Differentiation of germ cells and gametes from stem cells.

BACKGROUND: Advances in stem cell research have opened new perspectives for regenerative and reproductive medicine. Stem cells (SC) can differentiate under appropriate in vitro and in vivo conditions into different cell types. Several groups have reported their ability to differentiate SCs into germline cells, and some of them have been successful in obtaining male and female gamete-like cells by using different methodologies. METHODS: This review summarizes the current knowledge in this field and emphasizes significant embryological, genetic and epigenetic aspects of germ cells and gametes in vitro differentiation in humans and other species, highlighting major obstacles that need to be overcome for successful gametogenesis in culture: studies reporting development of germ cell-like cells from murine and human embryonic (ESC) and somatic SCs are critically reviewed. RESULTS: Published studies indicate that germ cells can be consistently differentiated from mouse and human ESC. However, further differentiation of germ cells through gametogenesis still has important genetic and epigenetic obstacles to be efficient. CONCLUSIONS: Differentiation of germ cells from SCs has the potential of becoming a future source of gametes for research use, although further investigation is needed to understand and develop the appropriate niches and culture conditions. Additionally, if genetic and epigenetic methodological limitations could be solved, therapeutic opportunities could be also considered.

In vitro maturation and intracytoplasmic sperm injection of oocytes collected from hormonally stimulated common wombats, Vombatus ursinus.

Porcine FSH/LH stimulation successfully induced development of multiple large (>or=4mm) antral follicles in 10 of 11 common wombats. A mean of 5.5 metaphase II (MII) oocytes were aspirated from wombats that were stimulated during the follicular phase of the oestrous cycle (n=3) or after pouch young removal (n=3). Three subadults (n=3) and two anoestrus adults did not produce MII oocytes despite pFSH/pLH administration. In vitro maturation of immature oocytes at the time of aspiration doubled the number of MII oocytes that could be collected from pFSH/pLH stimulated wombats. Immature oocytes with cumulus attached, matured more readily to the MII stage than immature oocytes without cumulus. Following intracytoplasmic sperm injection (ICSI), approximately 5% of the oocytes that were MII at the time of collection cleaved. Approximately 5% of those that were matured by in vitro maturation (IVM) formed two polar bodies following ICSI, although they not cleave. Parthenogenesis cannot be excluded. This demonstrates that assisted reproductive technologies may be applicable to the common wombat.

Effects of electrical stimulation and seminal plasma on the motility of mouse sperm.

The effects of electric current (in vivo and in vitro) and seminal plasma on epididymal and ejaculated sperm obtained from C57BL x CBA and C57BL/6J mice were investigated by studying motility parameters, fertilization and embryo development. Electroejaculates were obtained by applying a series of computer-generated sinusoidal alternating currents (0.25-3.0 V at 50 Hz) delivered for 1, 2 and 3 s with 1-s rest periods using a four-electrode rectal probe for 4 min. Epididymal sperm obtained from the same mice were either subjected to electric current in vitro in a Plexiglass chamber or incubated in a medium containing seminal plasma for 2 h. In vitro electric current application and incubation in a medium containing seminal plasma significantly (P < 0.01) decreased sperm motility. Neither electroejaculates nor epididymal spermatozoa incubated with seminal plasma could fertilize oocytes by conventional IVF (P < 0.001), whereas sperm subjected to in vitro electric current had lost little of their ability to fertilize oocytes. Following transfer of embryos generated by intracytoplasmic sperm injection (ICSI), the number of live pups obtained from electroejaculated sperm (10.2%; 6/59) was significantly (P < 0.01) lower than from epididymal sperm (50.0%; 22/42). Electroejaculation using a rectal probe had little effect on motility and fertilization capacity of mouse epididymal sperm, whereas the presence of seminal plasma decreased motility and prevented fertilization.

Characterization of the fertility of male aromatase knockout mice.

