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.     

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.     

嘉庚蛸雌性生殖系统组织学观察

对象山港自然海区中的嘉庚蛸(Octopus tankahkeei)雌性生殖系统的组织学结构进行了研究.结果表明,雌性生殖系统由卵巢、输卵管、输卵管腺组成.卵巢单个、球形,内包裹滤泡细胞围成的卵子,输卵管1对,开口于外套腔中部,每条输卵管中部膨大形成圆球状的输卵管腺.近端输卵管内具两瓣蘑菇状突起,上有不规则短指状分枝,突...     

Endogenous ghrelin and 5-HT regulate interdigestive gastrointestinal contractions in conscious rats

Endogenous ghrelin causes interdigestive contractions of the stomach in rats. In contrast, previous studies showed that 5-HT(3) and 5-HT(4) receptors were involved in regulating intestinal interdigestive contractions. We studied the possible role of endogenous ghrelin and 5-HT regulating interdigestive gastrointestinal (GI) contractions in rats. Four strain gauge transducers were implanted on the antrum, duodenum, and proximal and distal jejunum. After an overnight fast, GI contractions were recorded in freely moving conscious rats and ghrelin receptor antagonists [(d-lys3)GHRP6; 1 micromol/kg], 5-HT(3) antagonists (Ondansetron; 0.5 mg/kg) and 5-HT(4) antagonists (GR 125,487; 1 mg/kg) were administered (bolus iv). To evaluate the relationship between the luminal concentrations of 5-HT and phase III-like contractions of the duodenum, duodenal juice was collected via the intraduodenal catheter. 5-HT content of the duodenal juice was measured by HPLC. (d-lys3)GHRP6 significantly attenuated the occurrence and amplitude of phase III-like contractions of the antrum, but not the duodenum and jejunum. 5-HT(4) antagonists significantly reduced spontaneous phase III-like contractions of the jejunum, without affecting those of the antrum and duodenum. In contrast, 5-HT(3) antagonists did not affect phase III-like contractions in GI tract. Luminal concentration of 5-HT at the phase III-like contraction (36.0 +/- 13.3 ng/ml, n = 9) was significantly higher than that at the phase I-like contractions of the duodenum (4.9 +/- 1.6 ng/ml, n = 9, P < 0.05). It is suggested that released ghrelin from the gastric mucosa mediates gastric phase III-like contractions, whereas 5-HT released from enterochromaffin cells of the duodenal mucosa mediates intestinal phase III-like contractions via 5-HT(4) receptors.     

Central effects of the peptides,SchistoFLRFamide and proctolin,on locust oviduct contraction

We have developed a semi-intact preparation-consisting of an isolated oviduct with abdominal ganglia VII and VIII intact and attached-with which to characterize the effects on oviduct contraction, of peptides that are bath applied to CNS tissues. The work presented here offers a qualitative analysis of the central effects of SchistoFLRFamide and proctolin upon action potentials recorded from the oviducal nerves and upon oviduct contraction. In the process of this, we hope to demonstrate that a previously characterized putative CNS SchistoFLRFamide receptor [Peptides 23 (2002) 765] is a functional receptor.SchistoFLRFamide (10(-6)M), bath applied to abdominal ganglion VII, caused an increase in action potential frequencies recorded from the oviducal nerves, as well as an increase in the frequency of phasic contractions of the oviduct. Although the function of this response is not known, these results further support the possibility that the putative CNS SchistoFLRFamide receptors are functional receptors.Proctolin (10(-6)M), bath applied to abdominal ganglion VIII, altered the rhythmic bursting of action potentials recorded from the oviducal nerve and changed the appearance and cycle duration of neurogenic oviduct contractions.     

