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Endocrinology, doi:10.1210/en.2008-0323
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Endocrinology Vol. 149, No. 9 4346-4356
Copyright © 2008 by The Endocrine Society

A Novel Biological Role of Tachykinins as an Up-Regulator of Oocyte Growth: Identification of an Evolutionary Origin of Tachykininergic Functions in the Ovary of the Ascidian, Ciona intestinalis

Masato Aoyama1, Tsuyoshi Kawada1, Manabu Fujie, Kohji Hotta, Tsubasa Sakai, Toshio Sekiguchi, Kotaro Oka, Nori Satoh and Honoo Satake

Suntory Institute for Bioorganic Research (M.A., T.K., T.Sa., T.Se., H.S.), Osaka 618-8503, Japan, Division of Innovative Research, Creative Research Initiative (M.F.), Hokkaido University, Sapporo 060-1113, Japan; Department of Bioscience and Informatics (K.H., K.O.), Faculty of Science and Technology, Keio University, Yokohama 232-8522, Japan; and Department of Zoology (N.S.), Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan

Address all correspondence and requests for reprints to: Honoo Satake, Suntory Institute for Bioorganic Research, Wakayamadai 1-1-1, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan. E-mail: satake{at}sunbor.or.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tachykinins (TKs) and their receptors have been shown to be expressed in the mammalian ovary. However, the biological roles of ovarian TKs have yet to be verified. Ci-TK-I and Ci-TK-R, characterized from the protochordate (ascidian), Ciona intestinalis, are prototypes of vertebrate TKs and their receptors. In the present study, we show a novel biological function of TKs as an inducible factor for oocyte growth using C. intestinalis as a model organism. Immunostaining demonstrated the specific expression of Ci-TK-R in test cells residing in oocytes at the vitellogenic stage. DNA microarray and real-time PCR revealed that Ci-TK-I induced gene expression of several proteases, including cathepsin D, chymotrypsin, and carboxy-peptidase B1, in the ovary. The enzymatic activities of these proteases in the ovary were also shown to be enhanced by Ci-TK-I. Of particular significance is that the treatment of Ciona oocytes with Ci-TK-I resulted in progression of growth from the vitellogenic stage to the post-vitellogenic stage. The Ci-TK-I-induced oocyte growth was blocked by a TK antagonist or by protease inhibitors. These results led to the conclusion that Ci-TK-I enhances growth of the vitellogenic oocytes via up-regulation of gene expression and enzymatic activities of the proteases. This is the first clarification of the biological roles of TKs in the ovary and the underlying essential molecular mechanism. Furthermore, considering the phylogenetic position of ascidians as basal chordates, we suggest that the novel TK-regulated oocyte growth is an "evolutionary origin" of the tachykininergic functions in the ovary.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OVARIAN FUNCTIONS, including growth of oocytes and follicles, are believed to involve coordinated and multistep biological events that undergo functional regulation by a wide range of endogenous factors. There have been an increasing number of reports suggesting that several neuropeptides and hormone peptides play crucial roles in the functional regulation of ovaries in endocrine, paracrine, and neurotransmission systems (1).

Tachykinins (TKs) are multifunctional vertebrate brain/gut peptides (2, 3, 4, 5, 6). The mammalian TK family consists of four major peptides: substance P (SP), neurokinin (NK) A, NKB, and hemokinin (HK)-1/endokinins (EKs). The TAC1 (or PPTA) gene generates four splicing variants that produce SP alone or SP and NKA, the TAC3 (or PPTB) gene yields only NKB, and the TAC4 (or PPTC) gene generates multiple splicing variants encoding various EKs (2, 3, 4, 5, 6, 7, 8). The N-terminally extended forms of NKA, neuropeptide K and neuropeptide-{gamma}, are generated from the TAC1 gene due to the lack of posttranslational endoproteolysis (2, 3, 4, 5, 6). Three TK receptors, namely NK1, NK2, and NK3, induced both elevation of intracellular calcium and production of cAMP with moderate ligand selectivity to SP plus HK-1/EK, NKA, and NKB, respectively (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). In addition, neuropeptide K and neuropeptide-{gamma} show the highest affinity to NK2. TKs are known to participate in smooth muscle contraction, vasodilation, nociception, inflammation, neurodegeneration, and neuroprotection in a neuropeptidergic or endocrine fashion (2, 3, 4, 5, 6, 7, 8), and thus, both central and peripheral tachykininergic systems have become attractive as targets for the development of various clinical agents (13, 14, 15). Recently, tissue distribution of TKs and TK receptors in genital organs was detected by RT-PCR, immunostaining, or RIA (16, 17, 18, 19, 20, 21, 22, 23, 24, 25). Several recent studies also demonstrated the physiological or pathological effects regulated by TKs in reproductive tracts, including progression of sperm motility by SP (18), uterus contraction by SP, NKA, and NKB (19, 20, 21, 22), and elevation of circulating NKB in preeclampsia or pregnancy via enhancement of expression of TAC3 gene in the placenta (23, 24). However, the molecular mechanisms or signaling pathways underlying such reproduction-related functions almost remain to be understood. In the ovary, the presence of TK peptides was observed in follicle fluids and capsaicin-sensitive afferent neurons (16, 17), and the expression of the TAC1, TAC3, and NK1–3 genes was detected in the mammalian ovary, oocytes, and granulosa cells by RT-PCR (16, 17, 25), but the rigorous localization of the peptides and the receptors in the ovary has yet to be determined. Several studies indicate the possibility that TKs affect release of sexual steroid hormones (17). Nevertheless, this biological effect and the functional correlation of TKs with sexual steroid hormones still remain controversial. Such inconclusive data resulted primarily from the difficulty in elucidating in vitro and in vivo physiological functions of TKs in the mammalian ovaries due to the heterogeneous quality of the oocytes (the very small number of successfully growing oocytes), the low basal levels of TK receptor expression, and complicated sexual periods (16, 17, 22, 25, 26, 27). These studies suggest a potential requirement for new approaches and model organisms.

In the previous study, we characterized a novel TK peptide, Ci-TK-I and the endogenous receptor, Ci-TK-R, from the ascidian Ciona intestinalis (28), a basal chordate, as an emerging deuterostome invertebrate model animal (29, 30). Ci-TK-I harbors the TK C-terminal consensus, and the structural organization of the Ci-TK gene displays considerable homology to those of vertebrate TAC1 genes (28). Moreover, Ci-TK-R was equipotently activated by Ci-TK, SP, and NKA. These findings, combined with the phylogenetic position of ascidians as basal chordates, provide evidence that the TK family is conserved in chordates, and that Ci-TK-I and Ci-TK-R are direct prototypes or evolutionary origins of vertebrate TKs and TK receptors, respectively. Ci-TK-R mRNA was also abundantly expressed in the ovary (28). Altogether, the functional analysis of Ci-TK-I in the Ciona ovary is expected to provide crucial clues to the clarification of not only the essential biological roles of TKs in the ovarian functions conserved in chordates but also the evolutionary origin of the tachykininergic regulatory system in the ovary. In this paper we present a novel function of TKs as an up-regulator of oocyte growth. Ci-TK-I enhances the growth of oocytes from the vitellogenic stage to the post-vitellogenic stage via up-regulation of gene expression and enzymatic activity of several proteases. To the best of our knowledge, this is the first report on the biological roles of TKs in the ovary and the underlying molecular mechanism for tachykininergic oocyte growth in all animal species.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adults of C. intestinalis were cultivated and collected at the Maizuru Fisheries Research Station of Kyoto University, and maintained in seawater at 18 C.

