Differential regulation of human 3β-hydroxysteroid dehydrogenase type 2 for steroid hormone biosynthesis by starvation and cyclic AMP stimulation: studies in the human adrenal NCI-H295R cell model

Human steroid biosynthesis depends on a specifically regulated cascade of enzymes including 3β-hydroxysteroid dehydrogenases (HSD3Bs). Type 2 HSD3B catalyzes the conversion of pregnenolone, 17α-hydroxypregnenolone and dehydroepiandrosterone to progesterone, 17α-hydroxyprogesterone and androstenedione in the human adrenal cortex and the gonads but the exact regulation of this enzyme is unknown. Therefore, specific downregulation of HSD3B2 at adrenarche around age 6-8 years and characteristic upregulation of HSD3B2 in the ovaries of women suffering from the polycystic ovary syndrome remain unexplained prompting us to study the regulation of HSD3B2 in adrenal NCI-H295R cells. Our studies confirm that the HSD3B2 promoter is regulated by transcription factors GATA, Nur77 and SF1/LRH1 in concert and that the NBRE/Nur77 site is crucial for hormonal stimulation with cAMP. In fact, these three transcription factors together were able to transactivate the HSD3B2 promoter in placental JEG3 cells which normally do not express HSD3B2. By contrast, epigenetic mechanisms such as methylation and acetylation seem not involved in controlling HSD3B2 expression. Cyclic AMP was found to exert differential effects on HSD3B2 when comparing short (acute) versus long-term (chronic) stimulation. Short cAMP stimulation inhibited HSD3B2 activity directly possibly due to regulation at co-factor or substrate level or posttranslational modification of the protein. Long cAMP stimulation attenuated HSD3B2 inhibition and increased HSD3B2 expression through transcriptional regulation. Although PKA and MAPK pathways are obvious candidates for possibly transmitting the cAMP signal to HSD3B2, our studies using PKA and MEK1/2 inhibitors revealed no such downstream signaling of cAMP. However, both signaling pathways were clearly regulating HSD3B2 expression.

The 17, 20-lyase activity of cytochrome p450c17 from human fetal testis favors the delta5 steroidogenic pathway.

Cytochrome P450c17 catalyzes both 17alpha-hydroxylation and 17,20-lyase conversion of 21-carbon steroids to 19-carbon precursors of sex steroids. P450c17 can mediate testosterone biosynthesis via the conversion of pregnenolone to dehydroepiandrosterone (the delta(5) pathway) or via conversion of progesterone to androstenedione (the delta(4) pathway). In many species, the 17, 20-lyase activity of P450c17 for one pathway dominates, reflecting the preferred steroidogenic pathway of that species. All studies of recombinant human P450c17 and of human adrenal microsomes have found high 17, 20-lyase activity only in the delta(5) pathway. Because the 17, 20-lyase activities in both the delta(4) and delta(5) pathways for testicular P450c17 have not been directly compared, however, it is not known if the delta(5) pathway dominates in the human testis. To resolve this issue, we assayed the conversion of 17alpha-hydroxypregnenolone to dehydroepiandrosterone (delta(5) 17, 20-lyase activity) and of 17alpha-hydroxyprogesterone to androstenedione (delta(4) 17, 20-lyase activity) by human fetal testicular microsomes. We obtained apparent Michaelis constant (K(m)) and maximum velocity (V(max)) values of 1.0 microM and 0.73 pmol.min(-1). microg(-1) for delta(5) 17, 20-lyase activity and of 3.5 microM and 0.23 pmol.min(-1). microg(-1) for delta(4) 17, 20-lyase activity. Catalytic efficiencies, expressed as the ratio V(max)/K(m), were 0.73 and 0.066 for the delta(5) and delta(4) reactions, respectively, indicating 11-fold higher preference for the delta(5) pathway. We conclude that the majority of testosterone biosynthesis in the human testis proceeds through the conversion of pregnenolone to dehydroepiandrosterone via the delta(5) pathway.

