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Insect water-specific aquaporins in developing ovarian follicles of the silk moth Bombyx mori: Role in hydration during egg maturation.

Abstract. Egg formation in terrestrial insects is an absorptive process, accommodated not only by packing proteins and lipids into yolk but also by filling chorions with water. An osmotic swelling of ovarian follicles takes place during oocyte maturation. This study investigated the role of the aquaporin (AQP) water channel in the osmotic uptake of water during oogenesis in the silk moth Bombyx mori Linnaeus, 1758. Using the antibodies that specifically recognize previously characterized AQPs, two water-specific subtypes--AQP-Bom1 and AQP-Bom3--belonging to the Drosophila integral protein (DRIP) and Pyrocoelia rufa integral protein (PRIP) subfamilies of the insect AQP clade, respectively, were identified in the developing ovaries of B. mori. During oocyte growth, Bombyx PRIP was distributed at the oocyte plasma membrane, where it likely plays a role in water uptake and oocyte swelling, and may be responsible for oocyte hydration during fluid absorption by ovarian follicles. During the transition from vitellogenesis to choriogenesis during oocyte maturation, Bombyx DRIP expression became abundant in peripheral yolk granules underlying the oocyte plasma membrane. The restricted DRIP localization was not observed in non-diapause-destined follicles, where DRIP was evenly distributed in medullary yolk granules. There was no difference in PRIP distribution between diapause- and non-diapause-destined follicles. The diapause-destined oocytes encase DRIP protein in the peripheral yolk granules, where DRIP might be inert. This would be reflected in the metabolic arrest associated with diapause after fertilization and egg oviposition.


Egg formation in insects involves vitellogenesis, which is characterized by yolk accumulation in developing oocytes and the import of vitellogenin, a female-specific hemolymph protein produced by the fat body (insect liver) and endocytosed by oocytes (Telfer, 2009). A series of pioneering studies elucidated the cellular basis of yolk accumulation in the ovarian follicle of the giant silk moth Hyalophora cecropia Linnaeus, 1758 (Telfer, 1960; Telfer and Rutberg, 1960; Telfer and Anderson, 1968). The absorption of fluids (including ions and water) accompanies protein and lipid uptake, leading to an increase in oocyte mass (Wang and Telfer, 1998). This osmotic swelling is caused by the activity of the [H.sup.+]-translocating vacuolar-type ([H.sup.+] V-) ATPase, a proton pump that establishes the electrochemical proton gradient in ovarian follicles (Janssen et al., 1995; Harvey et al., 1998).

Water transport across membranes is facilitated by aquaporins (AQPs), a type of integral membrane protein ubiquitously expressed in living organisms (Preston et al., 1992; Carbrey and Agre, 2010). In addition to transcellular water transport across the plasma membrane of epithelial and endothelial barriers, AQPs have been implicated in cell migration, proliferation, and volume regulation (Verkman, 2011). Insects generally survive on land and must preserve body water content. This subject has long been studied in the transport mechanisms underlying fluid secretion (O'Donnell and Maddrell, 1983; Maddrell, 2009), and recent research has focused on the role of AQPs in water movement and osmoregulation (Azuma et al., 2012).

Our previous phylogenetic analyses based on bioinformatic predictions of insect AQPs revealed four distinct subgroups (Kambara et al., 2009). Group 1 AQPs include Drosophila integral protein (DRIP) and Pyrocoelia rufa integral protein (PRIP) subfamilies, both of which are widespread across class Insecta (Azuma et al., 2012; Ishida et al., 2012; Nagae et al., 2013: Kambara et al., 2014). They share a common protein structure comprising six transmembrane domains (I-VI) connected by five loops (A-E), including two signature Asn-Pro-Ala (NPA) motifs at loops B and E to restrict proton conductance in the channel. Group 3 AQPs are heterogeneous, and some have been characterized as insect aquaglyceroporins that transport glycerol in addition to water (Kataoka et al., 2009a, b; Wallace et al., 2012); and Groups 2 and 4 are known as big brain protein and superaquaporins, respectively (Ishibashi, 2006; Ishibashi et al., 2014).

