Embryo-uterine interaction in implantation

Pregnant donor (day 3) and non-pregnant recipient rats were hypophysectomized and injected daily for 6 days with 2 mg of progesterone. A single dose of 20 ng of estradiol-17β in saline was administered via a tail vein to either the donor, the recipient, or to both animals; blastocysts were transferred 60 to 90 minutes after the latter injection. Twenty-four hours later uterine implantation sites were delineated by injection of Chicago Blue-B dye. The results indicate that both the blastocyst and the uterus must be exposed to estrogen to obtain normal implantation rates. While 43.2% of the embryos implanted when both the donor and the recipient received estrogen, only 6.3% implanted when only the recipient was injected with estrogen. No implantations were found in animals in which only the embryos had been exposed to estrogen, suggesting that if this steroid was synthesized by the embryo it was insufficient to induce implantation in the rat.     

The effect of antibiotic treatment on the in vivo selection of resistant haemolytic Escherichia coli clones in mice

Abstract The appearance of resistant haemolytic Escherichia coli clones was studied in mice treated with a synergistic combination of antibiotics. The R plasmid donor and the haemolytic recipient were given orally. Ten animals were treated i.p. with 2.5 mg carbenicillin and 2.5 mg kanamycin pro die for 7 days. Ten control mice were injected with saline. Out of 80 faecal samples of animals receiving antibiotic treatment resistant haemolytic transconjugants were isolated from 13 samples by direct plating, and from 27 samples after selective enrichment. Out of 80 parallel samples of control animals a single one yielded a positive culture — after enrichment.     

Stable and unstable transformation by microinjection of macronucleoplasm in Paramecium.

Transformation by microinjection of macronucleoplasm in Paramecium caudatum was investigated. Macronucleoplasm with three genetic markers (behavior, trichocyst, and mating type) was injected into the macronucleus. To facilitate microinjection, in most cases, paramecia were immobilized in a gelatin (7.5%) solution. The injected cells began to express a dominant gene (cnrA+ or cnrB+) of the donor 9-24 hr after injection. Expression did not require cell division suggesting injected macronucleoplasm was capable of expressing a phenotype. The amount of injected macronucleoplasm appears to correlate with the frequency of successful expression but not to correlate with the time required for expression. After a number of fissions, the injected cells produced clones which had cells expressing the phenotype of the donor. This suggests that injected macronucleoplasm was replicated and expressed in the recipient cell lines. The transformed clones were classified into two groups. In one group, transformation was stable. All cell lines derived from the injected cells expressed a phenotype similar to the heterozygote of donor and recipient cells. In the other group, transformation was unstable. During the first five to seven fissions after injection, at each division, cells produced one daughter cell which later reverted to the recipient phenotype. After this unstable period, cells no longer produced the recipient phenotype but produced the donor phenotype exclusively. Donor and recipient phenotypes were, thus, segregated in different cell lines. Observation of genetic markers and analysis by computer simulation shed light on the mode of transmission of injected macronucleoplasm. In stable transformation, injected macronucleoplasm appears to be distributed equally to daughter cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Modified Method for Heterotopic Mouse Heart Transplantion

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents available. A difficulty is presented due to the small size of the animal and the considerable technical challenges of the microsurgery involved in heart transplantation. In particular, a high rate of technical failure early after transplantation may result from recipient death and post-operative complications such as hind limb paralysis or a non-beating heart. Here, the complete technique for heterotopic mouse heart transplantation is demonstrated, involving harvesting the donor heart and its subsequent implantation into a recipient mouse. The donor heart is harvested immediately following in situ perfusion with cold heparinized saline and transection of the ascending aorta and pulmonary artery. The recipient operation involves preparation of the abdominal aorta and inferior vena cava (IVC), followed by end-to-side anastomosis of the donor aorta with the recipient aorta using a single running 10-0 microsuture and a similar anastomosis of the donor pulmonary artery with the recipient IVC. Following the operation the animal is injected with 0.6 ml normal saline subcutaneously and allowed to recover on a 37 °C heating pad. The results from 227 mouse heart transplants are summarized with a success rate at 48 hr of 86.8%. Of the 13.2% failures within 48 hr, 5 (2.2%) experienced hind limb paralysis, 10 (4.4%) had a non-beating heart due to graft ischemic injury and/or thrombosis, while 15 (6.6%) died within 48 hr.     

