36 research outputs found
Neurokinin 3 receptor antagonism decreases gonadotropin and testosterone secretion in healthy men
Objective
Patients with mutations of NKB and its receptor show hypogonadotrophic hypogonadism, but there is little evidence for the importance of this pathway in reproductive function in normal men, or its functional hierarchy with kisspeptin.
Design
An open label study wherein men (n=6) were administered the NK3R antagonist MLE4901 40mg orally twice a day for 7 days. Kisspeptin-10 (0.3 μg/kg iv bolus) was given before and on day 7 of NK3R antagonist treatment.
Patients
Subjects were healthy men.
Measurements
Reproductive hormones were measured before and during the NK3R antagonist administration, including frequent sampling on two occasions for analysis of pulsatile LH secretion.
Results
LH, FSH and testosterone secretion were decreased during NK3R antagonist administration. LH showed a biphasic response, being reduced after 24 hours of treatment (4.5±0.6 IU/l pre-treatment to 1.7±0.2 IU/l, p<0.05), with partial recovery thereafter; but it was again decreased on day 7 (2.5±0.6 IU/l, p<0.05 vs pre-treatment). FSH secretion was also suppressed, with a similar temporal pattern to that of LH. Testosterone secretion was decreased from 24 hours (18.4±1.6 pre-treatment vs 5.6±1.5 nmol/l, p<0.01) and remained suppressed throughout the treatment period. Analysis of LH pulsatility showed that both basal and pulsatile LH secretion were markedly suppressed but there was no detected change in LH pulse frequency. Kisspeptin-10 stimulated LH secretion, with similar responses before and during NK3R antagonist administration.
Conclusions
These data demonstrate a central role for NKB/NK3R in the physiological regulation of reproductive function in men, and that this is functionally upstream of kisspeptin-mediated GnRH secretion
Neurokinin B regulates gonadotropin secretion, ovarian follicle growth and the timing of ovulation in healthy women
The opioid peptide beta-endorphin stimulates acrosome reaction in human spermatozoa
The acrosome reaction occurs in vivo following sperm capacitation and is essential for the acquisition of sperm fertilization ability. However, little is known about the molecular identity of the physiological acrosome reaction regulators. In addition to progesterone, which is produced by cumulus oophorus cells and known to regulate acrosome reaction by activating the specific calcium channel CatSper, endogenous opioid peptides such as beta-endorphin and met-enkephalin are present at high concentrations in the follicular fluid suggesting that the opioid system may be involved in the mechanisms regulating the acrosome reaction in humans. By using Reverse Transcription-PCR, western blot and immunofluorescence approaches, we described the presence and localization of the beta-endorphin precursor, pro-opiomelanocortinin the middle section and in flagellum of human spermatozoa, and inside the seminiferous tubules of human testis. Flow cytometry and intracellular calcium analyses showed that beta-endorphin causes an inversely dose-dependent increase in the percentage of acrosome-reacted sperm cells by a calcium-independent protein kinase C pathway. These findings are important for future studies of sperm physiology and provide new insight into the function of the opioid system as a target of fertility management.Peer Reviewe
Intra-follicular kisspeptin levels are related to oocyte maturation and gonadal hormones in patients who are undergoing assisted reproductive technology
Effects of veratridine on human sperm motility in the presence of tetrodotoxin, A-803467 or ab-66743.
<p>The effects of veratridine (10 μM) after different incubation times were analyzed in the presence of (A) the VGSC inhibitor tetrodotoxin (TTX) (10 nM) (B) TTX (10 μM), (C) the selective Na <sub>v</sub>1.8 antagonist A-803467 (10 μM), (D) the Na <sub>v</sub>1.8 antibody ab-66743 (dilution 1:50) or the corresponding solvent. Bars are means with SEM of 6-8 different experiments and represent percentage changes in progressive motility (grade A+B sperm) relative to the value observed at the same time in the respective solvent-treated paired controls. *<i>P</i><0.05, significant difference vs. responses to veratridine in the presence of the antagonist or antibody solvent.</p
Immunolocalization of the Nav1.8 protein in human sperm.
