, GATA-4, and GATA-6; in contrast, inhibin-α expression and LPS concentrations were significantly higher than in healthy preovulatory follicles. The proportion of pathologic vaginal discharge within 25 d postpartum in cystic cows were higher than in the cyclic group. In conclusion, it is suggested that intrafollicular dysregulation of the transforming growth factor–β superfamily, growth, and transcriptional factors is affected by high intrafollicular LPS concentrations and systemic metabolic changes and these disturbances may be responsible for the generation of ovarian cysts.

Beta-estradiol and ethinylestradiol enhance RBC deformability dependent on their blood concentration.

Natural and synthetic estrogens seems to have opposite effects on thrombosis and female cardiovascular system, since natural estrogen was supposed to be protective against cardiovascular diseases and synthetic estrogen has been related to thrombosis and cardiovascular diseases. In this work we have investigated if these differences could be related with the effects on those hormones on some hemorheological parameters. The objective of this work was to investigate the hemorheological changes of different concentrations of beta-estradiol and ethinylestradiol, on RBC aggregation and RBC deformability. Samples of blood of healthy donors were added with different concentrations of natural beta-estradiol or synthetic ethinylestradiol and were analyzed for red blood cell (RBC) aggregation and RBC deformability. There were no significant changes in RBC aggregation. Both beta-estradiol and ethinylestradiol increase the RBC deformability in shear stresses above 3.0 Pa accordingly with the hormone's concentration. Beta-estradiol and ethinylestradiol enhance RBC deformability dependent of their concentration. These findings may explain the different patterns of thrombotic and cardiovascular effects in different phases of the menstrual cycle or different dosages of oral contraceptive or hormonal replacement therapy.

Relationship between Progesterone and Oestradiol-17beta Concentrations and Embryonic Loss in Frisian Cows under Egyptian Condition

The objective of this study was to determine the association between circulating (Progesterone, oestradiol-17β and P4/E2 ratio) concentrations and embryonic loss in Friesian cows under Egyptian condition. On day 25 by both progesterone (P4) level and sonography, 65.71% (69/105) cows were continued their gestation normal and 17.14% (18/105) cows not conceived. While, 17.14% (18/105) of inseminated cows showed high P4 level firstly and decline before day 25 suspecting early pregnancy loss, EPL. Also, 5 cows (7.25%) of the pregnant cows lost their feti from day 25 to day 45 (late pregnancy loss, LPL) were detected. Progesterone concentrations was noticed that increased P4 level starting from day 4 to day 7after AI in cows diagnosed pregnant (P

Expression pattern of HIF1alpha and vasohibins during follicle maturation and corpus luteum function in the bovine ovary.

The aim of this study was to characterize expression patterns of hypoxia-inducible factor-1alpha (HIF1A) and vasohibin family members (VASH1 and VASH2) during different stages of ovarian function in cow. Experiment 1: Antral follicle classification occurred by follicle size and estradiol-17beta (E2) concentration in the follicular fluid into 5 groups (<0.5, 0.5-5, 5-40, 40-180 and >180 E2 ng/ml). Experiment 2: Corpora lutea (CL) were assigned to the following stages: days 1-2, 3-4, 5-7, 8-12, 13-16 and >18 (after regression) of oestrous cycle and of pregnancy (months 1-2, 3-4, 6-7, >8). Experiment 3: Cows on days 8-12 were injected with a prostaglandin F2alpha (PGF) analogue and CL were collected before and 0.5, 2, 4, 12, 24, 48 and 64hr after PGF injection. Expression of mRNA was measured by qPCR, steroid hormone concentration by EIA and localization by immunohistochemistry. HIF1A mRNA expression in our study increases significantly in follicles during final maturation. The highest HIF1A mRNA expression was detected during the early luteal phase, followed by a significant decrease afterwards. In contrast, the mRNA of vasohibins in small follicle was high, followed by a continuous and significant downregulation in preovulatory follicles. The obtained results show a remarkable inverse expression and localization pattern of HIF1A and vasohibins during different stages of ovarian function in cow. These results lead to the assumption that the examined factors are involved in the local mechanisms regulating angiogenesis and that the interactions between proangiogenic (HIF1A) and antiangiogenic (vasohibins) factors impact all stages of bovine ovary function.

