Application study of intracytoplasmic sperm injection for golden hamster and cattle production

This paper describes several technical improvements and our results in hamster intracytoplasmic sperm injection (ICSI), hamster round spermatid injection (ROSI) and bovine ICSI. The hamster is the mammalian species in which ICSI was first tried to produce fertilized oocytes. However, until recently, no live offspring following ICSI have ever been obtained. We reported the birth of live offspring following hamster ICSI. Improved points to success were 1) performing hamster ICSI in a dark room with a small incandescent lamp and manipulating both oocytes and fertilized eggs under microscope with a red light source and 2) injecting sperm heads without acrosomes. Under controlled illumination, the majority of the oocytes injected with acrosomeless sperm heads were fertilized normally, cleaved, and developed into morulae. Nine live offspring (19%) were born by transfer of hamster ICSI-derived embryos. Furthermore, we reported the birth of live offspring following hamster ROSI. About 70% of oocytes injected with round spermatids broken before injection were fertilized normally and about half of them developed to morulae and blastocysts. Three (5%) live young were born by transfer of hamster ROSI-derived embryos. On the other hand, in cattle, the main improvements were 1) injection of spermatozoa immobilized by scoring their tail just before injection into oocytes, and 2) additional ethanol activation 4 h after ICSI. About 70% of oocytes injected were activated 4 h after ICSI, and about 30% of them developed to blastocysts. Twenty-four live calves (39%) were born by non-surgical transfer of ICSI-derived embryos. Those results shows that, at present, live offspring are able to be obtained following hamster ICSI, ROSI and bovine ICSI, but further improvement is required due to higher production efficiency of offspring.     

Birth of normal mice following round spermatid injection without artificial oocyte activation

For fertilization using round spermatid injection (ROSI) in mice, oocytes need to be artificially preactivated because of the lack of oocyte-activating capacity in round spermatids of this species. However, when round spermatids were frozen-thawed before microinjection, 11-71% of injected oocytes developed into 2-cell embryos without any artificial activation. After being transferred into recipient females, 5-27% of these embryos reached term. At least some of the injected oocytes showed intracellular Ca(2+) oscillations, which normally occur after fertilization by mature spermatozoa. Thus, these round spermatids could transmit a sperm-borne oocyte-activating factor, which might have been released from spermatozoa and elongated spermatids in the same suspension by freezing and thawing. This possibility was further supported by activation of intact oocytes following transplantation of the pronuclei from ROSI-generated embryos. Thus, one-step ROSI can be achieved in mice simply by injecting frozen-thawed round spermatids into intact oocytes. Clearly, there is a need for careful interpretation of microinjection experiments when assessing the oocyte-activating capacity of spermatogenic cells, especially when they are derived from frozen-thawed stocks.     

Microinsemination with first-wave round spermatids from immature male mice

In several mammalian species, including mice, round spermatids have been used to produce normal offspring by means of microinsemination techniques. In this study, we examined whether mouse round spermatids retrieved from immature testes undergoing the first wave of spermatogenesis had acquired fertilizing ability comparable to cells from mature adults. Microinsemination with round spermatids was performed by direct injection into preactivated oocytes, as previously reported. About 60-85% of the successfully injected oocytes developed to the morula/blastocyst stage after 72 h in culture, irrespective of the age of the males (17-25 days old). After embryo transfer, normal pups were obtained from all age groups, including the day-17 group, the stage at which the first round spermatids appeared. A high correlation (r=0.90) was found between the birth rate and male age (P<0.01, Spearman rank correlation), indicating that the efficiency of producing offspring was dependent on the age of the donor males. Imprinted genes (H19, Igf2, Meg3, and Igf2r) were expressed from the correct parental alleles (maternal, paternal, maternal, and maternal, respectively) in all (n=12) day-9.5 fetuses derived from day-20 spermatids. These results clearly indicate that at least some first-wave spermatogenic cells have a normal haploid genome with the correct paternal imprint and are capable of supporting full-term embryo development, as do mature spermatozoa from adults. The use of male germ cells from immature animals may save time in the production of inbred/congenic strains and rescue male-factor infertility of early onset.     

