Plasma LH Research Articles

The local technical validation of new plasma tube with a mechanical separator

Abstract Background Unsuitable samples are common problem for laboratories. The blood collection tubes need to be validated or verified prior to their being used in the routine laboratory for reducing this situation. Objective We aim to compare the technical qualifications of routinely used BD Vacutainer® Serum Separator Tubes™ II Advance Plus with BD Vacutainer® Barricor™ LH Plasma Tubes for local technical validation. Materials and methods Apparently healthy 150 voluntary subjects were enrolled in the study. Samples were collected in two separated tubes by a single phlebotomist. Twelve quality indicators were used to compare these two different types of tubes for local technical validation. Differences (%) between them were calculated with the formula proposed by EFLM. In case of any difference of less than 1% for each indicator, the evaluated tube was considered as non-inferior. Results Indicators, such as tubes with physical defects, that fail to create vacuum, not properly fitting into the blood collection device, under filling (10%), cracked tubes, tubes exterior surface contaminated with blood, hemolysed specimens, including fibrin strand/mass in the sample, red blood cell adhesion, poor/incomplete barrier formation were found non-inferior in Barricor™ tubes. White particulate matter (WPM) was observed in 24.6% of Barricor™. Therefore, the last indicator (tubes including gel/foreign material/WPM in sample after centrifugation) was found inferior for Barricor™. Conclusion Technical local validation studies should be encouraged in terms of quality management. It was thought that WPM would not cause any interference in a properly filled tube. In addition to, Barricor™ was also found to be technically acceptable when evaluated through using all other indicators.

157 Effect of a slow-release gonadotrophin-releasing hormone analogue on ovarian activity and oestrous behaviour in mares

Oestrus behaviour in mares can contribute to problems in their handleability and reduced performance in equestrian sports. Therefore, methods of transient suppression of oestrous cyclicity in mares are of interest. The aim of our study was to determine whether treatment of mares with slow-release implants containing the gonadotrophin-releasing hormone (GnRH) analogue deslorelin downregulates pituitary GnRH receptors and reduces ovarian function and oestrous behaviour. Shetland mares (age=11.0±1.4 years; bodyweight=185.5±7kg) were oestrous synchronised with two injections of the prostaglandin F2α analogue luprostiol (3.725mg) at an interval of 12 days. One day after the second injection (Day 0), mares were randomly assigned to three groups: slow-release implant with 9.4mg of deslorelin (Suprelorin, Virbac; group D1; n=6), implant with 4.7mg of deslorelin (group D2; n=5), and intramuscular injection of 1.25mg of short-acting deslorelin (control, group C; n=5). Collection of blood samples for analysis of progesterone, LH, and anti-Müllerian hormone (AMH) using established and validated enzyme immunoassays (Scarlet et al. 2018 Theriogenology 117, 72-77), testing for oestrus-like behaviour with a Shetland stallion, and ultrasonography of the genital tract were performed at 2-day intervals until Day 10 after treatment and at 5-day intervals from there. On Days 10 and 45 after treatment, LH stimulation tests with the GnRH agonist buserelin (4µg IV) were performed. Data were normally distributed; differences among groups were analysed using analysis of variance and subsequent Tukey test. Values are means±s.e.m. In all mares without a corpus luteum on Day 0 (progesterone <1ngmL−1; one mare in group D1 and two in group C), ovulation was detected within 9 days after deslorelin treatment. These ovulations were classified as deslorelin induced, whereas ovulations after Day 10 were classified as spontaneous ovulations. The mean interval from deslorelin until the first spontaneous ovulation was 62.0±8.6, 44.2±14.1, and 22.2±3.1 days in groups D1, D2, and C (P<0.05), respectively. Subsequent oestrous cycles were regular. Oestrus-like behaviour until day 50 was reduced in groups D1 (2.0±0.9 days) and D2 (2.4±1.3 days) compared with group C (6.4±1.2 days; P<0.05). Concentration of plasma LH and AMH decreased in group D1 (P<0.05) but not in groups D2 and C. The GnRH stimulation test on Day 10 resulted in an increase (P<0.001) in plasma LH concentration in group C but not in groups D1 and D2 (treatment×time P<0.05). On Day 45, LH concentration increased in all mares in response to buserelin (NS among groups). Within 100 days of treatment, LH concentrations but not AMH concentrations in mares of group D1 returned to baseline. In conclusion, deslorelin slow-release implants transiently suppress ovarian function and oestrus behaviour in mares. Spontaneous ovulation is delayed in a dose-dependent manner. A decrease in AMH concentration suggests inhibitory effects of deslorelin on small antral follicles. Long-term effects on follicular dynamics and fertility in larger horses also need to be assessed.

