Plasma Estradiol Concentrations Research Articles

Interrelationship Between Alcohol Intake and Endogenous Sex-Steroid Hormones on Diabetes Risk in Postmenopausal Women

Objective: We examined whether circulating concentrations of sex hormones, including estradiol, testosterone, sex hormone–binding globulin (SHBG), and dehydroepiandrosterone sulfate (DHEAS), were associated with alcohol intake or mediated the alcohol–type 2 diabetes (T2D) association.Methods: Among women not using hormone replacement therapy and free of baseline cardiovascular disease, cancer, and diabetes in the Women's Health Study, 359 incident cases of T2D and 359 matched controls were chosen during 10 years of follow-up.Results: Frequent alcohol intake (≥1 drink/day) was positively and significantly associated with higher plasma estradiol concentrations in an age-adjusted model (β = 0.14, 95% confidence interval [CI], 0.03, 0.26), compared to rarely/never alcohol intake. After adjusting for additional known covariates, this alcohol–estradiol association remained significant (β = 0.19, 95% CI, 0.07, 0.30). Testosterone (β = 0.13, 95% CI, −0.05, 0.31), SHBG (β = 0.07, 95% CI, −0.07, 0.20), and DHEAS (β = 0.14, 95% CI, −0.04, 0.31) showed positive associations without statistical significance. Estradiol alone or in combination with SHBG appeared to influence the observed protective association between frequent alcohol consumption and T2D risk, with a 12%–21% reduction in odds ratio in the multivariate-adjusted models.Conclusions: Our cross-sectional analysis showed positive associations between alcohol intake and endogenous estradiol concentrations. Our prospective data suggested that baseline concentrations of estradiol, with or without SHBG, might influence the alcohol–T2D association in postmenopausal women.

Short communication: Comparison of estrus characteristics in Holstein heifers by 2 activity monitoring systems

Two activity monitoring systems—Heatime (SCR Engineers Ltd., Netanya, Israel) and IceTag (IceRobotics Ltd., Edinburgh, UK)—were compared on their ability to detect and quantify estrus expression. Holstein heifers (n=57) were fitted with Heatime (HT) and IceTag (IT) sensors from 12 mo of age until confirmation of pregnancy. Upon detection of high activity by HT, ovaries were scanned by ultrasound, a blood sample was collected for analysis of plasma estradiol, and signs of estrus (clear vaginal mucus, uterine muscle tone, visual mounting activity, standing to be mounted, or rump showing signs of repeated acceptance of mounts) were recorded. Because only estrus episodes detected by HT (n=111) were further evaluated, only the positive predictive value was measured. Heifers were housed in groups of 24 in a freestall pen. Data were analyzed using Proc CORR and GLM of SAS (SAS Institute Inc., Cary, NC). The positive predictive value was 84.7% (94/111) for HT and 98.7% (74/75) for IT. Estrus duration was recorded by HT as 14.3±4.1h [mean ± standard deviation (SD)] and by IT as 15.0±4.0h; duration measurements were correlated (r=0.60). The mean duration difference was 0.74±3.52h. Recordings of onset and end of estrus by IT were 3.5±4.3h and 2.9±4.9h earlier than those by HT. The overlap in duration was 9h. Measurements of estrus intensity were correlated (r=0.63). Peak activity was 77.3±19.5 index value (approximately 7.7 SD from basal activity) on HT. The relative increase in activity measured by IT was 360±170% baseline value. Measurements of intensity and duration from HT were correlated (r=0.64) but those from IT were not (r=0.13). Plasma estradiol concentration (11.2±4.6pg/mL) was not correlated with preovulatory follicle diameter or with duration or intensity of estrus. Diameter of preovulatory follicle (15.7±2.6mm) had no correlation with duration of estrus and was only weakly correlated with intensity measured by either system. Baseline steps/hour was negatively correlated with intensity from both sensors (r=−0.37 and −0.70 for HT and IT). Estrus episodes accompanied by 2 or 3 of the monitored signs of estrus had greater intensity and duration on HT but not on IT. Preovulatory follicle diameter and plasma estradiol concentration did not influence occurrence of estrus signs. Results indicate that both systems identified estrus precisely, with correlated characterization and similar timing. In contrast, relationships with plasma estradiol concentration and signs of estrus require further investigation.