Nuclear Transfer of Freeze-Dried Somatic Cells into Enucleated Sheep Oocytes

Lyophilization has been used since long time to preserve yeast and bacteria strains. Subsequently, a great deal of efforts has been dedicated to the preservation in a dry state of red blood cells and platelets. However, despite more than 30 years passed by, no significant progress has been achieved. Recently, it has been reported that freeze-dried mice spermatozoa were able to generate normal offspring following injection into the mature mice oocytes. In this work, we prompted to apply the lyophilization protocol developed for mice spermatozoa to sheep somatic cells (lymphocytes and granulosa cells). More than 350 enucleated sheep oocytes were injected with granulosa cells, and freeze dried using the protocol developed for mice sperm cells. Transplanted nuclei organized large pronuclei with fragmented DNA, but none of them entered the first mitosis. In the second part of the experiments, trehalose and EGTA were found to reduce significantly the extent of nuclear damage (65% and 55% intact nuclei in lymphocyte and granulosa cells, respectively) following freeze drying. Granulosa cells lyophilized with EGTA/trehalose and stored at room temperature for 3 years were used for nuclear transfer, and the injected oocytes were cultured in vitro for 7 days. Approximately 16% of the oocyte injected with freeze-dried cells developed into blastocysts. To conclude, we demonstrated for the first time that nucleated cells maintain genomic integrity after prolonged storage in a dry state, and we were able to achieve early embryonic development following injection of these cells into enucleated sheep oocytes.     

The migration of larval Toxocara canis in mice II. Post-intestinal migration in primary infections

Following oral infection of NIH mice with Toxocara canis embryonated eggs the L₂ pass the visceral phase of migration during the first week of infection. Larvae reach the liver and lungs and peak in number in these organs 2 and 3 days after infection, respectively. Larvae are then dispersed throughout the body and enter the myotropic—neurotropic phase by the 7th day of infection. Larvae injected directly into the brain are capable of migrating into the viscera and musculature. Considerable pathology occurs due to larval migrations, especially through the liver and lungs, and both acute and chronic disease are recorded. Studies of infections extending over a year show that the number of recoverable larvae declines gradually with periods of stable populations.On Days 3, 4 and 5 after infection, larvae were demonstrable in the faeces of infected mice. Prenatal infection was observed in a third of the offspring of mice infected the same day as conception.     

Transgenic mouse offspring generated by ROSI

The production of transgenic animals is an important tool for experimental and applied biology. Over the years, many approaches for the production of transgenic animals have been tried, including pronuclear microinjection, sperm-mediated gene transfer, transfection of male germ cells, somatic cell nuclear transfer and the use of lentiviral vectors. In the present study, we developed a new transgene delivery approach, and we report for the first time the production of transgenic animals by co-injection of DNA and round spermatid nuclei into non-fertilized mouse oocytes (ROSI). The transgene used was a construct containing the human CMV immediate early promoter and the enhanced GFP gene. With this procedure, 12% of the live offspring we obtained carried the transgene. This efficiency of transgenic production by ROSI was similar to the efficiency by pronuclear injection or intracytoplasmic injection of male gamete nuclei (ICSI). However, ICSI required fewer embryos to produce the same number of transgenic animals. The expression of Egfp mRNA and fluorescence of EGFP were found in the majority of the organs examined in 4 transgenic lines generated by ROSI. Tissue morphology and transgene expression were not distinguishable between transgenic animals produced by ROSI or pronuclear injection. Furthermore, our results are of particular interest because they indicate that the transgene incorporation mediated by intracytoplasmic injection of male gamete nuclei is not an exclusive property of mature sperm cell nuclei with compact chromatin but it can be accomplished with immature sperm cell nuclei with decondensed chromatin as well. The present study also provides alternative procedures for transgene delivery into embryos or reconstituted oocytes.     

