Identification of SepCRP analogues in the cuttlefish Sepia officinalis: a novel family of ovarian regulatory peptides

In the cuttlefish, Sepia officinalis, the ovary appears to be one of the main sources of regulatory peptides involved in the successive steps of egg-laying. Following the identification of the SepCRP-1, which is a peptide extracted from ovary and involved in egg capsule secretion, investigations were focused on the identification of related peptides. Seven related-Sepia Capsule Releasing Peptides (R-SepCRPs) were identified by means of mass spectrometry and characterized using MS/MS spectra and/or Edman degradation. Finally, primary structures were verified by the comparison of MS/MS spectra from endogenic and synthetic peptides. This new ovarian peptide family exhibits a conserved SLXKD tag involved in the biological activity. LC-MS/MS screening clearly demonstrates that R-SepCRPs are restricted to the female genital tract. Expressed during vitellogenesis, they are released by vitellogenic follicles and full-grown oocytes (FGO) in the genital coelom. Biological activities suggest that R-SepCRPs would be responsible for the storage of FGO before mating and would take part in the mechanical secretion of egg capsule products, as previously described for SepCRP-1.     

Egg capsule secretion in invertebrates: a new ovarian regulatory peptide identified by mass spectrometry comparative screening in Sepia officinalis

Mass spectrometry comparative screening was used to identify ovarian regulatory peptides involved in the successive steps of egg-laying in the cuttlefish Sepia officinalis. The peptide content of full-grown oocytes (FGO) was compared with that of oocyte-conditioned medium, which resulted in the detection of peptides that were present in both samples. These peptides, which are suspected of being released by the oocyte in the genital tract, were submitted to a structural analysis. This strategy led to the characterization of a new ovarian regulatory peptide (EISLDKD) able to inhibit the contractions of the whole female genital tract and of the main nidamental glands (MNG). As EISLDKD appeared to be the first regulatory peptide directly involved, at physiological concentrations, in the secretion of the egg capsule by the main nidamental glands, it was named SepCRP for Sepia Capsule Releasing Peptide. Mass spectrometry analysis clearly demonstrated that SepCRP was expressed during vitellogenesis by the ovarian follicles and released by the FGO in the lumen of the female genital tract. In association with the ovarian 5-HT, SepCRP would be responsible for the storage of FGO to avoid the spawning of unfertilized oocytes before mating. Released by the distal oviduct in the mantle cavity, SepCRP probably in association with a cocktail of ovarian regulatory factors targets the MNG to regulate the egg capsule secretion. Thus, the ovary appeared to be one of the main sources of regulatory peptides involved in the successive steps of egg-laying in the cephalopod mollusk S. officinalis.     

The SepOvotropin: a new ovarian peptide regulating oocyte transport in Sepia officinalis

In the cuttlefish Sepia officinalis, the successive steps of egg laying are controlled by multiple neuropeptides. Recent experiments led us to suppose that there was possible involvement of a second regulation pathway by the release of ovarian regulatory peptides in the genital tract. Using HPLC fractionation and an in vitro biological test, a C-terminal amidated peptide modulating the motility of the Sepia officinalis oviduct was isolated from an extract of vitellogenic ovarian follicles. The mass of this peptide as determined by MALDI-TOF (1501.8 Da) and analysis by Edman degradation led to the following sequence: Pro-Lys-Asp-Ser-Met-Leu-Leu-Leu-Gln-Val-Pro-Val-Tyr-amide. The peptide mapping performed by LC/MS revealed a distribution restricted to the follicles, the full grown oocytes and the eggs. This new peptide, called SepOvotropin, modulated contractions of the whole genital tract in physiological conditions from a threshold concentration between 10(-20) and 10(-19) M, demonstrating for the first time the occurrence of a specific peptidergic control of egg-laying in cephalopods.     