Immunohistochemistry
The Ciona ovaries were fixed overnight at 4 C in Bouin fluid, embedded in paraffin, and cut into 10-µm sections. Anti-Ci-TK-R chicken antiserum was ordered from Operon Biotechnologies Inc. (Tokyo, Japan). Immunostaining using Ci-TK-R antiserum diluted to 1:1000 was performed as previously described (31). The specificity of the Ci-TK-R antibody was confirmed by Western blotting on membrane proteins prepared from the Ciona ovary (data not shown). The immunoreactivity was visualized by an indirect immunofluorescence technique using 1:1000 diluted secondary antibody 568 goat antichicken IgG (Molecular Probes Inc., Eugene, OR). Coverslips were mounted in Fluorosafe mounting medium (Calbiochem, San Diego, CA) and viewed using a Nikon Eclipse TE2000-S photomicroscope (Nikon Corp., Tokyo, Japan) equipped with epifluorescence. Ciona oocytes are known to mature through four characteristic stages (29, 32): small pre-vitellogenic oocytes (stage I), vitellogenic oocytes (stage II), post-vitellogenic oocytes (stage III), and germinal vesicle breakdown (GVBD) mature oocytes (stage IV). Each growth stage of Ciona oocytes was classified based on morphological characteristics such as structure of follicular cells and oocyte size.

Tissue preparation
All Ciona ovaries used for the following studies were cut longitudinally into symmetrical half-pieces to minimize experimental errors resulting from used individual ovaries.

DNA microarray analysis
Total RNA was isolated from the half-portion of ovary untreated or treated with 0.1 µM Ci-TK-I for 16 h as previously described (33, 34). A Ciona 44K custom oligo DNA microarray chip (Agilent Technologies, Inc., Palo Alto, CA) containing 42034 oligonucleotides was used for comparative analysis of ovary genes regulated by Ci-TK. Preparation of Cy3- or Cy5-labeled RNA probes, hybridization, signal scanning, image analysis, and data extraction were performed as previously described (33, 34).

Real-time PCR
Total RNA was isolated from the ovary half-portion ovary incubated with or without Ci-TK-I for 16 h as previously described (33, 34). The real-time PCR was performed using Power SYBR Green PCR Master Mix and ABI Prism 7000 (Applied Biosystems, Foster City, CA). Quantification of each Ci-TK-I-up-regulated gene was used as a barometer for the induction level for transcriptional up-regulation, whereas quantification of Ci-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as a control for data normalization. The primers for genes (supplemental Table 1, which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org) were designed using Primer Express 1.5 Software (Applied Biosystems). Total volume of reaction mixtures was 50 µl, consisting of 1 µl template cDNA solution, each 100 nM primers and 25 µl SYBR Green Master Mix solution. The real-time PCR was performed for initial steps at 50 C for 2 min and at 95 C for 10 min, followed by 45 cycles at 95 C for 15 sec and at 60 C for 1 min. The melting curve analysis was performed to confirm the absence of primer dimers. Cycle threshold (Ct) and difference of Ct ({Delta}Ct) values were calculated according to the manufacturer’s instruction. Ct values represent the PCR cycle number when the PCR product arrived at the detectable level, and {Delta}Ct values show the induction level of target genes of the half-portion of the ovary treated with Ci-TK-I over the untreated half-portion.

Functional expression of Ci-TK-R in Xenopus oocytes
Preparation of the Ci-TK-R cRNA, the electrophysiological assays, and the data analyses were performed exactly as previously reported (28).

Measurement of protease activity in the ovary
The half-piece ovary was incubated in sterile seawater with Ci-TK-I or a Ci-TK-R agonist [Sar9, Met(O2)11]SP in the presence or absence of 1 µM GR94800 at 18 C for 16 h. The Ci-TK-I-treated or untreated ovaries were homogenized in 50 mM Tris HCl (pH 7.6) (for total protease assay) or cathepsin D assay buffer (Sigma-Aldrich, St. Louis, MO; for cathepsin D assay) and centrifuged at 15,000 x g at 4 C for 5 min. The supernatants were frozen with liquid nitrogen, and stored at –80 C until used for assays. Measurement of total protease activity of the Ciona ovary extracts was performed as previously described (35). Protein concentrations of the samples were determined using a BGA protein Assay Reagent Kit (Pierce, Rockford, IL) with BSA as a standard. The cathepsin D activity was quantified using the Cathepsin D Assay Kit (Sigma-Aldrich) according to the manufacturer’s instruction.

Morphological change of oocytes by Ci-TK-I
Vitellogenic (stage II) oocytes and post-vitellogenic (stage III) oocytes were classified based on morphological characteristics such as structure of follicle cells, oocyte size, organization, and localization of mitochondria (29, 32). Approximately 20 cells per well of stage II oocytes were isolated from the ovary, transferred into 200 µl filtered sterile seawater in a 96-well plate, and incubated at 18 C for 15 h after addition of Ci-TK-I (final concentration, 0.1 µM) or [Sar9, Met(O2)11]SP (final concentration, 0.1 µM), with or without a protease inhibitor (1 µM leupeptin, 1 µM pepstatin A, and 1 µM of EDTA) or GR94800 (1 µM). Each oocyte was fixed using 4% paraformaldehyde/PBS and washed three times with PBS containing 0.01% Triton X-100 (PBST). The fixed oocytes were incubated with 0.25 µl/ml MitoTracker Red (Molecular Probes) in PBST for 20 min at room temperature. After washing three times by PBST, the oocytes were incubated with Alexa Fluor 488 phalloidin (Molecular Probes) in PBST for 4 C, overnight. Mitochondria localization of each oocyte was observed using a confocal microscopy LSM510 META NLO (Carl Zeiss, Tokyo, Japan). To reconstruct the three-dimensional (3D) images, 70–150 cross-section images (1-µm each) of Alexa Fluor 488 phalloidin-stained oocytes from top to bottom per sample were collected as previously described (36).

