Pattern of Gonadal Sex Differentiation in the Rice Field Frog Hoplobatrachus rugulosus (Anura: Dicroglossidae)

© 2020 Academia Sinica, Taiwan Open Access BACKGROUND Sex differentiation is a diverse (e.g., protandric hermaphroditism, protogynous hermaphroditism, heterogametic sexual system; Tsai et al. 2011; Aneesh et al. 2018; Kottarathil and Kappalli 2019; Pewphong et al. 2020) and crucial process of animal development. In amphibians, genetic sex determination generally occurs after fertilization, which is directly influenced by sexdetermining genes (Hayes 1998; Eggert 2004; Nakamura 2010) and environmental temperature (Eggert 2004). Sex differentiation continues during metamorphosis, but observable differences may vary among different species. Sexual differentiation and development in amphibians have been studied for decades in several species, including Lithobates catesbeianus/Rana (R.) catesbeiana (Swingle 1925), R. temporaria (Witschi 1929b), R. ornativentris (Iwasawa 1969), Pelophylax Pattern of Gonadal Sex Differentiation in the Rice Field Frog Hoplobatrachus rugulosus (Anura: Dicroglossidae) Thrissawan Traijitt1,2, Noppadon Kitana2,3, and Jirarach Kitana2,3,* 1Biological Sciences Program, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. E-mail: [email protected] (Traijitt) 2Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. E-mail: [email protected] (N. Kitana) 3BioSentinel Research Group (Special Task Force for Activating Research), Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. *Correspondence: E-mail: [email protected] (Kitana). Tel: +662-218-5253. Received 29 July 2019 / Accepted 27 August 2020 / Published 30 October 2020 Communicated by Benny K.K. Chan Sex differentiation during gonadal development is diversified among anuran amphibian species. In this study, the anuran experimental species Hoplobatrachus rugulosus was examined. The pattern of gonadal sex differentiation was observed by morphological and histological approaches. The gonad was observed morphologically at Gosner stage 33, while distinct testis and ovary were evident from 3–4 weeks after metamorphosis ended. Histological analysis showed that genital ridge formation began at stage 25 and ovarian differentiation began at stage 36. The developing ovary appeared with numerous primary oogonia, which developed into oocytes, while the medulla regressed to form an ovarian cavity. During metamorphosis, only an ovary was observed. Testicular differentiation seemed to begin later, during the first week after metamorphosis, and occurred via an intersex condition. The intersex gonads contained developing testicular tissue with both normal and atretic oocytes. The fully developed testis was first identified at 6 weeks after metamorphosis. Comparing the times of gonadal differentiation and somatic development revealed that the ovary exhibited a basic rate of differentiation while the testis exhibited a retarded one. These results establish that males of this species develop later than do females, and the testis develops through an intersex gonad, as is evident from its seminiferous cord formation, the presence of testis-ova, and atretic oocytes in the tissue. Thus, the pattern of gonadal sex differentiation in H. rugulosus is an undifferentiated type, in which only female gonads are observed during metamorphosis and intersex and male gonads are observed later. These results are crucial for further research on the sexual development of anurans. Key words: Gonad, Sex differentiation, Development, Anuran, Hoplobatrachus rugulosus. Citation: Traijitt T, Kitana N, Kitana J. 2020. Pattern of gonadal sex differentiation in the rice field frog Hoplobatrachus rugulosus (Anura: Dicroglossidae). Zool Stud 59:51. doi:10.6620/ZS.2020.59-51. Zoological Studies 59:51 (2020) doi:10.6620/ZS.2020.59-51 1 © 2020 Academia Sinica, Taiwan nigromaculatus/R. nigromaculata and R. japonica (Shirane 1986), Bufo japonicus formosus (Tanimura and Iwasawa 1987), Bombina orientalis (Lopez 1989), Rhacophorus arboreus (Tanimura and Iwasawa 1989), Pelophylax ridibundus/R. ridibunda (Ogielska and Wagner 1990), Xenopus laevis (Kelley 1996), R. rugosa (Nakamura 2009), R. curtipes (Gramapurohit et al. 2000), Bombina variegata (Piprek et al. 2010), Euphlyctis cyanophlyctis (Phuge and Gramapurohit 2013), Scinax fuscovarius (Goldberg 2015), and Dendropsophus labialis (Pinto-Erazo et al. 2016). The different sex differentiation processes can be categorized into different patterns. Anuran species can be divided into three sex differentiation patterns, or types (Witschi 1929a; Gramapurohit et al. 2000; Saidapur et al. 2001; Eggert 2004; Ogielska 2009; Piprek et al. 2010; Phuge and Gramapurohit 2013). The first is the differentiated type, in which gonadal development begins with the formation of indifferent gonads, which develop into ovaries or testes. Female and male gonads differentiate after metamorphosis. This pattern is common and has been reported in Bombina orientalis (Lopez 1989), Bombina variegata (Piprek et al. 2010), Lithobates catesbeianus, Microhyla ornate, Pelophylax nigromaculatus, Xenopus leavis (Gramapurohit et al. 2000), and others. The second is the semi-differentiated type, in which the ovaries and intersex gonads can be found after metamorphosis (Flament 2016). This type is uncommon and has only been found in a few species, e.g., Lithobates sylvaticus/R. sylvatica (Witschi 1929a), Rhacophorus arboreus (Tanimura and Iwasawa 1989), and R. curtipes (Gramapurohit et al. 2000). The third is the undifferentiated type, in which the indifferent gonads develop into ovaries after metamorphosis. Testicular development occurs later, after the intersex gonads have differentiated (Flament 2016). This pattern was reported in Bufo bufo and R. ornativentris (Gramapurohit et al. 2000). While it is known that anurans undergo sexual differentiation via these three patterns, the distinction between the undifferentiated and semi-differentiated types is still not clear. The rice field frog, Hoplobatrachus rugulosus, is a frog native to Thailand and is widely distributed throughout the wetlands from south-central China to the Thai-Malay Peninsula (Diesmos et al. 2004). This frog is an economically important food species and has the potential to be an experimental species in many fields of research (Pariyanonth et al. 1985; Tokur et al. 2008). As an experimental animal, H. rugulosus also plays a key role in many fields, such as biodiversity and geographic variation (Khonsue and Thirakhupt 2001; Schmalz and Zug 2002; Bain and Truong 2004; Hasan et al. 2012) and the taxonomic study of phylogenetic relationships (Pansook et al. 2012) and cryptic species complexes (Yu et al. 2015). In physiology, this species has been used as a model for studying reproductive systems (Ratanasaeng et al. 2008), including the effects of environmental endocrine disruptors (Ruamthum et al. 2011; Trachantong et al. 2013). Although there have been a number of reports using H. rugulosus, there is still no information about the species’ gonadal development. Our preliminary study demonstrated that ovarian differentiation in this species may occur earlier than testicular differentiation (Traijitt et al. 2010), and so could be categorized into either the undifferentiated or semi-differentiated type. Therefore, in this study, the process of gonadal sex differentiation in H. rugulosus was studied chronologically using morphological and histological approaches to clarify its pattern of gonad development. MATERIALS AND METHODS Animal procurement Four pairs of adult male and female H. rugulosus were obtained from the Amphibian and Reptile Research Unit, Chulalongkorn University and used as the breeders for artificial fertilization. These frogs were originally obtained from a northern Thailand population that represents a single clade (Pansook et al. 2012). Subcutaneous injection of a GnRH analogue (Suprefact, Frankfurt am Main) was used to induce spermination, ovulation, and mating as reported in Pariyanonth et al. (1985). All tadpoles were raised in 100-L plastic containers under natural light (12L: 12D) and a water temperature of 27.5–28.1°C. Water was renewed daily to remove waste and maintain the oxygen level. Tadpoles and frogs were fed with commercial fish pellets once daily. The