Anandamide induces sperm release from oviductal epithelia through nitric oxide pathway in bovines

Mammalian spermatozoa are not able to fertilize an egg immediately upon ejaculation. They acquire this ability during their transit through the female genital tract in a process known as capacitation. The mammalian oviduct acts as a functional sperm reservoir providing a suitable environment that allows the maintenance of sperm fertilization competence until ovulation occurs. After ovulation, spermatozoa are gradually released from the oviductal reservoir in the caudal isthmus and ascend to the site of fertilization. Capacitating-related changes in sperm plasma membrane seem to be responsible for sperm release from oviductal epithelium. Anandamide is a lipid mediator that participates in the regulation of several female and male reproductive functions. Previously we have demonstrated that anandamide was capable to release spermatozoa from oviductal epithelia by induction of sperm capacitation in bovines. In the present work we studied whether anandamide might exert its effect by activating the nitric oxide (NO) pathway since this molecule has been described as a capacitating agent in spermatozoa from different species. First, we demonstrated that 1 μM NOC-18, a NO donor, and 10 mM L-Arginine, NO synthase substrate, induced the release of spermatozoa from the oviductal epithelia. Then, we observed that the anandamide effect on sperm oviduct interaction was reversed by the addition of 1 μM L-NAME, a NO synthase inhibitor, or 30 μg/ml Hemoglobin, a NO scavenger. We also demonstrated that the induction of bull sperm capacitation by nanomolar concentrations of R(+)-methanandamide or anandamide was inhibited by adding L-NAME or Hemoglobin. To study whether anandamide is able to produce NO, we measured this compound in both sperm and oviductal cells. We observed that anandamide increased the levels of NO in spermatozoa, but not in oviductal cells. These findings suggest that anandamide regulates the sperm release from oviductal epithelia probably by activating the NO pathway during sperm capacitation.     

Anandamide capacitates bull spermatozoa through CB1 and TRPV1 activation

Anandamide (AEA), a major endocannabinoid, binds to cannabinoid and vanilloid receptors (CB1, CB2 and TRPV1) and affects many reproductive functions. Nanomolar levels of anandamide are found in reproductive fluids including mid-cycle oviductal fluid. Previously, we found that R(+)-methanandamide, an anandamide analogue, induces sperm releasing from bovine oviductal epithelium and the CB1 antagonist, SR141716A, reversed this effect. Since sperm detachment may be due to surface remodeling brought about by capacitation, the aim of this paper was to investigate whether anandamide at physiological concentrations could act as a capacitating agent in bull spermatozoa. We demonstrated that at nanomolar concentrations R(+)-methanandamide or anandamide induced bull sperm capacitation, whereas SR141716A and capsazepine (a TRPV1 antagonist) inhibited this induction. Previous studies indicate that mammalian spermatozoa possess the enzymatic machinery to produce and degrade their own AEA via the actions of the AEA-synthesizing phospholipase D and the fatty acid amide hydrolase (FAAH) respectively. Our results indicated that, URB597, a potent inhibitor of the FAAH, produced effects on bovine sperm capacitation similar to those elicited by exogenous AEA suggesting that this process is normally regulated by an endogenous tone. We also investigated whether anandamide is involved in bovine heparin-capacitated spermatozoa, since heparin is a known capacitating agent of bovine sperm. When the spermatozoa were incubated in the presence of R(+)-methanandamide and heparin, the percentage of capacitated spermatozoa was similar to that in the presence of R(+)-methanandamide alone. The pre-incubation with CB1 or TRPV1 antagonists inhibited heparin-induced sperm capacitation; moreover the activity of FAAH was 30% lower in heparin-capacitated spermatozoa as compared to control conditions. This suggests that heparin may increase endogenous anandamide levels. Our findings indicate that anandamide induces sperm capacitation through the activation of CB1 and TRPV1 receptors and could be involved in the same molecular pathway as heparin in bovines.     

