Plasma Estradiol-17β Concentrations Research Articles

Oestrogen, Progesterone and Oxytocin Receptors and COX‐2 Expression in Endometrial Biopsy Samples from the Induction of Ovulation to Luteolysis in Llamas (Lama glama)

Endometrial expression of oestrogen (ERα), progesterone (PR) and oxytocin receptor (OR) and cyclooxygenase-2 (COX-2) was evaluated from the induction of ovulation to luteolysis in llamas. Ovarian activity was daily assessed by ultrasonography in five females. Ovulation was induced immediately after the detection of an ovulatory follicle by a GnRH injection (Day 0). Endometrial samples were obtained by transcervical biopsies from the left and right horns on day 0 and days 4, 8, 10 and 12 post-GnRH. Blood samples were collected daily for progesterone and estradiol-17β determinations by RIA. An immunohistochemical technique was used to study receptors population and COX-2 expression which were then evaluated by two independent observers. The expression of ERα and PR was highest on day 0 in the luminal epithelium and stroma in association with high plasma estradiol-17β concentrations. Thereafter, a decrease in ERα population was registered on day 4 and a new increase of its expression was observed between days 8 and 12 in those cell types. Conversely, PR population was gradually down-regulated until its lowest expression was reached on day 10 post-GnRH in the luminal epithelium. Content of OR was similar throughout the study in all cell types. The expression of COX-2 was highest from day 8 to 12 post-GnRH in the luminal epithelium, in relation to the time of maximal PGF2α release. Both steroid receptors populations and COX-2 expression were similar between horns. Meanwhile, OR expression was higher in the right than in the left uterine horn. In summary, this study showed that the loss of endometrium sensitivity to progesterone by days 8-10 post-induction of ovulation and the concomitant increase of COX-2 expression could play a key role in the mechanism of luteolysis and somehow be related to the short corpus luteum lifespan of llamas.

Evaluation of the efficacy and safety of single administration of 4.7-mg deslorelin acetate implants on egg production and plasma sex hormones in Japanese quail (Coturnix coturnix japonica)

To evaluate the effects of 4.7-mg deslorelin acetate implants on egg production and plasma concentrations of 17β-estradiol and androstenedione in Japanese quail (Coturnix coturnix japonica) over 180 days and assess safety of the implants in quail via gross and histologic examination. 20 female Japanese quail. Following a 7-day period of consistent egg laying, quail were anesthetized and received a 4.7-mg deslorelin implant (treatment group; n = 10) or identical placebo implant (control group; 10) SC between the scapulae. Egg production was monitored daily. Plasma concentrations of 17β-estradiol and androstenedione were measured on days 0 (immediately prior to implant injection), 14, 29, 62, 90, 120, 150, and 180 via radioimmunoassay. Birds were weighed periodically and euthanized at day 180 for complete necropsy. Egg production was significantly decreased in the treatment group, compared with the control group, from 2 to 12 weeks after implant injection. Egg production ceased in 6 of 10 quail in the treatment group (mean duration of cessation, 70 days). Plasma androstenedione and 17β -estradiol concentrations were significantly lower on day 29 in the treatment group than in the control group. On day 180, 17β -estradiol concentration was lower in control than in treated birds.No clinically relevant lesions were detected in either group at necropsy [corrected]. 4.7-mg deslorelin acetate implants reversibly decreased egg laying for approximately 70 days in most of the Japanese quail evaluated. Further studies evaluating implants containing different concentrations of the drug are needed in quail and other avian species.

Identification of follicular vitellogenesis stage by monitoring of plasma calcium and estradiol-17β concentrations in the cultured Caspian brown trout, Salmo trutta caspius Kessler, 1877

To identify a simple indicator for detection of follicular vitellogenesis stage of cultured Caspian brown trout, the changes of plasma calcium and estradiol-17β along with ovarian histology were investigated over a reproductive cycle. For this purpose, the blood and gonad samples were taken monthly over a six months period. According to results, the concentrations of plasma calcium increased significantly during October and November but then declined steadily until the end of experiment. Estradiol-17β concentrations were low throughout the experiment, a clear peak was observed in October. Histological observations showed that all examined females were in vitellogenesis when the peak of plasma calcium and estradiol-17β were observed. Also, significant positive relationship was found between plasma calcium and estradiol. The good fitness of plasma calcium and estradiol-17β peak with histological observations suggests that plasma calcium could be a good and simple indicator for detection of follicular vitellogenesis stage in the cultured Caspian brown trout.