Ovarian jelly-peptides (OJPs), a new family of regulatory peptides identified in the cephalopod Sepia officinalis

In marine invertebrates, numerous water-borne peptides involved in reproductive behavior have been characterized. In this study, we focused on three ovarian water-borne peptides, released by full-grown oocytes (FGO) in the genital coelom and in the lumen of the oviduct in the cuttlefish Sepia officinalis. The first one (DQVKIVL), was characterized by the monitoring of HPLC purified fraction using a myotropic bioassay. Subsequently, a peptidomic approach consisting of a mass spectrometry comparative screening performed between the peptide content of FGO with that of FGO-conditioned medium, led to the identification of two additional water-borne peptides. The second peptide identified (DEVKIVL) was characterized by MS/MS and the primary structure of the third one (DEVKIVLD) was elucidated by a combination of Edman degradation, acid hydrolysis and MS/MS analysis. Sequence homology, tissue mapping and bioactivity demonstrate that these peptides belong to the same family. DQVKIVL-related-peptides strictly localized in the female genital tract modulate the whole female genital tract and the main nidamental gland contractions. Furthermore, these peptides form a jelly, when resuspended in water. This particular property could play an important role in the kinetics of peptide diffusion in the external medium. Thus, these regulatory peptides were named ovarian jelly-peptides (OJPs).     

The effect of proctolin analogues and other peptides on locust oviduct muscle contractions

The locust oviduct bioassay system was used to assess the ability of a variety of peptides to induce oviducal contractions. Proctolin analogues were three orders of magnitude less potent than proctolin. Proctolin supra-analogue and Arg-Tyr-Leu-Ala-Thr demonstrated high activity. Perhaps the most significant finding was the discrepancy between the high binding capacity of the proctolin analogue Arg-Tyr-Ser-Pro-Thr and its relatively low myotropic activity. This observation argues for a crucial role for the leucine residue in activating the proctolin receptor. Several other myotropic peptides were tested for their effect on oviduct contractions. FMRFamide caused contractions at doses several orders of magnitude higher than proctolin. The FLRFamide leucomyosuppressin inhibited proctolin-induced contractions. In addition, myomodulin and catch relaxing peptide caused oviducal contractions at low concentrations. The enkephalins had no effect when applied alone but potentiated proctolin-induced oviduct contractions. The mechanism of the potentiation is not known. The data argue for the presence of several binding sites on the oviduct membrane.     

Spontaneous and neurally evoked contractions of visceral muscles in the oviduct of Locusta migratoria

The oviducts of Locusta migratoria are innervated by a pair of nerves which arise from, the seventh abdominal ganglion. A distinctive network of striated muscle fibres occurs in the oviducts. The lateral oviducts and common oviduct consist of an inner circular layer of muscle and an outer longitudinal layer of muscle. At the junction of the lateral and common oviduct an additional thin longitudinal layer is found adjacent to the basement epithelium. The oviducts contracted spontaneously when isolated from the central nervous system. These myogenic contractions took the form of peristaltic contractions in the lateral oviduct, and intermittent phasic-like contractions of the posterior regions of the lateral oviduct and the common oviduct. These phasic-like contractions were associated with individual complex potentials recorded extracellularly from the muscle fibres. In locusts that had been interrupted in the process of egg laying, there were large-amplitude action potentials, firing in a bursting pattern, in the oviducal nerves. These large action potentials were absent in locusts that had not been egg-laying. These action potentials were associated with both bioelectric potentials and mechanical events in the posterior region of the lateral oviduct and the common oviduct. Electrical stimulation of the oviducal nerve mimicked the effects of spontaneous action potentials, resulting in the appearance of monophasic potentials and contractions. The contractions were graded and dependent upon both frequency and duration of stimulation. It is concluded that the oviducts of Locusta are both myogenic and neurogenic. The role of these contractions in oviposition is discussed.     