Sulphated glycosaminoglycans (S-GAGs) and syndecans in the bovine oviduct

In vivo, bull sperm capacitation seems to occur mainly in the oviduct. Capacitation of bull spermatozoa can be triggered in vitro by exposure to heparin, a heavily sulphated glycosaminoglycan (S-GAG). We determined the concentration of S-GAGs in oviductal fluid from dairy heifers, collected over the course of several oestrous cycles via surgically implanted intraluminal catheters. We also investigated the presence of syndecans, i.e. heparan sulphate proteoglycans, in the bovine oviductal epithelium of Swedish dairy cattle during standing oestrus and the luteal phase of the oestrous cycle, using immunohistochemistry for three different polyclonal antibodies raised against human syndecan-2 and rat syndecan-1 and syndecan-2, respectively. The concentration of S-GAGs in oviductal fluid obtained from the ampullar segment of the oviduct was significantly higher (P=0.0026) than it was in fluid from the isthmic segment during the functional period, i.e. from prooestrus to metaoestrus (73.5+/-10.49 mg/L in ampullar ODF, compared to 43.2+/-10.74 mg/L in isthmic ODF); least square mean (L.S.M.)+/-standard error of the mean (S.E.M.). There was also a significantly higher concentration of S-GAGs in the fluid from the oviduct ipsilateral to the ovulation side 73.5+/-10.54 mg/L on the ovulation side, compared to 43.1+/-10.71 mg/L in the oviduct on the contralateral side (L.S.M.+/-S.E.M., P=0.0026) during this period. Both syndecan-1 and syndecan-2 were present in the epithelial cells lining all studied segments of the bovine oviduct, i.e. the UTJ, isthmus and ampulla, during both standing oestrus and dioestrus. The syndecans and S-GAGs found may influence the gametes, while they reside in the oviduct; the amounts of S-GAGs found in the bovine oviduct seem sufficient to act as capacitating factors in vivo.     

Identification of norepinephrine in bovine oviductal fluid by high performance liquid chromatography.

The final stages of sperm maturation, fertilization and early embryonic development take place in the microenvironment of the oviduct and are essential for successful reproduction in mammals. Although catecholamines have been shown to have beneficial effects on mammalian gametes in vitro, identification of catecholamines in native bovine oviductal fluid has not been studied. The objective of this research was to identify catecholamines in bovine oviductal fluid and to determine whether concentrations of catecholamines change with stage of the estrous cycle. Oviductal fluid was collected via indwelling oviductal cannulae and assayed for the presence of catecholamines by high performance liquid chromatography. Norepinephrine was the only catecholamine detected, in concentrations ranging from 0.828 ng/ml - 1117 ng/ml. The presence of norepinephrine in oviductal fluid corresponded to a period of time just prior to, during, and after ovulation, when serum progesterone levels were low. This was a consistent finding in ODF collected from normally cycling cows. Potential functions of norepinephrine in oviductal fluid include regulation of fluid formation, induction of capacitation and the acrosome reaction in spermatozoa, and cleavage of the early embryo.     

In vitro-cultured bovine oviductal cells bind acrosome-intact sperm and retain this ability upon sperm release

The mammalian oviduct plays a key role in sperm storage, capacitation, and selection. Specific oviduct secretions and/or binding to oviductal cells are thought to be responsible for the extension of the fertile life span of sperm. In this in vitro study, a quantitative assay for sperm binding was developed to analyze the mechanisms of sperm-oviductal cell adhesion and release in the bovine species. Distribution and acrosomal status of sperm bound to in vitro-cultured ampullary and isthmic cell monolayers were followed until the time of sperm release by means of fluorescence labeling techniques. In order to understand whether release is due to surface changes of sperm or oviductal cells, double incubation experiments with unlabeled and Hoechst-labeled sperm have been performed. Main findings demonstrate that (1) only acrosome-intact sperm bind specific bovine oviductal epithelial cells; (2) acrosomes of bound sperm are preserved intact over time; and (3) release of unreacted sperm is likely to be due to changes of the sperm surface, probably triggered by capacitation. These findings support the hypothesis that binding to oviductal cells is essential for preserving the sperm fertilization competence during the interval from the onset of estrus to ovulation.     

