A scanning electron microscopical study of sperm development and activation in Arenicola marina L. (Annelida: Polychaeta)

Abstract

The lugworm, Arenicola marina L. has an annual cycle of reproduction with epidemic spawning and external fertilisation. The spermatozoa of Arenicola are unusual in that they are held immotile (as plates of several hundred cells known as morulae) in the coelomic fluid until activated just prior to spawning. Activation of Arenicola sperm is brought about by a sperm maturation factor (SMF) from the prostomium and can be carried out in vitro using an assay technique developed by Bentley (1985). Scanning electron microscopy is used here to examine the changes which occur during in vitro activation. This revealed that the bundles of flagella of inactive sperm become disorganised as flagella beating commences but the flagella at this stage are still bound together at their tips. The sperm heads then become separated from the cytophore and finally the distal binding of the flagella is broken to give free-swimming spermatozoa. Coelomocytes present in the coelomic fluid resorb unspawned gametes prior to the initiation of the next gametogenic phase.     

Aspects of the reproductive biology of the South African polychaete,Arenicola loveni loveni (Kinberg 1866)

Summary

The annual cycles of gametogenesis, spawning and fertilization biology were investigated in the South African polychaete Arenicola loveni loveni (Kinberg, 1866). Sexes are strictly separate (gonochoric) with a 1:1 ratio and gametogenesis takes place in the coelomic cavity. Reproduction is iteroparous and occurs during a synchronized annual epidemic spawning event during the summer months of February or March, which lasts no longer than 2 weeks at around the time of highest annual seawater temperatures. A slight inter-annual variation in spawning time was observed between 2003 and 2004. Annual fecundity was estimated as 311,200 (± 82,040 standard deviations) oocytes per female per year. Oocytes are arrested at prophase I of the first meiotic division and require a maturation step prior to spawning and subsequent fertilization. Mean spawned oocyte diameter was 141.16 μm ± 0.43 SE. Sperm develop as morulae in the coelomic cavity and also require a maturation step prior to spawning. Spawning can be achieved by injection of prostomial homogenate in both males and females and by injection with 8,11,14- e cosatrienoic acid for males and can be delayed by temperature manipulation. Oocyte maturation and sperm maturation can be induced in vitro by incubating in prostomial homogenate.     

Sperm traits in relation to male quality in colonial spawning bluegill

Sperm traits (morphology, motility and concentration within ejaculates) and various correlates of male quality (age, body condition, spawning location and timing) were studied in bluegill Lepomis macrochirus , breeding in both the interior and periphery of six colonies in Lake Opinicon, Ontario, Canada. Sperm traits varied significantly more among than within males suggesting that some aspect of male phenotype might influence sperm morphology and behaviour. No measures of male body condition or size were correlated with any sperm or ejaculate traits, when controlling statistically for confounding variables. Sperm swimming speed increased significantly with male age and varied significantly among spawning bouts (controlling for sperm tail length) suggesting that some unknown aspects of male quality might influence the fertilization capacity of spermatozoa. Sperm concentration in ejaculates was significantly higher in males nesting in the interior rather than the periphery of a colony suggesting that those males might also have higher fertilization capacity correlated with their superior dominance status or the lower risk of sperm competition. Thus, older males nesting in the interior of a colony during the first spawning bout of the season are expected to be the best sperm competitors in this population, but the physiological reasons for this increased fertilization capacity remain unknown.     

Calcium signaling and the MAPK cascade are required for sperm activation in Caenorhabditis elegans

In nematode, sperm activation (or spermiogenesis), a process in which the symmetric and non-motile spermatids transform into polarized and crawling spermatozoa, is critical for sperm cells to acquire fertilizing competence. SPE-8 dependent and SPE-8 independent pathways function redundantly during sperm activation in both males and hermaphrodites of Caenorhabditis elegans. However, the downstream signaling for both pathways remains unclear. Here we show that calcium signaling and the MAPK cascade are required for both SPE-8 dependent and SPE-8 independent sperm activation, implying that both pathways share common downstream signaling components during sperm activation. We demonstrate that activation of the MAPK cascade is sufficient to activate spermatids derived from either wild-type or spe-8 group mutant males and that activation of the MAPK cascade bypasses the requirement of calcium signal to induce sperm activation, indicating that the MAPK cascade functions downstream of or parallel with the calcium signaling during sperm activation. Interestingly, the persistent activation of MAPK in activated spermatozoa inhibits Major Sperm Protein (MSP)-based cytoskeleton dynamics. We demonstrate that MAPK plays dual roles in promoting pseudopod extension during sperm activation but also blocking the MSP-based, amoeboid motility of the spermatozoa. Thus, though nematode sperm are crawling cells, morphologically distinct from flagellated sperm, and the molecular machinery for motility of amoeboid and flagellated sperm is different, both types of sperm might utilize conserved signaling pathways to modulate sperm maturation.     