Sex-peptides bind to two molecularly different targets in Drosophila melanogaster females

Sex-Peptide (SP) and the peptide DUP99B elicit two postmating responses in Drosophila melanogaster females: receptivity is reduced and oviposition is increased. Both are synthesized in the male genital tract and transferred into the female during copulation. To elucidate their function, we characterized the binding properties of SP and DUP99B in females. Cryostat sections of adult females were incubated with alkaline phosphatase (AP)-tagged peptides. In virgin females, both peptides have specific target sites in the nervous system and in the genital tract. The binding pattern is almost identical for both peptides. Incubation of sections of mated females confirm that some of these target sites correspond to the in vivo targets of the two peptides. Neuronal binding is dependent on an intact C-terminal sequence of SP, binding in the genital tract is less demanding in terms of amino acid sequence requirement. On affinity blots the AP-SP probe binds to membrane proteins extracted from abdomen and head plus thorax, respectively. The binding proteins in the nervous system and the genital tract differ in their molecular properties. Calculation of dissociation constants (K(d)), and also determination of the minimal peptide concentrations necessary for binding, indicate that SP is the more important peptide inducing the postmating responses. Our results suggest that binding of SP in the nervous system is responsible for eliciting the postmating responses, whereas binding in the genital tract reflects the presence of a peptide transporter.     

Sex‐peptides bind to two molecularly different targets in Drosophila melanogaster females

Sex‐Peptide (SP) and the peptide DUP99B elicit two postmating responses in Drosophila melanogaster females: receptivity is reduced and oviposition is increased. Both are synthesized in the male genital tract and transferred into the female during copulation. To elucidate their function, we characterized the binding properties of SP and DUP99B in females. Cryostat sections of adult females were incubated with alkaline phosphatase (AP)‐tagged peptides. In virgin females, both peptides have specific target sites in the nervous system and in the genital tract. The binding pattern is almost identical for both peptides. Incubation of sections of mated females confirm that some of these target sites correspond to the in vivo targets of the two peptides. Neuronal binding is dependent on an intact C‐terminal sequence of SP, binding in the genital tract is less demanding in terms of amino acid sequence requirement. On affinity blots the AP–SP probe binds to membrane proteins extracted from abdomen and head plus thorax, respectively. The binding proteins in the nervous system and the genital tract differ in their molecular properties. Calculation of dissociation constants (K_d), and also determination of the minimal peptide concentrations necessary for binding, indicate that SP is the more important peptide inducing the postmating responses. Our results suggest that binding of SP in the nervous system is responsible for eliciting the postmating responses, whereas binding in the genital tract reflects the presence of a peptide transporter. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 372–384, 2003     

Peptide and protein pheromones in molluscs

Pheromones have been implicated in the control of a number of behaviors in molluscs, but few peptide pheromones have been characterized in these animals. Peptide pheromones include: (1) a family of water-borne peptide pheromonal attractants (attractins) in the gastropod Aplysia that are released during egg laying and attract other Aplysia to form egg-laying and mating aggregations; (2) a tetrapeptide (ILME) in the cephalopod Sepia that elutes from egg masses and is thought to be involved in the transport of oocytes in the genital tract during egg laying; and (3) a Sepia sperm-attracting peptide (SepSAP; PIDPGVamide) that is released from oocytes during egg laying to facilitate external fertilization.     

Peptidomics and peptide hormone processing in the Drosophila midgut

Peptide hormones are key messengers in the signaling network between the nervous system, endocrine glands, energy stores and the gastrointestinal tract that regulates feeding and metabolism. Studies on the Drosophila nervous system have uncovered parallels and homologies in homeostatic peptidergic signaling between fruit flies and vertebrates. Yet, the role of enteroendocrine peptides in the regulation of feeding and metabolism has not been explored, with research hampered by the unknown identity of peptides produced by the fly's intestinal tract. We performed a peptidomic LC/MS analysis of the fruit fly midgut containing the enteroendocrine cells. By MS/MS fragmentation, we found 24 peptides from 9 different preprohormones in midgut extracts, including MIP-4 and 2 forms of AST-C. DH(31), CCHamide1 and CCHamide2 are biochemically characterized for the first time. All enteroendocrine peptides represent brain-gut peptides, and apparently are processed by Drosophila prohormone convertase 2 (AMON) as suggested by impaired peptide detectability in amon mutants and localization of amon-driven GFP to enteroendocrine cells. Because of its genetic amenability and peptide diversity, Drosophila provides a good model system to study peptide signaling. The identification of enteroendocrine peptides in the fruit fly provides a platform to address functions of gut peptide hormones in the regulation of feeding and metabolism.     

