Influence of embryonic conditioned media upon the endometrial beta-adrenergic receptor.

The main objective of this study was to evaluate the influence of the products secreted by the human embryo upon the three subtypes of beta-AR (beta1, beta2, beta3). Cell cultures were developed using endometrial biopsies, taken on day 7 after ovulation, from four healthy women <35 years of age, with regular cycles and infertility due only to male factors. Embryos from women with a normal uterine cavity and endometrial lining were incubated until they reached the 4-cell stage, before being transferred to their mother's uterus. Culture media for embryo incubation were derived from two groups: (i) embryos that achieved pregnancy, (ii) embryos which failed to implant. Control and experimental endometrial cell culture plates were treated with the two embryo culture media, with or without 10(-9) mol/l oestradiol and 10(-7) mol/l progesterone for 48 h. Expression of the three subtypes of beta-AR was assessed by RT-PCR. Beta1-AR was expressed in both control and experimental plates; beta2-AR was expressed only in plates incubated with embryonic culture media of embryos which achieved pregnancy, in both hormonal conditions, with or without oestradiol and progesterone. Beta3-AR was not expressed in any condition. Thus secretory products of human embryos may influence gene expression of beta2-AR concentrations in the human endometrium, and this subtype of beta-AR may be involved in implantation.

Vascular smooth muscle reactivity to norepinephrine in ovariectomized rats: relationship to vascular PGE2/PGF2 alpha ratio.

1. We have recently demonstrated that, in normal female rats, vascular reactivity (VR) and vascular prostaglandin-E2 (PGE2) and prostacyclin production are influenced by the ovarian cycle. 2. In this study, we investigated the vascular reactivity (VR) of isolated rat thoracic aorta to norepinephrine (NE 10(-12)-10(-6)M) in ovariectomized rats (OVX-rats), 48 hr and 8 days after surgical removal of the ovaries and in normally cycling rats (NR) at the proestrus stage of the estrous cycle, when the level of circulating estrogen was higher. 3. In addition, we determined the vascular synthesis of PGE2 and protaglandin-F2 alpha (PGF2 alpha) in both groups of OVX-rats, and in NR during the proestrous stage. 4. The results showed that VR to NE 10(-12)-10(-10) M was similar between OVX-rats and normal rats. 5. However, aortic rings obtained from both groups of OVX-rats showed a significant increase of the contraction response induced by NE 10(-9)-10(-6) M. 6. Furthermore, the contractile response to NE 10(-7)-10(-6) M was greater in the aortic rings from OVX-rats 8 days after ovariectomy compared to OVX-rats 48 hr (p < 0.001). 7. Vascular PGE2 and PGF2 alpha synthesis (ng/mg protein/hr) was significantly higher in both groups of OVX-rats than NR. But the vascular PGF2 alpha synthesis increased more than PGE2 in these OVX-rats, thus the ratio PGE2/PGF2 alpha was decreased significantly in both groups of OVX-rats (p < 0.01 and p < 0.001 OVX-rats 48 hr and 8 days after surgery, respectively) compared to NR-ratio.

Influence of the estrous cycle on the norepinephrine-induced contraction of rat aorta: relationship to vascular prostanoids biosynthesis.

Since ovarian sex steroids (estradiol and progesterone) may affect both blood pressure and prostanoids synthesis, and because prostaglandin-E2 (PGE2) and prostacyclin (PGI2) can modulate the vascular action of pressor hormones, we investigated the vascular reactivity to norepinephrine during the estrous cycle of the rat. In addition, we determined the vascular biosynthesis of PGE2 and 6-keto-PGF1 alpha (the stable metabolite of PGI2) at different stages of the estrous cycle. Cumulative dose-response curves were obtained by a stepwise increase in the concentration of norepinephrine. The contraction of thoracic aortic rings induced by norepinephrine did not change significantly between estrus, metestrus and diestrus. However, aortic rings obtained on proestrus showed a significant reduction in the maximal contraction (Emax) induced by norepinephrine (p < 0.001). In addition, we found significant increases in vascular synthesis of PGE2 and PGI2 on proestrus (p < 0.001). These results indicate that vascular reactivity and vascular prostanoids synthesis are influenced by the hormonal changes occurring during the estrous cycle of normal female rats. It is possible that prostanoids generated locally may play an important role in the regulation of vasomotor tone in the systemic vascular bed throughout the estrous cycle.