Metabolism of intratesticular spermatozoa of a tropical teleost fish (Clarias gariepinus)

Sperm metabolism of a tropical fish species, the African catfish, Clarias gariepinus, was studied by measurements of sperm enzyme activity and metabolite levels. We also analysed the effect of metabolites, co-enzymes and enzymatic blockers on sperm motility behaviour and viability. Similar to other teleostean species, African catfish spermatozoa have the capacity for glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, lipid catabolism, beta-oxidation and osmoregulation. In immotile spermatozoa, lipid catabolism, beta-oxidation, the tricarboxylic acid cycle and oxidative phosphorylation were important primary energy-delivering pathways; sperm oxygen consumption was 0.39-0.85 microg O(2)/min/ ml of testicular semen. During motility, glycolysis, lipid catabolism and beta-oxidation of fatty acids occurred simultaneously, which is atypical for teleosts, and the spermatozoal respiration rate increased drastically by 15-25-fold. Also in contrast to other teleostean sperm cells, ATP levels remained stable during motility and immotile storage. The sperm cell status was unstable in the African catfish. Although the spermatozoa have osmoregulation ability, and even though balanced physiological saline solutions were used for sperm motility activation and sperm incubation, the motility and viability of spermatozoa quickly decreased at 28 degrees C, the spawning temperature of the African catfish. Cyclic AMP and inhibition of phosphodiesterase activity could not prolong sperm motility and viability. In contrast, at 6-10 degrees C motility was prolonged from approximately 30 s to >5 min, probably due to decreased metabolic rates.     

Sperm release behaviour and fertilization in the grass goby

Nesting males of the grass goby Zosterisessor ophiocephalus showed bouts, with intervals of c. 30 min duration, of upside-down movements while rubbing its genital papilla onto the ceiling of its burrow. Such behaviour was shown during female courting and spawning, and even after female removal. Observations showed that this behaviour was associated with the release of a sperm trail on the substratum and clumped spermatozoa in water, agglutinated with a mercury-bromophenol blue and PAS positive glycoprotein secretion of the sperm duct glands. Agglutination in the secretion delayed the activation of spermatozoa and contributed a steady supply, for up to 40 min, of motile spermatozoa during prolonged egg laying by the female. Sperm released before egg laying achieved c. 50% fertilization success compared with nearly 100% obtained if the sperm was released during egg laying. The sperm release behaviour may improve the nest owner's reproductive success against intruders or sneakers. It also allows defence of the nest while the female is spawning and may allow the male to court other females in the proximity.     

Changes in motility, ATP content, morphology and fertilisation capacity during the movement phase of tetraploid Pacific oyster (Crassostrea gigas) sperm

Changes in sperm features during the movement phase are especially interesting to study in external fertilization species whose sperm duration movement is long because this implies a significant adaptation of moving cells to the external medium. This study describes the changes in tetraploid Pacific oyster sperm characteristics in relation to time post activation.Sperm individually collected on three tetraploid males were activated in seawater. Their features were analysed over a 24 h period and compared to a sperm pool collected on three diploid males as a reference. The percentage of motile spermatozoa, the intracellular ATP content, and the fine structure of spermatozoa were studied in relation to time post activation. Furthermore, the fertilisation capacity of sperm individually collected on five diploid males was assessed after 1 and 24 h post activation.A forward progressive movement was maintained for at least a 20 h duration. Compared to diploid males, the percentage of motile spermatozoa was lower in tetraploid males. The intracellular ATP concentration was higher in spermatozoa from tetraploid males than in spermatozoa from diploid males. A decrease in ATP content was observed in the first 6 h post activation and severe alterations were observed in sperm morphology after 24 h. Then, a lower fertilisation capacity of sperm from diploid males was observed at the end of the movement phase.The cessation of Pacific oyster sperm motility was unlikely caused by ATP consumption as ATP concentration was still high at the end of sperm movement but rather caused by drastic changes in sperm morphology. Compared to sperm collected on diploid males, the lower quality of sperm from tetraploid males was emphasized by a shorter movement duration and deeper morphological alterations at the end of the movement phase.     

