A cytologic analysis of the bag cell control of egg laying in Aplysia

A fine structural analysis of the ovotestis in Aplysia was undertaken in order to analyze the site of action of the bag cell hormone. Five stages of oocyte development are described. Of particular interest is the fact that the yolk seems to be synthesized primarily by the granular endoplasmic reticulum. In addition, small muscle cells whose long, thin processes surround the follicle of the ovotestis have been pointed out. This paper suggests that bag cell extract has a direct action on these small muscle cells causing them to contract and thus expel oocytes from the ovotestis. The evidence for this suggestion is that (1) these muscle cells are the most obvious effector cells in the ovotestis, (2) there are no signs of neural innervation of these muscles, (3) the time course for the liberation of the oocytes is so short that any other method of oocyte release is unlikely, (4) there is no cytologic evidence for any other expulsion process except muscular contraction, and (5) the ripe oocytes are attached to other cells of the wall of the ovotestis only by very small, simple junctions, thus making them the most likely cells to be expelled by muscular contraction.     

DEVELOPMENT OF ACCESSORY SEXUAL ORGANS IN BIOMPHALARIA GLABRATA (PLANORBIDAE) IN RELATION TO TIMING OF INFECTION BY SCHISTOSOMA MANSONI: CONSEQUENCES FOR ENERGY UTILIZATION PATTERNS BY THE PARASITE

The growth rates of ovotestis and individual accessory sexualorgans (ASO) of Biomphalaria glabrata snails were studied forcontrols and for immature and mature snails infected with Schistosomamansoni. The infection of immature B. glabrata strongly delaysgrowth of the ovotestis and inhibits the development of theaccessory sexual organs. There is no significant differenceup to 2 weeks post infection in the volume of the ovotestisand the ASO between mature infected B. glabrata and controlsnails. From 3 to 4 weeks post infection there was a reductionin the volume of the ovotestis and the ASO of infected matureB. glabrata; then growth of the ovotestis, albumen gland andfemale organs was stopped, but the effect of infection was lessconsistent for the male organs. For a parasite, immature andmature snails have to be considered as two different resourceenvironments, each having at infection time a particular patternof resource allocation, towards growth for juvenile and towardsreproduction for adult snails, changing the possible energyutilization patterns which can be used by the trematode. (Received 29 January 1993; accepted 22 April 1993)     

Differential hormonal action of the bag cell neurons on the arterial system ofAplysia

Summary The peptide-secreting bag cell neurons ofAplysia californica activate a long-lasting, complex behavior called egg laying. During egg laying some organ systems (reproductive) are more active than others (digestive) suggesting that blood flow to these tissues may change in accordance with their activities during egg laying. To examine this possibility we used a semi-intact preparation of the three major arteries innervated by the abdominal ganglion. We found that electrically stimulated bursts of bag cell activity triggered a long-lasting (>1 h) increase in contractile activity in two arteries, the anterior and gastroesophageal, but did not affect contractions of the third (abdominal) artery. The arterial responses were not affected either in form or duration by denervation of the arteries, suggesting that the increase in contractile activity was mediated by hormonal actions of bag cell transmitters on vasoconstrictor muscles. In intact animals this differential action on the arterial system may cause a long-term decrease in blood flow to relatively inactive tissues (digestive and locomotory organs) while increasing circulation to tissues involved in egg production (ovotestis and oviduct).Abbreviations ASW artificial sea water - BCA bag cell activation - ELH egg laying hormone     

Studies on the Neurosecretory Control of Egg Laying in Aplysia

Studies are reviewed on the electrophysiological and endocrinologicalfeatures of a group of neurosecretory cells (the big cells)in the abdominal ganglion of Aplysia. Electrophysiological studiessuggest that the bag cells are involved in the regulation ofa phasic behavioral function such as egg laying. Egg layingoccurs approximately one hour following the injection into thehemocele of a crude extract of the bag cells or of the perfusateof an abdominal ganglion in which a pleural abdominal connectiveis electrically stimulated. The bag cells appear to act as aunit, releasing a measured dose of egg laying hormone when theyare triggered into activity.     