Statistical analysis.
Results are shown as mean ± SEM. Data were analyzed by one-way ANOVA with Dunnett error protection. Differences were accepted as significant for P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distribution of Ci-TK-R in the Ciona ovary
The Ci-TK-R gene was expressed with abundance in the ovary, whereas the expression of gene encoding Ci-TK-I, its endogenous ligand, was not detected (28). This finding indicated the possibility that Ci-TK-R protein is also present in the ovary. In the ovary of C. intestinalis, the large part is occupied by pre-GVBD oocytes that are readily classified into three major growth stages on the basis of their diameter and organization of oocyte complexes, even using 10-µm sections: stage I (pre-vitellogenic stage), stage II (vitellogenic stage), and stage III (post-vitellogenic stage) (29, 32). Ciona oocytes are equipped with test cells (TCs), an acellular vitelline coat (VC), inner follicular cells (IFCs), and outer follicular cells (OFCs). Oocytes at stage I (<50 µm in diameter) contain the smallest germinal vesicle (GV) and cytoplasm, and are surrounded by envelope organs consisting of undifferentiated primary follicular cells (Fig. 1AGo). Stage II oocytes (50–70 µm in diameter) have prominently individualized cube-shaped follicle cells surrounding the oocytes (Fig. 1BGo). Stage III oocytes (~100 µm in diameter) have a larger cytoplasm and more outstanding inner follicle structure (Fig. 1CGo), and automatically cause GVBD when exposed to seawater (29, 32). In addition, stage IV (after GVBD) oocytes, predominantly present in the oviduct, have the ability of fertilization. Based on the histological characteristics of Ciona oocytes, distribution of Ci-TK-R in the ovary was observed by immunohistochemistry of 10-µm serial sections of the Ciona ovaries using the anti-Ci-TK-R serum. As shown in Fig. 1Go, D and E, the immunoreactivity of Ci-TK-R was detected exclusively in TCs residing in oocytes, which are believed to be functionally and cytologically related to mammalian granulosa, and to be involved in the growth of oocyte bodies and follicle cells (29, 37, 38). In contrast, no significant immunoreactivity was observed in any organs in the ovary. Intriguingly, the immunohistochemical studies demonstrated that Ci-TK-R-immunoreactive TCs were found specifically in stage II oocytes in the ovary (Fig. 1EGo), whereas no immunostaining was observed in TCs residing in oocytes at other stages (Fig. 1DGo). No positive signals were observed when antigen-absorbed antibodies were applied (Fig. 1FGo), confirming the specificity of immunostaining. These results verified the specific expression of Ci-TK-R in TCs residing in oocytes at stage II, and indicated that biological functions of Ci-TK-I in the ovary are evoked exclusively by the TCs expressing Ci-TK-R. In addition, this is the first localization of TK receptors to ovaries of any animal species.


Figure 1
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  		FIG. 1. Classification of Ciona oocytes at pre-GVBD stages and localization of Ci-TK-R on paraffin section of the ovary and localization of Ci-TK-R to TCs residing with in vitellogenic [stage (St) II] oocytes. Ciona oocytes have cuboidal vacuolated IFCs and thin OFCs. Each growth stage of oocytes was classified according to the follicle structure and diameter. A, Pre-vitellogenic (stage I) oocytes (<50 µm in diameter). B, Vitellogenic (stage II) oocyte (50–70 µm in diameter). C, The post-vitellogenic (stage III) oocyte (~100 µm in diameter). D, Immunoreactivity of Ci-TK-R on the section of the ovary (magnification, x200). E, Immunoreactivity of Ci-TK-R in TCs of the vitellogenic oocytes (magnification, x400). D and E, The red fluorescent signals indicate the immunoreactivity of Ci-TK-R. F, No positive signal was observed when the antigen-absorbed Ci-TK-R antibody was used. Y, Yolk.

 
Global transcriptional changes after Ci-TK treatment of the Ciona ovary
To identify the effects of Ci-TK-I on the ovary through the Ci-TK-R specifically expressed in TCs in stage II oocytes, we compared the gene expression profile between the untreated ovary and ovary treated with Ci-TK-I. The whole ovary was longitudinally cut into two symmetrical half-pieces, and the half-pieces were incubated in the presence or absence of 0.1-µM Ci-TK-I, respectively. Cy3- or Cy5-labeled RNA probes prepared from each half-piece were then hybridized with an Agilent Ciona 44K oligo-DNA microarray chip that contains 44,000 spots of 60-mer nonredundant oligonucleotides corresponding to 17,834 independent cDNA clusters (90% predicted or cloned cDNAs). In the ovary treated with Ci-TK-I, 21 oligonucleotide probes were shown to be up-regulated (>2-fold; P < 0.05), whereas no down-regulation of gene expression was detected (Fig. 2Go). The gene expression profiles have been deposited in the Gene Expression Omnibus database under the accession no. GSE8821. Moreover, these positive oligonucleotides were found to correspond to 10 individual cDNA clusters deposited in the C. intestinalis genomic/Expressed Sequence Tags database (Ghost Database: http://ghost.zool.kyoto-u.ac.jp/indexr1.html), given that more than two spots corresponded to a single gene. Identification (ID) numbers of the genes up-regulated by Ci-TK-I are as follows: 2785rl, 15917rl, 16332rl, 16601rl, 16624rl, 32073rl, 32548rl, 33874rl, 37777rl, and 37790rl. In addition, reproducibility of the data was confirmed by two microarray analyses with swapped-dye labeling (Fig. 2Go). In an attempt to reveal the features of the aforementioned genes, we determined all the sequences of open reading frames from the ovary (supplemental data), confirming that all the genes yield functional proteins. To annotate the putative functions of these genes, the deduced amino acid sequences were subjected to Basic Local Alignment Search Tool search in the DNA Data Bank of Japan, European Molecular Biology Laboratory, and GenBank database. The Ci-TK-I-up-regulated genes were categorized into two major groups on the basis of their sequence homologies: protease genes and functionally unidentified genes (Table 1Go and supplemental data). The former group includes the genes with ID nos. 2785rl, 16601rl, 16624rl, 32073rl, and 37777rl. The 2785rl and 32073rl genes display high-sequence similarity to vertebrate chymotrypsinogen (a precursor of a typical serine protease chymotrypsin) family genes, the 16601rl and 37777rl genes share high-sequence similarity to carboxy-peptidase B1, and 16624rl is highly homologous to cathepsin D, a lysosomal acidic protease that is believed to be involved in apoptosis and vitellogenesis in vertebrates (39). Because several proteases are known to be responsible for oocyte growth, maturation of follicle cells, and ovulation (39, 40, 41, 42, 43, 44), we focused on functions of these Ciona protease genes in the following studies.


Figure 2
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  		FIG. 2. Comparative scatter plot analysis of expression level of Ci-TK-I-treated and untreated half-piece of the ovary. A total of 21 spots was detected as significantly up-regulated oligonucleotides (>2-fold; P < 0.001) by both oligo-DNA chips on dye-swapped microarray analyses. A, Scatter plot analysis using Cy5-labeled RNA probes for the Ci-TK-I-treated half-piece of the ovary and Cy3-labeled RNA probes for the untreated one. B, Scatter plot analysis using Cy3-labeled RNA probes for the Ci-TK-I-treated half-piece of the ovary and Cy5-labeled RNA probes for the untreated one.

 

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  		TABLE 1. List of the protease genes up-regulated by Ci-TK-I in the ovary

 
Subsequently, real-time PCR was performed to confirm the fidelity of the result obtained by the microarray analyses. As shown in Fig. 3Go and Table 1Go, the real-time PCR analysis demonstrated that treatment of the ovary with Ci-TK-I resulted in significantly elevated expressions of the five protease genes, whereas expression of a housekeeping gene, Ci-GAPDH gene, was not affected. These results provide evidence that Ci-TK-I specifically up-regulates the gene expression of chymotrypsin, carboxy-peptidase B1, and cathepsin D.


Figure 3
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  		FIG. 3. Validation of microarray data by real-time PCR. The expressional level of each gene up-regulated in the half-ovary treated or untreated with Ci-TK-I is calculated from the {Delta}Ct values (also see Table 1Go). Data are shown as the means of three independent experiments ± SE (P < 0.05).