Behavioral and hormonal pattern of repeat breeder cows around estrus.

Repeat breeder (RB) cows were compared with normal (CTRL) ones with respect to behavioral estrus intensity, endocrine patterns and concentrations of plasma estradiol, progesterone and LH around estrus, and ovulation timing. A total of 27 and 31 cycles in 12 RB and 18 CTRL cows, respectively, were synchronized by means of the Ovsynch protocol followed by a single PG injection after 7 days. Behavioral estrus and ovulation were observed in 81.5 and 83.8% of the synchronized cycles in the RB and CTRL cows respectively. The RB and CTRL groups had similar estrus durations of 21.4 and 19.6 h respectively, but estrus was more intense in the RB, as indicated by numerically higher overall activity indexes and higher peak neck activity. The interval from PG injection to estrus onset (considered as proestrus) was 8.2 h shorter in RB than in CTRL cows, at 47.9 and 56.1 h respectively (P<0.007), but the average preovulatory follicle size was similar. The estradiol concentration at peak was numerically higher (21%) and the AUC tended to be higher in the RB cows than in the CTRL cows. LH secretion during the period from 18 to 3 h before the LH peak was also lower in RB than in CTRL cows: 2.5 and 4.6 ng/ml respectively (P<0.01). In conclusion, the behavioral estrus was more intense in the RB cows; nevertheless, short proestrus and subdued LH concentrations before the LH peak, which could impair oocyte competence and development, were first reported in RB cows.

The prevalence of subclinical endometritis and intrauterine infections in repeat breeder cows

The objectives of this study were to assess the prevalence of subclinical endometritis and the presence of common uterine pathogens in repeat breeder cows. A total of 121 cows with three or more consecutive artificial inseminations without conception and no clinical signs of disease were defined as repeat breeder cows and were enrolled in this trial. Intrauterine samples were collected with the cytobrush technique to determine the prevalence of subclinical endometritis and bacteriologic infections. Blood samples were analyzed for concentrations of progesterone and estradiol in plasma to assess ovarian activity. Furthermore, breed, parity, history of calving and postpartum uterine infection, clinical findings of transrectal palpation, and backfat thickness were analyzed as potential factors for the prevalence of subclinical endometritis in repeat breeder cows. The prevalence of subclinical endometritis in repeat breeder cows was 12.7%; but common uterine pathogens, Escherichia coli and Trueperella pyogenes, were found in only one and three cows, respectively. Ovarian activity was determined in 95.0% of all cows. Recorded variables had no effect on the prevalence of subclinical endometritis in repeat breeder cows. In conclusion, subclinical endometritis and uterine infections linked to common pathogens were playing a minor role as a cause for repeat breeder cows in this study. Alternative reasons for failure to conceive in these cows are discussed.

Premature pubarche before one year of age: distinguishing between mini-puberty variants and precocious puberty.

BackgroundThe aim of this study was to facilitate the distinction between the benign “mini-puberty of early infancy” and precocious puberty (PP).Material/MethodsWe compared 59 patients (21 boys and 38 girls) seen for pubic hair development before one year of age diagnosed as mini-puberty to 13 patients (2 boys) in whom pubertal development before one year revealed a PP.ResultsThe boys with mini-puberty presented with pubic hair development and prepubertal testicular volume, with low plasma testosterone concentrations. Their gonadotropin responses to gonadotropin releasing hormone (GnRH) test showed predominant luteinising hormone increase in 9/13. The girls presented with pubic hair development that was accompanied by breast development in 47% of cases, with low plasma estradiol concentrations. Their gonadotropin responses showed predominant follicle-stimulating hormone increase in the 17 evaluated.The patients with PP had organic central PP (5 hypothalamic hamartoma) or idiopathic central PP (n=6), or peripheral PP (one ovarian tumor and one congenital adrenal hyperplasia). The diagnosis was challenging only in 3 girls with idiopathic central PP presenting with prepubertal plasma estradiol concentrations and responses to GnRH test.ConclusionsThe diagnosis of PP was easily determined based on the clinical presentation and the pubertal concentrations of testosterone in boys or of estradiol in girls, as was the diagnosis of central or peripheral origin of PP based on gonadotropin response to the GnRH test. Once PP is excluded, these patients need careful follow–up and physician consultation is needed if clinical pubertal signs progress.