Luminally released serotonin stimulates colonic motility and accelerates colonic transit in rats

Enterochromaffin (EC) cells of the epithelial cells release 5-HT into the lumen, as well as basolateral border. However, the physiological role of released 5-HT into the lumen is poorly understood. Concentrations of 5-HT in the colonic mucosa, colonic lumen, and feces were measured by HPLC in rats. To investigate whether intraluminal 5-HT accelerates colonic transit, 5-HT and (51)Cr were administered into the lumen of the proximal colon, and colonic transit was measured. To investigate whether 5-HT is released into the lumen, we used an ex vivo model of isolated vascularly and luminally perfused rat proximal colon. To investigate whether luminal 5-HT is involved in regulating stress-induced colonic motility, the distal colonic motility was recorded under the stress loading, and a 5-HT(3) receptor antagonist (ondansetron, 10(-6) M, 0.5 ml) was administered intraluminally of the distal colon. Tissue content of 5-HT in the proximal colon (15.2 +/- 4.3 ng/mg wet tissue) was significantly higher than that in the distal colon (3.3 +/- 0.7 ng/mg wet tissue), while fecal content and luminal concentration of 5-HT was almost the same between the proximal and distal colon. Luminal administration of 5-HT (10(-6)-10(-5) M) significantly accelerated colonic transit. Elevation of intraluminal pressure by 10 cmH(2)O significantly increased the luminal concentration of 5-HT but not the vascular concentration of 5-HT. Stress-induced stimulation of the distal colonic motility was significantly attenuated by the luminal administration of ondansetron. These results suggest that luminally released 5-HT from EC cells plays an important role in regulating colonic motility in rats.     

GABA modulation of cholinergic transmission in rat oviduct

The effects of electrical stimulation, γ-aminobutyric acid (GABA), acetylcholine (ACh), norepinephrine (NE), 5-hydroxytryptamine (5-HT), GABA agonists and bicuculline were studied on spontaneous movements of isolated rat oviduct. The tissue did not respond to electrical stimulation or to GABA, NE and 5-HT when added to the incubation medium. ACh produced contractions related to its concentration which were maximal at the diestrous-1 phase when GABA caused a 20% rise in the ACh contraction. This effect was mimicked by GABA agonists whereas it was suppressed by bicuculline. β-Estradiol benzoate (EB) increased ACh contractions in diestrous-1 and in the late proestrous phases. GABA did not modify the EB effect. Progesterone did not modify ACh contractions in any of the studied phases. These findings suggest a possible modulatory role for GABA on ACh responses in the isolated rat oviduct.     

输卵管妊娠时输卵管壁肥大细胞的研究

目的　探讨肥大细胞在输卵管妊娠中的数量变化及其与血清性激素的关系。方法 :取输卵管妊娠时的输卵管及月经周期的增生期、分泌期和正常宫内早孕时的输卵管 ,常规石蜡切片 ,用甲苯胺蓝染色法显示肥大细胞 ;用酶免疫分析法检测输卵管妊娠患者、正常育龄未孕妇女 (增生期和分泌期 )及正常宫内早孕妇女血清雌二醇和孕酮水平。结果 :输卵管妊娠患者血清雌二醇和孕酮水平均高于正常育龄未孕妇女 (增生期和分泌期 ) ,低于正常宫内早孕妇女 ,四组间两两比较差异均有显著性 (P <0 0 5 ) ;肥大细胞主要分布于输卵管肌层 ,其数量变化为 :输卵管妊娠组较增生期和分泌期这两组均少 ,差异均有显著性 (P <0 0 5 ) ,而增生期和分泌期这两组肥大细胞数量变化不明显 ,差异无显著性 (P >0 0 5 ) ;两例正常宫内早孕时的输卵管壁肥大细胞数量明显比输卵管妊娠组多 ,与增生期和分泌期这两组比较 ,肥大细胞数量变化不明显。结论 :1 人输卵管壁内肥大细胞的数量不受血清性激素水平的影响。 2 输卵管妊娠时肥大细胞数量减少     

A estradiol-17beta receptor in the reproductive system of the female of Octopus vulgaris: characterization and immunolocalization