Previous studies employing the male aromatase knockout (ArKO) mouse have indicated that local expression of estrogens appears to be important for the progression of spermatogenesis. In the absence of estrogen biosynthesis round spermatids are observed to undergo apoptosis and thus fail to differentiate into mature, elongated spermatids. This lesion appears to arise between the ages of 18 weeks and 1 year. To ultimately determine if the disruption to spermatogenesis arises earlier than 18 weeks, we performed an intensive study to examine the fertility of younger male ArKO mice. This involved an analysis of their mating capacity together with an extensive stereological analysis, determination of the in vitro potential of mature sperm, and sexual behavior. ArKO and wild-type (w/t) males at 7 weeks of age were placed with w/t females for 7 weeks. At age 14 weeks, the males were killed and the testes removed. ArKO mice were observed to sire significantly fewer litters than the w/t mice; 5 out of the 10 sired no litters at all. Stereological analysis performed on the removed testes found a significant decrease in round spermatid numbers between w/t and ArKO mice at this age; however, there were no differences in all other germ cells and Sertoli cell numbers. When mature spermatozoa were analyzed, sperm from 15-week-old ArKO mice had a significant reduction in motility. This was further reduced by 1 year of age with a decrease in concentration. A preliminary examination of sexual behavior found that ArKO mice did not attempt to mount the females, in contrast to the w/t mice, which mounted consistently during the time period. In conclusion, we observed that ArKO mice have reduced fertility at age 14 weeks. This may be due in part to a disruption in spermatogenesis because the phenotype does appear to arise earlier than 18 weeks, possibly leading to abnormalities in the mature spermatozoa. Or, in part, this may be attributable to an impairment in the development of copulatory behavior, which is consistent with the available evidence that points to a crucial role for estrogens in the neural development and initiation of male sexual behavior.

Nuclear transfer of adult and genetically modified fetal cells of the rat.

The present study examines the handling, activation, and micromanipulation of rat eggs in an attempt to produce live young using nuclear transfer (NT) of adult and genetically modified rat fetal cells. Mature rat eggs cultured in calcium-free medium showed reduced rates (24%) of chromosomal dispersion ("spontaneous activation" characteristic of this species) compared with eggs cultured in calcium-containing medium (47%), but failed to survive micromanipulation procedures. High rates of parthenogenetic cleavage were obtained with chemical activation using ethanol/cycloheximide (65%) compared with other standard chemical activation methods (4-28%). This type of activation was also effective in reestablishing cleavage capability (19-71%), in a time-dependent manner, of spontaneously activated eggs arrested at a second prophase-like state. At most, two of four tested micromanipulation procedures were effective in producing NT embryos capable of morula or blastocyst development (14-16%) in vivo following transfer to mouse oviducts. NT blastocysts produced from cumulus cells and transfected rat fetal fibroblasts appeared morphologically and karyotypically normal (2n = 42). Nocodazole-assisted metaphase enucleation and piezoelectric-assisted donor cell injection produced significant and equivocal effects on survival and cleavage rates of reconstructed embryos but failed to significantly improve in vivo morula/blastocyst development rates (16-28%) compared with unassisted micromanipulation (16%). Live births have not yet been obtained from early cleavage stage embryos (n = 269) transferred to pseudopregnant recipient rat oviducts. Improvements in reconstituted NT embryo culture and transfer are required for these methods to be an effective means of transgenic rat production.

Cell synchronization for the purposes of nuclear transfer in the bovine.

We have examined the reprogramming ability of donor fibroblast nuclei in various phases of the cell cycle, upon transfer to cytoplasts, using a bovine nuclear transfer (NT) model. Bovine fetal fibroblasts were cultured in reduced serum and conditioned medium to induce quiescence (G0) and treated with nocodazole to induce M phase arrest. Unsynchronized actively dividing cells (control) were mainly in G1. Cells synchronized in G0, M, and G1 phase were transferred to enucleated bovine MII oocytes by direct injection using the Piezo-Drill microinjector. NT oocytes were artificially activated following injection. Cells at the M phase were also transferred to enucleated oocytes after artificial activation. Cells induced into quiescence by serum starvation and unsynchronized donor cells produced the highest rates of development to the morula/blastocyst stage (20% and 18%, respectively). Development to blastocyst was significantly higher in parthenogenetic controls compared to NT embryos. The transfer of M phase nuclei to MII cytoplasts was not associated with high development to the blastocyst stage. Nevertheless, determining the viability of these embryos requires transfer to recipient animals and assessment of in vivo development.