Relationships between reproductive characteristics in male Vimba vimba L. and the effects of osmolality on sperm motility

In Vimba vimba, GSI, sperm volume, and spermatozoa concentration range from 3.4-7.4 %, 0.1-1.1 ml, and 13.3-34.8 × 10⁹ spz ml⁻¹, respectively. Gonad mass (r = 0.90) and sperm volume (r = 0.35) significantly correlated with weight of males. Significant correlation was also found between gonad mass and length of males (r = 0.85). Sperm motility (r = 0.99) and velocity (r = 098) significantly decreased after activation in Tris-HCl 20 mM, pH 8.5. Osmolality of the seminal plasma was 273.2 mOsmol kg⁻¹. Sperm motility and velocity were significantly affected by the osmolality of the activation medium (P < 0.01). Hyper-osmolality compared to seminal plasma osmolality totally suppressed the sperm activation. At 15 s post-activation, the sperm motility significantly decreased at 240 mOsmol kg⁻¹ in KCl or NaCl media. The highest sperm motility and velocity (at 60 s post-activation) were observed at 200 mOsmol kg⁻¹ in NaCl, KCl, or sucrose media. In all treatments, the tip of the flagellum of spermatozoa became curled into a loop shape after activation of sperm in distilled water containing 20 mM Tris-HCl, pH 8.5 that shortened the flagellum.     

Spawning synchrony in Arenicola marina: evidence for sex pheromonal control

Chemical communication systems controlling reproductive behaviour have been shown in a number of marine polychaetes. This study investigated the use of sex pheromones to coordinate spawning behaviour in gravid lugworms (Arenicola marina). Lugworms typically reproduce in the autumn, during low water of spring tides, and often exhibit epidemic spawning. Females release gametes within the burrow whereas males deposit spermatozoa on to the beach surface. The incoming tide dilutes the spermatozoa and transports them to the females'' burrows. Sperm is diluted rapidly and sperm concentrations fall below the minimum required for fertilization within a few minutes. The present investigation establishes the existence of chemical signals synchronizing spawning for the first time in an iteroparous polychaete. The process can be divided into two steps, the induction of gamete release by waterborne chemical cues and burrow irrigation behaviour in females: burrow irrigation representing the means by which spermatozoa are carried to the eggs. In both sexes, the release of gametes can be induced by exposure to sea water into which other individuals had previously spawned. Males also respond to odour compounds from other males. The overall effect of the chemical signals results in synchronized, mass spawning of a population.     

Capacitation of hamster spermatozoa with the divalent cation chelators D-penicillamine,L-histidine,and L-cysteine in a protein-free culture medium

By using a chemically defined (protein-free) culture medium that supports sperm viability but not capacitation or the acrosome reaction, we have determined that hamster spermatozoa can be chemically capacitated in vitro by the divalent cation chelators D-penicilla-mine, L-histidine, and L-cysteine in the absence of bovine serum albumin (BSA). Washed cauda epididymal spermatozoa were preincubated (1–2 × 10⁶ sperm/ml) for 3, 4, or 6 hr at 37°C in 5% CO₂ in air. The basic culture medium used for sperm preincubation and for sperm:egg coincubation was a modified Tyrode's solution (protein-free) containing 10 mM sodium lactate, 100 μM sodium pyruvate, and 1.0 mg/ml polyvinylalcohol (TLP-PVA). Sperm viability was maintained in all preincubation and coincubation media with PHE (20 μM D-penicillamine, 100 μM hypotaurine, and 1.0 μM epinephrine). The low control sperm preincubation medium consisted of TLP-PVA. In some cases the high control preincubation medium also contained 3 mg/ml BSA (TALP-PVA). The experimental preincubation medium was TLP-PVA with additional D-penicillamine (125 or 500 μM), or L-histidine (10, 100, or 1,000 μM) or L-cysteine (25, 75, or 125 μM). After preincubation, sperm were coincubated (2 × 10⁴ sperm/ml) with cumulus-free hamster eggs in TALP-PVA ± additional D-penicillamine, L-histidine, or L-cysteine for 1.5 hr, fixed, and evaluated for percent egg penetration as an index of sperm capacitation. The results demonstrate that hamster spermatozoa can be chemically capacitated in vitro with D-penicillamine (500 μM: range of mean penetration values, 53.6%–84.3%), L-histidine (100 μM: range of mean values, 24.8%–56.3%) or L-cysteine (75 μM: 51.3%) in the absence of exogenous protein.     

