Small Blubber Samples (50 mg) Sufficient for Analyses of 10 Stress and Reproductive Steroid Hormones in Gray and Fin Whales via Liquid Chromatography Mass Spectrometry

Information on stress, reproductive fitness, and health is difficult to obtain in wild cetaceans but critical for conservation and management. The goal of this study was to develop a methodology requiring minimal blubber mass for analysis of reproductive and stress steroid hormones and, hence, suitable for cetacean biopsies. Blubber biopsies and samples were collected from free-ranging and stranded gray and fin whales. Steroid hormones were extracted from blubber samples as small as 50 mg using liquid-liquid extraction methodology developed to handle the high fat content of blubber. Samples were analyzed via liquid chromatography with tandem mass spectrometry for 10 hormones: aldosterone, androstenedione, cortisol, cortisone, corticosterone, 17β-estradiol, estrone, 17α-hydroxyprogesterone, progesterone, and testosterone. As part of the optimization, homogenization via bead beating and blade dispersion were compared, and the former found superior. To investigate optimal yet minimal tissue mass required, hormone panels were compared among paired 50, 150, and 400 mg samples, the latter two being commonly reported masses for hormone blubber analysis. Results indicated that 50 mg of blubber was suitable and sometimes superior. Additionally, significant differences in precision values were observed between species, possibly stemming from differences in blubber composition, and relevant to homogenization technique selection and calibration methods that use blubber matrix matches obtained from a species other than the study species. Based on recovery and precision values, our methodology was accurate and precise in the measurement of spiked known quantities for all 10 hormones, confirming the methodology capabilities in 50 mg blubber mass in both species. Altogether, and in our specific sample sets, all endogenous hormones, except corticosterone, were identified above the detection limit in 50 mg gray whale blubber samples while all endogenous hormones, except aldosterone, cortisone, estrone, and progesterone, were detected in 50 mg fin whale blubber samples. We present a robust methodology for the analysis of multiple reproductive and stress steroid hormones in minimal masses of cetacean blubber compatible with small biopsies. Finally, we identified statistically significant differences in corticosteroid concentrations between stranded and free ranging animals.

Reduction of Early Fruit Abscission by Main-branch-girdling in Macadamia Is Related to the Favorable Status of Carbohydrates and Endogenous Hormones

Fruit abscission occurring severely in the early fruit development affects macadamia yield. Developing effective methods to improve fruit retention is a priority for macadamia cultivation and production. Girdling is an important horticultural practice that has been widely used to increase fruit yield. Previous studies have shown that girdling fails to increase macadamia yield despite enhancing the early fruit set, but few have examined the effect of girdling on its related physiological mechanism. The objective of this study was to investigate the effects of main-branch girdling (MBG) on early fruit retention and also on the levels of carbohydrates and endogenous hormones in the leaves, bearing shoots and fruit of macadamia. Herein, MBG was performed at fruit set using a single-blade knife on 9-year-old macadamia trees (Macadamia integrifolia). Results showed that MBG significantly reduced young fruit drop, concurrent with significant increases in the contents of starch in both the leaves and the bearing shoots and in glucose, fructose, and sucrose levels in the husk and seed. It was suggested that the availability of carbohydrate for fruit retention was improved by MBG. Additionally, MBG increased indole-3-acetic acid (IAA), gibberellin (GA3), and zeatin-riboside (ZR, a type of cytokinin) concentrations and decreased abscisic acid (ABA) contents in the husk and the seed, indicating that MBG reduced the early fruit drop by modifying the balance of endogenous hormones. Therefore, a positive interplay between carbohydrates and endogenous hormones induced by MBG was involved in the reduction of early fruit abscission in macadamia.

Glucocorticoid receptor antagonist alters corticosterone and receptor sensitive mRNAs in the hypoxic neonatal rat

Hypoxia, a common stressor with preterm birth, increases morbidity and mortality associated with prematurity. Glucocorticoids (GC) are administered to the preterm infant to improve oxygenation; prolonged use of GCs remains controversial. We evaluated a selective glucocorticoid receptor (GR) antagonist (CORT113176) in our neonatal rat model of human prematurity to assess how fasting and hypoxia-induced increases in neonatal corticosterone affects endogenous hormones and endocrine pancreas function. Neonatal rat pups at postnatal day (PD) 2, PD8, and PD15 were pretreated with CORT113176 and, after 60 min of separation and fasting, exposed to hypoxia (8% O2) or control (normoxia) for 30 or 60 minutes while fasting was continued. Plasma corticosterone, ACTH, glucose, and insulin were measured and fasting HOMA-IR (index of insulin resistance) calculated. Glucocorticoid and insulin receptor sensitive gene mRNAs were analyzed in liver, muscle, and adipose to evaluate target tissue biomarkers. CORT113176 pretreatment augmented baseline and hypoxia-induced increases in corticosterone and attenuated hypoxia-induced increases in insulin resistance at PD2. Normoxic and hypoxic stress increased the hepatic GR sensitive gene mRNAs, Gilz and Per1; this was eliminated by pretreatment with CORT113176. CORT113176 pretreatment decreased baseline insulin receptor sensitive gene mRNAs Akt2, Irs1, Pik3r1, and Srebp1c at PD2. We show that CORT113176 variably augments the stress-induced increases in corticosterone concentrations (attenuation of negative feedback) and that GR is critical for hepatic responses to stress in the hypoxic neonate. We also propose that measurement of Gilz and Per1 mRNA expression may be useful to evaluate the effectiveness of GR antagonism.

