Spermatophore production and spermatheca content in Tenebrio molitor infected with Hymenolepis diminuta

Male and female Tenebrio molitor act as intermediate hosts for metacestodes of the rat tapeworm, Hymenolepis diminuta. It is known that the bean-shaped accessory glands of infected males exhibit an extended growth period and are significantly larger than those from noninfected males by day 10 after emergence. We wished to determine whether more material is transferred from these glands into the spermatophores. Here we report that the protein content and trehalase activity of spermatophores produced by bean-shaped accessory glands from infected males is elevated. However, protein transferred to the female spermatheca during mating was not affected by the infection status of the male. No evidence of transfer of trehalase to the spermatheca was detected but spermatheca from virgin, infected females contained significantly greater trehalase activity than those from noninfected females.     

Extracellular localization of cyclic GMP in the house cricket male accessory reproductive gland and its fate in mating

The male accessory reproductive gland (ARG) of the house cricket, Acheta domesticus (L.), contains an exceedingly high concentration of cyclic GMP, about 1,000 pmol/mg protein. Immunofluorescent localization and radioimmunoassay measurements show that cyclic GMP is concentrated in a small number of tubules. It accumulates in the tubule lumina where it is protected from degradation by phosphodiesterases. Cyclic GMP is secreted by the ARG and is incorporated into spermatophores. Over 80% of spermatophore cyclic GMP is found in the handle-capillary tube, a thin conduit through which sperm pass during transfer to the female. The concentration of cyclic GMP in the insemination fluid is about 20 microM but does not appear to be specifically associated with the sperm. Cyclic GMP enters the female spermatheca during insemination but disappears rapidly. Physiological effects of cyclic GMP on sperm were not observed nor was an effect of cyclic GMP observed on egg laying by mated females. Cyclic AMP was localized on sperm flagella in the spermatophore and in the spermatheca. These studies indicate that cyclic nucleotides have important roles in insect reproduction and that the house cricket is a good model for elucidating these functions.     

Pattern of sperm storage and migration in the reproductive tract of the swallowtail butterfly Papilio xuthus: cryptic female choice after second mating

Females of the swallowtail butterfly Papilio xuthus L. (Lepidoptera: Papilionidae) mate multiply during their life span and use the spermatophores transferred to increase their longevity as well as fecundity. Sperm from different males may be stored in the sperm storage organs (bursa copulatrix and spermatheca). To clarify the pattern of sperm storage and migration in the reproductive tract, mated females are dissected after various intervals subsequent to the first mating, and the type and activity of sperm in the spermatheca are observed. When virgin females are mated with virgin males, the females store sperm in the spermatheca for more than 10 days. Sperm displacement is found in females that are remated 7 days after the first mating. Immediately after remating, these females flush out the sperm of the first male from the spermatheca before sperm migration of the second male has started. However, females receiving a small spermatophore at the second mating show little sperm displacement, and the sperm derived from the small spermatophore might not be able to enter the spermatheca. Females appear to use spermatophore size to monitor male quality.     

Paternity analysis of wild‐caught females shows that sperm package size and placement influence fertilization success in the bushcricket Pholidoptera griseoaptera

In species where females store sperm, males may try to influence paternity by the strategic placement of sperm within the female's sperm storage organ. Sperm may be mixed or layered in storage organs, and this can influence sperm use beyond a ‘fair raffle’. In some insects, sperm from different matings is packaged into discrete packets (spermatodoses), which retain their integrity in the female's sperm storage organ (spermatheca), but little is known about how these may influence patterns of sperm use under natural mating conditions in wild populations. We examined the effect of the size and position of spermatodoses within the spermatheca and number of competing ejaculates on sperm use in female dark bushcrickets (Pholidoptera griseoaptera) that had mated under unmanipulated field conditions. Females were collected near the end of the mating season, and seven hypervariable microsatellite loci were used to assign paternity of eggs laid in the laboratory. Females contained a median of three spermatodoses (range 1–6), and only six of the 36 females contained more than one spermatodose of the same genotype. Both the size and relative placement of the spermatodoses within the spermatheca had a significant effect on paternity, with a bias against smaller spermatodoses and those further from the single entrance/exit of the spermatheca. A higher number of competing males reduced the chances of siring offspring for each male. Hence, both spermatodose size and relative placement in the spermatheca influence paternity success.     