Acute Effect of High‐dose Isoflavones from Pueraria lobata (Willd.) Ohwi on Lipid and Bone Metabolism in Ovariectomized Mice

We investigated the acute metabolic effects of isoflavones from Pueraria lobata (Willd.) Ohwi (IPL) in ovariectomized (OVX) mice. After 4 weeks of IPL feeding at 500 mg/day/kg body weight (OVX500), plasma 17β-estradiol concentrations were significantly higher (+25%, p < 0.05), whereas plasma triglyceride levels were significantly lower in OVX mice (-15%, p < 0.05) compared with controls. Abdominal adipose tissue weight was marginally reduced in IPL-fed groups compared with OVX controls and the plasma levels of liver enzymes were unchanged. In addition, IPL significantly inhibited the reduction of bone mineral density in the femurs of OVX mice (OVX200, +22%; OVX500, +26%; p < 0.05) compared with controls after 4 weeks of IPL feeding. In quantitative polymerase chain reaction analysis the expression of aromatase was significantly suppressed and SULT1E1 was increased by IPL feeding, showing that IPL feeding may not alter the risk for breast cancer in mice. Our results suggest that IPL could ameliorate menopausal symptoms in mice. Further studies will confirm the effects of IPL in humans.

A differentially methylated single CpG-site is correlated with estrogen receptor alpha transcription

DNA methylation of the promoter region of estrogen receptor alpha (ESR1) is recognized as an epigenetic mechanism that regulates its mRNA abundance. We questioned whether tissues in male growing piglets were influenced in terms of DNA methylation by the developmentally occurring distinct plasma estradiol-17β (E2) concentrations. Additionally, we aimed at broadening the currently limited understanding of the epigenetic regulation of ESR1 in physiological settings. Three distinct genetic regions of ESR1 were analyzed using a combination of methylation-sensitive high resolution melting (MS-HRM) and pyrosequencing. Unexpectedly, major E2 concentration differences were only marginally associated with minor variations in DNA methylation and mRNA abundance. However, by analyzing two tissues showing the greatest differences in transcript abundance, we were able to find one single CpG site in the +1kb intragenic region of ESR1 strikingly differently methylated between heart vs. epididymis. Interestingly, this single CpG-site was identified as a putative binding site for the transcriptional repressor TG-interacting factor 1 (TGIF) which can recruit histone deacetylase 1 (HDAC1) leading to chromatin condensation. Indeed, chromatin immunoprecipitation confirmed a reduced histone H3 presence at the specific ESR1 location in case of higher DNA methylation. We therefore hypothesize that ESR1 expression may be manifested by a single-CpG-site based methylation difference impairing transcription factor binding.

Lake sturgeon population attributes and reproductive structure in the Namakan Reservoir, Minnesota and Ontario

Summary Quantified were the age, growth, mortality and reproductive structure of lake sturgeon (Acipenser fulvescens) collected in the US and Canadian waters of the Namakan Reservoir. The hypotheses were tested that (i) age and growth of lake sturgeon in the Namakan Reservoir would differ by sex and reproductive stage of maturity, and (ii) that the relative strength of year-classes of lake sturgeon in the reservoir would be affected by environmental variables. To quantify age, growth and mortality of the population, existing data was used from a multi-agency database containing information on all lake sturgeon sampled in the reservoir from 2004 to 2009. Lake sturgeon were sampled in the Minnesota and Ontario waters of the Namakan Reservoir using multi-filament gillnets 1.8 m high and 30–100 m long and varying in mesh size from 178 to 356 mm stretch. Reproductive structure of the lake sturgeon was assessed only during spring 2008 and 2009 using plasma testosterone and estradiol-17β concentrations. Ages of lake sturgeon >75 cm ranged from 9 to 86 years (n = 533, mean = 36 years). A catch-curve analysis using the 1981–1953 year classes estimated total annual mortality of adults to be 4.8% and annual survival as 95.2%. Using logistic regression analysis, it was found that total annual precipitation was positively associated with lake sturgeon year-class strength in the Namakan Reservoir. A 10 cm increase in total annual precipitation was associated with at least a 39% increase in the odds of occurrence of a strong year class of lake sturgeon in the reservoir. Plasma steroid analysis revealed a sex ratio of 2.4 females: 1 male and, on average, 10% of female and 30% of male lake sturgeon were reproductively mature each year (i.e. potential spawners). Moreover, there was evidence based on re-captured male fish of both periodic and annual spawning, as well as the ability of males to rapidly undergo gonadal maturation prior to spawning. Knowledge of lake sturgeon reproductive structure and factors influencing recruitment success contribute to the widespread conservation efforts for this threatened species.