morphological stage of each individual was examined under a stereomicroscope and estimated according to the Gosner staging system (Gosner 1960). The samples at each stage were euthanized in 0.25% (w/v) MS-222 (tricaine methanesulfonate) and photographed. The experimental protocol was approved by the Animal Care and Use Committee of Faculty of Science, Chulalongkorn University (Protocol Review No. 1623002). Morphological study After reaching stage 25 (two days after fertilization; daf.), eight tadpoles were randomly sampled at each stage until stage 46, when metamorphosis was completed (29–31 daf.). After this, page 2 of 12Zoological Studies 59:51 (2020) © 2020 Academia Sinica, Taiwan eight frogs were collected weekly until four months after metamorphosis. After euthanization, the gonads of each sample were dissected and the morphological characters of the gonad were examined under a stereomicroscope and photographed. Histological study Sixteen individuals of each tadpole stage (stages 25–46) and 8–14 individuals for each week after metamorphosis to the adult stage were fixed in Davidson’s fixative for 24–48 h. The gonad-kidney complex was carefully dissected, then dehydrated in an alcohol series (70, 90, 95, and 100% (v/v) ethanol). The tissues were then cleared in xylene and embedded in a paraffin block following the standard procedure (Kiernan 2008) before being serially sectioned at 6-µm thickness and stained with periodic acid and Schiff reagent (PAS stain) and hematoxylin. PAS stain was used to demonstrate the basement membrane such as the boundary of the germ cell group and the boundary of the seminiferous cord. Slides of gonad tissues were examined under light microscopy and photographed (Carl Zeiss-Axio Scope A1). The histological data on the developmental stages of the gonad were compared to the results of the external and gonad morphology and reported as the chronology of gonadal development for this species. The pattern of gonadal sex differentiation was examined according to the definitions of Witschi (1929a), Ogielska (2009), and Flament (2016). RESULTS Gonad morphological sex differentiation The gonads of Hoplobatrachus rugulosus were observed under light stereomicroscopy starting at stage 33 (11 daf.), appearing as strands of a long white structure lining the anteromedial region of the kidneys (Fig. 1A). During metamorphosis, the gonads increased in size but were still not differentiated between sexes until 3 to 4 weeks after metamorphosis. The gonads were covered by a peritoneum, which was fused together and attached the gonad to the dorsal cavity wall. In the female, the gonad expanded to form a multiple-lobed structure composed of ovarian sacs (Fig. 1B), which was distinct in juvenile females to adults. The shape of the ovary gradually changed due to the production of oocytes. For males, under the peritoneum, the connective tissue capsule (tunica albuginea) was formed surrounding the developing testis. The distinctive character of the testes was their long oval shape with a smooth surface (Fig. 1C). Finger-like fat bodies were present at the cephalic end of both the ovary and testis. Gonad histological sex differentiation and development Genital ridge formation: Gosner stages 25 (2 daf.) – 26 (3 daf.) Fig. 1. Representative stereomicroscope photographs of the gonad morphology and location in H. rugulosus showing (A) the developing gonads at stage 33 (arrow heads), (B) ovaries at 4 weeks after metamorphosis with numerous ovarian sacs (arrow heads), and (C) oval elongate-shaped testes at 4 weeks after metamorphosis (arrow heads). page 3 of 12Zoological Studies 59:51 (2020) © 2020 Academia Sinica, Taiwan The genital ridge was noticeable by histology at stage 25. Two thickenings of the germinal epithelium formed ventral to the dorsal mesentery at the anteromedial region of the kidneys. At this stage, the genital ridge was composed of 1–2 large primordial germ cells (PGCs) surrounded by somatic cells (Fig. 2A). The distinctive character of the PGCs was their large irregular cell shape with a prominent and palestained spherical nucleus and yolk platelets in the cytoplasm. Development of