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In birds, sperm storage tubules (SST) located in the utero-vaginal junction are thought to be a site of sperm selection; however, the exact mechanism of sperm selection is poorly understood. Here, we investigated sperm entry into the SST and subsequent fertilization success under a competitive situation created by artificial insemination of a sperm mixture obtained from 2 males. We employed 2 quail strains, a wild-type and a dominant black (DB) type, as this allows easy assessment of paternity by feather coloration. We found paternity of embryos was biased toward DB males when a sperm mix with similar sperm numbers from the 2 males strains was artificially inseminated into females. Our novel sperm staining method with 2 different fluorescent dyes showed that the DB-biased fertilization was because of the better ability of DB sperm to enter the SST. Moreover, we found that DB sperm had a longer flagellum and midpiece. These characteristics probably allow sperm to swim faster in a high viscosity medium, which may be a similar environment to the lumen of the female reproductive tract. Our results indicated that sperm competition occurs to win a place in the SST and that filling the SST with their own spermatozoa is a critical step to achieve better fertilization success for the male Japanese quail.

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.

Fertilization is indispensable for zygotic formation leading to the birth of animals and the species-specific sperm-egg binding thought to be the initial step in this important process. In birds, the oocyte, which encounters the spermatozoa at the time of fertilization, is enclosed in a perivitelline membrane (pvm) constructed of several zona pellucida glycoproteins (ZP proteins: ZP1, ZP2, ZP3, ZP4 and ZPD). The aim of this study was to determine the ZP protein in the pvm responsible for sperm-pvm binding in Japanese quail. We tested the effects of anti-ZP protein antibodies on in vitro sperm perforation in the pvm. The results showed that the anti-ZP1 and ZP3 antibody significantly blocked hole formation by sperm, whereas anti-ZP2, ZP4 and ZPD as well as normal rabbit serum had no such effect. When the sperm acrosome reaction was inhibited in the presence of pertussis toxin, sperm-pvm binding was observed. This sperm-pvm binding was significantly prevented when the purified ZP1 or ZP3 was included in the reaction mixture. Moreover, both digoxigenin-labeled ZP1 and ZP3 were found to interact with the sperm head by immunocytochemical observation. Our results indicate that sperm binding to the pvm is, at least in part, mediated by the interaction of ZP1 and ZP3 with the sperm head during fertilization in Japanese quail.

Because of the presence of sperm storage tubules (SSTs) in the utero-vaginal junction (UVJ) in the oviduct, once ejaculated sperm enter the female reproductive tract, they can survive for a prolonged period in domestic birds; however, the specific mechanisms involved in sperm maintenance within the SST remain to be elucidated. In this study, we showed that transferrin (TF) and albumin (ALB) are expressed in SSTs. When UVJ extracts were subjected to size-exclusion column chromatography, we obtained fractions that extend sperm longevity in vitro. LC-MS/MS analysis of the two major proteins in the fractions identified these proteins as TF and ALB. Immunohistochemical analysis using specific antisera against TF and ALB indicated that both proteins were localized not only in the SSTs, but also in the surface epithelium of the UVJ. When the ejaculated sperm were incubated with either purified TF or ALB, sperm viability increased after 24 h. These results indicated that oviductal TF and ALB are involved in the process of sperm storage in SSTs and may open a new approach for technological improvement to prolong sperm longevity in vitro.

Prolactin receptor (PRLR) is expressed in a wide variety of tissues and mediates diverse biological actions of prolactin (PRL). In mammals, PRL signaling is thought to be involved not only in the process of spermatogenesis and steroidogenesis in the testis, but also in the survival of ejaculated sperm. In avian species, although the expression of PRLR with several variants in the testis was reported, the role of PRL in testicular function is still unclear. The aim of this study was to examine the expression of PRLR in the testis and mature sperm in quail. It is revealed that PRLR was mainly localized in the round- and elongated-spermatid by immunohistochemical analysis on the testis suggesting that PRL signaling may participate in the spermatogenesis. Western blot analysis confirmed the presence of PRLR in the plasma membrane of the ejaculated sperm (SPML), whereas the size of PRLR in the sperm was smaller than that in the hypothalamus. Moreover, PRLR was detected on the surface of the midpiece and flagellum of sperm by immunostaining. To evaluate the functionality of the sperm PRLR, the dot blot assay was performed to test the binding of pituitary PRL to PRLR in the SPML, and resulted in the detection of specific binding of PRL to the component of SPML, most likely to sperm PRLR. Furthermore, when the ejaculates were incubated with pituitary PRL to investigate the role of PRL on the sperm, the occurrence of spontaneous acrosome reaction was significantly decreased. In addition, the expression of PRL on the surface of utero-vaginal junction of oviduct was detected by immunohistochemistry. These results may suggest a novel system that the interaction between oviductal PRL and sperm PRLR is involved in the maintenance of the fertilizability of the spermatozoa through the prevention of the spontaneous acrosome reaction in Japanese quail.

