Herbert Schuel

N-Acylethanolamines in human reproductive fluids

N-Acylethanolamines (NAEs) are an important family of lipid-signaling molecules. Arachidonylethanolamide (anandamide) (AEA), palmitoylethanolamide (PEA), and oleoylethanolamide (OEA) are co-produced from similar phospholipid precursors when neurons are stimulated. AEA is an endogenous agonist (endocannabinoid) for cannabinoid receptors. It binds with higher affinity to type CB1 than to type CB2 cannabinoid receptors. PEA does not bind to CB1, while the hypothesis that it reacts with putative CB2-like receptors has been questioned. OEA does not activate currently known cannabinoid receptors, but it mimics the effects of AEA and cannabinoids in reducing the fertilizing capacity of sea urchin sperm. OEA and PEA also act as entourage compounds by inhibiting the hydrolysis of AEA by fatty acid amide hydrolase. Cannabinoid receptors and/or AEA are present in mammalian reproductive organs including the testis, epididymis, prostate, ovary, uterus, sperm, preimplantation embryo and placenta, as well as prostatic and mammary carcinomas. We now report that analysis by high-performance liquid chromatography/mass spectrometry (HPLC/MS) shows the presence of AEA, PEA, and OEA in human seminal plasma, mid-cycle oviductal fluid, follicular fluid, amniotic fluid, milk, and fluids from malignant ovarian cysts. Previous studies showed that AEA-signaling via cannabinoid receptors regulates capacitation and fertilizing potential of human sperm, early embryonic development and blastocyst implantation into the uterine mucosa of rodents, as well as proliferation of human mammary and prostatic carcinomas. Current results imply that NAEs also may modulate follicular maturation and ovulation, normal and pathological ovarian function, placental and fetal physiology, lactation, infant physiology, and behavior. Collectively, these findings suggest that NAEs in human reproductive fluids may help regulate multiple physiological and pathological processes in the reproductive system, and imply that exogenous cannabinoids delivered by marijuana smoke might impact these processes. This study has potential medical and public policy ramifications because of the incidence of marijuana abuse by adolescents and adults in our society, previously documented reproductive effects of marijuana, and the ongoing debate about medicinal use of marijuana and cannabinoids.

Heterogeneous distribution of "lysosomal" hydrolases in yolk platelets isolated from unfertilized sea urchin eggs by zonal centrifugation.

Abstract Three typical “lysosomal” glycosidases, α-L-fucosidase, N-acetyl glucosaminidase and N-acetyl galactosaminidase, were localized within the yolk platelets of unfertilized Strongylocentrotus purpuratus eggs. Homogenates of eggs were fractionated by rate-zonal centrifugation, and the isolated particles were subjected to integrated biochemical and morphological (electron microscopic) analysis. Enzymatic markers were used to determine the distribution of mitochondria (cytochrome oxidase), yolk platelets (acid nitrophenyl phosphatase), and cortical granules (β-1,3 glucanase) in the sucrose density gradient. Yolk platelets were isolated in a high state of purity, with contamination by mitochondria and cortical granules at trace levels. Enzymatic heterogeneity exists within the yolk platelet population. Acid nitrophenyl phosphatase and α- l -fucosidase activities appear to be uniformly distributed within all the yolk platelets, while N-acetyl glucosaminidase and galactosaminidase activities appear to be preferentially distributed within the slower sedimenting sub-population of yolk platelets. Another band of hexosaminidase containing particles sedimented slightly slower than the bulk of the yolk platelets, coincident with the mitochondria. The acid hydrolases packaged in the yolk platelets may participate in the mobilization of yolk material after fertilization. The yolk platelet thus appears to be a highly complex and structured “lysosome-like” storage organelle.

A rapid sodium-dependent block to polyspermy in sea urchin eggs☆

The rapid electrical depolarization of the eggs plasma membrane which protects sea urchin ova against polyspermy in the interval between stimulation by the fertilizing spermatozoon and completion of the cortical reaction is believed to be mediated by the influx of sodium (Na+) ions. This hypothesis was tested in Arbacia punctulata and Strongylocentrotus purpuratus by inhibiting the rapid block to polyspermy with low-Na+ (choline-substituted) seawater, and the cortical granule secretion-mediated block with soybean trypsin inhibitor (SBTI). Eggs inseminated in low-Na+ seawater or SBTI became heavily polyspermic. Polyspermy elicited by low Na+ or SBTI was increased in dejellied Strongylocentrotus eggs. However, the severity of polyspermy was not enhanced in low Na+ plus SBTI because the fertilizing capacity of sperm and gamete binding were reduced in low-Na+ media. Since SBTI completely suppresses the cortical granule secretion-mediated block to polyspermy in Arbacia for about 3 min postinsemination, the rate at which SBTI-treated eggs became polyspermic was used to measure the duration and efficacy of the rapid block. The half-time for SBTI-treated Arbacia eggs to become polyspermic in natural (425 mM Na+) seawater was 89.9 ± 4.7 sec (N = 4). The plot of incidence of polyspermy vs time was essentially an inverse mirror image of electrophysiologic data on repolarization of the oolemma during fertilization. The rapid block is also Na+ dependent, since SBTI-treated eggs became polyspermic more rapidly in 26 mM Na+ seawater (half-time, 15.8 ± 1.6 sec; N = 3, P < 0.01).