Sperm quality, secondary sexual characters and parasitism in roach (Rutilus rutilus L.)

According to sperm competition models, a male spawning in a disfavoured role should have spermatozoa with higher velocity but shorter longevity compared with a male spawning in a favoured role. Moreover, immunosuppressive androgens are needed to produce both secondary sexual characters and sperm cells. The 'sperm protection' hypothesis suggests that the immunosuppressive action of androgens has evolved to protect haploid spermatozoa, which are antigenic, from autoimmune attacks. Therefore, a male with high sexual ornamentation may be more susceptible to diseases but may possess better quality ejaculate than his less ornamented rival. We studied sexual ornamentation (breeding tubercles), ejaculation quality (sperm concentration, longevity and spermatozoal velocity) and intensity of parasitism in the cyprinid, Rutilus rutilus . Sperm longevity and spermatozoal velocity were positively correlated. Males having elaborated sexual ornamentation had longer-lived sperm and more Myxobolus mülleri parasites in the liver compared with males with low ornamentation. However, no difference was found between males with different degrees of ornamentation with respect to sperm concentration, spermatozoal velocity or other parasites. Since the highly ornamented males had higher sperm longevity, the present results are partly consistent with the predictions of the sperm competition models and the 'sperm protection' hypothesis.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 81 , 111–117.     

Sperm quality evaluation in Solea senegalensis during the reproductive season at cellular level

Sperm quality seems to be one of the reasons for the reproduction constraints faced by Senegalese sole (Solea senegalensis) aquaculturists. Previous studies in this species indicated that the sperm quality of individuals kept in culture varies throughout the year and that different sperm subpopulations can be identified in ejaculates according to the motility pattern of spermatozoa. Aiming to better understand factors affecting sole sperm quality in captivity, sperm of 11 males was assessed during the reproductive season using different parameters: motility characteristics using CASA analysis; cell plasma membrane resistance to seawater hyperosmolarity; DNA fragmentation with single-cell gel electrophoresis; and early apoptosis, labeled with Annexin-V FITC. Computer-assisted sperm analyses motility data were treated using multivariate analysis to identify the presence of different spermatozoa subpopulations according to their motility pattern. Four distinct sperm subpopulations were obtained: Subpop1, which includes fast linear spermatozoa; Subpop2, made up of fast nonlinear spermatozoa; Subpop3, which includes slow linear spermatozoa; and Subpop4, which contains slow nonlinear spermatozoa. The sperm subpopulation structure varied with time after activation and with male. Low cell resistance to the seawater hyperosmotic conditions was noticed. The Annexin-V assay allowed the identification of an apoptotic population ranging from 6% to 20%. A high percentage of cells (64.1%) showed a DNA fragmentation level below 30%, but these values varied significantly between males. DNA fragmentation appears to be related to cell membrane resistance to hyperosmotic conditions faced by the cells when in contact with seawater. This condition seems to modulate the composition of the motile sperm population and performance after activation. This phenomenon could be related to the spermatozoa maturation process.     

Inhibition of the mitochondrial permeability transition pore reduces “apoptosis like” changes during cryopreservation of stallion spermatozoa

In order to evaluate to what extent the changes that occur during cryopreservation involve the mitochondrial permeability transition pore (PT-pore), a specific inhibitor was used during the cryopreservation process of stallion spermatozoa. Four ejaculates from each of 7 stallions were frozen using a standard protocol. Before freezing, each ejaculate was split into three subsamples. The first was supplemented with 2.5 μM bongkrekic acid (BA) and the second with 5 μM BA. The third subsample served as control. The BA significantly reduced the percentage of spermatozoa depicting active caspases after thawing, reduced the percentage of spermatozoa with increased membrane permeability, and increased the mitochondrial membrane potential of thawed sperm. Sperm motility was reduced as a result of the treatment. It is concluded that the mitochondrial pathway of apoptosis seems to be an important factor involved in the sublethal damage that equine spermatozoa experience after freezing and thawing, and that sperm motility in the equine species is largely dependent on mitochondrial ATP produced by oxidative phosphorylation.     