Ultrasonographic characterization of ovarian follicular development in the Indian blackbuck antelope (Antilope cervicapra)

Transrectal ultrasonography was used to monitor the ovarian follicular dynamics in the Indian blackbuck antelope (Antilope cervicapra). The blackbuck has a short oestrous cycle (mean length 15.3±0.8 days) and exhibits two follicular waves, with a mean time of 14.3±0.8 and 8.9±0.6 days for the first and second waves, respectively. The mean diameter of the preovulatory follicles was 0.54±0.04cm, and only a single ovulatory follicle resulted from the second follicular wave in all the hinds. Follicular activity and preovulatory follicles appeared alternatively on the ovaries in successive cycles. The plasma estradiol-17β (E2) concentrations also exhibited a wave-like pattern, varying between 1.3±0.01pgml−1 and 5.3±1.9pgml−1. A positive relationship (r=0.85) was recorded between the dominant follicle diameter and the plasma E2 concentrations during the oestrous cycle. Furthermore, this baseline ultrasound data, in particular the diameter of the preovulatory follicle, enabled the team to carry out fixed-time AI, leading to live offspring in this native threatened species. This study, for the first time, documents the use of transrectal ultrasonography to monitor ovarian follicular dynamics in the Indian blackbuck antelope. It was also successful in the application and the development of a protocol for fixed-time AI in the Indian blackbuck antelope.

Seasonal Changes in Immunoreactivity of Vascular Endothelial Factor and its Receptors VEGFR1 and VEGFR2 in the Uterus of Wild Ground Squirrels (&lt;i&gt;Citellus dauricus&lt;/i&gt; Brandt)

In this study, we investigated the immunoreactivity of vascular endothelial growth factor (VEGF) and its receptors flt-1 (VEGFR1) and the kinase domain receptor (KDR/Flk-1, VEGFR2) in the uteri of the wild ground squirrels during the estrous period, early pregnancy and nonbreeding period. Cellular localizations of VEGF, VEGFR1 and VEGFR2 were detected by immunohistochemistry, and total proteins were extracted from uterine tissue in the estrous period, early pregnancy and nonbreeding period for Western blotting analysis. In addition, plasma estradiol-17β and progesterone concentrations were measured by radioimmunoassay. Stronger positive staining of VEGF was found in luminal epithelial cells and glandular cells, and its receptors (VEGFR1 and VEGFR2) were observed in stromal cells in the estrous period and early pregnancy compared with the nonbreeding period. The protein levels of VEGF, VEGFR1 and VEGFR2 were significantly higher in the estrous period and early pregnancy as compared with the nonbreeding period. Besides, plasma estradiol-17β and progesterone concentrations were higher in the estrous period and early pregnancy than in the nonbreeding period, suggesting that the immunoreactivities of VEGF, VEGFR1 and VEGFR2 were correlated with changes in plasma estradiol-17β and progesterone concentrations. These results suggested that VEGF and its receptors may be involved in the regulation of seasonal changes in the uterine functions of wild female ground squirrels.