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.

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.

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 vitropenetration 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.

DEAD-box helicase 4 (DDX4; also known as vasa) is essential for the proper formation and maintenance of germ cells. Although DDX4 is conserved in a variety of vertebrates and invertebrates, its roles differ between species. This study investigated the function of DDX4 in chicken embryos by knocking down its expression using retroviral vectors that encoded DDX4-targeting microRNAs. DDX4 was effectively depleted invitro and invivo via this approach. Male and female gonads of DDX4-knockdown embryos contained a decreased number of primordial germ cells, indicating that DDX4 is essential to maintain a normal level of these cells in chicken embryos of both sexes. Expression of doublesex and mab-3 related transcription factor 1 (DMRT1) and sex determining region Y-box 9 (SOX9), which are involved in testis determination and differentiation, was normal in male gonads of DDX4-knockdown embryos. In contrast, expression of cytochrome P450 family 19 subfamily A member 1 (CYP19A1), which encodes aromatase and is essential for ovary development, was significantly decreased in female gonads of DDX4-knockdown embryos. Expression of forkhead box L2 (FOXL2), which plays an important role in ovary differentiation, was also slightly reduced in DDX4-knockdown embryos, but not significantly. Based on several pieces of evidence FOXL2 was hypothesised to regulate aromatase expression. The results of this study indicate that aromatase expression is also regulated by several additional pathways.

This study was conducted to establish a new method of avian transgenesis by intracytoplasmic sperm injection (ICSI). First, we evaluated the fertilization ability of quail oocytes after microinjection of Triton X-100 (TX-100)-treated quail sperm with PLCZ cRNA. The quail oocytes were cultured for 24 h, and blastoderm development was examined by histological observation. The TX-100 treatment induced damage to the quail sperm membrane and interfered with fertilization of oocytes injected with sperm. On the other hand, when quail oocytes were injected with TX-100-treated sperm and PLCZ cRNA simultaneously, 43.5% (10/23) of the oocytes developed into blastoderms. This rate of development was comparable to that for oocytes injected with sperm without TX-100 treatment but with PLCZ cRNA (6 [42.9%] of 14). Second, we evaluated the rate of transduction of the enhanced green fluorescent protein (EGFP) gene in quail oocytes injected with TX-100-treated sperm and PLCZ cRNA. The EGFP expression was assessed by histological observation of fluorescence emission in the embryos. The intracytoplasmic injection of sperm without TX-100 treatment but with PLCZ cRNA and EGFP vector induced blastoderm development in 40% (4/10) of the oocytes, but those oocytes showed no fluorescence emission. In contrast, the intracytoplasmic injection of TX-100-treated sperm and PLCZ cRNA induced blastoderm development in 43.8% (7/16) of the oocytes, and, importantly, 85.7% (6/7) of oocytes showed fluorescence emission. In addition, PCR analysis detected GFP fragments in 50% (3/6) of GFP-expressing blastoderms. These results indicate that this ICSI method with additional treatments described herein may be the first step toward the production of transgenic birds.

The present study was conducted to show a profile of mRNA expression of sex-differentiation related genes in the duck gonad. Fertile duck eggs (Cherry-valley) were incubated (hatching at day 28 of incubation) and the gonads were collected at days 7, 8, 10, 12 and 15 of incubation after taking photos of the gonads. Left gonad was used for identification of genetic sex and for total RNA extraction. Reverse transcription-polymerase chain reaction (PCR) was performed using appropriate forward and reverse primers for each of FOXL2, P450arom, DMRT1, AMH, P450c17, SF1, ER α and AR genes of the chicken and the corresponding cDNA fragments were sequenced for duck. Subsequently, real-time PCR was performed to detect mRNA expression of those genes in the duck embryo. Male bilateral gonads assumed symmetry in appearance throughout the examined period, whereas the female gonads were noticeably asymmetric at 10 days of incubation with a smaller size of the right. Female asymmetry was distinct by day 15 of incubation. In females, FOXL2 and P450arom mRNA expression began at day 8 of incubation and remained high thereafter, but in males only negligible expression was detected. In males, mRNA expression of DMRT1 and AMH was detected at day 7 of incubation and tended to increase gradually thereafter, whereas in females the expression remained low throughout the examined period. Though the slight expression of P450c17 mRNA started at day 7 only in males, the expression was low throughout the examined period, whereas in female the expression was distinct at day 8 of incubation and remained high thereafter. Expressions of the other genes were variable with no differences in each expression between the sexes. The results indicate that FOXL2 and P450arom play an important role for the ovarian formation.

