Tamao Ono

Phospholipase Cζ mRNA expression and its potency during spermatogenesis for activation of quail oocyte as a sperm factor

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 Cζ (PLCζ) 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 PLCζ 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 Ca2+ chelator) before SE injection. On the other hand, when the oocytes were injected with PLCζ cRNA at 60 µg/ml, not only pronuclear formation but also cytoplasmic segmentation were optimally induced. However, PLCζ 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 PLCζ cRNA can induce development. In addition, RT‐PCR revealed that PLCζ mRNA is expressed in elongated spermatids and testicular sperm but not in round spermatids. It is concluded that PLCζ 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. Mol. Reprod. Dev. 76: 1200–1207, 2009.

A Novel Method to Isolate Primordial Germ Cells and Its Use for the Generation of Germline Chimeras in Chicken

Abstract A novel method was developed to isolate chick primordial germ cells (PGCs) from circulating embryonic blood. This is a very simple and rapid method for the isolation of circulating PGCs (cPGCs) using an ammonium chloride-potassium (ACK) buffer for lysis of the red blood cells. The PGCs were purified as in vitro culture proceeded. Most of the initial red blood cells were removed in the first step using the ACK lysis buffer. The purity of the cPGCs after ACK treatment was 57.1%, and the recovery rate of cPGCs from whole blood was 90.3%. The ACK process removed only red blood cells and it did not affect cPGC morphology. In the second step, the red blood cells disappeared as the culture progressed. At 7 days of in vitro culture, the purity of the PGCs was 92.9%. Most of these cells expressed germline-specific antibodies, such as those against chicken vasa homolog (CVH). The cultured PGCs expressed the Cvh and Dazl genes. Chimeric chickens were produced from these cultured PGCs, and the donor cells were detected in the gonads, suggesting that the PGCs had biological function. In conclusion, this novel isolation system for PGCs should be easier to use than previous methods. The results of the present study suggest that this novel method will become a powerful tool for germline manipulation in the chicken.

Germline replacement by transfer of primordial germ cells into partially sterilized embryos in the chicken.

We report a novel technique for almost complete replacement of the recipient germline with donor germ cells in the chicken. Busulfan solubilized in a sustained-release emulsion was injected into the yolk of fertile eggs before incubation. A dose of 100 μg was found to provide the best outcome in terms of reducing the number of endogenous primordial germ cells (PGCs) in embryonic gonads (0.6% of control numbers) and hatchability (36.4%). This was applied for preparing partially sterilized embryos to serve as recipients for the transfer of exogenous PGCs. Immunohistochemical analysis showed that the proportion of donor PGCs in busulfan-treated embryos was significantly higher than in controls (98.6% vs. 6.4%). Genetic cross-test analysis revealed that the germline transmission rate in busulfan-treated chickens was significantly higher than in controls (99.5% vs. 6.0%). Of 11 chimeras, 7 produced only donor-derived progenies, suggesting that these produced only donor-derived gametes in the recipients gonads. This novel germline replacement technique provides a powerful tool for studying germline differentiation, for generating transgenic individuals, and for conserving genetic resources in birds.

Restriction of proliferation of primordial germ cells by the irradiation of Japanese quail embryos with soft X-rays.

Primordial germ cells (PGCs) are the progenitor cells for the gametes. Avian PGCs are located in the central region of the area pellucida at the blastoderm stage. Shortly after further incubation, they migrate to the extra-embryonic germinal crescent, and then as soon as the blood vessels form, they enter the circulation and finally settle in the gonadal primordium. We have developed a simple method using soft X-ray irradiation (18 kV power, 20 cm distance) to reduce the number of PGCs in Japanese quail embryos, which should be useful in preparing recipient embryos for PGC-transfer studies. When embryos were exposed to the soft X-rays for 40 s before incubation, the concentration of circulating PGCs was less than one-fifth that in controls after 2 days of incubation. Embryos at day 6 of incubation contained approximately half the number of PGCs compared to controls when they were exposed before or at day 2 of incubation. Irradiation for 40 s is recommended taking into consideration the restriction of proliferation of PGCs, and viability and hatchability.