Hyaluronan and its binding proteins in the epithelium and intraluminal fluid of the bovine oviduct

Hyaluronan (HA) is involved in several important steps of sperm storage and of fertilization. This study investigates the presence and concentration of HA in oviductal fluid (ODF), together with the localization of HA and the presence of hyaluronan-binding proteins (HABPs) in the oviductal epithelium of normally cycling dairy heifers and cows. The concentration and amount of HA in ODF, collected over the course of several oestrous cycles via catheters placed in the isthmic and ampullar tubal segments, were measured using an ELISA. The concentration and amount of HA in ODF did not vary significantly between these anatomical regions, nor between the stages of the oestrous cycle (p > 0.05), although the amount of HA seemed to peak during oestrous. The most HA per day (2.9 +/- 0.64 microg, least square mean +/- SEM) was produced on the day of ovulation, whereas the lowest amount (1.25 +/- 0.68 microg) was produced 4 days before ovulation. To investigate the localization of HA, tissue samples were retrieved at well-defined stages of the oestrous cycle and from corresponding regions of the oviduct. Sections and protein extracts from the tissue samples were studied histochemically using biotinylated HABP and immunoblotted with fluorescein isothiocyanate (FITC)-HA, respectively. Presence of HA labelling in the oviductal epithelium was restricted to the sperm reservoir, a localization that seemed to be cycle-independent. The immunoblotting of samples from the lining epithelium revealed seven bands of HABPs. We confirm that the bovine oviduct produces HA and its binding proteins, and that HA is mainly localized to the epithelium of the sperm reservoir.     

Ovulatory follicular fluid induces sperm phagocytosis by neutrophils,but oviductal fluid around oestrus suppresses its inflammatory effect in the buffalo oviduct in vitro

We have recently shown that the conditioned media from bovine oviductal epithelial cell culture suppress sperm phagocytosis by neutrophils, suggesting that the oviduct around oestrus supplies the anti‐inflammatory microenvironment. To investigate the immune response of neutrophils toward the sperm at ovulation in the buffalo oviduct, we examined (a) a detailed distribution of neutrophils in the oviduct in buffaloes, (b) the effect of ovulatory follicular fluid (FF) and oviductal fluid (OF) on sperm phagocytosis by neutrophils, and (c) the interaction of the ovulatory FF with OF on sperm phagocytosis by neutrophils in vitro. Buffalo oviducts were collected from healthy reproductive tracts at a local slaughterhouse. A detailed observation by histological examination and transmission electron microscopy revealed that neutrophils exist in the oviduct epithelium and lumen throughout the oestrous cycle in buffaloes. The number of neutrophils at the oestrus stage was higher in ampulla compared with those in isthmus, whereas they remained relatively constant at the dioestrus stage. Two hours of preincubation of neutrophils with FF enhanced sperm phagocytosis through the formation of neutrophil extracellular traps (NETs) together with H₂O₂ production, whereas OF around oestrus (eOF) suppressed sperm phagocytosis, NETs formation, and H₂O₂ production and relieved the above FF‐induced inflammatory response. Our findings show that neutrophils exist in the healthy cyclic oviduct across bovine species, and the OF supplies a strong anti‐inflammatory environment that could minimize the inflammatory effect of the FF that flows into the oviduct lumen after ovulation and supports the occurrence of fertilization.     

Monosaccharides are not detected in whole or isthmic bovine oviductal fluid collected throughout the estrous cycle, as analyzed by HPLC

In the bovine oviduct, monosaccharides may play a role in the preparation of gametes for fertilization. Sperm are sequestered in the isthmic region of the oviduct where capacitation, requisite biochemical changes in sperm membranes, may take place. Retention of spermatozoa in the oviductal isthmus is dependent on a carbohydrate recognition system between oviductal epithelium and sperm membrane lectins. The monosaccharide, fucose, has been found to be important to this recognition system. However, both gametes and epithelium are also bathed in oviductal fluid (ODF), and fucose or other monosaccharides may be constituents of ODF and so may be important to sperm binding to oviductal epithelium and subsequent preparation for fertilization. In this study, ODF from dairy cows was analyzed by HPLC for the presence of 5 monosaccharides (fucose, galactose, glucosamine, mannose and xylose). Both whole ODF, collected by cannulation of the entire oviduct of 1 cow over a complete estrous cycle, and regional staged ODF, collected and pooled from 13 cows from the isthmic region only at estrus, were analyzed. We report negligible concentrations of all 5 monosaccharides in both types of ODF analyzed. Because the detection limit of our assay was 10(8) times lower than fucose concentrations found to be physiologically important in earlier in vitro studies, we conclude that bovine ODF does not contain physiologically active levels of free fucose or other, similar monosaccharides at any time of the estrous cycle.     