Differences in Apoptotic Status in the Bovine Placentome between Spontaneous and Induced Parturition

We conducted this study to analyze apoptotic changes in the bovine placentome at spontaneous and induced parturition. Cows delivered i) after the administration of dexamethasone followed by prostaglandin F(2α) and estriol, ii) after the administration of prostaglandin F(2α) and estriol or iii) spontaneously. Prepartum changes in plasma progesterone and estradiol-17β concentrations were similar between spontaneous and induced parturition. Messenger RNA of BCL2-related protein A1 (BCL2A1), an antiapoptotic gene, was expressed by trophoblast binucleate cells and caruncular epithelial cells. Quantitative RT-PCR showed that the expression of BCL2A1 mRNA in cotyledonary and caruncular portions was significantly lower in spontaneous parturition than induced parturition. The expression of BCL2-associated X protein (BAX) mRNA, a proapoptotic gene, was significantly higher in cotyledons at spontaneous parturition than parturition induced without dexamethasone. Caspase-3 (CASP3) mRNA and pre-activated CASP3 protein were predominantly detected in caruncular epithelial cells regardless of how parturition proceeded. Activated CASP3 protein was found in trophoblast uninucleate cells and binucleate cells rather than caruncular epithelial cells. In spontaneous parturition, intense staining of activated CASP3 was detected in caruncular epithelial cells. Spontaneous and dexamethasone-induced parturition increased apoptotic cells in the placentome compared with parturition induced without dexamethasone. The number of binucleate cells was significantly decreased in spontaneous parturition. The present results suggest that although the clinical dose of dexamethasone induces apoptosis in the placentome at term, neither dexamethasone nor prostaglandin F(2α) evoke normal physiological changes in the placentome during delivery such as a change in the balance of apoptosis-related genes and disappearance of binucleate cells.

Expression of nerve growth factor and its receptors TrkA and p75 in the uterus of wild female ground squirrel (Citellus dauricus Brandt)

In this study, we investigated the presence of nerve growth factor (NGF) and its receptors tyrosine kinase A (TrkA) and p75 in the uterus of the wild ground squirrels during the estrous period, early pregnancy and non-breeding period. In the estrous period and early pregnancy, NGF and TrkA were immunolocalized in stromal cells, luminal epithelial cells, glandular cells and smooth muscle cells whereas in the non-breeding period, both of them were detected only in luminal epithelial cells and glandular cells, but not in stromal cells or smooth muscle cells. Stronger immunostaining of NGF and TrkA was observed in luminal epithelial cells and glandular cells in the estrous period and early pregnancy as compared to the non-breeding period. p75 was immunolocalized only in luminal epithelial and glandular cells during the estrous period, early pregnancy and non-breeding period. The intensity of the immunohistochemical signals for p75 did not vary significantly in the estrous period, early pregnancy and non-breeding period. The mean mRNA levels of NGF and TrkA and p75 were significantly higher in the estrous period and early pregnancy as compared to the non-breeding period. Besides, plasma estradiol-17β and progesterone concentrations were higher in the estrous period and early pregnancy than in the non-breeding period, suggesting that the expression patterns of NGF and TrkA are correlated with changes in plasma estradiol-17β and progesterone concentrations. These results indicate that NGF and its receptor TrkA may be involved in the regulation of seasonal changes in the uterine functions of wild female ground squirrels.

Patterns of metal composition and biological condition and their association in male common carp across an environmental contaminant gradient in Lake Mead National Recreation Area, Nevada and Arizona, USA

There is a contaminant gradient in Lake Mead National Recreation Area (LMNRA) that is partly driven by municipal and industrial runoff and wastewater inputs via Las Vegas Wash (LVW). Adult male common carp (Cyprinus carpio; 10 fish/site) were collected from LVW, Las Vegas Bay (receiving LVW flow), Overton Arm (OA, upstream reference), and Willow Beach (WB, downstream) in March 2008. Discriminant function analysis was used to describe differences in metal concentrations and biological condition of fish collected from the four study sites, and canonical correlation analysis was used to evaluate the association between metal and biological traits. Metal concentrations were determined in whole-body extracts. Of 63 metals screened, those initially used in the statistical analysis were Ag, As, Ba, Cd, Co, Fe, Hg, Pb, Se, Zn. Biological variables analyzed included total length (TL), Fulton's condition factor, gonadosomatic index (GSI), hematocrit (Hct), and plasma estradiol-17β and 11-ketotestosterone (11kt) concentrations. Analysis of metal composition and biological condition both yielded strong discrimination of fish by site (respective canonical model, p<0.0001). Compared to OA, pairwise Mahalanobis distances between group means were WB<LVB<LVW for metal concentrations and LVB<WB<LVW for biological traits. Respective primary drivers for these separations were Ag, As, Ba, Hg, Pb, Se and Zn; and TL, GSI, 11kt, and Hct. Canonical correlation analysis using the latter variable sets showed they are significantly associated (p<0.0003); with As, Ba, Hg, and Zn, and TL, 11kt, and Hct being the primary contributors to the association. In conclusion, male carp collected along a contaminant gradient in LMNRA have distinct, collection site-dependent metal and morpho-physiological profiles that are significantly associated with each other. These associations suggest that fish health and reproductive condition (as measured by the biological variables evaluated in this study) are influenced by levels of certain metals in the Lake Mead environment.