Dietary calcium deficiency in laying ducks impairs eggshell quality by suppressing shell biomineralization.

The objective of this study was to determine the effects of dietary calcium deficiency on the process of shell formation. Four hundred and fifty female ducks (Anas platyrhynchos) at 22 weeks were randomly assigned to three groups. Ducks were fed one of two calcium-deficient diets (containing 1.8% or 0.38% calcium, respectively) or a calcium-adequate control diet (containing 3.6% calcium) for 67 days (depletion period) and then all ducks were fed a calcium-adequate diet for an additional 67 days (repletion period). Compared with the calcium-adequate control, the average shell thickness, egg shell weight, breaking strength, mammillae density and mammillary knob thickness of shell from ducks that consumed the diet with 0.38% calcium were significantly decreased (P<0.05) during the depletion period, accompanied by reduced tibia quality. The mRNA expression of both secreted phosphoprotein 1 (SPP1) and carbonic anhydrase 2 (CA2) in the uterus was decreased after feeding calcium-deficient diets (1.8% or 0.38% calcium). mRNA transcripts of calbindin 1 (CALB1), an important protein responsible for calcium transport, and the matrix protein genes ovocalyxin-32 (OCX-32) and ovocleidin-116 (OC-116) were reduced in ducks fed 0.38% calcium but not 1.8% calcium. Plasma estradiol concentration was decreased by both of the calcium-deficient diets (P<0.05). The impaired shell quality and suppressed functional proteins involved in shell formation could be reversed by repletion of dietary calcium. The results of the present study suggest that dietary calcium deficiency negatively affects eggshell quality and microarchitecture, probably by suppressing shell biomineralization.

Associations of acrylamide intake with circulating levels of sex hormones and prolactin in premenopausal Japanese women.

It has been hypothesized that alteration of hormone systems is involved in the carcinogenesis of acrylamide. The aim of the present study was to examine the cross-sectional associations between dietary acrylamide intake and sex hormone levels in premenopausal Japanese women. Study subjects were 393 women who had regular menstrual cycles less than 40 days long. Acrylamide intake was assessed with a food-frequency questionnaire and was based on acrylamide concentration reported from analyses of Japanese foods. We measured the plasma concentrations of estradiol, testosterone, dehydroepiandrosterone sulfate, sex hormone-binding globulin, follicle-stimulating hormone, luteinizing hormone, and prolactin. After controlling for age, the phase of the menstrual cycle, and other covariates, acrylamide intake was statistically significantly inversely associated with total and free estradiol levels and statistically significantly positively associated with follicle-stimulating hormone level. Total and free estradiol levels were 18.2% and 19.3% lower, respectively, in women in the highest quartile of acrylamide intake than in those in the lowest quartile of intake. Follicle-stimulating hormone levels were 23.5% higher in women in the highest quartile of acrylamide intake than in those in the lowest quartile of intake. The data suggest that acrylamide intake may alter estradiol and follicle-stimulating hormone levels. High estradiol levels have been associated with an increased risk of breast cancer. Although the results need confirmation, they highlight the need to investigate the relationships among dietary acrylamide, sex hormones, and breast cancer risk. Cancer Epidemiol Biomarkers Prev; 24(1); 249-54. ©2014 AACR.

Extreme concentrations of endogenous sex hormones, ischemic heart disease, and death in women.

Sex hormones may be critical determinants of ischemic heart disease and death in women, but results from previous studies are conflicting. To clarify this, we tested the hypothesis that extreme plasma concentrations of endogenous estradiol and testosterone are associated with risk of ischemic heart disease and death in women. In a nested prospective cohort study, we measured plasma estradiol in 4600 and total testosterone in 4716 women not receiving oral contraceptives or hormonal replacement therapy from the 1981 to 1983 examination of the Copenhagen City Heart Study. During ≤30 years of follow-up, 1013 women developed ischemic heart disease and 2716 died. In women with a plasma estradiol below the fifth percentile compared with between the 10th and 89th percentiles, multifactorially adjusted risk of ischemic heart disease was 44% (95% confidence interval, 14%-81%) higher; however, plasma estradiol concentrations did not associate with death. Also, in women with a plasma testosterone concentration at or above the 95th percentile compared with between the 10th and 89th percentiles, multifactorially adjusted risk was 68% (34%-210%) higher for ischemic heart disease, 36% (18%-58%) higher for any death, and 38% (15%-65%) higher for death from other causes than cardiovascular disease and cancer. These results were similar for postmenopausal women alone. In women, extreme low concentrations of endogenous estradiol were associated with high risk of ischemic heart disease, and extreme high concentrations of endogenous testosterone were associated with high risk of ischemic heart disease and death.

