Introduction
Successful copulation with a male or artificial insemination is essential at the same time as ovulation for fertilisation in all mammals, except in bats that can store sperm in uterus for 225 days (Wang et al., Reference WANG, ZHANG, LI, MA, LIANG and WANG2005). However, in birds and reptiles a single successful copulation can produce many fertile eggs for prolonged periods. Female birds achieve this by storing spermatozoa for longer periods in specialised structures called sperm storage tubules (SSTs) in the oviduct (Lake, Reference LAKE1975; Bakst et al., Reference BAKST, WISHART and BRILLARD1994). The SSTs are present in all domesticated and wild birds studied to this date and the duration of storage varies from six to 42 days depending on the species (Birkhead and Moller, Reference BIRKHEAD and MOLLER1992). This storage capacity in the oviduct for days or weeks overcomes the need for repeated daily copulation throughout the period of egg production, and is referred as fertile period (Lake, Reference LAKE1975). The duration of the fertile period is up to 12 days in Japanese quail (Sittmann and Abplanalp, 1965; Reddish et al., Reference REDDISH, KIRBY and ANTHONY1996), 21 days in chicken and guinea-fowl (Brillard, Reference BRILLARD1993), and over 70 days in turkey (Lorenz, Reference LORENZ1950). Storage of spermatozoa in the female tract provides a natural basis of sperm selection. It is assumed that following natural mating or artificial insemination (AI) only the most fecund sperm survive in storage sites. This eliminates need for precise synchronisation of ovulation with copulation or insemination.
Reproductive physiology of the hen
The oviduct of mature birds comprises five morphologically and functionally distinct segments, namely the infundibulum, magnum, isthmus, uterus and bud-like invaginations at the utero-vaginal junction (UVJ) located at the cranial end of vagina. The UVJ of the oviduct contains SSTs, where the most fecund sperm are stored (Fujii and Tamura, Reference FUJII and TAMURA1963; Bobr et al., Reference BOBR, LORENZ and OQASAWARA1964; Bakst et al., Reference BAKST, WISHART and BRILLARD1994). Fertilisation in birds typically occurs in the infundibulum during a brief fertilisation window of only 15 to 30 minutes duration, shortly after ovulation (Howarth, Reference HOWARTH1974). The ovum then begins its descent down the oviduct where albumin and shell membranes are deposited. Unless protected in SSTs, sperm are unlikely to survive in the oviduct during egg formation and laying (Lorenz, Reference LORENZ1966). Thus, specialised sperm storage sites may have evolved in birds so that inseminations do not have to be synchronised with the fertilisation window for successful conception (Hatch, Reference HATCH1983).
Sperm storage tubules
The occurrence of anatomical structures ‘sperm nests’ associated with prolonged sperm storage in the infundibulum of the chicken (Gallus gallus) was first recognised by Van Drimmelen (Reference VAN DRIMMELEN1946). Further histological studies (Bobr, Reference BOBR1962; Bobr et al., Reference BOBR, LORENZ and OQASAWARA1964; Fujii, Reference FUJII1963; Fujii and Tamura, Reference FUJII and TAMURA1963) showed infundibular sperm storage sites to be of doubtful or secondary significance. Primary sperm storage was found to take place at the junction of the uterus (shell gland) and vagina. Since their discovery, sperm storage sites in the UVJ have been referred to by several names such as vaginal glands (Fujii, Reference FUJII1963), sperm glands (Van Krey et al., Reference VAN KREY, OGASAWARA and PANGBORN1967), UV sperm-host glands (Gilbert et al., Reference GILBERT, REYNOLDS and LORENZ1968), sperm-storage tubules (Mero and Ogasawara, Reference MERO and OGASAWARA1970) and UV sperm-storage glands (Burke et al., Reference BURKE, OQASAWARA and FUQUA1972). Sperm are maintained in a viable state within these tubules located at the junction of the uterus and vagina (Bobr et al., Reference BOBR, LORENZ and OQASAWARA1964; Zavaleta and