Luteinizing Hormone Amplitude Research Articles

Chemogenetic Activation of RFRP Neurons Reduces LH Pulse Frequency in Female but not Male Mice.

The neuropeptide RFRP-3 (RFamide-related peptide-3) is thought to play a role in the negative regulation of fertility. However, the exogenous administration of RFRP-3 yields varying results depending on the dose and route of administration, sex of the subject, and many other variables. Manipulation of in vivo neuronal activity using DREADDs (designer receptor exclusively activated by designer drugs) technology enables investigation of cell type-specific neuronal activation in a manner that better reflects endogenous neuronal activity. To test the effects of RFRP neuronal activation on pulsatile luteinizing hormone (LH) secretion. We generated mice expressing the stimulatory hM3Dq designer receptor exclusively in RFRP cells using 2 different Cre-loxP-mediated approaches: (1) we bred mice to express hM3Dq in all Rfrp-Cre-expressing cells, including some that transiently expressed Rfrp-Cre neonatally (RFRP × hM3Dq mice), and (2) we stereotaxically injected Cre-dependent hM3Dq into the dorsomedial nucleus of RFRP-Cre mice to drive hM3Dq expression exclusively in a subpopulation of adult Rfrp-Cre neurons (RFRP-AAV-hM3Dq mice). We then investigated the effects of acute hM3Dq activation on LH pulse frequency in RFRP × hM3Dq mice, RFRP-AAV-hM3Dq mice, and their respective controls. In both female RFRP × hM3Dq and RFRP-AAV-hM3Dq mice, chemogenetic activation of Cre-driven hM3Dq led to a significant 35% to 50% reduction in LH pulse frequency compared with controls, while no differences in pulse amplitude or mean LH concentration were observed. In marked contrast, RFRP activation did not cause any changes to LH pulse dynamics in male mice. These data show for the first time that activation of neurons that have expressed Rfrp, or of a subset of adult RFRP neurons, can independently suppress LH pulsatility in female, but not male mice.

FRI280 Partial Loss Of KNDy Neurons In Prenatally Androgen Treated Female Rats Alters The LH Secretion And Ovarian Morphology In A Model Of Polycystic Ovary Syndrome

Abstract Disclosure: N.S. Aquino: None. A. Campideli-Santana: None. L.M. Antunes: None. R. Araújo-Lopes: None. K.S. Silva: None. P.C. Henriques: None. D.D. Gusmão: None. M.P. Bernuci: None. R.E. Szawka: None. A.M. dos Reis: None. Polycystic Ovary Syndrome (PCOS) is a high-prevalent endocrine dysfunction, causing metabolic disorders and infertility. Women with PCOS show a disrupted hypothalamus-pituitary-gonadal (HPG) axis, which results in increased luteinizing hormone (LH) secretion, LH pulse frequency, and LH/follicle stimulating hormone (FSH) ratio. These changes are related to an impaired negative-feedback effect of ovarian steroids, estradiol and progesterone, on the gonadotrophin releasing-hormone (GnRH) neurons. Because the kisspeptin/neurokinin-B/dynorphin (KNDy) neurons of the arcuate (ARC) nucleus are responsible for mediating the ovarian steroid negative feedback, we investigated whether the ablation of KNDy neurons would change the LH secretion and ovarian morphology in a rat model of PCOS. Pregnant dams were treated with dihydrotestosterone (DHT, 3 mg/day; s.c) from days 16 to 19 of pregnancy. Three months after birth, the prenatally androgen-treated (PNA) offspring received intra-ARC stereotaxic injections of the neurokinin-3 receptor agonist conjugated with saporin (NK3-SAP) to induce the lesion of KNDy neurons and were submitted to serial blood sampling for LH measurement. PNA NK3-SAP rats had a moderate loss of ARC neurokinin-B-immunoreactive neurons (between 50-70%) compared with the Blank-SAP-injected PNA group. The PNA Blank-SAP rats displayed LH pulse frequency, pulse amplitude, and mean LH levels similar to control rats on diestrus. Remarkably, the partial loss of KNDy neurons increased the LH pulse amplitude and mean LH without changing the pulse frequency. Histological analysis was performed to investigate the outcome of the partial loss of KNDy neurons on the ovarian morphology in PNA rats. The numbers of primordial, primary, and secondary health follicles were decreased in PNA Blank-SAP compared with diestrus rats. Notably, the number of primordial follicles was partially restored in NK3-SAP rats. On the other hand, neither the number of atretic follicles nor the number of corpora lutea was changed in PNA Blank or NK3-SAP rats, indicating no change in the ovulatory function. Therefore, we provide evidence that KNDy neurons negatively modulate LH release and LH pulse amplitude in PNA rats. Moreover, the results suggest that KNDy neurons are involved in the reduction of the primordial follicle pool and ovarian reserve in PNA rats. Financial Support: Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Brazilian National Council for Scientific and Technological Development (CNPq), British Society for Neuroendocrinology (BSN). Presentation: Friday, June 16, 2023

FRI279 Submaximal Loss Of KNDy Neurons Accelerates And Amplifies Pulsatile LH Secretion In Female Rats