indifferent gonad: Gosner stages 27 (5 daf.) – 35 (15 daf.) Starting at stage 27, the size of the genital ridge increased after the proliferation of PGCs and somatic cells. It developed into the indifferent embryonic gonad protruding into the coelomic cavity. Once the yolk platelets in the PGCs were depleted (Fig. 2B), the germ-line cells became gonocytes. The indifferent gonad gradually increased in size with increasing numbers of gonocytes and somatic cells. During this period, the gonocytes were large spherical cells with a spherical nucleus. At stage 35, the gonad could be divided into two layers of cortex—a peripheral region with gonocytes—and the medulla, an internal region consisting of dark blue stained somatic cells (Fig. 2C). Ovarian differentiation: Gosner stage 36 (17 daf.) – 3 weeks after metamorphosis Starting at stage 36, the developing gonad expanded and gonocytes (primary oogonia from now) was spherical in shape with a pale stained cytoplasm Fig. 2. (Left) Somatic development, (Middle) gonad histology, and (Right) schematic representation of the gonads in H. rugulosus at (A) stage 25, showing the genital ridge formation; (B) stage 27, showing the indifferent gonad formation; and (C) stage 35, showing the indifferent gonad with an increased number of gonocytes and somatic cells. Cx: cortex, G: gonocyte, M: medulla, PGC: primordial germ cell, SC: somatic cell, YP: yolk platelet. page 4 of 12Zoological Studies 59:51 (2020) © 2020 Academia Sinica, Taiwan and a spherical nucleus. Some of the primary oogonia divided mitotically and stayed aggregated into a cluster called the secondary oogonia, surrounded by a cyst. The cyst was composed of developing germ cells at the same stage of cell division. The medulla at this stage was less developed and regressed into an ovarian cavity (Fig. 3A). This is the first stage at which the gonad was clearly distinguishable as an ovary based on the appearance of the ovarian cavity and location of the single primary oogonia and cysts, which were confined to the cortex. The cortex then developed and increased in size. Secondary oogonia entered the first meiotic prophase and formed nests of meiocytes at the leptotene and pachytene stages. At stage 42 (24 daf.), diplotene oocytes were seen for the first time, and the diplotene oocytes were large spherical cells with a pink or purple stained cytoplasm and a spherical nucleus consisting of several nucleoli. The oocyte was surrounded by flat follicular cells with an oval nucleus. The medulla region of the ovary regressed continuously, and the ovarian cavity enlarged (Fig. 3B). The development of the ovary continued until 4 weeks after metamorphosis, when the ovary consisted of numerous diplotene oocytes surrounded by flattened follicular cells that looked like a simple squamous epithelial lining. The diplotene oocytes developed from the outermost layer towards the innermost layer of the cortex. The secondary oogonia entered meiosis and formed a cyst of leptotene-pachytene oocytes. Therefore, earlier-stage developing germ cells were found in parts of the outermost area, such as single primary oogonia and cysts containing secondary oogonia or leptotenepachytene meiocytes (Figs. 4A and 5A). A group of them was enveloped by loose connective tissue forming the germ patch. During this period, different groups of somatic cells/tissues also developed, such as theca cells, connective tissues, epithelial tissues, and blood vessels. Development of the intersex gonad: one week after metamorphosis One week after metamorphosis, some individuals (36.4%) began to develop intersex gonads after a long period (starting at stage 36) of ovarian development. The histological character of the intersex gonad resembled both a developing ovary and a developing testis. The diplotene oocytes were usually found in the innermost area of the cortex. The primary oogonia and Fig. 3. (Left) Somatic development, (Middle) gonad histology, and (Right) schematic representation of the gonads in H. rugulosus at (A) stage 36, showing the ovarian differentiation with the presence of ovarian cavity and primary oogonia in the cortex and (B) stage 