Developmental competence of nuclear transfer cow oocytes after direct injection of fetal fibroblast nuclei.

We have examined the in vitro and in vivo development of cloned embryos produced by incorporation of fetal fibroblast into in vitro matured and enucleated cow oocytes by direct injection and by fusion. For injection, nuclei were either mechanically isolated using the microinjection needle or chemically isolated by treatment with NP-40 lysis buffer. Fetal fibroblasts were serum starved and treated with calcium ionophore before injection to induce chromatin condensation. A range of 8% to 16% of successfully injected oocytes developed to blastocysts in culture and a total of nine pregnancies resulted from transfer of cloned embryos produced by this method. Nuclear transfer by fusion resulted in 22% development to blastocysts. Unlike in mice, the embryos derived from injection did not result in viable pregnancies, which may suggest species differences. All pregnancies were terminated after 45 to 150 days from transfer. Two pregnancies resulted from transfer of cloned embryos obtained by fusion which produced two healthy female calves. The study proposes an alternative method for the production of cow cloned embryos. Further research, however, is required to optimize bovine cloning by injection.

Pregnancies produced from fertile and infertile stallions by intracytoplasmic sperm injection (ICSI) of single frozen-thawed spermatozoa into in vivo matured mare oocytes.

The use of intracytoplasmic sperm injection (ICSI) for in vitro fertilization of equine oocytes and the developmental potential of these oocytes after transfer to the Fallopian tubes of synchronized mares were examined. Oocytes were aspirated from mature follicles 39 h after injection of a GnRH analogue and transported 190 km at 39 degrees C. Semen from a fertile and an infertile stallion was frozen and prepared for injection. Successfully injected oocytes were transferred surgically into the ampulla of the Fallopian tube either: (i) 4-8 h after semen injection; or (ii) after 24-48 h culture before transfer. Oocytes (n=26) were treated by ICSI: three oocytes fragmented after injection (12%) and eight oocytes were returned for immediate transfer to recipient mares. Of the 15 cultured oocytes, eight (53%) had two polar bodies and cleaved to two-cell embryos 24 h after injection. Pregnancies were identified on day 12-14 after transfer in 4/16 (25%) recipients that had received either transferred embryos (1/8) or freshly injected oocytes (3/8). Two of the four pregnancies did not progress beyond day 30 and two mares gave birth to foals. There were no apparent differences in the establishment of pregnancies from oocytes injected with frozen-thawed spermatozoa from the fertile (3/14) or infertile stallion (1/2).

Reprogramming cattle somatic cells by isolated nuclear injection.

The development of a somatic cell nuclear transfer procedure for the production of blastocyst stage cattle embryos is described. Bovine fetal fibroblasts were used for fusion experiments with surgically enucleated oocytes (cytoplasts) following the establishment of optimal parameters for electrofusion from isofusion contours. Fusion rates were increased by decreasing size of the cytoplasts used but cleavage was decreased by decreasing size of the cytoplast used (quarter, half and whole cytoplasts). The use of double cytoplasts did not improve cleavage, and development to blastocysts could not be achieved. In a comparison of electrofusion of fibroblasts with cytoplasts in the subzonal perivitelline space with intracytoplasmic injection of nuclei and parthenogenetically activated oocytes, 2%, 14% and 24% developed to blastocysts respectively. In the group injected with isolated nuclei, the passage number (4 to 9) had no apparent influence on developmental competence to blastocysts. The embryos produced by nuclear injection of somatic cell nuclei showed the normal pattern of cell surface appearance of TEC-3 and TEC-4 stage-specific epitopes during development, as seen in fertilized oocytes. We conclude that the nuclear injection of somatic cell nuclei is a relatively efficient way to clone bovine embryos.

