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Light and electron microscopic analyses of Vasa expression in adult germ cells of the fish medaka
Gene 545 (2014) 15–22
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Light and electron microscopic analyses of Vasa expression in adult germ cells of the fish medaka Yongming Yuan, Mingyou Li, Yunhan Hong ⁎ Department of Biological Sciences, National University of Singapore, Science Drive 4, Singapore 117543, Singapore
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Article history: Received 12 March 2014 Received in revised form 20 April 2014 Accepted 1 May 2014 Available online 9 May 2014 Keywords: Germ plasm Immunoelectron microscopy Oogenesis Spermatogenesis Vasa
a b s t r a c t Germ cells of diverse animal species have a unique membrane-less organelle called germ plasm (GP). GP is usually associated with mitochondria and contains RNA binding proteins and mRNAs of germ genes such as vasa. GP has been described as the mitochondrial cloud (MC), intermitochondrial cement (IC) and chromatoid body (CB). The mechanism underlying varying GP structures has remained incompletely understood. Here we report the analysis of GP through light and electron microscopy by using Vasa as a marker in adult male germ cells of the fish medaka (Oryzias latipes). Immunofluorescence light microscopy revealed germ cell-specific Vasa expression. Vasa is the most abundant in mitotic germ cells (oogonia and spermatogonia) and reduced in meiotic germ cells. Vasa in round spermatids exist as a spherical structure reminiscent of CB. Nanogold immunoelectron microscopy revealed subcellular Vasa redistribution in male germ cells. Vasa in spermatogonia concentrates in small areas of the cytoplasm and is surrounded by mitochondria, which is reminiscent of MC. Vasa is intermixed with mitochondria to form IC in primary spermatocytes, appears as the free cement (FC) via separation from mitochondria in secondary spermatocyte and becomes condensed in CB at the caudal pole of round spermatids. During spermatid morphogenesis, Vasa redistributes and forms a second CB that is a ring-like structure surrounding the dense fiber of the flagellum in the midpiece. These structures resemble those described for GP in various species. Thus, Vasa identifies GP and adopts varying structures via dynamic reorganization at different stages of germ cell development. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In animals, germ cells undergo oogenesis and spermatogenesis to produce eggs in the ovary and sperm in the testis. These gonadal germ cells originate from primordial germ cells (PGCs), which are segregated from somatic cells early in development and migrate into the embryonic gonad where they become gonocytes (Raz, 2003; Wylie, 1999). Upon sex maturation, gonocytes undergo proliferation and become undifferentiated oogonia and spermatogonia, which contain germ stem cells capable of self-renewal and differentiation into functional gametes. Gametogenesis is a well-organized and very dynamic process. Oogenesis includes oogonial proliferation and differentiation during the mitotic phase and oocyte growth and maturation in the meiotic phase. Spermatogenesis proceeds in mitotic, meiotic and postmeiotic phases (Hong et al., 2004). In the adult testis of mouse and human, a seminiferous tubule contains male germ cells at various stages, with mitotic spermatogonia residing near the tubular wall, meiotic spermatocytes in the middle tubular region, post-meiotic spermatids towards the tubular Abbreviations: CB, chromatoid body; FC, free cement; GP, germ plasm; IC, intermitochondrial cement; iEM, immunoelectron microscopy; MC, mitochondrial cloud. ⁎ Corresponding author. E-mail address: [email protected] (Y. Hong).
http://dx.doi.org/10.1016/j.gene.2014.05.017 0378-1119/© 2014 Elsevier B.V. All rights reserved.
center, and mature sperm in the lumen (Brinster, 2007). Spermiogenesis, post-meiotic cytodifferentiation from round spermatids towards sperm, includes nuclear reorganization and marked changes in the volume and structure of cytoplasm and organelles. The mature sperm has a head containing highly condensed haploid nucleus, a midpiece with mitochondrial sheath and a long flagellum (Brinster, 2007). Mutations in genes involved in each aspect of germ cell development lead to infertility in animals and human. Germ genes exhibit preferential or exclusive expression throughout germ cell development. Many of germ genes encode RNA-binding proteins (Bhat and Hong, 2014; Li et al., 2012; Liu et al., 2009; Xu et al., 2007, 2009). One of the best studied germ genes perhaps is vasa, its sequence is highly conserved in the animal kingdom and its expression identifies embryonic and adult germ cells in diverse animal species including invertebrates and vertebrates (Xu et al., 2005, 2010, 2014). Its protein product Vasa is an ATP-binding RNA helicase essential for embryonic and adult germ cell development. In Drosophila, vasa mutations abolish PGC formation. In medaka, vasa knockdown affects PGC migration (Li et al., 2009). In mouse, targeted vasa gene disruption compromises the survival of post-migratory PGCs and leads to male infertility, because male homozygotes produce no sperm in the testes, where germ cells are arrested at the premeiotic stage and undergo apoptotic death (Tanaka et al., 2000).
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Fig. 1. Western blot of Vasa detection by αVas. An equal amount (20 μg) of crude protein was loaded for each of the organs. M, protein size markers; kD, kilodalton shown to the left.
