Premature follicle growth in Ciona

The vertebrate follicle development process is categorized into two pathways: the gonadotropin-independent pathway and gonadotropin-dependent pathway. In mammals, the former involves the growth of primordial, primary, and secondary follicles, and functions prior to puberty, including during the fetus, baby, and infancy stages (Robker et al., 2018; Duffy et al., 2019; Orisaka et al., 2021). The latter includes the growth of preantral, and antral follicles, maturation (GVBD) of Graafian follicles, and ovulation, and is triggered by the release of gonadotropins from the onset of puberty to menopause, as stated below (Robker et al., 2018; Duffy et al., 2019; Orisaka et al., 2021). In oviparous animals such as teleost fish, follicle development is classified based on vitellogenesis. For example, in medaka, the previtellogenic and early vitellogenic stages are gonadotropin-independent, and the later vitellogenic and postvitellogenic stages are gonadotropin-dependent (Takahashi et al., 2019; Takahashi and Ogiwara, 2023). In mammals, phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt), phosphatase and tensin homolog (PTEN), and anti-Müllerian hormone (AMH) are thought to be involved in activation of primordial follicles (Robker et al., 2018; Duffy et al., 2019; Orisaka et al., 2021). Moreover, the Transforming growth factor β (TGFβ) superfamily proteins, such as Growth/ differentiation factor 9 (GDF9) and Bone morphogenetic protein 15 (BMP15), and insulin-like growth factor 1 (IGF1) are considered to be involved in gonadotropin-independent follicle growth as well as gonadotropin-dependent growth (Robker et al., 2018; Duffy et al., 2019; Orisaka et al., 2021). However, no regulatory factor specific to gonadotropin-independent follicle growth has so far been identified.

In Ciona, the follicle development at stage I and stage II is expected, at least in part, to correspond to the gonadotropin-independent follicle growth in vertebrates. In the Ciona ovary, the only Ciona tachykinin (CiTK) cognate receptor, CiTKR, was observed exclusively in test cells of stage II follicles (Aoyama et al., 2008; Matsubara et al., 2016). Furthermore, as depicted in Fig. 2, CiTK was shown to up-regulate the gene expression and enzymatic activities of cathepsin D, chymotrypsin, and carboxypeptidase B1 (Aoyama et al., 2008; Matsubara et al., 2016), and to eventually induce the follicle growth from stage II to stage III (Aoyama et al., 2008; Matsubara et al., 2016). Of particular interest is that these three proteases are involved in proteolytic processing of oocyte components. Carboxypeptidase B1 is responsible for the proteolytic processing of several component proteins of zona pellucida in mammalian oocytes at an early growth stage (Litscher et al., 1999). Inhibition of chymotrypsin has been shown to suppress oocyte growth at preGVBD stages in a non-Ciona ascidian (Halocynthia roretzi) (Sakairi and Shirai, 1991), a starfish (Asterina pectinifera) (Takagi-Sawada et al., 1989; Tanaka et al., 2000), and the fruit fly (Drosophila melanogaster) (Jakobsen et al., 2005). Moreover, cathepsin D has been shown to proteolytically modify precursors of yolk proteins (e.g., vitellogenin) and follicular components in non-mammalian vertebrate oocytes at the preGVBD stages (Carnevali et al., 2006). These findings support the view that the three CiTK-induced proteases also participate in the enhancement of the growth of Ciona stage-II (namely, preGVBD stage) follicles via proteolysis of various oocyte and follicle proteins, including vitellogenin. Interestingly, cathepsin D is specifically co-localized with CiTKR in test cells of stage-II follicles, whereas gene expression of carboxypeptidase B1 and chymotrypsin was detected in the follicular cells of the stage-II and stage-III follicles (Aoyama et al., 2012). Furthermore, the cathepsin D gene expression occurred 3 h before the expression of carboxypeptidase B1 and chymotrypsin (Aoyama et al., 2012). These findings suggest that the cathepsin D gene expression is directly upregulated by CiTK in test cells at stage II, and that the two remaining protease genes are secondarily activated. It is also noteworthy that the CiTK-I-directed stage II follicle growth was shown to be suppressed by a Ciona-specific neurotensin-like peptide, CiNTLP-6 (Fig. 2), via downregulation of the gene expression of carboxypeptidase B1, chymotrypsin, and cathepsin D (Kawada et al., 2011; Matsubara et al., 2016; Kawada et al., 2022; Satake, 2023). Collectively, these studies have substantiated the molecular mechanism underlying tachykinergic premature (preGVBD) follicle growth in Ciona (Fig. 2), although investigation of the molecular mechanism by which signaling by CiTK and CiNTLP6 in test cells regulates the expression of carboxypeptidase B1 and chymotrypsin in inner follicular cells awaits further studies. To date, no experimental evidence for a biological role of TKs in follicle development in vertebrates has been provided. However, combined with the findings that the TK receptor genes are expressed in the ovary (Pintado et al., 2003; García-Ortega et al., 2014; Blasco et al., 2020, 2023), it is likely that tachykinergic early-stage follicle growth is conserved in vertebrates.