Isolation of an egg-laying hormone-binding protein from the gonad of Aplysia californica and its localization in oocytes

A protein solubilized from a membrane preparation of the gonad of Aplysia californica has been isolated by affinity chromatography, using bag cell egg-laying hormone (ELH) as the bound ligand, and partially purified and characterized by gel electrophoresis. The protein has an apparent molecular weight of 52 kDa and consists of two disulfide-linked subunits of about 30 kDa each. The protein is glycosylated and has an acidic pI. Approximately 10–15 g of this protein can be isolated from a single ovotestis, representing less than 1% of the total protein in the gonad; but the protein could not be detected in buccal mass or body wall, tissues which do not have apparent response to ELH.Antibodies generated against this ELH-binding protein (ELHBP) were used to localize sites in the ovotestis which might contain this molecule and thus represent targets for egg-laying hormone. Immunocytochemical results indicate that the oocytes are a rich source of this protein, since their cytoplasm was the only detectable site of immunoreactivity.Whether this binding protein represents an egg-laying hormone receptor is uncertain, but its prevalence in oocytes suggests that ELH plays a signaling role on these gametes.Abbreviations ConA convalin A - DAB diaminobenzidine - ELH egg-laying hormone - ELHBP ELH-binding protein - IEF isoelectric focusing - IGFR insulin-like growth factor - IgG immunoglobulin - NGS normal goat serum - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate     

Why the ovotestis of Helix aspersa is innervated

Although Schmalz described the innervation of the ovotestis in pulmonate snails as early as 1914, no functions have been attributed to it. In H. aspersa, the intestinal nerve branches profusely within the ovotestis and terminates in the walls of the acini and in the sheath surrounding the early portion of the hermaphroditic duct. We found both sensory and motor functions for this innervation. Significantly, there is a tonic sensory discharge generated by the mechanical pressure of growing oocytes, and the level of tonic afferent activity is strongly correlated with the number of ripe oocytes; this is probably a permissive signal that gates ovulation. Tactile stimulation of the ovotestis causes a phasic sensory discharge and a pronounced cardio activation. Also, an efferent discharge is elicited in the ovotestis branch of the intestinal nerve. To study the motor consequences of efferent activity, the ovotestis branch was electrically stimulated. We found that such stimulation evokes peristaltic contractions of the initial portion of the hermaphroditic duct and increases beat frequencies of the cilia that line the interior of the duct. These effects could facilitate the transport of oocytes down the duct. Still other functions of afferent activity are implied by changes in the spontaneous activity of mesocerebral cells following nerve stimulation. Putative sensory neurons and putative motoneurons have been identified in the visceral and right parietal ganglia.     

The Influence of Gaseous Environment on the Storage Life of Araucaria hunsteinii Seed

The effect of various gas mixtures on the longevity of hydratedseeds of Araucaria hunsteinii K. Schum. was assessed under controlledconditions. The length of storage life decreased as oxygen concentrationwas reduced from 21 to zero per cent. No effect of carbon dioxideon seed longevity was detected within the range 1–50 percent when combined with 10 or 21 per cent oxygen. Ethylene at0.01 per cent, and sealed foil or polyethylene bag storage reducedthe period of seed germinability compared with that for 21 percent oxygen. Ethanol accumulation took place in stored seedswhen the environmental oxygen concentration was below a thresholdvalue which lay between 1 and 5 per cent. It is proposed that the observed effects of gases on longevityof hydrated seeds may be mediated through an influence on aerobicrespiration rate. Practical implications of the results areconsidered. Araucaria hunsteinii, Klinkii pine, seed longevity, seed storage, gas environments, oxygen, carbon dioxide, ethanol accumulation     

Patterns of reproductive morphology in the genus Gobiodon (Teleostei: Gobiidae)