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     

The spe-42 gene is required for sperm-egg interactions during C. elegans fertilization and encodes a sperm-specific transmembrane protein

Fertilization, the union of sperm and egg to form a new organism, is a critical process that bridges generations. Although the cytological and physiological aspects of fertilization are relatively well understood, little is known about the molecular interactions that occur between gametes. C. elegans has emerged as a powerful system for the identification of genes that are necessary for fertilization. C. elegans spe-42 mutants are sterile, producing cytologically normal spermatozoa that fail to fertilize oocytes. Indeed, male mating behavior, sperm transfer to hermaphrodites, sperm migration to the spermatheca, which is the site of fertilization and sperm competition are normal in spe-42 mutants. spe-42 mutant sperm make direct contact with oocytes in the spermatheca, suggesting that SPE-42 plays a role during sperm-egg interactions just prior to fertilization. No other obvious defects were observed in spe-42 mutant worms. Cloning and sequence analysis revealed that SPE-42 is a novel predicted 7-pass integral membrane protein with homologs in many metazoan species, suggesting that its mechanism of action could be conserved.     

Functional anatomy of the sperm storage organs in Pulmonata: the simple spermatheca of Bradybaena fruticum (Gastropoda, Stylommatophora)

Data on sperm storage and paternity analyses in the pulmonate land snail Arianta arbustorum suggest that the complex, multitubular sperm storage organ, the spermatheca, may influence paternity after multiple matings. Ultrastructural investigations show that the spermatheca is provided with the morphological correlates to exert cryptic female choice. However, in order to understand the function of a multitubular spermatheca it is necessary to understand how a single spermathecal tubule functions. In order to explore the potential to serve as a model for such a simple system in future experiments, the fine structure of the unitubular spermatheca and its interaction with spermatozoa were investigated in Bradybaena fruticum, another member of the Helicoidea. The spermatheca of B. fruticum is only about one-half as long as the fertilization chamber. Its epithelium is densely ciliated throughout its length. Vacuole, Golgi complex, rough endoplasmic reticulum, various vesicles, wide intercellular spaces, and an extensive basal labyrinth indicate strong secretory activity, providing the environment for sperm storage and capacitation. Prior to transfer, sperm are characterized by a perinuclear sheath and an acrosome tilted at about 50°. In the spermatheca, the perinuclear sheath is dissolved and, probably as a consequence, the acrosome folds up in line with the nuclear longitudinal axis. The spermatheca is surrounded by a network of differently oriented smooth muscle cells, which are extensively connected with each other through dense plaques. The fine structure of the muscle cells suggests that they are neither very strong nor enduring. The main function of the spermathecal musculature is certainly expulsion of sperm prior to fertilization. The musculature around the spermathecal tubule of B. fruticum appears to be a highly integrated system not allowing for much functional flexibility compared to A. arbustorum, where the muscle cells are more individualized, permitting finely tuned operations. This restricted flexibility needs to be taken into consideration in future experiments using B. fruticum as a model for the simple, unitubular sperm storage system.     

Quantitative determination by derivative spectrophotometry of Triton X-100 in solubilized preparations of membrane proteins

The amount of the nonionic detergent Triton X-100 in protein preparations cannot be determined spectrophotometrically from the normal spectrum because the spectral bands of the detergent and proteins overlap, but it was found that the detergent could be determined accurately by 4th-derivative spectrophotometry. The intensity of the derivative absorbance difference between the positive and negative peaks at about 277.5 and 281 nm, respectively, increased linearly with increase in the detergent concentration at more than its critical micelle concentration (about 0.03%). Proteins had little effect on these spectral bands of the detergent. This method is very simple and accurate for determination of the concentration of Triton X-100 in solubilized preparations of membrane proteins.     

