Embryonic rather than extraembryonic tissues have more impact on the development of placental hyperplasia in cloned mice.

Somatic cell cloning by nuclear transfer (NT) in mice is associated with hyperplastic placentas at term. To dissect the effects of embryonic and extraembryonic tissues on this clone-associated phenotype, we constructed diploid (2n) fused with (<-->) tetraploid (4n) chimeras from NT- and fertilization-derived (FD) embryos. Generally, the 4n cells contributed efficiently to all the extraembryonic tissues but not to the embryo itself. Embryos constructed by 2n NT<-->4n FD aggregation developed hyperplastic placentas (0.33+/-0.22 g) with a predominant contribution by NT-derived cells. Even when the population of FD-derived cells in placentas was increased using multiple FD embryos (up to four) for aggregation, most placentas remained hyperplastic (0.36+/-0.13 g). By contrast, placentas of the reciprocal combination, 2n FD<-->4n NT, were less hyperplastic (0.15+/-0.02 g). These nearly normal-looking placentas had a large proportion of NT-derived cells. Thus, embryonic rather than extraembryonic tissues had more impact on the onset of placental hyperplasia, and that the abnormal placentation in clones occurs in a noncell-autonomous manner. These findings suggest that for improvement of cloning efficiency we should understand the mechanisms regulating placentation, especially those of embryonic origin that might control the proliferation of trophoblastic lineage cells.

Ultrastructure of placental hyperplasia in mice: comparison of placental phenotypes with three different etiologies.

Hyperplastic placentas have been reported in several experimental mouse models, including animals produced by somatic cell nuclear transfer, by inter(sub)species hybridization, and by somatic cytoplasm introduction to oocytes followed by intracytoplasmic sperm injection. Of great interest are the gross and histological features common to these placental phenotypes--despite their quite different etiologies--such as the enlargement of the spongiotrophoblast layers. To find morphological clues to the pathways leading to these similar placental phenotypes, we analyzed the ultrastructure of the three different types of hyperplastic placenta. Most cells affected were of trophoblast origin and their subcellular ultrastructural lesions were common to the three groups, e.g., a heavy accumulation of cytoplasmic vacuoles in the trophoblastic cells composing the labyrinthine wall and an increased volume of spongiotrophoblastic cells with extraordinarily dilatated rough endoplasmic reticulum. Although the numbers of trophoblastic glycogen cells were greatly increased, they maintained their normal ultrastructural morphology, including a heavy glycogen deposition throughout the cytoplasm. The fetal endothelium and small vessels were nearly intact. Our ultrastructural study suggests that these three types of placental hyperplasias, with different etiologies, may have common pathological pathways, which probably exclusively affect the development of certain cell types of the trophoblastic lineage during mouse placentation.

Germline niche transplantation restores fertility in infertile mice.

BACKGROUND: Stem cells interact closely with their microenvironment or niche, and abnormalities in niche compromise the self-renewing tissue. In testis, for example, Sertoli cells interact with germ cells, and defects in Sertoli cells compromises spermatogenesis, leading to male infertility. However, it has not been possible to restore spermatogenesis from endogenous stem cells in infertile testis with environmental defects. METHODS AND RESULTS: When healthy Sertoli cells from infertile white spotting (W) mouse were transplanted into the seminiferous tubules of infertile Steel (Sl) mouse testis that had defective Sertoli cells, spermatogenesis occurred from Sl stem cells in the recipient testis. On average, 1.1% of the recipient tubules showed spermatogenesis. Furthermore, in a microinsemination experiment with germ cells that developed in the testis, we obtained four normal offspring from 114 successfully injected oocytes. CONCLUSIONS: This study demonstrates that defects in male germline microenvironment can be corrected by Sertoli cell transplantation. Although further improvements are required to enhance the low efficiency of spermatogenesis, the ability to correct environmental defect by niche transplantation has important implications in developing new strategies for treating incurable disorders in self-renewing tissues.

Restoration of fertility in infertile mice by transplantation of cryopreserved male germline stem cells.