Steroid hormones in bovine oviductal fluid during the estrous cycle

Ovarian steroid hormones are major regulators of the physiology of the oviduct and reproductive events occurring within the oviduct. To establish a whole steroid profiling of the bovine oviductal fluid (OF) during the estrous cycle, contralateral and ipsilateral (to the corpus luteum or preovulatory follicle) oviducts were classified into four stages of the estrous cycle (n = 18–27 cows per stage): postovulatory (Post-ov), mid-luteal (Mid-lut), late luteal (Late-lut), and preovulatory on the basis of the ovarian morphology and intrafollicular steroid concentrations. Steroids were extracted from pools of 150 to 200 μL OF (three to 10 cows per pool; three to four pools per “stage × side” group), purified, fractioned by high-performance liquid chromatography, and analyzed by gas chromatography coupled with tandem mass spectrometry. The concentrations of progesterone (P4) in ipsilateral OF increased from Post-ov (56.9 ± 13.4 ng/mL) to Mid-lut (120.3 ± 34.3 ng/mL), then decreased from Late-lut (76.7 ± 1.8 ng/mL) to Pre-ov (6.3 ± 1.7 ng/mL), and were four to 16 times higher than in contralateral OF. Most P4 metabolites followed similar patterns of variation. Concentrations of 17beta-estradiol (E2) were significantly higher at Pre-ov (290.5 ± 63.2 pg/mL) compared with all other stages (<118.3 pg/mL), with no difference regarding the side of ovulation. Concentrations of androstenedione displayed a pattern similar to that of E2, whereas other androgens, estrone, and corticoids did not vary between stages or sides. In conclusion, a highly concentrated and fluctuating hormonal environment was evidenced in the bovine OF. These results could be useful to improve media for IVF, embryo development, and culture of oviductal cells.

Expression and localization of members of the thrombospondin family during final follicle maturation and corpus luteum formation and function in the bovine ovary.

The aim of this study was to characterize the expression patterns and localization of the thrombospondin family members (THBS1, THBS2) and their receptors (CD36 and CD47) in bovine ovaries. First, the antral follicles were classified into 5 groups based on the follicle size and estradiol-17beta (E2) concentration in the follicular fluid (< 0.5, 0.5–5, 5–40, 40–180 and >180 E2 ng/ml). Second, the corpus luteum (CL) was assigned to the following stages: days 1–2, 3–4, 5–7, 8–12, 13–16 and >18 of the estrous cycle and of pregnancy (month 1–2, 3–4, 6–7 and > 8). Third, the corpora lutea were collected by transvaginal ovariectomy before and 0.5, 2, 4, 12, 24, 48 and 64 h after inducing luteolysis by injecting a prostaglandin F2alpha analog. The mRNA expression of examined factors was measured by RT-qPCR, steroid hormone concentration by EIA, and localization by immunohistochemistry. The mRNA expression of THBS1, THBS2, CD36, and CD47 in the granulosa cells and theca interna was high in the small follicles and reduced in the preovulatory follicles. The mRNA expression of THBS1, THBS2, and CD47 in the CL during the estrous cycle was high, but decreased significantly during pregnancy. After induced luteolysis, thrombospondins increased significantly to reach the maximum level at 12 h for THBS1, 24 h for THBS2, and 48 h for CD36. The temporal expression and localization pattern of the thrombospondins and their specific receptors in the antral follicles and corpora lutea during the different physiological phases of the estrous cycle and induced luteolysis appear to be compatible with their inhibitory role in the control of ovarian angiogenesis.

Hyaluronic acid prevents immunosuppressive drug-induced ovarian damage via up-regulating PGRMC1 expression.

Chemotherapy treatment in women can frequently cause damage to the ovaries, which may lead to primary ovarian insufficiency (POI). In this study, we assessed the preventative effects of hyaluronic acid (HA) in immunosuppressive drug-induced POI-like rat models and investigated the possible mechanisms. We found that HA, which was reduced in primary and immunosuppressant-induced POI patients, could protect the immunosuppressant-induced damage to granulosa cells (GCs) in vitro. Then we found that HA blocked the tripterygium glycosides (TG) induced POI-like presentations in rats, including delayed or irregular estrous cycles, reduced 17 beta-estradiol(E2) concentration, decreased number of follicles, destruction of follicle structure, and damage of reproductive ability. Furthermore, we investigated the mechanisms of HA prevention effects on POI, which was associated with promotion of GC proliferation and PGRMC1 expression. In conclusion, HA prevents chemotherapy-induced ovarian damage by promoting PGRMC1 in GCs. This study may provide a new strategy for prevention and treatment of POI.

