The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, 505 East 70th Street, HT-336, New York, NY 10021, USA
Abstract
Although great progress has been made in both the investigation and treatment of infertility, a considerable number of patients still fail to conceive. Spermatogenic failure and/or oocyte ageing appear to be responsible for a large proportion of cases. The use of donor gametes may bring legal, ethical and even social problems of acceptance that can discourage infertile couples from the donor route. Fortunately, emerging reproductive technologies and preliminary results from animal experiments provide some hope for alternative sources of gametes through which these infertile patients can finally conceive their own genetic child. In conjunction with intracytoplasmic sperm injection (ICSI), fertilization of human oocytes with immature sperm precursors, e.g. spermatids and even secondary spermatocytes, has resulted in healthy babies. Pregnancies have also resulted from the use of spermatids derived from in-vitro spermatogenesis. In the mouse, even primary spermatocytes appear able to participate in normal embryogenesis. In view of the possibility for transplantation and even xenotransplantation of spermatogonia to a host testis in animals, a similar use of human male stem cells might provide an attractive source for the treatment of males with arrested spermatogenesis, as well as male cancer patients. Transplantation of somatic cell nuclei and their haploidization within oocytes may prove to be a practical way of eradicating age-related aneuploidy and so constitute an innovative source of healthy oocytes. Most importantly, however, the safety of the procedures described here needs to be proven before their application to the human arena. Finally, we discuss the implications of cytoplasmic quality and of genetic imprinting in the context of these manipulations.
Key words: alternative sources/assisted reproduction/gametes/oocytes/spermatozoa
Introduction
Until ~20 years ago, almost the only way to conceive was by natural intercourse. As a treatment option, artificial insemination was quite successful when donor spermatozoa were used, but this had limited results with homologous spermatozoa. Although the situation has changed radically since the first human IVF pregnancy in 1978, nonetheless ~40% of couples can still fail to achieve fertilization because of poor sperm quality (Van Uem et al., 1985). The establishment of intracytoplasmic sperm injection (ICSI) (Palermo et al., 1992
, 1995a
,b
, 1996
) has greatly enhanced the chance of fertilization in couples with previous fertilization failure after standard IVF, adding another conspicuous portion of the infertile to the reproductive population. The application of ICSI became even wider when it was observed that this made pregnancies possible for azoospermic patients. The rapid acceptance of ICSI in clinical practice has been furthered by its ethical acceptability and by the fact that it generally avoids any need to use donor spermatozoa.
The use of IVF and ICSI has not been problem-free. The confidence of baby-boomers that they can reproduce at `any age' has often postponed the wish to achieve pregnancy, and this is particularly evident in women aged 40 years. While infertility due to relatively advanced female age can now be successfully treated with oocyte donation, most couples are more interested in generating their own child, and the astounding accomplishments of reproductive medicine in recent years have raised their expectations.
The decreased fertility of older women stems in large part from a decline in the frequency of intercourse, in the number of primordial follicles and, in particular, from a higher incidence of oocyte aneuploidy (Tietze, 1957; Dailey et al., 1996
). In the metaphase II (MII) oocyte, the frequency of aneuploidy, primarily the result of a non-disjunction of bivalent chromosomes at meiosis I, is estimated to be 4.9, 11.5 and 29.8% in the 2534, 3539, and 4045 year old age groups respectively (Dailey et al., 1996
). That oocyte aneuploidy is one major reason for the low ongoing pregnancy rates in older women, is suggested by the higher pregnancy rate in this group where young donor oocytes are used. Of course, age is also a predisposing factor for autosomal trisomy in the offspring (Hassold and Jacobs, 1984
).
An interest in finding a treatment for azoospermia and for oocyte aneuploidy has stimulated many investigators to seek alternative sources of spermatozoa and oocytes for the infertile couples who specifically desire to conceive their own genetic child. In recent years, several leads commonly used in cloning science have opened routes, real or theoretical, for safe ways to `manufacture' gametes that will make that goal possible.
Here we discuss the prospects for techniques that may lead to the production of viable gametes with the same parental genome. We consider those that relate to male germ cell isolation and transplantation, the procedures of nuclear transplantation, somatic cell haploidization and finally issues involved in cytoplasmicheteroplasmic transfer.
Sources of sperm precursors
Spermatids
The effectiveness of ICSI with freshly ejaculated spermatozoa has been extended to spermatozoa collected directly from the obstructed epididymis. However, for patients whose azoospermia is not obstructive in origin, the only option is retrieval directly from the testis. These cases, identified as non-obstructive, generally present considerable maturation arrest of the spermatogenic line (Martin-Du Pan and Campana, 1993). Fortunately, this maturation failure often occurs only in some of the tubules, so allowing the collection of spermatozoa from others (Silber et al., 1997
). However, in some cases (e.g. Sertoli cell-only syndrome), a complete failure of sperm development extends to all tubules.
The apparent absence of even testicular spermatozoa in many azoospermic men has stimulated an interest in utilizing more immature stagesan approach first suggested by the success of Ogura and Yanagimachi in mice (1993). However, a necessary feature of spermatogenesis, and of a viable germ cell, is the process of haploidization, the youngest haploid cell being the round spermatid. Round spermatids from hamsters and mice are able to replicate their DNA, participate in syngamy and support complete development when incorporated into oocytes either microsurgically (Ogura and Yanagimachi, 1993) or by electrofusion (Ogura et al., 1993
, 1994
). Studies performed in the rabbit have given similar results (Sofikitis et al., 1994
).