Abstract Disclosure: A.C. Campideli-Santana: None. K.S. da Costa Silva: None. R. Araújo-Lopes: None. L.M. Antunes: None. R.E. Szawka: None. Kisspeptin is a powerful stimulator neuropeptide in the hypothalamus-pituitary-gonadal axis and is essential for fertility. Neurons in the arcuate nucleus (ARC) of the hypothalamus that coexpress kisspeptin, neurokinin B, and dynorphin (KNDy) play a crucial role in the control of luteinizing hormone (LH) secretion, but the neuroendocrine mechanisms involved in this regulation are not fully understood. Our previous study demonstrated that the moderate loss of KNDy neurons amplifies the LH surge induced by estradiol in ovariectomized rats (1). The current study aimed to perform a longitudinal evaluation of female rats in which KNDy neurons were ablated to determine the integrated role of this neuronal population in the control of pulsatile LH secretion. Adult female rats underwent a neurochemical ablation of KNDy neurons via intra-ARC stereotaxic injections of neurokinin-3 receptor agonist conjugated with saporin (NK3-SAP), while control animals received blank-saporin (Control). Estrous cyclicity was monitored over 21 days before and after the stereotaxic surgery. Three weeks after surgery, blood samples (10 µL) were withdrawn from the tail tip at 6-minute intervals for 180 minutes on the day of diestrus. Rats were then ovariectomized and the ovaries were processed for histological analysis. Whole blood LH levels were measured by ELISA and brains were immunohistochemically labeled for neurokinin B in the ARC. NK3-SAP rats bearing a submaximal loss of KNDy neurons (between 55-95%; n = 8) displayed irregular estrous cycles. The ovarian weight and the number of corpora lutea and atretic follicles were unaltered, whereas the number of healthy early antral follicles decreased in NK3-SAP rats. The frequency of LH pulses was increased and both pulse amplitude and mean LH levels were higher in NK3-SAP compared with Control rats. Thus, the submaximal loss of KNDy neurons affects the regularity of estrous cyclicity but does not preclude ovulation, while seems to stimulate the maturation of early antral follicles. Notably, KNDy neuron loss accelerates and amplifies LH pulsatile secretion during the rat estrous cycle. These results suggest a new role for KNDy neurons in restraining the frequency and amplitude of LH pulses in ovary-intact animals, with possible implications in the rate of recruitment of early antral follicles. Reference: Campideli-Santana et al., J Neuroendocrinol. 2022 Nov;34(11):e13204. Support: FAPEMIG, CNPq, CAPES, and Pro-reitoria de Pesquisa-UFMG. Presentation: Friday, June 16, 2023

P-684 Subclinical hypothyroidism and ovarian reserve indices in women with normogonadotropic anovulation

Abstract Study question Does subclinical hypothyroidism (SCH) affect ovarian reserve indices in women with Polycystic Ovary syndrome (PCOS) or other forms of Hypothalamic-Pituitary-Ovarian Axis Dysfunction (HPOD)? Summary answer SCH increases Anti-Müllerian hormone (AMH) concentration, but has no influence on Follicle-stimulating hormone (FSH). What is known already In PCOS, anovulation is caused by the arrest of growing follicles due to increased frequency and amplitude of luteinizing hormone (LH) pulses, resulting in increased number of pre-antral and antral follicles, and concentration of AMH. In other cases of HPOD, the causes of anovulation are more diverse and have not yet been clearly identified. Thyroid hormones can modify the function of the HPO axis, affecting the maturation of follicles. While overt hypothyroidism is treated to improve ovulation and fertility, the effect of subclinical hypothyroidism (SCH) and the presence of circulating antithyroid antibodies (ATA) on ovarian function is uncertain. Study design, size, duration A prospective cohort tertiary single-center study (consent no. 1072.6120.172.2022) included women aged 18-45, examined due to menstrual disorders and/or infertility, from July to August 2022. Only women without previously diagnosed thyroid dysfunction were included. All enrolled women gave informed written consent to participate in the study. Participants/materials, setting, methods Women underwent routine gynecological examination and pelvic ultrasound. Blood sample was tested for AMH, FSH, Luteinizing hormone (LH), Thyroid-stimulating hormone (TSH), anti-thyroid peroxidase (anti-TPO) and anti-thyroglobulin (anti-TG) antibodies concentrations. The Rotterdam-ESHRE-ASRM criteria were used to diagnose PCOS and its phenotypes. HPOD was diagnosed according to WHO classification. SCH was defined as TSH > 2.5 uIU/ml with normal thyroid function. The above parameters within subpopulations were calculated using regression analysis, Kruskal-Wallis and post-hoc tests. Main results and the role of chance The study included 51 euthyroid women aged 18-40. PCOS was diagnosed in 42/51 (82.35%) women, including PCOS-A phenotype in 31/51 (60.78%), PCOS-B in 5/51 (9.8%), PCOS-D in 6/51 (11.76%) women, and HPOD in 9/51 (17.65%) women. There was a significant positive correlation between the concentration of TSH and AMH in the studied population - with the increase of TSH value, the concentration of AMH increased (r = 0.4, p = 0.0035). The mean AMH concentration significantly differed between the groups and equaled to 55.4, 39.4, 31.1, 24.0 pmol/l in PCOS-A, PCOS-B, PCOS-D and HPOD (p = 0.05), respectively. The mean anti-TPO concentration was significantly different between the groups and was equal to 18.2, 19.0, 14.6, 10.9 U/ml in PCOS-A, PCOS-B, PCOS-D and HPOD (p = 0.05), respectively. There was no significant difference in FSH, LH, TSH, anti-TG concentrations between the groups. Limitations, reasons for caution The limitations of the study are small study group and single-center nature. Wider implications of the findings SCH increases the concentration of AMH, which in PCOS may exacerbate the symptoms of anovulation. Whether treatment of subclinical hypothyroidism affects ovarian reserve indices and improves ovarian function remain a subject of further research. Trial registration number 1072.6120.172.2022