In this study, for the first time we have identified an estradiol-17beta receptor (ER) in the reproductive system of the female of Octopus vulgaris. Scatchard analysis revealed that one binding component with high affinity and low capacity for the ligand was present in the cytosol, but not in the nuclear extract of the ovary and the oviduct. A steroid specificity competition assay showed that 3H-estradiol-17beta binding activity showed a preference for estradiol-17beta. DNA-cellulose chromatography confirmed the presence of one 3H-estradiol-17beta binding component. By using antibodies anti ER (578-595), we have localized by Western blotting one band of about 70 kDa. ER immunoreactivity has been localized in the nuclei of the follicle cells of the ovary, in the nuclei of the epithelium lining the proximal portion of the oviduct and in the nuclei, and in the cytoplasm of the inner region of the oviducal gland and in the cytoplasm of the outer region of the oviducal gland. These data, taken together, provide evidence that in Octopus vulgaris the ER has biochemical and immunohistochemical characteristics resembling those of ER in vertebrates.     

A serotonin receptor antagonist induces oocyte maturation in both frogs and mice: evidence that the same G protein-coupled receptor is responsible for maintaining meiosis arrest in both species

Accumulating evidence has indicated that vertebrate oocytes are arrested at late prophase (G2 arrest) by a G protein coupled receptor (GpCR) that activates adenylyl cyclases. However, the identity of this GpCR or its regulation in G2 oocytes is unknown. We demonstrated that ritanserin (RIT), a potent antagonist of serotonin receptors 5-HT2R and 5-HT7R, released G2 arrest in denuded frog oocytes, as well as in follicle-enclosed mouse oocytes. In contrast to RIT, several other serotonin receptor antagonists (mesulergine, methiothepine, and risperidone) had no effect on oocyte maturation. The unique ability of RIT, among serotonergic antagonists, to induce GVBD did not match the antagonist profile of any known serotonin receptors including Xenopus 5-HT7R, the only known G(s)-coupled serotonin receptor cloned so far in this species. Unexpectedly, injection of x5-HT7R mRNA in frog oocytes resulted in hormone-independent frog oocyte maturation. The addition of exogenous serotonin abolished x5-HT7R-induced oocyte maturation. Furthermore, the combination of x5-HT7R and exogenous serotonin potently inhibited progesterone-induced oocyte maturation. These results provide the first evidence that a G-protein coupled receptor related to 5-HT7R may play a pivotal role in maintaining G2 arrest in vertebrate oocytes.     

Ultrastructure function and regulation of the oviduct of the Rana ridibunda (author's transl)]

An investigation has been carried out into the structure, ultrastructure, function and of the oviduct on the adult female of Rana ridibunda. The most important part of the oviduct comprises tubulary glands and a luminal epithelium which is composed of ciliated cells and vesicular cells. The discharge processes of secretory substances were studied. Injection of the mature females with estrogens and progesterone have show that progesterone was the most effective in provoking jelly release. It is probably that in Rana ridibunda the pituitary hormones act on the follicle cells of ripe oocytes, causing them to secrete a progesterone-like hormone which provodes the maturation of the oocytel and jelly release from the oviducal glands.     

The mating and reproductive biology of the freshwater planktonic calanoid copepod Eudiaptomus gracilis