RNAs and/or proteins of many germ genes are often the components of germ plasm (GP), a conserved membrane-less organelle in the cytoplasm of germ cells of diverse animals including zebrafish PGCs (Knaut et al., 2000). GP exhibits varying structures (Voronina et al., 2011). In female, for example, GP forms a spherical structure called Balbiani's body in previtollogenic oocytes. In male, GP appears as mitochondrial cloud (MC) in spermatogonia, becomes “intermitochondrial cement” (IMC) in spermatocytes (Yokota, 2008), and in the postmeiotic phase, develops into the chromatoid body (CB) (Shang et al., 2010), which has been reported to be on the nuclear surface in round spermatids (Parvinen, 2005) or around the sperm neck between the head and midpiece in elongating spermatids of mouse and human (GinterMatuszewska et al., 2011). In fish, GP has been analyzed so far in early embryos and PGCs of zebrafish (Knaut et al., 2000) and medaka (Hamaguchi, 1982; Herpin et al., 2007), and in spermatogenic cells of medaka (Hamaguchi, 1993)
and tilapia (Peruquetti et al., 2010). We make use of the fish medaka as a lower vertebrate model to study stem cells (Hong et al., 1998; Yi et al., 2009), germ cells (Hong et al., 2004; Li et al., 2009) and artificial reproductive technologies (Hong et al., 2004; Liu et al., 2011; Yi et al., 2009). In this study, we examined the Vasa protein in adult medaka germ cells by immunofluorescence (IF) light microscopy (LM) and nanogold immunoelectron microscopy (iEM). We show that Vasa is a GP component and its subcellular redistribution identifies dynamic GP reorganization from spermatogonia over spermatocytes to spermatids. Our data demonstrate that GP adopts varying structures via dynamic reorganization at different stages of germ cell development.
2. Materials and methods 2.1. Fish Work with fish followed the guidelines on the Care and Use of Animals for Scientific Purposes of the National Advisory Committee for Laboratory Animal Research in Singapore and approved by this committee (permit number 27/09). Medaka was maintained under a photoperiod of 14-h/10-h light/darkness at 26 °C (Hong et al., 2011; Yi et al., 2009).
2.2. Western blot and immunofluorescence Western blot analysis, gonadal cryosectioning and IF were performed by using a rabbit polyclonal anti-Vasa antibody (αVas) essentially as described (Xu et al., 2005). Briefly, ovaries and testes as well
Fig. 2. Expression of Vasa protein in the adult ovary. Ovarian cryosections were stained with αVas (green) plus DAPI (blue) for nuclei and analyzed by fluorescence microscopy. I–VII, stages of oocytes; fo, follicle cells surrounding the oocytes; og, oogonia.
Y. Yuan et al. / Gene 545 (2014) 15–22
as somatic organs were dissected from 3-month-old fish and subjected to either Western blotting or cryosections for immunostaining.
2.3. Chemical fixation For standard TEM sample preparation, medaka testes were fixed with glutaraldehyde and osmium tetroxide (OsO4) as previously described (Yuan et al., 2013). Briefly, testes from 6-month-old medaka fish were dissected out and fixed in 2.5% glutaraldehyde overnight and post-fixed in 1% osmium tetraoxide for 1 h. After dehydration in increasing ethanol series, samples were infiltrated with LX112 resin at gradient concentration and finally loaded into flat molds for polymerization at 45 °C for 24 h and another 48 h at 60 °C. Polymerized blocks were trimmed with glass knife and sectioned with a diamond knife (Diatome) on an ultra-microtome (Leica EM FCS). Ultrathin sections of 90-nm thickness were loaded onto 100-mesh copper grids and double stained with uranyl acetate and lead citrate before TEM observation.
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2.4. High-pressure freezing fixation To prepare sections for immunostaining, samples were fixed with HPF and embedded with Lowicryl HM20 as described (Yuan et al., 2014). Briefly, dissected testes were loaded in aluminum carriers (0.3-mm in depth) and rapidly frozen at 2100 bar during a 320-ms duration in a high pressure freezing machine (M. Wohlwend HPF 01). Cryofixed samples were transferred into a 1.5-ml cryotube containing 0.1% (wt/vol) uranyl acetate in pure acetone for freeze substitution. All samples were processed to freeze substitution and embedding in a temperaturecontrolling unit (Leica AFS2). After incubation at −90 °C for 48 h, the samples were washed in prechilled acetone with 3 changes when temperature was gradually raised to −50 °C (4.5 °C/h). Lowicryl HM20 at increasing concentrations (25, 50 and 75%) was added for gradient infiltration, with each step lasting 4 h. After the temperature was raised gradually to −30 °C (1.6 °C/h), 100% Lowicryl HM20 was added to the samples with 3 changes, with each change lasting 10 h. The samples were polymerized with UV for 48 h at −30 °C and for 24 h at 20 °C.
Fig. 3. IF analysis of Vasa protein in male germ cells. Cryosections of the adult medaka testis were stained with αVas (red) and DAPI (blue) and analyzed by light and fluorescent microscopy. (A and B) Overview of Vasa protein expression in the adult testis, showing male germ cells at major stages of spermatogenesis and spermiogenesis. (C and D) Spermiogenesis. (E) Larger magnification of the framed area in (D), highlighting the Vasa signal in one pole and intercellular region (asterisks) of spermatid cells, and as dots (arrows) in elongated spermatids. sg, spermatogonia; sc1, primary spermatocytes; sc2, secondary spermatocytes; rs, round spermatid; st, spermatids; sm, sperm; rb, residual body; es, elongating spermatid; ets, elongated spermatid. Scale bars, 100 μm in (A and B) and 10 μm in (C–E).
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Diamond knife sectioned slices of 220-nm thickness were transferred onto 50-mesh nickel grids for immunostaining.
3. Results 3.1. Western blot analysis of gonad-specific Vasa expression
2.5. Nanogold immunostaining Immunostaining was performed by using αVas (Xu et al., 2005) essentially as described (Yuan et al., 2013). Briefly, ultrathin sections were blocked with 4% bovine serum albumin and 0.05% Tween-20 for 20 min. The blocked sections were incubated with αVas at 1:50 dilution for 3 h and washed thoroughly with PBS, followed by incubation for 3 h with secondary antibodies conjugated with 1.4-nm nanogold (Nanoprobe). After washing in distilled water with 3 changes, the ultrathin sections were processed to the gold-enlargement procedure and air-dried according to the supplier's instruction (Nanoprobe).