The control of lymphoid leukosis in a flock White Plymouth Rock chickens

Summary Lymphoid leukosis (LL) was successfully controlled in a commercial basic breeding line of White Plymouth Rock chickens. The control method has been developed for breeder flocks and consists of three elements: - In the flock under study, homogenates of embryos from all eggs collected during a number of I4-day periods are tested for the presence of LL viruses. - Only eggs from hens that have been shown not to shed virus in their eggs are used for the production of progeny. The offspring are reared in isolation during the first two months of life, at which time the age-related resistance against tumour formation by LL viruses appears to be sufficiently developed. - The chickens are subsequently inoculated intramuscularly with LL viruses of subgroups A and B transferred to a conventional chicken house. The vaccination raises a solid immunity to horizontal LL virus exposure and, due to the age-related resistance, tumour formation does not follow. No excretion of LL viruses could be detected in three generations of White Plymouth Rock chickens to which the three elements of the control procedure were applied. Clinical disease was not observed in any of the chickens under notice.     

The cycle of the seminiferous epithelium in the greater Japanese shrew mole, Urotrichus talpoides

Spermatogenesis and acrosomal formation in the greater Japanese shrew mole, Urotrichus talpoides, were studied by light microscopy. On the basis of acrosomal changes, morphology of spermatid head, nuclear shape, appearance of meiotic figures, location of spermatid and period of spermiation, the cycle of the seminiferous epithelium was classified into 12 stages, and developing spermatids could be divided into 15 steps. The mean relative frequencies of stages from I to XII were 10.9, 8.7, 9.8, 7.3, 8.5, 10.3, 12.5, 8.7, 5.8, 5.4, 5.1 and 7.1%, respectively. Similar to the case in the musk shrew, the spermatid nucleus of the greater Japanese shrew mole remained in the middle region of the seminiferous epithelium and only the acrosome extended towards the basement membrane. The elongation of the acrosome, however, was not prominent. The proacrosomal vesicle first appeared in stage II and then one large and round granule was seen in stage III. The acrosomal vesicle became flattened on the surface of the nucleus in stage IV. Spreading of the acrosomic system has been recognized from stage VII. In stage VII, spermiation occurred. In stage IX, the spermatid nucleus began to elongate. Elongation and condensation of the nucleus were clearly observed in stage X. In stage XII, pachytene spermatocytes divided into diplotene spermatocytes. In stage XII, meiotic figures and secondary spermatocytes were observed.     

A3启动子缺陷piggyBac转座子在家蚕中的转基因研究

构建了A3启动子缺陷piggyBac转座质粒,以增强型绿色荧光蛋白基因EGFP为标记基因对家蚕品种N is-tari进行转基因实验,发现其具有较高的表达EGFP的转化效率,G0代中EGFP阳性蛾区检出占总注射蚕卵的比率达0.618%,且EGFP的表达呈组织特异性。PCR实验分析也证实了标记基因的成功导入。该实验中标记基因及转基因阳性个体均易于检出,为启动子缺陷转基因方法在家蚕组织特异性启动子筛选研究上的应用奠定了基础。     

Molecular characteristics of horse phospholipase C zeta (PLCζ)

A sperm‐specific phospholipase C (PLC), PLCzeta (PLCζ), is thought to underlie the initiation of calcium ([Ca²⁺]_i) oscillations that induce egg activation in mammals. In large domestic species, only bovine, porcine and recently equine PLCζ have been cloned, and the physiological functions of these molecules have not been fully characterized. Here, we evaluated the physiological functions of equine PLCζ (ePLCζ) in mouse oocytes. ePLCζ was cloned from testis using RT‐PCR. The expression of ePLCζ messenger RNA was confirmed in testis but not in other tissues. Microinjection of ePLCζ complementary RNA (cRNA) into mouse oocytes induced long‐lasting [Ca²⁺]_i oscillations, and most of the injected oocytes formed pronuclei (PN). The injection of cRNAs encoding horse, mouse, human and cow PLCζ into mouse oocytes showed that ePLCζ had the highest [Ca²⁺]_i oscillation‐inducing activity among the species tested. Mutation of D202R, which renders the protein inactive, abrogated the activity of ePLCζ. The nuclear translocation ability of ePLCζ was defective when expressed in mouse oocytes. Taken together, our findings show for the first time that ePLCζ has highest activity of the mammalian species studied to date. Our findings will be useful for the improvement of reproductive technologies in the horse.     