Sperm binding, in vitro fertilization, and in vitro embryonic development of bovine oocytes fertilized with spermatozoa incubated with norepinephrine

The final stages of sperm maturation, fertilization, and early embryonic development occur within the oviduct and are essential for successful reproduction in mammals. Norepinephrine was previously identified in native bovine oviductal fluid and its in vitro effects on bull sperm capacitation and the acrosome reaction have been determined. It was unknown how physiological concentrations of norepinephrine influence sperm binding, fertilization, and embryo development. Therefore, the objective of this study was to determine if pre-incubating bovine spermatozoa with physiological concentrations of norepinephrine prior to insemination of bovine oocytes would improve sperm-oocyte binding, fertilization, and embryonic development in vitro. Norepinephrine, in concentrations representing those measured in bovine oviductal fluid, was used to treat bovine spermatozoa prior to insemination. Spermatozoa incubated in norepinephrine were used to inseminate bovine oocytes matured in vitro, and oocytes were evaluated for sperm binding and fertilization. Additional experiments were conducted to evaluate how early in the co-incubation period oocytes were fertilized by spermatozoa pre-incubated with norepinephrine, and to test the developmental competence of those oocytes fertilized with norepinephrine-treated sperm. Sperm binding to the zona pellucida was reduced by pre-incubation with norepinephrine. Rates of fertilization and embryo development did not increase as a result of pre-incubating spermatozoa with norepinephrine, but as early as 4h after insemination, spermatozoa treated with 20 ng/ml norepinephrine fertilized more oocytes than spermatozoa incubated in medium alone. Interestingly, this concentration of norepinephrine was found to capacitate spermatozoa in previous studies. These data suggest that oocytes fertilized by spermatozoa incubated in 20 ng/ml norepinephrine fertilize earlier in vitro than sperm pre-incubated in medium alone, and provide additional support for the role of norepinephrine in sperm capacitation and the acrosome reaction.     

Role of the oviduct in sperm capacitation

Following insemination of spermatozoa pre-ovulation, the mammalian oviduct ensures, by the formation of a functional sperm reservoir (SR), that suitable (low) numbers of viable and potentially fertile spermatozoa are available for fertilization at the ampullary isthmic junction (AIJ). As ovulation approaches, a proportion of the SR-stored spermatozoa is continuously distributed towards the AIJ and individually activated leading to step-wise capacitation and the attainment of hyperactivated motility. This paper reviews in vivo changes in the intra-luminal milieu of the oviduct of pigs and cows, in particular the SR and the AIJ which relate to the modulation of sperm capacitation around spontaneous ovulation. In vivo, most viable spermatozoa in the pre-ovulatory SR are uncapacitated. Capacitation rates significantly increase after ovulation, apparently not massively but concurrent with the individual, continuous sperm dislocation from the SR. Bicarbonate, whose levels differ between the SR and the AIJ, appears as the common primary effector of the membrane destabilizing changes that encompasses the first stages of capacitation. Sperm activation can be delayed or even reversed by co-incubation with membrane proteins of the tubal lining, isthmic fluid or specific tubal glycosaminoglycans, such as hyaluronan. Although the pattern of response to in vitro induction of sperm activation - capacitation in particular - is similar for all spermatozoa, the capacity and speed of the response is very individual. Such diversity in responsiveness among spermatozoa insures full sperm viability before ovulation and the presence of spermatozoa at different stages of capacitation at the AIJ, thus maximizing the chances of normal fertilization.     