Effects of time-restricted feeding on letrozole-induced mouse model of polycystic ovary syndrome

The present study aimed to investigate whether time-restricted feeding (TRF) ameliorates metabolic and reproductive phenotypes in a letrozole-induced mouse model of polycystic ovary syndrome (PCOS). Sixty female C57BL/6 N mice were randomly divided into two groups according to the type of food received: either a chow or a 60% high-fat diet. Those mice were subcutaneously implanted with letrozole or placebo pellets at four weeks of age. Then, letrozole-treated mice were randomly assigned to different feeding regimens: (1) TRF for 4 h (ZT12–ZT16) or (2) ad libitum diet. After 4 weeks of dietary intervention, estrous cycles were determined with daily vaginal smear examination, and serial tail-tip blood sampling was performed at 5-min intervals for 2 h to measure the luteinizing hormone (LH) pulse frequency, amplitude, and mean LH levels in the diestrus cycle stage. Letrozole-treated mice in the ad libitum group demonstrated multiple PCOS-like phenotypes including ovulatory dysfunction, polycystic ovaries, and increased body weight, parametrial fat weight, adipocyte size and inflammation, and higher expression of Cyp17, Cyp19, and Fshr in the ovary, and Kiss1r and Gnrh in the hypothalamus, elevated serum testosterone levels, and more rapid and elevated LH pulsatility, with increased pulse frequency, amplitude, and mean levels in the diestrus stage, compared with the controls. After TRF for 4 weeks, those phenotypes reverted to normal levels in letrozole-treated mice, except the percentage of diestrus cycles indicating the arrest of estrous cycling which did not differ between the TRF and ad libitum groups. Our results demonstrate that TRF has therapeutic effects on the reproductive and metabolic phenotypes of a letrozole-induced mouse model of PCOS.

Characterization of the Action of Tachykinin Signaling on Pulsatile LH Secretion in Male Mice.

The alternation of the stimulatory action of the tachykinin neurokinin B (NKB) and the inhibitory action of dynorphin within arcuate (ARH) Kiss1 neurons has been proposed as the mechanism behind the generation of gonadotropin-releasing hormone (GnRH) pulses through the pulsatile release of kisspeptin. However, we have recently documented that GnRH pulses still exist in gonadectomized mice in the absence of tachykinin signaling. Here, we document an increase in basal frequency and amplitude of luteinizing hormone (LH) pulses in intact male mice deficient in substance P, neurokinin A (NKA) signaling (Tac1KO), and NKB signaling (Tac2KO and Tacr3KO). Moreover, we offer evidence that a single bolus of the NKB receptor agonist senktide to gonad-intact wild-type males increases the basal release of LH without changing its frequency. Altogether, these data support the dispensable role of the individual tachykinin systems in the generation of LH pulses. Moreover, the increased activity of the GnRH pulse generator in intact KO male mice suggests the existence of compensation by additional mechanisms in the generation of kisspeptin/GnRH pulses.

Androgen Suppresses In Vivo and In Vitro LH Pulse Secretion and Neural Kiss1 and Tac2 Gene Expression in Female Mice.

Androgens can affect the reproductive axis of both sexes. In healthy women, as in men, elevated exogenous androgens decrease gonad function and lower gonadotropin levels; such circumstances occur with anabolic steroid abuse or in transgender men (genetic XX individuals) taking androgen supplements. The neuroendocrine mechanisms by which endogenous or exogenous androgens regulate gonadotropin release, including aspects of pulsatile luteinizing hormone (LH) secretion, remain unknown. Because animal models are valuable for interrogating neural and pituitary mechanisms, we studied effects of androgens in the normal male physiological range on in vivo LH secretion parameters in female mice and in vitro LH secretion patterns from isolated female pituitaries. We also assessed androgen effects on hypothalamic and gonadotrope gene expression in female mice, which may contribute to altered LH secretion profiles. We used a nonaromatizable androgen, dihydrotestosterone (DHT), to isolate effects occurring specifically via androgen receptor (AR) signaling. Compared with control females, DHT-treated females exhibited markedly reduced in vivo LH pulsatility, with decreases in pulse frequency, amplitude, peak, and basal LH levels. Correlating with reduced LH pulsatility, DHT-treated females also exhibited suppressed arcuate nucleus Kiss1 and Tac2 expression. Separate from these neural effects, we determined in vitro that the female pituitary is directly inhibited by AR signaling, resulting in lower basal LH levels and reduced LH secretory responses to gonadotropin-releasing hormone pulses, along with lower gonadotropin gene expression. Thus, in normal adult females, male levels of androgen acting via AR can strongly inhibit the reproductive axis at both the neural and pituitary levels.