1. Quantitative aspects of the mating and reproductive biology of the freshwater planktonic calanoid copepod Eudiaptomus gracilis, including duration and frequency of mating, duration of various phases of the oviducal cycle, egg production rate and adult longevity were studied under laboratory conditions. One set of copepods was fed the alga Chlamydomonas reinhardtii whose density was adjusted to 2 × 10⁵ cells mL^?1 (about 10 mg C L^?1), another set was fed a mixed diet consisting of natural plankton (copepod nauplii, small rotifers and large algae) in the size range of 50–150 μm (dry mass approximately 90 mg L^?1). 2. The entire mating process, from the grasping of the female by the male’s right geniculate antennule to the separation of the pair, lasted about 2 min. Spermatophore placement started at about 30 s to 1 min after mating began and took approximately 1 min. Immediately after the spermatophore had been fixed in the female’s genital segment, the pair separated. 3. The total oviducal cycle, including the gravid phase where the female carried ripe oocytes and the non‐gravid phase where the female did not carry ripe oocytes, lasted about 5–6 days. The non‐gravid phase was particularly long; it was longer than the gravid phase and constituted 62–72% of the total cycle. 4. Mating and spermatophore placement usually occurred with gravid females although occasionally (in 30 of 200 observations) spermatophores were attached in the genital segment of non‐gravid females. Generally two to four, but up to seven, spermatophores were observed at a female’s genital segment at the same time. 5. Clutch size, rate of egg production and adult longevity depended on food. When fed on C. reinhardtii, females carried 7–8 eggs clutch^?1, produced a mean of 1.3 clutches and lived 14 days on average. When fed natural mixed food, females carried 10 eggs clutch^?1, produced 5.6 clutches and lived 37 days on average. 6. Removal of males after the first clutch resulted in no further egg production. Re‐mating is necessary in E. gracilis for continuous clutch production and the production of fertile eggs. 7. Mating duration is comparatively short and the non‐gravid phase comparatively long in E. gracilis. This could be an adaption to the life in the pelagic zone of the lake, where fish predators are present. Fish select ovigerous females, pairs in copula and, probably, females with ripe oocytes which make them conspicuous. Thus, a short mating duration and a prolonged period without conspicuous oocytes, can be advantageous.     

rRNA accumulation and protein synthetic patterns in growing mouse oocytes

The rRNA contents of mouse primordial oocytes, three stages of growing oocytes, full-grown oocytes, and ovulated ova have been measured by hybridization of RNA samples to excess 3H-DNA complementary to rRNA. Since it was known from previous work that rRNA is stable, the results when plotted against days of oocyte growth indicated that rRNA was synthesized at a constant rate over the first 9 days of growth and about 1.5 times faster in the last 5 days. The maximum value of 0.3 ng per oocyte was attained by about 14 days of growth in oocytes 59 micrometers in diameter, well below the maximum diameter of 77 micrometers for full-grown oocytes. The stability of proteins synthesized in mid-growth phase oocytes was measured by labeling for 5 h with 35S-methionine and then following the decline of incorporated label during a 48h chase; 40% of the label decayed with a half-life of 11 h. and 60% was apparently stable. The two-dimensional electrophoretic patterns of labeled proteins synthesized by growing and full-grown oocytes were compared. The principal change was the appearance or great increase in intensity of several spots in full-grown oocytes as compared to growing oocytes. Egg proteins separated on a two-dimensional gel were visualized by silver staining. The cytoskeletal proteins actin, tubulin, and putative intermediate filament protein, as well as putative lactate dehydrogenase, were synthesized in growing and full-grown oocytes, and accumulated to form a significant portion of bulk egg protein.     

Increased mortality during early embryonic development after in-vitro fertilization of rat oocytes

Immature female rats (60-65 g) were injected with 4 i.u. PMSG on Day -2, and allocated to 3 groups. For Groups I and II, unmated donors were killed 67-69 h after PMSG injection, shortly after the expected time of ovulation. Oocytes were recovered from the oviducts and transferred immediately into the oviduct of mated recipients (Group I) whose ipsilateral ovary had been exposed by peeling back the bursa, preventing endogenous oocytes from entering the oviduct, or were fertilized in vitro (Group II) and were transferred 16-18 h later. Rats in Group III were allowed to mate and half were killed 6 h after mating. The fertilized oocytes were then incubated for 10-12 h until transfer. The remaining rats in Group III were killed 16-18 h after mating and fertilized oocytes were collected and transferred immediately. Recipient rats were killed on Days 2, 5, 8 and 20. Zygotes resulting from in-vitro fertilization (Group II) were as able as those fertilized in donors (Group III) or recipients (Group I) to develop to the 2-cell stage, but underwent significantly greater embryonic loss beyond this stage of development. There was a slower rate of development of such oocytes to the blastocyst stage (Day 5) and a lower mean weight of implantation sites (Day 8). Transfer of zygotes after in-vitro fertilization resulted in a loss of 35% of the embryos at the time of implantation. These results suggest that in-vitro fertilization of rat oocytes leads to defects in the embryos causing a delay in early embryo development and a large number of implantation losses.