Contingent role of phoenixin and nesfatin-1 on secretions of the male reproductive hormones.

Phoenixin (PNX) and nesfatin-1 are localised in the hypothalamus and the pituitary gland. Moreover, the most of the PNX-expressing neurons in the hypothalamus also co-express nesfatin-1. These outcomes may suggest that there is an interaction between PNX and nesfatin-1, at least in terms of neuroendocrine-mediated regulations. Hence, the study was planned to find out the effects of centrally delivered PNX and nesfatin-1 on male sex hormones or to show the interactive association of intracerebroventricularly (ICV) injected PNX+nesfatin-1 combination on the release of male hormones. PNX and nesfatin-1, single or together, were delivered ICV to different male Wistar Albino rat groups. Both PNX and nesfatin-1 induced a significant enhancement in plasma FSH, LH and testosterone without inducing any alteration in plasma GnRH in the rats. The central combinatorial treatment of both the neuropeptides produced a more potent rise in male plasma hormone levels than treating with single neuropeptide. In summary, our preliminary data show that centrally delivered PNX and nesfatin-1 can affect plasma male hormone levels. Moreover, that the combinatorial treatment with both the neuropeptides in male rats leading to a more potent effect on the plasma male hormone levels might suggest that both these neuropeptides act synergistically in terms of regulation of male HPGA.

P4503A multi-site coronary vein sampling study on cardiac troponin T degradation in non-ST-elevation myocardial infarction

Abstract Background High-sensitivity cardiac troponin (hs-cTn) assays have reduced specificity for myocardial infarction (MI). In conditions other than MI, cardiac troponin T (cTnT) elevations have been attributed to small cTnT fragments. In search of improved cTnT assay specificity for MI, determination of the in-vivo molecular appearance of cTnT during MI is pivotal. Purpose To determine cTnT composition close to its site of release and during the course of MI by multi-site coronary venous system (CVS) and peripheral sampling. Methods Lithium-heparinized (LH) plasma and serum samples were obtained from multiple CVS locations in NSTEMI patients. Additionally, samples were drawn from a peripheral artery and vein followed by collections at 6- and 12 hours post-catheterization. cTnT concentrations were measured using the hs-cTnT immunoassay (Roche). The molecular cTnT composition was determined by gel filtration chromatography and Western blotting in an early and late presenting patient. Results CVS hs-cTnT concentrations were 28% higher than in the peripheral artery sample (n=71, p<0.001). In contrast to LH plasma, serum caused pre-analytical cTn T-I-C complex disintegration and cTnT degradation. CVS samples demonstrated presence of cTn T-I-C complex, free intact cTnT, and 29 kDa and 15–18 kDa cTnT fragments in higher absolute concentrations than measured peripherally. While the proportion of cTn T-I-C complex decreased and disappeared over time, 15–18 kDa cTnT fragments increased. Moreover, cTn T-I-C complex was more prominent in the early versus the late presenting patient. Central illustration Conclusions In NSTEMI, cTnT is released as a combination of cTn T-I-C complex, free intact cTnT and multiple cTnT fragments. Over time, the composition of observed cTnT forms changes due to in-vivo degradation. These findings serve as a stepping stone to improve diagnostic accuracy of the hs-cTnT assay for MI.

Lame cows have a subdued hypothalamus-pituitary axis as revealed by LH release in response to exogenous endocrine challenges

Lameness is a serious concern in cows due to its multilevel effects and increasing incidences. The ability of anterior pituitary and hypothalamus to release LH, a prime reproductive hormone, is unknown in lame cows. Our study tested this ability by exogenous endocrine challenge using Buserelin acetate (GnRH analogue) and estradiol valerate (EV). The study included moderately lame (L; n=7) either cyclic (LC; n=3) or anestrus (LA; n=4); and normal cyclic, non–lame (NL; n=5) cows. Progesterone blocks LH release, so it was lowered by injecting PG in cyclic cows. After 14 h, all cows were given a walk for 5 min and immediately thereafter injected with 0.008 mg GnRH and 4 h later with 1 mg of EV. Blood plasma progesterone, cortisol and LH were quantified, periodically. There was a significant delay in the onset (105.0 vs 75.0±7.0 vs 54.0±9.0 min) and duration (22.5±5.3 vs 80.0±10.8 vs 117.0±2.7 min) of LH surge in LA vs LC vs NL cows; the surge was absent in two LA cows. The altered LH profile was attributed to a 3 fold (in L) vs 1.6 fold (in NL) rise in cortisol. LH released (ng/ml) in response to EV was also low in LA (31.5±2.4) and LC (69.9±7.2) than NL (79.2±7.0) cows. In conclusion, lame cows subjected to walk exhibited reduced pituitary response to release LH following exogenous GnRH as also a reduced ability of hypothalamus to stimulate LH release in response to exogenous estradiol.