High-Fat Diet Alters LH Secretion and Pulse Frequency in Female Mice in an Estrous Cycle-Dependent Manner.

Reproductive fitness in females is susceptible to obesogenic diets. Energy balance and reproduction are tightly regulated, in part, by hypothalamic neurons in the arcuate nucleus (ARC), and high-fat diet (HFD) can steadily increase estradiol levels in rodents. Estradiol regulates the reproductive axis via negative feedback mechanisms in ARC neurons by modulating pulsatile release of the gonadotropin luteinizing hormone (LH). However, it is unclear how the circulating estradiol milieu of adult females interacts with a state of high-caloric fat intake to alter LH pulse dynamics. Here, we used serial tail-tip blood sampling to measure pulsatile LH release at different estrous cycle stages in mice fed a HFD. Starting at 21 days of age, female C57BL/6J mice were freely fed with either regular chow diet (RD) or 60% kcal HFD for 12 weeks. Blood samples were collected once at diestrus, and then again at estrus. LH was measured in 10-minute intervals for 3 hours and analyzed for pulse frequency, amplitude, and mean and basal LH levels. Compared with RD-fed controls, mice fed HFD displayed significantly increased pulse frequency at diestrus, but not at estrus. HFD-fed mice also had lower mean and basal LH levels compared with RD-fed controls, but only during estrus. These data suggest that circulating estradiol can variably contribute to the impact that HFD has on LH pulsatile release and also provide insight into how obesity impacts women's reproductive health when ovarian estradiol levels drastically change, such as during menopause or with hormone replacement therapy.

Does the repeat dose of gonadotropin-releasing hormone agonist trigger in polycystic ovarian syndrome improve in vitro fertilization cycles outcome? A clinical trial study.

Background A repeat dose of Gonadotropin-releasing Hormone (GnRH) agonist could provide long duration of luteinizing hormone (LH) surge and amplitude appropriately.Objective Improvement in oocyte maturity could be obtained by a repeat dose of GnRH agonist.Materials and MethodsIn this randomized double-blinded study, 120 women with polycystic ovarian syndrome and serum estradiol level (E2) 3000 who were candidate for in vitro fertilization with Antagonist protocol were enrolled between July 2018 and July 2019. Participants were randomized in two groups - and final oocyte maturation was triggered with two doses: In group A, a repeat dose of 0.1 mg, 12 hr. after the first dose and in group B, 0.2 mg SC triptorelin (decapeptyl) 35 hr. prior to oocyte retrieval. Serum Estradiol, LH, and progesterone concentration were measured on the trigger day. Serum LH measurement was done three times in both groups. The outcomes were oocyte yield, meiosis (M) I, MII, Maturity rate, germinal vesicle (GV) rate, 2 pronuclear, embryo yield, ovarian hyper stimulation syndrome rates.ResultsMaturity rate (p = 0.89), MI (p = 0.38), MII (p = 0.89), and GV oocytes (p = 0.38) were not statistically different between the two study groups. LH levels measured at 12 hr post-trigger did not relate statistically significant with maturity rate in our participants (p = 0.96). No empty follicular syndrome was reported.ConclusionAlthough, the second dose of GnRH agonist after 12 hr since the first dose could provide duration of LH surge and amplitude and as a result no empty follicular syndrome was seen, the maturity rate, MI, MII, and GV oocytes were not different between the two study groups.

Estradiol Potentiates But Is Not Essential for Prolactin-Induced Suppression of Luteinizing Hormone Pulses in Female Rats.

Hyperprolactinemia causes infertility by suppressing gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion. Because effects of prolactin (PRL) on the hypothalamus usually require estradiol (E2), we investigated the role of E2 in PRL-induced suppression of LH pulses. Ovariectomized (OVX) rats treated with oil or E2 (OVX + E2) received a subcutaneous injection of ovine PRL (oPRL) 30 minutes before serial measurement of LH in the tail blood by enzyme-linked immunosorbent assay. E2 reduced pulsatile LH secretion. oPRL at 1.5 mg/kg further reduced LH pulse frequency in OVX + E2 but had no effect in OVX rats. The higher dose of 6-mg/kg oPRL decreased LH pulse frequency in both OVX and OVX + E2 rats, whereas pulse amplitude and mean LH levels were lowered only in OVX + E2 rats. Kisspeptin immunoreactivity and Kiss1 messenger ribonucleic acid (mRNA) levels were decreased in the arcuate nucleus (ARC) of OVX + E2 rats. oPRL decreased both kisspeptin peptide and gene expression in the ARC of OVX rats but did not alter the already low levels in OVX + E2 rats. In the anteroventral periventricular nucleus, oPRL did not change kisspeptin immunoreactivity and, paradoxically, increased Kiss1 mRNA only in OVX + E2 rats. Moreover, oPRL effectively reduced Gnrh expression regardless of E2 treatment. In this study we used tail-tip blood sampling to determine the acute effect of PRL on LH pulsatility in female rats. Our findings characterize the role of E2 in the PRL modulation of hypothalamic components of the gonadal axis and LH release, demonstrating that E2 potentiates but is not essential for the suppression of pulsatile LH secretion caused by hyperprolactinemia.

