Mei Matsuzaki

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.

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.

Sperm-Egg Interaction during Fertilization in Birds

Fertilization in animals that employ sexual reproduction is an indispensable event for the production of the next generation. A significant advancement in our understanding of the molecular mechanisms of sperm-egg interaction in mammalian species was achieved in the last few decades. However, the same level of knowledge has not been accumulated for birds because of egg size and the difficulty in mimicking the physiological polyspermy that takes place during normal fertilization. In this review, we summarize the current understanding of sperm-egg interaction mechanism during fertilization in birds, especially focusing on sperm-egg binding, sperm acrosome reaction and the authentic sperm protease required for the hole formation on the perivitelline membrane. We explain that the zona pellucida proteins (ZP1 and ZP3) in the perivitelline membrane play important roles in sperm-egg binding, induction of the acrosome reaction as well as sperm penetration by digestion of sperm protease. We anticipate that a deeper understanding of avian fertilization will open up new avenues to create powerful tools for a myriad of applications in the poultry industries including the production of transgenic and cloned birds.

Specific Mechanism of Sperm Storage in Avian Oviducts

The capability for sperm storage 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, including insects, fish, amphibians, reptiles, birds, and in mammals, 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 for sperm storage in the female reproductive tract has been poorly understood until recently. In this chapter, we report our recent findings on the mechanism of sperm storage in avian oviducts, especially data obtained from the Japanese quail (Coturnix japonica), as an experimental model. Because sperm storage in birds occurs at body temperature (i.e. 41 °C), elucidation of the mechanism of sperm maintenance in the avian oviduct may open up new avenues for the development of novel strategies for sperm storage in vitro without cryopreservation.

Female Japanese quail visually differentiate testosterone-dependent male attractiveness for mating preferences

Biased mating due to female preferences towards certain traits in males is a major mechanism driving sexual selection, and may constitute an important evolutionary force in organisms with sexual reproduction. In birds, although the role of male ornamentation, plumage coloration, genetic dissimilarity, and body size have on mate selection by females have been examined extensively, few studies have clarified exactly how these characteristics affect female mate preferences. Here, we show that testosterone (T)-dependent male attractiveness enhances female preference for males of a polygamous species, the Japanese quail. A significant positive correlation between female mating preference and circulating T in the male was observed. The cheek feathers of attractive males contained higher levels of melanin and were more brightly colored. The ability of females to distinguish attractive males from other males was negated when the light source was covered with a sharp cut filter (cutoff; < 640 nm). When females were maintained under short-day conditions, the expression of retinal red-sensitive opsin decreased dramatically and they became insensitive to male attractiveness. Our results showed that female preference in quail is strongly stimulated by male feather coloration in a T-dependent manner and that female birds develop a keen sense for this coloration due to upregulation of retinal red-sensitive opsin under breeding conditions.

Postcopulatory Reproductive Strategies in Spermatozoa

To reproduce sexually, males and females produce very different gametes (sperm and eggs) in many animals. This difference gives rise to very different strategies in the two sexes and in gamete cells from the two sexes. Sperm meet eggs in harmony; however, the male and female do not always have common interests in reproduction. The battle of the sexes continues even after copulation. Female promiscuity is key to the understanding of reproductive behaviors not only in male individuals but also in sperm cells, because sexual selection continues after mating through sperm competition. Here, we highlight multiple sperm traits—the sperm acrosome reaction in sea urchins, sperm storage in birds, and sperm dimorphism in squid—that are tightly associated with postcopulatory reproductive strategies.

Sperm Storage in the Female Reproductive Tract: A Conserved Reproductive Strategy for Better Fertilization Success

In internal fertilizers including mammals, fertilization success depends on the timely arrival of sperm and egg at the site of fertilization. Males should transfer their spermatozoa to the female reproductive tract by copulation during or prior to ovulation in order to achieve this aim. However, such a collaborative mating behavior is often disconnected from the efficiency of the sperm-egg encounter, i.e., ovulation by females occurs independently from insemination by males in many species. To compensate for this time lag, females are capable of storing spermatozoa in their reproductive tracts until the eggs are ready to be fertilized. In avian species, simple tubular invaginations referred to as sperm storage tubules (SSTs) are located between the vagina and uterus as sperm storage sites. Spermatozoa, once ejaculated, migrate to and are thereafter stored in the lumen of the SSTs without loss of fertilizing capacity for up to 15 weeks at a body temperature of 41 °C. This is astonishing, because terminally differentiated cells that lack new protein synthesis are still capable of being functional for a long period at a high temperature; however, the actual mechanism has been an enigma for more than half a century. In this chapter, we will first describe the physiological importance and adoptive benefits of sperm storage in the female genital tract for successful fertilization in animals, and next, we will describe our recent findings in birds with regard to the specific mechanism of sperm uptake into the SST, sperm maintenance within it, and controlled release from it.

Handling of Gametes for In Vitro Insemination in Birds

A characteristic biological property of avian gamete (e.g., extremely large egg and polyspermic fertilization) does not allow the direct observation of sperm-egg interactions in vitro, but recent research advances make it possible to manipulate the gamete in vitro. Here, we describe the techniques for the handling of gametes required for in vitro fertilization assay. In addition, we also introduce the procedures for sperm-perivitelline membrane assay, intracytoplasmic sperm injection, and ex ovo culture.