Kisspeptin induces ovulation in heifers under low plasma progesterone concentrations

The objective of the present study was to compare the effect of a single versus multiple doses of a 10-amino acid fragment of human (hKp) or murine (mKp) kisspeptin on LH secretion and the fate of the dominant follicle. In all experiments, a new wave was induced (Day 0) by ultrasound-guided ablation of >5 mm follicles, a progesterone device (CIDR) was placed in the vagina, animals given prostaglandin F2α analog im on Day 3.5 and 4, and hKp or mKp treatment given on Day 6. The experimental design maintained growth and ovulatory potential of the dominant follicle for 12 days and allowed hypothesis testing during the low-progesterone period (plasma progesterone ≤1.8 ng/ml on Day 6) wherein spontaneous wave emergence and ovulation did not occur between Day 6 and Day 12. In Experiment 1, heifers (n = 10/group) were given single iv dose of 45 mg hKp, 45 mg mKp, or 2 ml normal saline (control). Post-treatment plasma LH concentrations from 15 to 90 min were higher (P < 0.01) in hKp group than in the mKp and control groups. Two heifers ovulated in hKp group versus none in other groups. In Experiment 2, heifers (n = 6/group) were given 45 mg hKp over a 2 h period divided into multiple iv doses treatments or 2 ml normal saline (control). Post-treatment plasma LH concentrations were higher (P < 0.01) in all hKp treatment groups than in the control group. The ovulation rate was higher (P = 0.06) after hKp treatments (11/18) than in the control group (0/6). In Experiment 3, heifers (n = 6/group) were given 45 mg mKp over a 2 h period divided into multiple iv doses treatments or a single iv dose of gonadorelin acetate (positive control). Plasma LH concentration was higher (P < 0.01) and the ovulation rate was greater (P = 0.01) in the GnRH group (5/6) than mKp groups (1/12). In summary, hKp was more effective to induce ovulation than mKp. Human kisspeptin-10 given over a 2 h period induced ovulations at a rate similar to that of GnRH treatment in heifers under a low plasma progesterone state.

Endogenous Circadian Regulation of Female Reproductive Hormones.

Studies suggest that female reproductive hormones are under circadian regulation, although methodological differences have led to inconsistent findings. To determine whether circulating levels of reproductive hormones exhibit circadian rhythms. Blood samples were collected across ∼90 consecutive hours, including 2 baseline days under a standard sleep-wake schedule and ∼50 hours of extended wake under constant routine (CR) conditions. Intensive Physiological Monitoring Unit, Brigham and Women's Hospital. Seventeen healthy premenopausal women (22.8 ± 2.6 years; nine follicular; eight luteal). Fifty-hour CR. Plasma estradiol (E2), progesterone (P4), LH, FSH, SHBG, melatonin, and core body temperature. All hormones exhibited significant 24-hour rhythms under both standard sleep-wake and CR conditions during the follicular phase (P < 0.05). In contrast, only FSH and SHBG were significantly rhythmic during the luteal phase. Rhythm acrophases and amplitudes were similar between standard sleep-wake and CR conditions. The acrophase occurred in the morning for P4; in the afternoon for FSH, LH, and SHBG; and during the night for E2. Our results confirm previous reports of ∼24-hour rhythms in many female reproductive hormones in humans under ambulatory conditions but demonstrate that these hormones are under endogenous circadian regulation, defined as persisting in the absence of external time cues. These results may have important implications for the effects of circadian disruption on reproductive function.

Ovarian Expression of Adipokines in Polycystic Ovary Syndrome: A Role for Chemerin, Omentin, and Apelin in Follicular Growth Arrest and Ovulatory Dysfunction?