Individual evaluation of luteinizing hormone in aged C57BL/6J female mice.

In female mammals, reproductive senescence is a complex process involving progressive ovarian dysfunction associated with an altered central control of the hypothalamic-pituitary axis. The objective of this study was to compare the longitudinal change in preovulatory luteinizing hormone (LH) secretion as well as estrous cycle in individual C57BL/6J female mice at 3, 6, 9 and 12months. Amplitude and timing of LH secretion at the surge were similar from 3 to 9months but were altered in 12-month old mice with a significant decrease of more than 50% of peak LH value and a 2h delay in the occurrence of the LH surge as compared to younger mice. The analysis of two to three successive LH surges at 3, 6, 9 and 12months showed low and similar intra-individual variability at all ages. The estrous cycle length and intra/inter variability were stable over the age. This study shows that female mice in regular environmental conditions display stable LH surge timing and amplitude up to 9months, but at 12months, the LH surge is delayed with a reduced amplitude, however without overt modification in the estrous cycles. Analysis of individual preovulatory LH secretion and estrous cycle indicates that mice can be followed up to 9months to investigate the detrimental effects of various parameters on mouse reproductive activity.

GnRH-1, GnIH mRNA and Luteinizing Hormone in Domestic hens (Gallus gallus domesticus) Exposed to Different Wavelengths of light

The objective of this study was to establish the effects of red spectrum of light (650nm, treated n=12) and normal spectrum of light (450nm control=12) on GnRH-I and GnIH mRNA expression, amplitude and frequency of luteinizing hormone (LH) and egg production from 42 to 52 weeks of age in white leghorn hens. Blood samples were collected at weekly interval from both the groups. At the 47th week of age blood samples from both the groups were collected at every 3 h for 36h to study the pulsatile secretion of LH surges. GnRH and GnIH mRNA expression pattern was studied between control and treated birds. Egg production and pause days were calculated between the two groups. LH concentration in the plasma was increased significantly (P<0.01) in hens exposed to red spectrum of light. Plasma LH concentration was higher (P<0.01) in treated birds with more number of LH surges. The amplitude and frequencies of LH were advanced in birds exposed to red spectrum of light during 36 h of sampling at 3h intervals. GnRH-I mRNA concentration was significantly (P<0.01) higher, whereas GnIH mRNA was significantly (P<0.01) lower in birds exposed to red spectrum of light compared to controls. It is hypothesized that exposure of birds to red spectrum of light enhanced (P<0.01) GnRH-I mRNA, along with LH required for ovulation and egg lay. During 77 days (42-52 weeks of age) of the experimental period, egg production was increased (p<0.01) with lower incidence of pause days in the treated group. It is concluded that low GnIH mRNA and higher levels of GnRH-I mRNA, LH, lower number of pause days enabled the birds to lay more eggs by stimulating GnRH through red spectrum of light.

GnRH-1 mRNA, LH surges, steroid hormones, egg production, and intersequence pause days alter in birds exposed to longer wavelength of light in the later stages of production in Gallus gallus domesticus

The objective of this was to establish the effects of red spectrum of light (650 nm, treated n = 12) and normal spectrum of light (450 nm control = 12) on GnRH-I mRNA expression, amplitude and frequency of luteinizing hormone (LH), and egg production from 72-82 weeks of age in white leghorn hens. Birds exposed to red spectrum of wavelength significantly improved (P < 0.01) steroid hormone, and egg production improved over old laying 72 to 82 weeks. Weekly interval profiles followed the same pattern. At 77th weeks of age blood, samples from both the groups were collected at every 3 h for 36 h to study the pulsatile secretion of LH surges. Plasma LH concentration was higher (P < 0.01) in treated birds with more number of frequencies and amplitude LH surges in plasma of treated birds. LH frequencies were more pronounced and advanced during 36 h of sampling at 3 h interval in treated birds. Weekly interval of plasma LH, E2β, and P(4) concentrations increased (P < 0.01) in treated birds from 72 to 82 weeks of age. GnRH-I mRNA concentration was significantly (P < 0.01) higher in birds exposed to red spectrum of light compared to controls. It is hypothesized that exposure of birds to red spectrum of light-enhanced (P < 0.01) GnRH-I mRNA with more number of yellow yolky follicles was found in birds exposed to red spectrum of light during 77 days (72-82 weeks of age) of experimental period. It is concluded that higher levels of GnRH-I mRNA, LH, E2β, and P(4) concentration with lower incidence of pause days enabled the birds to lay more eggs even later in the productive period by modulating the wavelengths of light under normal husbandry conditions.