Mounting behaviour in finishing pigs: Stable individual differences are not due to dominance or stage of sexual development

Every year around 100 million male piglets are castrated in the EU, usually without anaesthesia or post-operative analgesia. This surgical intervention is painful and stressful. Several main players within the pig industry have voluntarily agreed to end the practice of surgical pig castration in the EU by 2018. One alternative to castration is entire male pig production. However, entire males behave differently than castrates, for example, by performing more mounting behaviour, which is suggested to be a welfare problem. The aim of our study was to develop a comprehensive ethogram of different types of mounting and to investigate properties, causes and consequences of mounting behaviour in finishing pigs. The study included 80 entire male and 80 female pigs from two farrowing batches born six weeks apart. Mixed sex and single-sex housing of pigs are both common in pig farming, so to ensure our study was representative, the 160 pigs were assigned to social groups of 20 in three treatments: entire male pigs only (MM, 2 groups, n = 40), entire females only (FF, 2 groups, n = 40) and entire males and females mixed together (MF, 4 groups, n = 80). Measurements took place during the final six weeks before slaughter (between 63.5 and 105.5 kg). Observations of mounting behaviour on 12 days per batch suggested that: (i) males mounted more than females, (ii) within sex, there was no effect of treatment on the amount of mounting (although the statistical power of the study to detect these effects was low), and (iii) there were individual differences in mounting that were stable over time (within sex). Classification of mounting into different categories revealed that sexual mounting was most common overall and in males but only rare in females. Compared to other types of mounting (e.g. caused by crowding or during a fight), sexual mounts lasted longer and provoked more screaming by the recipient. There were no relationships between mounting behaviour on the one hand and dominance rank in food competition tests, the circulating levels of sex hormones (oestradiol, testosterone and progesterone) at the end of the study, the health scores (lameness and scratches) or weight gain on the other hand. The stable individual differences of mounting over time suggest that mounting behaviour is a trait of the individual rather than the appearance of random outbreaks. However, these differences in mounting cannot be explained by dominance behaviour or by differences in sex hormone concentrations that could indicate the onset of puberty. Mounting behaviour and in particular sexual mounting provoked high pitched screaming of the recipients indicating that mounting is a welfare problem. For the welfare assessment of entire male pig production the performance of mounting behaviour should be considered. (C) 2013 Elsevier B.V. All rights reserved.

Dicer is required for female reproductive tract development and fertility in the mouse.

Dicer encodes a riboendonuclease required for microRNA biosynthesis. Dicer was inactivated in Müllerian duct mesenchyme-derived tissues of the reproductive tract of the mouse, using an Amhr2-Cre allele. Although Amhr2-Cre; Dicer conditional mutant males appeared normal and were fertile, mutant females were infertile. In adult mutant females, there was a reduction in the size of the oviducts and uterine horns. The oviducts were less coiled compared to controls and cysts formed at the isthmus near the uterotubal junction. Unfertilized, degenerate oocytes were commonly found within these cysts, indicating a defect in embryo transit. Beads transferred into the mutant oviduct failed to migrate into the uterus. In addition, blastocysts transferred directly into the mutant uterus did not result in pregnancy. Histological analysis demonstrated that the mutant uterus contained less glandular tissue and often the few glands that remained were found within the myometrium, an abnormal condition known as adenomyosis. In adult mutants, there was ectopic expression of Wnt4 and Wnt5a in the luminal epithelium (LE) and glandular epithelium (GE) of the uterus, and Wnt11 was ectopically expressed in GE. These results demonstrate that Dicer is necessary for postnatal differentiation of Müllerian duct mesenchyme-derived tissues of the female reproductive tract, suggesting that microRNAs are important regulators of female reproductive tract development and fertility.