The reproductive system of gobiid fishes (family Gobiidae), especially among hermaphroditic goby species, is morphologically diverse. Two hermaphroditic species in the genus Gobiodon, G. okinawae and G. oculolineatus, have several modifications of the gonoduct and gonad that are associated with secretion production and storage. In this study, an examination of six additional Gobiodon species, G. citrinus, G. fulvus, G. histrio, G. micropus, G. quinquestrigatus and G. rivulatus, revealed similar reproductive modifications. Among these six Gobiodon species, a number of features were found to be shared amongst each other and with G. okinawae and G. oculolineatus. All individuals had either an ovariform gonad or an ovotestis; no individuals had a purely testicular gonad. The gonadal lobes extended caudally past, rather than terminating at, their union with the gonoduct. Accessory secretory structures associated with the reproductive complex, termed accessory gonoduct structures, or AGdS, always originated from the gonoduct. The ovariform gonad was comprised entirely of ovarian tissue, while the ovotestis was divided into three morphologically distinct regions. Only one of the ovotestis regions was strictly gametogenic, consisting of both early stage oocytes and sperm-filled seminiferous lobules. The second region of the ovotestis was made up of stromal tissue surrounding some compressed lumina and a small number of early-stage oocytes. The third region was highly lobulated and acted as a storage region for eosinophilic secretions. Anteriorly, the stromal region of each of the two ovotestis lobes disappeared and the gametogenic and secretory storage regions of the ovotestis separated into two discrete lobes. In all of the examined Gobiodon species, all individuals having an ovotestis also had AGdS. However, AGdS presence among individuals having an ovariform gonad varied in a species-specific manner, with the AGdS being fully differentiated and well-developed in a number of species, and either in a very early stage of development, or absent, in others. The distribution of these AGdS states among Gobiodon species corresponds with that of several other morphological features that have been proposed by Harold et al. (Bull Mar Sci 82:119–136, 2008) as phylogenetically informative for intra-generic clade identification. Reproductive characters may prove informative in the development of hypotheses of relationships among gobiid fishes.     

Cyst development in the conchostracan shrimp,Eulimnadia texana (Crustacea: Spinicaudata)

The fertilized egg (or cyst) of branchiopods is a highly resistant stage in the life cycle of these aquatic crustaceans. Previous examinations of these cysts have determined that early embryonic development arrests at a late blastula stage, resulting in a small, crescent-shaped body within the egg shell of these shrimp. Herein, we examine the early development of these embryos by sectioning eggs in the ovotestis, brood chamber, and several time periods after exit from the brood chamber in the clam shrimp Eulimnadia texana Packard. The early sections find no evidence of internal fertilization in the ovotestis. Eggs in the ovotestis showed no signs of cell division, whereas eggs sectioned from the brood chamber were found to be undergoing early embryonic development. A number of empty egg shells and the lack of unfertilized eggs in the brood chamber suggested that egg yolks quickly degrade after egg extrusion from the ovotestis. Cysts that were allowed to develop for 24, 48, 72, and 96 h, 1 week and 1.5 years were sectioned, and embryonic development did not change after the 48 h time period. Thus, embryos appear to arrest development somewhere between 24 and 48 h after exiting the brood chamber.     

Neuroendocrine Regulation of Egg Laying in Aplysia californica

Two clusters of neurons, the bag cells, associated with thecentral nervous system of Aplysia californica play an essentialrole in the induction of egg laying by the animal. Studies concernedwith the morphology, electrophysiology, biochemistry, and functionof these cells are reviewed and discussed. The unusually favorablecharacteristics of this preparation suit it for developmentas a model neuroendocrine effector system.     