Nicotine-induced respiratory effects of cigarette smoke in dogs

We report that nicotine is responsible for both a blood-borne stimulation of the respiratory center and a direct effect on intrathoracic airway tone in dogs. We introduced cigarette smoke into the lungs of donor dogs and injected arterial blood obtained from them into the circulation of recipient dogs to show that a blood-borne material increased breathing and airway smooth muscle tone. Smoke from cigarettes containing 2.64 mg of nicotine was effective; that from cigarettes containing 0.42 mg of nicotine was not. Nicotine, in doses comparable to the amounts absorbed from smoke, also increased breathing and tracheal smooth muscle tension when injected into the vertebral circulation of recipient dogs. Finally, blockade of nicotine receptors in the central nervous system and in the airway parasympathetic ganglia inhibited the effects of inhaled cigarette smoke and intravenous nicotine on the respiratory center and on bronchomotor tone. We conclude that nicotine absorbed from cigarette smoke is the main cause of cigarette smoke-induced bronchoconstriction. It caused central respiratory stimulation, resulting in increased breathing and airway smooth muscle tension, and had a direct effect on airway parasympathetic ganglia as well.     

Production of chimeric loach by cell transplantation from genetically pigmented to orange embryos

To establish techniques for chimera formation and to obtain further knowledge of chimerism, chimeric loach were produced using the wild strain as the donor and the orange strain as the recipient by cell transplantation. Transplantation between embryos at two different stages was performed to achieve efficient chimera formation. In the combination of the early-mid-blastula as the donor and the late-blastula as the recipient, 100-150 blastomeres were injected into the blastoderm of the recipient and the rate of chimera formation was 46.2%. On the other hand, in the combination of early-mid-blastula and early-gastrula, only 30 blastomeres were injected and the rate of chimera formation was 80.0%. These results demonstrating the combination of embryonic stages may provide a key for efficient chimera formation. We also compared the number of melanophores on chimeric larvae with that on donor cells labelled with latex beads; it was found that the number of transplanted cells has a profound effect on chimerism, whereas the site of pigmentation is not always in agreement with the site of actual transplantation of donor cells.     

Transplantation of germ cells from rabbits and dogs into mouse testes.

Spermatogonial stem cells of a fertile mouse transplanted into the seminiferous tubules of an infertile mouse can develop spermatogenesis and transmit the donor haplotype to progeny of the recipient mouse. When testis cells from rats or hamsters were transplanted to the testes of immunodeficient mice, complete rat or hamster spermatogenesis occurred in the recipient mouse testes, albeit with lower efficiency for the hamster. The objective of the present study was to investigate the effect of increasing phylogenetic distance between donor and recipient animals on the outcome of spermatogonial transplantation. Testis cells were collected from donor rabbits and dogs and transplanted into testes of immunodeficient recipient mice in which endogenous spermatogenesis had been destroyed. In separate experiments, rabbit or dog testis cells were frozen and stored in liquid nitrogen or cultured for 1 mo before transplantation to mice. Recipient testes were analyzed, using donor-specific polyclonal antibodies, from 1 to >12 mo after transplantation for the presence of donor germ cells. In addition, the presence of canine cells in recipient testes was demonstrated by polymerase chain reaction using primers specific for canine alpha-satellite DNA. Donor germ cells were present in the testes of all but one recipient. Donor germ cells predominantly formed chains and networks of round cells connected by intercellular bridges, but later stages of donor-derived spermatogenesis were not observed. The pattern of colonization after transplantation of cultured cells did not resemble spermatogonial proliferation. These results indicate that fresh and cryopreserved germ cells can colonize the mouse testis but do not differentiate beyond the stage of spermatogonial expansion.     