The physiological functions of Group 1 AQPs have been characterized primarily in liquid-feeding insects such as the green leafhopper (Le Caherec et al., 1996; 1997), buffalo fly (Elvin et al., 1999), yellow fever mosquito (Pietrantonio et al., 2000; Duchesne et al., 2003; Drake et al., 2010), pea aphid (Shakesby et al., 2009), whitefly (Mathew et al., 2011), and pest bugs (Staniscuaski et al., 2013; Fabrick et al., 2014). In each of these species, DRIP is distributed in the alimentary tract (mid- and hindgut) and Malpighian tubules (insect kidney). Mosquitoes also express PRIP in digestive and excretory tissues (Drake et al., 2010; Liu et al., 2011; Marusalin et al., 2012; Tsujimoto et al., 2013). Both types of AQPs are water-specific and are presumed to enable small insects to extrude excess amounts of water or avoid salt overload through dietary intake.

Tissue-specific expression and the functional relationship between DRIP and PRIP were demonstrated in our study of the foliage-feeding domesticated silkworm Bombyx mori (Azuma et al., 2012). DRIP and PRIP at the apical and basal plasma membranes, respectively, act in concert to provide a major route for transcellular water movement in hindgut epithelial cells. Efficient water recovery through the hindgut wall enables larvae to circumvent water loss and tolerate desiccation and starvation (Kataoka et al., 2009a; Azuma et al., 2012).

The involvement of PRIP in egg formation has been suggested by studies in the malaria vector mosquito Anopheles gambiae. After a blood meal by female adults, mRNA expression of AgAQP1, the gene encoding PRIP, is upregulated in the fat body and ovary (Liu et al., 2011); the ovarian PRIP-type gene BgAQP1 has also been cloned in the cockroach Blattella germanica Linnaeus, 1767 (Herraiz et al., 2011). Although these reports provide evidence of PRIP mRNA expression in ovarian development, protein nature and function remain poorly characterized. In Anopheles, the splice variants AgAQP1A and AgAQP1B were independently expressed in the ovary and alimentary tract, respectively (Tsujimoto et al., 2013). Water is presumed to accompany the transepithelial movement of ions ([H.sup.+], [K.sup.+], [Na.sup.+], [Cl.sup.-]), thereby resulting in oocyte hydration; however, the precise functions of PRIP and DRIP in insect oogenesis remain unknown.

To address this question, we examined the temporal and spatial distributions of PRIP (AQP-Bom3) and DRIP (AQP-Bom1) in the ovarian follicle of B. mori. In this species, eggs are formed during pupal-adult metamorphosis rather than after eclosion, similar to H. cecropia. The role of AQPs in oocyte hydration was therefore investigated during the critical period between vitellogenesis and choriogenesis preceding adult emergence. A crucial difference between B. mori and H. cecropia in terms of egg development is that in the former, eggs enter diapause at an early embryonic stage after fertilization and around 24 h after oviposition, when the arrest of cell division in the embryo is induced by the diapause neuropeptide hormone (Yamashita, 1996), leading to diapause egg formation. In contrast, non-diapause egg formation occurs in the absence of this hormone, and embryogenesis begins just after oviposition. The present findings reveal distinct functions for B. mori DRIP (formerly AQP-Bom1), depending on this hormonal state.

Materials and Methods

Insects and tissues

Two hybrid strains of silk moth (Shunrei x Shogetsu and Kinshu x Showa) were used in this study. The silkworms were reared at 24 to 26 [degrees]C and maintained on an artificial diet as previously described (Miyake and Azuma, 2008; Kataoka et al., 2009a; Azuma et al., 2012). The final (5th) larval instar lasts 7 d for feeding before larvae enter the wandering and spinning phase (larval-pupal metamorphosis); pupal-adult development lasts 12 d at 25 [degrees]C.