Immunomagnetic purification of viable primordial germ cells of Japanese quail (Coturnix japonica).

Immunomagnetic cell sorting (MACS) with the monoclonal antibody (mAb) QCR1 was compared with the Ficoll density-gradient centrifugation system (FICS) in terms of the efficiency of enrichment of quail (Coturnix japonica) primordial germ cells (PGCs) from blood. The purified PGCs were tested for their ability to settle in the chick (Gallus domesticus) embryonic gonad. Blood containing 60-100 PGCs microliter-1 was taken from the dorsal aorta of quail embryos at Hamburger and Hamiltons stages 14-16. The amount and concentration of PGCs in the PGC-rich fraction purified by MACS were greater than in the fraction purified by FICS. Purified quail PGCs were transfused into chick embryos at stages 14-16 and immunohistochemically stained with mAb QCRI on day 8 of chick development. Transfused PGCs purified by either MACS or FICS were positively stained in the chick embryonic gonads.

Sex‐specific and left‐right asymmetric expression pattern of Bmp7 in the gonad of normal and sex‐reversed chicken embryos

A genetic switch determines whether the indifferent gonad develops into an ovary or a testis. In adult females of many avian species, the left ovary is functional while the right one regresses. In the embryo, bone morphogenetic proteins (BMP) mediate biological effects in many organ developments but their roles in avian sex determination and gonadal differentiation remains largely unknown. Here, we report the sex‐specific and left‐right (L‐R) asymmetric expression pattern of Bmp7 in the chicken gonadogenesis. Bmp7 was L‐R asymmetrically expressed at the beginning of genital ridge formation. After sexual differentiation occurred, sex‐specific expression pattern of Bmp7 was observed in the ovary mesenchyme. In addition, ovary‐specific Bmp7 expression was reduced in experimentally induced female‐to‐male reversal using the aromatase inhibitor (AI). These dynamic changes of expression pattern of Bmp7 in the gonad with or without AI treatment suggest that BMP may play roles in determination of L‐R asymmetric development and sex‐dependent differentiation in the avian gonadogenesis.

Fertilization and development of quail oocytes after intracytoplasmic sperm injection.

Abstract The present study was conducted to establish the intracytoplasmic sperm injection (ICSI) method for in vitro fertilization and development in quail. The efficiency of fertilization of oocytes was compared 1) between spontaneous and premature ovulation and 2) among testicular round spermatids, elongated spermatids, and immature and mature spermatozoa. The oocytes were injected with a single spermatozoon or spermatid and cultured for 24 h. Cell division was histologically observed with hematoxylin-eosin (HE) and a nucleus-specific fluorescent dye (DAPI). Five of 30 (16.6%) and 4 of 30 (13.3%) oocytes injected with mature sperm were fertilized in the spontaneous and induced ovulation group, respectively. Those embryos showed development at stages II–VII. Half the number (three of six) of the oocytes injected with testicular spermatozoa were fertilized and developed to stages IV–VII, and two of five oocytes injected with elongated spermatids were fertilized and developed to stage VI. All ooocytes injected with round spermatids were unfertilized. The results demonstrate that intracytoplasmic injection of a single sperm into quail oocyte can activate the oocyte and lead to fertilization. Oocytes prematurely ovulated are capable of fertilizing with mature sperm as are those spontaneously ovulated. In addition, the results suggest that the testicular round spermatids may not possess sufficient oocyte-activating potency but that the elongated spermatids and immature spermatozoa are competent to participate in fertilization and early embryonic development in quail.