Adiposity and sex hormones across the menstrual cycle: the BioCycle Study

ObjectiveTo investigate the influence of adiposity on patterns of sex hormones across the menstrual cycle among regularly menstruating women.SubjectsThe BioCycle Study followed 239 healthy women for 1–2 menstrual cycles, with up to 8 visits per cycle timed using fertility monitors.MethodsSerum estradiol (E2), progesterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and sex hormone binding globulin (SHBG) were measured at each visit. Adiposity was measured by anthropometry and by dual energy x-ray absorptiometry. Differences in hormonal patterns by adiposity measures were estimated using nonlinear mixed models, which allow for comparisons in overall mean levels, amplitude (i.e. lowest to highest level within each cycle), and shifts in timing of peaks while adjusting for age, race, energy intake, and physical activity.ResultsCompared to normal weight women (n=154), obese women (BMI≥30 kg/m2, n=25) averaged lower levels of progesterone (−15%, P=0.003), LH (−17%, P=0.01), FSH (−23%, P=0.001) and higher free E2 (+22%, p=0.001) across the cycle. To lesser magnitudes, overweight women (BMI: 25–30, n=60) also exhibited differences in the same directions for mean levels of free E2, FSH, and LH. Obese women experienced greater changes in amplitude of LH (9%, p=0.002), and FSH (8%, p=0.004), but no differences were observed among overweight women. Higher central adiposity by top compared to bottom tertile of trunk-to-leg fat ratio by DXA was associated with lower total E2 (−14%, p=0.005) and FSH (−15%, p=0.001). Peaks in FSH and LH occurred later (~0.5 day) in the cycle among women with greater central adiposity.ConclusionGreater total and central adiposity were associated with changes in mean hormone levels. The greater amplitudes observed among obese women suggest compensatory mechanisms at work to maintain hormonal homeostasis. Central adiposity may be more important in influencing timing of hormonal peaks than total adiposity.

EL ESTÍMULO DEL MACHO CABRÍO INCREMENTA LA FUNCIÓN REPRODUCTIVA DE LAS CABRAS CRIOLLAS DEL SEMIDESIERTO MEXICANO INDEPENDIENTEMENTE DEL RÉGIMEN DE FOTOPERIODO

Influence of a sexually-active male buck was evaluated on the onset of reproductive function (hypothalamic and ovarian activity) and estrous cycles progression in Criollo goats from the northen Mexican desert, exposed to an alternated (decreasing/increasing) artificially controlled photoperiod fluctuating within 13.4 to 10.6 light hours per day until fulfilling 6 photoperiodic cycles of 90 d each cycle: ascending (n=3); and descending (n=3). Mexican-native Criollo goats (n=30) were randomly assigned into 2 treatment groups: 1) goats exposed to a sexually-active male buck (n=15); 2) goats not exposed to a male buck (absence of a male buck; n=15). Each experimental group of goats included ovariectomized goats (OVX, n=5), ovariectomized and estradiol-implanted goats (OVX + E2, n=5), and intact-ovaries goats (Control, n=5). Blood samples were taken from OVX and OVX + E2 goats, every four weeks, during 6 h at 15 min intervals (i.e., 24 samples/day), to determine frequency (FREQ), amplitude (AMP), and concentration (CONC) of luteinizing hormone (LH). For Control goats, blood samples were taken twice every week in order to quantify serum-progesterone levels through radioimmunoanalysis (RIA). Goats implanted with E2 (OVX + E2-goats) showed an increased LH pulse frequency compared to OVX-goats without an E2 implant (2.0 ± 0.5 vs. 0.7 ± 0.1 LH pulses/ 6 h). Presence of a sexually-active male buck increased frequency, amplitude and concentration of LH in OVX goats compared to goats not exposed to males (Frequency: 3.2 ± 0.4 vs. 0.7 ± 0.1 pulses/6 h; Amplitude: 1.6 ± 0.1 vs. 0.8 ± 0.3 ng•mL-1; Concentration: 5.3 ± 0.6 vs. 2.0 ± 0.9 ng•mL-1) (P0.05). In conclusion, presence of a sexually-active male buck induced a greater ovarian activity in Criollo goats, shortening seasonal anestrous even during an ascendent controlled-photoperiod. Such strategy, using a sexually-active male buck might be helpful to increase ovarian activity and reproductive function during the seasonal anestrous of Criollo goats from dry and arid areas of northern Mexico and similar regions of the world.

Influence of ovarian hormones on endocrine activity of gonadotroph cells in the adenohypophysis of lambs during the postnatal transition to prepuberty

