Ejaculated mammalian sperm require several hours exposure to secretions in female reproductive tracts, or incubation in appropriate culture medium in vitro, before acquiring the capacity to fertilize eggs. Arachidonylethanolamide (AEA), also known as anandamide, is a novel lipid-signal molecule that is an endogenous agonist (endocannabinoid) for cannabinoid receptors. We now report that AEA is present in human seminal plasma, mid-cycle oviductal fluid, and follicular fluid analyzed by high-performance liquid chromatography/mass spectrometry. Sperm are sequentially exposed to these reproductive fluids as they move from the vagina to the site of fertilization in the oviduct. Specific binding of the potent cannabinoid agonist [3 H]CP-55,940 to human sperm was saturable (K D 9.71 AE 1.04 nM), suggesting that they express cannabinoid receptors. R-methanandamide , a potent and metabolically stable AEA analog, and (À)D 9 tetrahydrocannabinol (THC), the major psychoactive constituent of Cannabis, modulated capacitation and fertilizing potential of human sperm in vitro. AM-356 elicited biphasic effects on the incidence of hyperactivated sperm motility (HA) between 1 and 6 hr of incubation: at (2.5 nM) it inhibited HA, while at (0.25 nM) it stimulated HA. Both AM-356 and THC inhibited morphological alterations over acrosomal caps between 2 and 6 hr (IC 50 5.9 AE 0.6 pM and 3.5 AE 1.5 nM, respectively). Sperm fertilizing capacity, measured in the Hemizona Assay, was reduced 50% by (1 nM) AM-356. These findings suggest that AEA-signaling may regulate sperm functions required for fertilization in human reproductive tracts, and imply that smoking of marijuana could impact these processes. This study has potential medical and public policy ramifications because of the incidence of marijuana abuse by adults in our society, previously documented reproductive effects of marijuana, and the ongoing debate about medicinal use of marijuana and cannabinoids.
Capacitation and capacitation-related hyperactivated motility do not occur spontaneously in cynomolgus monkey (Macaca fascicularis) spermatozoa; instead, both have an absolute requirement for exogenous stimulation with caffeine and dibutyryl (db)cAMP. In the present study, we 1) defined sorting criteria for automated analysis of macaque sperm exhibiting hyperactivated motility (HA) and 2) investigated protein tyrosine phosphorylation involvement in dbcAMP- and caffeine-stimulated capacitation and HA. Motion characteristics were assessed by computer-assisted motion analysis. Tyrosine phosphorylation of sperm tail proteins was determined by immunocytochemistry with PY-20 antiserum. Automated sorting criteria for HA were curvilinear velocity (VCL) >/= 150 microm/sec; amplitude of lateral head displacement (ALH) >/= 8.0 microm, and linearity (LIN) = 60%. Using these criteria, caffeine and dbcAMP significantly stimulated HA (61 +/- 8%) compared to control conditions (12 +/- 2%), p < 0.01, with a concomitant increase in PY-20 labeling (88 +/- 12%) vs. control (13 +/- 2%), p < 0.01. PY-20 labeling significantly correlated with HA (r = 0.75, p < 0.01) and with some motion characteristics used for HA sorting including ALH (r = 0.86, p = 0.0013) and LIN (r = -0.88, p < 0.001) but not VCL (r = 0.21). Treatment with genistein (10 microM) had no effect on HA or PY-20 immunocytochemistry in the absence of caffeine and dbcAMP, but the tyrosine kinase inhibitor significantly decreased caffeine- and dbcAMP-stimulated HA and PY-20 labeling in a dose-dependent manner (p < 0.01). These results demonstrate that tyrosine phosphorylation of sperm tail proteins is an integral signaling pathway modulating some but not all of the motion characteristics associated with cAMP- and caffeine-stimulated HA in cynomolgus monkey spermatozoa.
These experiments show that H2O2 directly affects sperm functions crucial to fertilization in a dose- and time-dependent fashion. Low concentrations maintain capacitation, whereas higher concentrations have deleterious effects, as determined by the end points of the capacitation process. The latter effects are probably dependent on modifications of plasma membrane and intracellular homeostasis by the oxidative process.
BackgroundThis prospective, Phase IV, multicenter, observational registry of assisted reproductive technology clinics in the USA studied outcomes of first cycles using thawed/warmed cryopreserved (by slow-freezing/vitrification) oocytes (autologous or donor).MethodsPatients were followed up through implantation, clinical pregnancy, and birth outcomes. The main outcome measure was live birth rate (LBR), defined as the ratio of live births to oocytes thawed/warmed minus the number of embryos cryopreserved for each cycle, averaged over all thawing cycles. Clinical pregnancy rate (CPR) was also evaluated, and was defined as the presence of a fetal sac with heart activity, as detected by ultrasound scan performed on Day 35–42 after embryo transfer.ResultsA total of 16 centers enrolled 204 patients; data from 193 patients were available for analyses. For donor oocytes, in the slow-freezing (n = 40) versus vitrification (n = 94) groups, respectively, CPR and LBR were significantly different: 32.4% versus 62.6%, and 25.0% versus 52.1%; outcomes from Day 3 transfers did not differ significantly. For vitrified oocytes, in the autologous (n = 46) versus donor (n = 94) group, respectively, CPR and LBR were significantly different: 30.0% versus 62.6% and 17.4% versus 52.1%. This was largely due to a significant difference in CPR with Day 5/6 transfers.ConclusionsIn two subgroup data analyses, in women who received cryopreserved oocytes from donors, CPR and LBR were significantly higher in cycles using oocytes cryopreserved via vitrification versus slow-freezing, reflecting differences in methodologies and more Day 5/6 transfers; in women who received vitrified oocytes, CPR and LBR were significantly higher in cycles using donor versus autologous oocytes with Day 5/6 transfers.Trial registrationClinicalTrials.gov: NCT00699400. Registered June 13, 2008.
The objective of these studies was to evaluate the modulatory effect(s) of progesterone on sperm functions crucial to fertilization in infertile men with abnormal sperm parameters. A prospective, controlled study applying a sequential diagnostic analysis capable of identifying specific dysfunctions of the male gamete was performed. Patients (n = 14) were allocated to the study group if they had a history of infertility of > 1 year duration and after semen evaluation showed teratozoospermia (< 14% normal sperm forms as diagnosed by strict criteria) or terato-asthenozoospermia (< 50% progressive motility). After swim-up separation of the motile sperm fraction, the following functions were assessed with and without previous exposure to progesterone (1.0 micrograms/ml): acrosome reaction (using Pisum sativum agglutinin), hyperactivated motility (using a computerized semen analyser), sperm-zona pellucida binding (in the hemizona assay), sperm-zona pellucida penetration (in a sperm-zona penetration assay), and sperm-oocyte penetration (using the hamster zona-free oocyte/sperm penetration assay). Progesterone did not affect the percentage of acrosome-reacted spermatozoa after 1 or 3 h of incubation. Hyperactivated motility was significantly enhanced by progesterone after 1 h (12 +/- 4 versus 6 +/- 2% in controls; P < 0.02). Although progesterone did not affect sperm-zona binding, it significantly enhanced both sperm-zona pellucida penetration (27 versus 12% in controls; P = 0.03) and sperm-oocyte penetration (15 versus 8% in controls; P < 0.05). Because those sperm functions enhanced by progesterone are crucial to fertilization, the steroid may have value in the treatment of some male-factor patients undergoing assisted reproductive therapy.
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