Developmentally and physiologically distinct phases were examined by dissecting ovarioles from pharate adults 6 d and 10 d after larval-pupal ecdysis in phosphate-buffered saline (PBS; 0.15 mol [1.sup.-1] NaCl, 10 mmol [1.sup.-1] sodium phosphate buffer, pH 7.4). In this report, the former stage is referred to as the vitellogenic phase, or Day 6 ovary, and the latter as the choriogenic phase, or Day 10 ovary. In a preliminary experiment, each ovariole in the vitellogenic phase was divided in half to determine the wet weight for early (premature) follicle and fully vitellogenic (terminal 20) follicle segments (Fig. 1). The precise staging of follicles was confirmed by staining with vital dye (Telfer and Anderson, 1968) consisting of 50 [micro]l of 1% (w/v) Trypan Blue dissolved in PBS, which was injected into female pupae prior to dissection. Vitellogenic follicles incorporated the dye into oocytes, giving the follicle a blue color, in contrast to colorless post-vitellogenic oocytes that did not retain the dye (Fig. 1A). For membrane separation studies, ovarioles were similarly collected in homogenization buffer consisting of 0.3 mol [1.sup.-1] NaCl, 0.3 mol [1.sup.-1] mannitol, 5 mmol [1.sup.-1] ethylenediamine-N,N,N',N'-tetra acetic acid (EDTA), and 10 mmol [1.sup.-1] 2-[4-(2-hydroxyethyl)-l-piperazinyl] ethanesulfinic acid (FIEPES)-NaOH (pH 7.5). Aliquots of ovaries (1 or 2 g wet weight) were collected and stored at -30 [degrees]C until use.

The hybrid moth strains used in this study produce diapause eggs under the control of the diapause hormone (Yamashita, 1996). To obtain non-diapause-destined eggs, the suboesophageal ganglion (SOG), which is the diapause hormone-secreting endocrine organ, was surgically extirpated with fine forceps on the day of pupation, when the pupal cuticle was a light brown color. This operation had no effect on ovarian development (i.e., the size or number of eggs) or on other adult tissues, and did not delay eclosion. Ovarian follicles are destined for non-diapause under the diapause hormone-free condition during oogenesis, when all eggs that are laid will immediately proceed to embryogenesis. Day 6 and Day 10 ovaries from SOG-removed pupae were collected as described above.

Membrane fractionation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting

Developing follicles accumulate large amounts of yolk protein, some of which is packaged in yolk granules at different densities. In contrast to the major yolk proteins, AQP is an integral membrane protein and a minor constituent of the total protein content of ovarian extracts. Thus, the AQP-enriched membrane fraction was isolated by sucrose density gradient centrifugation. Furthermore, since yolk proteins such as vitellin and lipophorin readily aggregate under low ionic conditions, organellar membranes of a reasonable quality were isolated by including 0.3 mol [1.sup.-1] NaCl at each step (Azuma and Yamashita, 1985).

Several membrane fractions were prepared depending on their densities using sucrose instead of sorbitol (Fig. 1B). Briefly, 1 or 2 g of vitellogenic (Day 6) or choriogenic (Day 10) ovaries from female pupae were homogenized with a Teflon glass homogenizer in 10 volumes (w/v) of 10 mmol [1.sup.-1] HEPES-NaOH buffer (pH 7.5) containing 0.3 mol [1.sup.-1] NaCl, 0.3 mol [1.sup.-1] mannitol, and 5 mmol [1.sup.-1] EDTA. To inhibit serine and cysteine proteinase activities, a complete, EDTA-free proteinase inhibitor cocktail mini-tablet (Roche Diagnostics GmbH, Mannheim, Germany) was added to the buffer solution (one tablet/10 ml). The 10% (w/v) homogenate was first centrifuged at 1,000 x g ([R.sub.max]) for 10 min and the supernatant was centrifuged at 100,200 x g ([R.sub.av]) for 120 min. The pellet was resuspended with the same buffer using the Teflon glass homogenizer. This membrane suspension (approximately 1 ml) was layered onto discontinuous gradients consisting of 20%, 30%, and 40% (w/v) sucrose (2.7 ml each) and 2.5 ml of 50% (w/v) sucrose plus 0.3 mol [1.sup.-1] NaCl, 5 mmol [1.sup.-1] EDTA, and 10 mmol [1.sup.-1] HEPES buffer (pH 7.5) with proteinase inhibitors and centrifuged at 93,900 x g ([R.sub.av]) for 18 h at 4 [degrees]C in a swinging-bucket rotor of a Model RPS-40T ultracentrifuge (Hitachi, Tokyo, Japan). After centrifugation, three whitish bands (at the 20%/30%, 30%/40%, and 40%/50% interfaces, referred to as Bands 2, 3, and 4, respectively) were collected and pooled in a manner similar to hindgut preparations (Azuma et al., 2012). Additionally, the upper 20% sucrose zone beneath the sample-loaded layer was collected as Band 1 (Fig. 1B). Given that the basal plasma membrane of hindgut epithelial cells was recovered in Band 2 (Azuma et al., 2012), it was predicted that ovarian Band 2 preparations would contain the basal plasma membrane of follicular epithelial cells and the oocyte plasma membrane. Other bands in the sucrose zone with certain organellar membranes were not identified, because the developing ovaries are differentiating tissues with various densities of yolk granules. A part of each fraction was used to determine protein concentration with the Bradford Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA) using bovine [gamma]-globulin as the standard.