Adipokines are a potential link between reproduction and energy metabolism and could partly explain some infertilities related to some pathophysiology, such as polycystic ovary syndrome (PCOS). However, adipokines were predominantly assessed in blood samples, while very little is known concerning their variations in follicular fluid (FF) and ovarian granulosa cells (GCs) of PCOS women. Thus, the objectives of our study were to investigate adiponectin, chemerin, resistin, visfatin, omentin, and apelin ovarian expression in PCOS women in comparison with controls and women with only a polycystic ovary morphology. In total, 78 women undergoing an in vitro fertilization procedure were divided into three groups: 23 PCOS women, 28 women presenting only ≥12 follicles per ovary (ECHO group), and 27 control women. Each group almost equally included normal weight and obese women. Follicular fluid (FF) concentration and granulosa cells (GCs) mRNA expression of adipokines and their receptors were assessed by ELISA and RT-qPCR, respectively. Omentin levels in FF and GC were higher in PCOS than in ECHO and control women, while apelin expression was increased in both PCOS and ECHO groups. FF chemerin concentration was predominant in normal-weight PCOS women compared to BMI (Body Mass Index)-matched ECHO and control women, while GC mRNA levels were higher in the obese PCOS group than in the ECHO one. Compared to PCOS, ECHO women had increased FF adiponectin concentrations and lower plasma AMH levels. The FF concentration of all adipokines was higher in obese subjects except for adiponectin, predominant in normal-weight women. In conclusion, women with PCOS expressed higher GC chemerin and omentin, whereas the ECHO group presented higher levels of FF adiponectin and apelin and lower plasma AMH and LH concentrations. Chemerin, omentin, and apelin expression was differently regulated in women with PCOS, suggesting their possible role in follicular growth arrest and ovulatory dysfunction characterizing PCOS pathogenesis.

Searching for an inhibitory action of blood-borne [beta]-endorphin on LH release

Concentrations of beta-endorphin were quantified in peripheral blood plasma of sheep by a radioimmunoassay that cross-reacted with beta-lipotrophin. Plasma concentrations of beta-endorphin increased abruptly after physical confinement, bacteraemia, and electroacupuncture treatment for induction of analgesia. In these experimental situations in which plasma concentrations of beta-endorphin increased, plasma concentrations of LH often decreased. To test the hypothesis that increases in blood-borne beta-endorphin actually caused the decrease in LH release, naloxone was administered to antagonize the opioid receptors at which blood-borne beta-endorphin might act. In no case did administration of naloxone disrupt the temporal correlation between experimentally induced increases in plasma beta-endorphin and decreases in plasma LH. It was concluded that the increases in blood-borne beta-endorphin did not cause the decrease in LH release. Other research investigated whether beta-endorphin might be delivered via blood from pituitary to hypothalamus in locally enriched concentrations. Even when pituitary release of beta-endorphin was acutely stimulated, it was not possible to demonstrate retrograde delivery of beta-endorphin to the hypothalamus without dilution in the systemic circulation. In conclusion, it is unlikely that blood-borne beta-endorphin inhibits the release of LH, and beta-endorphin should not be classified as a hormone until blood concentrations of the peptide can be shown to exert some effect at a location distant from its site of secretion.

Short light cycles induce persistent reproductive activity in Ile-de-France rams

European breeds of rams appear to be responsive to photoperiodic changes even though there are large differences between breeds in the timing and amplitude of endocrine (LH and testosterone) and gametogenetic variations before the sexual season. Light regimens such as 6-month light cycles or alternations of constant short and long days every 12-16 weeks are able to entrain the parameters of sexual activity. In these regimens in which the period of the light cycle is shortened, LH release is markedly stimulated during decreasing daylength and evidence is presented, from the relationship of LH and testosterone patterns, that the dampening of LH stimulation could simply result from the effect of steroid feedback. However, there is a gap of several weeks between the maximum LH and testosterone concentrations during which testis growth occurs. Experiments were conducted with Ile-de-France rams, markedly seasonal breeders, in which the period of the light cycle was decreased, in different groups of animals, from 6 to 4, 3, 2 or 1 month. Rams submitted to the three light regimens with the longest periods presented testicular variations which paralleled those of the photoperiod, but those kept in the two regimens with the shortest periods had a progressive increase in testicular weight up to the maximum value (300-350 g) with no further major changes. Therefore, in rams kept in 2-month light cycles, testicular weight remained constant for twelve successive cycles (2 years). LH and testosterone plasma measurements indicated that LH was sufficiently stimulated to maintain testicular development during each decreasing daylength phase but that the stimulation was shifted before testosterone could reach levels at which feedback effects could be exerted. However, all the measures of sperm production were at values characteristic of the sexual season. Similar testicular weight maintenance was also obtained in rams submitted to a regimen in which short days (8L:16D) alternated every month with a split photoperiod interpreted as a long day (7L:8D:1L:8D). It is concluded that short light cycles are able to induce persistent reproductive activity in Ile-de-France rams, which may have practical applications in sheep production systems.