Ogasawara, Reference ZAVALETA and OGASAWARA1987). The SSTs are lined by a single layer of non-ciliated cells that are formed by the invagination and differentiation of mucosal surface epithelium (Tingari and Lake, Reference TINGARI and LAKE1973). The proximal part (neck) of the glands is lined by a pseudo-stratified epithelium consisting of high columnar ciliated cells and small basal cells that do not reach the lumen. The ciliated cells are tall and wedge-shaped and display the same features as the apical cells of the surface epithelium of the utero-vaginal region (Frieb et al., Reference FRIEB, SINOWATZ, WROBEL and SCKLEK-WINNISCH1978). The spermatozoa are arranged in bundles, and lie freely in the glandular lumen. The heads of the sperm cells are oriented towards the base of the glands and do not penetrate the intercellular spaces, as described by Fujii and Tamura (Reference FUJII and TAMURA1963) in the domestic hen. The spermatozoa receive energy yielding substances not by an intimate association with the cytoplasm of the glandular cells, but rather from glandular secretions. The cells of the SSTs are characterised by a comparatively well-developed Golgi complex and the accumulation of numerous vesicles in the supranuclear and apical cell region. In domestic hens and quail, lipid droplets are found juxta-nuclearly in the cells of SSTs (Fujii, Reference FUJII1963; Gilbert et al., Reference GILBERT, REYNOLDS and LORENZ1968). Besides its function as a storage site for spermatozoa (Fujii and Tamura, Reference FUJII and TAMURA1963; Bobr et al., Reference BOBR, LORENZ and OQASAWARA1964; Burke et al., Reference BURKE, OQASAWARA and FUQUA1972; Tingari and Lake, Reference TINGARI and LAKE1973), absorption of non-viable spermatozoa has been shown to take place in the utero-vaginal region of the oviduct (Degen and Hawes, Reference DEGEN and HAWES1972) and the apical tubular system could be involved in absorption (Frieb et al., Reference FRIEB, SINOWATZ, WROBEL and SCKLEK-WINNISCH1978; Birkhead and Moller, Reference BIRKHEAD and MOLLER1992). Further, study on the number of SSTs in four strains of broilers and one strain of large white turkeys was conducted by Bakst et al. (Reference BAKST, DONOGHUE, YOHO, MOYLE, WHIPPLE, CAMP, LIN and BRAMWELL2010). Unlike the numbers of SST in the chicken (13,533) and turkey (20,000) reported by Birkhead and Moller (Reference BIRKHEAD and MOLLER1992), Bakst et al. (Reference BAKST, DONOGHUE, YOHO, MOYLE, WHIPPLE, CAMP, LIN and BRAMWELL2010) observed that broiler lines averaged 4,900 SSTs per hen whereas turkey averaged 30,600 SSTs per hen. Thus, the difference in the length of the fertile period between broiler and turkey is predominantly due to the increased numbers of SSTs and the resultant increase in sperm storage capacity of the turkey compared with broilers.
Mechanism of sperm storage and release
sperm transport
Semen is ejaculated into the vagina and sperm rely on their motility to reach the numerous SSTs located at the UVJ (Bakst et al., Reference BAKST, WISHART and BRILLARD1994). Because of selection during their migration through the vagina, only 1-2% of inseminated sperm enter the SSTs, the rest probably are ejected the next time that the female defecates (Birkhead and Brillard, Reference BIRKHEAD and BRILLARD2007). In the UVJ, SSTs accept only morphologically and physiologically normal sperm which passes the selection process in vagina. In the turkey and the chicken, spermatozoa take up to three days to fill the SSTs after artificial insemination (Brillard and Bakst, Reference BRILLARD and BAKST1990; Bakst et al., Reference BAKST, WISHART and BRILLARD1994). Vaginal contractions associated with oviposition can affect sperm transfer in the vagina (Brillard, Reference BRILLARD1993). Bakst et al. (Reference BAKST, WISHART and BRILLARD1994) stated that intrinsic sperm motility coupled with a fluid transport mechanism in the troughs between tightly apposed mucosal folds were responsible for rapid sperm transport to the UVJ at the distal end of the vagina.