Purification of cortical granules from unfertilized sea urchin egg homogenates by zonal centrifugation.

Abstract Cortical granules were purified from homogenates of unfertilized sea urchin, Strongylocentrotus purpurataus, eggs by rate-zonal centriguation. Purification was evaluated by integrated biochemical and morphological (electron microscopic) analysis. The enzyme β-1,3-glucanase was employed as the biochemical marker for cortical granules. Cortical granule preparations obtained in sucrose gradients constructed in 0.5 M KCl contained acid phosphatase activity and were heavily contaminated by yolk platelets. Highly purified cortical granules isolated in sucrose gradients constructed in 0.5 M KCl with 10−3 M EDTA at pH 7.0 were almost completely free of contamination by yolk and other cytoplasmic particles, and contained only trace levels of acid phosphatase activity. The purified cortical granules contained 40–45% of the total β-1,3-glucanase activity in the eggs, and 6.7 ± 1.9% of the total egg protein. The isolated cortical granules retained another known chemical constituent, sulfated acid mucopolysaccharides.

Polyspermic fertilization of sea urchin eggs treated with protease inhibitors: Localization of sperm receptor sites at the egg surface☆

The normal elevation of the fertilization membrane and the establishment of the block to polyspermy are retarded in Arbacia punctulata eggs by specific protease inhibitors, soybean trypsin inhibitor (SBTI), leupeptin, and antipain. Ultrastructural observations show that the vitelline layer remains attached to the plasma membrane of fertilized SBTI treated eggs at numerous sites (cortical projections). Quantitive morphometric analysis indicates that the vitelline layer elevates from about 65% of the surface of SBTI treated eggs during the first 3 min post insemination. However, the vulnerability of SBTI treated eggs to refertilization (polyspermy) only declined during the subsequent gradual detachment of the vitelline layer from the cortical projections over the next 15 min. Antipain and leupeptin (10−5 to 10−3 M) also promoted polyspermy in Arbacia eggs by a process of refertilization extending for a 10- to 15-min period after the initial monospermic insemination. Normal cleavage and development was obtained when eggs were placed in leupeptin and antipain (10−3 M) after the fertilization membrane had elevated. The data indicate that the normal secretory function (or functions) of the cortical granule protease in establishing the block to polyspermy is retarded by these protease inhibitors, and that the vitelline layer is transformed into a mechanical barrier to prevent penetration by supernumerary sperm during its detachment from the plasma membrane of the egg. Furthermore, the vitelline layer in unfertilized eggs appears to be a mosaic structure, with sperm receptor sites localized in regions of the eggs surface, which give rise to cortical projections in the presence of SBTI.

Cannabinoids inhibit fertilization in sea urchins by reducing the fertilizing capacity of sperm

Delta-9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) inhibit fertilization in the sea urchin Strongylocentrotus purpuratus by reducing the fertilizing capacity of the sperm. Sperm fertility depends upon their motility, and their capacity to undergo the acrosome reaction upon encountering a specific ligand derived from the eggs jelly coat. The acrosome reaction involves exocytosis of the acrosomal granule at the apex of the sperm head and elongation of the acrosomal filament. This process exposes the sperm membrane that will attach to and fuse with the egg. Pretreatment of sperm with THC prevents the triggering of the acrosome reaction by solubilized egg jelly in a dose and time dependent manner. Motility of THC-treated sperm is not reduced compared to control sperm in sea water or vehicle dissolved in sea water. The adverse effects of THC on the acrosome reaction and sperm-fertilizing capacity are reversible. Studies with ionophores suggest that THC blocks the acrosome reaction by affecting event(s) in the stimulation-secretion coupling mechanism in the sperm preceding the opening of ion channels. Ultrastructural studies show that THC, CBD and CBN block the membrane fusion reaction between the sperms plasma membrane and the acrosomal membrane that normally is elicited in response to stimulation by egg jelly to initiate the acrosome reaction. However, lipid deposits are found in the subacrosomal and centriolar fossae of cannabinoid treated sperm. The nuclear envelope is fragmented in close proximity to the lipid deposits within the subacrosomal fossa. These morphological observations suggest that cannabinoids may activate phospholipase(s) within the sperm. Biochemical studies show that THC activates phospholipase A2 activity in sperm homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of soybean trypsin inhibitor induced polyspermy as determined by an analysis of refertilized sea urchin (Arbacia punctulata) eggs.