Immunizing potential of various stages of Taenia ovis eggs injected subcutaneously into neonatal or sixteen-week-old lambs

When viable eggs of Taenia ovis were given orally to 1-week-old lambs, infection occurred only in those lambs that had been deprived of colostrum. When viable eggs were injected subcutaneously into 1-week-old lambs, no larvae developed at the injection site, and no resistance was stimulated against an oral challenge of T. ovis eggs given 11 weeks later. However, when eggs, oncospheres or developing cysticerci were subcutaneously injected into 16-week-old lambs, all grew at the injection site and stimulated a high degree of immunity to oral infection. Colostrum-derived antibodies against T. ovis apparently suppressed the immunizing potential of T. ovis eggs injected subcutaneously into neonatal lambs.     

The control of lymphoid leukosis in a flock White Plymouth Rock chickens

Summary

Lymphoid leukosis (LL) was successfully controlled in a commercial basic breeding line of White Plymouth Rock chickens. The control method has been developed for breeder flocks and consists of three elements: - In the flock under study, homogenates of embryos from all eggs collected during a number of I4‐day periods are tested for the presence of LL viruses.

- Only eggs from hens that have been shown not to shed virus in their eggs are used for the production of progeny. The offspring are reared in isolation during the first two months of life, at which time the age‐related resistance against tumour formation by LL viruses appears to be sufficiently developed.

- The chickens are subsequently inoculated intramuscularly with LL viruses of subgroups A and B transferred to a conventional chicken house.

The vaccination raises a solid immunity to horizontal LL virus exposure and, due to the age‐related resistance, tumour formation does not follow.

No excretion of LL viruses could be detected in three generations of White Plymouth Rock chickens to which the three elements of the control procedure were applied. Clinical disease was not observed in any of the chickens under notice.     

The developmental ability of vitrified oocytes from different mouse strains assessed by parthenogenetic activation and intracytoplasmic sperm injection

Assessment of the developmental ability of oocytes following freezing and thawing is an important step for optimizing oocyte cryopreservation techniques. However, the in vitro fertilization of frozen-thawed mouse oocytes is often inefficient because of incomplete capacitation of spermatozoa in the absence of surrounding cumulus cells. This study was undertaken to determine whether the oocyte cryopreservation efficiency of different strains of mice could be assessed from the development of oocytes following parthenogenetic activation and intracytoplasmic sperm injection (ICSI). Oocytes were collected from hybrid (C57BL/6 x DBA/2) F1 or inbred (C57BL/6J, C3H/HeN, DBA/2J and BALB/cA) strains and were vitrified in a solution containing ethylene glycol, DMSO, Ficoll and sucrose. In the first series of experiments, oocytes were activated parthenogenetically by Sr(2+) treatment after warming. The oocytes from the inbred strains, but not those of the F1 hybrid, were diploidized by cytochalasin treatment to obtain a sufficient number of blastocysts. In all strains tested, parthenogenetic embryos derived from vitrified oocytes developed into blastocysts at rates between 23 and 68%. In the second series of experiments, vitrified oocytes from each strain were injected with homologous spermatozoa after warming. Normal offspring were obtained from all strains at rates between 5 and 26% per embryo transferred. Thus, the feasibility of oocyte cryopreservation protocols can be assessed easily by in vitro development of parthenogenetic embryos or by in vivo development of ICSI embryos. Moreover, the oocytes of these four major inbred strains of mice can be cryopreserved safely for production of offspring.     