Four different membrane fractions (20 [micro]g protein) were solubilized and mildly denatured by incubation at 37 [degrees]C for 60 min in a buffer containing 62.5 mmol [1.sup.-1] tris (hydroxymethyl) aminomethane (Tris)-HCl (pH 6.8), 10% glycerol, 2% SDS, and 5% 2-mercaptoethanol. Treated samples (20 [micro]g protein per lane) were separated with SDS-PAGE using Mini-PROTEAN TGX Precast Gels (12%, Bio-Rad Laboratories, Inc.). Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Trans-Blot Turbo Transfer System Transfer Pack; Bio-Rad Laboratories, Inc.) and processed for immunodetection using the enhanced chemiluminescence (ECL) Western Blotting Analysis System (GE Healthcare, Buckinghamshire, UK). After blocking the membrane for 2-3 h with 5% ECL blocking reagent dissolved in PBS with 0.1% (w/v) Tween 20 (PBST), the membranes were incubated overnight at 4 [degrees]C with immunoglobulin (Ig)G preparations from anti-(AQP-Bom3) antiserum, which was raised against the C-terminal sequence, C+[E.sub.225]KELKRLDGGKLD[D.sub.268]. For AQP-Bom1 (DRIP) detection, rabbit polyclonal antibodies were raised against a synthetic peptide corresponding to part of the most hydrophilic D loop region of the protein, namely amino acid residues 165-179 ([C.sub.165][DPQRNDLKGSAPLA.sub.179]) (Azuma et al., 2012). The IgG fraction was passed through a Protein G HP SpinTrap column (GE Healthcare) to reduce nonspecific binding of the antibody to the PVDF membrane.

The IgG fraction was diluted 1:1000 in PBS containing 3% bovine serum albumin (A7030, minimum 98%; Sigma, St Louis, MO) to block non-specific binding sites. Following three 10-min washes with PBST, membranes were incubated for 2-3 h with ECL horseradish peroxidase-conjugated anti-rabbit IgG (from donkey) diluted 1:10,000 in PBST. After washing the membranes with PBST, specifically bound antibodies were incubated for several minutes in the ECL detection reagent (GE Healthcare). Images were acquired using the luminescent image analyzer (LAS-4000 mini; Fujifilm Corp., Tokyo, Japan) according to the manufacturer's instructions. An apparent molecular mass was estimated using 15-150 kDa ECL DualVue Western Blotting Markers (GE Healthcare) as a reference and was calibrated with Multi Gauge software v.3.2 (Fujifilm Corp.). In control experiments, an absorption test was performed by incubating the antibody (IgG fraction) with the antigen peptide (500 [micro]g [ml.sup.-1]) to verify the specificity of the immunoreaction. The IgG fraction preabsorbed with the antigen peptide was processed as described above.

Immunohistochemistry of Bombyx PRIP (AQP-Bom3) and DRIP (AQP-Bom1)