Molecular basis of maturation promoting factor

Maturation promoting factor (MPF), which is functionally defined by its ability to induce Xenopus oocyte maturation, is an M phase (meiosis and mitosis) specific activity that is present in all species tested. It was hypothesized that MPF is a universal trigger of the interphase to M phase transition during the cell cycle. The current model for the molecular basis of MPF is that MPF is a protein kinase having the cdc2 protein as its catalytic subunit and is identical to the M phase-specific histone H1 kinase. In the present study, I have shown that more than just cdc2 kinase contributes to MPF activity, and M phase-specific H1 kinase is composed of at least two entities, instead of just cdc2 kinase. Therefore, the simple model of MPF = cdc2 kinase = M phase-specific H1 kinase should be ruled out.^ My study began with the characterization of the mitosis-specific monoclonal antibody MPM-2. MPM-2 reacts specifically with M phase cells from different species by recognizing a discrete set of proteins once they are phosphorylated at the G$\sb2$/M transition. I found that phosphorylation of MPM-2 antigens coincided with the appearance of MPF activity during oocyte maturation stimulated by progesterone. If MPM-2 was injected into oocytes before the stimulation, MPF activity failed to appear, and the oocytes could not mature. Furthermore, MPM-2 was able to deplete MPF activity from M phase extracts. These results identified MPM-2 as a probe that recognizes either MPF itself or a regulator of MPF.^ Since M phase-specific H1 kinase was believed to be identical to cdc2 kinase and MPF, I proceeded to determine whether MPM-2 recognized the M phase-specific H1 kinase. I found that MPM-2 did recognize an M phase-specific H1 kinase. However, this kinase was not cdc2 kinase. This kinase (MPM-2 kinase) is present in a latent form in immature oocytes and is activated in tandem with the activation of MPF during oocyte maturation. It appears to accelerate progesterone-induced oocyte maturation. Therefore, MPM-2 kinase may be a novel positive regulator of MPF activation.^ MPM-2 depletes MPF activity, but not cdc2 kinase activity. This discrepancy caused me to question the equivalency of MPF with cdc2 kinase. I found that when a high percentage of MPF activity was recovered from gel filtration of mature oocyte extract, the recovered MPF activity was due to two factors, cdc2 kinase and a factor recognized by MPM-2. This factor might activate and stabilize cdc2 kinase. Identification of this factor in the present study may contribute to the understanding of the autoactivation of MPF. ^

A study of factors regulating the autonomous proliferation of the steroid hormone-induced LJ6195 murine reproductive tract tumor

Neonatal estrogen treatment of BALB/c mice results in the unregulated proliferation of the cervicovaginal epithelium and eventually tumorigenesis. The conversion of the normally estrogen responsive cyclic proliferation of the vaginal epithelium to a continuous estrogen-independent pattern of growth is a complex phenomenon. The aim of this study was to gain an understanding of the mechanism(s) by which steroid hormone administration during a critical period of development alters the cyclic proliferation of vaginal epithelium, ultimately leading to carcinogenesis in the adult animal.^ The LJ6195 murine cervicovaginal tumor was induced by treating newborn female BALB/c mice with 20 $\mu$g 17$\beta$-estradiol plus 100 $\mu$g progesterone for the first 5 days after birth. In contrast to proliferation of the normal vaginal epithelium, proliferation of LJ6195 is not regulated by estradiol. Northern blot analysis of RNA from vaginal tracts of normal mice, neonatal-estrogen treated mice, and LJ6195 indicate that there is an alteration in the expression of several genes such as the estrogen receptor, c-fos, and HER2/neu. In response to neonatal estrogen treatment, the estrogen receptor is down regulated in the murine vaginal tract. Therefore, the estrogen-independent nature of this tissue is established as early as 3 months after treatment. There is strong evidence that the proliferation of LJ6195 is regulated through an autocrine growth pathway. The LJ6195 tumor expresses mRNA for the epidermal growth factor receptor. In addition, conditioned medium from the LJ6195 tumor cell line contains a growth factor(s) with epidermal growth factor-like activity. Conditioned medium from the LJ6195 cell line stimulated the proliferation of the EGF-dependent COMMA D mouse mammary gland cell line in a dose-dependent manner. The addition of an anti-mEGF-antibody to LJ6195 cell cultures significantly decreased growth. These results suggest that the EGF-receptor mediated growth pathway may play a role in regulating the estrogen-independent proliferation of the LJ6195 tumor. ^