The present study was conducted to investigate (1) the effective day of treatment with Fadrozole, nonsteroidal aromatase inhibitor (AI), during incubation for sex reversal, (2) the mRNA expression of aromatase (P450arom), anti-Mullerian hormone (AMH) and estrogen receptor α (ERα) in quail gonad and gonadal structures after the AI treatment before hatching and (3) the effects of AI on gonad growth, left-right asymmetry of the gonad and body weight gain between 1 to 7 weeks posthatch. (1) Females AI-treated at 0, 2 and 4 days of incubation had symmetrical gonads at hatch similar to normal males, whereas those treated at 6 and 8 days of incubation had asymmetrical gonads. (2) In females AI-treated at day 0 of incubation, the left gonad became an ovotestis at day 15 of incubation, displaying seminiferous tubules with testicular-like cords and ovarian follicle with ovum. In the control group, P450arom mRNA expression was significantly higher in females than males. However, AI-treated females showed 2 response-patterns of either higher or lower expression. The latter was similar to that of control males. No significant differences in levels of AMH and ERα mRNA between sexes either in control or AI-treated groups was observed. (3) A gradual increase in body weight of the control males and females without sex difference was observed up to 5 weeks of age. However, at 6-7 weeks the average weight of females was heavier than males each in control and treated group. These results indicate that shortly before day 6 of incubation P450arom mRNA expression, at least in a part, is important for asymmetrical formation of gonads in female quail. In AI-treated females, the high responder show similar level of P450arom mRNA in control males and the low responder do not respond to the AI. Body weight is controlled non-gonadally in quail.

Although many studies have shown that P450aromatase (P450arom), anti-Mullerian hormone (AMH) and estrogen receptor α (ERα) play pivotal roles in sexual differentiation of the gonads during early embryonic development in chickens and quail, few studies have been reported for other domestic birds. Furthermore, little information is available in relation to the mRNA expression in gonads after sexual differentiation in posthatching birds. The present study was conducted to assay mRNA expression of P450arom, AMH and ERα in gonads at day of hatch in turkey and duck and 2 days after hatching in goose using the real-time PCR. The mRNA expression was also determined at one week of age in gonads of these birds. At the time of collection of the left gonads for total RNA extraction, the external appearance of the left and right gonads of male and female was documented by digital camera. Clear asymmetry was observed in the female ovary in which the right ovary was regressed completely by 1-2 days in posthatching turkey, duck and goose. Although gonadal asymmetry was as remarkable as in females, the left testis was larger than the right one in males. Remarkable expression of P450arom mRNA was observed only in females in all the 3 species but substantially no expression was detected in males. Significantly higher expression of AMH mRNA was detected in males than females only in goose but there was no sex difference in turkey and duck at 1-2 days posthatching and one week of age. Weak ERα mRNA expression without a sex difference was detected in the 3 birds. These results suggest that estrogen plays a key role for ovarian development via P450arom mRNA expression after hatching, whereas absence of its expression in males leads to testis development in turkey, duck and goose.

The present study was conducted to improve the rate and stage of blastoderm and embryo development of quail oocytes by intracytoplasmic sperm injection (ICSI) with quail phospholipase Cζ (PLCζ) cRNA. Blastoderm development of quail oocytes cultured for 24 or 72h were staged under a stereomicroscope. The blastoderms were stained with 4,6-diamidino-2-phenylindole (DAPI) for cell division progress. A quail PLCζ cDNA clone was isolated from sperm by RT-PCR. The 1978bp nucleotide sequence contained an ORF encoding 637 amino acids of protein. The deduced quail PLCζ protein consists of EF-hand domain, X and Y catalytic domain and C2 domain, but lacked a plextrin-homology (PH) domain at the N-terminus. RT-PCR analysis revealed that PLCζ mRNA is expressed only in the testis. ICSI into a quail oocyte without PLCζ cRNA induced blastodermal development in 16% (4/25) of the oocytes which reached stages III-VI 24h after culture. In contrast, ICSI together with PLCζ cRNA (in 3nl at 60μg/ml) into a quail oocyte induced blastoderm development more than 2 fold (36.8%, 7/19) with embryos reaching stages VI-VII. Furthermore, 72h after culture ICSI into a quail oocyte without PLCζ cRNA induced embryo development in 15.4% (2/13) of the oocytes and reached stages V-VII. In contrast, ICSI together with PLCζ cRNA (in 3nl at 60μg/ml) into a quail oocyte induced 3 fold more embryo development (50%, 9/18) with embryos between stages VI and over X. The microinjection of PLCζ cRNA significantly improved the conventional ICSI method by enhancing the proportion and the stages of embryo development. Accordingly, the present PLCζ cRNA microinjection method in birds may contribute to assist in the production of transgenic birds and protection of endangered species of birds.

