Andreas Vernunft

Anti-Muellerian hormone levels in plasma of Holstein-Friesian heifers as a predictive parameter for ovum pick-up and embryo production outcomes

The aim of this study was to investigate whether plasma anti-Muellerian hormone (AMH) levels of Holstein-Friesian heifers could be used to predict ovum pick-up (OPU) and embryo production outcomes. Plasma samples and data were collected from 64 heifers, which underwent repeated OPU with subsequent in vitro embryo production followed by embryo flushing after superovulation. AMH levels were significantly positively correlated with the number of follicles aspirated per OPU session (r = 0.45), recovered oocytes per OPU (r =0.43) and in vitro produced embryos per OPU (r = 0.28). No significant correlations between AMH and in vivo produced embryos were ascertained. Our results suggest that correlations between AMH and outcomes of an OPU-IVF program are too low to use AMH as a precise predictive parameter for the success of a particular OPU procedure in Holstein-Friesian heifers. However, AMH can help to identify groups of very good or very poor oocyte donors.

LOX-1 regulates estrogenesis via intracellular calcium release from bovine granulosa cells.

Estradiol produced by ovarian granulosa cells triggers the luteinizing hormone surge which in turn initiates ovulation in female mammals. Disturbances in estradiol production from granulosa cells are a major reason for reproductive dysfunctions in dairy cows. Endogenous estradiol production might be altered by reactive oxygen species (ROS) such as oxidized low‐density lipoprotein (ox‐LDL). Inhibition of lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1), a receptor of ox‐LDL, leads to increased estrogenesis in granulosa cells. This activity is mediated by calcium release from endoplasmic reticulum (ER)‐dependent and ER‐independent calcium pools. Inhibition of the LOX‐1 signal transduction pathway is followed by mitochondrial alterations. The membrane potential ΔΨ increases and the ROS production decreases in mitochondria after blocking LOX‐1. Our data indicate that blocking the LOX‐1 receptor signal pathway might be a promising way to improve steroid hormone concentrations in metabolically highly active female mammals and, therefore, to defend against reproductive dysfunctions in humans and animals.

Postpartum levels of 8-iso-prostaglandin F2α in plasma and milk phospholipid fractions as biomarker of oxidative stress in first-lactating dairy cows

F2-isoprostanes such as 8-iso-prostaglandin F2 (8-iso-PGF2α) are formed by free radical-catalyzed mechanisms from membrane phospholipids and from low density lipoproteins through peroxidation of arachidonic acid. Esterified 8-iso-PGF2α is cleaved by phospholipases, circulates in blood and is excreted as putatively harmful oxidatively modified lipid via the kidney into urine. In this study we demonstrate that 8-iso-PGF2α concentrations in plasma samples from heifers are higher (p<0.005) compared to those from first-lactating dairy cows at 71 days postpartum. Furthermore, plasma 8-iso-PGF2α concentrations vary with ovarian activity and differ in response to luteolytic initiation as well as activation of the hypothalamic-pituitary-gonadal axis between heifers and first-lactating cows. Sustainable concentrations of 8-iso-PGF2α (50-150 pg/ml) are detectable in the phospholipid fraction of milk, suggesting milk as an additional excretion route for 8-isoprostanes. Plasma levels largely paralleled levels in milk (p<0.001). Plasma phospholipid 8-iso-PGF2α concentrations in cyclic cows decreased (p<0.05) from day 38 to day 71 postpartum, whereas milk phospholipid 8-iso-PGF2α rather increased (p<0.05). Cyclic cows tend to have higher 8-isoprostane levels compared to acyclic animals. In contrast to lipohydroperoxides, concentration of 8-iso-PGF2α were not correlated with milk yield (p>0.05). Our data indicate 8-iso-PGF2α may be a novel biomarker of oxidative stress in dairy cow, which is detectable in blood as well as in milk.