水平显微注射法制备转基因小鼠的研究

由于传统显微注射法具有操作繁琐、技术要求高和外源基因导入率低等局限性,本试验对常规显微注射法进行改进,旨在建立一套比较完善的水平显微注射方法。采用水平微注射系统,将外源DNA片段导入胚胎的雄原核中,获得子代鼠,分娩后3周提取鼠尾基因组DNA,PCR法筛选转基因阳性鼠。本试验采用Puc/BLG IN S和Chym os in基因,共使用710枚鼠原核胚进行研究,胚胎经注射后移植了25只受体,产下仔鼠77只;妊娠率分别为33.3%(5/15)和50%(5/10),胚胎成活率分别为9.47%(41/433)和l3.00%(36/277),阳性率分别为4.88%(2/41)和8.33%(3/36)。将阳性雌鼠配种妊娠后,对外源基因整合情况进行检测,结果表明初步获得了转基因阳性小鼠。     

Optimized cryopreservation of mouse sperm based on fertilization rate

Although procedures for in vitro fertilization with cryopreserved sperm have been published there is a lack of data indicating that the cryoprotectant and cryopreservation procedures used for those procedures were optimal. To redress this, fertilization rate of eggs exposed to sperm in vitro was used as the outcome in the optimization of raffinose concentration in the cryoprotectant (raffinose in water), volume of cryoprotectant, and freezing conditions for C57BL/6J mouse sperm. Sperm were frozen in a cylindrical Dewar with an internal diameter and height of 14.0 cm and 36.0 cm respectively. The optimal concentration of raffinose was 23-24% (510-540 mOsm/kg). The optimal volume of cryoprotectant used to prepare the sperm suspension from a single mouse was 180-400 μl, and sperm proved most fertile when frozen 13-25 mm above liquid nitrogen. Raffinose in the fertilization medium did not inhibit fertilization. Fertilized eggs transferred to oviducts of recipient mice developed into viable offspring.     

A new ENU-induced mutant mouse with defective spermatogenesis caused by a nonsense mutation of the syntaxin 2/epimorphin (Stx2/Epim) gene

Repro34 is an N-ethyl-N-nitrosourea (ENU)-induced mutation in mice showing male-specific infertility caused by defective spermatogenesis. In the present study, we investigated pathogenesis and molecular lesions in relation to spermatogenesis in the repro34/repro34 homozygous mouse. Histological examination of the testis showed that the seminiferous epithelium of the repro34/repro34 mouse contained spermatogonia and spermatocytes but no round and elongating spermatids. Instead of these haploid cells, multinucleated giant cells occupied the niche of the seminiferous tubules. Immunohistochemical staining for Hsc70t, an elongating spermatid specific protein, confirmed the absence of elongating spermatids. Furthermore, RT-PCR showed that there were significantly reduced expressions of the marker genes specifically expressed in the spermatid and that there was no difference in the expressions of the spermatocyte specific marker genes. These findings indicated interruption of the spermatogenesis during transition from the spermatocyte to spermatid in the repro34/repro34 mouse. The repro34 locus has been mapped on a 7.0-Mb region of mouse chromosome 5 containing the Syntaxin 2/Epimorphin (Stx2/Epim) gene, and targeted disruption of this gene has been reported to cause defective spermatogenesis. We therefore sequenced the entire coding region of the Stx2/Epim gene and found a nucleotide substitution that results in a nonsense mutation of this gene. The expression pattern of the Stx2/Epim gene during the first wave of spermatogenesis, increased expression at later stages of spermatogenesis, was in agreement with the affected phase of spermatogenesis in the adult repro34/repro34 testis. We therefore concluded that the male infertility of the repro34/repro34 mouse is caused by the interruption of spermatogenesis during transition from the spermatocyte to spermatid and that the nonsense mutation of the Stx2/Epim gene is responsible for the interruption of spermatogenesis.     