A polyclonal anti-Vasa antibody (αVas) was used for immunostaining. This antibody was generated by immunizing the rabbit with a recombinant protein containing the 310-aa N-terminal sequence of the gibel carp Vasa and was able to specifically detect Vasa protein in several fish species including zebrafish and grouper (Xu et al., 2005). In medaka, this antibody detected a band on a Western blot exclusively in the ovary and testis but not in the liver (Fig. 1). The protein detected is approximately 75 kDa, in accordance with the calculated molecular mass of 76 kDa as predicted for the medaka Vasa. This result suggests that αVas is capable of specifically detecting the Vasa protein in adult medaka gonads.
2.6. Light and transmission electron microscopy
3.2. Vasa in the adult ovary by light microscopy
The cryosections with or without immunofluorescent staining were photographed with Zeiss Axioskop 2 plus upright microscope with a Zeiss AxioCam M5Rc digital camera (Zeiss Corp., Germany). The ultrathin sections immunostained with nanogold particles were examined on a 120 kV transmission EM (FEI Tecnai T12) and photographed on the Gatan 4K × 4K CCD camera (Gatan Corporation) as described (Yuan et al., 2014).
Temporospatial Vasa expression was first examined on cryosections of the adult ovary. The medaka female produces eggs daily, and the mature ovary comprises oogonia and oocytes at ten ontogenic stages (Iwamatsu et al., 1988). On ovarian sections, the Vasa signal is the most intense in oogonia and early oocytes at stages I–II, remains high in oocytes at stages III–VI and becomes reduced in oocytes from stage VII onwards (Fig. 2). The Vasa signal is seen widely in the cytoplasm
Fig. 4. Nanogold immunoelectron microscopy of germ plasm during spermatogenesis. Ultrathin sections of the adult medaka testis were incubated with αVas, stained with a nanogoldconjugated second antibody and analyzed by transmission EM. (A) Spermatogonium. (B) Larger magnification of the framed area in (A), highlighting that nanogold particles are surrounded by mitochondria. (C) Primary spermatocyte, showing that nanogold particles form intermitochondrial cement (IC) or an aggregate (arrowhead) close to mitochondria. (D) Secondary spermatocyte, showing close association of nanogold particles and mitochondria. mt, mitochondrium; nu, nucleus. Scale bars, 1 μm.
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of oogonia and early oocytes of stages I–II, concentrated in the peripheral cytoplasm of advanced oocytes at stages IV–V. Localization or concentration in a particular cytoplasmic area of early oocytes was not observed for Vasa. Therefore, Vasa is expressed in mitotic and meiotic female germ cells and shows dynamic subcellular distribution during oogenesis. 3.3. Vasa in the adult testis by light microscopy The Vasa was then examined in the adult testis. In medaka, the adult testis consists of seminiferous cysts that contain germ cells at various stages of spermatogenesis. Spermatogonia reside at the testicular periphery. Spermatogenesis proceeds synchronously within each cyst, and cysts of germ cells at progressively advanced stages of development are usually positioned closer to the efferent duct in the central region. This ordered arrangement of developmental stages facilitates distinction of various spermatogenetic stages. FISH on testicular sections revealed the Vasa signal in male germ cells (Fig. 3). At a low magnification, the signal is the strongest in spermatogonia, remains moderate in primary and secondary spermatocytes, reduced in spermatids and disappears in sperm
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(Fig. 3A and B). At a higher magnification, the signal is easily detectable in round spermatids and barely detectable in elongating spermatids (Fig. 3C and D). A closer observation under an oil lens revealed that the Vasa signal was positioned in one half of round spermatids and concentrated in one to three small dots (Fig. 3E). These dots are reminiscent of the ring-formed chromatoid body (CB) described in spermatids of diverse animals including mouse (Shang et al., 2010), revealing for the first time that Vasa is a component of CB in medaka. Therefore, Vasa is expressed in mitotic, meiotic and postmeiotic male germ cells and shows dynamic subcellular distribution during spermatogenesis and spermiogenesis. 3.4. Vasa expression during spermatogenesis by electron microscopy Male germ cell in the adult testis undergoes spermatogenesis and spermiogenesis, culminating in the production of mature sperm. Spermatogenesis includes mitosis of spermatogonia for self-renewal and proliferation and meiosis. Spermiogenesis is a postmeiotic process of cytodifferentiation. We examined Vasa expression first during spermatogenesis. To this end, the Vasa was detected by a nanogold-conjugated antibody on testicular ultrathin sections. This procedure in the absence
Fig. 5. Nanogold immunoelectron microscopy of germ plasm during spermiogenesis. Ultrathin sections of the adult medaka testis were incubated with αVas, stained with a nanogoldconjugated second antibody and analyzed by transmission EM. (A) Chemically fixed sample showing spermatids at different stages. (B and D) Round spermatids. (C and E) Elongating spermatids. (F–K) Elongated spermatids. (B′ and C′) Higher magnification of the area framed in (B) and (C) respectively. Gold particles (arrows) delineating the Vasa protein are concentrated in the area separating the nucleus and mitochondria. df, dense fiber; mt, mitochondria; nu, nucleus. Scale bars, 1 μm in (A); 0.5 μm in (B–M); 0.2 μm in (B′ and C′).
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of αVas did not produce nanogold particles that were distributed exclusively or preferentially in particular structures or areas of male germ cells on testicular ultrathin sections (Fig. S1), demonstrating the specificity of iEM procedure. In the presence of αVas, nanogold particles delineating the Vasa protein were found in male germ cells (Fig. 4). These nanogold particles were closely associated with mitochondria in male germ cells at various stages. Intriguingly, subcellular Vasa distribution varied when spermatogenesis proceeds. In spermatogonia, the nanogold particles were found in a cytoplasmic compartment of 2–5 μm in size, which is surrounded by mitochondria in spermatogonia (Fig. 4A and B). This architecture is reminiscent of MC. In primary spermatocytes, the nanogold particles became intermixed with mitochondria, forming IC (Fig. 4C). In secondary spermatocytes, the nanogold particles were located in areas that are separated from, and close to, mitochondrial aggregates (Fig. 4D). Hence, Vasa undergoes dynamic changes in subcellular distribution in mitotic and meiotic male germ cells.