Reproductive endocrine disruption in smallmouth bass (Micropterus dolomieu) in the Potomac River basin: spatial and temporal comparisons of biological effects

A high prevalence of intersex or testicular oocytes (TO) in male smallmouth bass within the Potomac River drainage has raised concerns as to the health of the river. Studies were conducted to document biomarker responses both temporally and spatially to better understand the influence of normal physiological cycles, as well as water quality and land-use influences. Smallmouth bass were collected over a 2-year period from three tributaries of the Potomac River: the Shenandoah River, the South Branch Potomac and Conococheague Creek, and an out-of-basin reference site on the Gauley River. The prevalence of TO varied seasonally with the lowest prevalence observed in July, post-spawn. Reproductive maturity and/or lack of spawning the previous spring, as well as land-use practices such as application of manure and pesticides, may influence the seasonal observations. Annual, seasonal, and site differences were also observed in the percentage of males with measurable concentrations of plasma vitellogenin, mean concentration of plasma vitellogenin in females, and plasma concentrations of 17β-estradiol and testosterone in both sexes. Bass collected in the South Branch Potomac (moderate to high prevalence of TO) had less sperm per testes mass with a lower percentage of those sperm being motile when compared to those from the Gauley River (low prevalence of TO). An inverse relationship was noted between TO severity and sperm motility. An association between TO severity and wastewater treatment plant flow, percent of agriculture, total number of animal feeding operations, the number of poultry houses, and animal density within the catchment was observed.

Temporal evaluation of effects of a model 3β‐hydroxysteroid dehydrogenase inhibitor on endocrine function in the fathead minnow

Inhibition of enzymes involved in the synthesis of sex steroids can substantially impact developmental and reproductive processes controlled by the hypothalmic-pituitary-gonadal (HPG) axis. A key steroidogenic enzyme that has received little attention from a toxicological perspective is 3β-hydroxysteroid dehydrogenase (3β-HSD). In these studies, we exposed reproductively-active fathead minnows (Pimephales promelas) to the model 3β-HSD inhibitor trilostane at two test concentrations (300 and 1,500 µg/L) over a 16-d period that included both 8-d exposure and 8-d recovery phases. Plasma concentrations of 17β-estradiol (E2) in females were depressed within hours of exposure to the drug and remained decreased at the highest trilostane concentration throughout the 8-d exposure. Reductions in E2 were accompanied by decreases in plasma concentrations of the estrogen-responsive protein vitellogenin (VTG). During the recovery phase of the test, plasma E2 and VTG concentrations returned to levels comparable to those of controls, in the case of E2 within 1 d. Up-regulation of ovarian expression of gene products for follicle-stimulating hormone receptor (fshr) and aromatase (cyp19a1a) suggested active compensation in trilostane-exposed animals. Effects of trilostane on HPG-related endpoints in exposed males were less pronounced, although, as in females, up-regulation of gonadal fshr was seen. Data from these time-course studies provide insights as to direct impacts, compensatory responses, and recovery from effects associated with perturbation of a comparatively poorly characterized enzyme/pathway critical to sex steroid synthesis. This information is important to the design and interpretation of approaches for assessing the occurrence and effects of HPG-active chemicals in both the laboratory and the field.