Influence of the estrous cycle on the norepinephrine-induced contraction of rat aorta: relationship to vascular prostanoids biosynthesis

Since ovarian sex steroids (estradiol and progesterone) may affect both blood pressure and prostanoids synthesis, and because prostaglandin-E2 (PGE2) and prostacyclin (PGI2) can modulate the vascular action of pressor hormones, we investigated the vascular reactivity to norepinephrine during the estrous cycle of the rat. In addition, we determined the vascular biosynthesis of PGE2 and 6-keto-PGF1 alpha (the stable metabolite of PGI2) at different stages of the estrous cycle. Cumulative dose-response curves were obtained by a stepwise increase in the concentration of norepinephrine. The contraction of thoracic aortic rings induced by norepinephrine did not change significantly between estrus, metestrus and diestrus. However, aortic rings obtained on proestrus showed a significant reduction in the maximal contraction (Emax) induced by norepinephrine (p < 0.001). In addition, we found significant increases in vascular synthesis of PGE2 and PGI2 on proestrus (p < 0.001). These results indicate that vascular reactivity and vascular prostanoids synthesis are influenced by the hormonal changes occurring during the estrous cycle of normal female rats. It is possible that prostanoids generated locally may play an important role in the regulation of vasomotor tone in the systemic vascular bed throughout the estrous cycle

Regulation of cyclic adenosine monophosphate synthesis in human ejaculated spermatozoa. I. Experimental conditions to quantitate membrane-bound adenylyl cyclase activity.

Although cyclic adenosine monophosphate (cAMP) is an important regulator of motility and metabolism in human spermatozoa, little is known on the cellular system responsible for its synthesis. Here, we investigated the experimental conditions directly to quantitate adenylyl cyclase (AC) activity synthesizing cAMP in human ejaculated spermatozoa and analysed the general properties of the enzyme. A 10,000 g membrane fraction was prepared from washed sperm cells homogenized by sonication. AC activity was monitored by the direct conversion of [alpha-32P]adenosine triphosphate (ATP) into [32P]cAMP. Using a nucleoside triphosphate regenerating system to ensure availability of ATP substrate, the human sperm AC showed a steady production of cAMP for at least 1 h. The assay was optimized for pH, buffer concentration, membrane protein and substrate concentration. Activity was dependent upon the presence of Mn2+ as a divalent cation and showed a pH optimum between 7.0 and 8.5. Optimal activity required 5 mM ATP, 1 mM ethylenediamine tetraacetic acid (EDTA) and 20-40 mM total MnCl2. Dependence on Mn2+ was not mandatory; Mg2+ at 5-40 mM also supported significant activity, but the activity was 4-6 times lower than that with Mn2+. Regardless of the presence of Mn2+ or Mg2+ as divalent cation in the assay, human sperm AC was insensitive to the regulatory ligands NaF, guanine nucleotide or forskolin. Insensitivity to these ligands supports the proposal that this enzyme system does not contain a stimulatory guanine nucleotide-binding regulatory protein and that its catalytic component is unique and different from that of somatic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of cyclic adenosine monophosphate synthesis in human ejaculated spermatozoa. II. The role of calcium and bicarbonate ions on the activation of adenylyl cyclase.