Immunocontraceptive effect of Izumo and enhancement by combination vaccination

Sperm proteins are being investigated for their applications in the development of contraceptive vaccines (CV) in several laboratories. In the present study, various synthetic peptides based upon four sperm proteins, namely Izumo, fertilization antigen-1 (FA-1), YLP(12), and SP56 that are involved in various steps of the fertilization cascade were examined for their immunocontraceptive effect. The synthetic peptides were conjugated to four carrier proteins namely keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), chicken gamma-globulin, and chicken ovalbumin. Female mice were immunized with various peptide vaccines and each booster injection was given with the peptide conjugated to a different carrier protein. Two different fertility trials with different doses of the peptide vaccines were conducted to examine the contraceptive effect. Injection of 150 microg of the peptides (Trial II) caused a significantly higher immune response in serum as well as in the vaginal tract causing enhanced contraceptive effect than those observed after injection with 75 microg of the peptides (Trial I). Immunization with the peptide vaccines based upon Izumo, which is involved in sperm-egg plasma membrane fusion, caused a significant reduction in fertility. The contraceptive effect was enhanced by immunizing with the peptides based upon other antigens (FA-1, YLP(12), and SP56), resulting in an overall 73.33% reduction in fertility. When the antibodies against the peptides disappeared after >9-10 months from circulation and genital tract, all the animals regained fertility. These findings indicate for the first time that the immunization with Izumo and other sperm peptides namely FA-1, YLP(12), and SP56 induces antibodies in serum and genital tract that cause a reversible long-term contraceptive effect in female mice. The data further indicate that the proteins involved in sperm-egg fusion can also be used for contraceptive vaccine development. The contraceptive effects are enhanced by immunizing with the multipeptide vaccines.     

MUC1 Positive,Kras and Pten Driven Mouse Gynecologic Tumors Replicate Human Tumors and Vary in Survival and Nuclear Grade Based on Anatomical Location

Activating mutations of Kras oncogene and deletions of Pten tumor suppressor gene play important roles in cancers of the female genital tract. We developed here new preclinical models for gynecologic cancers, using conditional (Cre-loxP) mice with floxed genetic alterations in Kras and Pten. The triple transgenic mice, briefly called MUC1KrasPten, express human MUC1 antigen as self and carry a silent oncogenic Kras^G12D and Pten deletion mutation. Injection of Cre-encoding adenovirus (AdCre) in the ovarian bursa, oviduct or uterus activates the floxed mutations and initiates ovarian, oviductal, and endometrial cancer, respectively. Anatomical site-specific Cre-loxP recombination throughout the genital tract of MUC1KrasPten mice leads to MUC1 positive genital tract tumors, and the development of these tumors is influenced by the anatomical environment. Endometrioid histology was consistently displayed in all tumors of the murine genital tract (ovaries, oviducts, and uterus). Tumors showed increased expression of MUC1 glycoprotein and triggered de novo antibodies in tumor bearing hosts, mimicking the immunobiology seen in patients. In contrast to the ovarian and endometrial tumors, oviductal tumors showed higher nuclear grade. Survival for oviduct tumors was significantly lower than for endometrial tumors (p = 0.0015), yet similar to survival for ovarian cancer. Oviducts seem to favor the development of high grade tumors, providing preclinical evidence in support of the postulated role of fallopian tubes as the originating site for high grade human ovarian tumors.     

Binding sites of Drosophila melanogaster sex peptide pheromones

Drosophila melanogaster sex peptide (SP) and Ductus ejaculatorius peptide (DUP99B) are male pheromones transferred in the seminal fluid to the female during copulation. Both reduce sexual receptivity and stimulate oviposition in females. The presence of high-affinity SP and DUP99B binding sites in the female were investigated by incubation of cryostat tissue sections with (125)I-iodinated peptides and subsequent autoradiography. We found that in adult females radiolabeled SP and DUP99B bind to peripheral nerves, the subesophageal ganglion, the cervical connective, to discrete parts of the thoracic ganglion, and to the genital tract. Weak and uniform labeling was detected in the neuropil of the brain and the thoracic ganglion. The labeling pattern in the nervous system suggests binding of the peptides to sensory afferents or glial cells. Scatchard analysis of the binding of (125)I-DUP99B to antennal nerves yielded a dissociation constant K(d) of 6.4 nM. Competition experiments with peptide fragments show that the peptides bind with their homologous C-terminal regions. Binding sites in the nervous system of females are established throughout sexual maturation. Prominent binding of the peptides to afferent nerves suggests modification of sensory input.     