sperm selection
The initial selection of avian sperm in the vagina is a pre-requisite to their entry into the SSTs. Studies performed in chickens and turkeys have revealed that only about 1% of the initial sperm population deposited intravaginally reaches the SSTs (Brillard and Bakst, Reference BRILLARD and BAKST1990; Brillard, Reference BRILLARD1993). Birkhead and Brillard, (Reference BIRKHEAD and BRILLARD2007) postulated that the overall population of pre-selected sperm transported to the sites of fertilisation does not exceed 1/10000th of the initial population deposited intravaginally since ~1% of the sub-population of pre-selected sperm can be recovered in the perivitelline membrane of eggs. This intense selection of normal sperm is attributed to relatively low embryonic mortality compared to mammals (Birkhead and Brillard, Reference BIRKHEAD and BRILLARD2007). Based on the current body of knowledge, the first vaginal barrier is of a mechanical nature, i.e. it limits sperm migration to those capable of progressive motility, therefore rejecting dead and immobile sperm and those with impaired motility (Allen and Grigg, Reference ALLEN and GRIGG1957).
The surface epithelium of the vagina consists of a ciliated pseudo-stratified epithelium unlike in mammals. The number of polymorph nuclear leukocytes and lymphocytes increased in the vagina and UVJ after copulation and some of the immune cells were closely associated with the spermatozoa (Higaki et al., Reference HIGAKI, YOSHIMURA, TAMURA and OKAMOTO1995). There are reports showing significant increases in the expression of MHC class II in the infundibulum of hen after AI (Zheng et al., Reference ZHENG, NISHIBORI, ISOBE and YOSHIMURA2001) and the presence of antibody-producing plasma cells in the SST epithelium of infertile turkey hens (Van Krey et al., Reference VAN KREY, SCHUPPIN, DENBOW and HULET1987). These reports suggest the possibility of immune-responses to sperm in the vagina of the hen oviduct. Such responses to sperm in the normal process of transport to the vagina may be responsible for the selection of good-quality sperm and only these may ascend the vagina to UVJ and enter the SST, ultimately participating in fertilisation (Das et al., Reference DAS, ISOBE and YOSHIMURA2008). The glycoprotein composition of the sperm plasma membrane has a profound influence on the fate of sperm following insemination (Wishart and Steele, Reference WISHART and STEELE1990). Removal of at least some component associated with the sperm plasma membrane notably neuraminic acid prior to vaginal insemination results in failure of sperm to be accepted by the SST (Wishart and Steele, Reference WISHART and STEELE1990). Spermatozoa are transported from the SSTs to the infundibulum largely via the contractions of the uterus (Allen and Grigg, Reference ALLEN and GRIGG1957), and it is possible that some sperm are lost or diluted by the large oviduct size relative to the losses that occur when sperm transport is dependent upon sperm motility (e.g. when swimming up the vagina to the SSTs or penetrating the ovum membrane; Allen and Grigg, Reference ALLEN and GRIGG1957). Sperm mobility is the dominant factor in sperm selection within the vagina (Froman et al., Reference FROMAN, FELTMANN, PENDARVIS, COOKSEY, BURGESS and RHOADS2011). If vaginal insemination is performed two hours before or after oviposition, oviduct sperm transport is altered and sperm filling of the SST is reduced (Birkhead et al., Reference BIRKHEAD, CUNNINGHAM and CHENG1996). The vaginal insemination of heterologous semen into chickens resulted in few sperm reaching the SST, presumably due to the absence of the specific sperm surface glycoprotein array compatible with the oviductal sperm selection mechanism in the hen. However, when added to explants of UVJ folds, heterologous sperm entered the SST, indicating that the vagina and not the SST (Wishart and Horrocks, Reference WISHART and HORROCKS2000) orchestrate the selection process.