Seasonal expressions of COX-1, COX-2 and EP4 in the uteri of the wild Daurian ground squirrels (Spermophilus dauricus)

Prostaglandins (PGs) play a pivotal role in uterine reproductive process including maternal recognition of pregnancy, cell proliferation, and myometrium contractions in mammals. In this study, we investigated the immunolocalizations and expression levels of Prostaglandin E2 synthases cyclo-oxygenase (COX)-1 and COX-2, as well as one of PGE2 receptor subtypes 4 (EP4) in the uteri of the wild Daurian ground squirrels (Spermophilus dauricus) during the breeding and non-breeding seasons. Histologically, the thickness of endometrium: myometrium ratio in the uteri of the breeding season was higher than that of the non-breeding season. The immunostainings of COX-1, COX-2 and EP4 were observed in stromal cells, glandular cells and myometrium cells in the breeding and non-breeding seasons. The protein and mRNA expression levels of COX-1, COX-2 and EP4 were higher in the uteri of the breeding season than those of in the non-breeding season. The mean mRNA levels of COX-1, COX-2 and EP4 were positively correlated with uterine weights. In addition, the PGE2 concentration of uterine tissues as well as plasma PGE2, 17β-estradiol, progesterone, LH and FSH levels were also significantly higher in the breeding season compared to those of the non-breeding season. These results suggested that PGE2 might play an important autocrine or paracrine role in the regulation of seasonal changes in the uterine functions of the wild Daurian ground squirrels during the breeding and non-breeding seasons.

The effect of food restriction on the regulation of gonadotropin-releasing hormone in male house finches (Haemorhous mexicanus)

Seasonal activation of the vertebrate hypothalamic-pituitary-gonadal (HPG) axis and gonadal development is initiated by gonadotropin-releasing hormone-I (GnRH) release from the hypothalamus. In photoperiodic species, the consistent annual change in photoperiod is the primary environmental signal affecting GnRH cell activity, including changes in the synthesis and secretion of this neuropeptide. Non-photoperiodic environmental cues such as energy availability also influence HPG axis activity, but the mechanisms mediating this influence, in particular on the GnRH system, are unclear. Understanding how the neuroendocrine system integrates environmental information is critical in determining the plasticity and adaptability of physiological responses to changing environments. The primary objective of this study was to investigate GnRH-mediated changes in HPG axis activity and gonadal development in response to energy availability in a wild bird. We hypothesized that negative energy balance inhibits HPG axis activity by affecting GnRH secretion. Moderate food restriction for several weeks in male house finches, Haemorhous mexicanus, decreased body condition and inhibited photoinduced testicular growth compared to birds fed ad libitum. Food restriction did not affect plasma luteinizing hormone (LH; a correlate of GnRH release) or plasma testosterone, but it enhanced the plasma LH response to an injection of the glutamatergic agonist, N-methyl-D-aspartate (NMDA). Thus, food restriction may decrease photoinduced HPG axis activation by acting centrally, in particular by attenuating the release of accumulated GnRH stores.

Continuous presence of females in estrus does not prevent seasonal inhibition of LH and androgen concentrations in bucks

In male goats, being in permanent visual contact with females in estrus does not prevent seasonal variation in certain endocrine hormone levels and sexual activities. In this study, we tested whether continuous and full contact with females in estrus prevented seasonal endocrinological variation in bucks. In 1 experiment (Exp. 1), we verified that the sudden introduction of goats in estrus increased the plasma concentrations of androgen in bucks during the nonbreeding season under our experimental conditions. In another experiment (Exp. 2), we tested the ability of estrous goats to prevent seasonal inhibition of LH and androgen secretions in bucks kept in permanent and full contact with them. In Exp. 1, 3 groups of bucks (n = 5 in each group) were isolated from females from the months of July to January. On January 27, one group continued being isolated from females; a second group was exposed to ovariectomized, untreated goats; and a third group was exposed to ovariectomized goats with induced estrus. Plasma androgen concentrations were determined every 2 h from 8 h before to 8 h after the introduction of females. The introduction of estrus-induced goats significantly increased androgen concentrations, which were higher than in the isolated bucks, as well as in those exposed to untreated goats (P < 0.05). In Exp. 2 (n = 5 per group), one group of bucks was isolated from females from October to July, whereas two other groups remained in contact with ovariectomized goats, either untreated or regularly induced to estrus. In the three groups of bucks, plasma concentrations of LH were determined once during the months of October, February, March, and June, whereas androgen concentrations were determined weekly from October to July. The mean plasma LH and androgen concentrations were low and did not differ among the groups of bucks during the normal seasonal period of sexual inactivity (P > 0.05). We conclude that full contact and sexual interactions with estrus-induced goats failed to stop the seasonality of LH and androgen plasma concentrations of bucks, although bucks could respond to the introduction of females by acute increases in plasma LH and androgen.