The interaction between air pollution and diet does not influence the DNA damage in lymphocytes of pregnant women

The aim of the study was to evaluate the risk of DNA damage in lymphocytes of pregnant women with respect to hormonal and nutritional status and to air pollution in Lesser Poland. The study was performed on 39 healthy pregnant women. The oxidative DNA damage, alkali-labile sites and uracil in DNA of lymphocytes were measured by using the comet assay. The concentration of 17beta-estradiol, progesterone, DHEA, cholesterol, vitamin B12 and folates were determined. Dietary data were assembled from food diaries. Voivodeship Inspectorate for Environmental Protection in Krakow using automatic pollution monitoring system provided the air pollution information, such as concentrations of PM10, PM2.5, NO, NO2, SO2, CO and O3. Many statistical correlations between DNA damage and air pollutants concentration were found however their biological meaning is still to be explained. It should be taken under consideration, that the protective effect of air pollutants is a result of hormesis, as the measured amounts of air pollutants during the study did not exceed the admissible levels. There was found no diet-and air pollution interaction.

SAT0440 Serum Sclerostin is Higher in Men with Severe Osteophytes at the Spine – the Minos Study

BackgroundSclerostin is a negative regulator of bone formation that might be involved in osteoarthritis (OA) pathophysiology.ObjectivesOur aim was to analyze the association between spine OA severity and serum sclerostin concentration...

Evidence for the presence of sex steroid hormones in Zhikong scallop, Chlamys farreri

To obtain evidence of the presence of sex steroid hormones in mollusks, hormone variation in the gonads of the Zhikong scallop Chlamys farreri was analyzed using UPLC-MS/MS. These were found, as expected, with concentrations of estrone (E1), 17beta-estradiol (E2), and testosterone (T) in the testes ranging from not detected (ND) to 0.07±0.10, ND to 3.10±2.00, and ND to 2.67±1.55ng/g wet weight, respectively. In the ovaries, these hormones ranged from ND to 2.45±1.22, ND to 27.90±4.23, and ND to 2.38±1.56ng/g ww, respectively. The levels of T in males and E2 in females followed a trend similar to the gonadal-somatic index over the course of the reproductive period. In addition, the gene expression of vitellogenin and calmodulin-2 showed similar patterns to T and E2, while the estrogen receptors and calmodulin-1 did not. These results indicate that sex steroids are present in the scallop and that they may regulate endocrine functions during the reproductive process.

Endocrine and metabolic mechanisms of embryo and fetal intrauterine growth retardation in dairy cows

This research was conducted to study hormonal and metabolic statuses and to determine ultrasound criteria for early diagnosis of fetal growth retardation syndrome (FGRS) in dairy cows of Black-motley (n = 41) and Red-motley (n = 56) Holstein breeds. Concentrations of progesterone, testosterone, 17beta-estradiol, dehydroepiandrosterone sulfate, cortisol, and triiodothyronine in blood serum were assessed by immune-enzyme analysis. Concentrations of plasma L-ascorbic acid and nitrogen oxides, serum immune globulin, middle molecular peptides, and bactericidal and lysozyme activity were determined by spectrophotometry. On days 38-40 and 60-65 of gestation, FGRS in cows was associated with hypoprogesteronemia, decrease of adrenal and thyroid gland functions, decreased nitric oxide synthesis, low immune responsiveness, and high endogenous intoxication. In conclusion, body length less than 16 mm on days 38-40 and less than 45 mm on days 60-65 are the criteria for designating underdevelopment of embryos and fetuses in dairy cows. FGRS genesis in dairy cows is determined by the embryo's and fetus's malnutrition at the early implantation and placentation stages, caused by the imbalance of endocrine regulation, nitric oxide systems, and endogenous intoxication.

The effect of short-term lupin (Lupinus angustifolius) feed supplementation on serum steroid hormones, insulin-like growth factor I, and ovarian follicular development and atresia in Merino ewes

The effects of short-term lupin supplementation on ovulation rate in ewes during oestrous season are well known. In this study, we tested the effects of an 8-day lupin supplementation on folliculogenesis during anoestrus applied over the hormonal treatment of ewes. Fourteen anoestrous Merino ewes were assigned into 2 groups (n = 7 for each) in which the control group was fed a basal diet and the experimental group was supplemented with lupin groats (500 g per head per day). Both groups were treated intramuscularly with 12.5 µg/head lecirelinum (luteinising hormone releasing hormone super analogue) on the first day of supplementation and 5 days later with 37.5 µg/head cloprostenolum D (prostaglandin F2 alpha analogue). Serum sexual steroid and insulin-like growth factor I (IGF-I) concentrations were assessed using radioimmunoassay and enzyme-linked immunosorbent assay methods, respectively. Folliculogenesis was evaluated using a light microscope. Lupin feeding did not affect progesterone or IGF-I, but it significantly decreased the concentration of oestradiol-17beta. Lupin significantly increased the number of follicles of 3-5 mm in diameter and the total rate of atresia. The follicles mainly collapsed and underwent late atresia. The size of healthy and collapsed follicles increased in the lupin-fed group. These results indicated that 8-day lupin supplementation affected follicular development and atresia through an IGF-I/oestradiol feedback system.