 
Up-regulation of protease activity in the ovary by Ci-TK-I
We measured the activity of the protease up-regulated by Ci-TK-I. The half-piece of the ovary was treated with Ci-TK-I under the same conditions as in the DNA microarray analysis (Fig. 2Go) and real-time PCR (Fig. 3Go). The lysosomal protease cathepsin D is known to exert its maximal activities under mild acidic condition (pH 4.0), whereas neutral pH (pH 7.6) is optimal for activity of the chymotrypsin and carboxy-peptidase B1. Thus, we observed the acidic protease activity and neutral protease activity in separate experiments. Incubation of the half-piece of the ovary with Ci-TK-I at higher than 10 nM led to a 2-fold increase in the activity of cathepsin D in a dose-dependent fashion, compared with that of the untreated half-piece (Fig. 4AGo). Likewise, the total activity of neutral proteases was evaluated using Azo-casein as a substrate, which is susceptible to degradation by any protease (35). As expected, an approximate 5-fold higher total activity of neutral proteases was induced in the Ci-TK-I-treated half-piece of the ovary compared with the untreated half-piece (Fig. 4BGo). The Ci-TK-I-induced total neutral proteases are partially inhibited by the pretreatment of the half-piece of the ovary with either a chymotrypsin inhibitor leupeptin or a metal protease inhibitor EDTA, but not by the treatment with an acidic protease inhibitor pepstatin A, showing that cathepsin D was inactive under neutral condition (Fig. 4CGo). In addition, the inhibitory effect of EDTA is 3-fold higher than that of leupeptin, and pretreatment of the half-ovary piece with both leupeptin and EDTA completely abolished the elevation of the total neutral protease activity induced by Ci-TK-I. These results, combined with the findings that the chymotrypsin genes and carboxy-peptidase B1 genes are up-regulated by Ci-TK-I, indicate that the Ci-TK-I-induced neutral protease activity resulted from the increase in activity of chymotrypsin and carboxy-protease B1.


Figure 4
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  		FIG. 4. Stimulatory effects of Ci-TK-I on the activity of cathepsin D (A) and total neutral protease (B) in the ovary. C, The neutral protease activity was completely abolished by addition of leupeptin (an inhibitor against chymotrypsin) and EDTA (an inhibitor against carboxy-peptidase B1). Data are shown as the means of three independent experiments ± SE (*, P < 0.05).

 
To confirm the specificity of Ci-TK-I, the effects of synthetic TK receptor agonists or antagonists on the elevation of the ovarian protease activity were examined. Because Ci-TK-R, unlike mammalian NK1 and NK2, was activated equipotently by SP and NKA (28), Ci-TK-R was likely to exhibit the agonist- or antagonist-selectivity distinct from mammalian TK receptors. Moreover, various submammalian neuropeptide receptors are known to exhibit the pharmacological profiles different from those of the mammalian counterparts (45). To identify an effective Ci-TK-R agonist and antagonist, we examined the pharmacological activity of numerous commercially available synthetic TK receptor agonists and antagonists at Ci-TK-R expressed in Xenopus oocytes. As shown in Fig. 5AGo, an NK1 agonist [Sar9, Met(O2)11]SP was found to elicit significant activation of Ci-TK-R. Likewise, an NK2 antagonist, GR94800, was found to block effectively the activity of Ci-TK-I at Ci-TK-R (Fig. 5BGo).


Figure 5
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  		FIG. 5. Effects of a synthetic TK receptor agonist and antagonist. A, A NK1 agonist, [Sar9, Met(O2)11]SP, elicited activation of Ci-TK-R expressed in Xenopus oocytes. B, A NK2 antagonist completely blocked activation of the Ci-TK-R by Ci-TK-I. Maximum membrane currents elicited by ligands are plotted, and the current caused by 10–7 M Ci-TK-I was taken as 100%. Each point represents the mean of three independent experiments ± SE (P < 0.05). [Sar9, Met(O2)11]SP elevated activities of both cathepsin D (C) and total neutral proteases (D). No significant increase in activity of cathepsin D (E) or total neutral protease (F) was detected in the ovary in the presence of the Ci-TK-R antagonist, GR94800. Each point represents the mean of three independent experiments ± SE (*, P < 0.05), compared with cathepsin D and total neutral protease activity in the absence of Ci-TK-I.

 
Subsequently, we examined the effects of [Sar9, Met(O2)11]SP and GR94800 on the ovarian protease activities. As depicted in Fig. 5Go, C and D, induction of activities of both cathepsin D and total neutral proteases was detected by treatment of the half-piece ovary with [Sar9, Met(O2)11]SP. In contrast, the elevation of cathepsin D activity or total neutral protease activity was blocked by treatment with GR94800 (Fig. 5Go, E and F). These data confirm that such significant increases in the protease activities were not due to nonspecific or artifactual action by small peptides. In combination, a series of protease assays indicated that up-regulation of gene expression of the proteases by Ci-TK-I leads to elevation of their enzymatic activities in the ovary.

Effects of Ci-TK on Ciona oocyte growth
Whole stage II (vitellogenic) and stage III (post-vitellogenic) oocytes are discriminated by their morphological features and mitochondria localization. Vitellogenic oocytes have a 50- to 70-µm diameter and a cube-shaped follicle structure (Fig. 6AGo, right and left images on upper panel). Moreover, small mitochondria are scattered throughout the cytoplasm (Fig. 6AGo, middle image on upper panel). At stage III, oocytes have a 100-µm diameter and grown follicles with more outstanding and larger petal-like structures (Fig. 6AGo, right and left images on lower panel), and mitochondria are localized to the oocyte periphery (Fig. 6AGo, middle image on lower panel). Based on this morphological information, we observed the morphological effects of Ci-TK-I or Ci-TK-I-up-regulated protease on the vitellogenic oocytes where Ci-TK-R is specifically expressed (Fig. 1Go). The vitellogenic oocytes were incubated with 0.1-µM Ci-TK-I for 15 h, and the morphological changes were monitored by confocal microscopical cross-sections and 3D images. Trypan blue-based cell staining confirmed viability of more than 90% of the isolated oocytes at each stage during incubation with all reagents under this condition. The vitellogenic oocytes displayed no significant morphological change or mitochondria localization after 15-h incubation in seawater (Fig. 6BGo). The striking feature is that the 15-h incubation with Ci-TK-I caused the growth of approximately 50% oocytes from the vitellogenic stage to the post-vitellogenic stage (Fig. 6CGo and Table 2Go). The mitochondria gradually dispersed from the cytoplasm, and almost completely localized to the periphery of the oocytes after incubation with Ci-TK-I for 15 h (Fig. 6CGo, left panels), as seen in naturally occurring post-vitellogenic oocytes (Fig. 6AGo). Such Ci-TK-I-induced oocyte growth was also confirmed by 3D image observation of the follicle structure and the oocyte size. Treatment of vitellogenic oocytes with Ci-TK-I for 15 h resulted in morphological changes, including an approximate 100-µm diameter, and the follicle cells with more grown and outstanding structure (Fig. 6CGo, right panels), which is characteristic of naturally occurring post-vitellogenic oocytes (Fig. 6AGo).