Immunohistochemistry was performed as described in our previous studies (Miyake and Azuma, 2008; Azuma et al., 2012; Nagae et al., 2013). Ovarioles from Day 6 (terminal 20 follicles) or Day 10 female pupae were dissected in PBS and immediately placed in Bouin's fixative on ice for 6-8 h depending on tissue mass and follicular stage. The specimens were then dehydrated through a graded ethanol series, cleared, and embedded in Histosec pastilles (without dimethylsulfoxide) (Merck KGaA, Darmstadt, Germany). Sections about 5 [micro]m thick were cut and collected on 3-aminopropyltriethoxysilane-coated glass slides (Matsunami Glass Ind., Ltd., Tokyo, Japan) and dried at 40 [degrees]C overnight. Sections were then deparaffinized in xylene and rehydrated through an ethanol series with PBS as the final solution. Sections were incubated with 10% normal goat serum (NGS) in PBS for 2-4 h at room temperature in a humid chamber, followed by the IgG fraction of each primary antibody (diluted at 1:100 with 10% NGS) at 4 [degrees]C overnight to reduce nonspecific staining. After four 10-min washes in PBS, sections were incubated for 2-3 h with Alexa Fluor 488 goat anti-rabbit IgG (H+L) or Alexa Fluor 555 goat anti-rabbit IgG (H+L) (Molecular Probes, Eugene, OR) diluted 1:1000 with PBS. Following four PBS washes, sections were mounted in ProLong Gold antifade reagent with 4',6-diamidino-2-phenylindole (Molecular Probes). In some experiments, sections were treated in succession with biotinylated anti-rabbit secondary antibody and avidin-biotinylated peroxidase complex regent (VECTASTAIN Elite ABC kit; Vector Laboratories, Burlingame, CA). Reaction products were observed as deposits of 3,3'-diaminobenzidine (DAB). The sections were observed under a BX51 microscope (Olympus Optical Co., Tokyo, Japan) equipped with a differential interference contrast device and a BX-epifluorescence attachment (Olympus), and images were captured with a DP-70 digital camera and cellSens imaging software v. 1.4.1 (Olympus).

Follicle/oocyte swelling test

Vitellogenic ovarioles were dissected from Day 6 pupae with modified Barth's saline (MBS) consisting of 0.33 mmol [1.sup.-1] Ca[([NO.sub.3]).sub.2], 0.4 mmol [1.sup.-1] Ca[Cl.sub.2], 88 mmol [1.sup.-1] NaCl, 1 mmol [1.sup.-1] KCl, 2.4 mmol [1.sup.-1] NaH[CO.sub.3], 0.82 mmol [1.sup.-1] MgS[O.sub.4], and 10 mmol [1.sup.-1] HEPES (pH 7.5). A chain of 10 follicles with an ovariole sheath was collected from the most terminal to Trypan Blue-stained follicle (Fig. 1A). Selected ovarioles were immersed in 0.1% Type II collagenase (Gibco/Invitrogen, Carlsbad, CA) dissolved in MBS without 0.4 mmol [1.sup.-1] Ca[Cl.sub.2] for 30 min at room temperature (20 [degrees]C-25 [degrees]C). After several changes of MBS, follicles with an ellipsoidal shape (> 1 mm in longer diameter) were released from the ovariole sheath. Using a Pasteur pipette with an inner diameter slightly larger than the size of a follicle, an intact follicle was placed in the well of a [Nunclon.sup.TM] Surface 60-well cell culture mini-tray (Nunc A/S, Roskilde, Denmark) filled with 50 [micro]l of 10-fold diluted MBS (1/10 MBS). The follicle was monitored and imaged with a DP-25 charge-coupled device camera (Olympus) attached to an Olympus SZX10 stereo microscope at room temperature (20 [degrees]C-25 [degrees]C) up to 60 min. Surrounding follicular epithelial cells were partially removed by repeated pipetting; however, harsh pipetting or stronger collagenase treatment frequently rendered oocytes very fragile, causing leakage of oocyte contents. Impaired oocytes were removed; those that were intact (i.e., with a significant amount of follicular epithelia still attached) were used for experiments. Since mercuric chloride (at about 1 mmol [1.sup.-1] Hg[Cl.sub.2]) blocks water channel function, 0.9 mmol [1.sup.-1] Hg[Cl.sub.2] was included in the 1/10 MBS solution in some experiments. In contrast to the Xenopus oocyte AQP swelling assay (Preston et al., 1992) that takes 2-3 min due to the puncture of the oocyte. 60 min was typically required to obtain an intact oocyte in the present experiment. Since changes in shape depend on adherent follicular epithelia, quantitative analyses could not be carried out.


A PRIP-type AQP (AQP-Bom3) is localized at the oocyte plasma membrane in vitellogenic ovaries of Bombyx mori

Recent reports have suggested that PRIP-type AQPs are important for oocyte development irrespective of insect ovary structure (panoistic or meroistic) (Herraiz et al., 2011; Liu et al., 2011). The prevalence of AQP-Bom3, a PRIP-type AQP was examined in the B. mori ovary by membrane fractionation and immunoblotting. Trypan Blue staining revealed that, with the exception of several terminal follicles, most follicles in each ovariole of Day 6 female pupae were in the fluid-absorbing growth or vitellogenic phase (Fig. 1A). Day 6 ovarioles were classified as early or fully vitellogenic follicles, and whole ovarioles from Day 10 female pupae were also examined. There were no differences among the three different preparations in terms of banding pattern in the sucrose density gradient (Fig. 1B).