sperm storage
Ashizawa and Nishiyama (Reference ASHIZAWA and NISHIYAMA1983) reported that chicken sperm cultured with the infundibulum or UVJ cells showed better motility than those cultured with other oviduct cells. This suggests that sperm interact with cells of specific oviduct segments where SST exists. However, the mechanism by which spermatozoa are stored in SSTs has not been completely established. Previously, regulation of sperm metabolism (Barna et al., Reference BARNA, BOLDIZSAR and ZSOLNAI-HARCZI1996) and the maintenance of the SST microenvironment (Holm et al., Reference HOLM, EKWALL, WISHART and RIDDERSTRALE2000) that involve calcium, zinc, sodium, phosphorus, sulphur, chlorine and potassium, have been associated with sperm storage. Calcium increases chicken sperm motility and oxygen consumption in vitro (Ashizawa et al., Reference ASHIZAWA, HASHIGUCHI and TSUZUKI1994). Zinc is present in the cells and tubular fluid of the sperm storage tubules of turkey, but it was not detected in the cells or in the luminal fluid of sperm storage tubules in chickens or quail. This may provide the reason for the fact that duration of sperm storage in turkeys is greater than that in other birds (Holm et al., Reference HOLM, EKWALL, WISHART and RIDDERSTRALE2000). Zinc may function as a metabolic inhibitor, as supported by the high concentration of zinc found in female reproductive tract of some hibernating bats that store spermatozoa for up to six months (Crichton et al., Reference CRICHTON, KRUTZSCH and CHVAPIL1982).
Presence of lipid components (Bakst et al., Reference BAKST, WISHART and BRILLARD1994), carbonic anhydrase (Holm et al., Reference HOLM, EKWALL, WISHART and RIDDERSTRALE2000), and aquaporins (Zaniboni and Bakst, Reference ZANIBONI and BAKST2004) and expression of multiple cytoskeletal protein genes (Long et al., Reference LONG, SONSTEGARD, LONG, VAN TASSELL and ZUELKE2003) in the SST cells suggest that these components play essential physiological functions for prolonged sperm storage. It is assumed that resident sperm metabolise endogenous fatty acids (Froman et al., Reference FROMAN, FELTMANN, PENDARVIS, COOKSEY, BURGESS and RHOADS2011) or other lipids derived from the apical microvilli of the SST epithelium (Bakst et al., Reference BAKST, WISHART and BRILLARD1994). Turkey intraluminal SST epithelial cells contain relatively high levels of carbonic anhydrase, suggesting that intraluminal pH might play a significant role in modulating sperm motility during oviduct sperm storage and transport (Holm et al., Reference HOLM, EKWALL, WISHART and RIDDERSTRALE2000). Aquaporins mainly located in apical portion of SSTs in turkeys participate in the processes of elimination of sperm catabolites accumulated in the lumen during prolonged storage (Zaniboni and Bakst, Reference ZANIBONI and BAKST2004). As the local immunity responses in the UVJ may affect the sperm survivability, one of the requirements for prolonged sperm storage and sperm survivability in the SST may be to suppress this activity. Das et al. (Reference DAS, ISOBE and YOSHIMURA2008) indicated that the storage of sperm in the SST necessitates an immunosuppression of sperm antigenicity. Transforming growth factor-beta (TGF-β) is one of the possible candidates that suppress the immune-response and protect the sperm (Das et al., Reference DAS, ISOBE, NISHIBORI and YOSHIMURA2006; Reference DAS, ISOBE and YOSHIMURA2007). Furthermore, the population of lymphocytes in UVJ was higher in repeated AI flocks than virgin groups. This suggested that the immune responses in the UVJ of these hens occurred more strongly compared to the virgin bird.
Swelling of the SST structure and thinning of epithelial cells can be caused by AI. One of the causes of this may be an inflammatory reaction due to repeated AI may cause SST destruction which may result a decline in fertility. A complex antioxidant system including Se-dependent enzymes (e.g. GSH-Px) is present in the SSTs to prevent or repair membrane damage caused by peroxidation during storage (Bréque et al., Reference BRÉQUE, SURAI and BRILLARD2003).