Adenosine 3′,5′-monophosphate dependent phosphorylation of ribosomes and ribosomal subunits from bovine corpus luteum

In a previous publication the purification and properties of two protein kinases (KI and KII) from a soluble fraction of bovine corpus luteum and the stimulation of the latter fraction by cyclic AMP and luteinizing hormone was reported (Menon, K.M.J. (1973) J. Biol. Chem. 248, 494–501). We have now studied the effects of cyclic AMP and luteinizing hormone on ribosomal protein phosphorylation of corpus luteum by protein kinase II. Protein kinase II catalyzed the phosphorylation of ribosomes by transfer of terminal phosphate of ATP to ribosomal proteins. Extraction with hot trichloroacetic acid and non-aqueous solvent revealed that about 80% of total radioactivity incorporated remain associated with the protein residue. Radioactivity was identified in the phosphoserine and phosphothreonine residues of polypeptides by high voltage paper electrophoresis. The extent of phosphorylation was stimulated by cyclic AMP but not by luteinizing hormone. At least 9 proteins of 80-S ribosomes and 12 proteins of the 60-S ribosomal subunit were phosphorylated in the presence of cyclic AMP as resolved by urea polyacrylamide gel electrophoresis. However, only one major and four minor bands were phosphorylated in the case of 40-S ribosomal subunit under the influence of cyclic AMP. The ribosomal protein phosphorylation catalyzed by protein kinase II is regulated by cyclic AMP whereas luteinizing hormone has no effect on ribosome phosphorylation.     

Molecular evolutionary analysis of seminal receptacle sperm storage organ genes of Drosophila melanogaster

Sperm storage organs are common and broadly distributed among animal taxa. However, little is known about how these organs function at the molecular level. Additionally, there is a paucity of knowledge about the evolution of genes expressed in these organs. This investigation is an evolutionary expressed sequence tag (EST) study of genes expressed in the seminal receptacle, one of the sperm storage organs in Drosophila. The incidence of positive selection is higher for the seminal receptacle genes than Drosophila reproductive genes as a whole, but lower than genes associated with the spermatheca, a second type of Drosophila sperm storage organ. By identifying overrepresented classes of proteins and classes for which sperm storage function is suggested by the nature of the proteins, candidate genes were discovered. These candidates belong to protein classes such as muscle contraction, odorant binding and odorant receptor, protease inhibitor and immunity.     

Regulation of the leptin content of obese human adipose tissue

The objective of this study was to determine whether obese human adipose tissue contains preformed stores of leptin and their relationship to secreted leptin. Detergent increased detectable leptin by about twofold, suggesting that leptin is stored in a membrane-bound location. Subcutaneous tissue leptin was approximately 1.6-fold higher than omental, paralleling known differences in leptin secretion and expression. The amount of leptin secreted during a 3-h incubation was similar to that of extractable tissue leptin. Tissue leptin levels were maintained over the incubation. Inhibition of protein synthesis decreased tissue leptin content but did not decrease leptin secretion until after 3 h of incubation. Culture of adipose tissue for 2 days with the combination of insulin and dexamethasone, but not with either hormone alone, increased tissue leptin content about twofold in both depots. Although insulin did not affect tissue leptin content, it potentiated leptin secretion (as a % of tissue stores). These data suggest that adipose tissue leptin storage and secretion per se are regulated. Regulation of the release of preformed leptin may modulate serum leptin levels in obese humans.     

Eupyrene sperm migrates to spermatheca after apyrene sperm in the swallowtail butterfly, Papilio xuthus L. (Lepidoptera: Papilionidae)

When swallowtail butterflies, Papilio xuthus, are mated by the hand-pairing method, both types of sperm, eupyrene and apyrene sperm, are transferred from the male to the spermatheca via the spermatophore in the bursa copulatrix. This mechanism is demonstrated by two different kinds of experiments. The first set of experiments employed interrupted copulation, and the second set was examination of the sperm in the spermatophore and spermatheca after the termination of copulation. The sperm was transferred 30 min after the start of copulation. The eupyrene sperm was still in the bundle; the number of the bundles ranged from 9 to 108 (mean, 42.7; n = 27). The bundles were gradually released after the completion of copulation, and the free eupyrene spermatozoa then remained in the spermatophore at least 2 h before migrating to the spermatheca. On the other hand, about 160 000 apyrene spermatozoa were transferred to the spermatophore and remained there for more than 1 h. We observed 11 000 apyrene spermatozoa in the spermatheca 12 h after the completion of copulation, but most of this type of sperm disappeared shortly thereafter. In contrast, the eupyrene sperm arrived in the spermatheca more than 1 day after the completion of copulation and remained there at least 1 week. Therefore, these findings suggest that apyrene sperm migrate from the spermatophore to the spermatheca earlier than eupyrene sperm. Accordingly, if females mated multiply, the time difference might avoid the mixing of sperm. In addition, the predominance of sperm from the last mating session may occur not in the bursa copulatrix but in the spermatheca. Received: January 7, 2000 / Accepted: May 24, 2000     