Membrane fractions were analyzed by SDS-PAGE and immunoblotting with an antibody recognizing the C-terminus of AQP-Bom3. The antibody reacted strongly with a single band of molecular mass 109 kDa in the 20%/30% sucrose interface membrane fraction (Fig. 1C), indicating the oligomeric nature of insect PRIP subfamily members and suggesting the presence of a homotetrameric AQP-Bom3 in vitellogenic and choriogenic ovaries. Notably, AQP-Bom3 monomers (theoretically 27.7 kDa; Azuma et al., 2012) were not detected in the ovarian extracts, in contrast to basal plasma membrane preparations recovered from the 20%/30% sucrose interface fraction of hindgut extracts (Azuma et al., 2012).

Immunoblotting revealed that the PRIP-type AQP was constitutively expressed during oogenesis in specific organellar membranes. The subcellular localization of AQP-Bom3 in developing follicles was assessed by immunohistochemistry. Specific signals were weakly detected at the oocyte surface in early vitellogenic follicles (Fig. 2A, 2C), but immunoreactivity around the oocyte surface was intensified in fully vitellogenic follicles (Fig. 2B). At higher magnification (Fig. 2D), PRIP was distributed at the oocyte plasma membrane and was absent in nurse and follicular epithelial cells. Patency between follicular epithelial cells was evident by immunohistochemistry and bright field microscopy (Fig. 2E). No immunoreactivity was observed when the preimmune rabbit serum was used (Fig. 2F). A PRIP signal was detected at the oocyte plasma membrane in choriogenic follicles, albeit at reduced intensity (Fig. 2G, 2H), in contrast to the immunoblotting data (Fig. 1C). The difference may be explained by the variation in detection sensitivity between the experimental methods.

Partially naked oocytes were prepared by incubating fully vitellogenic follicles with collagenase in order to examine actual swelling under hypo-osmotic conditions. Although follicular epithelial cells were partially removed, oocytes gradually swelled and changed shape; this effect was abolished in the presence of 0.9 mmol [1.sup.-1] Hg[Cl.sub.2], a potent AQP inhibitor (Fig. 3). While various types of membrane protein likely function at the oocyte plasma membrane, these results indicate that water uptake by developing oocytes occurs through AQP-Bom3 (PRIP).

A DRIP-type AQP (AQP-Bom1) is localized at the oocyte cortex in choriogenic ovaries of Bombyx mori

AQP-Bom1 (DRIP) is another water-specific AQP that has been characterized in B. mori (Miyake and Azuma, 2008; Kataoka et al., 2009a). AQP-Bom1 was detected in membrane preparations from developing follicles of Day 6 female pupae in the upper 20% sucrose zone (Band 1) containing lighter organellar membranes than the oocyte plasma membrane fraction (Band 2). A band at approximately 27.8 kDa was observed that potentially represented a monomeric form of AQP-Bom1 (Kataoka et al., 2009a). The intensity of the monomer band was greater in the same density fraction (Band 1) prepared from mature follicles of Day 10 female pupae (Fig. 4A, right panel). The 27.8-kDa band was scarcely visible when anti-(AQP-Bom1) antibody was preabsorbed with the peptide used as an immunogen. Minor bands at 57.0 kDa and 82.4 kDa were likely nonspecific, since they were still present after preincubation of the antibody with the synthetic peptide (Fig. 4B, lane 1). The 57.0-kDa band seen in Band 2 was also nonspecific (Fig. 4B, lane 2). These data indicate that the expression of AQP-Bom1 (DRIP) increases during oocyte maturation.

DRIP expression was negligible in vitellogenic follicles, as observed by immunohistochemistry (Fig. 5A, 5D). A strong positive signal was observed around the oocyte cortex in choriogenic follicles (Fig. 5B), in which the protein was localized in granules underlying the oocyte plasma membrane (Fig. 5E). Follicular epithelial cells also showed a weak positive signal at the basal surface. Immunoreactivity was lost when the anti-(AQP-Bom1) antibody was preabsorbed with the peptide used as an immunogen (Fig. 5H), confirming the specificity of the signal.