Expression and hormonal regulation of Muc-1 at the apical surface of mouse uterine epithelial cells and its role in modulating embryo implantation

Previous studies from our lab have established that large molecular weight mucin glycoproteins are major apically-disposed components of mouse uterine epithelial cells in vitro (Valdizan et al., (1992) J. Cell. Physiol. 151:451-465). The present studies demonstrate that Muc-1 represents one of the apically-disposed mucin glycoproteins of mouse uterine epithelia, and that Muc-1 protein and mRNA expression are regulated in the peri-implantation stage mouse uterus by ovarian steroids. Muc-1 expression is high in the proestrous and estrous stages, and decreases during diestrous. Both Muc-1 protein and mRNA levels decline to barely detectable levels by day 4 of pregnancy, i.e., prior to the time of blastocyst attachment. In contrast, Muc-1 expression in the cervix and vagina is maintained during this same period. Delayed implantation was established in pregnant mice by ovariectomy and maintained by administration of exogenous progesterone. Initiation of implantation was triggered by coinjection of progesterone maintained mice with a nidatory dose of 17$\beta$-estradiol. Muc-1 levels in the uterine epithelia of progesterone maintained mice declined to similar low levels as observed on day 4 of normal pregnancy. Coinjection of estradiol did not alter Muc-1 expression suggesting that down-regulation of Muc-1 is a progesterone dominated event. This was confirmed in ovariectomized, non-pregnant mice which displayed stimulation of Muc-1 expression following 6 hr of estradiol injection. Estradiol stimulated Muc-1 expression was inhibited by the pure antiestrogen, ICI 164,384. While progesterone alone had no effect on Muc-1 expression, it antagonized estradiol action in this regard. Injection of pregnant mice with the antiprogestin, RU 486, a known implantation inhibitor, on day 3 of pregnancy restored high level expression of Muc-1 mRNA on day 4, indicating that down-regulation of Muc-1 is progesterone receptor-mediated. Muc-1 appears to function as an anti-adhesive molecule at the apical cell surface of mouse uterine epithelial cells. Treatment of polarized cultures of mouse uterine epithelial cells with O-sialoglycoprotein endopeptidase reduced mucin expression in vitro, by about 50%, and converted polarized uterine epithelia to a functionally receptive state. Similarly, ablation of Muc-1 in Muc-1 null mice resulted in polarized uterine epithelia that were functionally receptive as compared to their wild-type counterparts in vitro. Collectively, these data indicate that Muc-1 and other mucins function as anti-adhesive molecules and that reduction or removal of these molecules is a prerequisite for the generation of a receptive uterine state. ^

A novel form of CYP3A from rat brain: cDNA cloning, protein characterization and gene regulation of cytochrome P450 3A9