Effects of porcine pre-ovulatory oviductal fluid on boar sperm function

Sperm storage within the oviductal isthmus prior to ovulation typically involves binding to oviductal epithelial cells, which are thought to modulate sperm functions including internal calcium concentration, membrane fluidity, and motility. Around the time of ovulation the spermatozoa are gradually released so that they eventually encounter the oocytes within the oviductal ampulla. Previous studies have shown that the oviductal epithelial cells selectively sequester high quality spermatozoa, but the role of oviductal fluid as a selective modulator of sperm function has been investigated to a lesser extent. Here we address the hypothesis that oviductal fluid is also likely to modulate sperm function. Using samples of porcine oviductal fluid collected in the follicular phase of the estrus cycle, we show that short exposure (20 min to 50 μg/mL of oviductal fluid proteins) to either of two separate proteins fractions (> or < 100 kDa) promotes boar sperm viability and acrosomal integrity, decreases sperm plasma membrane fluidity (measured using merocyanine S540), and increases zona binding and polyspermy during in vitro fertilization. Exposure to the lower molecular fraction significantly inhibited, but did not abolish, the bicarbonate-induced stimulation of motility. The results show that subpopulations of spermatozoa respond differentially to oviductal fluid, and suggest that exposure to oviductal fluid in vivo could exert a further level of functional sperm selection.     

Boar spermatozoa in the oviduct

In the pig, a functional tubal sperm reservoir (SR) is established before ovulation to ensure availability of suitable numbers of viable spermatozoa for fertilization. The boar's large ejaculate is split: most spermatozoa are delivered in a sperm-rich fraction (SRF) followed by a post-SRF fraction containing increasing amounts of the spermadhesin PSP-I/PSP-II-rich seminal vesicle secretion. This heterodimer acts as leukocyte chemoattractant both in vitro and in vivo, contributing to the phagocytosis of those spermatozoa not reaching the SR. Sequential ejaculate deposition of marked spermatozoa and SR screening showed that most spermatozoa in the SR arose from the fortuitous PSP-poor, first portion of the SRF fraction, escaping phagocytosis and replenishing the SR within 2-3 h. The SR-sperm numbers diminish gradually in relation to ovulation, spermatozoa being continuously redistributed toward the upper isthmus. In vitro, only uncapacitated spermatozoa bind to epithelial explants, suggesting that the SR influences sperm capacitation. In vivo, most viable spermatozoa--usually harbored in the deep furrows in the pre- or peri-ovulatory SR during spontaneous standing estrus--are uncapacitated, but capacitation significantly increases after ovulation. Pre-/peri-ovulatory SR spermatozoa promptly capacitate in vitro when exposed to the effector bicarbonate, an influence that can be reversed by co-incubation with SR fluid or its component hyaluronan. Fluid collected from the ampullar segment (rich in bicarbonate) induces capacitation in vitro. In conclusion, the lack of massive sperm capacitation in the SR and the diverse individual response to capacitation shown by tubal spermatozoa would relate both to the insurance of full sperm viability before ovulation and the presence of spermatozoa at different stages of capacitation in the upper oviduct, thus maximizing the chances of normal fertilization.     

Distribution, morphology and epithelial interactions of bovine spermatozoa in the oviduct before and after ovulation: a scanning electron microscope study.

In cows undergoing spontaneous oestrous cycles and mated during the first 6 hours of oestrus, the distribution of spermatozoa in the oviduct isthmus and changes in their surface membranes and neighbouring epithelium have been examined shortly before and after ovulation. In agreement with previous histological studies, relatively few spermatozoa were detected in the oviduct lumen: most were located in the caudal isthmus before ovulation, frequently among folds and in the presence of a viscous secretion. A majority of spermatozoa in this region showed strands and droplets of secretory material distributed over the anterior portion of an intact head before ovulation, whereas distribution of material over the post-nuclear cap of spermatozoa close to vesiculation or already acrosome-reacted was characteristic of the post-ovulatory situation. These changes in sperm head membranes were viewed as an expression of the completion of capacitation, and seemingly permit microvillous engagement with the rostral tip of the head. In conjunction with a narrow lumen and viscous secretions in the caudal isthmus, microvilli may thus serve to regulate periovulatory sperm progression towards the site of fertilisation, and be the basis of intermittent phases of adhesion to the oviduct epithelium as seen by phase-contrast microscopy. Although cilia do not similarly engage the heads of bull spermatozoa (cf. boar spermatozoa), they may act to regulate progression of capacitated spermatozoa by contacting the principal piece of the flagellum. In the light of these observations, changes in the molecular composition of sperm surface domains during the process of capacitation in vivo now require specific definition.     