SUN-LB002 CYP11A1 Inhibition as a Therapeutic Approach for the Treatment of Castration Resistant Prostate Cancer

Background: Prostate cancer is a major global challenge due to the increasing number of aging population and frequency of diagnosis. During the past decade new treatments have been targeted to the androgen signaling axis either by inhibiting the binding of androgens to androgen receptor (AR) and AR nuclear translocation, or by inhibiting androgen production via CYP17A1 enzyme. Despite the significant progress on the research and new therapies, CRPC is still incurable and there is urgent need for better, more effective treatments. ODM-208 is an oral, non-steroidal and selective inhibitor of CYP11A1 enzyme, suppressing the synthesis of all steroid hormones that could be potential agonistic AR ligands. Methods: The inhibition of CYP11A1 was measured in vitro by the formation of radiolabelled isocapronic acid in a human adrenal cortex cell line (H295R). The tumor growth inhibition of ODM-208 was studied in VCaP castration-resistant prostate cancer (CRPC) xenograft model. At the end of the xenograft study, plasma ACTH and LH, and key steroid hormone concentrations were analysed from plasma and target tissues. In addition, full length AR (AR-FL) and AR-V7 were analysed from the tumors by western blot and key enzymes of androgen biosynthesis, CYP17A1, AKR1C3, SRD5A1 were quantified by qPCR. In dogs an ACTH stimulation test was done. Results: ODM-208 potently inhibits CYP11A1 enzyme in vitro. In addition, in vivo in the VCaP CRPC xenograft model ODM-208 significantly inhibited tumor growth. Importantly, the amount of AR-FL and AR-V7 levels remained unchanged in the tumors after ODM-208 treatment. Slight increase of CYP17A1 and SRD5A1 enzyme levels was observed, indicating the activation of steroidogenesis in VCaP tumors in vivo. Treatment had no effect on plasma LH, whereas ACTH was significantly increased demonstrating reduction in glucocorticoid levels by negative feedback. In line with ACTH, all measured steroid hormones were significantly reduced both in plasma and target tissues. In dogs ACTH-stimulated cortisol production was significantly inhibited after single oral dose of ODM-208. Conclusions: ODM-208 shows promising antitumor activity in preclinical CRPC models and has favorable toxicological profile. Thus, ODM-208 might have potential for treating patients with CRPC. Based on the nonclinical results, a phase 1/2 clinical trial has been initiated. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Endocrine patterns of the post-partum cow

Summary. Milked dairy cows generally have a shorter post-partum interval to ovarian cyclicity than suckling dairy or beef cows. In milked and suckling cows, there is a strong seasonal influence with spring-calving cows remaining anoestrous longer. Increasing the suckling intensity further delays the onset of ovarian cyclicity, probably by increasing the frequency or strength of its inhibitory influence on hypothalamic activity. Plasma FSH levels rise in most cows 5–10 days after calving and thereafter the random changes observed have little relationship to the onset of cycles. Recovery of FSH release therefore occurs earlier post partum than recovery of LH release. Hyperprolactinaemia is not a cause of reproductive failure in milked or suckling cows because there is no correlation between plasma prolactin levels and the onset of ovarian cycles. Plasma LH concentrations undergo significant changes directly related to the initiation of ovarian cycles, with low plasma levels immediately post partum, followed by an increase in basal secretion and the development of clear LH episodes. This pulsatile pattern appears earlier in dairy than in beef cows and is further delayed by suckling compared to milking. Before the first ovulation there is an increased frequency and peak height of LH episodes leading to a rise in plasma LH levels and eventually to a preovulatory-type LH surge which results in the first ovulation. These changes in the pattern of LH release appear definitive in the initiation of ovarian activity in post-partum cows.