脂质体转染胚盘细胞制备携pEGFP—C2基因的转基因鹌鹑

利用脂质体转染第X期胚盘细胞的方法将外源质粒pEGFP—C2经脂质体包裹后注射到鹌鹑种蛋X期胚盘下腔,处理种蛋110枚,封口后孵化。出壳9只G0代,出壳率为8．18％。经检验6只鹌鹑中有4只为阳性,阳性率为66．67％。对成年的4只转基因鹌鹑中的1只内脏组织进行PCR分析以及切片荧光检测,证实外源基因在G0代成年鹌鹑组织中成功表达。将3只成年转基因鹌鹑分别与野生型鹌鹑进行杂交,得到G1代阳性率分别为33．96％、20．59％、10％。再将3只成年转基因公母鹌鹑之间进行杂交,得到G,代阳性率分别为10．87％、42．86％。证实利用脂质体转染胚盘细胞制备转基因鹌鹑是可以将外源基因整合到G0代生殖系中,且外源基因能遗传给后代。     

Xenografting of gonadal tissues into mice as a possible method for conservation and utilization of porcine genetic resources

In vitro production of embryos, including in vitro maturation, fertilization of oocytes and their subsequent culture to the embryo stage, has become the most popular method of studying gametogenesis and embryogenesis in pigs. As well as their utility for basic studies, these procedures now enable us to generate viable embryos and offspring as a means of conserving genetic resources and rare animal breeds. Recently, more advanced technologies such as xenografting of gonadal (testicular and ovarian) tissues into immunodeficient experimental animals have been developed. In combination with in vitro embryo production techniques, this approach may provide many benefits. We have been carrying out studies to acquire basic information about the application of this method to porcine species, and to improve the existing techniques. Recently, we obtained oocytes from ovarian tissue xenografted and grown in nude mice that had the capacity to be fertilized and the ability to develop into early‐stage embryos. We also obtained spermatozoa from the xenografted testicular tissues and injected them intracytoplasmically into in vitro‐matured oocytes to produce piglets. Here we discuss the further possibilities of conservation and utilization of porcine gonadal tissue by xenografting into immunodeficient mice.     

Non‐invasive sampling technique for DNA extraction from captive Japanese Crested Ibis on Sado Island

The Japanese Crested Ibis Nipponia nippon is a critically threatened bird. The post‐hatch eggs of the current captive population of this species on Sado Island have been stored at room temperature for the long‐term. In this study, we investigated the suitability of the vascularized chorioallantois membrane from the eggs as a non‐invasive DNA source. Using microsatellite loci developed for the Japanese Crested Ibis, we performed three experiments for comparison of genotypes obtained among DNA. First, DNA from five different sites of the identical membrane showed the same genotypes at either of two loci examined. Second, DNA from the membrane of each full‐sibling birds and blood of their parents showed the genotypes that were consistent with Mendelian parent–offspring relationships at any of eight loci examined. Third, DNA from the membrane and blood of the same bird showed the matched genotypes at any of eight loci examined. These results indicate that the vascularized chorioallantois membrane from post‐hatch eggs stored at room temperature for the long‐ term can be used as a reliable DNA source of offspring that had hatched from the egg. This study will promote a molecular genetics study on genetic diversity of the current captive Japanese Crested Ibis population on Sado Island.     

In ovo injection of branched‐chain amino acids: Embryonic development,hatchability and hatching quality of turkey poults

In this study, the influence of a branched‐chain amino acid blend (BCAA composed of 3 l ‐leucine:1 l ‐valine:2 l ‐isoleucine) injected into the amniotic fluid was evaluated for embryonic growth, yolk‐sac (YS) utilization and development of gastrointestinal tract (GIT) and skeletal muscles of turkey embryos from day 24 of incubation (24E) to hatching, together with hatchability, poult quality and liver L* (lightness), a* (redness) and b* (yellowness) values at hatch. At day 22 of incubation, embryonated eggs (n = 240) were assigned to three treatments, that is, eggs were not injected (control, NC) or injected with 1.5 ml sterile solution with 0.9% salt (SA) or 0.2% BCAA blend (BCAAb). These solutions were injected manually into the amniotic fluid of the embryonated eggs. To determine weights and lengths (where appropriate) of the studied organs and tissues, four embryonated eggs and poults per treatment were selected at 24E and at hatch. While the BCAAb decreased the YS and embryo weight, hatchability and the liver L* value, it increased the weight and quality of poults and the weights of breast and thigh muscles at hatch. In conclusion, the in ovo feeding of the BCAA blend negatively affected hatchability but positively affected hatching weight and poult quality by improving development of skeletal muscles and by regulating energy metabolism.