Long-term follow-up of porcine male germ cells transplanted into mouse testes

This study investigated the effect of increased phylogenetic distance on the outcome of spermatogonial transplantation, with porcine donors and mice recipients. It was designed to develop a technique for detecting foreign donor cells in recipient animals. Porcine male germ cells were harvested from postnatal male testes and incubated with the lipophilic membrane dye PKH-26. For transplantation, approximately 10(6) PKH-26-labelled porcine male germ cells were injected into the efferent ducts of mouse testes. Animals were sacrificed at post-graft days 1, 10, 30, 45, 60 and 150 (n = 5 each). Serial frozen sections of explanted testes were prepared for detecting labelled cells. Transplanted porcine donor cells were easily detected in the recipient tubules for 8 weeks. After transplantation, we could detect both incorporation into the basement membrane and differentiation of grafted porcine donor cells by our double detection system, using PKH staining and slide PCR. However, our RT-PCR and apoptosis results revealed that most of the grafted porcine male donor cells could not differentiate past early-meiotic spermatocytes. We could induce partial differentiation of xenografted porcine donor cells in mouse testes, but not full induction of spermatogenesis. We have developed a very reliable technique for detecting foreign donor cells in recipient animals using a combination of PKH staining and slide PCR methods. Our results provide a valuable experimental model for applying and evaluating this technology in other species.     

Translocation of Gunnison's prairie dogs from an urban and suburban colony to abandoned wildland colonies

Translocating prairie dogs from areas in or near human developments to wildlands can reduce conflicts with humans or supplement wild populations, but translocation methods differ in cost and fate of translocated individuals is often difficult to assess. We translocated 74 Gunnison's prairie dogs from 1 source colony in downtown Flagstaff, Arizona (urban) and 75 from 1 source colony in lower density housing outside the city (suburban) to 2 abandoned, recipient colonies on open grasslands 50 km north of the city (wildland). We released animals into uncaged, pre-existing burrow entrances (hard release) or into temporary cages over pre-existing burrow entrances (soft release). We captured 15 (10%) marked animals 1 year post-translocation at the 2 recipient colonies, 7 from soft release treatments and 8 from hard release treatments but visual surveys indicated a minimum of 57 adult prairie dogs and 76 pups present. Adult prairie dogs in all photographs taken by automated cameras placed at burrow entrances at each recipient colony had ear tags, suggesting that most animals at these colonies were survivors from translocation and that survival was likely higher than 10%. By 1 year post-release, recipient colonies occupied an area roughly 9–18 times that of source colonies. Urban Gunnison's prairie dogs can be successfully translocated to abandoned wildland colonies without using soft release methods, but animals may disperse widely. Given the cost and effort translocation requires, and the fact that all 6 confirmed mortalities were from human shooting, we recommend temporary restrictions on shooting at recipient colonies until populations have met management goals. © 2011 The Wildlife Society.     

A cloned toy poodle produced from somatic cells derived from an aged female dog

To date, dogs have been cloned with somatic cell nuclear transfer (SCNT), using donor cells derived from large-breed dogs 2 months to 3 years of age. The objective of the present study was to use SCNT to produce a small-breed dog from ear fibroblasts of an aged poodle, using large-breed oocyte donors and surrogate females, and to determine the origin of its mitochondrial DNA (mtDNA) and the length of its telomeres. Oocytes were derived from large-breed donors, matured in vivo, collected by flushing oviducts, and reconstructed with somatic cells derived from an aged (14-year-old) female toy poodle. Oocytes and donor cells were fused by electric stimuli, activated chemically, and transferred into the oviducts of large-breed recipient females. Overall, 358 activated couplets were surgically transferred into the oviducts of 20 recipient dogs. Two recipients became pregnant; only one maintained pregnancy to term, and a live puppy (weighing 190 g) was delivered by Caesarean section. The cloned poodle was phenotypically and genetically identical to the nuclear donor dog; however, its mtDNA was from the oocyte donor, and its mean telomere length was not significantly different from that of the nuclear donor. In summary, we demonstrated that a small-breed dog could be cloned by transferring activated couplets produced by fusion of somatic cells from a small-breed, aged donor female with enucleated in-vivo-matured oocytes of large-breed females, and transferred into the oviduct of large-breed recipient female dogs.     

