Heinrich H. D. Meyer

Sex hormones originating from different livestock production systems: fate and potential disrupting activity in the environment

Abstract Endogenous hormones of human or animal origin have been reaching the environment for thousands of years, even though to an increasing extent due to growing population and more intensive farming. During the last decade the hormonal disrupting activity of different substances of both natural and anthropogenic origin, has been discussed for wildlife populations in various ecosystems and even for human fertility. So far, natural recycling has not been causally linked to any known severe adverse effect on wildlife or human endocrine system, but discussion on environmental endocrine disrupters has to be extended by this important aspect. The amount of sex steroids excreted by humans and livestock seems in the same order of magnitude, but the available data on their importance is still limited. Besides endogenous hormones, exogenous sex steroids used as anabolics in animals are excreted and reach the environment. The environmental fate of steroids originating from livestock excreta seems to be strongly influenced by storage conditions and also by the soil type of the fields where the dung is spread. Particle size and organic components strongly affect adsorption and migration in the soil. Our studies indicate that low concentrations of trenbolone and melengestrol acetate are very mobile in agricultural soils. However, both hormones have a high affinity to the organic fraction of the immobile phase leading to a high retardation within soil materials.

The novel mTOR inhibitor RAD001 (everolimus) induces antiproliferative effects in human pancreatic neuroendocrine tumor cells.

Background/Aim: Tumors exhibiting constitutively activated PI(3)K/Akt/mTOR signaling are hypersensitive to mTOR inhibitors such as RAD001 (everolimus) which is presently being investigated in clinical phase II trials in various tumor entities, including neuroendocrine tumors (NETs). However, no preclinical data about the effects of RAD001 on NET cells have been published. In this study, we aimed to evaluate the effects of RAD001 on BON cells, a human pancreatic NET cell line that exhibits constitutively activated PI(3)K/Akt/mTOR signaling. Methods: BON cells were treated with different concentrations of RAD001 to analyze its effect on cell growth using proliferation assays. Apoptosis was examined by Western blot analysis of caspase-3/PARP cleavage and by FACS analysis of DNA fragmentation. Results: RAD001 potently inhibited BON cell growth in a dose-dependent manner which was dependent on the serum concentration in the medium. RAD001-induced growth inhibition involved G0/G1-phase arrest as well as induction of apoptosis. Conclusion: In summary, our data demonstrate antiproliferative and apoptotic effects of RAD001 in NET cells in vitro supporting its clinical use in current phase II trials in NET patients.

Monozygotic Twin Model Reveals Novel Embryo-Induced Transcriptome Changes of Bovine Endometrium in the Preattachment Period

Abstract Initiation and maintenance of pregnancy are critically dependent on an intact embryo-maternal communication in the preimplantation period. To get new insights into molecular mechanisms underlying this complex dialog, a holistic transcriptome study of endometrium samples from Day 18 pregnant vs. nonpregnant twin cows was performed. This genetically defined model system facilitated the identification of specific conceptus-induced changes of the endometrium transcriptome. Using a combination of subtracted cDNA libraries and cDNA array hybridization, 87 different genes were identified as upregulated in pregnant animals. Almost one half of these genes are known to be stimulated by type I interferons. For the ISG15ylation system, which is assumed to play an important role in interferon tau (IFNT) signaling, mRNAs of four potential components (IFITM1, IFITM3, HSXIAPAF1, and DTX3L) were found at increased levels in addition to ISG15 and UBE1L. These results were further substantiated by colocalization of these mRNAs in the endometrium of pregnant animals shown by in situ hybridization. A functional classification of the identified genes revealed several different biological processes involved in the preparation of the endometrium for the attachment and implantation of the embryo. Specifically, elevated transcript levels were found for genes involved in modulation of the maternal immune system, genes relevant for cell adhesion, and for remodeling of the endometrium. This first systematic study of maternal transcriptome changes in response to the presence of an embryo on Day 18 of pregnancy in cattle is an important step toward deciphering the embryo-maternal dialog using a systems biology approach.