Is the acrosome reaction a prerequisite for sperm incorporation after intra-cytoplasmic sperm injection (ICSI)?

There is debate as to whether the acrosome reaction is necessary for sperm incorporation after intra-cytoplasmic sperm injection (ICSI). Ultrastructural evidence is presented to show that the acrosome reaction could occur in the ooplasm before sperm incorporation in mature human oocytes or the acrosome could be discarded intact before sperm incorporation in immature oocytes, matured in vitro. Both germinal vesicle and growing follicular oocytes showed sperm chromatin decondensation, with discarded acrosomes close to the sites of incorporation, and were able to form male pronuclei. This is probably the first report of microfertilization of a growing oocyte with a reticulate nucleolus by ICSI. The acrosome reaction, when it occurs, is preceded by acrosome swelling and is followed by vesiculation of surface membranes exposing the inner acrosome membrane, as observed on the surface of the zona during IVF or in the perivitelline space after subzonal sperm injection. These sperm were probably capacitated at the time of ICSI. There was subtle evidence of leaching of the acrosomal matrix from intact discarded acrosomes and from partially depleted acrosomes attached to decondensing spermheads. These sperm were probably not fully capacitated at the time of ICSI. It is concluded that both the acrosome reaction and acrosome deletion are possible prerequisites to sperm incorporation after ICSI.

Blastocyst development and birth after in-vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching.

Immature oocyte recovery followed by in-vitro oocyte maturation and in-vitro fertilization is a promising new technology for the treatment of human infertility. The technology is attractive to potential oocyte donors and infertile couples because of its reduced treatment intervention. Immature oocytes were recovered by ultrasound-guided transvaginal follicular aspiration. Oocytes were matured in vitro for 36-48 h followed by intracytoplasmic sperm injection (ICSI). Embryos were cultured in vitro for 3 or 5 days before replacement. Assisted hatching was performed on a day 5 blastocyst stage embryo. Embryo and uterine synchrony were potentially enhanced by luteinization of the dominant follicle at the time of immature oocyte recovery. Mature oocyte and embryo production from immature oocyte recovery were similar to the previous IVF results of the patients. A blastocyst stage embryo, produced as a result of in-vitro maturation, ICSI, in-vitro culture and assisted hatching, resulted in the birth of a healthy baby girl at 39 weeks of gestation.

Intracytoplasmic sperm injection in mice: increased fertilization and development to term after induction of the acrosome reaction.

A technique has been developed for intracytoplasmic sperm injection (ICSI) in the mouse with a relatively low rate of lysis of oocytes (range 5-25% across experiments) and pronuclear formation in around one-third of the intact oocytes (range 30-38% across experiments) for untreated spermatozoa. The treatment of spermatozoa with calcium ionophore, to induce the acrosome reaction (increases acrosome-free spermatozoa from 28 to 58%) before ICSI, increased pronuclear formation to approximately 60% (range 59-62% across experiments) in intact oocytes. The pronuclear oocytes developed to blastocysts in vitro and to term when transferred to recipient foster mothers at rates equivalent to zygotes formed after insemination in vitro. There was no benefit for fertilization rates of activating oocytes with 8% ethanol before or after ICSI, nor was there any evidence of parthenogenetic activation by the sperm solution used for ICSI. This technique adds to other in-vitro fertilization techniques which can be used to explore gamete interactions and to recover breeding in infertile strains and reproductively unfit mice.

Embryo development capacity of oocytes fertilized by immature sperm and sperm treated with motility stimulants.