四氧嘧啶诱发糖尿病模型效果的性别差异

目的了解性别因素对四氧嘧啶诱发糖尿病动物模型的影响,为提高动物模型的复制效率提供实验依据。方法分别给雌、雄比格犬和昆明小鼠注射不同剂量的四氧嘧啶,药后3、7、14、21 d测定血糖值,同时统计实验期间动物的死亡情况。结果给予同等剂量的四氧嘧啶,雌性比雄性动物的血糖升高更快,浓度更高。雌性犬四氧嘧啶的最适造模剂量为40 mg/kg,而雄性犬在此剂量下的模型成功率只有40%,二者差异极显著（70%VS40%,P〈0.01）;雄性犬的最适使用剂量为50 mg/kg,但在此剂量下有高达30%的雌性犬因高血糖而死亡。四氧嘧啶对小鼠的影响与犬基本一致,雌雄鼠的最佳剂量分别为200 mg/kg和250 mg/kg。结论雌性动物对四氧嘧啶的敏感性较雄性动物高,雄性动物在使用四氧嘧啶复制糖尿病模型时,其剂量通常需要较雌性动物高20%左右。     

Neurite extension by peripheral and central nervous system neurons in response to substratum-bound fibronectin and laminin

Small groups of blastoderm cells were transplanted from wild-type donor embryos into genetically marked host embryos of the same age. Donor cells were injected either into an homologous or an ectopic region of the recipient, and both donor and recipient embryos were allowed to develop. Donor flies were examined for defects in external structures. Recipients were scored for patches of donor-type marked tissue derived from the injected cells. After ectopic transfer, the donor cells recovered in chimaeric recipients differentiated structures consistent with the donor site of cell removal. No apparent fate change was observed. In the rare cases when both individuals of a donor/host pair survived, a direct correspondence could be made between the deleted region in the donor and the chimaeric patch in the host. The results show that blastoderm cells are stably determined to within a segment.     

Dogs cloned from fetal fibroblasts by nuclear transfer

Fetal fibroblasts have been considered as the prime candidate donor cells for the canine reproductive cloning by somatic cell nuclear transfer (SCNT) in regard to the future production of transgenic dogs, mainly due to their higher developmental competence and handling advantage in gene targeting. In this study, the cloning efficiency with canine fetal fibroblasts as donor cells was determined. A total of 50 presumptive cloned embryos were reconstructed, activated and transferred into the oviducts of naturally synchronous recipient bitches. While the fusion rate (76.9%) was similar to those of our earlier studies with adult fibroblasts as donor cells (73.9–77.1%), a high cloning efficiency (4.0%; 2 births/50 embryos transferred) was found compared to the previous success rate with adult fibroblasts (0.2–1.8%). The cloned beagles were healthy and genotypically identical to the donor fibroblast cells. This study shows that a fetal fibroblast cell would be an excellent donor for future production of transgenic dogs via gene targeting in this cell followed cloning using SCNT technology.     

Intratesticular injection of a zinc-based solution as a contraceptive for dogs

The objective of the present study was to evaluate, by light and transmission electron microscopy, the efficacy of a single intratesticular injection of a novel zinc-based solution, as a contraceptive for male dogs. Fifteen mongrel dogs were assigned to three groups (five dogs/group). Group 1, the control group, which consisted of animals ranging from 8 mo to 4 yr, was injected with saline solution. Group 2, which consisted of animals ranging from 8 mo to 1 yr old and Group 3, animals ranging from 2 to 4 yr old, were injected with a zinc-based solution (0.2-1.0mL; volume based on testicular width). There were no histopathological changes detected in testes from control dogs. Histological examination of treated groups revealed degeneration, vacuolation, fewer germ cells, formation of multinucleated giant cells, and a lack of elongated spermatids in atrophic seminiferous tubules. Leydig cells had varying degrees of lipid degeneration and necrosis. The majority of seminiferous tubules in all zinc-treated dogs were lined only by Sertoli cells, which were vacuolated. Ultrastructure of testis of treated groups had degenerate Sertoli and Leydig cells, characterized by numerous mitochondria with the lack of a matrix and agglomeration of lysosomal bodies. The cytoplasm of elongated spermatids was characterized by tubules of hyperplastic and hypertrophic smooth endoplasmic reticulum and numerous Golgi apparati. Round spermatids in Golgi phase had lysis of acrosomal vesicles. The degree of histological changes suggested irreversibility. In conclusion, intratesticular injection of a zinc-based solution effectively impaired spermatogenesis.     