Alcohol consumption in relation to plasma sex hormones, prolactin, and sex hormone-binding globulin in premenopausal women.

Alcohol consumption is a consistent risk factor for breast cancer, and evidence suggests premenopausal plasma hormones are associated with breast cancer. Plasma concentrations of estradiol, estrone, estrone sulfate, testosterone, androstenedione, progesterone, prolactin, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), and sex hormone-binding globulin (SHBG) were measured in samples collected in 1996-99. Average alcohol intake was calculated from semiquantitative food frequency questionnaires collected in 1995 and 1999. We used generalized linear models to calculate geometric mean hormone concentrations across alcohol categories and the percentage difference for the highest versus lowest category. Comparing women who consumed >20 g/d with nondrinkers, levels were 25.7% higher for luteal estrone (geometric mean, 106 vs. 84.5 pg/mL; Ptrend = 0.001), 27.2% higher for luteal estradiol (182 vs. 143 pg/mL; Ptrend = 0.006), and 16.8% higher for SHBG (85.6 vs. 73.3 nmol/L; Ptrend = 0.03); concentrations of free testosterone were 17.9% lower (0.16 vs. 0.20 ng/dL; Ptrend = 0.002). Women consuming >10 g/d compared with nondrinkers had 26.5% higher concentrations of follicular estrone sulfate (950 vs. 751 pg/mL; Ptrend = 0.04). We did not observe significant associations between alcohol and the other sex hormones evaluated. Significant positive associations were observed with beer intake, but not other alcohol types, for DHEA (Pinteraction = 0.003) and androstenedione (Pinteraction = 0.006). Alcohol consumption was significantly positively associated with plasma luteal estrogen concentrations, but not with androgen levels, nor estrone or estradiol measured in the follicular phase. Differences in premenopausal estrogen levels may contribute to the association between alcohol and breast cancer.

Raloxifene modulates regulators of osteoclastogenesis and angiogenesis in an oestrogen deficiency periapical lesion model.

To analyse the local regulatory mechanisms of osteoclastogenesis and angiogenesis during the progression of periapical lesions in female rats with oestrogen deficiency and treatment with raloxifene (RLX). Female Wistar rats were distributed into groups: SHAM-veh, subjected to sham surgery and treated with a vehicle; OVX-veh, subjected to ovary removal and treated with a vehicle; and OVX-RLX, subjected to ovary removal and treated with RLX. Vehicle or RLX was administered orally for 90 days. During treatment, the dental pulp of mandibular first molars was exposed to the oral environment for induction of periapical lesions, which were analysed after 7 and 30 days. After the experimental periods, blood samples were collected for measurement of oestradiol, calcium, phosphorus and alkaline phosphatase. The rats were euthanized and the mandibles removed and processed for immunohistochemical detection of receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG), hypoxia-inducible factor-1 alpha (HIF-1α) and bone-specific alkaline phosphatase (BALP). Data were compared using Kruskal-Wallis followed by Dunn test (nonparametric values) and anova followed by the Tukey's test (parametric values). The plasma concentration of oestradiol showed hypo-oestrogenism in the rats subjected to ovary removal. On day 7, alkaline phosphatase activity, calcium and phosphorus were higher in the OVX-RLX group than in the OVX-veh group (P < 0.001), but immunolabelling for RANKL and HIF-1α was lower in OVX-RLX group (P < 0.001). On day 30, the OVX-veh group had higher immunolabelling for RANKL than the OVX-RLX group (P < 0.05). There were no significant differences in the immunoreactivity of OPG and BALP between any groups at either time-point (P > 0.05). RLX therapy reversed the increased levels of the local regulators of both osteoclastogenesis and angiogenesis induced by oestrogen deficiency.