The maximum filling of SST cells is higher during the first part of the reproductive season but declines thereafter. It can be partly compensated by Se and/or vitamin E dietary supplementation (Bréque et al., Reference BRÉQUE, SURAI and BRILLARD2006). Briskie and Montgomerie (Reference BRISKIE and MONTGOMERIE1993) examined the relationship between number of SSTs and clutch size, reasoning that species with larger clutches might be expected to store larger quantities of sperm since they have more eggs to fertilise. Recently, Huang et al. (Reference HUANG, ISOBE, OBITSU and YOSHIMURA2016) suggested that the fatty acids present in the SSTs cell might be responsible for survival of sperm in the oviduct of bird.
role of hormone receptors in sperm storage
The growth, differentiation and secretory functions of the oviduct are primarily regulated by sex steroids (Oka and Schimke, Reference OKA and SCHIMKE1969a; Reference OKA and SCHIMKE1969b; Palmiter and Wrenn, Reference PALMITER and WRENN1971). Progesterone is a central regulator of all aspects of female reproductive activity in birds (Conneely et al., Reference CONNEELY, DOBSON, TSAI, BEATTIE, TOFY, HUCKABY, ZARUCKIA, SCHRADER and O'MALLEY1987). It plays an important role in the regulation of several functions such as ovulation, gonad differentiation, and sexual and nesting behaviours. In birds, the functional ovary produces progesterone and oestrogen (Tilly et al., Reference TILLY, KOWALSKI and JOHNSON1991), which stimulate SST development by acting via receptors (Pescatore and Marquez, Reference PESCATORE and MARQUEZ1977). Blood levels of oestrogen, which increases to maximum values two to three weeks before the first egg is laid, is temporally correlated with the development of the oviduct (Peterson and Webster, Reference PETERSON and WEBSTER1974; Senior, Reference SENIOR1974). Therefore, the development and functions of the SST may be controlled by the sex steroids. Sex steroids (oestrogen and progesterone) are thought to play a significant role in the formation and maintenance of the SST structure and in the regulation of the sperm storage function (Yoshimura et al., Reference YOSHIMURA, KOIKE and OKAMOTO2000). The biological actions of these hormones are mediated via oestrogen receptors (ER) and progesterone receptors (PR). The expression of these receptors in the SST cells of laying hens suggests that these cells are the target sites of progesterone and oestrogen (Das et al., Reference DAS, NAGASAKA and YOSHIMURA2005b; Yoshimura et al., Reference YOSHIMURA, KOIKE and OKAMOTO2000). Das et al. (Reference DAS, NAGASAKA and YOSHIMURA2005a) observed decreased ER-α mRNA expression in SSTs after repeated AI in hens. A reduction in the expression of ER-α mRNA in SST may result in the failure to maintain normal SST functions due to insufficient effects of oestrogen contributing to the decreased fertility in hens (Das et al., Reference DAS, NAGASAKA and YOSHIMURA2005b). However, decreased mRNA expression of oestrogen receptor-α coupled with the increased numbers of immuno-competent cells in and around SST, may be directly related to the absence of significant sperm storage in low fertility hens (Das et al., Reference DAS, ISOBE, NISHIBORI and YOSHIMURA2006). Sasanami et al. (Reference SASANAMI, IZUMI, SAKURAI, HIRATA, MIZUSHIMA, MATSUZAKI, HIYAMA, YORINAGA, YOSHIMURA, UKENA and TSUTSUI2015) reported that PGF2α and cloacal gland secretions contributed to the vaginal contraction and opening of SSTs.
role of enzymes
The presence of alkaline phosphatases (AP) is known in quail SSTs (Sinowatz et al., Reference SINOWATZ, WROBEL and FRIES1976). The APs are a class of cell surface zinc metallo-enzymes that hydrolyse phosphate ester groups at an alkaline pH. In birds, AP activity was present in the regions thought to be involved in the mid-portion of the vaginal differentiation and proliferation during oviduct maturation (Aire and Steinbach, Reference AIRE and STEINBACH1976). AP possibly reflects cell proliferation of surface epithelia and lipid transfer activities in the SST of turkeys (Bakst and Akuffo, Reference BAKST and AKUFFO2007). Liposome-like vesicles present on the microvillar tip of SST epithelium and those vesicles show the interaction with luminal sperm. This region of the SST epithelium is AP positive (Bakst and Akuffo, Reference BAKST and AKUFFO2007) and corresponds to the localisation of AP in the rat intestinal luminal epithelium (Narisawa et al., Reference NARISAWA, HUANG, IWASAKI, HASEGAWA and ALPERS2003). These authors suggested that AP may function in the transfer of lipid across the enterocyte brush border, and speculated that AP may have a similar role in the SST epithelium (Bakst and Akuffo, Reference BAKST and AKUFFO2007).