Influence of oocyte donor and embryo recipient conditions on cloning efficiency in dogs

To determine factors that affect the efficiency of dog cloning by somatic cell nuclear transfer, the present study was performed to investigate 1) the effects of surgical history (non-operated/operated) and parity (nullipara/multipara) on the recovery of in vivo canine oocytes; 2) the effects of surgical history and parity of recipients on the pregnancy and delivery; and 3) the effects of synchronization state (AA, advanced asynchrony; SY, synchrony; RA, retarded asynchrony) between oocytes donor and recipient on the pregnancy and delivery. Oocyte recovery rate was significantly higher in non-operated dogs compared to operated dogs (93.8 vs. 89.6%, P < 0.05) and not different between nulliparous dogs and multiparous dogs. Delivery rate was also significantly higher in non-operated dogs compared to operated dogs (2.8 vs. 1.0%, P < 0.05) and in nulliparous dogs than multiparous dogs (3.0 vs. 1.7%, P < 0.05). Even though SY showed increased pregnancy and delivery rate (20.0% and 3.0%) compared to AA (15.0% and 2.0%) and RA (0.0% and 0.0%), there was no significant difference. In conclusion, we recommend non-operated dogs as experimental dogs and nulliparous dogs as recipient dogs to increase delivery rate after transfer of somatic cell nuclear transferred embryos, but further study is needed to find out appropriate synchrony status at the transfer.     

Germ cell transplantation in an azoospermic Klinefelter bull

Germ cell transplantation is a technique that transfers donor testicular cells into recipient testes. A population of germ cells can colonize the recipient testis, initiate spermatogenesis, and produce sperm capable of fertilization. In the present study, a nonmosaic Klinefelter bull was used as a germ cell recipient. The donor cell suspension was introduced into the rete testis using ultrasound-guided puncture. A pulsatile administration of GnRH was performed to stimulate spermatogenesis. The molecular approach to detect donor cells was done by a quantitative polymerase chain reaction with allele discrimination based on a genetic mutation between donor and recipient. Therefore, a known genetic mutation, associated with coat-color phenotype, was used to calculate the ratio of donor to recipient cells in the biopsy specimens and ejaculates for 10 mo. After slaughtering, meiotic preparations were performed. The injected germ cells did not undergo spermatogenesis. Six months after germ cell transplantation, the donor cells were rejected, which indicates that the donor cells could not incorporate in the testis. The hormone stimulation showed that the testosterone-producing Leydig cells were functionally intact. Despite subfertility therapy, neither the recipient nor the donor cells underwent spermatogenesis. Therefore, nonmosaic Klinefelter bulls are not suitable as germ cell recipients. Future germ cell recipients in cattle could be mosaic Klinefelters, interspecies hybrids, bulls with Sertoli cell-only syndrome, or bulls with disrupted germ cell migration caused by RNA interference.     

Effect of telophase enucleation on bovine somatic nuclear transfer

Telophase enucleation has been proven to be an efficient method for preparing recipient cytoplasts in bovine embryonic nuclear transfer (2, 11). This research was designed to study in vitro development of bovine oocytes containing transferred somatic cell nuclei, reconstructed by using enucleated in vitro-matured oocytes 32 h of age at telophase II stage as recipient cytoplasts, compared with those 24 h of age at metaphase II stage. Two protocols for donor cell injection were adopted, i.e., subzonal injection (SUZI) and intracytoplasmic injection (ICI). Bovine oviduct epithelial cells (BOECs) and bovine cumulus cells (BCCs) from an adult cow were used as nuclear donors for these experiments. In SUZI groups, the fusion rate of donor cells, both BOECs and BCCs, with MII enucleated oocytes were higher than those with TII enucleated oocytes (54% vs. 41% and 53% vs. 39%, respectively; P<0.05), but the development rates to morula plus blastocyst stage in MII groups were lower than those in TII groups (22% vs. 39% and 21% vs. 41%, respectively; P<0.05). In ICI groups, about 26% of enucleated MII oocytes injected with BOECs or BCCs cleaved and only small parts of them developed to blastocyst stage (4% and 3%, respectively; P>0.05). When BOECs or BCCs were intracytoplasmically injected into oocytes enucleated at TII stage, no blastocyst was formed in either donor cell group and no cleavage occurred in BOEC group. Our data demonstrated that telophase enucleation is beneficial to early embryo development when bovine somatic nuclei are transferred by subzonal injection. However, it is harmful when donor cells are directly injected into the cytoplast of the enucleated oocytes.     