Manifestation of estrous behavior and subsequent progesterone concentration at timed-embryo transfer in cattle are positively associated with pregnancy success of recipients

Plasma estradiol and progesterone (P4) concentrations during the peri-ovulatory period are positively correlated with pregnancy success in cattle. The aims of this study were to assess the effects of estrus occurrence and early diestrus P4 concentrations on pregnancy per timed-embryo transfer (P/TET). A total of 267 crossbred beef heifers [222 with corpus luteum (CL) and 45 without a CL but with a follicle >8mm at beginning of the estrous synchronization protocol) received an intra-vaginal P4 device and intramuscular administration of estradiol benzoate. Progesterone devices were removed 8 days later (Day 0), and heifers received d-cloprostenol, eCG and estradiol cypionate. Estrous behavior was monitored twice daily for 3 days after P4 device removal. Plasma P4 concentration was measured by radioimmunoassay at Day 7 and Day 9. At Day 9, heifers with a CL (n=236; i.e. submission rate of 85.5%; 236/276) undergoing TET received an in vitro-produced embryo. Heifers expressing a standing behavioral estrus had a greater P/TET than heifers that did not express a standing estrus [62.4% (106/170) compared with 47.0% (31/66)]. The probability of pregnancy was positively correlated with plasma P4 concentration at TET. When heifers were grouped by quartiles of P4 concentration at TET (Q1=0.64±0.16, Q2=1.70±0.04, Q3=2.90±0.07 and Q4=5.52±0.27ng/mL) the P/TET were 45.8% (Q1; 27/59)c, 52.25% (Q2; 31/59)bc, 66.1% (Q3; 39/59)ab and 67.8% (Q4; 40/59)a. Additionally, heifers that became pregnant had greater P4 concentrations at TET (2.87±0.16ng/mL; n=137) than heifers that did not become pregnant (2.45±0.24ng/mL; n=99). No statistical difference was observed regarding P4 concentrations on Day 7, regardless of standing estrus or pregnancy status. In cattle, manifestation of estrous behavior and plasma P4 concentration at TET increase the probability of pregnancy in in vitro-produced embryo recipients.

Induced spawning of wild-caught adult lake sturgeon: assessment of hormonal and stress responses, gamete quality, and survival

Summary This research assessed the endocrine and physiological effects of Ovaprim™ and synthetic homologous sturgeon gonadotrophic releasing hormone (GnRH) on spawning male and female adult lake sturgeon, Acipenser fulvescens (Rafinesque, 1817). Wild-caught adults were administered Ovaprim, GnRH, or Ringers solution (control), and serial blood samples were collected for up to 27 days following capture and hormone administration. Circulating cortisol levels appeared to increase following administration of both hormonal treatments. However, circulating cortisol was higher and plasma pH lower at capture than at any other time for all treatments, indicating that capture and transport stress were the largest disrupting factors, more so than the effects of induced spawning. Plasma concentrations of estradiol and testosterone in both sexes were similar to previously reported values. In all treatments the circulating estradiol and testosterone never significantly exceeded the values at capture prior to endocrine manipulation of the reproductive axis. Similarly, testosterone in males and estradiol in females in the muscle tissue were consistently at the same or lower concentrations than at capture. The apparent survival of all of the study animals following return to their native river indicated that the stress effects from capture and induced spawning were not severe. In concert, our results suggest that hormonal injections can be used to aid in gamete collection from wild A. fulvescens, without negative effects on long-term broodstock survival. Hatching success, larval survival to the juvenile stage, egg diameter, and concentrations of protein, triglyceride, and glucose in the egg, sperm, and ovarian fluid (but not seminal plasma), were the same or greater in GnRH-treated fish than gametes from the Ovaprim-treated group, suggesting that utilization of GnRH may be preferable to other common methods of spawning induction.

Infusion of Gabrα6 siRNA into the trigeminal ganglia increased the myogenic orofacial nociceptive response of ovariectomized rats treated with 17β-estradiol