Ovarian and sperm regulatory peptides regulate ovulation in the oyster Crassostrea gigas

For more than six decades, several studies have shown that genital products to entering the mantle cavity via the incurrent siphon, initiate in oyster, strong and rhythmic contractions of the adductor muscle (AM). In order to characterize the regulatory peptides capable of triggering AM contractions, we focused on the identification of putative myotropic peptides from genital products. Two experimental approaches were developed. The first one, based on a mass spectrometry screening of the male genital products, led to the identification of the tetrapeptide APGWamide. This neuropeptide was also detected in the seminal secretions of the cephalopod Sepia officinalis. In this species, APGWamide is directly involved in the oocyte transport. In Crassostrea, in vitro bioassay demonstrated that APGWamide modulates the AM contractions that insure the release of oocytes in the external medium. Exposure of oysters to a physiological concentration of APGWamide triggered repetitive shell closures. The second experimental approach was based on the monitoring of HPLC purification by a myotropic bioassay using the cuttlefish oviduct contractions as a target. The successive purification steps of the acidic extraction of ovaries from mature female oysters, led to the characterization of the hexapeptide PIESVD. When applied to mature female oysters, this peptide triggered the increase of shell closure frequency. The activity of these two regulatory peptides is the first experimental evidence of a peptidergic control of egg-laying in oyster. APGWamide and PIESVD could be used, in commercial and experimental hatcheries, for the identification of mature females to be selected for in vitro fertilization.     

Identification of multiple antimicrobial peptides from the skin of fine-spined frog, Hylarana spinulosa (Ranidae)

In this study, peptidomics and genomics analyses were used to study antimicrobial peptides from the skin of Hylarana spinulosa. Twenty-nine different antimicrobial peptide precursors were characterized from the skin of H. spinulosa, which produce 23 mature antimicrobial peptides belonging to 12 different families. To confirm the actual presence and characteristics of these antimicrobial peptides in the skin tissue extractions from H. spinulosa, we used two distinct methods, one was peptide purification method that combined gel filtration chromatography and reversed-phase high performance liquid chromatography (RP-HPLC), and the other was peptidomics approach based on liquid chromatography in conjunction with tandem mass spectrometry (LC–MS/MS). In the peptidomics approach, incomplete tryptic digestion and gas-phase fractionation (GPF) analysis were used to increase peptidome coverage and reproducibility of peptide ion selection. Multiple species of microorganisms were chosen to test and analyze the antimicrobial activities and spectrum of these antimicrobial peptides.     

Mass spectrometric investigation of the neuropeptide complement and release in the pericardial organs of the crab, Cancer borealis

The crustacean stomatogastric ganglion (STG) is modulated by both locally released neuroactive compounds and circulating hormones. This study presents mass spectrometric characterization of the complement of peptide hormones present in one of the major neurosecretory structures, the pericardial organs (POs), and the detection of neurohormones released from the POs. Direct peptide profiling of Cancer borealis PO tissues using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) revealed many previously identified peptides, including proctolin, red pigment concentrating hormone (RPCH), crustacean cardioactive peptide (CCAP), several orcokinins, and SDRNFLRFamide. This technique also detected corazonin, a well-known insect hormone, in the POs for the first time. However, most mass spectral peaks did not correspond to previously known peptides. To characterize and identify these novel peptides, we performed MALDI postsource decay (PSD) and electrospray ionization (ESI) MS/MS de novo sequencing of peptides fractionated from PO extracts. We characterized a truncated form of previously identified TNRNFLRFamide, NRNFLRFamide. In addition, we sequenced five other novel peptides sharing a common C-terminus of RYamide from the PO tissue extracts. High K+ depolarization of isolated POs released many peptides present in this tissue, including several of the novel peptides sequenced in the current study.     