BACKGROUND: The development of a spermatogonial transplantation technique has provided new possibilities for the treatment of male infertility. Previous studies have shown that spermatogonial stem cells could reinitiate spermatogenesis after cryopreservation and reintroduction into the seminiferous tubules of infertile recipient males, and this raised the possibility of banking frozen stem cells for male infertility treatment. It remains unknown, however, whether germ cells from freeze-thawed stem cells are fertile, leaving the possibility that the procedure compromises the integrity of the stem cells. METHODS AND RESULTS: Dissociated mouse testis cells were cryopreserved and transplanted into infertile recipient testes. The freeze-thawed testis cell populations contained higher concentrations of stem cells than fresh testis cell populations. Offspring were obtained from freeze-thawed stem cells transplanted into infertile males, and fertility restoration was more efficient in immature (5-10 days old) than in mature (6-12 weeks old) recipients. However, offspring were also obtained from infertile adult recipients using in-vitro microinsemination. CONCLUSIONS: This first successful application of frozen stem cell technology in the production of offspring by spermatogonial transplantation suggests the superiority of immature recipients for clinical applications. Thus, the combination of cryopreservation and transplantation of stem cells is a promising approach to overcome male infertility.

New microinsemination techniques for laboratory animals.

Since the development of a reliable mouse intracytoplasmic sperm injection (ICSI) technique in 1995, microinsemination techniques have been widely applied in several laboratory species. As gametes and embryos have specific biological and biochemical features according to the species, technical improvements are necessary for successful microinsemination that subsequently leads to normal fetal development in several species. Recent advanced reproductive research involving genetic engineering often depends on microinsemination techniques that require a high degree of skill, and new human assisted reproductive technology (ART) requires experimental models using laboratory animals. The accumulation of technical improvements in these fields should accelerate the development of microinsemination techniques in mammals, including humans.

Birth of offspring following transplantation of cryopreserved immature testicular pieces and in-vitro microinsemination.

BACKGROUND: Fertility protection is an urgent clinical problem for prepubertal male oncology patients who undergo either chemotherapy or radiotherapy. As these patients do not have mature sperm to be frozen, there is as yet no effective method to preserve their fertility. METHODS AND RESULTS: Single pieces of immature mouse (1.5 x 1.5 x 1.5 mm) or rabbit (2.0 x 2.0 x approximately 3.0 mm) testis were cryopreserved, thawed and transplanted into mouse testes. Histological techniques were used to determine the presence of spermatogenesis, which was restored in both mouse and rabbit testicular pieces, and led to the production of mature sperm after both cryopreservation and syngeneic or xenogeneic transplantation into mouse testes. Using sperm developed in the frozen-thawed transplants, mouse offspring were born after in-vitro microinsemination. Furthermore, rabbit offspring were obtained using rabbit sperm that developed in fresh transplants in a xenogeneic surrogate mouse. CONCLUSIONS: This approach of 'testicular tissue banking' is a promising technique for the preservation of fertility in prepubertal male oncology patients. Xenogeneic transplantation into immunodeficient mice may provide a system for studying spermatogenic failure in infertile men.

Phenotypic effects of somatic cell cloning in the mouse.

Although a variety of phenotypes and epigenetic alterations have been reported in animals cloned from somatic cells, the exact nature and consequences of cloning remain unclear. We cloned mice using fresh or short-term cultures of donor cells (cumulus cells, immature Sertoli cells, and fetal or adult fibroblast cells) with defined genetic backgrounds, and then compared the phenotypic and epigenetic characteristics of the cloned mice with those of fertilization-derived control mice. Irrespective of the nucleus-donor cell type, about 50% of the reconstructed embryos developed to the morula/blastocyst stage, but about 90% of these clones showed arrested development between days 5 and 8, shortly after implantation. Most of the clones were alive at term, readily recovered respiration, and did not show any malformations or overgrowths. However, their placentas were two- to threefold larger than those of the controls, due to hyperplasia of the basal (or spongiotrophoblast) layer. Although there was significant suppression of a subset of both imprinted and non-imprinted placental genes, fetal gene suppression was minimal. The seven imprinted genes that we examined were all expressed correctly from the parental alleles. These findings were consistent for every cell type from the midgestation through term stages. Therefore, cloning by nuclear transfer does not perturb the parent-specific imprinting memory that is established during gametogenesis, and the phenotypic and epigenetic effects of cloning are restricted to placental development at the midgestation and term stages. Twelve male mice that were born in a normal manner following nuclear transfer with immature Sertoli cells (B6D2F1 genetic background) were subjected to long-term observation. They died earlier than the genotype-matched controls (50% survival point: 550 days vs. 1028 days, respectively), most probably due to severe pneumonia, which indicates that unexpected phenotypes can appear as a result of the long-term effects of somatic cell cloning.