High levels of 17β-estradiol (E2) have been found to reduce inflammatory temporomandibular joint (TMJ) pain. A search for genes effected by a high concentration of estradiol showed an increase in GABAA receptor subunit alpha 6 (Gabrα6) in the trigeminal ganglia (TG). Blockade of Gabrα6 expression in the TG increases masseter muscle nociception in male rats, but the relationship between estradiol’s effect on nociception and Gabrα6 expression remains unclear in females. To address this knowledge gap we hypothesized that reducing Gabrα6 expression in the TG will increase the orofacial nociceptive response of ovariectomized female rats treated with estradiol. To administer hormone osmotic pumps were placed in rats that dispensed a low diestrus plasma concentration of 17β-estradiol, in addition, 17β-estradiol was injected to produce a high proestrus plasma concentration of estradiol. A ligature was then placed around the masseter tendon to induce a nociceptive response; a model for TMJ muscle pain. Gabrα6 small interfering RNA (siRNA) was later infused into the TG and the nociceptive response was measured using von Frey filaments and a meal duration assay. GABAA receptor expression was measured in the TG and trigeminal nucleus caudalis and upper cervical region (Vc-C1). Ligature significantly increased the nociceptive response but a high proestrus concentration of 17β-estradiol attenuated this response. Gabrα6 siRNA infusion decreased Gabrα6 expression in the TG and Vc-C1 but increased the nociceptive response after 17β-estradiol treatment. The results suggest estradiol decreased the orofacial nociceptive response, in part, by causing an increase in Gabrα6 expression.

Testosterone or oestradiol implants in the medial preoptic area induce mating in noncopulating male rats.

Noncopulating (NC) male rats are those males that do not mount, intromit or ejaculate when repeatedly tested with receptive females. The lack of sexual behaviour in these males is not associated with alterations in testosterone or oestradiol (E2) plasma concentrations. Instead, it has been shown that androgen receptors are higher and oestrogen receptors are lower in the medial preoptic area (MPOA) of NC male rats than those observed in copulating (C) male rats. We have also observed reduced aromatase activity in the MPOA (but not in other brain regions) of NC male rats. The aim of the present study was to determine whether testosterone or E2 implants in the MPOA of NC male rats could induce sexual behaviour. Accordingly, in Experiment 1, we evaluated the long-term effects of testosterone or E2 implants in the MPOA, the ventromedial nucleus of the hypothalamus or the medial amygdala with respect to inducing sexual behaviour in castrated C male rats. Male rats were bilaterally implanted with a guide cannula, either empty or containing testosterone or E2. Starting 1 week later, all male rats were mated once weekly for 5 months. As described previously, the site where hormone implants most consistently induced sexual behaviour in castrated C male rats was the MPOA. Experiment 1 extended these findings showing that the males continued mating even 5 months after the implant. In the second experiment, NC males were implanted in the MPOA with a guide cannula empty or filled with testosterone or E2. One week after the testosterone or E2 implant, the percentage of males that mounted and intromitted started to increase and, 5 weeks after the implant, 50% of the subjects displayed mounts and intromissions. All NC males implanted with testosterone ejaculated consistently from week 11 after the implant until the end of testing (5 months), whereas all subjects implanted with E2 ejaculated from week 16 after the implant until the end of testing. These results support the hypothesis that, in the MPOA of NC male rats, there is a hormonal alteration associated with the lack of sexual behaviour.

Identification of a Novel Kisspeptin with High Gonadotrophin Stimulatory Activity in the Dog

Kisspeptin (KISS1) and its receptor (KISS1r) are essential for normal reproductive function in many species, but the role of kiss1/kiss1r signalling in the dog has not yet been elucidated. The aims of this study were to identify the canine kiss1 and kiss1r genes and to determine gonadotrophin and oestradiol stimulatory activity of KP-10, the shortest biologically active form of KISS1. Canine kiss1 and kiss1r genes were localized by comparing the reference dog genome with relevant human cDNA sequences, using BLASTn software. The amino acid sequence of canine KP-10 (YNWNVFGLRY) differs at two positions from human KP-10 (YNWNSFGLRF). A single bolus of canine KP-10 was administered intravenously to anoestrous Beagle bitches in dosages of 0, 0.1, 0.2, 0.3, 0.5, 1, 5, 10, and 30 μg/kg. Blood samples were collected before and after canine KP-10 administration for the measurement of plasma luteinizing hormone (LH, all doses), follicle-stimulating hormone (FSH) and oestradiol (1-30 μg/kg). From 0.2 μg/kg onwards, canine KP-10 resulted in a rapid and robust rise in plasma LH concentration (max. at 10 min). KP-10 also resulted in a rapid and robust rise in plasma FSH concentration (max. at 10-20 min). Plasma oestradiol concentration increased significantly after dosages of 1, 5, and 10 μg/kg and reached a maximum at 60-90 min. In conclusion, canine KP-10 is a potent kisspeptin which elicits robust gonadotrophin and oestradiol responses in anoestrous bitches, suggesting that canine kiss1/kiss1r are cogent targets for modulating reproduction in dogs.