3.5. Vasa expression during spermiogenesis by electron microscopy We then examined subcellular Vasa distribution during spermiogenesis. Transmission EM without immunostaining clearly revealed morphologies of spermatids at different stages of spermiogenesis (Fig. 5A). In round spermatids, the immediate meiotic product, nanogold particles were concentrated in the caudal pole, in which they were located in a cap-like CB, which was sandwiched by the nucleus and mitochondria
(Fig. 5B and D). In elongating and elongated spermatids, the nanogold particles were found in two compartments: One remained as the cap-like CB that turned out to be at the base of the forming and growing flagellum, and the other moved posteriorly to the developing midpiece and formed a second CB, a ring-like structure surrounding the dense fiber of the flagellum (Fig. 5C and E–K). Thus, Vasa shows subcellular redistribution during spermiogenesis and forms two distinct CBs in advanced spermatids.
4. Discussion In this study we have analyzed Vasa expression and subcellular distribution in adult medaka germ cells by light and electron microscopy. We show that Vasa expression is specific to germ cells of both sexes, similar to that reported for vasa RNA (Shinomiya et al., 2000; Xu et al., 2009). Specifically, Vasa differs not only at the level of expression at different stages of oogenesis and spermatogenesis but also in subcellular distribution in female and male germ cells, suggesting its dynamic redistribution during medaka gametogenesis. This expression pattern has similarly described for Vasa in gibel carp, zebrafish and grouper (Xu et al., 2005), and thus appears to be conserved in fish. A striking observation in this study is the localization of Vasa to CBs in round and elongated spermatids (Fig. 6A). Many germ genes' RNA and protein products are components of germ plasm (Xu et al., 2010). Germ plasm is a nucleoprotein complex
Fig. 6. Vasa protein in adult male medaka germ cells. (A) Immunofluorescence light microscopy. sg, spermatogonium; sc1 and sc2, primary and secondary spermatocytes; st, round spermatid; st2, elongated spermatid; sm, sperm. (B) Nanogold immunoelectron microscopy. FC, free cement; IC, intermitochondria cement; MC, mitochondrial cloud; nu, nucleus.
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and dynamic in composition and structure. Germ cells in diverse animal species develop two unique structures, namely the Balbinani body (BB) in female germ cells at the early oocyte stages and CB in male germ cells at the postmeiotic phase. Both BB and CB have been thought of as being sex- and stage-specific forms of germ plasm (Xu et al., 2010). In medaka, mRNAs of dazl (Xu et al., 2009), mitf1 and mitf2 (Li et al., 2013) are components of BB, whereas those of boule and vasa are not (Shinomiya et al., 2000; Xu et al., 2009). Strikingly, our IF analysis in this study reveals for the first time that Vasa is a component of CB but is absent in BB of medaka. These data clearly demonstrate an apparent difference between BB and CB in composition and support CB as a spermatid-specific form of germ plasm. Whether BB is the oocyte-specific germ plasm remains to be determined in the future. Polymorphic structures as a strand-like structure and amorphous fibrous body have been described for GP or nuage in medaka spermatogonia by standard transmission EM (Hamaguchi, 1993). In this study, we have analyzed medaka Vasa expression by nanogold iEM in adult male germ cells. We show for the first time in medaka that Vasa exhibit subcellular GP redistribution during spermatogenesis and spermiogenesis. Specifically, Vasa appears as MC in spermatogonia, transforms to IC in primary spermatocyte, forms FC in secondary spermatocytes via separation from mitochondria and culminates in two distinct CBs in spermatids (Fig. 6B). Since these features are reminiscent of those described for GP in mammalian spermatogenesis and spermiogenesis (Fawcett et al., 1970; Parvinen, 2005; Shang et al., 2010; Susi and Clermont, 1970), we conclude that Vasa is a GP component and its expression identifies dynamic GP redistribution and reorganization in medaka. Therefore, GP undergoes reorganization and thus forms varying fine structures during different stages of male germ cell development in medaka, which suggests that the mechanisms underlying GP structures and CB formation are highly conserved in diverse vertebrate species. CB has been extensively studied in mammals, where apparent differences have been reported in the number, shape, location and behavior of CB. According to Fawcett et al. (1970), a single CB as a ring appears around the anterior base of the forming flagellum in early elongating spermatids and moves to the caudal end of the forming middle piece in elongated spermatids. It has been reported in rat spermiogenesis that CB first appears as an arc around the base of the flagellum, and the bulk of its content subsequently condenses into a dense body and moves away from the nucleus and is fragmented (Susi and Clermont, 1970). A striking finding in this study is that GP is able to form two distinct CBs. One is present first in round spermatids where it appears as a cap or arc around the flagellum base, similar to the murine situation (Fawcett et al., 1970; Susi and Clermont, 1970). However, there is also a second CB, which is newly formed in elongating spermatids where it takes the shape of a ring around the dense fiber of flagellum in the midpiece. The presence of two CBs has recently been identified also by iEM in mouse, where a ring-shaped CB resides around the base of the flagellum and a satellite-like CB resides in the midpiece of elongating spermatids (Shang et al., 2010). Consequently, the presence of two CBs may be a conserved feature of spermatogenesis from fish to mammals. It has been reported that a premature loss of the CB-derived ring structure is associated with male infertility due to production of abnormal sperm (Shang et al., 2010). Our finding that Vasa is an integral component of both GP and CBs suggests an important role for this protein also in postmeiotic cytodifferentiation and flagellum formation. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.05.017.