An Examination of the Value of 14C-urea as a Source of 14CO2 for Studies of Assimilate Distribution

Twenty-four hours after leaf 3 of a plant of Lolium multiflorumLam, was supplied with a droplet of ¹⁴C-urea and the plant enclosedin a polyethylene bag with an untreated plant, there were significantamounts of radiocarbon recovered from the untreated plant. Theleaf treated with ¹⁴C-urea was the major source of ¹⁴C leakagebut significant losses were also recorded from other parts ofthe plant. Reducing the humidity within the bag decreased theamount of ¹⁴CO₂ which escaped. Losses of radiocarbon from CO₂-treated plants were very low compared with those from urea-treatedplants but the pattern of assimilate distribution within thetwo types of plants was very similar. The possible causes ofthese effects are considered and the usefulness of ¹⁴C-ureaas a source of ¹⁴CO₂ discussed.     

Etude exploratoire de la spermatogenèse induite par la chaleur chez l'escargot Helix aspersa en hibernation: Rôle du cerveau / An exploratory study on temperature-induced spermatogenesis in the hibernating snail Helix aspersa: the role of the brain

Summary

The male cells in the ovotestis of hibernating snails undergo multiplication when the temperature of the environment is raised from 5°C to 25°C. If the temperature is maintained at 25°C for 4 weeks the process of spermatogenesis is completed but the rate of spermatogenesis (DNA synthesis) starts decreasing from the 3rd week (Table 1; Fig. 1).

Brain ablation in hibernating snails maintained at 25°C causes a significant increase in DNA synthesis exclusively in male cells of the ovotestis. This suggests that the brain exerts an inhibitory influence on spermatogenic multiplication. This influence is effective only during the first and the fourth week of exposure of hibernating snails to 25°C (Fig. 1; Table 1) indicating the existence of an endogenous cyclical control. Spermiogenesis is, however, not affected by brain extirpation (Fig. 4 A,B,C).

When reimplanted in the head haemocoel the brain appears normal histologically (Fig. 5 A,B) and it reestablishes the inhibitory influence on DNA synthesis in the ovotestis only during the first day of temperature-induction. During the 1st, 3rd and 4th week the reimplanted brain, deprived of its neural connections, fails to exert its inhibitory influence suggesting that for this influence to function neural connections to the brain are essential. Surprisingly, the implanted brain seems to inhibit DNA synthesis during the 2nd week of temperature-induction (Table 1).

These experiments show that the brain control temperature-induced spermatogenic multiplication in the ovotestis in snails at the onset of hibernation and this control is exerted by one or more inhibitory factors originating from the brain which may function in concert or independently to produce the neuroendocrine effect. It seems therefore justified to consider spermatogenesis in hibernating snails as being neuroendocrinologically controlled.     

Influence of the mesonephros on the development of fetal mouse ovaries following transplantation into adult male and female mice

We previously reported that fetal mouse ovaries frequently develop testicular structure following transplantation into adult male mice. The mechanism involved in gonadal sex reversal of ovarian grafts is not known. In the present study, we examined the influence of the adjacent mesonephros on development of the ovarian grafts. The results show that (1) when fetal ovaries were transplanted with the attached mesonephros, the frequency of ovotestis development was higher in male hosts than in female hosts, (2) the fetal ovaries that had been separated from mesonephros developed testicular structures more frequently than those with the mesonephros, and the incidence of ovotestis development was comparable in male and female hosts, (3) removal of the cranial or caudal half of the mesonephros resulted in a similar frequency of ovotestis development, and (4) when fetal ovaries were separated and reattached to the mesonephros, they developed testicular structures at a frequency similar to that of ovaries left attached to the mesonephros, and the sex of mesonephroi reattached to ovarian grafts did not influence the incidence of ovotestis development. These findings suggest that fetal ovaries can develop testicular structures after transplantation regardless of the sex of host, and that the adjacent mesonephros protects ovarian grafts from masculinizing stimuli more efficiently in female host than male hosts.     