Bovine oviductal fluid (bOF) collected in the follicular or luteal phase of the estrous cycle exerts similar effects on ram sperm kinematics and acrosome reactivity in vitro

This study examined the effects of bovine oviductal fluid (bOF) obtained during the follicular or luteal phase of the estrous cycle on ram sperm kinematics, capacitation status and plasma membrane (PM) integrity at various time points during the 24-h incubation period. Fresh ram spermatozoa were selected using the swim-up technique and then incubated separately with either follicular phase (FbOF) or luteal phase (LbOF) bovine oviductal fluid added to Fert-TALP medium (positive control - POSControl) or in Fert-TALP medium without capacitating agents (negative control - NEGControl) at 38 °C under 5% CO₂. Incubation with FbOF or LbOF for 2 h and 4 h promoted an increase (P < 0.05) in most of the sperm motility parameters as compared with the NEGControl group, and bOF-induced changes in sperm kinematics were similar (P > 0.05) to those seen in the POSControl group. After 6 h of incubation, the stimulatory effect of FbOF or LbOF on ram sperm kinematics was no longer observed (P > 0.05). Sperm PM integrity was not affected (P > 0.05) by incubation in bOF-supplemented media or in absence of capacitating factors (NEGControl). Although neither FbOF nor LbOF had any effect on sperm capacitation rates, the proportion of acrosome-reacted spermatozoa was greater (P < 0.05) for bOF-containing media compared with the NEGControl group during the long incubation periods (18 h and 24 h). In conclusion, bOF from either follicular or luteal phase of the estrous cycle enhances ram sperm motility for up to 4 h and the rate of acrosome reaction after long (18–24 h) incubation periods without affecting sperm viability.     

Endocannabinoids measurement in human saliva as potential biomarker of obesity

Background

The discovery of the endocannabinoid system and of its role in the regulation of energy balance has significantly advanced our understanding of the physiopathological mechanisms leading to obesity and type 2 diabetes. New knowledge on the role of this system in humans has been acquired by measuring blood endocannabinoids. Here we explored endocannabinoids and related N-acylethanolamines in saliva and verified their changes in relation to body weight status and in response to a meal or to body weight loss.

Methodology/Principal Findings

Fasting plasma and salivary endocannabinoids and N-acylethanolamines were measured through liquid mass spectrometry in 12 normal weight and 12 obese, insulin-resistant subjects. Salivary endocannabinoids and N-acylethanolamines were evaluated in the same cohort before and after the consumption of a meal. Changes in salivary endocannabinoids and N-acylethanolamines after body weight loss were investigated in a second group of 12 obese subjects following a 12-weeks lifestyle intervention program. The levels of mRNAs coding for enzymes regulating the metabolism of endocannabinoids, N-acylethanolamines and of cannabinoid type 1 (CB₁) receptor, alongside endocannabinoids and N-acylethanolamines content, were assessed in human salivary glands.The endocannabinoids 2-arachidonoylglycerol (2-AG), N-arachidonoylethanolamide (anandamide, AEA), and the N-acylethanolamines (oleoylethanolamide, OEA and palmitoylethanolamide, PEA) were quantifiable in saliva and their levels were significantly higher in obese than in normal weight subjects. Fasting salivary AEA and OEA directly correlated with BMI, waist circumference and fasting insulin. Salivary endocannabinoids and N-acylethanolamines did not change in response to a meal. CB₁ receptors, ligands and enzymes were expressed in the salivary glands. Finally, a body weight loss of 5.3% obtained after a 12-weeks lifestyle program significantly decreased salivary AEA levels.