Chromatin and cytoplasmic characteristics of equine oocytes recovered by transvaginal ultrasound-guided follicle aspiration are influenced by the developmental stage of their follicle of origin

Dynamic follicular changes occur during the equine estrus cycle, but little is known about their impact on the properties of recovered oocytes. The aim of this study was to characterize the cytoplasmic and chromatin status of equine oocytes in relation to the time of recovery during the follicle wave. Transvaginal ultrasound-guided follicle aspiration was performed two times in relation to the follicle wave: estrus-subordinate, from the subordinate follicles of mares in estrus, 24 hours after human chorionic gonadotropin stimulation of a dominant preovulatory follicle, and new-wave, from the follicles of the subsequent induced follicular wave, at the time of dominant follicle divergence (largest follicle 23 mm diameter). A total of 1011 follicles were aspirated. The oocyte recovery rate in the new-wave group was significantly lower than that for the estrus-subordinate group (12% vs. 26%, respectively); this was associated with a significantly higher proportion of oocytes with compact cumuli (44% vs. 27%, respectively). Estradiol concentrations were markedly higher in follicular fluid from new-wave follicles (885.6 ± 123.2 ng/mL vs. 54.3 ± 18.9 ng/mL, for estrus-subordinate; P < 0.001), indicating greater viability. Aspiration group did not affect glucose-6-phosphate dehydrogenase activity in recovered oocytes. Fibrillar (more juvenile) chromatin was more prevalent in new-wave oocytes, whereas estrus-subordinate oocytes showed more condensed chromatin or resumption of meiosis (P < 0.05). Mitochondrial activity was higher in oocytes with expanded cumuli in the new-wave group, but not in the estrus-subordinate group. In conclusion, our results clearly showed that the time of aspiration in relation to the follicle wave is associated with significant differences in follicle status and oocyte characteristics: new-wave oocytes were from a more viable follicle population and had more juvenile chromatin and cytoplasmic characteristics, whereas estrus-subordinate oocytes were from a more atretic follicle population and exhibited signs of atresia-related acquisition of meiotic and cytoplasmic competence. These findings will help in effective scheduling of oocyte recovery for equine-assisted reproduction techniques.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) activity decreases estrogenesis in ovarian granulosa cells

THE reproductive cycle in mammalian females is initiated by a surge of luteinizing hormone (LH). It is secreted from the pituitary gland in response to hypothalamic gonadotropin releasing hormone (GnRH). An increase in the activity of GnRH neurons predominantly results from rising secretion of estrogens from granulosa cells of the maturing ovarian follicle. The transient exposure to intense LH amplitudes instigates changes in the follicular expression of genes which are essential for the release or expulsion of the oocyte (ovulation). Prerequisite for its fertilization is a rupture of the apical follicle wall. The LH surge ceases (dependent on the species almost or completely) the proliferation and estrogenesis of mural granulosa cells while activates pathways also evident in challenging inflammatory response, such as the expression of cyclooxygenase-2 and the expression of the genomic progesterone receptor. It acts as a transcription factor that stimulates the transcription of genes which encode proteases capable of hydrolyzing follicular wall matrix, including cathepsin L and metalloproteinases such as ADAMTS. The transcription factor is also involved in the expression of the epidermal growth factor superfamily signaling for expansion of the granulosal cumulus cells (1). Recently, receptors known to play a role in the innate immunity, such as Toll-like 4 (2), and in an inflammatory process, including LOX-1 (3), have been identified in subpopulations of mural granulosa cells. Toll-like 4 receptor belongs to pattern-recognition receptor which have the capacity to recognize conserved motifs in complexes of lipids, proteins, and nucleic acids in contrast to selective antigen recognition by cells of the adaptive immunity. Expression and functionality of LOX-1 have been extensively studied in endothelial cells. They respond to LOX-1 activators, such as oxidized low-density lipoprotein (oxLDL), with raising the formation of reactive oxygen metabolites (ROM). The increase amplifies oxLDL binding and LOX-1 expression. An expression of LOX-1 has been recently detected in human ovarian follicular granulosa cells and LOX-1 has been reported to be involved in the granulosal reparative autophagy (4). Our analyses confirmed the LOX-1 expression using mural granulosa cells from bovine follicles. They were induced by a GnRH analogue after regressing the corpus luteum as described elsewhere (5). The concentration of transcripts was quantified by RT-PCR using the I-script-I-cycler technique (Biorad, Hamburg, Germany). LOX-1 protein was detected by immunochemistry and immunocytofluorimetry using a selective rabbit antibody against LOX-1 (LOX-1 AB) (BioVision, San Francisco, CA, USA), an anti-rabbit Alexa 488-conjugated antibody (Invitrogen, Darmstadt, Germany), and single cell analysis (Gallios, Beckman-Coulter, Krefeld, Germany) on the analogy of a described approach (6,7). We found an increase in LOX-1 mRNA in response to LH (5 ng/ml) following a culture (4 h) of granulosa cells in serum-free Mega cell medium (Sigma, Deisenhofen, Germany) and a correlation between the mRNA level and the LOX-1 protein (r 5 0.8, P < 0.05). A decline in mRNA for LOX-1 was observed in granulosa cells freshly prepared 20 h post GnRH (late ovulatory phase) in comparison with cells from follicles sampled 5 h post GnRH. These data are indicative of gonadotropin-dependent LOX-1 expression. To test the functionality of LOX-1, the granulosa cells from the late ovulatory phase were exposed to the selective rabbit antibody to LOX-1 (1.5 or 3.0 lg/ml). In comparison with an unspecific rabbit antibody (control), the mRNA for