Spermatozoa in the reproductive system of a hermaphroditic marine tardigrade,Orzeliscus belopus (Tardigrada: Arthrotardigrada)

Orzeliscus belopus has long been regarded as the only hermaphroditic marine tardigrade, yet there has been no published detailed information on its internal anatomy. Our study elucidates the ultrastructure of the ovotestis and the spermatozoa of Orzeliscus cf. belopus from Bermuda. The ovotestis had no septum to separate male and female germ cells and, while early stages of spermatogenesis were not observed, many spermatozoa were found at the periphery in both anterior and posterior areas of the gonad. The nucleus and the mitochondria of the spermatozoa in the ovotestis extend backward from the centriole region, forming a half-headed arrow-shape with the nucleus on the outer side of the ‘arrowhead’. The cross section of a long vesicular body (the paranuclear vesicle) is dumbbell- or horseshoe-shaped and attached to almost the entire length of the nucleus. In the seminal receptacle, we found both a complete spermatozoon and some which started to degrade. There is an indication that sperm is further modified after discharge as within the receptacle duct the sperm is no longer half-headed arrow-shaped but has a straight nucleus. This modification might be correlated with the degeneration of the paranuclear vesicle. Our observations clearly show that O. belopus is a simultaneous hermaphrodite, and suggests that the reproductive mode includes copulation and cross-fertilization.     

Activation of Nitrite Reductase in a Cell-Free System from the Denitrifier Alcaligenes sp. by Freeze-Thawing

Nitrite reductase (NiR) activity of the cell-free extract orthe soluble fraction prepared from cells of Alcaligenes sp.NC1B 11015 grown anaerobically in the presence of nitrate wasexamined by measuring the rate of nitrite disappearance withdithionitemethyl viologen (MV) as an electron donor. Freezingat — 20?C and subsequent thawing of the fraction resultedin 5-40 times increase of the specific activity of NiR. Fromthe experiments on the effect of freezing conditions on theactivation, the phase change of solvent water due to freezingis considered to play an important role in the activation. Thisactivation occurred with the preparation in the exponentialgrowth phase, but not that in the stationary growth phase. Clearly,the low-molecular-weight (< 12,000) component which was obtainedfrom the soluble fraction through a collodion bag participatedin the activation. The activated enzyme proved to be the dissimilatory NiR, becauseNO production from nitrite, one of the typical characteristicsof the dissimilatory NiR, was also activated when assayed withascorbate-tetramethyl-p-phenylene diamine (TMPD) as an electrondonor. Nevertheless, the reaction products of nitrite reductionwere identified as hydroxylamine and ammonia with dithionite-MV.The possible pathway of nitrite reduction with this electrondonor is discussed. (Received May 26, 1983; Accepted February 2, 1984)     

Ultrastructure of gametogenesis in the ovotestis of an estuarine pulmonate slug,Onchidium tigrinum (Stoliczka, 1869)

The pulmonate slug Onchidium tigrinum (Stoliczka, 1869) is an estuarine protandrous gastropod. Transmission electron microscopy of both the gonadal and somatic cell populations of the ovotestis of the slug is documented. The acini of smaller slugs are comprised of developing spermatogenic cells and three to four small ill-developed oocytes. Details of the microscopic structures of Sertoli cells, interacinar cells and acinar boundary are described in-depth, revealing their secretory function. Sertoli cells are more numerous in the ovotestes of smaller slugs than in those of larger slugs. Tunnelling nanotubes of 200–400?nm in diameter are described for the first time in the Sertoli cells of molluscan ovotestis. These nanotubes may help to supply various cellular materials into distantly developing spermatogenic cells. The acini of larger slugs possess 2–3 mature oocytes along with a few spermatogenic cells. Sertoli cells, interacinar cells and spermatogonial cells are fewer in number in the acini of the ovotestis of larger individuals establishing the predominance of oogenesis in this phase of life. The number of oocytes per acinus is analysed in relation to the habitat of the pulmonates.     