Fig. 2.

Follicle growth from stage II to stage III. CiTK directly induces the gene expression of Cathepsin D in test cells. Moreover, CiTK secondarily upregulates the gene expression of Chymotrypsin and Carboxypeptidase B1 in inner follicular cells. Activation of these proteases is required for progression from stage II to stage III. Red dots indicate mitochondria.

Oocyte maturation in Ciona

Oocyte maturation, a process involving drastic nuclear and cytoplasmic reorganization including resumption of meiosis I, is featured by GVBD upon pro-phase arrest of meiosis. In mammals, LH and FSH, secreted by the pituitary in response to a hypothalamic hormone, GnRH, stimulate steroidogenesis in the preantral follicles, leading to preantral-to-antral transition; FSH stimulates the synthesis of testosterone in theca cells, and the latter induce 17β-estradiol (estrogen, E2) in granulosa cells using testosterone synthesized by theca cells (Robker et al., 2018; Duffy et al., 2019; Takahashi et al., 2019; Orisaka et al., 2021; Takahashi and Ogiwara, 2023). Quite recently, cholecystokinin, instead of GnRH, was shown to induce the secretion of FSH in medaka (Uehara et al., 2023). E2 enters the circulatory system, and induces LH surge (Robker et al., 2018; Duffy et al., 2019; Takahashi et al., 2019; Orisaka et al., 2021; Takahashi and Ogiwara, 2023). LH surge activates mitogen-activated protein kinase (MAPK) cascades, and then the phosphorylation of ERK1/2 in antral follicles, which in turn activates multiple signaling cascades, including the phosphorylation of maturation-promoting factor (MPF, CDK1/CYB). Subsequently, MPF triggers GVBD, namely, oocyte maturation via activation of various factors (Robker et al., 2018; Duffy et al., 2019; Takahashi et al., 2019; Jessus et al., 2020; Takahashi and Ogiwara, 2023).

Unlike vertebrates, invertebrates lack glycoproteins homologous to FSH and LH, although an ortholog of another glycoprotein, thyrostimulin, which is not involved in follicular development, is conserved in various invertebrates as well as in vertebrates (Nakabayashi et al., 2002; Dos Santos et al., 2009; Sun et al., 2010; Bassett et al., 2015; Wang et al., 2018; Yang et al., 2023). In non-ascidian invertebrates, several species-specific neuropeptides or compounds function as maturation-inducing factors: W/RPRPamide in jellyfish (Takeda et al., 2018), juvenile hormones in insects (Roy et al., 2018), and 1-methyladenine in starfish (Kanatani, 1985; Mita et al., 2009; Mita, 2023). Collectively, these findings show that oocyte maturation trigger molecules vary among phyla, but the intraovarian MAPK-MPF signaling cascade is conserved throughout metazoans (Jessus et al., 2020).