The progestin levonorgestrel disrupts gonadotropin expression and sex steroid levels in pubertal roach (Rutilus rutilus)

The aim of the present study was to investigate the effects of the synthetic progestin levonorgestrel (LNG) on the reproductive endocrine system of a teleost fish, the roach (Rutilus rutilus). Pubertal roach were exposed for 28 days in a flow-through system to four concentrations of LNG (3, 31, 312, and 3124ng/l). Both males and females treated with 3124ng/l LNG exhibited the upregulated levels of vitellogenin and oestrogen receptor 1 mRNA in the liver. At the same concentration, LNG caused a significant upregulation of the mRNA expression of the gene encoding luteinising hormone β-subunit (lhβ) and the suppression of the mRNA expression of the gene encoding follicle-stimulating hormone β-subunit (fshβ) in the pituitary of both male and female roach. A lower LNG concentration (312ng/l) suppressed mRNA expression of fshβ in males only. Females treated with 3124ng/l LNG exhibited significantly lower plasma 11-ketotestosterone (11-KT) and oestradiol (E2) concentrations, whereas their testosterone (T) level was higher compared with the control. Females exposed to 312ng/l LNG presented significantly lower plasma E2 concentrations. Males exposed to ≥31ng/l LNG exhibited significantly reduced 11-KT levels. As determined through a histological analysis, the ovaries of females were not affected by LNG exposure, whereas the testes of males exposed to 31 and 312ng/l LNG exhibited a significantly higher percentage of spermatogonia B compared with the control. The results of the present study demonstrate that LNG disrupts the reproductive system of pubertal roach by affecting the pituitary gonadotropin expression and the sex steroid levels. This disruption was determined to occur in males after exposure to an environmentally relevant concentration (31ng/l). Moreover, the highest tested concentration of LNG (3124ng/l) exerted an oestrogenic effect on fish of both sexes.

Estrous cycle fluctuations in sex and ingestive behavior are accentuated by exercise or cold ambient temperatures

This article is part of a Special Issue “Energy Balance”.In female Syrian hamsters (Mesocricetus auratus), low circulating levels of ovarian steroids are associated with increased food hoarding and decreased sexual motivation, but these effects are exaggerated in food-restricted females. To determine whether cold ambient temperature has the same effects as food restriction, groups of hamsters were fed ad libitum while they were housed at either 5°C or 22°C, and then tested for behavior for 90min on each day of the estrous cycle. In females housed at 22°C, high levels of sexual motivation and low levels of food hoarding were seen every day of the estrous cycle. In females housed at 5°C, high levels of sexual motivation were restricted to the periovulatory day. On the three nonestrous days, these females showed high levels of food hoarding, but not food intake. A separate cohort of females were provided with access to running wheels and housed at 22°C. They showed high levels of sexual motivation restricted to the periovulatory day, similar to the pattern of sexual motivation seen in cold-housed females. Unlike cold-housed females, those with running wheels showed low levels of food hoarding and high levels of food intake. Food restriction, cold housing, and access to wheels had no significant effect on plasma estradiol or progesterone concentrations, but significantly decreased plasma leptin concentrations. All three energetic challenges unmask estrous cycle fluctuations in sexual motivation that are obscured in laboratory conditions, i.e., isolation in a small cage with an overabundance of food.

Effect of leptin on in vivo goat embryo production.