Our previous study demonstrated that elongated spermatids and sperm carrying the female-specific W-chromosome of the sex-reversed domestic fowl can activate the mouse oocyte, but whether they can fertilize the avian oocyte and lead to a developing zygote remains undetermined. A single sperm isolated from the semen and testis of normal rooster and from a testis of sex-reversed hen was microinjected into a quail oocyte and cultured for 20 to 24 h. Blastoderms were fixed, cleaved, nuclei stained by 4',6'-diamidino-2-phenylin-dole, and developmental stages were assessed. In the normal rooster group, ejaculated and testicular sperm induced blastodermal development in 22.6 and 20% of the quail oocytes, respectively. The developmental stages ranged from IV to VII. In the sex-reversal group, 20% of injected testicular sperm induced blastodermal development. The blastodermal stages varied from stage III to VI. Blastoderms after 4',6'-diamidino-2-phenylindole staining were assayed by PCR to identify the W chromosome of either chicken sperm or quail oocyte. The PCR assay results showed that 2 out of 9 developed blastoderms microinjected with sperm of sex-reversed hen were identified containing the female-specific W chromosome derived from sex-reversed hen. From these results, it is concluded that chicken sperm bearing the W chromosome possess fertilizing ability and can function to stimulate blastoderm development similar to that of normal chicken sperm carrying the Z chromosome.

The biological activities of androgen are largely mediated via androgen receptor (AR). Although a partial sequence AR cDNA was revealed in canaries and zebra finch, AR cDNA has not been cloned in quail and chickens. To understand the physiological function of androgen and to apply it for other purposes, AR cDNA is necessary. Hence, this study was aimed to isolate the cDNA of AR and to reveal changes in mRNA expression using RT-PCR analysis in male reproductive organs of quail under different day-lengths and castration treatment. We found that a partial length of quail AR cDNA contains a 1385bp sequence encoding 343 amino acid residues and had high homology to other vertebrates. Quail were raised under continuous light regimen from 4 to 6 weeks old (6LL) and they were divided into 3 groups under the following conditions up to 9 weeks old : (1) continuous light regimen group (24h light ; 9LL), (2) short days regimen group (8h light and 16h darkness ; 9SD) and (3) castration group in which testis were surgically removed at 6 weeks old and raised under continuous light regimen (9CAS). Although weekly changes in the cloacal gland protrusion area showed significantly progressive increase at 7-9 weeks old in 9LL group and a progressive decrease in 9SD group, there were no changes in AR mRNA levels in the gland. In contrast, in 9CAS group, although the cloacal gland protrusion area decreased after castration treatment, AR mRNA levels increased in the gland. On the other hand, AR mRNA levels in epididymis and vas deferens increased in 9LL group, but there were no differences in 9SD and 9CAS groups from 6LL. The testicular AR mRNA levels did not change after long or short days treatment. These results indicate there is tissue-specific regulation in AR mRNA expression in quail.

Intracytoplasmic sperm injection (ICSI) technology in birds has been hampered due to opacity of oocyte. We developed ICSI-assisted fertilization and gene transfer in quail. This paper reviews recent advances of our ICSI experiments. The oocyte retrieved from the oviduct and a quail sperm was injected into the oocyte under a stereomicroscope. The oocyte was cultured for 24h at 41°C under 5% CO2 in air. The fertilization and development was assessed by microscopic observation. The fertility rate ranged 12-18% and development varied from stage II to V in trials. To improve the fertility rate, phospholipase C (PLC) zeta was injected with a sperm. It was increased to 37-50%. Furthermore, injection of inositol trisphosphate increased to over 85%. Quail oocyte can be fertilized with chicken sperm and so can testicular elongated spermatid. To extend embryonic development, chicken eggshell was used as a surrogate culture at 37°C after the 24h incubation at 41°C under 5% CO2 in air. It survived up to 2days thereafter. Finally, gene transfer was attempted in quail egg. The sperm membrane was disrupted with Triton X-100 (TX-100) and was injected with PLCzeta cRNA and enhanced green fluorescent protein (EGFP) gene in oocyte. The GFP expression was evaluated at 24h incubation at 41°C under 5% CO2 in air in the embryos. While the expression was not detected in the control oocytes, the experimental treatment induced blastoderm development (44%) of the oocytes and 86% of blastoderm showed fluorescent emission. In addition, PCR analysis detected EGFP fragments in 50% of GFP-expressing blastoderm. Our ICSI method may be the first step toward the production of transgenic birds.