One full length cDNA clone, designated 3aH15, was isolated from a rat brain cDNA library using a fragment of CYP3A2 cDNA as a probe. 3aH15 encoded a protein composed of 503 amino acid residues. The deduced amino acid sequence of 3aH15 was 92% identical to mouse Cyp3a-13 and had a 68.4% to 76.5% homology with the other reported rat CYP3A sequences. Clone 3aH15 was thus named CYP3A9 by Cytochrome P450 Nomenclature Committee. CYP3A9 seems to the major CYP3A isozyme expressed in rat brain. Sexual dimorphism of the expression of CYP3A9 was shown for the first time in rat brain as well as in rat liver. CYP3A9 appears to be female specific in rat liver based on the standards proposed by Kato and Yamazoe who defined sex specific expression of P450s as being a 10-fold or higher expression level in one sex compared with the other. CYP3A9 gene expression was inducible by estrogen treatment both in male and in female rats. Male rats treated with estrogen had a similar expression level of CYP3A9 mRNA both in the liver and brain. Ovariectomy of adult female rats drastically reduced the mRNA level of CYP3A9 which could be fully restored by estrogen replacement. On the other hand, only a two-fold induction of CYP3A9 expression by dexamethasone was observed in male liver and no significant induction of CYP3A9 mRNA was observed in female liver or in the brains. These results suggest that estrogen may play an important role in the female specific expression of the CYP3A9 gene and that CYP3A9 gene expression is regulated differently from other CYP3A isozymes. ^ P450 3A9 recombinant protein was expressed in E. coli using the pCWOri+ expression vector and the MALLLAVF amino terminal sequence modification. This construct gave a high level of expression (130 nmol P450 3A9/liter culture) and the recombinant protein of the modified P450 3A9 was purified to electrophoretic homogeneity (10.1 nmol P450/mg protein) from solubilized fractions using two chromatographic steps. The purified P450 3A9 protein was active towards the metabolism of many clinically important drugs such as imipramine, erythromycin, benzphetamine, ethylmorphine, chlorzoxazone, cyclosporine, rapamycin, etc. in a reconstituted system containing lipid and rat NADPH-P450 reductase. Although P450 3A9 was active towards the catabolism of testosterone, androstenedione, dehydroepiandrosterone (DHEA) and 17β-estradiol, P450 3A9 preferentially catalyzes the metabolism of progesterone to form four different hydroxylated products. Optimal reconstitution conditions for P450 3A9 activities required a lipid mixture and GSH. The possible mechanisms of the stimulatory effects of GSH on P450 3A9 activities are discussed. Sexually dimorphic expression of P450 3A9 in the brain and its involvement in many neuroactive drugs as well as neurosteroids suggest the possible role of P450 3A9 in some mental disorders and brain functions. ^

Cell-type specific expression and regulation of apolipoprotein D and E in human endometrium.

OBJECTIVE To assess the expression and regulation of antilipoprotein D (ApoD) and antilipoprotein E (ApoE) in human endometrium. STUDY DESIGN Endometrial biopsies from healthy, regularly cycling women were collected during the late proliferative and mid-secretory phase. mRNA gene expression of ApoD and ApoE was determined using real-time PCR in whole tissue, in isolated stromal (ESC), epithelial (EEC) and CD45(+) leukocytes (EIC), as well as after hormonal stimulation of ESC and EEC in vitro. Protein expression was analyzed using immunohistochemistry. RESULTS ApoD and ApoE mRNA was expressed in all cell types examined. A rise in ApoD mRNA expression was seen in whole endometrium, ESC, and EEC in the secretory phase, as well as after hormonal stimulation of ESC and EEC in vitro. ApoE mRNA was significantly upregulated in whole endometrium of secretory phase biopsies, while its expression was not altered by progesterone in vitro. Immunohistochemistry of whole endometrial tissue localized ApoD mainly in ESC and EEC. While ApoE was localized slightly in ESC, it was particularly noted on the surface of secretory phase endothelial cells. CONCLUSION We demonstrate for the first time the cell-type and cycle dependent expression of ApoD and ApoE within human endometrium, suggesting their role in endometrial modulation.

Tocolysis for preterm labor: expert opinion

Tocolysis is an important treatment in the improvement of outcome in preterm labor and preterm birth, provided that its use follows clear evidence-based recommendations. In this expert opinion, the most recent evidence about efficacy and side effects of different tocolytics is being reviewed and evidence-based recommendation about diagnosis and treatment of preterm labor is given. Further aspects such as progesterone administration or antibiotic treatment for the prevention of preterm birth are included. Our review demonstrates that an individualized choice of different tocolytics and additional treatments is necessary to improve short- and long-term neonatal outcome in preterm labor and preterm birth.