A computational model of the hypothalamic - pituitary - gonadal axis in female fathead minnows (Pimephales promelas) exposed to 17α-ethynylestradiol and 17β-trenbolone

BackgroundEndocrine disrupting chemicals (e.g., estrogens, androgens and their mimics) are known to affect reproduction in fish. 17α-ethynylestradiol is a synthetic estrogen used in birth control pills. 17β-trenbolone is a relatively stable metabolite of trenbolone acetate, a synthetic androgen used as a growth promoter in livestock. Both 17α-ethynylestradiol and 17β-trenbolone have been found in the aquatic environment and affect fish reproduction. In this study, we developed a physiologically-based computational model for female fathead minnows (FHM, Pimephales promelas), a small fish species used in ecotoxicology, to simulate how estrogens (i.e., 17α-ethynylestradiol) or androgens (i.e., 17β-trenbolone) affect reproductive endpoints such as plasma concentrations of steroid hormones (e.g., 17β-estradiol and testosterone) and vitellogenin (a precursor to egg yolk proteins).ResultsUsing Markov Chain Monte Carlo simulations, the model was calibrated with data from unexposed, 17α-ethynylestradiol-exposed, and 17β-trenbolone-exposed FHMs. Four Markov chains were simulated, and the chains for each calibrated model parameter (26 in total) converged within 20,000 iterations. With the converged parameter values, we evaluated the model's predictive ability by simulating a variety of independent experimental data. The model predictions agreed with the experimental data well.ConclusionsThe physiologically-based computational model represents the hypothalamic-pituitary-gonadal axis in adult female FHM robustly. The model is useful to estimate how estrogens (e.g., 17α-ethynylestradiol) or androgens (e.g., 17β-trenbolone) affect plasma concentrations of 17β-estradiol, testosterone and vitellogenin, which are important determinants of fecundity in fish.

Comparison of Plasma Concentrations of Estradiol-17.BETA. and Progesterone, and Conception in Dairy Cows with Cystic Ovarian Diseases between Ovsynch and Ovsynch plus CIDR Timed AI Protocols

The objectives of this study were 1) to determine the effects of adding a CIDR to the Ovsynch protocol on plasma concentrations of estradiol-17β and progesterone and conception in dairy cows with cystic ovarian diseases and 2) to examine associations among the estradiol-17β and progesterone concentrations and conception. Cows were diagnosed as having cystic ovarian diseases if they were found to have a cystic follicle (diameter ≥25 mm) without a corpus luteum by two palpations per rectum with an interval for 7 to 14 days. They were treated with either the Ovsynch (GnRH on Day 0, PGF(2α) on Day 7 and GnRH on Day 9, with AI on Day 10; n=15) or Ovsynch+CIDR protocol (Ovsynch protocol plus a CIDR from Day 0 to Day 7; n=23). Plasma estradiol-17β concentrations were determined on Days 0, 7 and 9, and plasma progesterone concentrations were determined on Days 0, 7, 9 and 17. The plasma estradiol-17β and progesterone concentrations at all of the days examined and conception rates did not differ significantly between the two timed AI protocols. The progesterone concentrations on Day 17 and conception rates were lower (P<0.05) for cows with low concentrations of estradiol-17β (<2 pg/ml) on Day 9 than for cows with high concentrations of estradiol-17β (≥2 pg/ml). The present study suggests that, in dairy cows with cystic ovarian diseases, addition of a CIDR to the Ovsynch protocol had no remarkable effects on plasma estradiol-17β and progesterone concentrations during and after the treatments or on conception after timed AI. This study indicates that the low plasma estradiol-17β concentration at the second administration of GnRH in the protocols can be a predictor for impaired luteal formation and lower likelihood of pregnancy in dairy cows with cystic ovarian diseases.

Ovarian follicular dynamics and hormonal secretory profiles in guanacos ( Lama guanicoe)

The objective of the present study was to describe ovarian activity in 11 adult non-mated guanacos, evaluated every second day for 40 days by transrectal ultrasonography and by plasma estradiol-17β and progesterone concentrations. An inverse relationship ( r = −0.29, P < 0.001) was observed between the diameter of the largest ovarian follicle and the total number of follicles indicating that follicular growth in guanacos occurs in waves. The mean duration of follicular wave was 15.1 ± 4.2 days with a range from 9 to 26. The follicular growth phase was 7.0 ± 2.4 days, the static phase 3.0 ± 1.2 days, the regression phase 5.2 ± 2.1 days and the inter-wave interval was 12.6 ± 5.6 days. The maximum follicular diameter in each follicular wave was 10.2 ± 2.1 mm with a range from 7.2 to 16.1 mm. Inter-wave intervals of longer duration were associated with a larger maximum follicle diameter ( P < 0.001). Follicular activity alternated between ovaries in 93% of the waves with 48% of dominant follicles occurring in the left and 52% in the right ovary without differences ( P > 0.05). Plasma estradiol-17β concentrations showed a wave-like pattern, varying from 20.0 to 92.1 pmol/L. Plasma progesterone concentrations remained below 1 nmol/L without any ultrasonic evidence of ovulation during the study. These results in guanacos suggest a follicular wave pattern more similar to the llama ( Lama glama) than previously described in other South American and Old World camelid species.