In this study, we have applied our previous data describing the experimental conditions necessary for expression of cyclic adenosine monophosphate (cAMP)-synthesizing adenylyl cyclase in human ejaculated spermatozoa, to investigate the direct effects of calcium (Ca2+) and bicarbonate (HCO3-) upon activation of the enzyme in vitro. We report that the effects of Ca2+ and HCO3- were significantly dependent on the status of the enzyme activity. Thus, at a near saturating (10 mM) concentration of MnCl2 giving high enzyme activity, addition of less than 10 mM HCO3- did not affect adenylyl cyclase activity and higher concentrations inhibited the enzyme, with 50 mM HCO3- reducing the activity by 33%. Also, addition of less than 20 mM CaCl2 alone or in combination with 10 mM HCO3- did not significantly change the enzyme activity. In great contrast, enzyme activation was highly responsive to Ca2+ and HCO3- when MnCl2 was present at a concentration giving submaximal enzyme activity. Thus, at 2 mM MnCl2, adenylyl cyclase was markedly increased by CaCl2 concentrations between 10 and 100 mM. The activation was further enhanced by increasing concentrations of HCO3-, with 50 mM HCO3- giving the highest activity at 50-100 mM CaCl2. Activation by CaCl2 was also observed in the absence of added MnCl2, being significantly greater than basal activity at 10 mM CaCl2 and maximal at 100 mM CaCl2.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the posterior hypophysis and ovary on pheromonal production in normal females and in the reproductive behavior of male rats treated with D,L-propranolol.

Posterior hypophysis homogenates of female rats stimulated with epinephrine subcutaneously contain a pheromonal factor that increments significantly the incidence of fertile coitus after having been placed in the oronasal groove of rats in proestro (69.2% fertile coitus vs. 36.6% of control). These same homogenates obtained from spayed rats increment reproductive activity even more (80.0% fertile coitus). The homogenates do not act by an intraperitoneal route. Male rats under the effect of high doses of D,L-propranolol reduce reproductive activity (21.2% fertile coitus). This effect is counteracted by posterior hypophysis homogenates from female rats stimulated with epinephrine, placed in the oronasal groove (72.2% fertile coitus). The homogenates of anterior hypophysis do not increase the reproductive capacity of rats in proestro, and do not modify the propranolol effects in male rats.

Effects of opioid peptides on aldosterone production: stimulatory effect of leu-enkephalin.

The effects of several opioid peptides (Leu-enkephalin, Met-enkephalin, D-Ala2,D-Leu5-enkephalin, and peptide E) on aldosterone secretion have been studied in isolated bovine adrenal glomerulosa cells. Leu-enkephalin significantly increased aldosterone production in a dose-dependent manner (10(-10)-10(-7) M). Similar results on steroidogenesis were obtained with the synthetic opioid D-Ala2,D-Leu5-enkephalin, whereas much higher concentrations of Met-enkephalin were required. Peptide E did not affect steroidogenesis. Naltrexone or naloxone (10(-5) M) potentiated the effect of Leu-enkephalin on aldosterone secretion. The effect of a general opioid antagonist (diprenorphine) or an agonist (etorphine) was also studied. The stimulation by Leu-enkephalin of aldosterone secretion was only partially blocked by diprenorphine. Etorphine (10(-6) and 10(-8) M) had no effect on basal steroidogenesis. It is proposed that the stimulatory effect of Leu-enkephalin on aldosterone production could be mediated by a different receptor than the classical opioid receptors presently known.

Changes in reproductive behavior and urinary pheromone induction in rats through beta-adrenergic stimulation.

Treating rats in proestro with beta-adrenergic stimulants (epinephrine and phenotherol bromhydrate) significantly increases the instances of fertile coitus. Concurrently, it induces the excretion through the urine of compounds with pheromonal action that affect males and females of the same strain, increasing their reproductive activity.

Effect of theophylline on nuclear retention of oestrogenreceptor complexes: correlation with oestrogen responses.