Fertilization without spermatozoa.

Mammalian spermatozoa first acquire the ability to fertilize oocytes as they pass through the epididymis to mature. Due to recent advances in microinsemination techniques, not only mature spermatozoa, but also immature sperm cells at certain stages in the testis, have been used to construct diploid zygotes, some of which subsequently develop to normal offspring. Using round spermatids, the most youngest haploid male germ cells, normal births have been reported in the mouse, rabbit, and human. Furthermore, in the mouse, secondary and primary spermatocytes also support full term development after incorporation into immature or mature homologous oocytes. Spermatogenic cells of several species can be cryopreserved easily in simple cryoprotectant solutions. Thus, the microinsemination techniques using spermatogenic cells give us a way to treat infertility, and provide valuable information on gametogenesis, including spermatogenesis, meiosis, and genomic imprinting.

Activity of a sperm-borne oocyte-activating factor in spermatozoa and spermatogenic cells from cynomolgus monkeys and its localization after oocyte activation.

It is widely accepted that mature mammalian oocytes are induced to resume meiosis by a sperm-borne oocyte-activating factor(s) (sperm factor, SF) immediately after normal fertilization or intracytoplasmic sperm injection. The SF is most likely a soluble factor that is localized within the cytoplasm of mature spermatozoa, but the exact stage at which it appears during spermatogenesis and its localization after oocyte activation is not fully understood, except in the mouse. First, we injected mature spermatozoa and spermatogenic cells from cynomolgus monkeys into mouse oocytes to assess their oocyte-activating capacity. More than 90% of mouse oocytes were activated after injection of monkey spermatozoa. Round spermatids and primary spermatocytes (late pachytene to diplotene) also activated oocytes (93% and 79%, respectively). Injection of monkey spermatozoa and spermatids induces intracellular Ca(2+) oscillations in a pattern similar to that seen following normal fertilization. Most spermatocytes did not produce typical intracellular Ca(2+) oscillations. Second, we transferred pronuclei or cytoplasts from mouse oocytes that had been activated by monkey spermatozoa or spermatids into intact mature mouse oocytes by electrofusion in order to examine the localization of the SF after pronuclear formation. Some of the SF was localized within the pronuclei, but some stayed in the ooplasm. This study demonstrated that spermatogenic cells of cynomolgus monkeys acquire oocyte-activating capacity at much earlier stages than those of mice, and that the monkey SF has a pronucleus-directing nature, although to a lesser extent than the mouse SF.

Short-term nonfrozen storage of mouse epididymal spermatozoa.

Six simple methods for short-term (up to 8 d), nonfrozen (5 to 20 degrees C) storage of mouse epididymides were compared with respect to the motility and fertility of spermatozoa. A high percentage of progressively motile spermatozoa was obtained from epididymis stored for 8 d at 5 degrees C in mineral oil (78.3%), covered with body fat (80.0%), or stored in the intact body of the euthanized donor animal (77.5%). Fertilized eggs (6.4% fertilization rate) were obtained by IVF using spermatozoa that had been stored in mineral oil at 5 degrees C for at least 8 d, and offspring were obtained from 77.5% of transferred eggs that were fertilized by spermatozoa stored for 2 d. These methods inhibited moisture loss from the preserved epididymal spermatozoa, thereby allowing spermatozoa to be stored for a few days without loss of either motility or fertility. These methods make possible such wide-ranging applications as the long-distance transport of epididymis spermatozoa. While in storage at 5 degrees C, the tail of each recovered spermatozoon was bent midway along the tail, possibly owing to damage to the plasma membranes and due to the spermatozoa's hardening in the phospholipid by exposure to the low temperature.