Figure 6
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  		FIG. 6. Effects of Ci-TK-I on Ciona oocyte growth. A, A naturally occurring vitellogenic oocyte (stage II, upper panel) and post-vitellogenic oocyte (stage III, lower panel). The left, middle (indicated by mitochondria), and right panels (indicated by 3D) showed optical microscopical images of an oocyte at each stage, the cross-sections of mitochondria localization by fluorescent confocal microscopical observation using MitoTracker Red, and 3D images of the whole oocytes obtained by reconstruction of 70–150 cross-sections using AlexaPhalloidin488, respectively. Vitellogenic oocytes (50–70 µm in diameter) have a cube-shaped follicle (FC) structure surrounding the oocytes, and the mitochondria, stained in red, are abundantly observed throughout the oocyte cytoplasm. Post-vitellogenic (stage III) oocytes have grown follicles with more outstanding petal-like structures surrounding 100-µm diameter oocytes, and the fluorescent intensity of mitochondria is markedly lowered in the cytoplasm and localized to the oocyte periphery. No change was observed after incubation in seawater for 15 h (B), whereas incubation of vitellogenic oocytes with Ci-TK-I resulted in an gradual increase in size and reached 100 µm in diameter 15 h after addition of Ci-TK-I (C, right panel). The cube-shaped follicles also grew in size and showed the outstanding appearance, which is typical of post-vitellogenic oocytes (C, right panel). Moreover, during the incubation the mitochondria gradually dispersed from the cytoplasm to the periphery (C, left panel). CP, Cytoplasm of oocytes; hr, h.

 

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  		TABLE 2. Effects of Ci-TK-I, [Sar9, Met(O2)11]SP, GR94800, and protease inhibitors on the Ciona oocyte growth from stage II (vitellogenic stage) to stage III (post-vitellogenic stage)

 
To confirm the specificity of the Ci-TK-I-induced oocyte growth, we evaluated the effects of the same TK agonist and antagonist used for the aforementioned protease activity assays (Fig. 5Go, C–F). [Sar9, Met(O2)11]SP was found to enhance the oocyte growth comparable to Ci-TK-I (Fig. 7AGo). In contrast, pretreatment of the vitellogenic oocytes with GR94800 markedly reduced the Ci-TK-I-dependent oocyte growth (Fig. 7BGo). Together, these results revealed that Ci-TK-I induced growth of oocytes from the vitellogenic stage to the post-vitellogenic stage via Ci-TK-R. Subsequently, we examined the functional correlation of protease activities with the Ci-TK-I-induced growth of the oocytes, given that Ci-TK-I up-regulated not only gene expression, but also activity of the proteases (Figs. 3–5GoGoGo). As depicted in Fig. 7CGo, the Ci-TK-I-induced oocyte growth was completely blocked in the presence of either the protease inhibitor leupeptin (against chymotrypsin), pepstatin (against cathepsin D), or EDTA (against carboxy-peptidase B1). These results provided evidence that the elevation of each protease activity is a requisite for the Ci-TK-I-dependent growth of the vitellogenic oocytes, and absolutely excluded the possibility that the oocyte growth induced the gene expression and enzymatic activities of these proteases. Collectively, the immunohistological analyses (Fig. 1Go), gene expression analyses (Figs. 2Go and 3Go, and Table 1Go), protease activity assays (Figs. 4Go and 5Go), and morphological change observations (Figs. 6Go and 7Go, and Table 2Go) lead to the establishment of a novel tachykininergic pathway for the stage-specific growth of oocytes (Fig. 8Go) where Ci-TK-I specifically acts at Ci-TK-R in TCs of the vitellogenic oocytes (stage II), induces gene expression and activity of proteases, and eventually enhances the growth of oocytes from the vitellogenic stage (stage II) to the post-vitellogenic stage (stage III).


Figure 7
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  		FIG. 7. Evidence for induction of the vitellogenic oocyte growth via Ci-TK-I-triggered protease activities. A, The Ci-TK-R agonist, [Sar9, Met(O2)11]SP, induces the oocyte growth almost equipotently to Ci-TK-I. B, The Ci-TK-I-induced oocyte growth was abolished by cotreatment with the Ci-TK-R antagonist GR94800. C, Treatment of the vitellogenic oocytes with each protease inhibitor against chymotrypsin (leupeptin), carboxy-peptidase B1 (EDTA), and cathepsin D (pepstatin) resulted in the complete loss of the Ci-TK-induced oocyte growth. hr, h.

 

Figure 8
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  		FIG. 8. Scheme of the essential molecular mechanism underlying the growth of vitellogenic oocytes triggered by Ci-TK-I. Ci-TK-I activates Ci-TK-R specifically expressed in TCs residing within vitellogenic oocytes, and then up-regulates gene expressions and enzymatic activities of chymotrypsin, carboxy-peptidase B1, and cathepsin D. The resultant increased protease activities enhance the growth of oocytes from the vitellogenic stage (stage II) to the post-vitellogenic stage (stage III).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Elucidation of biological functions of TKs in the ovaries is expected to lead to advances in tachykininergic drug development in two regards: exploration of new drug targets in the ovary and reduction of side effects on ovarian functions. However, the biological roles of TKs in the ovary have not been verified either in vitro or in vivo. In the present study, we for the first time revealed the ovarian function of TKs using the cosmopolitan ascidian, C. intestinalis. Ci-TK-I enhances the growth of oocytes from the vitellogenic stage to the post-vitellogenic stage via activation of cathepsin D, chymotrypsin, and carboxy-peptidase B1 at both the transcriptional and enzymatic level (Fig. 8Go). The evolutionary position of ascidians as basal chordates strongly suggests that ascidians and vertebrates share common functions of nervous and endocrine systems, including ovarian functions (29, 30), and that ascidians are excellent model animals for functional analyses and evolutionary studies of vertebrate neuropeptides and hormones. Indeed, we previously showed that Ci-TK-I and Ci-TK-R are prototypes of the ligand-receptor pair of the vertebrate TK family (6, 28). In contrast, authentic TKs are not conserved in any other invertebrates, including insects, mollusks, annelids, and nematodes (6, 46). Consequently, we have used a basal chordate, C. intestinalis, as a model animal for vertebrates.

Ci-TK was shown to induce activation of ovarian chymotrypsin, carboxy-peptidase B1, and cathepsin D (Figs. 2–4GoGoGo), which in turn enhances the oocyte growth (Figs. 6Go and 7Go). These proteases were found to be responsible for multiple biological events in the growth of oocytes and follicle cells in various animal species. Leupeptin, which has the ability of inhibiting activity of chymotrypsin, suppressed progression of oocyte growth at pre-GVBD stages in an ascidian Halocynthia roretzi (40) and a starfish Asterina pectinifera (41, 42). In Drosophila, fluorescent-conjugated chymotrypsin inhibitors were localized to the growing oocyte-somatic follicle cells (43). Carboxy-peptidase B1 also played a crucial role in the proteolytic processing of several component proteins for zona pellucida in mammalian oocytes at an early growth stage (44). Recently, cathepsin D has participated in the processing of yolk proteins and follicular component in various vertebrate oocytes at the stage before GVBD (39). These findings are in good agreement with our data that the Ci-TK-I-induced gene expression (Figs. 2Go and 3Go) followed by the enzymatic activity (Fig. 4Go) of the three proteases enhanced growth of Ciona oocytes at the vitellogenic stage (Fig. 6Go and Table 2Go), and addition of inhibitors for these proteases resulted in the loss of the oocyte growth triggered by Ci-TK-I (Fig. 7Go and Table 2Go). In contrast, no endogenous factors have ever been found to elevate the gene expression or enzymatic activity of any proteases involved in oocyte growth at pre-GVBD stages. In combination, our present study shows the first ID of TKs as an inducible factor for several ovarian proteases essential for oocyte growth. It is also noteworthy that only one inhibitor for each of the three Ci-TK-I-induced proteases completely suppressed the oocyte growth (Fig. 7Go). Cathepsin D is a lysosomal acidic protease, whereas chymotrypsin and carboxy-peptidase B1 are neutral proteases. These findings support the notion that the three proteases are responsible for proteolysis of distinct protein substrates, but each proteolysis is functionally concerted for the TK-induced oocyte growth. Characterization of substrates for each of the proteases will lead to the establishment of a functional correlation of proteolytic processes with the TK-induced oocyte growth pathway.