In the ovariectomized adult rat uterine oedema induced by 0.01 and 0.1 micrograms oestradiol-17 beta/100g body weight increased further in the presence of theophylline. Nuclear retention of oestrogen-receptor complexes also increased in response to theophylline both in vivo and in vitro. Theophylline decreased the number of eosinophils in the blood and concurrently decreased oestrogen-induced uterine eosinophilia at doses of 0.001, 0.01, 0.1, 1, 10 or 30 micrograms oestradiol/100 g body weight, through a mechanism independent of glucocorticoids. There was, therefore, no correlation between changes in the number of uterine eosinophils and changes in uterine wet weight induced by theophylline and oestrogen. It is suggested that the presence of oestrogen-receptor complexes in the nucleus for at least 4 h is a prerequisite for the induction of uterine oedema and growth in the presence of theophylline and oestradiol-17 beta.

Effect of topically applied adrenergic blockers on fertility.

The contraceptive properties of two adrenergically active drugs, propranolol and reserpine, were measured in vivo and in vitro. Propranolol became effective as a spermicide for human sperm at a concentration of about 2 x 10(-3) M and for rat sperm at 2 x 10(-2) M. Reserpine was also more effective on human than rat sperm and about 20 times less effective than propranolol. Both propranolol and reserpine prevented implantation when instilled into the uteri of rats. The anti-implantation effect persisted for up to 5 days after instillation of propranolol. When freely breeding rats eventually became pregnant after daily vaginal instillation of propranolol, the number of uterine implantations decreased as the concentration of vaginally instilled propranolol increased. The implantations tended to be toward the fundal end of the uteri, away from the source of propranolol, thus indicating a topical rather than a systemic anti-implantation effect.

Endocrinologic basis of electrochemical vaginal potential in estrous cycle of rats.

This sequel to an earlier report on the correlation of vagino-oral voltage measurements in rats with their estrous cycles deals with the endocrinologic activation of these voltages. The normally sinusoidal voltages, positive during estrus and negative during diestrus, were absent in spayed rats but were restored with a single intraperitoneal injection of 5 microgram or more of 17beta-estradiol or with 0.1 mg of adrenaline injected subcutaneously every 12 hours. Estrogen antagonists, drugs that inhibit catecholamine synthesis or adrenergic beta blockers, totally inhibit the negative potential recorded during estrus. Vaginal estrous potentials in the rat reflect an important reproductive state possibly linked to sexual behavior (heat) or to ovulation. Triggering of the cyclic potential by adrenaline without estrogenic medication suggests that adrenaline belongs to the group of sexual-specific hormones.

Electrochemical vaginal-oral potentials during the menstrual cycle in women.

Electrochemical potentials were measured with non-polarizable salt electrodes (agar KCl-AgCL) during successive menstrual cycles in eight women. Four of them did not use any contraceptive, two were TCu users and the other two took oral contraceptives. Vaginal-tongue potentials presented biphasic cyclic changes. The vagina became positive in the middle of the menstrual cycle in all the women except those who were using contraceptive pills. Vaginal-oral potentials became positive (+) 24 hours after ingestion of oral contraceptives and up to 24 hours after discontinuation.

PIP: Nonpolarizable salt electrodes (agar (KC1-AgC1) were used to measure electrochemical potentials during successive menstrual cycles in 8 women, 4 of whom were using no contraception, 2 of whom were using copper T IUDs, and 2 of whom were taking oral contraceptives. The results are shown as a series of curves representing the different voltages measured every 2-3 days. Subjects using no contraceptives, as well as T-device users, showed a change in the direction of the current in the vaginal electrode around the 14th day of the cycle. Subjects using oral contraceptives showed a positive (+) potential 24 hours after taking the first pill, and this was maintained throughout the period of pill ingestion; the charge became negative (-) 24 hours after pill ingestion was discontinued. In sum, vaginal-oral potentials presented biphasic cyclic changes in women using IUDs or no contraception, and the biphasic nature was lost during oral contraceptive use.