Protein and peptide degradation in the intestine of the common brushtail possum (Trichosurus vulpecula)

A protein (bovine serum albumin: BSA) and a peptide (luteinizing hormone releasing hormone: LHRH) were used to evaluate proteolytic activity in the intestine of common brushtail possums (Marsupiala, Trichosurus vulpecula). Luminal and mucosal extracts were isolated from the duodenum, jejunum, ileum, caecum, proximal colon and distal colon, their protein content assessed and specific activities in metabolising LHRH and BSA determined in vitro. The degradation of LHRH by luminal extracts was compared with that by the pancreatic enzymes, chymotrypsin, trypsin, and elastase. The protein concentration (microg x mg-1) of mucosal extract in the duodenum was higher ( P<0.05) than in the proximal colon, but that of luminal extracts did not differ significantly between regions. Proteolytic activity of luminal extracts was greater ( P<0.01) in the jejunum and ileum than in the hindgut. In the small intestine, proteolytic activity of luminal enzymes far exceeded that of mucosal enzymes ( P<0.05). All three pancreatic enzymes hydrolysed LHRH, but chymotrypsin had the greatest activity. This study has demonstrated that, in possums, proteolysis occurs primarily in the small intestine through luminal enzymes, with chymotrypsin playing a major role. The possum hindgut contributes little to the metabolism of peptides and proteins, identifying it as a potential site to target for their absorption following oral delivery.     

Ultrastructural analysis of the morphology and function of the spermatheca of the pulmonate slug Arion subfuscus

A study of the histology, histochemistry and ultrastructure of the spermatheca of Arion subfuscus in different stages of its life cycle shows that the spermatheca, an accessory organ of the femal genital tract of pulmonates, is a spherical-shaped organ lined with a columnar and non-ciliated epithelium surrounded by a thin network of connective tissue with some muscle fibres. The narrow epithelial cells possess numerous microvilli that, excepting young specimens, are provided with long, thin and generally curved, membranous process. Golgi apparatus, RER and mitochondria are abundant. Basal infolds are not very deep. In specimens killed two days after-copula, the spermatheca is swollen and its lumen is full of degenerating spermatozoa, mucus masses and spermatophore fragments. The apex of the cell is rich in granules with varied content, including multivesicular bodies. In specimens killed two weeks after-copula there are numerous endocytic vesicles in the apex and big vacuoles containing lipid. It is considered that after mating the excess of exogenous spermatozoa and copulatory seminal fluids are digested in the spermatheca. First, there is extracellular digestion that may be carried out by the enzymes contained in the multivesicular bodies exocytosed to the lumen, as well as by the enzymes secreted in apocrine vesicles. The partially digested materials would then be absorbed by endocytosis and further digested intracellularly. The great accumulation of lipid in the epithelial cells two weeks after mating suggest that the spermatheca could be involved in lipid synthesis, acting as a reserve organ.     

Adenosine 3'5'-m onophosphate dependent phosphorylation of ribosomes and ribosomal subunits from bovine corpus luteum.

In a previous publication the purification and properties of two protein kinases (KI and KII) from a soluble fraction of bovine corpus luteum and the stimulation of the latter fol. Chem. 248,494-501). We have now studied the effects oc cyclic AMP and luteinizing hormone on ribosomal protein phosphorylation of corpus luteum by protein kinase II. Protein kinase II catalyzed the phosphorylation of ribosomes by transfer of terminal phosphate of ATP to ribosomal proteinsmextraction with hot trichloroacetic acid and non-aqueous solvent revealed that about 80% of total radioactivity incorporated remain associated with the protein residue. Radioactivity was identified in the phosphoserine and phosphothreonine residues of polypeptides by high voltage paper electrophoresis; The extent of phosphorylation was stimulated by cyclic AMP but not by luteinizing hormonemat least 9 proteins of 80-S ribosomes and 12 proteins of the 60-S ribosomal subunit were phosphorylated in the presence of cyclic AMP as resolved by urea polyacrylamide gel electrophoresis. However, only one major and four minor bands were phosphorylated in the ase of 40-S ribosomal subunit under the influence of cyclic AMP. The ribosomal protein phosphorylation catalyzed by protein kinase II is regulated by cyclic AMP wherease luteinizing hormone has no effect on ribosome phosphorylation.     