Hormonal state of breeding bulls after a long winter period of operation in the conditions of the Udmurt Republic

Relevance. The study will help to identify new perspectives in breeding; get new data about the biological processes occurring in the body of animals according to the endogenous hormones of breeding bulls. The hormonal-immunobiological relationship in the body of bulls and their adaptive ability in different climatic conditions is a very important issue today.Methods. The work was performed in a temperate continental climate with a long, snowy winter and warm summer in the conditions of the Udmurt Republic in the period from 2020 to 2021 on breeding bulls (n = 27) of modern breeding at the age of 15–69 months. The state of endogenous hormones testosterone, estradiol, thyroxine and cortisol after a long winter period was studied, depending on the age, breed and selection in the normal physiological functioning of breeding bulls. The average serum values of estradiol — 0.517 nmol/l, testosterone — 25.7 nmol/l, thyroxine — 74.7 nmol/l, cortisol — 420 nmol/l, and their cholesterol precursor at the level of 3.2 nmol/l were established in the blood of breeding bulls located in this region. It was found that, depending on age, there are differences in all the studied indicators, however, there is a reliability for thyroxine and cortisol. The concentration of cortisol by selection significantly differs in imported bulls of European selection from the Netherlands at the level of p > 0.05, which characterizes the presence of a certain stress adaptation factor in this group. In terms of variability, a significant difference between min and max indicators was noted for the concentration of testosterone, which varied between 2.4 nmol/l and 60 nmol/l, indicating the individuality of these hormones in breeding bulls in this region.Results. Based on the data obtained, the need to take these data into account when operating breeding bulls and analyzing sperm products, taking into account the concentration of these hormones, as well as the implementation of appropriate preventive measures, is revealed.

Role of Spiral Steroids in Pregnancy and Pre-Eclampsia

My laboratory discovered a new type of steroids. The structure of these steroids is unique in three ways: (i) they have 23, 24 or 25 carbon atoms – no other known vertebrate steroid has more than 21 carbon atoms; (ii) they are phosphodiesters – no other steroid phosphodiesters are known; and (iii) some of them have a spiral steroid at carbon 17 – no other endogenous spiral steroids are known. In total, our laboratory had elucidated the structure and path of biosynthesis for more than 20 related compounds. We have developed an LC–MS method and a MS–MS method to measure the compounds in small samples (< 1 ml). Synthetic compounds with similar spiral steroids (e.g., spironolactone) function as potassium sparing hormones but there were no known endogenous hormones with this function. We propose that the natural spiral steroids have that function. Endogenous compounds with these functions would have important roles in the physiology of pregnancy, pre-eclampsia, and eclampsia. This chapter will review the proposed physiology and pathology of the spiral steroids during pregnancy. There are many details to confirm but this is a useful paradigm.

Polymorphism of the cytochrome P450 CYP1A1 (Ile462Val) gene in the populations of Balkars and Karachays

BACKGRAUND: Cytochrome P450 is an enzyme involved in the metabolism of phase I xenobiotics, toxins, endogenous hormones and pharmaceuticals. AIM: Is studying the polymorphism of the CYP1A1 gene (Ile462Val, rs1048943) in the Turkic-speaking populations of the central part of the North Caucasus Region (Balkars and Karachays). MATERIALS AND METHODS: We analyzed DNA isolated from leukocytes of peripheral venous blood of a total of 177 unrelated Balkars and Karachays (104 Balkars, 73 Karachays). RESULTS: The ethnicity of the studied individuals was established, indicating their ancestors up to the third generation. The method of genotyping was performed by real-time PCR using competing TaqMan probes. As a result: the frequency of the 462Val variant in the sample of Balkars was 8,6% (95% CI 5,2113,33), slightly above the frequency range found in European populations. In the sample of Karachays, the frequency of the 462Val allele was 7,5% (95% CI 9,8213.08), which is the upper limit of values typical for European populations. CONCLUSION: In the Turkic-speaking populations of the central part of the North Caucasus, the CYP1A1 462Val allele occurs with a frequency characteristic of the Near Eastern and European populations.