Tomohiro Sasanami

Effects of Testosterone on Production of Perivitelline Membrane Glycoprotein ZPC by Granulosa Cells of Japanese Quail (Coturnix japonica)

Abstract Avian perivitelline membrane, an investment homologous to the zona pellucida of mammalian oocytes, is composed of at least two glycoproteins. Previous studies have indicated that one of the components, a glycoprotein homologous to mammalian ZPC, is produced and secreted by the granulosa cells of developing follicles of the chicken ovary. In the present study, we evaluated the expression and regulation of ZPC in Japanese quail (Coturnix japonica) granulosa cells both in vivo and in vitro. Western blot analysis of the SDS-solubilized granulosa layer using anti-quail ZPC antiserum showed that the amount of ZPC increased in parallel with follicular development. Northern blot analysis of total RNA using cDNA of quail ZPC showed that the increase in mRNA expression was also correlated with follicular development. To investigate the regulation of ZPC production, the granulosa cells were cultured in a medium containing steroid hormones such as progesterone, estradiol-17β, or testosterone. By measuring ZPC protein and mRNA with Western and Northern blot analyses, respectively, we found that addition of testosterone maintained ZPC contents in the culture of the granulosa cells, and that ZPC mRNA expression was high in the culture with testosterone compared to the control. These results suggest that testosterone stimulates ZPC protein production at the gene transcription level.

Sperm Storage in the Female Reproductive Tract in Birds

Abstract The ability to store sperm in the female genital tract is frequently observed in vertebrates as well as in invertebrates. Because of the presence of a system that maintains the ejaculated sperm alive in the female reproductive tract in a variety of animals, this strategy appears to be advantageous for animal reproduction. Although the occurrence and physiological reasons for sperm storage have been reported extensively in many species, the mechanism of sperm storage in the female reproductive tract has been poorly understood until recently. In avian species, the specialized simple tubular invaginations referred to as sperm storage tubules (SSTs) are found in the oviduct as a sperm storage organ. In this review, we summarize the current understanding of the mechanism of sperm uptake into the SSTs, maintenance within it, and controlled release of the sperm from the SSTs. Since sperm storage in avian species occurs at high body temperatures (i.e., 41 C), elucidation of the mechanism for sperm storage may lead to the development of new strategies for sperm preservation at ambient temperatures, and these could be used in a myriad of applications in the field of reproduction.

Progesterone Is a Sperm-Releasing Factor from the Sperm-Storage Tubules in Birds

Because of the presence of sperm-storage tubules (SST) in the utero-vaginal junction (UVJ) in the oviduct, once ejaculated sperm have entered the female reproductive tract, they can survive for a prolonged time in domestic birds, although the specific mechanisms involved in the sperm uptake into, maintenance within, and controlled release from the SST remain to be elucidated. In this report, we provide evidence that progesterone triggers the release of the resident sperm from the SST in the UVJ. The ultrastructural observation of the SST indicated that the resident sperm are released from the SST around 20 h after oviposition. When laying birds were injected with progesterone, most of the sperm were released from the SST within 1 h of injection. In situ hybridization analyses demonstrated the presence of the transcripts of membrane progestin receptor α in the UVJ, and the translated proteins were detected in the UVJ extracts by Western blotting. Moreover, the number of secretory granules in the SST epithelial cells fluctuates during the ovulatory cycle, and the progesterone administration mimics this phenomena. A binding assay using [(3)H]-progesterone indicated the presence of a high affinity, limited capacity, saturable and single binding site for [(3)H]-progesterone in the membrane fraction of the UVJ, and this receptor did not interact with the synthetic antiprogestin RU486. These results demonstrated for the first time that the progesterone stimulates the release of the resident sperm from the SST and that the release of the sperm might occur via membrane progestin receptor α-mediating signal transduction.

Induction of sperm acrosome reaction by perivitelline membrane glycoprotein ZP1 in Japanese quail (Coturnix japonica)

The extracellular matrix surrounding avian oocytes, called the perivitelline membrane (PL), consists of at least two major glycoproteins, ZP3 and ZP1. Our previous study using Japanese quail had demonstrated that the PL obtained from the preovulatory follicles was incubated in vitro with spermatozoa, and perforations were observed. This result indicated that the PL might contain a constituent that possesses activity to initiate the acrosome reaction (AR) in quail. In order to elaborate upon our previous findings, we evaluated the effects of ZP3 and ZP1 on the induction of sperm AR in Japanese quail. Ejaculated sperm were incubated with or without the purified PL glycoprotein, and their acrosome status was determined based on the presence or absence of the acrosome. Treatment of spermatozoa with increasing doses of the purified monomeric ZP1 led to a concentration-dependent stimulation of AR. The purified dimeric ZP1 had similar effect. Moreover, we found that the ZP1-induced AR was significantly blocked by the digestion of the PL protein with PNGaseF. In contrast, the addition of purified ZP3 failed to induce AR at any doses tested. These results indicate that N-linked glycans on ZP1 play an important role in triggering the AR in Japanese quail.