Tissue‐specific expression pattern of estrogen receptors (ER): Quantification of ERα and ERβ mRNA with real‐time RT‐PCR

We have examined the tissue‐specific mRNA expression of ERα and ERβ in various bovine tissues using real‐time RT‐PCR. The goal of this study was to evaluate the deviating tissue sensitivities and the influence of the estrogenic active preparation RALGRO on the tissue‐specific expression and regulation of both ER subtypes. RALGRO contains Zeranol (α‐Zearalanol), a derivative of the mycotoxin Zearalenon, shows strong estrogenic and anabolic effects, and exhibits all symptoms of hyperestrogenism, in particular reproductive and developmental disorders. Eight heifers were treated over 8 weeks with multiple‐dose implantations (0X, 1X, 3X, 10X) of Zeranol. Plasma Zeranol concentration, measured by enzyme immunoassay, of multiple treated heifers was elevated. To quantify ERα and ERβ transcripts also in low‐abundant tissues, sensitive and reliable real‐time RT‐PCR quantification methods were developed and validated on the LightCycler. Expression results indicate the existence of both ER subtypes in all 15 investigated tissues. All tissues exhibited a specific ERα and ERβ expression pattern and regulation. With increasing Zeranol concentrations, a significant downregulation of ERα mRNA expression could be observed in jejunum (p<0.001) and kidney medulla (p<0.05). These data support the hypothesis that ERβ may have different biological functions than ERα, especially in kidney and jejunum.

Biochemistry and physiology of anabolic hormones used for improvement of meat production

A number of hormones are involved in endocrine regulation of growth. In general, these hormones enhance body protein accretion and metabolise fat stores resulting in increased lean growth rates. Most practical importance was obtained by sex hormones (oestrogens and androgens), β‐agonists and growth hormone — whether legally or illegally. Efficiency of growth promotion ranges between 0% and +20% depending on the prerequisites such as species, breed, gender, age, reproductive status, body score or feeding of the animals. Oestrogens and androgens mediate their activity via intracellular receptors — directly in muscular tissue as well as indirectly via stimulation of growth hormone from the hypophysis and other growth factors from liver plus several further organs. In addition, mineral absorption in the gut is improved. The outstanding efficiency of trenbolone is based on its androgenic plus antiglucocorticoid activity. Melengestrolacetate is thought to act indirectly via stimulation of endogenous ovarian oestradiol in non‐pregnant heifers. The necessary dosages and residue formations depend on the pharmacokinetic parameters of each substance and extrapolations between compounds are hardly possible. Growth hormone and β2‐agonists use independent pathways for growth promotion not related to steroid biochemistry.

Characterisation of the affinity of different anabolics and synthetic hormones to the human androgen receptor, human sex hormone binding globulin and to the bovine progestin receptor

For the steroidal growth promoters trenbolone acetate (TBA) and melengestrol acetate (MGA) neither the complete spectrum of biological activities nor the potential endocrine disrupting activity of their excreted metabolites in the environment is fully understood. The potency of these substances in [3H]‐dihydrotestosterone ([3H]‐DHT) displacement from the recombinant human androgen receptor (rhAR) and from human sex‐hormone binding globulin (hSHBG) was evaluated. In addition, the potency for [3H]‐ORG2058 displacement from the bovine uterine progestin receptor (bPR) was tested. For comparison, different anabolics and synthetic hormones were also tested for their binding affinities. For 17β‐trenbolone (17β‐TbOH), the active compound after TBA administration, an affinity the rhAR similar to dihydrotestosterone (DHT) and a slightly higher affinity to the bPR than progesterone were demonstrated. The affinity of the two major metabolites, 17α‐trenbolone and trendione, was reduced to less than 5% of the 17β‐TbOH‐value. The affinity of these three compounds and of MGA to the hSHBG was much lower compared with DHT. MGA showed a 5.3‐fold higher affinity than progesterone to the bPR but only a weak affinity to the rhAR. The major MGA metabolites have an affinity to the bPR between 85% and 28% of the affinity of progesterone. In consequence, MGA and TBA metabolites may be hor‐monally active substances, which will be present in edible tissues and in manure. We conclude that detailed investigations on biodegradation, distribution and bio‐efficacy of these substances are necessary.

Nuclear magnetic resonance and mass spectrometry-based milk metabolomics in dairy cows during early and late lactation

Milk production in dairy cows has dramatically increased over the past few decades. The selection for higher milk yield affects the partitioning of available nutrients, with more energy being allocated to milk synthesis and less to physiological processes essential to fertility and fitness. In this study, the abundance of numerous milk metabolites in early and late lactation was systematically investigated, with an emphasis on metabolites related to energy metabolism. The aim of the study was the identification and correlation of milk constituents to the metabolic status of the cows. To investigate the influence of lactation stage on physiological and metabolic variables, 2 breeds of different productivity were selected for investigation by high-resolution nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. We could reliably quantify 44 different milk metabolites. The results show that biomarkers such as acetone and beta-hydroxybutyrate are clearly correlated to the metabolic status of the individual cows during early lactation. Based on these data, the selection of cows that cope well with the metabolic stress of early lactation should become an option.