Efficient system for preservation and regeneration of genetic resources in chicken: concurrent storage of primordial germ cells and live animals from early embryos of a rare indigenous fowl (Gifujidori)

The unique accessibility of chicken primordial germ cells (PGCs) during early development provides the opportunity to combine the reproduction of live animals with genetic conservation. Male and female Gifujidori fowl (GJ) PGCs were collected from the blood of early embryos, and cryopreserved in liquid nitrogen for >6 months until transfer. Manipulated GJ embryos were cultured until hatching; fertility tests indicated that they had normal reproductive abilities. Embryos from two lines of White Leghorn (24HS, ST) were used as recipients for chimera production following blood removal. The concentration of PGCs in the early embryonic blood of 24HS was significantly higher than in ST (P < 0.05). Frozen-thawed GJ PGCs were microinjected into the bloodstream of same-sex recipients. Offspring originating from GJ PGCs in ST recipients were obtained with a higher efficiency than those originating from GJ PGCs in 24HS recipients (23.3% v. 3.1%). Additionally, GJ progeny were successfully regenerated by crossing germline chimeras of the ST group. In conclusion, the cryogenic preservation of PGCs from early chicken embryos was combined with the conservation of live animals.

Increased proportion of donor primordial germ cells in chimeric gonads by sterilisation of recipient embryos using busulfan sustained-release emulsion in chickens.

The aim of the present study was to improve the efficiency of endogenous primordial germ cell (PGC) depletion and to increase the ratio of donor PGCs in the gonads of recipient chicken embryos. A sustained-release emulsion was prepared by emulsifying equal amounts of Ca(2+)- and Mg(2+)-free phosphate-buffered saline containing 10% busulfan solubilised in N,N-dimethylformamide and sesame oil, using a filter. Then, 75 microg per 50 microL busulfan sustained-release emulsion was injected into the yolk. To determine the depletion and repopulation of PGCs in the gonads after 6 days incubation, whole-mount immunostaining was performed. The busulfan sustained-release emulsion significantly reduced the number of endogenous PGCs compared with control (P < 0.05). Moreover, the busulfan sustained-release emulsion significantly depleted endogenous PGCs compared with other previously reported busulfan delivery systems (P < 0.05), but with less variation, suggesting that the sustained-release emulsion delivered a consistent amount of busulfan to the developing chicken embryos. The PGC transfer study showed that the proportion of donor PGCs in the gonads of busulfan sustained-release emulsion-treated embryos after 6 days incubation increased 28-fold compared with control. In conclusion, the results demonstrate that exogenous PGCs are capable of migrating and settling in gonads from which endogenous PGCs have been removed using a busulfan sustained-release emulsion.

Vitamin D and chick embryonic yolk calcium mobilization: identification and regulation of expression of vitamin D-dependent Ca2(+)-binding protein, calbindin-D28K, in the yolk sac.

The developing chick embryo acquires calcium from two sources. Until about Day 10 of incubation, the yolk is the only source; thereafter, calcium is also mobilized from the eggshell. We have previously shown that during normal chick embryonic development, vitamin D is involved in regulating yolk calcium mobilization, whereas vitamin K is required for eggshell calcium translocation by the chorioallantoic membrane. We have studied here the biochemical action of 1,25-dihydroxy vitamin D3 in the yolk sac by examining the expression and regulation of the cytosolic vitamin D-dependent calcium-binding protein, calbindin-D28K. Two types of embryos are used for this study, normal embryos developing in ovo and embryos maintained in long-term shell-less culture ex ovo, the latter being dependent solely on the yolk as their calcium source. Our findings are (1) calbindin-D28K is expressed in the embryonic yolk sac, detectable at incubation Days 9 and 14; (2) the embryonic yolk sac calbindin-D28K resembles that of the adult duodenum in both molecular weight (Mr 28,000) and isoelectric point, as well as the presence of E-F hand Ca2(+)-binding structural domains; (3) systemic calcium deficiency caused by shell-less culture of chick embryos results in enhanced expression of calbindin-D28K in the yolk sac during late development; (4) yolk sac calbindin-D28K expression is inducible by 1,25-dihydroxy vitamin D3 treatment in vivo and in vitro; and (5) immunohistochemistry revealed that yolk sac calbindin-D28K is localized exclusively to the cytoplasm of the yolk sac endoderm. These findings indicate that the chick embryonic yolk sac is a genuine target tissue of 1,25-dihydroxy vitamin D3.