Conflict of interest The authors declare that there is no conflict of interest.
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Light and electron microscopic analyses of Vasa expression in adult germ cells of the fish medaka Yongming Yuan, Mingyou Li, Yunhan Hong ⁎ Department of Biological Sciences, National University of Singapore, Science Drive 4, Singapore 117543, Singapore
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Article history: Received 12 March 2014 Received in revised form 20 April 2014 Accepted 1 May 2014 Available online 9 May 2014 Keywords: Germ plasm Immunoelectron microscopy Oogenesis Spermatogenesis Vasa
a b s t r a c t Germ cells of diverse animal species have a unique membrane-less organelle called germ plasm (GP). GP is usually associated with mitochondria and contains RNA binding proteins and mRNAs of germ genes such as vasa. GP has been described as the mitochondrial cloud (MC), intermitochondrial cement (IC) and chromatoid body (CB). The mechanism underlying varying GP structures has remained incompletely understood. Here we report the analysis of GP through light and electron microscopy by using Vasa as a marker in adult male germ cells of the fish medaka (Oryzias latipes). Immunofluorescence light microscopy revealed germ cell-specific Vasa expression. Vasa is the most abundant in mitotic germ cells (oogonia and spermatogonia) and reduced in meiotic germ cells. Vasa in round spermatids exist as a spherical structure reminiscent of CB. Nanogold immunoelectron microscopy revealed subcellular Vasa redistribution in male germ cells. Vasa in spermatogonia concentrates in small areas of the cytoplasm and is surrounded by mitochondria, which is reminiscent of MC. Vasa is intermixed with mitochondria to form IC in primary spermatocytes, appears as the free cement (FC) via separation from mitochondria in secondary spermatocyte and becomes condensed in CB at the caudal pole of round spermatids. During spermatid morphogenesis, Vasa redistributes and forms a second CB that is a ring-like structure surrounding the dense fiber of the flagellum in the midpiece. These structures resemble those described for GP in various species. Thus, Vasa identifies GP and adopts varying structures via dynamic reorganization at different stages of germ cell development. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In animals, germ cells undergo oogenesis and spermatogenesis to produce eggs in the ovary and sperm in the testis. These gonadal germ cells originate from primordial germ cells (PGCs), which are segregated from somatic cells early in development and migrate into the embryonic gonad where they become gonocytes (Raz, 2003; Wylie, 1999). Upon sex maturation, gonocytes undergo proliferation and become undifferentiated oogonia and spermatogonia, which contain germ stem cells capable of self-renewal and differentiation into functional gametes. Gametogenesis is a well-organized and very dynamic process. Oogenesis includes oogonial proliferation and differentiation during the mitotic phase and oocyte growth and maturation in the meiotic phase. Spermatogenesis proceeds in mitotic, meiotic and postmeiotic phases (Hong et al., 2004). In the adult testis of mouse and human, a seminiferous tubule contains male germ cells at various stages, with mitotic spermatogonia residing near the tubular wall, meiotic spermatocytes in the middle tubular region, post-meiotic spermatids towards the tubular Abbreviations: CB, chromatoid body; FC, free cement; GP, germ plasm; IC, intermitochondrial cement; iEM, immunoelectron microscopy; MC, mitochondrial cloud. ⁎ Corresponding author. E-mail address: [email protected] (Y. Hong).
http://dx.doi.org/10.1016/j.gene.2014.05.017 0378-1119/© 2014 Elsevier B.V. All rights reserved.
center, and mature sperm in the lumen (Brinster, 2007). Spermiogenesis, post-meiotic cytodifferentiation from round spermatids towards sperm, includes nuclear reorganization and marked changes in the volume and structure of cytoplasm and organelles. The mature sperm has a head containing highly condensed haploid nucleus, a midpiece with mitochondrial sheath and a long flagellum (Brinster, 2007). Mutations in genes involved in each aspect of germ cell development lead to infertility in animals and human. Germ genes exhibit preferential or exclusive expression throughout germ cell development. Many of germ genes encode RNA-binding proteins (Bhat and Hong, 2014; Li et al., 2012; Liu et al., 2009; Xu et al., 2007, 2009). One of the best studied germ genes perhaps is vasa, its sequence is highly conserved in the animal kingdom and its expression identifies embryonic and adult germ cells in diverse animal species including invertebrates and vertebrates (Xu et al., 2005, 2010, 2014). Its protein product Vasa is an ATP-binding RNA helicase essential for embryonic and adult germ cell development. In Drosophila, vasa mutations abolish PGC formation. In medaka, vasa knockdown affects PGC migration (Li et al., 2009). In mouse, targeted vasa gene disruption compromises the survival of post-migratory PGCs and leads to male infertility, because male homozygotes produce no sperm in the testes, where germ cells are arrested at the premeiotic stage and undergo apoptotic death (Tanaka et al., 2000).
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Fig. 1. Western blot of Vasa detection by αVas. An equal amount (20 μg) of crude protein was loaded for each of the organs. M, protein size markers; kD, kilodalton shown to the left.