An ultrastructural study of spermatogenesis and sperm morula breakdown inArenicola marina (L.) (Annelida: Polychaeta)

Spermatogenesis in the lugwormArenicola marina, in common with other members of Arenicolidae, occurs in the coelomic fluid and results in the formation of discs of mature spermatozoa known as a morulae. Within a morula, individual spermatozoa are connected by a common mass of cytoplasm called the cytophore and therefore make up a syncitium. Immediately prior to spawning, and in response to an endocrine substance known as “Sperm Maturation Factor” (SMF), the structure of the sperm morulae breaks down and free spermatozoa are liberated. These are subsequently spawned from the body cavity. The investigation described here uses transmission electron microscopy to investigate the ultrastructural changes, which accompany spermatogenesis and the breakdown of sperm morulae in response to SMF in vitro. The study demonstrates that the cytophore appears to have a key role both during spermatogenesis and during sperm morula breakdown. The ultrastructure of sperm morulae and of mature spermatozoa is described. The structure of spermatozoa is shown to be primitive with a single flagellum which appears to be coiled at its distal end. The phagocytosis of free spermatozoa by coelomocytes is also described and it is suggested that these may play a role in the resorption of unspawned gametes in vivo.     

Relationship between sperm density, spermatocrit, sperm motility and spawning date in wild and cultured haddock

Semen was collected repeatedly from captive haddock Melanogrammus aeglefinus and the effect of seasonality on various sperm parameters was investigated. No differences in sperm traits were observed for wild and cultured haddock. A highly significant positive relationship existed between spermatocrit and spermatozoa density. A significant increase in mean spermatocrit occurred throughout the spawning season but the amount of variability explained by collection date was low (35·1%) due to variability between males. Each of 10 males sampled repeatedly throughout the spawning season demonstrated an increase in spermatocrit. No relationship existed between spermatocrit and proportion of motile spermatozoa when spermatocrit was ≤70%. Motility was reduced in semen samples with spermatocrits >70%. The proportion of spermatozoa that were motile decreased with time since activation. Some motility was still observed after 60 min in sea water (0·1–15·2%) for sperm collected at all times within the spawning season. Of those spermatozoa that were motile, the proportion that exhibited forward swimming motion decreased and the proportion that had only vibratory movement increased with time post‐activation. The speed of forward swimming spermatozoa showed no significant relationship with spermatocrit at any time between 0 and 60 min after activation. Swimming speed was negatively related to time since activation, decreasing from 174–240 μm s⁻¹ at 0 min to 80–128 μm s⁻¹ at 60 min after activation.     

Motility activation and metabolism characteristics of spermatozoa of the black-lip-pearl oyster Pinctada margaritifera var: cumingii (Jameson, 1901)

Motility of Pinctada margaritifera (Linnaeus, 1758); var: cumingii (Jameson, 1901) (P. margaritifera) spermatozoa collected from gonads are not immediately activated at spawning in seawater (SW) but motility occurs when spermatozoa are transferred into alkaline seawater (pH ranging from 9.0 to 11.4). This motility-activating effect of alkaline pH is reversed when pH is shifted back to more acidic values. In both cases, activity of sperm (% motile cells) increases gradually after alkaline pH activation then lasts for several minutes. The characteristics of these fully motile spermatozoa are described in details at the level of flagella: the wave amplitude and wave-length range 5 to 6 μm and 15 μm respectively, while the flagellar beat frequency is approximately 49 Hz. The velocity of sperm displacement is from 220 to 230 μm/sec. The general swimming pattern is almost circular: the head trajectories describe portions of circles intercalated with small linear segments. Spermatozoa saved in natural seawater at 4°C retain potent motility for several days and can be subsequently activated by alkaline seawater. Respiration and ATP concentration were measured in 3 conditions: regular seawater (pH 7.8), artificial diluent (pH 8.2), and alkaline Tris-buffered seawater (pH 10.5). Results show that sperm respiration rates are higher whereas ATP levels are lower in the latter two media.     