The aim of this study was to evaluate the effect of leptin administration during superovulation on in vivo goat embryo production. Ten mature does were superovulated with 133mg follicle-stimulating hormone (FSH) i.m. in six descending doses at 12-h intervals. The goats received 4.8μg/kg human recombinant leptin s.c. (leptin group, n=5) or phosphate-buffered saline (PBS) (control group, n=5) with the first and second FSH doses. The does were mated and subjected to embryo collection by transcervical technique 6days later. The total number of cells per embryo and the number of cells with fragmented DNA were assessed in selected blastocysts by combining Hoechst 33342 and terminal dUTP nick-end labelling (TUNEL) staining. Plasma concentrations of oestradiol (E2 ) and progesterone (P4 ) were determined by electrochemiluminescence from the day of FSH treatment, on the day of superovulatory oestrus and on the day before embryo collection. Compared with the control group, the does that received leptin had a higher number of transferable embryos (p<0.005), fewer embryos classified as degenerated (p<0.001) and fewer TUNEL-positive cells/blastocyst (p<0.001). The number of transferable embryos was positively correlated with E2 concentrations on day of oestrus (r=0.562; p<0.01) and P4 concentrations on the day of embryo collection (r=0.912; p<0.001). We concluded that in vivo leptin administration during FSH treatment improved embryo quality and affected ovarian steroidogenesis in superovulated goats.

Kisspeptin, c‐Fos and CRFR Type 2 Co‐expression in the Hypothalamus after Insulin‐Induced Hypoglycaemia

Normal reproductive function is dependent upon availability of glucose and insulin-induced hypoglycaemia is a metabolic stressor known to disrupt the ovine oestrous cycle. We have recently shown that IIH has the ability to delay the LH surge of intact ewes. In the present study, we examined brain tissue to determine: (i) which hypothalamic regions are activated with respect to IIH and (ii) the effect of IIH on kisspeptin cell activation and CRFR type 2 immunoreactivity, all of which may be involved in disruptive mechanisms. Follicular phases were synchronized with progesterone vaginal pessaries and at 28h after progesterone withdrawal (PW), animals received saline (n=6) or insulin (4IU/kg; n=5) and were subsequently killed at 31h after PW (i.e., 3h after insulin administration). Peripheral hormone concentrations were evaluated, and hypothalamic sections were immunostained for either kisspeptin and c-Fos (a marker of neuronal activation) or CRFR type 2. Within 3h of treatment, cortisol concentrations had increased whereas plasma oestradiol concentrations decreased in peripheral plasma (p<0.05 for both). In the arcuate nucleus (ARC), insulin-treated ewes had an increased expression of c-Fos. Furthermore, the percentage of kisspeptin cells co-expressing c-Fos increased in the ARC (from 11 to 51%; p<0.05), but there was no change in the medial pre-optic area (mPOA; 14 vs 19%). CRFR type 2 expression in the lower part of the ARC and the median eminence was not altered by insulin treatment. Thus, disruption of the LH surge after IIH in the follicular phase is not associated with decreased kisspeptin cell activation or an increase in CRFR type 2 in the ARC but may involve other cell types located in the ARC nucleus which are activated in response to IIH.

Ovarian surface epithelium receptors during pregnancy and estrus cycle of rats with emphasis on steroids and gonadotropin fluctuation

The present study is designed to demonstrate the ovarian surface epithelial cells’ (OSE) estrogen receptor α (ERα) and progesterone receptor (PR) during pregnancy and estrous cycle in rat. Moreover, determination of the levels of plasma progesterone, estradiol, FSH and LH was also made. The levels of plasma progesterone, estradiol, FSH and LH concentrations were determined on days 7 (n=5), 14 (n=5), and 21 (n=5) of pregnancy in three groups of rats and during the estrous cycle (n=5) using an ELISA kit. Immunohistochemical method for PR and ERα expressions was also made on the ovary. During pregnancy, FSH and LH remained low except at term when LH levels began to increase from 16ng/ml to 47ng/ml. Progesterone levels significantly exceeded estradiol values in all pregnant rats with a peak value of 202ng/ml on day 14. Elevated progesterone levels were associated negatively with LH and estradiol levels during pregnancy. The levels of estradiol surged significantly on day 21. Immunohistochemistry of the ovary showed low levels of OSE cells staining positive for ERα expression. ERα positive cells were absent on day 7 and 14 of pregnancy, only day 21 recorded a very low percentage of immunostaining (0.5%) within the nuclei of OSE cells. On the contrary, immunostaining of PR was not observed within the nuclei of OSE cells in all groups of study. In conclusion, these results may suggest that the progesterone effect during pregnancy seems to be overriding the positive effect of estrogens on OSE cells. High progesterone levels may have a direct negative effect on gonadotropin production and thereby it might inhibit events leading to both follicular development and OSE proliferation. Understanding the factors affecting OSE proliferation may help elucidating the mechanism(s) of assisted diseases such as ovarian cancer.