RNAs and/or proteins of many germ genes are often the components of germ plasm (GP), a conserved membrane-less organelle in the cytoplasm of germ cells of diverse animals including zebrafish PGCs (Knaut et al., 2000). GP exhibits varying structures (Voronina et al., 2011). In female, for example, GP forms a spherical structure called Balbiani's body in previtollogenic oocytes. In male, GP appears as mitochondrial cloud (MC) in spermatogonia, becomes “intermitochondrial cement” (IMC) in spermatocytes (Yokota, 2008), and in the postmeiotic phase, develops into the chromatoid body (CB) (Shang et al., 2010), which has been reported to be on the nuclear surface in round spermatids (Parvinen, 2005) or around the sperm neck between the head and midpiece in elongating spermatids of mouse and human (GinterMatuszewska et al., 2011). In fish, GP has been analyzed so far in early embryos and PGCs of zebrafish (Knaut et al., 2000) and medaka (Hamaguchi, 1982; Herpin et al., 2007), and in spermatogenic cells of medaka (Hamaguchi, 1993)
and tilapia (Peruquetti et al., 2010). We make use of the fish medaka as a lower vertebrate model to study stem cells (Hong et al., 1998; Yi et al., 2009), germ cells (Hong et al., 2004; Li et al., 2009) and artificial reproductive technologies (Hong et al., 2004; Liu et al., 2011; Yi et al., 2009). In this study, we examined the Vasa protein in adult medaka germ cells by immunofluorescence (IF) light microscopy (LM) and nanogold immunoelectron microscopy (iEM). We show that Vasa is a GP component and its subcellular redistribution identifies dynamic GP reorganization from spermatogonia over spermatocytes to spermatids. Our data demonstrate that GP adopts varying structures via dynamic reorganization at different stages of germ cell development.
2. Materials and methods 2.1. Fish Work with fish followed the guidelines on the Care and Use of Animals for Scientific Purposes of the National Advisory Committee for Laboratory Animal Research in Singapore and approved by this committee (permit number 27/09). Medaka was maintained under a photoperiod of 14-h/10-h light/darkness at 26 °C (Hong et al., 2011; Yi et al., 2009).
2.2. Western blot and immunofluorescence Western blot analysis, gonadal cryosectioning and IF were performed by using a rabbit polyclonal anti-Vasa antibody (αVas) essentially as described (Xu et al., 2005). Briefly, ovaries and testes as well
Fig. 2. Expression of Vasa protein in the adult ovary. Ovarian cryosections were stained with αVas (green) plus DAPI (blue) for nuclei and analyzed by fluorescence microscopy. I–VII, stages of oocytes; fo, follicle cells surrounding the oocytes; og, oogonia.
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as somatic organs were dissected from 3-month-old fish and subjected to either Western blotting or cryosections for immunostaining.
2.3. Chemical fixation For standard TEM sample preparation, medaka testes were fixed with glutaraldehyde and osmium tetroxide (OsO4) as previously described (Yuan et al., 2013). Briefly, testes from 6-month-old medaka fish were dissected out and fixed in 2.5% glutaraldehyde overnight and post-fixed in 1% osmium tetraoxide for 1 h. After dehydration in increasing ethanol series, samples were infiltrated with LX112 resin at gradient concentration and finally loaded into flat molds for polymerization at 45 °C for 24 h and another 48 h at 60 °C. Polymerized blocks were trimmed with glass knife and sectioned with a diamond knife (Diatome) on an ultra-microtome (Leica EM FCS). Ultrathin sections of 90-nm thickness were loaded onto 100-mesh copper grids and double stained with uranyl acetate and lead citrate before TEM observation.
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2.4. High-pressure freezing fixation To prepare sections for immunostaining, samples were fixed with HPF and embedded with Lowicryl HM20 as described (Yuan et al., 2014). Briefly, dissected testes were loaded in aluminum carriers (0.3-mm in depth) and rapidly frozen at 2100 bar during a 320-ms duration in a high pressure freezing machine (M. Wohlwend HPF 01). Cryofixed samples were transferred into a 1.5-ml cryotube containing 0.1% (wt/vol) uranyl acetate in pure acetone for freeze substitution. All samples were processed to freeze substitution and embedding in a temperaturecontrolling unit (Leica AFS2). After incubation at −90 °C for 48 h, the samples were washed in prechilled acetone with 3 changes when temperature was gradually raised to −50 °C (4.5 °C/h). Lowicryl HM20 at increasing concentrations (25, 50 and 75%) was added for gradient infiltration, with each step lasting 4 h. After the temperature was raised gradually to −30 °C (1.6 °C/h), 100% Lowicryl HM20 was added to the samples with 3 changes, with each change lasting 10 h. The samples were polymerized with UV for 48 h at −30 °C and for 24 h at 20 °C.
Fig. 3. IF analysis of Vasa protein in male germ cells. Cryosections of the adult medaka testis were stained with αVas (red) and DAPI (blue) and analyzed by light and fluorescent microscopy. (A and B) Overview of Vasa protein expression in the adult testis, showing male germ cells at major stages of spermatogenesis and spermiogenesis. (C and D) Spermiogenesis. (E) Larger magnification of the framed area in (D), highlighting the Vasa signal in one pole and intercellular region (asterisks) of spermatid cells, and as dots (arrows) in elongated spermatids. sg, spermatogonia; sc1, primary spermatocytes; sc2, secondary spermatocytes; rs, round spermatid; st, spermatids; sm, sperm; rb, residual body; es, elongating spermatid; ets, elongated spermatid. Scale bars, 100 μm in (A and B) and 10 μm in (C–E).
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Diamond knife sectioned slices of 220-nm thickness were transferred onto 50-mesh nickel grids for immunostaining.
3. Results 3.1. Western blot analysis of gonad-specific Vasa expression
2.5. Nanogold immunostaining Immunostaining was performed by using αVas (Xu et al., 2005) essentially as described (Yuan et al., 2013). Briefly, ultrathin sections were blocked with 4% bovine serum albumin and 0.05% Tween-20 for 20 min. The blocked sections were incubated with αVas at 1:50 dilution for 3 h and washed thoroughly with PBS, followed by incubation for 3 h with secondary antibodies conjugated with 1.4-nm nanogold (Nanoprobe). After washing in distilled water with 3 changes, the ultrathin sections were processed to the gold-enlargement procedure and air-dried according to the supplier's instruction (Nanoprobe).