<p>(A) Flow cytometry plots of spermatozoa after overnight labeling with a Na <sub>v</sub>1.8 antibody (red plot) and the negative control treated with secondary antibody alone (black plot), <i>n</i> = 9. (B) Immunofluorescence images of sperm cells stained with a primary antibody against Na <sub>v</sub>1.8. Bar graph represents the distribution of this voltage-gated sodium channel in different sperm regions and the percentage of sperm cells showing each specific localization, <i>n</i> = 9. Scale bar, 10 μM. (C) Western blot analysis of Nav1.8 in human sperm homogenates using the Nav1.8 rabbit polyclonal antibody ab-66743. Molecular mass is indicated on the right side of the panel. The figure is representative of results obtained in 6 separate protein preparations from 6 different donors.</p
Effects of veratridine on intracellular free Ca<sup>2+</sup> ([Ca<sup>2+</sup>]<sub>i</sub>) and intracellular free Na<sup>+</sup> ([Na<sup>+</sup>]<sub>i</sub>) levels in human sperm cells.
<p>(A,B) For [Ca<sup>2+</sup>]<sub>i</sub> measurement, cells were loaded with Fura-2 and responses to veratridine (10 μM) were determined in the presence of (A) the Na <sub>v</sub>1.8 antibody ab-66743 (dilution 1:50) (red line) or its solvent (black line) or (B) in the presence of the Na <sub>v</sub>1.8 antagonist A-803467 (1 μM) (red line) or its solvent (black line). The X axis shows time in seconds and the Y axis shows [Ca<sup>2+</sup>]<sub>i</sub> data expressed by the F340/F380 ratio. Traces are representative of typical results obtained in 5-7 different experiments for each blocker. (C) For [Na<sup>+</sup>]<sub>i</sub> measurement, cells were loaded with SBFI and responses to veratridine (10 μM) were determined in the presence of A-803467 (1 μM) (red line) or its solvent (black line). The X axis shows time in seconds and the Y axis shows [Na<sup>+</sup>]<sub>i</sub> data expressed by the F340/F385 ratio. Traces are representative of typical results obtained in 6 different experiments.</p
Effects of veratridine on human sperm motility in Ca<sup>2+</sup>-containing and Ca<sup>2+</sup>-free mHTF solution.
<p>(A) Effects of veratridine (10 μM) on progressive motility (grade A+B sperm), non-progressive motility (grade C sperm) and immotility (grade D sperm) at different times of incubation in Ca<sup>2+</sup>-containing mHTF solution. Bars are means with SEM of 36 different experiments and represent percentage changes in motility in samples treated with veratridine relative to the value observed at the same time in solvent-treated paired controls. *<i>P</i><0.05, significant difference vs. control responses. (B) Time-dependent inactivation of sperm motility after incubation in a Ca<sup>2+</sup>-free mHTF solution. Afer capacitation for 2 h at 37°C in 5% CO<sub>2</sub>, the sperm suspension was divided in two aliquots, one of them incubated in Ca<sup>2+</sup>-containing solution and the other one in Ca<sup>2+</sup>-free solution. Data points are means with SEM of 7 different experiments and represent % motile sperm (grade A+B sperm). (C) Effects of veratridine (10 μM) at different times of incubation in Ca<sup>2+</sup>-containing and Ca<sup>2+</sup>-free mHTF solution. Bars are means with SEM of 7 different experiments and represent percentage changes in progressive motility (grade A+B sperm) relative to the value observed at the same time in solvent-treated paired controls.</p
Time-dependent effects of veratridine on sperm hyperactivation and acrosome reaction (AR).
<p>(A) Effect of veratridine (10 μM) and its solvent on hyperactivated sperm motility. Capacitated sperm were treated with veratridine or its solvent for different times and hyperactivation evaluated by computer-assisted sperm analysis (CASA). Bars are means with SEM of 7 different experiments and represent % hyperactivated sperm. (B) Effect of veratridine and A-23187 on AR. Capacitated sperm were treated with veratridine (10 μM) or A-23187 (10 μM) for different times and the acrosomal status was assessed by staining sperm with FITC-PSA. Bars are means with SEM of 5 different experiments and were calculated as: (%AR reacted spermatozoa in veratridine- or A23187-treated aliquots) – (%AR reacted spermatozoa in the corresponding solvent-treated aliquots at the same time). <sup>*</sup><i>P</i><0.05, significant difference vs. values at time =0.</p