Mass spectrometric assay and physiological–pharmacological activity of androgenic neurosteroids

Steroid hormones play a key role in the pathophysiology of several brain disorders. Testosterone modulates neuronal excitability, but the underlying mechanisms are obscure. There is emerging evidence that testosterone-derived “androgenic neurosteroids”, 3α-androstanediol and 17β-estradiol, mediate the testosterone effects on neural excitability and seizure susceptibility. Testosterone undergoes metabolism to neurosteroids via two distinct pathways. Aromatization of the A-ring converts testosterone into 17β-estradiol. Reduction of testosterone by 5α-reductase generates 5α-dihydrotestosterone, which is then converted to 3α-androstanediol, a powerful GABA A receptor-modulating neurosteroid with anticonvulsant properties. Although the 3α-androstanediol is an emerging neurosteroid in the brain, there is no specific and sensitive assay for determination of 3α-androstanediol in biological samples. This article describes the development and validation of mass spectrometric assay of 3α-androstanediol, and the molecular mechanisms underlying the testosterone modulation of seizure susceptibility. A liquid chromatography–tandem mass spectrometry assay to measure 3α-androstanediol is validated with excellent linearity, specificity, sensitivity, and reproducibility. Testosterone modulation of seizure susceptibility is demonstrated to occur through its conversion to neurosteroids with “anticonvulsant” and “proconvulsant” actions and hence the net effect of testosterone on neural excitability and seizure activity depends on the levels of distinct testosterone metabolites. The proconvulsant effect of testosterone is associated with increases in plasma 17β-estradiol concentrations. The 5α-reduced metabolites of testosterone, 5α-dihydrotestosterone and 3α-androstanediol, had powerful anticonvulsant activity. Overall, the testosterone-derived neurosteroids 3α-androstanediol and 17β-estradiol could contribute to the net cellular actions of testosterone in the brain. Because 3α-androstanediol is a potent positive allosteric modulator of GABA A receptors, it could serve as an endogenous neuromodulator of neuronal excitability in men. The 3α-androstanediol assay is an important tool in this area because of the growing interest in the potential to use adjuvant aromatase inhibitor therapy to improve treatment of epilepsy.

Changes in numbers of large ovarian follicles, plasma luteinizing hormone and estradiol-17β concentrations and egg production figures in farmed ostriches throughout the year

In this study we described and analysed changes in the numbers of large ovarian follicles (diameter 6.1–9.0 cm) and in the plasma concentrations of luteinizing hormone (LH) and estradiol-17β (E 2β) in relation to individual egg production figures of farmed ostriches ( Struthio camelus spp.) throughout one year. Ultrasound scanning and blood sampling for plasma hormone analysis were performed in 9 hens on a monthly basis during the breeding season and in two periods of the non-breeding season. Our data demonstrated that: (1) large follicles were detected and LH concentrations were elevated already 1 month before first ovipositions of the egg production season took place; (2) E 2β concentrations increased as soon as the egg production season started; (3) numbers of large follicles, LH and E 2β concentrations were elevated during the entire egg production season; and that (4) numbers of large follicles, LH and E 2β concentrations decreased simultaneous with or following the last ovipositions of the egg production season. By comparing these parameters during the egg production season with their pre-and post-seasonal values, significant differences were found in the numbers of large follicles and E 2β concentrations between the pre-seasonal, seasonal and post-seasonal period; while LH concentrations were significantly different between the seasonal and post-seasonal period. In conclusion, our data demonstrate that changes in numbers of large follicles and in concentrations of LH and E 2β closely parallel individual egg production figures and provide some new cues that egg production in ostriches is confined to a marked reproductive season. Moreover, our data provide indications that mechanism, initiating, maintaining and terminating the egg production season in farmed breeding ostriches are quite similar to those already known for other seasonal breeding bird species.