42, showing the developing ovary with germ cells at different stages. Cx: cortex, DiO: diplotene oocyte, F: follicle, M: medulla, OC: ovarian cavity, PG: primary oogonia, SC: somatic cell, SG: cyst with secondary oogonia. page 5 of 12Zoological Studies 59:51 (2020) © 2020 Academia Sinica, Taiwan cysts with meiocytes were still observed during this stage in the outermost area of the cortex. The ovarian cavity regressed (Fig. 4B). Some diplotene oocytes showed abnormal characters, which indicated atresia, such as hyperbasophilic cytoplasm, vacuolation, and an irregular nuclear shape. The others were normal during the diplotene stage. Later, the outermost area of the cortex was replaced by developing testicular tissue with evidence of forming seminiferous tubules (Fig. 4B). These findings implied that the transformation of the gonadal sex had occurred and the ovary had seemingly transformed into the testis (Fig. 5B). During the development of the testis, the diplotene oocytes were found inside the testicular tissue and called testis-ova. Additionally, in terms of the external morphology (Fig. 6A), the intersex gonad was a long cylindrical organ, indistinguishable from the testis in figure 1C. Although the external morphology showed a testislike character, the histology revealed ovarian tissue mixed together with the developing testicular tissue. We found that the intersex gonads were still observed in some individuals until the adult stage at 16 weeks after metamorphosis. In juvenile males at 5 weeks after metamorphosis, diplotene oocytes were present in the innermost area surrounded by the area of testicular formation, with a seminiferous tubule containing spermatogonia and cysts of spermatogenic cells present at every stage (Fig. 6B). However, the degree of intersex tissue occupying the gonad varied among individuals, from 1 to 12 diplotene oocytes per cross section. In some individuals, the external morphology was similar to that of the testis, but small ovarian sacs were still present (Fig. 6C). The histology of the above-mentioned gonads revealed a mixture of ovarian tissue containing numerous diplotene oocytes and some degenerating oocytes in the testicular tissue. They had a thickening connective tissue layer that surrounded the outermost area of the cortex. This character was similar to that of the tunica albuginea, which is normally formed in the developing male gonad. The outer surface was smooth. This character was not found in females at the same stage of development (Fig. 6D). Fig. 4. (Left) Somatic development, (Middle) gonad histology, and (Right) schematic representation of the gonads in H. rugulosus from 1 to 3 weeks after metamorphosis. (A) Developing ovary with numerous diplotene oocytes surrounded by follicular cells and (B) intersex gonad with regressed ovarian cavity, diplotene oocytes and degenerating oocytes at the innermost layer, and early seminiferous tubules (containing spermatogonia and some cysts) in the outermost layer of the cortex. C: sex cord, ePC: early primary oocyte (leptotene-pachytene), DiO: diplotene oocyte, DO: degenerating oocyte, F: follicle, OC: ovarian cavity, PG: primary oogonia, RC: regressed cavity, Spg: spermatogonia. page 6 of 12Zoological Studies 59:51 (2020) © 2020 Academia Sinica, Taiwan Testicular differentiation: 1–6 weeks after metamorphosis After the intersex gonad was observed (one week after metamorphosis), the testicular differentiation seemed to occur continuously. At 2–4 weeks after metamorphosis, the seminiferous cord was composed of dividing gonocytes (spermatogonia). Clusters of germ cells at different stages of spermatogenesis were found, including spermatogonia, secondary spermatogonia, and primary spermatocytes during prophase. Up to 6 weeks after metamorphosis, the testis was found to be welldeveloped and contain cysts of spermatogenic cells at every stage inside the seminiferous tubule (Fig. 5C). The spermatogenic cell types—including spermatogonium, spermatocyte, spermatid, and spermatozoa—were easily identifiable. Some mesenchymal cells differentiated into Leydig cells in the intertubular space. Other somatic cells developed into