Vital marker for muscle nuclei in myoblast transfer

A new method is developed using Fluoro-Gold (FG) as a vital stain to label the nuclei of donor myoblasts in myoblast transfer studies. In vitro incubation with 0.01% FG for 16 h resulted in 100% nuclei labelling. Intensive fluorescence persisted following 9 days of subculture, when the human myoblasts were injected into the quadriceps of mouse recipients immunosuppressed with cyclosporine. Injected muscles showed mosaicism of host and donor nuclei 25 days after injection, indicating (i) survival and fusion among donor myoblasts, and (ii) fusion between host and donor cells. FG labelling was not observed in control muscles injected with an equal volume of FG-labelled dead myoblasts, 0.01% FG medium, or phosphate-buffered saline.     

Stable and unstable transformation by microinjection of macronucleoplasm in Paramecium

Transformation by microinjection of macronucleoplasm in Paramecium caudatum was investigated. Macronucleoplasm with three genetic markers (behavior, trichocyst, and mating type) was injected into the macronucleus. To facilitate microinjection, in most cases, paramecia were immobilized in a gelatin (7.5%) solution. The injected cells began to express a dominant gene (cnrA⁺ or cnrB⁺) of the donor 9–24 hr after injection. Expression did not require cell division suggesting injected macronucleoplasm was capable of expressing a phenotype. The amount of injected macronucleoplasm appears to correlate with the frequency of successful expression but not to correlate with the time required for expression. After a number of fissions, the injected cells produced clones which had cells expressing the phenotype of the donor. This suggests that injected macronucleoplasm was replicated and expressed in the recipient cell lines. The transformed clones were classified into two groups. In one group, transformation was stable. All cell lines derived from the injected cells expressed a phenotype similar to the heterozygote of donor and recipient cells. In the other group, transformation was unstable. During the first five to seven fissions after injection, at each division, cells produced one daughter cell which later reverted to the recipient phenotype. After this unstable period, cells no longer produced the recipient phenotype but produced the donor phenotype exclusively. Donor and recipient phenotypes were, thus, segregated in different cell lines. Observation of genetic markers and analysis by computer simulation shed light on the mode of transmission of injected macronucleoplasm. In stable transformation, injected macronucleoplasm appears to be distributed equally to daughter cells. In unstable transformation, injected macronucleoplasm is distributed only to one of the daughter cells at every division until about the fifth to seventh fission after injection and then begins to assort equally to daughter cells. The cell cycle stage at injection may influence the mode of transformation. Interspecific microinjection of macronucleoplasm from P. multimicronucleatum and P. tetraurelia to P. caudatum. resulted in the expression of foreign genes in P. caudatum. In one case, injection of macronucleoplasm of P. tetraurelia produced a stable transformant indicating replication of foreign macronucleoplasm in P. caudatum. This work reveals the mode of transformation by injected macronucleoplasm and shows the possibility of transformation among Paramecium species, which is significant in the study of the conservation of gene products and the mechanism of gene expression in different species. © 1992 Wiley-Liss, Inc.     

Therapeutic effect of a saline solution with sodium lactate (lactasol) in traumatic shock

Acute experiments on 67 adult dogs have demonstrated that infusions of lactasol in a dose of 25 ml/kg in the early period and in a dose of 50 ml/kg in the late period of shock are not efficacious enough. Injection of the increased amount of the saline in the early and late period of shock (up to 50 and up to 100 ml/kg, respectively) makes it possible to save from death almost all the animals. A conclusion is made that the therapeutic effect of lactasol depends on the amount of the saline injected and on the period of shock during which the saline is injected, as well as on the specific action of sodium lactate.