A polyclonal anti-Vasa antibody (αVas) was used for immunostaining. This antibody was generated by immunizing the rabbit with a recombinant protein containing the 310-aa N-terminal sequence of the gibel carp Vasa and was able to specifically detect Vasa protein in several fish species including zebrafish and grouper (Xu et al., 2005). In medaka, this antibody detected a band on a Western blot exclusively in the ovary and testis but not in the liver (Fig. 1). The protein detected is approximately 75 kDa, in accordance with the calculated molecular mass of 76 kDa as predicted for the medaka Vasa. This result suggests that αVas is capable of specifically detecting the Vasa protein in adult medaka gonads.
2.6. Light and transmission electron microscopy
3.2. Vasa in the adult ovary by light microscopy
The cryosections with or without immunofluorescent staining were photographed with Zeiss Axioskop 2 plus upright microscope with a Zeiss AxioCam M5Rc digital camera (Zeiss Corp., Germany). The ultrathin sections immunostained with nanogold particles were examined on a 120 kV transmission EM (FEI Tecnai T12) and photographed on the Gatan 4K × 4K CCD camera (Gatan Corporation) as described (Yuan et al., 2014).
Temporospatial Vasa expression was first examined on cryosections of the adult ovary. The medaka female produces eggs daily, and the mature ovary comprises oogonia and oocytes at ten ontogenic stages (Iwamatsu et al., 1988). On ovarian sections, the Vasa signal is the most intense in oogonia and early oocytes at stages I–II, remains high in oocytes at stages III–VI and becomes reduced in oocytes from stage VII onwards (Fig. 2). The Vasa signal is seen widely in the cytoplasm
Fig. 4. Nanogold immunoelectron microscopy of germ plasm during spermatogenesis. Ultrathin sections of the adult medaka testis were incubated with αVas, stained with a nanogoldconjugated second antibody and analyzed by transmission EM. (A) Spermatogonium. (B) Larger magnification of the framed area in (A), highlighting that nanogold particles are surrounded by mitochondria. (C) Primary spermatocyte, showing that nanogold particles form intermitochondrial cement (IC) or an aggregate (arrowhead) close to mitochondria. (D) Secondary spermatocyte, showing close association of nanogold particles and mitochondria. mt, mitochondrium; nu, nucleus. Scale bars, 1 μm.
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of oogonia and early oocytes of stages I–II, concentrated in the peripheral cytoplasm of advanced oocytes at stages IV–V. Localization or concentration in a particular cytoplasmic area of early oocytes was not observed for Vasa. Therefore, Vasa is expressed in mitotic and meiotic female germ cells and shows dynamic subcellular distribution during oogenesis. 3.3. Vasa in the adult testis by light microscopy The Vasa was then examined in the adult testis. In medaka, the adult testis consists of seminiferous cysts that contain germ cells at various stages of spermatogenesis. Spermatogonia reside at the testicular periphery. Spermatogenesis proceeds synchronously within each cyst, and cysts of germ cells at progressively advanced stages of development are usually positioned closer to the efferent duct in the central region. This ordered arrangement of developmental stages facilitates distinction of various spermatogenetic stages. FISH on testicular sections revealed the Vasa signal in male germ cells (Fig. 3). At a low magnification, the signal is the strongest in spermatogonia, remains moderate in primary and secondary spermatocytes, reduced in spermatids and disappears in sperm
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(Fig. 3A and B). At a higher magnification, the signal is easily detectable in round spermatids and barely detectable in elongating spermatids (Fig. 3C and D). A closer observation under an oil lens revealed that the Vasa signal was positioned in one half of round spermatids and concentrated in one to three small dots (Fig. 3E). These dots are reminiscent of the ring-formed chromatoid body (CB) described in spermatids of diverse animals including mouse (Shang et al., 2010), revealing for the first time that Vasa is a component of CB in medaka. Therefore, Vasa is expressed in mitotic, meiotic and postmeiotic male germ cells and shows dynamic subcellular distribution during spermatogenesis and spermiogenesis. 3.4. Vasa expression during spermatogenesis by electron microscopy Male germ cell in the adult testis undergoes spermatogenesis and spermiogenesis, culminating in the production of mature sperm. Spermatogenesis includes mitosis of spermatogonia for self-renewal and proliferation and meiosis. Spermiogenesis is a postmeiotic process of cytodifferentiation. We examined Vasa expression first during spermatogenesis. To this end, the Vasa was detected by a nanogold-conjugated antibody on testicular ultrathin sections. This procedure in the absence
Fig. 5. Nanogold immunoelectron microscopy of germ plasm during spermiogenesis. Ultrathin sections of the adult medaka testis were incubated with αVas, stained with a nanogoldconjugated second antibody and analyzed by transmission EM. (A) Chemically fixed sample showing spermatids at different stages. (B and D) Round spermatids. (C and E) Elongating spermatids. (F–K) Elongated spermatids. (B′ and C′) Higher magnification of the area framed in (B) and (C) respectively. Gold particles (arrows) delineating the Vasa protein are concentrated in the area separating the nucleus and mitochondria. df, dense fiber; mt, mitochondria; nu, nucleus. Scale bars, 1 μm in (A); 0.5 μm in (B–M); 0.2 μm in (B′ and C′).
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of αVas did not produce nanogold particles that were distributed exclusively or preferentially in particular structures or areas of male germ cells on testicular ultrathin sections (Fig. S1), demonstrating the specificity of iEM procedure. In the presence of αVas, nanogold particles delineating the Vasa protein were found in male germ cells (Fig. 4). These nanogold particles were closely associated with mitochondria in male germ cells at various stages. Intriguingly, subcellular Vasa distribution varied when spermatogenesis proceeds. In spermatogonia, the nanogold particles were found in a cytoplasmic compartment of 2–5 μm in size, which is surrounded by mitochondria in spermatogonia (Fig. 4A and B). This architecture is reminiscent of MC. In primary spermatocytes, the nanogold particles became intermixed with mitochondria, forming IC (Fig. 4C). In secondary spermatocytes, the nanogold particles were located in areas that are separated from, and close to, mitochondrial aggregates (Fig. 4D). Hence, Vasa undergoes dynamic changes in subcellular distribution in mitotic and meiotic male germ cells.