connective tissues and blood vessels. After this period, the testis became large and the number of seminiferous tubules increased. DISCUSSION Hoplobatrachus rugulosus exhibited an undifferentiated type of gonadal sex differentiation, and all of the indifferent gonads differentiated initially into Fig. 5. (Left) Somatic development, (Middle) gonad histology, and (Right) schematic representation of the gonads in H. rugulosus from 4 to 6 weeks after metamorphosis. (A) Developing ovary with numerous diplotene oocytes surrounded by follicular cells; (B) intersex gonad with testis ova at the innermost area and testicular tissue at the outermost area of the cortex, inset showing spermatogenic cells at every stage (scale bar = 20 µm); and (C) testis with seminiferous tubules containing cysts of spermatogenic cells at every stage, inset showing spermatogonium, group of spermatocytes, spermatozoa, Sertoli cell, and Leydig cells (scale bar = 20 µm). DiO: diplotene oocyte, F: follicle, GP: germ patch, L: Leydig cell, OC: ovarian cavity, S: Sertoli cell, Spc: spermatocyte, Spg: spermatogonium, Spz: spermatozoa, ST: seminiferous tubule, TO: testis-ovum. page 7 of 12Zoological Studies 59:51 (2020) © 2020 Academia Sinica, Taiwan ovaries during metamorphosis, and later the ovaries of more than one-third of the frogs transformed into testes, as evident by the seminiferous cord formation, presence of testis-ova, and atretic oocytes in the tissue. During this period, a prolonged intersex condition and testis-ova was found starting at one week after metamorphosis until the individuals reached maturity (16 weeks after metamorphosis). The intersex condition found was similar to that reported in some other anurans that undergo this type of gonadal differentiation (Gramapurohit et al. 2000; Eggert 2004; Saidapur et al. 2001; Flament 2016), including Bufo bufo, R. curtipes, and R. ornativentris (Gramapurohit et al. 2000); Hyla japonica (Nakamura 2009); Euphlyctis cyanophlyctis (Phuge and Gramapurohit 2013); and Scinax fuscovarius (Goldberg 2015). In H. rugulosus, the first evidence of ovarian differentiation appears at Gosner stage 36. Several studies have reported that it can occur at various Gosner stages among undifferentiated species, such as stage 25 in R. curtipes (Gramapurohit et al. 2000), stage 26 in Scinax fuscovarius (Goldberg 2015), stage 27 in Euphlyctis cyanophlyctis (Phuge and Gramapurohit 2013), and stages 31–35 in R. temporaria (Ogielska and Kotusz 2004). At this point, Ogielska and Kotusz (2004) studied 12 species of anuran (R. lessonae, R. ridibunda, R. temporaria, R. arvalis, R. pipiens, R. catesbeiana, Bombina bombina, Hyla arborea, Bufo bufo, B. viridis, Xenopus laevis, Pelobates fuscus) and reported that their ovarian differentiation is independent of somatic development, and that tadpole age is a more important factor. Female H. rugulosus reached maturity Fig. 6. Comparison of gonad morphology and histology in juvenile male H. rugulosus at (A, B) 5 and (C, D) 7 weeks after metamorphosis. Representative (A) stereomicroscope photograph of the testes; (B) photomicrograph of A, the intersex gonad with oocytes, gonial cysts and groups of meiocytes; (C) stereomicroscope photograph of the gonad at 7 weeks with a mixed appearance of male gonad and lobular structure of female gonad (arrows); (D) photomicrograph of C, the ovary. DiO: diplotene oocyte, DO: degenerating oocyte, F: follicle, GP: germ patch, OC: ovarian cavity, Spg: spermatogonia, TO: testis-ova. page 8 of 12Zoological Studies 59:51 (2020) © 2020 Academia Sinica, Taiwan at 16 weeks after metamorphosis, as evidenced by the presence of ripe diplotene oocytes (full of yolk) and signs of spawning. According to the Ogielska and Kotusz (2004), the pattern of female gonadal differentiation in 12 species of anurans, as described using the morphology and histology of developing ovaries, separated ovarian differentiation into 10 stages. Stages I–III involve an undifferentiated gonad and stages IV–X are ovarian. By comparing the rate of somatic development with the rate of ovarian differentiation, Ogielska and Kotusz (2004) derived three types of female gonad