Gen Hiyama

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.

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.

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.

Changes in post-translational modifications of prolactin during development and reproductive cycles in the chicken.

Changes in proportion of glycosylated prolactin in the anterior pituitary glands of chickens were assessed using one- and two-dimensional western blotting analysis during the perihatch stage of embryos and reproductive cycles. Multiple isoforms of prolactin were detected by one-dimensional analysis and glycosylated (G) and non-glycosylated (NG) isoforms were identified by N-glycosidase and neuraminidase treatment. Increases of ratio of G to NG isoforms were observed in both embryonic stages and reproductive cycles by the one-dimensional analysis. Although a similar tendency of increase of proportion of G prolactin was obtained, different values of proportion were observed between one-dimensional and two-dimensional analysis. Since two-dimensional analysis may better resolve isoforms differing slightly in molecular size of G prolactin, the results from two-dimensional analysis may reflect the actual proportion of prolactin isoforms. Furthermore, isoforms differing in isoelectric points were detected after N-glycosidase and neuraminidase treatment. These results indicate that prolactin may also be additionally post-translationally modified such as by phosphorylation. Thus function and biological activity of prolactin were, at least in part, regulated by post-translational modification in the various physiological stages.

Sperm activation by heat shock protein 70 supports the migration of sperm released from sperm storage tubules in Japanese quail (Coturnix japonica)

Systems for maintaining the viability of ejaculated sperm in the female reproductive tract are widespread among vertebrates and invertebrates. In birds, this sperm storage function is performed by specialized simple tubular invaginations called sperm storage tubules (SSTs) in the uterovaginal junction (UVJ) of the oviduct. Although the incidence and physiological reasons for sperm storage in birds have been reported extensively, the mechanisms of sperm uptake by the SSTs, sperm maintenance within the SSTs, and control of sperm release from the SSTs are poorly understood. In this study, we demonstrated that the highly conserved heat shock protein 70 (HSP70) stimulates sperm motility in vitro and also that HSP70 expressed in the UVJ may facilitate the migration of sperm released from the SSTs. Quantitative RT-PCR analysis demonstrated that the expression of HSP70 mRNA in the UVJ increases before ovulation/oviposition. Gene-specific in situ hybridization and immunohistochemical analysis with a specific antibody to HSP70 demonstrated that HSP70 is localized in the surface epithelium of the UVJ. Furthermore, injection of anti-HSP70 antibody into the vagina significantly inhibited fertilization in vivo. In addition, we found that recombinant HSP70 activates flagellar movement in the sperm and that the binding of recombinant HSP70 to the sperm surface is mediated through an interaction with voltage-dependent anion channel protein 2 (VDAC2). Our results suggest that HSP70 binds to the sperm surface by interacting with VDAC2 and activating sperm motility. This binding appears to play an important role in sperm migration within the oviduct.

Lactic acid is a sperm motility inactivation factor in the sperm storage tubules

Although successful fertilization depends on timely encounters between sperm and egg, the decoupling of mating and fertilization often confers reproductive advantages to internally fertilizing animals. In several vertebrate groups, postcopulatory sperm viability is prolonged by storage in specialized organs within the female reproductive tract. In birds, ejaculated sperm can be stored in a quiescent state within oviductal sperm storage tubules (SSTs), thereby retaining fertilizability for up to 15 weeks at body temperature (41 °C); however, the mechanism by which motile sperm become quiescent within SSTs is unknown. Here, we show that low oxygen and high lactic acid concentrations are established in quail SSTs. Flagellar quiescence was induced by lactic acid in the concentration range found in SSTs through flagellar dynein ATPase inactivation following cytoplasmic acidification (<pH 6.0). The long-term preservation of sperm morphology under hypoxic and high temperature conditions indicates that a combination of these factors enables sperm cells to survive during the ovulation cycles. Our findings suggested a novel physiological role for lactic acid in promoting sperm quiescence in SSTs and opened up a new opportunity for technological improvement in prolonging sperm longevity at ambient or body temperature.

A unique mechanism of successful fertilization in a domestic bird

Fertilization is an indispensable step for formation of a zygote in sexual reproduction, leading to species survival. When mating occurs, sperm is transported to the female reproductive tracts via the seminal plasma (SP). SP is derived from male accessory sex glands and it plays pivotal roles for fertilization in animals. However, molecular mechanisms of SP or a fluid derived from male accessory sex glands for successful fertilization remain unclear. Here, we report that in male quail the cloacal gland (CG) produces prostaglandin F2α (PGF2α) that contributes to successful fertilization. PGF2α, as well as the secretion of CG (CGS), induced vaginal contractions and caused the opening of the entrance of the sperm storage tubules, the structures responsible for the long-term sperm storage and fertilization. The removal of CGS from the male before mating reduced the fertility, but the supplementation of CGS or PGF2α rescued the subfertility. We further showed that male CG contains glucose that is utilized as energy source for the intrinsic sperm mobility after transportation to female vagina. This mechanism, in concert with the excitatory effects of PGF2α enables successful fertilization in the domestic bird.

Vectorial Secretion of Perivitelline Membrane Glycoprotein ZPC of Japanese Quail (Coturnix japonica) in Polarized Madin-Darby Canine Kidney Cells

The avian perivitelline membrane (PL), which is an investment homologous to the mammalian zona pellucida, is found between the surface of the oocyte and the apical surface of ovarian granulosa cells. Our previous study demonstrated that ZPC, one of the components of PL, is synthesized in ovarian granulosa cells. However, how the secretion of ZPC is regulated in the cells has been insufficiently investigated. We studied the secretion of quail ZPC expressed in polarized Madin-Darby canine kidney (MDCK) cells in a dual-chamber apparatus. Western blot analyses of the conditioned medium demonstrated that the majority of the secreted ZPC were distributed in the apical compartment. When ZPC lacking N-linked oligosaccharides was transfected into the cells, the 31-kDa immunoreactive band was detected in both the apical and the basolateral medium. Interestingly, immunohistochemical observations of the follicular wall demonstrated that the predominant intracellular form of ZPC in the cells localized in the apical side of the perinuclear region apposed to the PL, but not the basolateral side, indicating the possibility that ZPC could be selectively transported toward the apical surface in vivo. Taken together, these results indicated that ZPC expressed in MDCK cells are selectively released to the apical compartment, and that the N-linked carbohydrates might possess information that causes the efficient transport of ZPC to the apical surface of the cells.