Conclusions/Significance

Endocannabinoids and N-acylethanolamines are quantifiable in saliva and their levels correlate with obesity but not with feeding status. Body weight loss significantly decreases salivary AEA, which might represent a useful biomarker in obesity.     

Inhibiting Sperm Pyruvate Dehydrogenase Complex and Its E3 Subunit,Dihydrolipoamide Dehydrogenase Affects Fertilization in Syrian Hamsters

Background/Aims

The importance of sperm capacitation for mammalian fertilization has been confirmed in the present study via sperm metabolism. Involvement of the metabolic enzymes pyruvate dehydrogenase complex (PDHc) and its E3 subunit, dihydrolipoamide dehydrogenase (DLD) in hamster in vitro fertilization (IVF) via in vitro sperm capacitation is being proposed through regulation of sperm intracellular lactate, pH and calcium.

Methodology and Principal Findings

Capacitated hamster spermatozoa were allowed to fertilize hamster oocytes in vitro which were then assessed for fertilization, microscopically. PDHc/DLD was inhibited by the use of the specific DLD-inhibitor, MICA (5-methoxyindole-2-carboxylic acid). Oocytes fertilized with MICA-treated (MT) [and thus PDHc/DLD-inhibited] spermatozoa showed defective fertilization where 2^nd polar body release and pronuclei formation were not observed. Defective fertilization was attributable to capacitation failure owing to high lactate and low intracellular pH and calcium in MT-spermatozoa during capacitation. Moreover, this defect could be overcome by alkalinizing spermatozoa, before fertilization. Increasing intracellular calcium in spermatozoa pre-IVF and in defectively-fertilized oocytes, post-fertilization rescued the arrest seen, suggesting the role of intracellular calcium from either of the gametes in fertilization. Parallel experiments carried out with control spermatozoa capacitated in medium with low extracellular pH or high lactate substantiated the necessity of optimal sperm intracellular lactate levels, intracellular pH and calcium during sperm capacitation, for proper fertilization.

Conclusions

This study confirms the importance of pyruvate/lactate metabolism in capacitating spermatozoa for successful fertilization, besides revealing for the first time the importance of sperm PDHc/ DLD in fertilization, via the modulation of sperm intracellular lactate, pH and calcium during capacitation. In addition, the observations made in the IVF studies in hamsters suggest that capacitation failures could be a plausible cause of unsuccessful fertilization encountered during human assisted reproductive technologies, like IVF and ICSI. Our studies indicate a role of sperm capacitation in the post-penetration events during fertilization.     

Detection of Fas ligand in the bovine oviduct

Presence of a Fas-Fas ligand (FasL) system defines the immune-privileged status of certain tissues such as placenta. This study examined the fluids and tissue(s) of the bovine oviduct, where both spermatozoa and early embryos escape elimination by the female immune system, for the presence and the distribution of Fas and FasL, which might provide an explanation for the immune-privileged site of this organ. In the present study, the immunolocalisation of FasL and Fas, as well as the gene expression of FasL, were determined in the uterotubal junction (UTJ), isthmic (I) and ampullar (A) segments of the oviduct during oestrus and the luteal phase of the oestrous cycle. The degree of apoptosis of oviductal epithelium was examined by the TUNEL method. Oviductal fluid (ODF), collected chronically via indwelling catheters from the I or A segments during both non-luteal and luteal phases of the cycle, was analysed for the presence of FasL. The Fas immunostaining was scattered along the epithelium of all regions of the oviduct and cycle stages investigated, whereas FasL immunolabelling was more conspicuous in oestrous samples. This staining disappeared during the luteal phase, which was particularly evident in the sperm reservoir (UTJ and I). There were fewer TUNEL-positive cells than Fas- or FasL-positive cells in the oviductal epithelium, suggesting that tubal Fas and FasL are not directly involved in epithelial apoptosis. Western blot analyses detected FasL in ODF collected from both I and A, most conspicuously as a 24-27kDa band but also at a 40-45kDa band level. FasL mRNA was expressed in the epithelial cells from the sperm reservoir and A during both non-luteal and luteal phases. However, the level of expression differed significantly between segments during the luteal phase. The results provide novel evidence that the Fas-FasL system is present in the bovine oviduct and could be involved in mediating survival of spermatozoa and early embryos.     