Immunization against gonadotropin-releasing hormone in dairy cattle: Antibody titers, ovarian function, hormonal levels, and reversibility

Suppression of cyclic activity in cattle is often desired in alpine farming and for feedlot cattle not intended for breeding. A cattle-specific anti-GnRH vaccination (Bopriva, Zoetis Australia Ltd., West Ryde, Australia) is approved for use in heifers and bulls in New Zealand, Australia, Mexico, Brazil, Argentina, Turkey, and Peru. Eleven healthy, cyclic Swiss Fleckvieh cows were included in the study and vaccinated twice with Bopriva 4wk apart. Injection site, rectal body temperature, and heart and respiratory rates were recorded before and 3d following each vaccination. Blood samples were taken weekly for progesterone and estrogen analysis and to determine GnRH antibody titer. Ovaries were examined weekly, using ultrasound to count the number of follicles and identify the presence of a corpus luteum. Thirty weeks after the first vaccination, the cows were subjected to a controlled internal drug-releasing device-based Select-Synch treatment. The GnRH antibody titers increased after the second vaccination and peaked 2wk later. Estrogen levels were not influenced by vaccination, and progesterone level decreased in 7 of 11 cows up to 3wk after the second vaccination and remained low for 10 to 15wk following the second vaccination. The number of class I follicles (diameter ≤5mm) was not influenced by vaccination, whereas the number of class II follicles (diameter 6-9mm) decreased between 7 and 16wk after the first vaccination. Class III follicles (diameter >9mm) were totally absent during this period in most cows. The median period until recurrence of class III follicles was 78d from the day of the second vaccination (95% confidence interval: 60-92d). After vaccination, all cows showed swelling and pain at the injection site, and these reactions subsided within 2wk. Body temperature and heart and respiratory rates increased after the first and second vaccinations and returned to normal values within 2d of each vaccination. The cows in our study were not observed to display estrus behavior until 30wk after the first vaccination. Therefore, a Select-Synch protocol was initiated at that time. Ten cows became pregnant after the first insemination (the remaining cow was reinseminated once until confirmed pregnancy). Bopriva induced a reliable and reversible suppression of reproductive cyclicity for more than 2mo. The best practical predictor for the length of the anestrus period was the absence of class III follicles.

Menstrual cycle phase affects discrimination of infant cuteness

Recent studies have shown that women are more sensitive than men to subtle cuteness differences in infant faces. It has been suggested that raised levels in estradiol and progesterone may be responsible for this advantage. We compared young women's sensitivity to computer-manipulated baby faces varying in cuteness. Thirty-six women were tested once during ovulation and once during the luteal phase of their menstrual cycle. In a two alternative forced-choice experiment, participants chose the baby which they thought was cuter (Task 1), younger (Task 2), or the baby that they would prefer to babysit (Task 3). Saliva samples to assess levels of estradiol, progesterone and testosterone were collected at each test session. During ovulation, women were more likely to choose the cuter baby than during the luteal phase, in all three tasks. These results suggest that cuteness discrimination may be driven by cyclic hormonal shifts. However none of the measured hormones were related to increased cuteness sensitivity. We speculate that other hormones than the ones measured here might be responsible for the increased sensitivity to subtle cuteness differences during ovulation.