Lactate dehydrogenase is one of the important enzymes of energy metabolism and is a tetramer with two different polypeptide chains/subunits in different combinations, forming five major isozymes. The H-type is important for conversion of lactate to pyruvate, and therefore is predominantly present in tissues like heart, kidney and spleen while the M-type is localised mostly in muscle tissue that converts pyruvate to lactate. The seminal plasma of Japanese quail exhibits high concentrations of H-type LDH isozyme (Singh et al., Reference SINGH, SASTRY, PANDEY, SHIT, AGRAWAL, SINGH, MOHAN, SAXENA and MOUDGAL2011a), which is important for lactate utilisation by quail sperm, which acts as an energy substrate (Singh et al., Reference SINGH, SASTRY, PANDEY, SHIT, AGRAWAL, SINGH, MOHAN, SAXENA and MOUDGAL2011b).
sperm release
The sperm present in SSTs are invariably positioned with their heads directed towards the distal end of the tubule which suggests that egress from the SSTs is passive. Sperm are lost from the SSTs more or less continuously at a constant per capita rate (Birkhead and Brillard, Reference BIRKHEAD and BRILLARD2007). The SSTs are blind-end tubules and the fluid secreted epithelial cells can generate a current. Sperm with ample velocity can move against the current and remain within the tubule. In contrast, motile sperm emerge from the tubule once velocity is decreased to a point at which position cannot be maintained against the current (Hodges, Reference HODGES1974).
Burke and Ogasawara (Reference BURKE and OGASAWARA1969) reported that the release of sperm from SSTs is a slow and continuous process that occurs during the ovulatory cycle. In addition to this, after vaginal insemination in chicken, sperm present in the SSTs were released from these tubules at the time of oviposition or ovulation (Bobr et al., Reference BOBR, LORENZ and OQASAWARA1964). It has been observed that when mobility of sperm falls below the flow rate of the fluid, at the same time a uni-directional fluid current produced would propel sperm out of the SST (Froman, Reference FROMAN2003). Van Krey et al. (Reference VAN KREY, BALANDER and COMPTON1981) proposed that sperm release from the SST was due to the inability of individual sperm to remain agglutinated in the sperm bundle in the SST lumen. Sperm release from the SST takes place when, possibly as a result of waning ATP when sperm motility decreases, they are carried out with the luminal fluids (Froman et al., Reference FROMAN, FELTMANN, PENDARVIS, COOKSEY, BURGESS and RHOADS2011). The localisation of aquaporin-3 in the apical region of the SST epithelium (Zaniboni and Bakst, Reference ZANIBONI and BAKST2004) would support the suggestion that there is a transfer of fluids from the SST epithelium to the lumen.
The rate of loss of spermatozoa from the sperm storage tubules is an important determinant of the duration of sperm storage in naturally mated Japanese quail (Birkhead and Fletcher, Reference BIRKHEAD and FLETCHER1994). Sperm enter the uterus and is carried passively to the infundibulum. Sperm accumulate or move relatively slowly through the infundibulum so that there is usually a population available to fertilise each ovum as it is ovulated. Recently, Ito et al. (Reference ITO, YOSHIZAKI, TOKUMOTO, ONO, YOSHIMURA, TSUKADA, KANSAKU and SASANAMI2011) demonstrated that progesterone is a factor responsible for sperm release from the SST via membrane progestin receptor α-mediating signal transduction. Hiyama et al. (Reference HIYAMA, MATSUZAKI, MIZUSHIMA, DOHRA, IKEGAMI, YOSHIMURA, SHIBA, INABA and SASANAMI2014) suggested that heat shock protein 70 binds to the sperm surface via voltage-dependent anion channel protein 2 and activates the sperm in UVJ for release from SST to infundibulum.
Conclusions
Sperm storage tubules play an important role in production of continuous fertile eggs and consequently in fertility of the bird. However, the exact mechanism of sperm storage and their release from SSTs is yet to be elucidated. Functional genomic studies may provide a better insight in identifying pathways that regulate SSTs function when they are filled with sperm.