The relation between ultrasonographic observations in the oviduct and plasma progesterone, luteinizing hormone and estradiol during the egg laying cycle in ostriches

In this study we investigated the temporal relationship between ovulation, egg formation, oviposition and the changes in plasma concentrations of progesterone, luteinizing hormone and estradiol-17β during the egg laying cycle in farmed ostriches. In 10 egg-producing birds, transcutaneous ultrasound scanning was performed at 3 h intervals and blood sampling at hourly intervals during a period of at least 48 h (one egg laying cycle). In hens ( n = 8) that ovulated during the observational period, the ovulated egg was first detected 2 h after oviposition; thus, ovulation occurred shortly after oviposition in all birds. During the period between two consecutive ovipositions, the developing egg remained for 9 h in the proximal part (infundibulum, magnum or isthmus) and for 39 h in the distal part of the oviduct (uterus). In ovulating hens, plasma progesterone concentrations showed a characteristic and consistent profile: from basal levels of around 0.1 ng/ml concentrations started to increase 12 h before oviposition, reached an average maximum of 3.5 ng/ml at 3 h before oviposition and returned to basal levels 3 h and 30 min after oviposition. Changes in plasma luteinizing hormone and estradiol-17β concentrations showed comparable patterns of elevation and decline relative to the timing of oviposition and ovulation. However, variation in their individual basal concentrations was generally larger and peak values were less conspicuous than those of progesterone. In non-ovulating hens ( n = 2) neither progesterone, nor luteinizing hormone nor estradiol-17β showed elevations to peak concentrations before oviposition. These data demonstrate that during the egg laying cycle of ostriches, events such as ovulation, egg development and oviposition evolve according to a rather strict time schedule, and that progesterone, luteinizing hormone and estradiol-17β reach peak concentrations shortly before ovulation. Additionally, our findings also show that on-farm ultrasound scanning is a useful technique to discriminate between ovulating and non-ovulating hens.

Temporal relations between plasma concentrations of luteinizing hormone, follicle-stimulating hormone, estradiol-17β, progesterone, prolactin, and α-melanocyte-stimulating hormone during the follicular, ovulatory, and early luteal phase in the bitch

Compared with other domestic animals, relatively little is known about the changes in, and temporal relations between, reproductive hormones around the time of ovulation in the domestic bitch. Therefore, plasma concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol-17β, progesterone, prolactin (PRL), and α-melanocyte-stimulating hormone (α-MSH) were determined one to six times daily from the start of the follicular phase until 5 days after the estimated day of ovulation in six Beagle bitches. In all bitches, the pre-ovulatory LH surge was accompanied by a pre-ovulatory FSH surge. A pre-ovulatory PRL or α-MSH surge was not observed. The pre-ovulatory FSH and LH surges started concomitantly in four bitches, but in two bitches the FSH surge started 12 h earlier than the LH surge. The FSH surge (110 ± 8 h) lasted significantly longer than the LH surge (36 ± 5 h). In contrast with the pre-ovulatory FSH surge, the pre-ovulatory LH surge was bifurcated in four of six bitches. The mean plasma LH concentrations before (1.9 ± 0.4 μg/L) and after (1.9 ± 0.3 μg/L) the LH surge were similar, but the mean plasma FSH concentration before the FSH surge (1.6 ± 0.3 U/L) was significantly lower than that after the FSH surge (3.1 ± 0.2 U/L). In most bitches the highest plasma estradiol-17β concentration coincided with or followed the start of the pre-ovulatory LH surge. In five of the six bitches the plasma progesterone concentration started to rise just before or concurrently with the start of the LH surge. In conclusion, the results of this study provide evidence for the differential regulation of the secretion of LH and FSH in the bitch. In addition, the interrelationship of the plasma profiles of estradiol-17β and LH suggests a positive feedback effect of estradiol-17β on LH surge release. The start of the pre-ovulatory LH surge is associated with an increase in the plasma progesterone concentration in this species.