Sperm proteasome degrades egg envelope glycoprotein ZP1 during fertilization of Japanese quail (Coturnix japonica).

At the time of fertilization, the extracellular matrix surrounding avian oocytes, termed the perivitelline membrane (pvm), is hydrolyzed by a sperm-borne protease, although the actual protease that is responsible for the digestion of the pvm remains to be identified. Here, we show evidence that the ubiquitin-proteasome system is functional in the fertilization of Japanese quail. The activities for the induction of the acrosome reaction and binding to ZP3 as revealed by ligand blotting of purified serum ZP1 are similar to those of pvm ZP1. Western blot analysis of purified ZP1 and ZP3 by the use of the anti-ubiquitin antibody showed that only pvm ZP1 was reactive to the antibody. In vitro penetration assay of the sperm on the pvm indicated that fragments of ZP1 and intact ZP3 were released from the pvm. Western blot analysis using the anti-20S proteasome antibody and ultrastructural analysis showed that immunoreactive proteasome was localized in the acrosomal region of the sperm. Inclusion of specific proteasome inhibitor MG132 in the incubation mixture, or depletion of extracellular ATP by the addition of apyrase, efficiently suppressed the sperm perforation of the pvm. These results demonstrate for the first time that the sperm proteasome is important for fertilization in birds and that the extracellular ubiquitination of ZP1 might occur during its transport via blood circulation.

Zona Pellucida Domain of ZPB1 Controls Specific Binding of ZPB1 and ZPC in Japanese Quail (Coturnix japonica)

The extracellular matrix surrounding avian oocytes, referred to as the perivitelline membrane (PL), exhibits a three-dimensional network of fibrils between granulosa cells and the oocyte. We previously reported that one of its components, ZPC, is synthesized in granulosa cells that are specifically incorporated into the PL; this incorporation might be mediated by a specific interaction with ZPB1, another PL constituent, which is synthesized in the liver. In order to extend our previous findings, we established an expression system for quail ZPB1 using a mammalian cell line, and several ZPB1 mutants lacking the zona pellucida (ZP) domain or the glutamine-rich repeat region were produced. Western blot analysis of the immunoprecipitated materials with anti-ZPC antiserum indicated that ZPB1 was coimmunoprecipitated with the antiserum in the presence of ZPC. Ligand blotting also revealed the specific binding of ZPC and ZPB1 and indicated that the binding of these two components might be mediated via an ionic interaction. An analysis using recombinant ZPB1 demonstrated that the ZPB1 lacking the ZP domain did not bind to ZPC, whereas the mutant missing the glutamine-rich repeat region retained its capacity for binding. Furthermore, although the ZPB1 lacking the N-terminal half of the ZP domain was able to bind to ZPC, the deletion of the C-terminal half completely abolished ZPB1 binding to ZPC. These results suggested that the C-terminal half of the ZP domain of ZPB1 contains a binding site for ZPC, and that it appears to be involved in insoluble PL fibril formation in the quail ovary.

Characterization of progressive changes in ZPC of the vitelline membrane of quail oocyte following oviductal transport

The inner layer of the vitelline membrane of avian oocyte is equivalent to the zona pellucida of mammalian oocytes or to the vitelline envelope of amphibian oocytes. One of the two major glycoproteins in the inner layer of quail vitelline membrane, formerly called 33‐kDa glycoprotein, is homologous to mammalian ZPC, one of the components of zona pellucida. Quail ZPC is found to have different mobilities on SDS‐polyacrylamide gel electrophoresis depending on whether it is obtained from the preovulatory follicle or from the laid eggs. In order to characterize the progressive changes in the molecular size of quail ZPC during the oviductal transport, the inner layer isolated from the follicle was incubated in vivo in various regions of the oviduct and subjected to Western blot analysis with anti‐quail ZPC antiserum. The quail ZPC of the inner layer incubated in infundibulum reduced its apparent molecular weight, exhibiting the same electrophoretic mobility as that of laid eggs. The similar reduction in molecular weight was observed after the in vitro incubation of the inner layer with the extracts of infundibulum. From the comparison of the N‐terminal amino acid sequences, it was found that the first 26 residues of the quail ZPC in follicular oocytes are missing from the ZPC of laid eggs. In addition, lectin blot analysis suggested the modification of oligosaccharide chains during the oviductal transport. These results represent the first description in the avian oviduct of the presence of protease, which is similar to oviductin, a trypsin‐like protease involved in the hydrolysis of a major component of the vitelline envelope of amphibian oocytes. Mol. Reprod. Dev. 55:175–181, 2000.