3.5. Vasa expression during spermiogenesis by electron microscopy We then examined subcellular Vasa distribution during spermiogenesis. Transmission EM without immunostaining clearly revealed morphologies of spermatids at different stages of spermiogenesis (Fig. 5A). In round spermatids, the immediate meiotic product, nanogold particles were concentrated in the caudal pole, in which they were located in a cap-like CB, which was sandwiched by the nucleus and mitochondria
(Fig. 5B and D). In elongating and elongated spermatids, the nanogold particles were found in two compartments: One remained as the cap-like CB that turned out to be at the base of the forming and growing flagellum, and the other moved posteriorly to the developing midpiece and formed a second CB, a ring-like structure surrounding the dense fiber of the flagellum (Fig. 5C and E–K). Thus, Vasa shows subcellular redistribution during spermiogenesis and forms two distinct CBs in advanced spermatids.
4. Discussion In this study we have analyzed Vasa expression and subcellular distribution in adult medaka germ cells by light and electron microscopy. We show that Vasa expression is specific to germ cells of both sexes, similar to that reported for vasa RNA (Shinomiya et al., 2000; Xu et al., 2009). Specifically, Vasa differs not only at the level of expression at different stages of oogenesis and spermatogenesis but also in subcellular distribution in female and male germ cells, suggesting its dynamic redistribution during medaka gametogenesis. This expression pattern has similarly described for Vasa in gibel carp, zebrafish and grouper (Xu et al., 2005), and thus appears to be conserved in fish. A striking observation in this study is the localization of Vasa to CBs in round and elongated spermatids (Fig. 6A). Many germ genes' RNA and protein products are components of germ plasm (Xu et al., 2010). Germ plasm is a nucleoprotein complex
Fig. 6. Vasa protein in adult male medaka germ cells. (A) Immunofluorescence light microscopy. sg, spermatogonium; sc1 and sc2, primary and secondary spermatocytes; st, round spermatid; st2, elongated spermatid; sm, sperm. (B) Nanogold immunoelectron microscopy. FC, free cement; IC, intermitochondria cement; MC, mitochondrial cloud; nu, nucleus.
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and dynamic in composition and structure. Germ cells in diverse animal species develop two unique structures, namely the Balbinani body (BB) in female germ cells at the early oocyte stages and CB in male germ cells at the postmeiotic phase. Both BB and CB have been thought of as being sex- and stage-specific forms of germ plasm (Xu et al., 2010). In medaka, mRNAs of dazl (Xu et al., 2009), mitf1 and mitf2 (Li et al., 2013) are components of BB, whereas those of boule and vasa are not (Shinomiya et al., 2000; Xu et al., 2009). Strikingly, our IF analysis in this study reveals for the first time that Vasa is a component of CB but is absent in BB of medaka. These data clearly demonstrate an apparent difference between BB and CB in composition and support CB as a spermatid-specific form of germ plasm. Whether BB is the oocyte-specific germ plasm remains to be determined in the future. Polymorphic structures as a strand-like structure and amorphous fibrous body have been described for GP or nuage in medaka spermatogonia by standard transmission EM (Hamaguchi, 1993). In this study, we have analyzed medaka Vasa expression by nanogold iEM in adult male germ cells. We show for the first time in medaka that Vasa exhibit subcellular GP redistribution during spermatogenesis and spermiogenesis. Specifically, Vasa appears as MC in spermatogonia, transforms to IC in primary spermatocyte, forms FC in secondary spermatocytes via separation from mitochondria and culminates in two distinct CBs in spermatids (Fig. 6B). Since these features are reminiscent of those described for GP in mammalian spermatogenesis and spermiogenesis (Fawcett et al., 1970; Parvinen, 2005; Shang et al., 2010; Susi and Clermont, 1970), we conclude that Vasa is a GP component and its expression identifies dynamic GP redistribution and reorganization in medaka. Therefore, GP undergoes reorganization and thus forms varying fine structures during different stages of male germ cell development in medaka, which suggests that the mechanisms underlying GP structures and CB formation are highly conserved in diverse vertebrate species. CB has been extensively studied in mammals, where apparent differences have been reported in the number, shape, location and behavior of CB. According to Fawcett et al. (1970), a single CB as a ring appears around the anterior base of the forming flagellum in early elongating spermatids and moves to the caudal end of the forming middle piece in elongated spermatids. It has been reported in rat spermiogenesis that CB first appears as an arc around the base of the flagellum, and the bulk of its content subsequently condenses into a dense body and moves away from the nucleus and is fragmented (Susi and Clermont, 1970). A striking finding in this study is that GP is able to form two distinct CBs. One is present first in round spermatids where it appears as a cap or arc around the flagellum base, similar to the murine situation (Fawcett et al., 1970; Susi and Clermont, 1970). However, there is also a second CB, which is newly formed in elongating spermatids where it takes the shape of a ring around the dense fiber of flagellum in the midpiece. The presence of two CBs has recently been identified also by iEM in mouse, where a ring-shaped CB resides around the base of the flagellum and a satellite-like CB resides in the midpiece of elongating spermatids (Shang et al., 2010). Consequently, the presence of two CBs may be a conserved feature of spermatogenesis from fish to mammals. It has been reported that a premature loss of the CB-derived ring structure is associated with male infertility due to production of abnormal sperm (Shang et al., 2010). Our finding that Vasa is an integral component of both GP and CBs suggests an important role for this protein also in postmeiotic cytodifferentiation and flagellum formation. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.05.017.
Conflict of interest The authors declare that there is no conflict of interest.
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