Immunohistological analysis demonstrated that Ci-TK-R is localized to TCs situated in vitellogenic (stage II) oocytes (Fig. 1Go). TCs, like mammalian granulosa cells, are believed to play pivotal roles in the growth of oocytes, although the functional mechanisms have yet to be well elucidated (29, 37, 38). These findings lead to the presumption of two possible pathways for activation of the protease functions. First, the Ci-TK-I-up-regulated ovarian proteases are produced in TCs, and secreted to oocytes and follicle cells in a paracrine fashion, followed by participation in proteolytic processing of oocytic and follicular components. Alternatively, TCs may secrete secondary signaling molecules to oocytes and follicle cells, which in turn elevate the up-regulation of the protease gene expression. To address these issues, localizations of the transcripts and proteins of the proteases are currently in progress.

The ascidian ovary harbors numerous oocytes at each growth stage that are readily characterized and abundantly isolated (Figs. 1Go, 6Go, and 7Go). This advantage enables investigation of precise morphological changes of oocytes regulated by Ci-TK-I. The abundant expression of the Ci-TK-R gene in the ovary (28) also allowed us to detect the localization of Ci-TK-R to TCs residing in the vitellogenic (stage II) oocytes (Fig. 1Go). Such specific expression of Ci-TK-R clearly proved that TCs of the vitellogenic oocytes are the sole targets of Ci-TK-I in the ovary, and, thus, all biological actions of Ci-TK-I in the ovary should be mediated by the TCs expressing Ci-TK-R in the vitellogenic oocytes. These results led to the elucidation of the molecular mechanisms underlying biological functions of Ci-TK-I via the TCs by DNA microarray analyses (Fig. 2Go), real-time PCR (Fig. 3Go and Table 1Go), and enzymatic activity assays (Figs. 4Go and 5Go). Furthermore, we reproducibly detected the growth of approximately 50% vitellogenic oocytes treated with Ci-TK-I (Fig. 6Go and Table 2Go), which was completely blocked by addition of a TK antagonist and protease inhibitors (Fig. 7Go and Table 2Go). Detection of the Ciona oocyte growth at such a high ratio led to the characterization of the biological role of TKs as up-regulators of the oocyte growth. In contrast, only a few mammalian primordial oocytes are recruited to the following growth steps during the pre-GVBD process and are available to in vitro functional analyses, although numerous inactive primordial oocytes are present in the ovary (26, 27). Furthermore, no procedure for detection of oocytes competent in growth has been developed. These disadvantages hindered clarification of biological roles of TKs in the mammalian ovaries and oocytes in vitro and in vivo (17, 26, 27). Combined with the evolutionary position of ascidians as basal chordates, our present study supports the view that the TK-dependent protease-associated oocyte growth at pre-GVBD stages (Fig. 8Go) is conserved in mammals and that tachykininergic agents affect some oocyte growth at pre-GVBD stages. Intriguingly, neonatal treatment with capsaicin (an SP-depleting compound) leads to a low percentage of successful fertilization, and ip injection of an NK3 antagonist resulted in reduction of the litter size in rats (25). These findings indicate the possibility that such abnormalities in fertilization and reproduction are attributed to disturbance of TK-regulated oocyte growth at pre-GVBD stages by tachykininergic compounds. Unfortunately, in vivo administration of tachykininergic ligands or protease inhibitors to the ascidian body at present seems technically quite difficult due to their open circulatory system. In contrast, mammals possess the closed circulatory system. Therefore, our present study using the Ciona ovary and oocytes is expected to contribute a great deal to in vivo functional analysis of the tachykininergic protease-associated oocyte growth in mammals. In vivo and in vitro studies on TK-dependent protease-associated growth of mammalian oocytes are now being attempted.

There are three major pathways for biological functions of TKs: paracrine within the peripheral tissues, endocrine through the hypothalamic-pituitary-peripheral gland, and neurotransmission via nerve fibers (2, 3, 4, 5, 6, 7, 8, 16, 17, 23, 24, 25). Previously, we showed that the expression of Ci-TK gene is absent in the ovary, excluding the possibility that Ci-TK-I plays any paracrine role in the Ciona ovary (28). In contrast, mammalian TK genes are expressed in nonneuronal cells of various genital organs, including the ovary (2, 3, 4, 5, 6, 7, 8, 16, 17, 23, 24, 25), which confers TKs with biological roles as paracrine factors. These findings suggest that tachykininergic paracrine systems in genital organs might have been established during the evolution of ancestral vertebrates. Ascidians have possessed orthologs or prototypes for vertebrate neuropeptides and/or hypothalamic hormones (28, 47, 48, 49, 50), including GnRHs and TKs, which have not ever been identified in traditional protostome model organisms, e.g. Drosophila and Caenorhabditis elegans (6, 46, 51, 52). Nevertheless, the hypothalamic-pituitary-periphery endocrine system is believed to have been established in vertebrates, given that the organs corresponding to the hypothalamus and pituitary have not ever been characterized in ascidians (51, 52, 53, 54, 55), and that no putative genes encoding pituitary hormone orthologs, including LH and FSH, were found in the genome of C. intestinalis (51, 53). Consequently, Ci-TK-I cannot serve via the hypothalamic-pituitary-periphery. Instead, GnRH or TKs were identified in neurons of the central nervous system of C. intestinalis (28, 49, 50, 51, 52), which projects several nerve fibers to the ovary (28, 29, 49). These findings indicate that the ascidian counterparts for mammalian hypothalamic peptides serve as neuropeptides, and Ci-TK-I induces the oocyte growth in the ovary in a neuropeptidergic fashion. Altogether, we presume that the neuropeptidergic TK-protease-oocyte growth cascade (Fig. 8Go) was established in common ancestral chordates before the hypothalamic-pituitary-peripheral gland endocrine and paracrine systems in the ovary had been acquired in ancestral vertebrates.

In conclusion, we have substantiated a novel biological role of TKs as an inducer of oocyte growth in the ovary. Our present data not only establish the unprecedented tachykininergic pathway responsible for oocyte growth but also presume an evolutionary origin of ovarian functions regulated by TKs.


   Acknowledgments
 
We thank Ms. Kazuko Hirayama and all members of the Maizuru Fisheries Research Station for cultivation of the ascidians. All ascidians (Ciona intestinalis) were provided by Kyoto University through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan.