Steroidogenesis of the testis - new genes and pathways

Defects of androgen biosynthesis cause 46,XY disorder of sexual development (DSD). All steroids are produced from cholesterol and the early steps of steroidogenesis are common to mineralocorticoid, glucocorticoid and sex steroid production. Genetic mutations in enzymes and proteins supporting the early biosynthesis pathways cause adrenal insufficiency (AI), DSD and gonadal insufficiency. The classic androgen biosynthesis defects with AI are lipoid CAH, CYP11A1 and HSD3B2 deficiencies. Deficiency of CYP17A1 rarely causes AI, and HSD17B3 or SRD5A2 deficiencies only cause 46,XY DSD and gonadal insufficiency. All androgen biosynthesis depends on 17,20 lyase activity of CYP17A1 which is supported by P450 oxidoreductase (POR) and cytochrome b5 (CYB5). Therefore 46,XY DSD with apparent 17,20 lyase deficiency may be due to mutations in CYP17A1, POR or CYB5. Illustrated by patients harboring mutations in SRD5A2, normal development of the male external genitalia depends largely on dihydrotestosterone (DHT) which is converted from circulating testicular testosterone (T) through SRD5A2 in the genital skin. In the classic androgen biosynthetic pathway, T is produced from DHEA and androstenedione/-diol in the testis. However, recently found mutations in AKR1C2/4 genes in undervirilized 46,XY individuals have established a role for a novel, alternative, backdoor pathway for fetal testicular DHT synthesis. In this pathway, which has been first elucidated for the tammar wallaby pouch young, 17-hydroxyprogesterone is converted directly to DHT by 5α-3α reductive steps without going through the androgens of the classic pathway. Enzymes AKR1C2/4 catalyse the critical 3αHSD reductive reaction which feeds 17OH-DHP into the backdoor pathway. In conclusion, androgen production in the fetal testis seems to utilize two pathways but their exact interplay remains to be elucidated.

Continuous milking of dairy cows disrupts timing of peak IgG concentration appearance in mammary secretions

The length of the dry period in commercial dairy production is under close scrutiny. While the main concern is the composition and volume of milk produced, the evaluation of colostrum quality under these new paradigms has suggested a decline in IgG concentrations, while some reports indicate no change. Colostrum quality has been defined as an adequate concentration (>50 mg/ml) of immunoglobulin in the secretions to provide the newborn with maximal disease resistance. We investigated the appearance of IgG in mammary pre- and post partum secretions in cows without a dry period (continuously milked, Dry0) and compared the secretions with cows that experienced a dry period of 60 d (Dry60). Blood was collected during the experimental period and plasma analysed for progesterone (P4) and prolactin (Prl). Approximately -6 d relative to parturition, the Dry0 animals exhibited increased concentration of IgG in their secretions to an average of ∼35 mg/ml that remained rather constant through subsequent pregnancy and following parturition. Dry0 cows were producing an average IgG concentration in parturition colostrum of 44·2±17·6 mg/ml that was not different than that of controls (66·86±16·8 mg/ml). However, Dry0 cows exhibited high variation, different peak times (day) of IgG concentration including times that occurred both pre and post parturition. IgG mass of the Dry0 cows remained rather constant pre- and post partum and did not show the same declining mass following parturition that was shown for the Dry60 cows. The change in plasma P4 and Prl were shown to have no timing effect on colostrum IgG concentration.

A quantitiative LC-MS/MS method for the measurement of arachidonic acid, prostanoids, endocannabinoids, N-acylethanolamines and steroids in human plasma.

Free arachidonic acid is functionally interlinked with different lipid signaling networks including those involving prostanoid pathways, the endocannabinoid system, N-acylethanolamines, as well as steroids. A sensitive and specific LC-MS/MS method for the quantification of arachidonic acid, prostaglandin E2, thromboxane B2, anandamide, 2-arachidonoylglycerol, noladin ether, lineoyl ethanolamide, oleoyl ethanolamide, palmitoyl ethanolamide, steroyl ethanolamide, aldosterone, cortisol, dehydroepiandrosterone, progesterone, and testosterone in human plasma was developed and validated. Analytes were extracted using acetonitrile precipitation followed by solid phase extraction. Separations were performed by UFLC using a C18 column and analyzed on a triple quadrupole MS with electron spray ionization. Analytes were run first in negative mode and, subsequently, in positive mode in two independent LC-MS/MS runs. For each analyte, two MRM transitions were collected in order to confirm identity. All analytes showed good linearity over the investigated concentration range (r>0.98). Validated LLOQs ranged from 0.1 to 190ng/mL and LODs ranged from 0.04 to 12.3ng/mL. Our data show that this LC-MS/MS method is suitable for the quantification of a diverse set of bioactive lipids in plasma from human donors (n=32). The determined plasma levels are in agreement with the literature, thus providing a versatile method to explore pathophysiological processes in which changes of these lipids are implicated.