There is juvenile hiatus during maturation of larger mammals with relatively long life spans. Using histomorphological and functional criteria we describe the feedback mechanisms which could play a role in the regulation of the gonadotrophic axis during the postnatal transition to the quiescent prepubertal period in sheep. The aim of this study was to determine the influence of ovarian factors on the endocrine activity of gonadotroph cells, the site of synthesis, storage and release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), in adenohypophyses of weanling and weaned prepubertal lambs. The examination was made in (i) 9-week-old infantiles, suckling lambs undergoing weaning, ovary-intact (OVI) and ovariectomised (OVX) at the 6th week of age, and (ii) 16-week-old juveniles OVI and OVX at the 12th week of age ( n = 5 per group). Changes in gonadotrophs were assayed with hybridohistochemistry, immunohistochemistry and radioimmunoassay. The percentage of the adenohypophyseal area (PA) occupied by gonadotrophs containing LHβ-mRNA and immunoreactive for LHβ was lower ( P < 0.05), whereas the PA occupied by cells containing FSHβ-mRNA and immunoreactive for FSHβ was higher ( P < 0.05) in the 16-week-old OVI lambs in comparison with the 9-week-old ones. The mean concentration and basal level of LH in the peripheral blood plasma were greater ( P < 0.05) in the 16-week-old OVI lambs in comparison with the 9-week-old group, whereas the circulating FSH was not different. In the OVX 9-week-old lambs, the PA occupied by gonadotrophs containing LHβ-mRNA and the plasma LH concentration, basal level, pulse frequency and amplitude were greater ( P < 0.05), whereas the PA occupied by cells immunoreactive for LHβ was lower ( P < 0.05) in comparison with the OVI group. In the OVX 16-week-old lambs, the PA occupied by gonadotrophs containing LHβ-mRNA and immunoreactive for LHβ, the LH plasma concentration, basal level and pulse frequency were ( P < 0.05) greater in comparison with the OVI group. The PA occupied by gonadotrophs containing FSHβ-mRNA and the plasma FSH concentration were greater ( P < 0.05) in the OVX 9- and 16-week-old lambs in comparison with the OVI ones. The ovariectomy had no effect on the PA occupied by cells immunoreactive for FSHβ in both age stages. In conclusion, ovarian factors may play inhibitory role in regulating both LH and FSH synthesis rate and release and stimulatory role in regulating LH storage in adenohypophyseal gonadotrophs in infantile lambs. In lambs at the beginning of the juvenile period, ovarian factors may play only inhibitory role in regulating both LH and FSH synthesis and release and LH storage. The effects of ovarian hormones on the gonadotrophin storage, i.e. releasable pools in adenohypophyseal cells, are specific for LH, no such effects are apparent on FSH in lambs during the postnatal transition to prepuberty. Thus, the initiation of the juvenile period in female sheep is a function of change of the stimulatory role of ovarian hormones in regulating LH storage to the inhibitory one.

18 EFFECT OF GnRH TREATMENT IN LH SECRETION IN INTACT AND OVARIECTOMIZED NELLORE HEIFERS

The gonadotropin-releasing hormone (GnRH) and its analogues become control reproductive function in farm animals. The GnRH agonist (GnRHa), which is used in veterinary practice, has a longer half-life and higher affinity to GnRH receptors than the native decapeptide. GnRH or GnRHa effects depend on the species, dose, route, and the moment of administration. As Nellore is the main bovine breed in Brazil, corresponding to 70% of Brazilian herds, more studies on its reproductive physiology are needed. The objective was to study luteinizing hormone (LH) secretion in intact (I, n = 4) and ovariectomized (Ov, n = 4) prepubertal Nellore heifers (18 months) after gonadorelin acetate administration (25 μg, iv; Lecirelina, Gestran Plus, ARSA S.R.L., Buenos Aires, Argentina) from samples collected every 15 min for 10 h. LH was quantified by RIA, with sensitivity of 0.03 ng mL-1 and CV of 11%. Maximum LH amplitude was considered as the peak for each period. Peaks were identified as an increase 2 times higher than intraassay CV and involving at least 3 samples. The time for the highest peak occurrence was determined by GraphPad Prism program (Prism 3.00 for Windows, GraphPad Software, San Diego CA, USA). Results were compared by the ANOVA followed by t-test either paired or not using GraphPad InStat (3.06 version). After GnRH treatment, maximum LH peak amplitude was higher in Ov (12.48 ± 1.41 ng mL-1) (P ≤ 0.05) than in intact heifers (6.42 ± 1.30 ng mL-1), but there was no significant difference (P ≥ 0.05) on LH pulse frequency, with only one peak in both groups during the 10-h period. Although there was a small delay in peak occurrence in I (63.75 ± 21.48 min) compared with Ov heifers (45 ± 16.43 min), there was no difference (P ≥ 0.05) between groups. The results suggested a higher hypothalamus sensitivity to GnRH in absence of gonadal steroids or a higher LH storage during the hypergonatropism period. Support by FAPESP (São Paulo, Brazil); fellowship from CAPES.

Duration of the reproductive period decreases the frequency of preovulatory luteinizing hormone surges in heavy weight-sire line turkey hens