This study was conducted to investigate the role of a sperm-borne compound in oocyte activation in special reference to the time when oocyte activation is required by testicular cells during spermatogenesis in quail. First, effects of a microinjection of quail sperm extract (SE) and quail phospholipase Czeta (PLCzeta) cRNA into quail oocytes were assessed by observation of pronuclear formation and cytoplasmic segmentation, respectively. Secondly, the effects of a microinjection of round spermatids with or without PLCzeta cRNA into quail oocytes were studied by observation of development. When the oocytes were injected with SE at 0.13 mg protein/ml, both pronuclear formation and cytoplasmic segmentation were optimally induced. However, pronuclear formation was blocked when SE was pretreated with heat or when the oocyte was pretreated with BAPTA (a Ca(2+) chelator) before SE injection. On the other hand, when the oocytes were injected with PLCzeta cRNA at 60 microg/ml, not only pronuclear formation but also cytoplasmic segmentation were optimally induced. However, PLCzeta cRNA-induced pronuclear formation was blocked by pretreatment with cycloheximide (an inhibitor of protein synthesis) or with BAPTA. Most interestingly, round spermatids alone cannot induce blastodermal development but microinjection of a round spermatid with PLCzeta cRNA can induce development. In addition, RT-PCR revealed that PLCzeta mRNA is expressed in elongated spermatids and testicular sperm but not in round spermatids. It is concluded that PLCzeta is a functional sperm factor for oocyte activation to initiate resumption of meiotic division in quail and its potency is acquired after elongated spermatid formation during the spermatogenesis.

Intracytoplasmic sperm injection (ICSI) is an important technique in animal biotechnology for animal cloning and conservation of genetic resources, but has been a challenge for avian species. In the present study, we investigated the ability of cryopreserved quail spermatozoa to achieve fertilisation and embryo development. Female quail were killed 70–120 min after previous oviposition to collect unfertilised oocytes from the oviduct. Fresh or cryopreserved–thawed spermatozoa were injected into the cytoplasm of unfertilised oocytes, and the manipulated oocytes were incubated in quail surrogate eggshells. Injection of fresh spermatozoa supplemented with inositol 1,4,5-trisphosphate (IP3) resulted in a significantly increased rate of embryo development compared with injection of fresh spermatozoa alone (90% vs 13%, respectively). Although >80% of embryos stopped cell division and development before Hamburger and Hamilton (HH) Stage 3, approximately 15% of embryos from the fresh sperm injection developed to past HH Stage 4, and one embryo survived up to HH Stage 39 (11 days of incubation). In the case of cryopreserved spermatozoa, the embryo development rate was 30% after ICSI, and this increased significantly to 74% with IP3 supplementation. In conclusion, cryopreserved spermatozoa combined with ICSI followed by surrogate eggshell culture can develop quail embryos.

Although a rise in intracellular calcium concentration of vertebrate oocytes plays a pivotal role for the initiation of fertilization or oocyte activation, no study on this subject has been reported in birds. This study was conducted to study the role of intracellular calcium in relation to fertilization in avian oocytes. First, immediately after a quail oocyte was injected with a sperm, it was treated with strontium chloride as an inducer for intracellular calcium rise at doses of 0, 2.5, 5, 7.5, 10 mM for 4 hr in the culture medium and was followed by 20-hr culture. Treatment with 5 mM of strontium chloride induced blastodermal development in 24.2% of injected eggs, although no oocytes developed without strontium treatment. Second, quail oocytes were injected with a sperm and 0.1 M calcium chloride or a sperm and saline solution, cultured without calcium for 4 hr and was followed by 20-hr culture without strontium. The calcium solution induced blastodermal development in 20.5% of the oocytes, although no oocytes developed without calcium treatment. Third, quail oocytes were injected with 1,2-bis (o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) as a calcium chelator, cultured with strontium (5 mM) for 4 hr followed by 20-hr culture without strontium. Only one oocyte developed after BAPTA and strontium treatment of 36 oocytes examined. Developmental stages of all the oocytes ranged from IV to VII. These results suggest that intracellular calcium rise may participate in quail oocyte activation and allow fertilization and blastodermal development. J. Exp. Zool. 307A:647–653, 2007. © 2007 Wiley-Liss, Inc.

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.