Block of hamster fertilization by anti-ovary antibody.

Antibody produced in rabbits against hamster ovary blocked fertilization of golden hamster eggs in vitro by binding to the surface of the zona pellucida and rendering it impenetrable to spermatozoa. The antibody was also effective in blocking fertilization in vivo. An intraperitoneal injection of the antibody into females resulted in the complete inhibition of fertilization for approximately 12 days, i.e. three oestrous cycles. This temporary sterility was apparently due to the binding of the antibody to the zona pellucida of oviducal and ovarian oocytes, and dot due to a failure of sperm ascent or capacitation in the female genital tract. Neither the oestrous cycle nor ovulation was affected by the antibody injection.     

A tale of two cells: endocannabinoid-signaling regulates functions of neurons and sperm

Sea urchin and human sperm contain receptors for neurotransmitters and psychoactive drugs, including cannabinoid receptors (CNRs). Anandamide, arachidonoylethanolamide (AEA), is a lipid-signal molecule that is an endogenous agonist for CNRs. AEA is enyzmatically released from membrane phospholipids when neurons are stimulated. Retrograde AEA signals from depolarized postsynaptic neurons inhibit neurotransmitter release at synapses in mammalian brain. Analogous processes regulate sperm functions during fertilization in sea urchins. AEA and (-)delta9tetrahydrocannabinol [(-)delta9THC], the major psychoactive constituent of marijuana, inhibit fertilization by blocking acrosomal exocytosis/acrosome reactions (AR) stimulated by egg jelly. The acrosome is a Golgi-derived secretory granule in sperm analogous to synaptic vesicles in neurons. AEA and (-)delta9THC do not block ionophore-induced AR, suggesting that they inhibit AR by modulating signal transduction event(s) before opening of ion channels. Unfertilized sea urchin eggs have enzymes required to release AEA from membrane phospholipids. These results indicate that sea urchin eggs may release AEA after activation by the fertilizing sperm. Released AEA may then react with CNRs in nearby sperm to block AR, thereby helping to prevent polyspermy. AEA is present in human seminal plasma, midcycle oviductal fluid, and follicular fluid. Sperm are sequentially exposed to these fluids as they move from the vagina to the site of fertilization in the oviduct. R-methanandamide (AM-356), a metabolically stable AEA analog, and (-)delta9THC modulate capacitation and fertilizing potential of human sperm in vitro. These findings suggest that AEA signaling directly affects sperm functions required for fertilization and provide additional evidence for common signaling processes in neurons and sperm.     

Redox regulation of sperm surface thiols modulates adhesion to the fallopian tube epithelium

Sperm that adhere to the fallopian tube epithelium are of superior quality and adhesion extends their fertile life. It has been postulated that periovulatory signals, as yet undefined, promote sperm release. In the in vitro studies described here, we examined the effects of several antioxidants, reportedly present within oviductal fluid, on the modulation of sperm-oviduct adhesion in bovine species. Results showed that 1) the cell-permeant thiols (penicillamine, beta mercaptoethanol, cysteine, and dithiotreitol), as well as the nonpermeant thiol, reduced glutathione, cause adhering spermatozoa to release from the epithelium; 2) thiol action is exerted on spermatozoa; and 3) oxidized glutathione, as well as the non-thiol antioxidants (dimethylthiourea, trolox, superoxide dismutase, and catalase) have no effect. Sperm surface sulfhydryls labeled with iodoacetamide fluorescein showed that spermatozoa devoid of sulfhydryls on the head surface adhered to the fallopian epithelium in vitro, whereas thiol-induced release increased the exposure of sulfhydryls on the sperm head surface. Finally, analysis of capacitation status demonstrated that uncapacitated spermatozoa adhered to the oviduct, and that thiol-induced release of spermatozoa was accompanied by capacitation. In conclusion, thiol-reducing agents in the oviductal fluid may modulate the redox status of sperm surface proteins, leading to the release of spermatozoa selected and stored through adhesion to the fallopian tube epithelium in the bovine species.