The frequency of preovulatory luteinizing hormone (LH) surges is an important determinant of ovulation and oviposition rates in turkeys. Egg production rate is relatively poor in heavy weight-sire line type turkey hens and declines with advancing duration of the reproductive period. The purpose of this study was to measure frequency and characteristics of preovulatory LH surges in turkey hens of a heavy weight-sire line type early, at peak of egg production (Early), and late, after egg production rate had declined (Late), in a reproductive period. The Early hens were photostimulated with a continuous photoperiod [24 h light (L):0 h dark (D)] at 40 weeks of age and sampled during peak egg production at about 47 weeks of age. The Late hens were photostimulated at 40 weeks of age with a long day photoperiod (14L:10D). After a 27-week egg production period, the Late hens were switched to the 24L:0D photoperiod and sampled at 74 weeks of age. Continuous lighting was used during blood sampling to allow the rhythm of preovulatory LH surges to free run. All hens were cannulated 3–5 days before starting sampling and hourly blood samples were collected for 200 h. All hens were necropsied and ovarian and oviductal morphologies were measured after serial bleeding. The Late hens had a longer interval between intra-clutch preovulatory LH surges than the Early hens, and a higher incidence of atretic ovarian follicles. The Early hens had higher baseline and surge amplitude LH concentrations but lower progesterone (P 4) surge amplitude concentrations than the Late hens. The duration of preovulatory LH surges, incidence of “blind” preovulatory LH surges, baseline P 4 concentrations, and overall estradiol-17β (E 2) concentrations were not different between Early and Late hens. In conclusion, a longer interval between preovulatory LH surges, lower LH baseline and surge amplitude concentrations, a higher incidence of atretic follicles, and higher P 4 surge amplitude concentration were associated with the decline in egg production late in the reproductive period in a heavy weight-sire line of turkey hens.

Suppression of pulsatile luteinizing hormone secretion but not luteinizing hormone surge in leptin resistant obese Zucker rats.

The adipose tissue-derived hormone leptin may be a primary mediator linking nutritional status and reproduction. The present study used the leptin-resistant obese female Zucker rat to investigate whether leptin signalling is required for normal pulsatile luteinizing hormone (LH) secretion and/or generation of the LH surge. For the pulsatile LH secretion study, an indwelling atrial catheter was implanted and a low dose of oestrogen given as a subcutaneous implant to lean and obese ovariectomized (OVX) Zucker rats. One week following OVX, blood samples were collected every 10 min for 3 h during the morning. Plasma LH concentrations were measured by radioimmunoassay. For the LH surge study, lean and obese OVX rats were given a high dose of oestrogen as a subcutaneous implant. Two days later, rats were given progesterone at 09.00 h to induce a proestrus-like LH surge. Blood samples were collected from an indwelling atrial catheter throughout that and the following day and plasma LH concentrations were measured by radioimmunoassay. LH pulse amplitude and mean LH secretion were profoundly attenuated in obese Zucker rats compared with lean littermates, whereas LH pulse frequency was not significantly different between phenotypes. The opioid receptor antagonist naloxone did not affect the pattern of pulsatile LH secretion in obese rats, suggesting that leptin does not exert its facilitatory effects on LH secretion through an opioidergic pathway. Both lean and obese rats showed characteristic steroid-induced LH surges. It therefore appears that a leptin signal is required for generation of a normal pattern of pulsatile LH secretion, but is not a necessary component of the steroid-induced LH surge.

Decreased androgen levels in massively obese men may be associated with impaired function of the gonadostat.

In obese men, sex hormone-binding globulin (SHBG) as well as total testosterone (TT) levels are decreased. Data concerning serum free testosterone (FT) levels in obese men are discordant. FT levels are decreased in only some morbidly obese men, consistent with an impairment of the feedback regulatory mechanism. In this study we aimed to verify serum levels of TT and FT in two groups of obese men (BMI < 35.0 kg/m2 and BMI > 35.1 kg/m2) before and after weight loss. Two groups of obese men (group 1: BMI < or = 35 kg/m2; and group 2: BMI > or =35.1 kg/m2) were studied before and after 6 months of a low energy diet (1200 kcal/day). Every patient received a therapeutic prescription of dexfenfluramine (15 mg b.i.d.) that was maintained for 6 months. Thirty-seven obese men and 20 normal weight men. Serum sex hormones (TT and FT), serum luteinizing hormone (LH) and insulin were analyzed by RIA assays. Plasma insulin levels, serum TT, FT and LH concentrations were obtained before and after weight loss. Moderately obese men (BMI = 32.3+/- 1.9 kg/m2) presented significantly decreased TT levels (390+/-120ng/dl) as well as FT (mean+/-s.d.:16.0+/-4.8pg/ml) as compared with normal controls. FT serum levels had a significant and negative correlation with body mass index (BMI), whereas for TT concentrations this correlation was not significant. Serum LH concentrations (4.5+/-2.9mlU/ml) were normal. Insulin levels were elevated in all patients (46.3+/-30.1 microU/ml). After weight loss there was a significant (P< 0.01) increase in TT, FT and LH levels, whereas insulin concentrations significantly decreased. In massively obese men (BMI = 43.0 6.7 kg/m2), TT (320+/-110ng/dl), FT (11.0+/-2.1 pg/ml) and LH (3.1+/-1.3mlU/ml) were decreased and significantly lower as compared with the previous group and normal controls. As expected, after weight loss TT, FT and LH levels increased significantly while insulin concentrations decreased. We concluded that FT levels are dependent on the degree of obesity, massively obese men (BMI > or =35.1 kg/m2) being considered as candidates for consistently low FT levels. A functional decrease of LH pulse amplitude and serum LH levels as well as a possible negative action of excess of circulating leptin on the steroidogenesis may be related to the decreased androgens levels in massively obese men.