A comparison of the neuropeptides from the retrocerebral complex of adult male and female Manduca sexta using MALDI-TOF mass spectrometry

The occurrence of neuropeptides in the retrocerebral complexes of adult male and females of the tobacco hawkmoth, Manduca sexta, was investigated using matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectrometry (MS), post source decay (PSD) and collision-induced dissociation (CID) MS/MS. From fractions of methanol extracts of corpora cardiaca (CC)/corpora allata (CA), separated by reversed-phase high performance liquid chromatography (RP-HPLC), a total of 11 mass ions were assigned to known peptides from M. sexta. These peptides were adipokinetic hormone (AKH), FLRFamides I, II and III, crustacean cardioactive peptide (CCAP), cardioactive peptide 2b (CAP(2b)), three myoinhibitory peptides, corazonin, and M. sexta allatostatin (Manse-AS). A further six masses were in agreement with Y/FXFGLamide allatostatins identified from other Lepidoptera. The sequence identities of FLRFamide I and AKH were confirmed using post source decay analysis. Fragmentation by collision-induced dissociation MS/MS identified an extended AKH peptide. The apparent differences in the peptides present in male and female retrocerebral complexes are most likely quantitative rather than sex specific.     

The host-seeking inhibitory peptide, Aea-HP-1, is made in the male accessory gland and transferred to the female during copulation

Male accessory glands (MAGs) of insects are responsible for the production of many of the seminal fluid proteins and peptides that elicit physiological and behavioral responses in the post-mated female. In the yellow fever mosquito, Aedes aegypti, seminal fluid components are responsible for stimulating egg production, changing female behavior away from host-seeking toward egg-laying and mating refractoriness, but hitherto no behavior-modifying molecule from the MAGs has been structurally characterized. We now show using mass spectrometry and HPLC/ELISA that the MAG is a major site of synthesis of the biologically active decapeptide, Aea-HP-1 (pERPhPSLKTRFamide) that was first characterized by Matsumoto and colleagues in 1989 from mosquito head extracts and shown to have host-seeking inhibitory properties. The peptide is localized to the anterior portion of the MAG, occurs at high concentrations in the gland and is transferred to the female reproductive tract on copulation. Aea-HP-1 has a pyroglutamic acid at the N-terminus, an amidated carboxyl at the C-terminus and an unusual 4-hydroxyproline in position 4 of the peptide. The structure of the peptide with its blocked N- and C-termini confers resistance to metabolic inactivation by MAG peptidases; however the peptide persists for less than 2h in the female reproductive tract after copulation. Aea-HP-1 is not a ligand for the mosquito sex peptide/myoinhibitory peptide receptor. A. aegypti often mate close to the host and therefore it is possible that male-derived Aea-HP-1 induces short-term changes to female host-seeking behavior to reduce potentially lethal encounters with hosts soon after insemination.     

A new algorithm for the evaluation of shotgun peptide sequencing in proteomics: support vector machine classification of peptide MS/MS spectra and SEQUEST scores

Shotgun tandem mass spectrometry-based peptide sequencing using programs such as SEQUEST allows high-throughput identification of peptides, which in turn allows the identification of corresponding proteins. We have applied a machine learning algorithm, called the support vector machine, to discriminate between correctly and incorrectly identified peptides using SEQUEST output. Each peptide was characterized by SEQUEST-calculated features such as delta Cn and Xcorr, measurements such as precursor ion current and mass, and additional calculated parameters such as the fraction of matched MS/MS peaks. The trained SVM classifier performed significantly better than previous cutoff-based methods at separating positive from negative peptides. Positive and negative peptides were more readily distinguished in training set data acquired on a QTOF, compared to an ion trap mass spectrometer. The use of 13 features, including four new parameters, significantly improved the separation between positive and negative peptides. Use of the support vector machine and these additional parameters resulted in a more accurate interpretation of peptide MS/MS spectra and is an important step toward automated interpretation of peptide tandem mass spectrometry data in proteomics.     