In early studies on oocyte maturation in ascidians, intracellular calcium ion mobilization and/or influx of calcium ions were believed to play a pivotal role in triggering GVBD in several marine invertebrates, including Ciona and other ascidians, although cAMP is responsible for GVBD in vertebrates and other invertebrates (Lambert, 2011; Deguchi et al., 2015). However, an ascidian GVBD inducer remained to be identified until 2019. Matsubara et al. (2019) demonstrated that the receptor for CiVP (CiVPR) is expressed predominantly in late stage II and early stage III (i.e., the preGVBD stages). Moreover, CiVPergic neurons in the cerebral ganglion were found to innervate the ovary by neuroanatomical analysis using the CiVP gene promoterKaede-transgenic Ciona (Kawada et al., 2021). These findings suggested that CiVP, secreted from the cerebral ganglion, participated in the regulation of GVBD. Indeed, treatment of Ciona pre-GVBD follicles with CiVP resulted in prompt induction of GVBD in the original in vitro follicle maturation/ovulation assays (Matsubara et al., 2019, 2020). Furthermore, CiVP was shown to activate ERK1/2, inducing the phosphorylation of Ciona MPF (Fig. 3). Eventually, phosphorylated MPF triggered GVBD (Matsubara et al., 2019; Satake, 2023) (Fig. 3). These findings are in good agreement with those of previous studies that showed that intracellular calcium ions are prerequisites for GVBD of ascidian oocytes (Lambert, 2011; Deguchi et al., 2015), given that CiVP induces intracellular calcium ion mobilization, but not production of cAMP, via the cognate receptor CiVPR (Kawada et al., 2008). Also of interest is that considerably fewer stage III follicles (post-GVBD follicles) were developed and, instead, more numerous stage I and stage II follicles were present in the ovaries of the TALEN-based CiVP-gene edited Ciona ovary, compared with the wild type (Kawada et al., 2021). Collectively, these studies have substantiated the molecular mechanism underlying Ciona oocyte maturation (Fig. 3).

These studies provided three major novel findings about Ciona follicle maturation. Firstly, GVBD is triggered by a VP family peptide in Ciona. Secondly, CiVP is a GVBD inducer in Ciona, whereas LH is responsible for induction of GVBD in vertebrates. This role of CiVP in the induction of GVBD is compatible with the findings that ascidians possess no orthologs of vertebrate pituitary gonadotropins FSH and LH (Dehal et al., 2002; Satake, 2023; Yang et al., 2023). Thirdly, in contrast with the species-specificity of a GVBD trigger, the basal MPF phosphorylation signaling cascade is evolutionarily conserved throughout metazoans, including ascidians. In combination, they support the conclusion that Ciona employs a VP family peptide as an inducer of oocyte maturation, and shares the intraovarian MPF phosphorylation-directed GVBD induction with other metazoans.

Fig. 3.

Induction of oocyte maturation (GVBD). CiVP activates Erk1/2 in oocytes at stage III, and promotes GVBD via activation of MPF, leading to GVBD. Red dots indicate mitochondria.

Ovulation in Ciona

In vertebrates, LH surge also almost simultaneously induces ovulation as well as oocyte maturation. In mammals, LH stimulates multiple signaling pathways, including the cAMP-protein kinase A (PKA)-CREB, inositol triphosphate 3-intracellular calcium mobilization- ERK1/2, and PI3K/AKT pathways (Robker et al., 2018; Duffy et al., 2019). Activation of ERK1/2 results in the gene expression and secretion of epidermal growth factor (EGF)–like ligands in granulosa cells, leading to the formation of the cumulus oocyte complex (COC) that functions in the regulation of oocyte condition and fertilization (Robker et al., 2018; Duffy et al., 2019). These multiple signaling cascades also upregulate COX2, the synthase of prostaglandin H (PGH), which is a precursor of all PGs in granulosa cells (Robker et al., 2018; Duffy et al., 2019). PGE2, a dominant PG in granulosa cells, mediates follicular rupture and angiogenesis via its cognate receptor, EP2 (Robker et al., 2018; Duffy et al., 2019). Furthermore, the increase in progesterone synthesis by these signaling cascades up-regulates the gene expression of a matrix metalloprotease, ADAMTS-1, in granulosa cells, which is responsible for degradation of an extracellular matrix, versican, and release of COC from the follicle (Robker et al., 2018; Duffy et al., 2019). Integration of such complex molecular mechanisms is required for the drastic remodeling of follicles, leading to ovulation of fertile oocytes.

In teleosts, including medaka, LH surge has been shown to upregulate the cAMP-PKA-CREB pathway, leading to the gene expression of MMP15 and a PGE2 receptor, PTGER4b, via non-MAPK signaling pathways (Takahashi et al., 2019; Ogiwara et al., 2023; Takahashi and Ogiwara, 2023). Furthermore, MMP15 is responsible for extracellular matrix degradation, and PTGER4b is a prerequisite for intracellular actin filament reorganization; both processes eventually induce ovulation (Takahashi et al., 2019; Ogiwara et al., 2023; Takahashi and Ogiwara, 2023).