Abstract Soybean trypsin inhibitor (SBTI) retards the separation of the vitelline layer from the surface of Arbacia eggs and the formation of the fertilization membrane. Numerous loci of vitelline layer (cortical projections) remain associated with the plasma membrane of SBTI-treated zygotes immediately after insemination. Gradually the attached portions of vitelline layer decrease in number and by approximately 15 min postinsemination the cortical projections are sparse or nonexistent. Monospermic SBTI-treated zygotes show a corresponding decline in both the incidence and degree of refertilization (polyspermy) upon reinsemination during this period. Ultrastructural analysis demonstrates that refertilizing spermatozoa are able to fuse with and penetrate SBTI-treated zygotes only at the apical surface of the cortical projections, i.e., where the vitelline layer remains attached to the plasma membrane of the zygote. These data indicate that the vitelline layer lining the apical surface of the cortical projections retains structural and functional properties characteristic of the surface of the unfertilized ovum.

SEPARATION OF INTACT CORTICAL GRANULES FROM HOMOGENATE OF UNFERTILIZED SEA URCHIN EGGS BY ZONAL CENTRIFUGATION

Cortical granules are localized in the peripheral cytoplasm of mature, unfertilized eggs. The rupture of these granules constitutes one of the initial morphologically observable responses of the egg to fertilization or parthenogenetic activation. These organelles were separated from homogenates of unfertilized sea urchin eggs (Strongylocentrotus purpuratus) by fractionation in the A-XII zonal centrifuge using sucrose density gradients constructed in (0.5 M) KCl. Cortical granules were abundant in electron micrographs of the most rapidly sedimenting population of particles. This population was rich in acid nitrophenylphosphatase activity, contained particles which stained metachromatically with toluidine blue and also contained yolk platelets. Acid phosphatase activity was also associated with several populations of slower sedimenting particles. Yolk platelets were widely distributed in the gradient, and were found in all fractions examined. The mitochondria and cytochrome oxidase activity were sedimented a short distance from the starting boundary.

Cannabinoid Receptors in Sperm

Mammalian and invertebrate sperm contain receptors for a wide variety of neurotransmitters that regulate sperm functions required for fertilization such as motility and the initiation of the acrosome reaction. The acrosome reaction is a ligand-stimulated secretory event in sperm that enables the sperm to penetrate the egg’s investments and to fuse with the egg’s plasma membrane. Previous studies in our laboratory using [3H]CP-55,940 showed that sea urchin sperm contain cannabinoid receptors that are remarkably similar to cannabinoid receptors found in mammalian brain and peripheral organs. Cannabinoid agonists and anandamide (an endogenous ligand for cannabinoid receptors in mammalian tissues) inhibit fertilization in sea urchins by blocking the acrosome reaction. These findings, taken together with other studies showing that the gene for the human brain cannabinoid receptor also is expressed in the human testis and that anandamide is synthesized in the female reproductive tract in mammals, suggested to us that human sperm may contain cannabinoid receptors. We have obtained preliminary evidence that [3H]CP-55,940 binds to putative cannabinoid receptors in live human sperm in a saturable manner, and that cannabinoid ligands affect in vitro capacitation of human sperm. These findings show that functional cannabinoid receptors are present in sperm, suggest that sperm cannabinoid receptors and their endogenous ligands may regulate normal sperm functions required for fertilization within the female reproductive tract in humans, and also imply that smoking marijuana may affect these processes in vivo.

UREA PARTHENOGENETICALLY ACTIVATES THE CORTICAL REACTION AND ELONGATION OF MICROVILLI IN EGGS OF THE SEA URCHIN, STRONGYLOCENTROTUS PURPURATUS

Isotonic urea is believed to activate sea urchin eggs by triggering event(s) that normally follow cortical granule secretion at fertilization, particularly surface perturbations that result in elongation of microvilli (Mazia et al., 1975). However, Moser (1940) reported that urea triggered the cortical reaction. Transmission electron microscopy showed that unfertilized Strongylocentrotus purpuratus eggs discharge their cortical granules in isotonic urea (containing 1.0 to 0.1 mM CaCl2 or 25 mM EGTA) to form incipient fertilization envelopes and hyaline layers. These investments quickly disperse in urea. Elongation of microvilli follows cortical granule discharge. Urea-activated eggs can be fertilized after return to sea water and fail to elevate fertilization envelopes but do form hyaline layers. Hyalin must be secreted from a secondary reservoir in these eggs, since the cortical granule store is discharged during the prior urea activation. Cortical granule secretion and elongation of microvilli do not oc...