Intensity dependent disruptive effects of light at night on activation of the HPG axis of tree sparrows (Passer montanus)

Artificial light at night (ALAN) has become increasingly recognized as a disruptor of the reproductive endocrine process and behavior of wild birds. However, there is no evidence that ALAN directly disrupt the hypothalamus-pituitary-gonadal (HPG) axis, and no information on the effects of different ALAN intensities on birds. We experimentally tested whether ALAN affects reproductive endocrine activation in the HPG axis of birds, and whether this effect is related to the intensity of ALAN, in wild tree sparrows (Passer montanus). Forty-eight adult female birds were randomly assigned to four groups. They were first exposed to a short light photoperiod (8 h light and 16 h dark per day) for 20 days, then exposed to a long light photoperiod (16 h light and 8 h dark per day) to initiate the reproductive endocrine process. During these two kinds of photoperiod treatments, the four groups of birds were exposed to 0, 85, 150, and 300 lux light in the dark phase (night) respectively. The expression of the reproductive endocrine activation related TSH-β, Dio2 and GnRH-I gene was significantly higher in birds exposed to 85 lux light at night, and significantly lower in birds exposed to 150 and 300 lux, relative to the 0 lux control. The birds exposed to 85 lux had higher peak values of plasma LH and estradiol concentration and reached the peak earlier than birds exposed to 0, 150, or 300 lux did. The lower gene expression of birds exposed to 150 and 300 lux reduced their peak LH and estradiol values, but did not delay the timing of these peaks compared to the control group. These results reveal that low intensity ALAN accelerates the activation of the reproductive endocrine process in the HPG axis, whereas high intensity ALAN retards it.

The kisspeptin analog C6 is a possible alternative to PMSG (pregnant mare serum gonadotropin) for triggering synchronized and fertile ovulations in the Alpine goat.

In temperate regions goat’s reproduction is seasonal. To obtain year-round breeding, hormonal treatments are currently applied. These treatments usually combine a progesterone analog with the pregnant mare serum gonadotropin (PMSG). However, their use has significant ethical and environmental drawbacks. Therefore, alternative methods to manage reproduction are needed. The discovery that in mammals the neuropeptide kisspeptin is a major positive regulator of hypothalamo-pituitary gonadal axis offered an attractive alternative strategy to control reproduction. We have previously designed a kisspeptin analog, called C6, which offers pharmacological advantages over endogenous kisspeptin. These include a longer lasting effect and enhanced activity following intramuscular injection. In the present work, we evaluated C6 effect on LH and FSH plasma concentrations in the Alpine goat breed and tested whether C6 could replace PMSG to trigger ovulation. An intramuscular injection of C6 (15 nmol/doe) given 24 hours after the end of progestogen treatment induced a surge-like peak of both LH and FSH. This was followed by an increase of progesterone, a hallmark of ovulation induction and corpus luteus formation. These results were obtained at three different time of the year: during the breeding season, the non-breeding season and at the onset of the breeding season. Furthermore, we compared the efficacy of C6 and PMSG to induce fertile ovulations when these treatments are given at the onset of the breeding season and are followed by artificial insemination. The results of this first attempt were extremely promising with gestation rates of 45% and 64% for C6 and PMSG respectively. Pending optimization of the treatment procedure in order to improve efficacy, kisspeptin analogs could be the long sought-after alternative to PMSG.

Photobiomodulation can improve ovarian activity in polycystic ovary syndrome-induced rats

Follicular cystic ovary disease is a common reproductive disorder in women and females of domestic animals, characterized by anovulation and the persistence of follicle is a common cause of reproductive failure in mammalian. Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism (HA), chronic anovulation and polycystic ovaries, and it is a common reproductive endocrine disease with clinical manifestations including hirsutism, acne, infertility and obesity that can affect 5–20% of women in their reproductive age. Photobiomodulation (PBM) has been investigated and used in clinical practice, related to biomodulatory influences on cellular functions in animals and humans, both in vivo and in vitro. In this study, we include endocrine and reproductive features in a rat model for PCOS and the effects of PBM on ovarian activities. Forty-five adult female Wistar rats PCOS-induced by a single dose of the estradiol valerate (EV) were used in the study. After the EV injection for PCO induction, rats were divided into 9 groups (n = 5/group) named C30, C45 and C60 (Control group), S30, S45 and S60 (PCO group) and L30, L45 and L60 (PCO/Laser group). The rats were irradiated with laser 3 times/week. The results shown that EV PCO-induced rats had increased body mass, reduced ovary mass, and reduced GSI. The plasma levels of P4 and T were increased, and the LH plasma level was decreased by PBM stimulation. The number of ovarian follicles and corpus luteum were increased, and the number of ovarian cysts was decreased by PBM stimulation. Thus, reproductive and endocrine characteristics were modulated by PBM.