   Footnotes
 
This study is in part financially supported by Japan Society for the Promotion of Science (to H.S. and T.Se.) and Japan Science and Technology Corporation (to N.S. and H.S.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 15, 2008

1 M.A. and T.K. equally contributed to this study. Back

Abbreviations: Ct, Cycle threshold; {Delta}Ct, difference in Ct; EK, endokinin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GV, germinal vesicle; GVBD, germinal vesicle breakdown; HK, hemokinin; ID, identification; IFC, inner follicular cell; NK, neurokinin; OFC, outer follicular cell; PBST, PBS containing 0.01% Triton X-100; SP, substance P; TC, test cell; TK, tachykinin; 3D, three-dimensional; VC, vitelline coat.

Received March 7, 2008.

Accepted for publication May 2, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Wojcik-Gladysz A, Nowak KW, Pierzchala-Koziec K, Wankowska M, Misztal T, Polkowska J, Nowak M, Kaczmarek P, Gorska T, Szczepankiewicz D, Zubel J 2006 Aspects of central and peripheral regulation of reproduction in mammals. Reprod Biol 6:89–103[Medline]
  2. Severini C, Improta G, Falconieri-Erspamer G, Salvadori S, Erspamer V 2002 The tachykinin peptide family. Pharmacol Rev 54:285–322[Abstract/Free Full Text]
  3. Page NM 2004 Hemokinins and endokinins. Cell Mol Life Sci 61:1652–1663[Medline]
  4. Almeida TA, Rojo J, Nieto PM, Pinto FM, Hernandez M, Martin JD, Candenas ML 2004 Tachykinins and tachykinin receptors: structure and activity relationships. Curr Med Chem 11:2045–2081[Medline]
  5. Page NM 2005 New challenges in the study of the mammalian tachykinins. Peptides 26:1356–1368[CrossRef][Medline]
  6. Satake H, Kawada T 2006 Overview of the primary structure, tissue-distribution, and functions of tachykinins and their receptors. Curr Drug Targets 7:963–974[CrossRef][Medline]
  7. Zhang Y, Lu L, Furlonger C, Wu GE, Paige CJ 2000 Hemokinin is a hematopoietic-specific tachykinin that regulates B lymphopoiesis. Nat Immunol 1:392–397[CrossRef][Medline]
  8. Page NM, Bell NJ, Gardiner SM, Manyonda IT, Brayley KJ, Strange PG, Lowry PJ 2003 Characterization of the endokinins: human tachykinins with cardiovascular activity. Proc Natl Acad Sci USA 100:6245–6250[Abstract/Free Full Text]
  9. Nakajima Y, Tsuchida K, Negishi M, Ito S, Nakanishi S 1992 Direct linkage of three tachykinin receptors to stimulation of both phosphatidylinositol hydrolysis and cyclic AMP cascades in transfected Chinese hamster ovary cells. J Biol Chem 267:2437–2442[Abstract/Free Full Text]
  10. Holst B, Hastrup H, Raffetseder U, Martini L, Schwartz TW 2001 Two active molecular phenotypes of the tachykinin NK1 receptor revealed by G-protein fusions and mutagenesis. J Biol Chem 276:19793–19799[Abstract/Free Full Text]
  11. Palanche T, Ilien B, Zoffmann S, Reck MP, Bucher B, Edelstein SJ, Galzi JL 2001 The neurokinin A receptor activates calcium and cAMP responses through distinct conformational states. J Biol Chem 276:34853–34861[Abstract/Free Full Text]
  12. Lecat S, Bucher B, Mely Y, Galzi JL 2002 Mutations in the extracellular amino-terminal domain of the NK2 neurokinin receptor abolish cAMP signaling but preserve intracellular calcium responses. J Biol Chem 277:42034–42048[Abstract/Free Full Text]
  13. Quartara L, Altamura M 2006 Tachykinin receptors antagonists: from research to clinic. Curr Drug Targets 7:975–992[CrossRef][Medline]
  14. Chahl LA 2006 Tachykinins and neuropsychiatric disorders. Curr Drug Targets 7:993–1003[CrossRef][Medline]
  15. Palma C 2006 Tachykinins and their receptors in human malignancies. Curr Drug Targets 7:1043–1052[CrossRef][Medline]
  16. Candenas L, Lecci A, Pinto FM, Patak E, Maggi CA, Pennefather JN 2005 Tachykinins and tachykinin receptors: effects in the genitourinary tract. Life Sci 76:835–862[CrossRef][Medline]
  17. Debeljuk L 2006 Tachykinins and ovarian function in mammals. Peptides 27:736–742[CrossRef][Medline]
  18. Ravina CG, Seda M, Pinto FM, Orea A, Fernandez-Sanchez M, Pintado CO, Candenas ML 2007 A role for tachykinins in the regulation of human sperm motility. Hum Reprod 22:1617–1625[Abstract/Free Full Text]
  19. Patak E, Candenas ML, Pennefather JN, Ziccone S, Lilley A, Martin JD, Flores C, Mantecon AG, Story ME, Pinto FM 2003 Tachykinins and tachykinin receptors in human uterus. Br J Pharmacol 139:523–532[CrossRef][Medline]
  20. Pennefather JN, Patak E, Ziccone S, Lilley A, Pinto FM, Page NM, Story ME, Grover S, Candenas ML 2006 Regulation of the stimulant actions of neurokinin a and human hemokinin-1 on the human uterus: a comparison with histamine. Biol Reprod 75:334–341[Abstract/Free Full Text]
  21. Pinto FM, Almeida TA, Hernandez M, Devillier P, Advenier C, Candenas ML 2004 mRNA expression of tachykinins and tachykinin receptors in different human tissues. Eur J Pharmacol 494:233–239[CrossRef][Medline]
  22. Patak E, Pinto FM, Story ME, Pintado CO, Fleming A, Page NM, Pennefather JN, Candenas ML 2005 Functional and molecular characterization of tachykinins and tachykinin receptors in the mouse uterus. Biol Reprod 72:1125–1133[Abstract/Free Full Text]
  23. Page NM, Woods RJ, Gardiner SM, Lomthaisong K, Gladwell RT, Butlin DJ, Manyonda IT, Lowry PJ 2000 Excessive placental secretion of neurokinin B during the third trimester causes pre-eclampsia. Nature 405:797–800[CrossRef][Medline]
  24. Page NM, Dakour J, Morrish DW 2006 Gene regulation of neurokinin B and its receptor NK3 in late pregnancy and pre-eclampsia. Mol Hum Reprod 12:427–433[Abstract/Free Full Text]
  25. Pintado CO, Pinto FM, Pennefather, JN, Hidalgo A, Baamonde A, Sanchez T, Candenas ML 2003 A role for tachykinins in female mouse and rat reproductive function. Biol Reprod 69:940–946[Abstract/Free Full Text]
  26. Krisher RL 2004 The effect of oocyte quality on development. J Anim Sci 82(Suppl):E14–E23
  27. Wang Q, Sun QY 2007 Evaluation of oocyte quality: morphological, cellular and molecular predictors. Reprod Fertil Dev 19:1–12[CrossRef][Medline]