Litter Decomposition in Four Woody Species in a Mediterranean Climate: Weight Loss, N and P Dynamics

Decomposition dynamics in leaves and needles of two Mediterraneanshrubs and two pine species growing in the Sierra de Filabres(Almería, Spain) was investigated during 2 years usingthe litter bag technique. The species studied are representativeof the vegetation of the study area and differ greatly in theirfoliar traits. Results are discussed in relation to the initiallitter quality (C, N and P) and through the application of theexponential decay model. The mass lost at the end of study variedin the order: Pinus pinaster < Pinus nigra < Cistus laurifolius< Adenocarpus decorticans. Differences in annual rates ofdecomposition among species are consistent with the particularchemical and structural attributes of their leaves. The massof decomposing litter remaining after 2 years was positivelyassociated with the initial C:N ratio. Weight loss and nutrientrelease were fastest in the leguminous A. decorticans. The resultssuggest the importance of both structure and elemental concentrationof initial litter for decomposition dynamics in Mediterraneanspecies. Copyright 2000 Annals of Botany Company Adenocarpus decorticans Boiss., C:N ratio, Cistus laurifolius L., litter decomposition, litter quality, Mediterranean environments, nutrient dynamics, Pinus pinaster Aiton, Pinus nigra Arnold, single exponential model, Southern Spain     

ECOLOGY AND TAXONOMIC STATUS OF THE APLYSIOMORPH AKERA BULLATA IN THE BRITISH ISLES

Dwarf forms of Akera bullata found in the Fleet (Dorset) andBalta Sound (Shetland) were given the subspecific name nanaby Jeffreys (1867); large Irish specimens were called farraniby Norman (1890). Dwarf Fleet akerids were studied in orderto decide whether (a) these geographical extremes ranked asvalid species, or (b) there was an ontogenetic series withoutany significant discontinuity. Monthly samples (1984–87) of A. bullata nana from LangtonHive Point (LHP) in the Fleet were analysed. These results werecompared with local hydrographic data, and with samples fromother Fleet localities, from Portland Harbour, Plymouth Sound,and Lough Ine. Salinity at LHP varied from 6. in winter to 35.5 in summer.Akerids were most at risk when a cold wind coincided with lowwater and rain. Akera was herbivorous in the summer, feedingon rafts of Enteromorpha, but during the remainder of the yearit fed directly upon the sediment. There was no evidence thatit attacked the leaves of Zostera or Ruppia. The biometric datashowed an annual life cycle, each generation growing to maturityduring the spring and early summer. Spawning took place at thetime of the Enteromorpha bloom; the spent adults then died.Moribund adults were common at LHP from April. Monthly ovotestis samples confirmed this interpretation of theannual life cycle, but it was disrupted catastrophically inFebruary, 1986 when the entire LHP population was destroyed.Recolonisation took many months and involved sporadic immigrationto LHP from adjacent, deeper parts of the Fleet. There was noevidence of recruitment from outside. Akerids of the subspecies nana from LHP reached 15 mm in shell-length,lacked defensive purple and the posterior pallial tentacle,and swam only in the juvenile phase. By contrast, Lough Inesamples of farrani reached 40 mm or more in shell-length, producedpurple when disturbed, exhibited an elongated posterior tentacleand engaged in swimming activity in the adult phase. Intermediatespecimens made it certain, however, that no significant discontinuitiesexisted in the cline; consequently all British akerids can beembraced by a single specific name, Akera bullata Müller,1776. Development-type and gamete mass were similar throughout thegeographic range of British Akera bullata. Fecundity, however,was lower in the dwarf subspecies nana. Features of the gametesand the early development confirm the necessity to transferthe Akeridae from the Bullomorptaa to the Aplysiomorpha. (Received 24 June 1988; accepted 24 November 1988)     

In vitro cultivation of cells from ovotestis tissue of pigmented Biomphalaria glabrata

Cells derived from ovotestis tissue of pigmented Biomphalaria glabrata, Puerto Rican strain were cultured in double diluted GIT medium supplemented with modification of amino acids components of pigmented B. glabrata, ovotestis and mid-gut region and 3% inactivated fetal calf serum. As a result, two types of cells, epithelial and fibroblastic like cells increased in number during the cultivation. It seem that the medium used in this study is a suitable medium for cultivation of cells from ovotestis of pigemeted B. glabrata. These two types of cells have been maintained by successive transplantation for over 3 passages.