Short-term preservation of mouse oocytes at 5 degrees C.

The temporary preservation of oocytes without freezing would be useful for some experiments. ICR mouse oocytes were kept in a preservation medium under mineral oil for 1, 2, 3, 4 or 7 days at 5 degrees C, and 1 or 2 days at 37 degrees C. In vitro fertilization was attempted on oocytes rinsed with TYH medium after preservation. More than 70% of morphologically normal oocytes were recovered from each preservation group. Fertilization rates of oocytes preserved for 1, 2, 3, 4 or 7 days at 5 degrees C were 69.9, 66.5, 45.3, 26.7 and 8.8% respectively. Fertilization rates of oocytes preserved for 1 or 2 days at 37 degrees C were 9.6 and 1.6%, respectively. Preservation of oocytes at 5 degrees C has some capability as a method of short-term storage without freezing.

Recent advances in the microinsemination of laboratory animals.

Microinsemination techniques were applied to laboratory animals, including mice, Mongolian gerbils, mastomys, guinea pigs and cynomolgus monkeys. After micro-insemination with spermatozoa or round spermatids, their oocytes were successfully fertilized and subsequently developed into 4-cells to blastocysts, depending on species. The efficiencies of microinsemination techniques for producing fertilized oocytes were comparable with, or superior to those of IVF in these species.

Production of male cloned mice from fresh, cultured, and cryopreserved immature Sertoli cells.

Although it is generally accepted that relatively high efficiencies of somatic cell cloning in mammals can be achieved by using donor cells from the female reproductive system (e.g., cumulus/granulosa, oviduct, and mammary gland cells), there is little information on the possibility of using male-specific somatic cells as donor cells. In this study we injected the nucleus of immature mouse Sertoli cells isolated from the testes of newborn (Days 3-10) males into enucleated mature oocytes in order to examine the ability of their nuclei to support embryonic development. After activation of the oocytes that had received the freshly recovered immature Sertoli cells, some developed into the morula/blastocyst stage, depending on the age of the donor cells (22.0-37.4%). When transferred into pseudopregnant females, 7 (3.3%, 7 of 215) developed into normal pups at term. Nuclear transfer of immature Sertoli cells after 1 wk in culture also produced normal pups after embryo transfer (3.1%, 2 of 65). Even after cryopreservation in a conventional cryoprotectant solution, their ability as donor cells was maintained, as demonstrated by the birth of cloned young (6.7%, 7 of 105). Immature Sertoli cells transfected with green fluorescent protein gene also supported embryo development into morulae/blastocysts, which showed specific fluorescence. This study demonstrates that immature Sertoli cells, male-specific somatic cells, are potential donors for somatic cell cloning.

Comparison of two methods of assisted fertilization in cynomolgus monkeys (Macaca fascicularis): intracytoplasmic sperm injection and partial zona dissection followed by insemination.

Intracytoplasmic sperm injection (ICSI) and partial zona dissection followed by insemination (PZD-I) were used to establish a microinjection system in cynomolgus monkeys (Macaca fascicularis), which are potential models for human reproduction. Two experimental systems were studied, in which either hamster oocytes or cynomolgus monkey oocytes were used as the vehicle. When hamster oocytes were used, 66 out of 81 ICSI-treated oocytes (82%) showed sperm head swelling or pronucleus formation. Following PZD-I of hamster oocytes the rates of spermatozoa penetration (85/114; 75%) and fertilization (71/114; 62%) were relatively high. When cynomolgus monkey oocytes were used, 19 out of 31 (61%) were fertilized by ICSI with cynomolgus monkey spermatozoa and, subsequently, two embryos (7%) developed to the morula stage. In total, 94% (15/16) of the PZD-I treated oocytes were penetrated by spermatozoa and 63% (10/16) were fertilized. These results demonstrate that both micromanipulation techniques can be used in assisted fertilization with cynomolgus monkeys.