Does kisspeptin participate in GABA-mediated modulation of GnRH and GnRH receptor biosynthesis in the hypothalamic-pituitary unit of follicular-phase ewes?

The inverse relationship between GnRH transcript level and GABA neurons activity has suggested that GABA at the hypothalamic level may exert a suppressive effect on subsequent steps of the GnRH biosynthesis. In the present study, we analyzed the effects of GABA type A receptor agonist (muscimol) or antagonist (bicuculline) on molecular mechanisms governing GnRH/LH secretion in follicular-phase sheep. ELISA technique was used to investigate the effects of muscimol and/or bicuculline on levels of post-translational products of genes encoding GnRH ligand and GnRH receptor (GnRHR) in the preoptic area (POA), anterior (AH) and ventromedial (VMH) hypothalamus, stalk/median eminence (SME), and GnRHR in the anterior pituitary (AP). Real-time PCR was chosen for determination of the effect of drugs on kisspeptin (Kiss 1) mRNA level in POA and VMH including arcuate nucleus (VMH/ARC), and on Kiss1 receptor (Kiss1r) mRNA abundance in POA-hypothalamic structures. These analyses were supplemented by RIA method for measurement of plasma LH concentration. The study demonstrated that muscimol and bicuculline significantly decreased or increased GnRH biosynthesis in all analyzed structures, respectively, and led to analogous changes in plasma LH concentration. Similar muscimol- and bicuculline-related alterations were observed in levels of GnRHR. However, the expression of Kiss 1 and Kiss1r mRNAs in selected POA-hypothalamic areas of either muscimol- and bicuculline-treated animals remained unaltered. Our data suggest that GABAergic neurotransmission is involved in the regulatory pathways of GnRH/GnRHR biosynthesis and then GnRH/LH release in follicular-phase sheep conceivably via indirect mechanisms that exclude involvement of Kiss 1 neurons.

Organic Zn and Cu interaction impact on sexual behaviour, semen characteristics, hormones and spermatozoal gene expression in bucks (Capra hircus)

The reproductive performance during the transition from prepubertal to pubertal stage was evaluated in Osmanabadi breed bucks supplemented with organic Zinc (Zn) and Copper (Cu). A total number of 40 bucks aged 20 weeks were randomly assigned to 10 groups (each n = 4). The control group was maintained with basal diet, without any additional mineral supplementation. The treatment groups were supplemented with graded doses of organic Zn (Zn 20 mg, Zn 40 mg and Zn 60 mg), Cu (Cu 12.5 mg, Cu 25 mg and Cu 37.5 mg) and a combination of Zn + Cu (Zn 20 mg + Cu 12.5 mg, Zn 40 mg + Cu 25 mg and Zn 60 mg + Cu 37.5 mg), respectively for a period of 26 weeks (up to the age of 46 weeks). Sexual behaviour and scrotal biometry were recorded periodically. Blood and semen samples were collected and processed for LH estimation in blood plasma, and testosterone, T3 and T4 hormones in the seminal plasma. The mounts with ejaculation were observed earlier (P < 0.05) in the treatment bucks (from 38th week of age) than the control group (43rd week onwards). A positive correlation was observed between blood plasma LH and testosterone with total mounts (r = 0.31, P < 0.05; r = 0.51, P < 0.01) and mounts without ejaculation (r = 0.40, P < 0.01; r = 0.52, P < 0.01). A negative correlation between T4 with sperm number per ejaculation (r = −0.31, P < 0.05) and sperm concentration (r = −0.35, P < 0.05) had been noticed. Different doses of minerals showed positive interaction (P < 0.05) with sperm functional and behavioural characteristics. The spermatozoal gene expression of ODF2 and ZCCHC6 were significantly influenced by the mineral supplementation in all doses. The ZCCHC6 gene expression was positively correlated with testosterone (r = 0.50, P < 0.001) and sperm number per ejaculation (r = 0.42, P < 0.001), and ODF2 gene with T3 hormone (r = 0.34, P < 0.05). The present study indicates that the diet supplemented with organic trace minerals cause intense sexual behaviour, enhancement in sperm number per ejaculate, total motility, spermatozoal genes expression and altered LH, testosterone and T4 hormones.