Female zebrafish (Danio rerio) are more vulnerable than males to microcystin-LR exposure, without exhibiting estrogenic effects

Microcystins (MCs) released during cyanobacterial blooms exert varied toxicity on fish. Up to now, the reproductive toxicity of MCs on fish has rarely been reported. The present study investigated the reproductive toxicity of microcystin-LR (MC-LR) on male and female zebrafish (Danio rerio) by subchronic immersion in 1, 5, 20μg/L for 30d. After MC-LR exposure, the hatchability and the 17 beta-estradiol (E2) concentration in gonads significantly decreased in the 20μg/L group. In the 5 and 20μg/L groups, the whole body vitellogenin (VTG) levels significantly increased in females, while considerably decreased in males. The VTG1 transcriptional level significantly reduced in the liver of both female and male treated fish. Marked histological lesions were observed in the livers, ovaries and testes in MC-LR treated fish. Apoptotic rate in the ovaries significantly increased. Significant down-regulation of Bcl-2 transcriptional level was found in the gonads of all MC-LR treated fish, while marked up-regulation of Bax transcription level was determined in the 20μg/L female treatment group, but a significant down-regulation in males. Although the transcriptional level of caspase-3 dropped in ovaries of the 5 and 20μg/L treatment groups, the significant increase of caspase-3 activation levels in the ovaries and testes were detected. The present findings indicate that MC-LR exposure exerts diverse reproductive toxicity in zebrafish with females exhibiting more sensitivity than males. The present study also confirmed for the first time that MC-LR does not cause any estrogenic effects in adult zebrafish.

Preweaning Over- and Underfeeding Alters Onset of Puberty in the Rat Without Affecting Kisspeptin1

The perinatal nutritional environment can permanently influence body weight, potentially leading to changes in puberty onset and reproductive function. We hypothesized that perinatal under- or overfeeding would alter puberty onset and influence concentrations of a neuropeptide crucial for successful puberty, kisspeptin. We manipulated Wistar rat litter sizes to derive small (SL), control (CL), and large (LL) litters containing 4, 12, and 20 rat pups respectively. This manipulation results in an overweight phenotype in SL rats and a lean phenotype in LL that persists throughout life. To investigate whether successful puberty onset is affected by neonatal under- or overfeeding, we examined indices of growth and development, including the onset of puberty, as well as the central expression of Kiss1 mRNA in these pups. Male LL rats reached puberty later than those from CL. These males also had reduced plasma testosterone and elevated 17beta-estradiol concentrations at puberty. The age at puberty onset was not affected in SL males despite accelerated growth. In females, puberty onset was not significantly delayed by having a lean phenotype, and steroid hormones were not affected. The age at onset was, however, younger in the SL females. Kiss1 mRNA in the hypothalamus was not affected by neonatal nutrition either at puberty or 7 days later. Our findings show early life underfeeding in males and overfeeding in females significantly affects puberty onset, altering steroid hormone concentrations in males, but this is not related to changes in hypothalamic kisspeptin.

Vitellogenin Expression in White Adipose Tissue in Female Teleost Fish1

In most oviparous animal species, oocyte growth occurs via the uptake of plasma egg yolk precursors, predominantly vitellogenins (Vtg). These glycolipoproteins are members of the large lipid transfer protein superfamily and key players in reproduction. While the vertebrate liver has been demonstrated to synthesize large amounts of Vtg, mostly under 17beta-estradiol control, the ability of other tissues to express significant amounts of Vtg has not been conclusively demonstrated. RT-PCR revealed vtg1 transcripts in female zebrafish and rainbow trout white adipose tissue (WAT). It was also found to coexpress mtp, known to perform the intracellular lipidation of Vtg prior to secretion. The liver and pancreas markers apobb2 and ins, or ela2, respectively, were not expressed in adipocytes. Whole-mount in situ hybridization and in situ RT-PCR tests of histological sections revealed vtg1 signal in adipocytes, whereas no signal was detected in infiltrated pancreatic islets. Transcript expression of vtg1 was induced in WAT of 17beta-estradiol-treated males, and the transcript and corresponding protein were detected in the thin rim of cytoplasm surrounding the adipocyte. Real-time quantitative RT-PCR showed that rainbow trout perivisceral WAT vtg1 transcript levels were high during early compared to late vitellogenesis. Taking normalized mRNA levels and tissue somatic index into account, vtg1 transcript levels at the beginning of oocyte yolk deposition were approximately 45 times lower in WAT than in liver, and these levels were not correlated to plasma Vtg and 17beta-estradiol concentrations. These findings suggest that WAT Vtg is implicated in providing components to the ovary during the early stages of vitellogenesis.