However, neither the ovulation of mature Ciona follicles nor the relevant molecular mechanisms have been investigated. The ovulation of mature Ciona follicles was observed in vitro for the first time in 2019, and CiVP was identified as an ovulation inducer as well as a GVBD trigger in Ciona (Matsubara et al., 2019, 2020). CiVP-activated ERK1/2 also up-regulates the gene expression of the matrix metalloproteinase MMP2/9/13, and the degradation of the extracellular matrix by MMP2/9/13 results in ovulation (Matsubara et al., 2019). These studies verified that CiVP plays a crucial role in induction of ovulation as well as GVBD in Ciona, instead of LH in vertebrates (Fig. 4). Interestingly, vasotocin (VT), a non-mammalian vertebrate VP family peptide, was shown to induce oocyte maturation and ovulation in catfish (Joy and Chaube, 2015; Singh et al., 2021), although the balance in contribution to the ovulation process between VT and LH and its conservation in teleost species remain unclear. Notably, the ovulation process in Ciona has been shown to share some characteristics with both teleosts and mammals. For example, the activation of ERK1/2 leading to ovulation is conserved between Ciona and mammals (Matsubara et al., 2019; Kawada et al., 2021; Satake, 2023). In contrast, no evidence for the involvement of MMPs in ovulation in mammals has been provided. Instead, another matrix metalloprotease, ADAMTS-1, participates in the proteolysis of the extracellular matrix including versican during the ovulation process (Robker et al., 2018). Moreover, upregulation of the gene expression and the enzymatic activity of MMPs is a common signaling cascade during ovulation of Ciona and teleosts; however, ERK1/2 phosphorylation has not been detected in the ovulation pathway of teleosts (Matsubara et al., 2019; Takahashi et al., 2019; Satake et al., 2023; Takahashi and Ogiwara, 2023). Taken together, these findings suggest that the Ciona intrafollicular pathway of ovulation induction appear to be a “hybrid” of mammalian and teleost ovulatory systems. In other words, ancestral chordates might have been endowed with a Ciona-like ovulation system, some parts of which have been conserved throughout vertebrates, while others have been newly acquired or diverged in each lineage of chordates.

Fig. 4.

Induction of ovulation. CiVP also upregulates MMP2/9/13 via activation of Erk1/2. Cionin also induces MMP2/9/13 expression via CioR2 in inner follicular cells and RTK signaling and subsequent ovulation in stage III follicles. Red dots indicate mitochondria.

The Ciona homolog of the vertebrate cholecystokinin (CCK), cionin (Johnsen and Rehfeld, 1990), has also been reported to elicit ovulation (Fig. 4). The cognate receptor, CioR2, is expressed specifically in inner follicular cells in early stage III follicles (Sekiguchi et al., 2012; Osugi et al., 2021). Moreover, cionin was found to increase the gene expression of receptor tyrosine kinase (RTK) signaling-related genes such as rora, fcol1, and gla3 (Osugi et al., 2021). Subsequently, RTK signaling upregulated gene expression, resulting in an increase in the enzymatic activity of MMP2/9/13, which eventually stimulates ovulation (Osugi et al., 2021; Kawada et al., 2022; Satake, 2023). These findings verified that cionin also serves as an ovulation inducer, and that Ciona possesses multiple distinct pathways for upregulation of MMPs leading to ovulation. Notably, unlike CiVP, cionin was found to induce only ovulation, but not GVBD (Osugi et al., 2021; Kawada et al., 2022; Satake, 2023). Furthermore, ERK1/2 phosphorylation, which leads to CiVP-directed GVBD and ovulation, was not implicated in the cionin-RTK-MMP cascade for ovulation (Fig. 4). In mammals, inhibition of RTK signaling resulted in suppression of ovulation (Bernard et al., 2016), although no endogenous stimulatory factor for the RTK signaling has yet been investigated. Moreover, CCK stimulated RTK signaling in non-ovarian cells (Zeng et al., 2020). These findings raise a question of whether CCK-RTK signaling is involved in vertebrate ovulation.