The fertilizing ability of mouse spermatozoa develops during maturation and coincides with the acquisition of motility. The lack of progressive motility of spermatozoa from the testis and precaudal segments of the epididymis interferes with their ability to fertilize oocytes after insemination in vitro. The removal of cumulus cells for insemination in vitro and the use of subzonal injection of a single spermatozoon resulted in a higher number of oocytes fertilized by immature caput and corpus spermatozoa. High rates of embryonic arrest and retarded development were observed in oocytes fertilized by caput and corpus spermatozoa when compared with oocytes fertilized by cauda spermatozoa. However, when the oocytes were enclosed in their cumulus cells or microinjected with a single spermatozoon, these effects were reduced. A block in embryonic development was also observed after human and mouse oocytes were exposed to the sperm motility stimulants pentoxifylline (PTF) and 2-deoxyadenosine (DOA). These observations suggest that exposure of oocytes to PTF and DOA should be avoided.

Microinjection: choice of embryo transfer technique.

Subzonal sperm microinjection (SUZI) is indicated in severe oligoasthenozoospermia, in which the total count of motile sperm is inadequate for in vitro fertilization (IVF), and in cases with repeated failure of fertilization. Sperm for microinjection are selected following centrifugation on a Percoll gradient and stimulation with pentoxifylline and 2-deoxyadenosine. Motile sperm (2-10 per egg) are injected into the perivitelline space and fertilized oocytes are then cultured for two days prior to transfer into the Fallopian tube (tubal embryo stage transfer, TEST) or uterus. During 1992, SUZI results showed a total fertilization rate of 30% (19% were 2 pronuclear, 11% were polyspermic), a transfer rate of 55% and pregnancy rates of 15.2% per transfer and 8.3% per cycle. Recent pregnancy data in mild-moderate male factor infertility showed that gamete intrafallopian transfer (GIFT) results were consistently superior to TEST or IVF, suggesting a beneficial effect of the tubal environment on fertilization and early embryonic development. Accordingly, the combination of SUZI followed by the immediate transfer of injected oocytes into the Fallopian tube, the MIFT procedure, was explored. An initial study of 21 consecutive microinjection candidates showed a clinical pregnancy rate of 24% per cycle. Information regarding fertilization and polyspermy rates was available from supernumerary oocytes in 90% of patients. A randomized, controlled trial comparing MIFT with SUZI or TEST in severe male factor infertility is required to confirm the improved pregnancy rate in MIFT cycles.

The effects of the sperm motility activators 2-deoxyadenosine and pentoxifylline used for sperm micro-injection on mouse and human embryo development.

The sperm motility stimulants 2-deoxyadenosine (DOA) and pentoxifylline (PTF), used to improve the success of insemination and sperm micro-injection for low motility sperm samples, were studied for their effects on the developmental capacity of mouse and human oocytes. When human oocytes were micro-injected with spermatozoa in 3 mM DOA 80% of them became blocked at the 1-cell pronuclear stage. However, when spermatozoa in 3 mM PTF were used for micro-injection or when spermatozoa were washed to remove DOA before micro-injection only a few oocytes (9-10%) were blocked. Pregnancies occurred in five of 14 patients into whom cleaving embryos from all three treatments had been transferred, indicating that once cleavage was initiated, development of embryos occurred at expected rates. Exposure of mouse oocytes to DOA for a short period during insemination (4-6 h) or a longer period during the pronuclear cell cycle (18-20 h) significantly reduced cleavage beyond the 2-cell stage, resulting in a dramatic reduction in blastocyst formation. PTF did not significantly reduce mouse embryo development. Similar results were obtained for oocytes inseminated in vitro or micro-injected with a spermatozoon into the perivitelline space. Neither DOA nor PTF increased fertilization of mouse oocytes. PTF reduced fertilization, particularly in cumulus-enclosed oocytes and oocytes micro-injected with spermatozoa in PTF. We conclude that DOA is a potent inhibitor of embryo development and oocytes should not be exposed to DOA. Exposure of oocytes to PTF had little effect on their subsequent development but treatment of cauda epididymal mouse spermatozoa can reduce their fertilizing capacity.