The effect of galanin, CGRP and ANP on spontaneous smooth muscle activity of rat uterus

In the present study the effect of the newly isolated peptides galanin, calcitonin gene-related peptide (CGRP) and atrial natriuretic peptide (ANP) was examined on spontaneous uterine smooth muscle activity on the rat in vitro. Galanin showed a slight and insignificant stimulatory effect on the amplitude, while both CGRP and ANP were found to be potent inhibitors of the uterine smooth muscle contractions. In connection with the recent demonstration of galanin and CGRP nerves in the genital tract, these pharmacological findings suggest that the peptides may participate in the control of nervous control of uterine muscular activity, although the exact physiological roles remain to be clarified.     

Efficacy of antibodies against a chimeric synthetic peptide encompassing epitopes of bonnet monkey (Macaca radiata) zona pellucida-1 and zona pellucida-3 glycoproteins to inhibit in vitro human sperm-egg binding

Immunocontraception achieved by immunization with zona pellucida (ZP) glycoproteins is invariably associated with ovarian dysfunction. Use of ZP glycoprotein-based synthetic peptides as immunogens has been proposed to overcome adverse side effects on ovaries. In the present study, a chimeric peptide encompassing the epitopes of bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1; amino acid residues 251-273) and ZP glycoprotein-3 (bmZP3; amino acid residues 324-347), separated by a tri-glycine spacer, was synthesized and conjugated to diphtheria toxoid (DT). Immunization of female BALB/cJ mice and bonnet monkeys with the chimeric peptide led to generation of antibodies that reacted with the chimeric peptide, individual bmZP1 & bmZP3 peptides, and also recombinant bmZP1 and bmZP3 proteins expressed by E. coli in an ELISA. Indirect immunofluorescence studies revealed that the immune serum also recognized human as well as bonnet monkey ZP. A significant inhibition of human sperm binding to ZP was observed with antibodies generated against the chimeric peptide in mice (P = 0.0001) as well as monkeys (P = 0.0002) in a hemizona assay (HZA). The inhibition efficacy was significantly higher than that observed by using antibodies against the individual bmZP1 and bmZP3 peptides. Interestingly, no ovarian pathology was observed in female bonnet monkeys immunized with the chimeric peptide. These studies have demonstrated that the chimeric peptide encompassing peptides of multiple ZP glycoproteins may be a promising candidate antigen for designing immunocontraceptive vaccines.     

ILME: a waterborne pheromonal peptide released by the eggs of Sepia officinalis

A novel tetrapeptide modulating the oviduct contractions was characterized from egg mass of Sepia officinalis. After two purification steps by rpHPLC, an apparent pure fraction containing the biological activity was submitted to MALDI-TOF analysis. The mass spectrum revealed 6 peaks of m/z 293, 505, 596, 613, 728, and 745. The tissue peptide mapping performed in LC-MS demonstrated the occurrence of the m/z 505 peptide in the follicles, the full-grown oocytes, and in the eggs. This peptide was also recovered in the seawater after the incubation of full grown oocytes or eggs, demonstrating a release in the genital tract and in the environment. Edman degradation gave the following sequence: Ileu-Leu-Met-Glu. The synthetic peptide applied to the whole genital tract triggered a cyclisation of the contractions at 10(-14) M. ILME appeared to be a chemical messenger released by the oocytes and the eggs, and was able to exert both paracrine and pheromonal activity.     

Expression of vasoactive intestinal peptide (VIP) receptors in human uterus

We show the existence of functional vasoactive intestinal peptide (VIP) receptors in normal human female genital tract (endometrium, myometrium, ovary and Fallopian tube) as well as in leiomyoma (a frequent uterine pathology). The correlation between VIP binding and stimulation of adenylyl cyclase activity for all studied tissues was linear (r = 0.86) suggesting the expression of VIP receptors throughout the human female genital tract. Immunodetection of VIP receptor subtypes gave different molecular weights for VPAC(1) (47 kDa primarily) and VPAC(2) (65 kDa), which may be due to different glycosylation extents. In conclusion, this study demonstrates the expression of both subtypes of VIP receptors and their functionality in human female genital tract, suggesting that this neuropeptide could play an important physiological and pathophysiological role at this level.