Individual variability in physiological adaptation to metabolic stress during early lactation in dairy cows kept under equal conditions

This study was conducted to investigate individual metabolic and endocrine adaptation to lactation under conditions of identical housing and feeding conditions in high-yielding dairy cows. Forty-five cows were studied on a research farm under standardized but practical conditions. From wk 2 before calving until wk 14 postpartum, blood samples were collected at weekly intervals and assayed for blood chemistry and various metabolites and hormones. Body weight, BCS, and backfat thickness were also recorded weekly. Milk yield, milk composition, and feed intake and energy balance were accordingly measured during the postpartum phase. The animals were retrospectively classified according to their plasma concentration of beta-hydroxybutyrate (BHB): cows in which a BHB threshold of 1 mM was exceeded at least once during the experiment were classified as BHB positive (BHB+); cows with BHB values consistently below this threshold were classified as BHB negative (BHB -). Using this classification, differences for NEFA and glucose concentrations were observed, but the mean calculated energy balance did not differ between the groups during the experimental period (-22.2 MJ of NE(1)/d +/- 4.7 for BHB+ and -18.9 MJ of NE(1)/d +/- 4.9 for BHB-). In BHB+ cows, the peripartum decrease (P < 0.05) of BW, BCS, and backfat thickness was more pronounced than in BHB- cows. Mean milk yields did not differ between groups. However, BHB+ cows had greater milk fat and lesser milk protein contents (P < 0.05), resulting in a greater (P < 0.05) fat:protein ratio than in BHB- cows. Thus, to some extent, cows were able to compensate for the negative energy balance by adjustments in performance. Milk acetone concentrations followed BHB concentrations in blood. Insulin-like growth factor-I and leptin concentrations were greater (P < 0.05) in BHB- cows during the time of observation than in the BHB+ cows. Comparing the reproductive variables recorded (first increase of progesterone, first service conception rate, number of services per conception, interval from calving to first AI, interval from first AI to conception, and days open) between the 2 groups yielded no significant differences. Our findings imply that despite comparable energy balance, there is considerable individual variation of the adaptive ability of cows during early lactation based on a variety of metabolic and endocrine variables.

Evolution of oestrogen functions in vertebrates

Steroidal oestrogens have been isolated from marine and terrestrial animals representative of all major classes of vertebrates including fish, amphibians, reptiles, birds and mammals. In general, oestrogens are responsible for most features characteristic of the female sex of a species, such as metabolic, behavioural and morphological changes during the stages of reproduction; they also support several processes in males. The evolution of the hormonal system always involves both the ligand and its sites of interaction. In the case of oestrogens, the steroid producing enzymes, mainly the aromatase complex, and the oestrogen receptor belong together within their co-evolution. The finding of oestrogenic steroids, the more recent identification of aromatase and receptor genes and their expression fit together, thereby confirming the importance for all vertebrates. Within the present paper, the evolution of the physiological functions of oestrogens from oviparous vertebrates to Eutherian mammals, oestrogen biosynthesis, metabolization and signalling pathways will be reviewed in detail.

The expression of leptin and its receptor during different physiological stages in the bovine ovary.

Leptin, the hormonal product of the obese (ob) gene, circulates in the blood at levels paralleling those of fat reserves and regulates satiety. In cattle, leptin has also been implicated in the control of ovarian function, but its local production in the ovary and role in the control of ovarian function in autocrine/paracrine manner is unknown. The aims of this study were to document the expression of leptin and its receptor (Ob‐R) in detail in bovine corpus luteum (CL) obtained from different stages of the oestrous cycle and during pregnancy—and to determine if the leptin/Ob‐R system is expressed clearly in bovine follicles during final growth to preovulatory follicles. Real‐time RT‐PCR (qPCR) was applied to investigate mRNA expression of examined factors. In general, we demonstrated leptin and its receptor transcripts and leptin protein are consistent with in vivo luteinisation of bovine CL and decline coincidental with luteal regression. The highest co‐expression of leptin/Ob‐R system was observed in TI and GC of the smallest follicles with E2 concentration <0.5 ng/ml followed by significant down regulation in growing follicles with the increase of follicular size and E2 content in the follicular fluid. Furthermore, expression of the leptin/Ob‐R system does not show any significant variation in the CL throughout pregnancy. To conclude, our results are the first to demonstrate the possible involvement of locally produced leptin/Ob‐R system in the bovine ovary, suggesting roles in the function and/or development of the CL and growth of small follicles in an autocrine/paracrine fashion. Mol. Reprod. Dev. 77: 174–181, 2010.