Platelet-Activating Factor-Stimulated Production of Reactive Oxygen Species in Ovarian Granulosa Cells from Periovulatory Follicles

The platelet‐activating factor (PAF) is a proinflammatory lipid present in the fluid of ovarian Graafian follicle. Ovarian blockage of the PAF receptor (PAFr) reduces ovulations in the rat whereas underlying mechanism is poorly understood. Mural granulosa cells (MGC) were mechanically isolated from the theca interna of bovine periovulatory follicle. The mRNA abundance for PAFr, progesterone receptor and cyclooxygenase‐2 were measured by real‐time PCR. Cytosolic calcium (Ca2+) concentration was assayed by microscopy using Fura‐2 AM as indicator, 8‐isoprostaglandin F2α (8‐isoPGF2α) by an ELISA kit. Fluorescent products arising from intracellular oxidation of hydroethidine (HE) and dihydrorhodamine (DHR) were quantified by flow cytometry. The cells expressed PAFr mRNA and PAFr protein and responded to cPAF (nonhydrolyzable form of PAF) with a pulsating increase in Ca2+, demonstrating functional PAFr. Elevation of Ca2+ was reversed by WEB‐2086, an inverse PAFr agonist. cPAF elevated the level of 8‐isoPGF2α in the medium of MGC cultured with luteinizing hormone (LH). cPAF alone had no significant influence on the oxidation of HE and DHR, or 8‐isoPGF2α level. In MGC from vital periovulatory follicle, PAF and LH signaling plays an important role in regulating the production of excessive oxidants. Blockage of PAFr seems to interfere with these regulatory processes essential for ovulation.

The Male Fetal Biomarker INSL3 Reveals Substantial Hormone Exchange between Fetuses in Early Pig Gestation.

The peptide hormone INSL3 is uniquely produced by the fetal testis to promote the transabdominal phase of testicular descent. Because it is fetal sex specific, and is present in only very low amounts in the maternal circulation, INSL3 acts as an ideal biomarker with which to monitor the movement of fetal hormones within the pregnant uterus of a polytocous species, the pig. INSL3 production by the fetal testis begins at around GD30. At GD45 of the ca. 114 day gestation, a time at which testicular descent is promoted, INSL3 evidently moves from male to female allantoic compartments, presumably impacting also on the female fetal circulation. At later time-points (GD63, GD92) there is less inter-fetal transfer, although there still appears to be significant INSL3, presumably of male origin, in the plasma of female fetuses. This study thus provides evidence for substantial transfer of a peptide hormone between fetuses, and probably also across the placenta, emphasizing the vulnerability of the fetus to extrinsic hormonal influences within the uterus.

Inactivation of the LOX‐1 Pathway Promotes the Golgi Apparatus during Cell Differentiation of Mural Granulosa Cells

In female mammals, granulosa cells of the ovarian follicle differentiate into the corpus luteum after ovulation of the pregnable oocyte into the fallopian tube. During these differentiation processes several morphological alterations have to occur and the molecular basis is not fully understood. As an endpoint estradiol production from granulosa cells has to switch off in favor for progesterone production from the proceeding corpus luteum to sustain the developing embryo. Previously, we demonstrated that the multiligand receptor LOX‐1 plays a critical role in steroid hormone synthesis of granulosa cells via intracellular calcium release from endoplasmic (ER)‐dependent and ER‐independent calcium pools. In the present study, we show that inhibition of LOX‐1 leads to a rearrangement of ceramide from the basal membrane toward the Golgi apparatus. This activity is accomplished by a calcium‐dependent phosphorylation of aromatase, the key step in estradiol production. Phosphorylated aromatase increased estradiol production in a dose‐dependent manner. Our data indicate that the ceramide cascade is essential for proper granulosa cell function and ceramide redistribution serves as a first step in order to proceed with the prosperous differentiation into a corpus luteum. J. Cell. Physiol. 229: 1946–1951, 2014.