The birth of quail chicks after intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) has been successfully used to produce offspring in several mammalian species including humans. However, ICSI has not been successful in birds because of the size of the egg and difficulty in mimicking the physiological polyspermy that takes place during normal fertilization. Microsurgical injection of 20 or more spermatozoa into an egg is detrimental to its survival. Here, we report that injection of a single spermatozoon with a small volume of sperm extract (SE) or its components led to the development and birth of healthy quail chicks. SE contains three factors – phospholipase Cζ (PLCZ), aconitate hydratase (AH) and citrate synthase (CS) – all of which are essential for full egg activation and subsequent embryonic development. PLCZ induces an immediate, transient Ca2+ rise required for the resumption of meiosis. AH and CS are required for long-lasting, spiral-like Ca2+ oscillations within the activated egg, which are essential for cell cycle progression in early embryos. We also found that co-injection of cRNAs encoding PLCZ, AH and CS support the full development of ICSI-generated zygotes without the use of SE. These findings will aid our understanding of the mechanism of avian fertilization and embryo development, as well as assisting in the manipulation of the avian genome and the production of transgenic and cloned birds.

Cellular analysis of cleavage-stage chick embryos reveals hidden conservation in vertebrate early development

Birds and mammals, phylogenetically close amniotes with similar post-gastrula development, exhibit little conservation in their post-fertilization cleavage patterns. Data from the mouse suggest that cellular morphogenesis and molecular signaling at the cleavage stage play important roles in lineage specification at later (blastula and gastrula) stages. Very little is known, however, about cleavage-stage chick embryos, owing to their poor accessibility. This period of chick development takes place before egg-laying and encompasses several fundamental processes of avian embryology, including zygotic gene activation (ZGA) and blastoderm cell-layer increase. We have carried out morphological and cellular analyses of cleavage-stage chick embryos covering the first half of pre-ovipositional development, from Eyal-Giladi and Kochav stage (EGK-) I to EGK-V. Scanning electron microscopy revealed remarkable subcellular details of blastomere cellularization and subgerminal cavity formation. Phosphorylated RNA polymerase II immunostaining showed that ZGA in the chick starts at early EGK-III during the 7th to 8th nuclear division cycle, comparable with the time reported for other yolk-rich vertebrates (e.g. zebrafish and Xenopus). The increase in the number of cell layers after EGK-III is not a direct consequence of oriented cell division. Finally, we present evidence that, as in the zebrafish embryo, a yolk syncytial layer is formed in the avian embryo after EGK-V. Our data suggest that several fundamental features of cleavage-stage development in birds resemble those in yolk-rich anamniote species, revealing conservation in vertebrate early development. Whether this conservation lends morphogenetic support to the anamniote-to-amniote transition in evolution or reflects developmental plasticity in convergent evolution awaits further investigation. Summary: Early chick embryos share previously unappreciated features with anamniote embryos such as the timing of zygotic gene activation and yolk syncytial layer formation.

Sperm acrosin is responsible for the sperm binding to the egg envelope during fertilization in Japanese quail (Coturnix japonica)

An antibody library against quail sperm plasma membrane components was established and a mAb, which strongly inhibits sperm perforations of the perivitelline membrane (PVM) was obtained from the library. The antigen molecule of the mAb showed an apparent molecular weight of 45  kDa, and was distributed both on the surface and in the acrosomal matrix of the sperm head. Periodate oxidation revealed that the epitope of the antigen includes a sugar moiety. Tandem mass spectrometry analysis of the antigen revealed that the mAb recognizes sperm acrosin. When sodium dodecyl sulfate-solubilized PVM immobilized on a polyvinylidene difluoride membrane was incubated with sperm plasma membrane lysates, the sperm acrosin was detected on the PVM immobilized on the membrane, indicating that the sperm acrosin interacts with the components of PVM. Indeed, the mAb effectively inhibited the binding of acrosome-intact sperm to the PVM. These results indicate that the 45  kDa sperm acrosin is involved in the binding of sperm to the PVM in fertilization of Japanese quail.