Sperm motility and fertilizing ability of frozen spermatozoa of males (XY) and neomales (XX) of perch (Perca fluviatilis)

The objective of this study was to freeze sperm of sex‐reversed females (neomales) of perch and to test their fertilization ability. Sperm used was testicular (TSN), collected from females that have been inverted by means of externally administered 17‐alpha methyltestosterone. Sperm collected from intact males (SSNM) of the same origin were used as control. Prior to freezing, both TSN and SSNM were diluted into 300 mm glucose solution at the ratio of 1 : 6 and DMSO was used as cryoprotectant (10% final concentration). Crypreservation was performed in 0.5 ml straws placed into a polystyrene box, three cm above the liquid nitrogen level for 10 min and thereafter transferred fully into liquid nitrogen. Samples were thawed in 40°C water bath for 8 s and used for the fertilization experiments. Spermatozoa concentration of fresh TSN and SSNM were estimated with 45.3 × 10⁹ and 37.8 × 10⁹ spermatozoa ml^?1, respectively. Both sperm velocity and motility showed significant decreases in the TSN (134.6 μm s^?1 and 12.8%) compared to the SSNM (203.2 μm s^?1 and 94.7%) at 10 s after sperm activation. However, no differences were observed in terms of hatching rates between fresh TSN and SSNM (42.5 vs 49.3%) at fertilization densities of 12 × 10⁵ spermatozoa per egg. Frozen/thawed SSNM exhibited similar hatching rates at 12 × 10⁵ and 2.4 × 10⁵ spermatozoa per egg (37.2% vs 29.1%). Hatching rates for frozen/thawed TSN were about 7.3% with 12 × 10⁵ spermatozoa per egg and did not show any difference at 2.4 × 10⁵ spermatozoa per egg (6.6%). Stripped sperm of normal perch can be successfully frozen. Squeezing of the testes is not a good method for collection of testicular sperm resulting into low velocity, motility and hatching rate. To understand the influences of neomales on sperm quality on reproductive success further studies should be performed addressing a full assay of motility and fertility criteria when using stripped sperm from normal males and neomales. Additionally, the results indicate that many of sex reversed perch neomales are not able to release sperm and that for further studies some well spermiating neomales must to be selected.     

Some environmental factors ifluencing the activity of spermatozoa of Mugil capito Cuvier, a grey mullet

The period of activity of mullet spermatozoa suspended in seawater was similar to that of sperm suspended in osmolar equivalent dextrose solution or artificially prepared seawater. Sperm activity was longest in solutions of pH and osmolarity closely approaching that of normal seawater. Temperatures lower than those at which spawning normally occurs greatly increased the period of activity. Undiluted milt stored at 10° C couid be activated by the addition of seawater for a period of 36 h.     

Nereis succinea nuptial behavior: Does size matter?

Summary

Although Darwin noted that size may affect sexual selection, the effect of size on reproduction is controversial. Mature Nereis succinea, a polychaete that mates in pheromone-mediated twilight nuptial swarms, varies greatly in size, ranging more than 10-fold in body weight from 30 mg to >300 mg. Swim speed of swarming male worms increases with worm size, with the fastest males swimming more than twice the speed of the slowest. The female-produced spawning pheromone, nereithione, stimulates both swimming speed and spawning in males at concentrations of 10^?6 M and above, and lower concentrations cause significant activation of swimming. Individual worms can be stimulated to release sperm up to 40 or more times in a single experimental session. Larger worms release cumulatively more sperm than smaller ones, resulting in significant loss of body mass from repetitive spawning activated by nereithione. Size may enhance mating success of male N. succinea due to encountering more females as a result of faster swimming speed and due to the higher sperm density and number of spawning responses of large animals.     

Unsaturated fatty acid effect on cyclic amp levels in human embryo lung fibroblasts

The addition of arachidonic acid at 250 μM to cultures of human embryo lung fibroblasts (IMR-90) increases cellular cyclic AMP levels within 5 minutes to approximately 15-fold over basal. Other unsaturated fatty acids, 11, 14, 17-eicosatrienoic, linoleic, 8, 11, 14-eicosatrienoic and oleic also cause similar rapid elevation of cellular cyclic AMP. During this time interval, no detectable conversion of the added linoleic or arachidonic acids to prostaglandin is observed. These cells produce prostaglandins at measurable concentrations in response to treatment with ascorbic acid or bradykinin. Saturated fatty acids have no influence on cyclic AMP levels in these cells. This effect of unsaturated fatty acids on cellular cyclic AMP levels varies with the cell type. For example, smooth muscle and endothelial cells obtained from the calf pulmonary artery show very little or no increase in cellular cyclic AMP upon exposure to arachidonic acid.