  28. Satake H, Ogasawara M, Kawada T, Masuda K, Aoyama M, Minakata H, Chiba T, Metoki H, Satou Y, Satoh N 2004 Tachykinin and tachykinin receptor of an ascidian, Ciona intestinalis: evolutionary origin of the vertebrate tachykinin family. J Biol Chem 279:53798–53805[Abstract/Free Full Text]
  29. Burighel P, Cloney RA 1997 Urochordata ascidiacea. In: Harrison FW, ed. Microscopic anatomy of invertebrates. Vol. 15. New York: Wiley-Liss; 221–347
  30. Satoh N, Levine M 2005 Surfing with the tunicates into the post-genome era. Genes Dev 19:2407–2411[Abstract/Free Full Text]
  31. Sakai T, Satake H, Minakata H, Takeda M 2004 Characterization of crustacean cardioactive peptide as a novel insect midgut factor: isolation, localization, and stimulation of {alpha}-amylase activity and gut contraction. Endocrinology 145:5671–5678[Abstract/Free Full Text]
  32. Prodon F, Chenevert J, Sardet C 2006 Establishment of animal-vegetal polarity during maturation in ascidian oocytes. Dev Biol 290:297–311[CrossRef][Medline]
  33. Ishibashi T, Usami T, Fujie M, Azumi K, Satoh N, Fujiwara S 2005 Oligonucleotide-based microarray analysis of retinoic acid target genes in the protochordate, Ciona intestinalis. Dev Dyn 233:1571–1578[CrossRef][Medline]
  34. Hamaguchi M, Fujie M, Noda T, Satoh N 2007 Microarray analysis of zygotic expression of transcription factor genes and cell signaling molecule genes in early Ciona intestinalis embryos. Dev Growth Differ 49:27–37[Medline]
  35. Sakai T, Satake H, Takeda M 2006 Nutrient-induced {alpha}-amylase and protease activity is regulated by crustacean cardioactive peptide (CCAP) in the cockroach midgut. Peptides 27:2157–2164[CrossRef][Medline]
  36. Hotta K, Mitsuhara K, Takahashi H, Inaba K, Oka K, Gojobori T, Ikeo K 2007 A web-based interactive developmental table for the ascidian Ciona intestinalis, including 3D real-image embryo reconstructions: I. From fertilized egg to hatching larva. Dev Dyn 236:1790–1805[CrossRef][Medline]
  37. Sugino YM, Tominaga A, Takashima Y 1987 Differentiation of the accessory cells and structural regionalization of the oocyte in the ascidian Ciona savignyi during early oogenesis. J Exp Zool 242:205–214[CrossRef]
  38. Wang J, Karabinos A, Zimek A, Meyer M, Riemer D, Hudson C, Lemaire P, Weber K 2002 Cytoplasmic intermediate filament protein expression in tunicate development: a specific marker for the test cells. Eur J Cell Biol 81:302–311[CrossRef][Medline]
  39. Carnevali O, Cionna C, Tosti L, Lubzens E, Maradonna F 2006 Role of cathepsins in ovarian follicle growth and maturation. Gen Comp Endocrinol 146:195–203[CrossRef][Medline]
  40. Sakairi K, Shirai H 1991 Possible MIS production by follicle cells in spontaneous oocyte maturation of the ascidian, Halocynthia roretzi. Dev Growth Differ 33:155–162[CrossRef]
  41. Takagi-Sawada M, Someno T, Hoshi M, Sawada H 1989 Inhibition of starfish oocyte maturation by leupeptin analogs, potent trypsin inhibitors. Dev Biol 133:609–612[CrossRef][Medline]
  42. Tanaka E, Takagi-Sawada M, Sawada H 2000 Enzymatic properties of the proteasome purified from starfish oocytes and its catalytic subunits involved in oocyte maturation. Comp Biochem Physiol C Toxicol Pharmacol 125:215–223[Medline]
  43. Jakobsen RK, Ono S, Powers JC, DeLotto R 2005 Fluorescently labeled inhibitors detect localized serine protease activities in Drosophila melanogaster pole cells, embryos, and ovarian egg chambers. Histochem Cell Biol 123:51–60[CrossRef][Medline]
  44. Litscher ES, Qi H, Wassarman PM 1999 Mouse zona pellucida glycoproteins mZP2 and mZP3 undergo carboxy-terminal proteolytic processing in growing oocytes. Biochemistry 38:12280–12287[CrossRef][Medline]
  45. Cho HJ, Acharjee S, Moon MJ, Oh DY, Vaudry H, Kwon HB, Seong JY 2007 Molecular evolution of neuropeptide receptors with regard to maintaining high affinity to their authentic ligands. Gen Comp Endocrinol 153:98–107[CrossRef][Medline]
  46. Satake H, Kawada T, Nomoto K, Minakata H 2003 Insight into tachykinin-related peptides, their receptors, and invertebrate tachykinins: a review. Zoolog Sci 20:533–549[CrossRef][Medline]
  47. Johnsen AH, Rehfeld JF 1990 Cionin: a disulfotyrosyl hybrid of cholecystokinin and gastrin from the neural ganglion of the protochordate Ciona intestinalis. J Biol Chem 265:3054–3058[Abstract/Free Full Text]
  48. Monstein HJ, Thorup JU, Folkesson R, Johnsen AH, Rehfeld JF 1993 cDNA deduced procionin. Structure and expression in protochordates resemble that of procholecystokinin in mammals. FEBS Lett 331:60–64[CrossRef][Medline]
  49. Adams BA, Tello JA, Erchegyi J, Warby C, Hong DJ, Akinsanya KO, Mackie GO, Vale W, Rivier JE, Sherwood NM 2003 Six novel gonadotropin-releasing hormones are encoded as triplets on each of two genes in the protochordate, Ciona intestinalis. Endocrinology 144:1907–1919[Abstract/Free Full Text]
  50. Tello JA, Rivier JE, Sherwood NM 2005 Tunicate gonadotropin-releasing hormone (GnRH) peptides selectively activate Ciona intestinalis GnRH receptors and the green monkey type II GnRH receptor. Endocrinology 146:4061–4073[Abstract/Free Full Text]
  51. Sherwood NM, Tello JA, Roch GJ 2006 Neuroendocrinology of protochordates: insights from Ciona genomics. Comp Biochem Physiol A Mol Integr Physiol 44:254–271
  52. Satake H, Kawada T 2006 Neuropeptides, hormones, and their receptors in ascidians–emerging model animals. In: Satake H, ed. Invertebrate neuropeptides and hormones: basic knowledge and recent advances. Trivandrum, India: Transworld Research Network; 253–276
  53. Campbell RK, Satoh N, Degnan BM 2004 Piecing together evolution of the vertebrate endocrine system. Trends Genet 20:359–366[CrossRef][Medline]
  54. Deyts C, Casane D, Vernier P, Bourrat F, Joly JS 2006 Morphological and gene expression similarities suggest that the ascidian neural gland may be osmoregulatory and homologous to vertebrate peri-ventricular organs. Eur J Neurosci 24:2299–2308[CrossRef][Medline]
  55. Dufour HD, Chettouh Z, Deyts C, de Rosa R, Goridis C, Joly JS, Brunet JF 2006 Precraniate origin of cranial motoneurons. Proc Natl Acad Sci USA 103:8727–8732[Abstract/Free Full Text]



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