The DRIP-type AQP is not assembled around the oocyte cortex in non-diapause-destined oocytes

B. mori females produce diapause eggs that survive over 6 months after oviposition (even at 25 [degrees]C); in contrast, non-diapause eggs undergo embryogenesis and larval hatching within 2 weeks of oviposition (Yamashita and Yaginuma, 1991; Yamashita, 1996). The latter phenotype was induced by surgically removing the SOG in female pupae and comparing DRIP distribution to that of diapause-destined oocytes. DRIP expression was not detected in the granules underneath the oocyte plasma membrane in non-diapause-destined oocytes (Fig. 5C, 5F). Immunoreactivity was observed throughout medullary yolk granules, but was absent in those granules surrounding the cortical region. Immunolabeling with DAB confirmed the presence of DRIP in yolk granules but not at the cortex (Fig. 51), while control staining with the preabsorbed immunogen showed no signal (data not shown). DRIP expression in non-diapause-destined oocytes from the Day 6 ovary was also similarly weak (Fig. 5G). There was no difference in PRIP expression between the diapause-destined (Fig. 2) and non-diapause-destined oocytes from the Day 6 ovary (Fig. 5J, 5K). In contrast to DRIP distribution in mature oocytes, PRIP expression showed the same localization irrespective of the hormonal milieu produced by the SOG. It may be reasonable to conclude that the diapause hormone produces no difference in the size of the matured egg or the number of eggs through oogenesis, increasing an oocyte mass in both destinies (diapause or non-diapause).

Given that the recovery of 27.8-kDa DRIP protein from the Band 1 fraction was similar for diapause- and non-diapause-destined ovaries from Day 10 pupae (Appendix), it is likely that the diapause hormone affects DRIP distribution and localization in yolk granules only during Bombyx egg development (shown in the schema in Fig. 5).


Bombyx mori and Hyalophora cecropia have polytrophic-meroistic ovaries, in which each follicle is a functional developing unit consisting of one oocyte and an anterior cap-forming cluster of seven nurse cells of germ cell origin encapsulated by a monolayer of thousands of follicular epithelial cells. The development of each follicle reaches a plateau after the vitellogenic phase before undergoing oocyte maturation (choriogenic phase), leading to the formation of the vitelline envelope/membrane, which is followed by chorion deposition (Swevers et al., 2005; Telfer, 2009) and the construction of an elaborate, watertight structure that protects the eggs from dehydration (Woods, 2010). The present study characterized the temporal and spatial distributions of Bombyx PRIP (AQP-Bom3) and DRIP (AQP-Bom1) in relation to their different physiological importance during oocyte maturation.

During the vitellogenic phase, the ample extracellular space between follicular epithelial cells is visible by microscopy. Water and many solutes, therefore, can readily access the oocyte surface when the surface area becomes enlarged for massive fluid absorption. At this stage, AQP-Bom3 (PRIP) is localized at the oocyte plasma membrane where [H.sup.+] V-ATPase is also present, as observed in the hawk moth Manduca sexta Linnaeus, 1763 (Janssen et al., 1995). During the post-vitellogenic (choriogenic) phase, follicular epithelial cells surrounding the oocyte form a tight epithelium, creating an epithelial diffusion barrier (Wang and Telfer, 1997). Although AQP-Bom3 (PRIP) was expressed even during the choriogenic phase, it is unlikely that it continues to function in water transport through the transcellular route across follicular epithelia. The downregulation of [H.sup.+] V-ATPase expression in M. sexta oocytes (Janssen et al., 1995) and the weak PRIP immunoreactivity at the oocyte plasma membrane observed here suggest that water transport concludes prior to egg maturation. Moreover, the end of vitellogenesis is followed by a large uptake of water by oocytes during a brief period just prior to choriogenesis (Telfer and Rutberg, 1960; Telfer and Anderson, 1968; Wang and Telfer, 1998). Thus, AQP-Bom3 (PRIP) functions in water transport during vitellogenesis immediately before choriogenesis and plays a role in hydration and osmotic swelling of oocytes.