Effects of grain species, genotype and starch quantity on the postprandial plasma amino acid response in horses

Postprandial alterations of plasma amino acid (PAA) levels partly reflect a temporal contribution of the feed. How cereal grains affect PAA levels is not known. We hypothesized that a meal of cereal grains causes a temporal increase of PAA, affected by grain species, grain genotype and meal size. Six mares were used in three consecutive trials, receiving four oats, barley and maize genotypes, respectively. Individual grain genotypes were provided as 3 meal sizes corresponding to 1.0, 1.5 or 2.0 g starch/kg body weight. Meadow hay (1.5 kg/100 kg body weight) was offered daily. At the test days, 1 kg hay was fed 60 min prior to the grain meal. Blood samples were taken before grain feeding (0 min) and 30, 60, 90, 120, 180, 240 and 300 min thereafter. Subsequently, the remaining hay was offered. The genotype × starch quantity (i.e., meal size) interaction had a major effect on postprandial PAA concentrations (P < 0.05). Availability of amino acids (AA), ingested from different grain genotypes, apparently differed at both the digestive and post-digestive level. Thus, AA supply from grain feeding can better be assessed on the genotype level. The concentrations of most PAA increased rapidly with a postprandial maximum at around 30 min. Hay feeding might have an underrated capability for AA provision because increases of PAA levels were initialized already by ingestion of a 1 kg hay. It remains unclear which portion of the PAA kinetics response originates from hay feeding and which one from the cereal grain meal.

Acute effects of general anesthesia with propofol, pentobarbital or isoflurane plus propofol on plasma metabolites and hormones in adult pigs

Experimental setups for physiological research, in which acute operative interventions need to be performed, can require inclusion of general anesthesia (GA), which may interfere or confound with the effects of the experimental factors of interest on measured variables. It was recently shown that the most commonly used sedatives/anesthetics in pigs (e.g., ketamine, xylazine, azaperone) affect physiological responses and thus the primary metabolic readouts have the potential to be confounded. To extend the search for a physiologically-friendly anesthesia regime for such studies, we investigated effects of GA induced by propofol (Prop) or pentobarbital (Pent) or propofol plus isoflurane (Prop + Isof) on plasma concentrations of commonly measured metabolites and hormones. In 2 experimental runs, 6 female pigs fitted with jugular vein catheters were used. Fasted pigs received either no drug (CON) or anesthetized rotationally either with Prop, Pent or Prop + Isof on different days, separated with washout periods of sufficient length (2 to 3 d). Six-h profiles of glucose, lactate, non-esterified fatty acids (NEFA), triglycerides (TG), cholesterol, urea as well as hormones including glucagon, insulin and cortisol were determined. Concentrations of cholesterol, urea and glucagon remained unaffected by any of the treatments ( > 0.05). Pent tended to increase cortisol from 30 to 90 min after drug administration. Glucose and lactate concentrations were increased ( < 0.05) by Prop and Pent within the first hour of GA ( < 0.05). Propofol and Pent reduced NEFA concentrations, which were more pronounced during the last 2 h of the studied period. Triglyceride concentrations were increased by all 3 agents within the first 45 min with Prop containing treatments exerting a stronger effect than Pent. Our data suggest that GA with Prop and particularly with Pent adulterate plasma metabolite and hormone profiles of pigs acutely, and thus has the potential to confound the effects of experimental factors of interest. Although Prop + Isof anesthesia did not differ from the controls, providing a physiologically-friendly GA, both single and the isoflurane-combined treatment of Prop induced hypertriglyceridemia due to the lipid adjuvant of the Prop drugs. It is concluded that readouts obtained under GA may be influenced both by physiological adulterations as response to anesthesia as well as by artifacts due to accompanying ingredients of the drug formulations.