development. The first is a basic rate, in which ovarian differentiation occurs during metamorphosis. The second is an accelerated rate, in which ovarian differentiation occurs before metamorphosis at the earlier tadpole stage. The third is the retarded rate, in which ovarian differentiation occurs after metamorphosis. Based on these criteria, H. rugulosus has a basic rate of ovarian differentiation (Table 1), like several other amphibian species, including R. temporaria (Ogielska and Kotusz 2004), Bombina orientalis (Lopez 1989), and Rhacophorus arboreus (Tanimura and Iwasawa 1989). The rate of gonadal differentiation is known to be different among amphibian species (Storrs-MNdez and Semlitsch 2010; Goldberg 2015; Pinto-Erazo et al. 2016). It should be noted that the pathways that control the development of the body and gonad vary among species, and might also be affected by environmental conditions, such as season, temperature, diet, or population density (Ogielska 2009). The testicular differentiation of species with differentiated gonad development begins with a centralization of the germ cells in the indifferent gonad. The germinal epithelium in the cortex degenerates while germ cells migrate to the central medulla to form sex cords, which then develop into seminiferous tubules. Testicular differentiation in undifferentiated species occurs through the process of gonad transformation from ovary into testis (Witschi 1921). Witschi (1921) also observed gonad transformation in R. curtipes. In these cases, the transformation of the ovary occurs at an early stage. Gonocytes at the same stage of cell division are present as the sex cord forms in the ovarian sac. The sex cord appears as a highly compact group of gonocytes. Spermatogonia migrate from the cortex to the central part of the gonad. Then, the cortex degenerates and the central region develops into the testis (Witschi 1929b) (Table 2). Recently, it was accepted that the testis in this group of amphibian develops from the intersex gonad (Flament 2016). Gonad transformation in the present study was different from that in the above-mentioned reports on H. rugulosus. The intersex condition was first found in the gonad when the diplotene oocytes were developed, at one week after metamorphosis. The cluster of gonocytes was first present in the cortex, after which the sex cords were formed. The testicular tissue in the intersex gonad was found in the outermost area of the cortex and became fully developed, which was characterized by complete spermatogenesis. The diplotene oocytes were found at the innermost area of Table 1. Comparison of the somatic (Gosner stage) and gonadal stages of ovarian development in H. rugulosus following the criteria of Ogielska and Kotusz (2004) Gonadal stage Characteristic of gonad Somatic stage I PGCs migrate into the genital ridge. 25–26 II Gonocyte present separately as a single cell in the indifferent gonad. 27–34 III Somatic cells migrate to the medial part. Gonad composed of cortex and medulla. Gonocyte division present in the cortex. 35 IV Sexual differentiation begins. Medulla degenerates and ovarian cavity develops. 36 VSecondary oogonia enters meiosis. Cortex composed of primary oogonia and nests of secondary oogonia. 37–41 VI First diplotene oocytes present. Cortex composed of primary oogonia, nests of secondary oogonia, nests of meiocytes and diplotene oocytes. 42–45 VII Diplotene oocytes increase in number and size and protrude into the ovarian cavity, which changes shape into a narrow space. 46 (metamorphosis complete) VIII Ovary composed of almost only diplotene oocytes. The outermost area filled with primary oogonia, nests of secondary oogonia while the nests of meiocytes decrease. 1 week after metamorphosis IX Fat body becomes finger-like shaped. Primary oogonia, nests of secondary oogonia and meiocytes at the outermost area of the cortex are presented as a thin area. 2 weeks after metamorphosis XFully developed ovary, the cortex is composed mostly of diplotene oocytes. Germ patches present in the outermost area of the cortex. 4 weeks after metamorphosis page 9 of 12Zoological Studies 59:51 (2020)