Ovarian AQP--specifically PRIP subfamily members--is expressed in the cockroach Blattella germanica (BgAQP1, Herraiz et al., 2011) and the mosquito Anopheles gambiae (AgAQP1, Liu et al., 2011). The transcript of AgAQP1A--an ovary-specific variant--was restricted to the female ovary, while the protein was detected in the oviduct but not in oocytes, although precise subcellular localization was unclear (Tsujimoto et al., 2013). Although mosquitoes also possess polytrophic-meroistic ovarioles, the ovarian function of PRIP likely differs in A. gambiae and in moths. In B. germanica, PRIP (BgAQP1) is expressed predominantly in mature oocytes. This PRIP is unique in that it transports modest amounts of urea (but not glycerol) in addition to water, and mRNA is rarely expressed in the gut and Malpighian tubules of adult females (Herraiz et al., 2011). The negligible phenotypic effects of RNA interference imply the presence of another AQP in oocyte development of B. germanica. Compared to the previous ovarian PRIP studies, we are able to provide a clearer picture of the function of AQP in insect egg physiology.

The DRIP-type AQP (AQP-Bom1) was identified and characterized as another functional AQP in choriogenic oocytes that is localized to a subset of yolk granules around the oocyte cortex. At this stage of development (2 days before eclosion), all pre-fertilization oocytes are already destined for diapause egg formation (Yamashita and Yaginuma, 1991). The significance of DRIP-containing peripheral granules is uncertain, but peripheral packaging or assembly may be related to developmental arrest (quiescence). In contrast, when all oocytes are in the diapause hormone-free state, the peripheral assembly of DRIP-positive yolk granules is not observed. We suggest that the egg mass, including yolk granules, is ready to use for embryonic development immediately after oviposition. Diapausing eggs such as those seen in B. mori circumvent evaporative water loss by limiting gas exchange and reducing metabolic activity (Yamashita and Yaginuma, 1991). Exploring the fate of DRIP in diapausing eggs will shed light on additional AQP function(s) in insect egg diapause, given that diapause-destined eggs are sufficiently watertight to protect them from desiccation during their long period of survival.


The authors thank Professors Jun Kobayashi (Yamaguchi University) and Yoshihisa Ozoe (Shimane University) for their continuous encouragement throughout this study. This work was supported by Grants-in-Aid for Scientific Research (nos. 20380035, 23580071, and 25292108) from the Japan Society for the Promotion of Science (JSPS). This paper is dedicated to the late William H. Telfer (Emeritus Professor of Biology, University of Pennsylvania) who conceptualized water uptake by oocytes, which was published in The Biological Bulletin.

Literature Cited

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Immunoblotting of AQP-Bom1 (DRIP) in membrane fractions prepared from choriogenic follicles from diapause-destined and non-diapause-destined pupae of the silk moth Bombyx mori. Lanes 1, 2, 3, and 4 represent membrane fractions recovered from Bands 1, 2, 3, and 4, respectively (see Fig. 1B). A 27.8-kDa polypeptide representing the AQP-Bom1 was detected in Band 1 from both preparations. The 57.8-kDa polypeptide is nonspecific. Mr, protein marker (150, 100, 75, 50, 35, 25, and 15 kDa).


(1) Laboratory of Insect Physiology, The United Graduate School of Agricultural Sciences, Tottori University; and (2) Faculty of Agriculture, Tottori University, Koyama-cho, Minami 4-101, Tottori 680-8553, Japan

Received 2 October 2014; accepted 13 January 2015.

(*) To whom correspondence should be addressed. E-mail:

([dagger]) Current address: Wood Protection Laboratory, Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan.

Abbreviations: AQP, aquaporin; DAB, 3,3'-diaminobenzidine, tetrahydrochloride; DRIP, Drosophila integral protein; ECL, enhanced chemiluminescence; EDTA, ethylenediamine-N,N,N',N'-tetra acetic acid; HEPES, 2-[4-(2-hydroxyethyl)-l-piperazinyl] ethanesulfinic acid; IgG, immunoglobulin G; MBS, modified Barth's saline; NGS, normal goat serum; PBS, phosphate-buffered saline; PRIP, Pyrocoelia rufa integral protein; PVDF, polyvinylidene difluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SOG, suboesophageal ganglion; Tris, tris (hydroxymethyl) aminomethane.
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Author:Maruyama, Mariya; Kambara, Kohei; Naka, Hideshi; Azuma, Masaaki
Publication:The Biological Bulletin
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Date:Aug 1, 2015
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