Circular dichroism studies on human chorionic somatomammotropin

The conformation of human chorionic somatomammotropin has been studied by means of circular dichroism spectra. The protein appears to contain about 45% α-helix in the native state. Circular dichroism bands in the region of side chain absorption have been assigned to phenylalanine and tryptophan residues. Tentative assignments has also been made to bands probably arising mostly from tyrosine residues. The stability of the native structure has been assessed by challenging the protein with four perturbing solvents. With the exception of 0.1 n NaOH which produced permanent denaturation, all conformational changes produced by the perturbants were fully reversible. In addition, the monomer molecular weight has been evaluated by gel filtration and osmotic pressure measurements. A value of 21,600 ± 900 was found by osmotic pressure at pH 8.4. The results have been compared with similar findings on human pituitary growth hormone and ovine pituitary lactogenic hormone.     

The effect of mating on phonotactic behaviour in Gryllus bimaculatus (De Geer)

Abstract. Phonotaxis of G. bimaculatus females is expressed by a course-consistent, directional approach to a conspecific model calling song emitted alternatingly at sound intensities of 50–90 dB SPL and appears at the age of 6–7 days, the onset of maturation. Mating abolishes phonotaxis after about 1 h. By that time, the content of the attached spermatophore has migrated into the female's sper-matheca. Removal or emptying of the spermatheca, or severing the ventral nerve cord, fully reinstates phonotaxis, whereas ovariectomy has no effect. We suggest that mechanoreceptors record the expansion of the filled spermatheca and that the inhibitory message is communicated via the ventral nerve cord anteriorly to an unknown control centre in the brain. The loss of phonotaxis is combined with reduced locomotor activity and results in the female no longer being attracted to another calling male.     

Sperm transfer during mating, movement of sperm in the female reproductive tract, and sperm precedence in the common cutworm Spodoptera litura

Abstract. Mating behaviour, sperm transfer and sperm precedence were studied in the moth Spodoptera litura (Fabr.) (Lepidoptera: Noctuidae). There existed a rhythmic, diel pattern of mating behaviour of this moth during the scotophase, presumably set with respect to an endogenous activity rhythm. Approximately 30 min after copulation had started, the formation of the corpus of the spermatophore began in the bursa copulatrix of the female moth, but full inflation of the corpus was not completed until 45–60 min after mating had started. The mature spermatophore contained about 350 eupyrene sperm bundles and a large number of individual (loose) apyrene spermatozoa. The mating status and the age of the male insect influenced the number of sperm transferred to the female within the spermatophore, and also affected the consequent fertility. There was no evidence of sperm reflux within the male tract. Within the female, dissociation of eupyrene sperm bundles was evident within the spermatophore less than 15 min after the completion of mating. Spermatozoa began to move from the bursa (in which the spermatophore is lodged) into the spermatheca 30–45 min after the end of the copulation, and the quantity of sperm in the spermatheca reached a plateau at 90 min after mating. Apyrene sperm reached the spermatheca first, followed by eupyrene sperm. Examination of total (apyrene plus eupyrene) sperm in the female tract showed that 86% of mated females received an apparently normal amount of total sperm from the male. Examination of eupyrene sperm alone showed that 81% of matings resulted in an apparently normal transfer of eupyrene sperm. A small proportion (approximately 8%) of the matings, however, were identified as transferring a clearly subnormal quantity of eupyrene sperm to the spermatheca. The eggs produced as a result of such pairings displayed much reduced fertility (about 43%) compared to those from matings confirmed to have transferred normal quantities of sperm, which showed about 92% fertility. This shows that the availability of eupyrene sperm in the spermatheca may be an important constraint on fertility in normal populations of insects. In the laboratory, S. litura females exhibited multiple matings. Of the females, 93% mated, and the mean frequency of mating was 1.69. Mating with a fertile male led to the oviposition of an increased number of eggs. This effect continued even when the female subsequently mated with an infertile male. Displacement of sperm from previous matings is known to be an important factor in the evolution of multiple mating strategies. Our results on sperm utilization by S. litura indicated that after a second mating, the sperm utilized for subsequent fertilization were almost exclusively from the last mating with little mixing. The proportion of eggs fertilized by sperm from the second mating (P₂) was calculated as 0.95, indicating almost complete sperm precedence from the last mating.