The success achieved with this approach in animals has stimulated an interest in the injection into human oocytes of immature spermatogenic cells retrieved from the testis. Fertilization and early cleavage have been observed following the use of human round spermatids in this way (Vanderzwalmen et al., 1995), and a healthy girl was born after ICSI with elongated spermatids (Fishel et al., 1995
, 1997
). In the same year, two normal infants were born after ICSI with round spermatids (ROSI) obtained from ejaculates of azoospermic men (Tesarik et al., 1995
; Tesarik, 1996
). However, the rates of fertilization and pregnancy with round spermatids have been disappointing (Tesarik and Mendoza, 1996
; Amer et al., 1997
; Antinori et al., 1997
; Vanderzwalmen et al., 1997
; Yamanaka et al., 1997
; Palermo et al., 1999
).
While the poor results with spermatids in these years have tended to discredit ROSI, there may be various reasons for this. It is still unclear whether elongating spermatids carry a much greater chance for success than round spermatids. One further problem in using round spermatids is a difficulty in distinguishing them from other round cells such as spermatocytes, monocytes and polymorphonuclear leukocytes. Furthermore, the acrosomal granule can sometimes be misidentified, even under the inverted microscope with Hoffman modulation contrast optics, the system most commonly used for identification and aspiration in wet preparations (Tesarik and Mendoza, 1996; Antinori et al., 1997
; Vanderzwalmen et al, 1997
; Silber and Johnson, 1998
; Veheyen et al, 1998). Recently, a more reliable method has been described using a phase-contrast objective adapted to an inverted microscope together with a Petri dish with an especially thin glass bottom (Verheyen et al., 1998
).
Another problem concerns oocyte activation. Calcium is the universal intracellular signal for triggering oocyte activation (Vitullo and Ozil, 1992; Homa and Swann, 1994
; Tesarik et al., 1994
; Palermo et al., 1997
; Fissore et al., 1999
), and at spermoocyte fusion a factor present in the spermatozoon initiates repetitive transient calcium fluxes in the oocyte (Vitullo and Ozil, 1992
; Ozil and Swann, 1995
). An absence of activation has been found to be the most common cause of fertilization failure after ICSI (Moomjy et al., 1998
), and suboptimal activation after sperm penetration can arrest the fertilization process (Fishel et al., 1996
). An insufficiency of the activation factor in round spermatids retrieved from men with complete failure of spermatogenesis appears to be at least partly responsible for the poor results of ROSI (Tesarik et al., 1998a
). This is in contrast to round spermatids from men with continuing spermiogenesis, which seem able to induce a calcium signalling similar to that brought by mature spermatozoa (Sousa et al., 1996
). It is possible that abnormal calcium signalling after ICSI might also result in chromosomally abnormal embryos (In't Veld et al., 1995
; Tesarik et al., 1998b
). The high frequency of apoptotic germ cells in patients with maturation arrest (Tesarik et al., 1998a
) may compromise ROSI by several mechanisms. In fact, caspase-activated hydrolases may cause damage both to spermatid DNA and to developmentally relevant cytoplasmic components, e.g. oocyte-activating factors, which may be an additional factor for the lower success with round spermatids.
Unfertilized human oocytes injected with mature spermatozoa apparently show a relatively high incidence of premature chromosome condensation (PCC) (Schmiady et al., 1996), which may occur even when spermatids are used for ICSI (Tesarik et al., 1998a
,b
). Unlike mature spermatozoa, whose condensed SS stabilized protamines prevent sperm nuclei being driven to metaphase prematurely by oocyte metaphase promoting factor (MPF), round spermatids feature a dispersed chromatin in which the histone/protamine substitution has hardly begun. From this viewpoint, nuclear maturity appears to be important and could explain the slightly higher fertilization rate using elongated rather than round spermatids (Antinori et al., 1997
). Interestingly, PCC may not always lead to developmental failure, as recently demonstrated by the birth of normal offspring after injection of mouse oocytes with round spermatids followed by delayed oocyte activation (Ogura et al., 1999
). Moreover, it is possible that PCC may be helpful in nuclear reprogramming of immature germ cells, similar to the observed behaviour of somatic cell nuclei (Cibelli et al., 1998
; Wells et al., 1999
).
While healthy babies have been delivered worldwide, some authors have questioned the utility of ROSI as the treatment for azoospermic patients. Silber and Johnson (1998) compared 143 consecutive cases involving testis biopsies of men with non-obstructive azoospermia due to spermatogenic failure, and 62 controls with obstructive azoospermia and normal spermatogenesis. In no case were round spermatids seen in the absence of elongated spermatozoa and maturation arrest was found always to be a failure of progression beyond meiosis, not merely from the round to the elongated spermatid. This suggests that one should search exhaustively for mature spermatozoa or elongated stages before resorting to the injection of spermatids. On the other hand, ROSI might serve as a strategy to avoid the harmful effect of extensive biopsy in searching for spermatozoa. Ischaemia of the testis can be produced by testicular sperm extraction (Schlegel and Su, 1997) and the risk is increased when the intervention involves multiple incisions of the tunica albuginea (Jarow, 1991
). Moreover, complete maturation arrest at the spermatid stage has been reported (Re et al., 1979
; Aumuller et al., 1987
), and round spermatids can be identified in many patients even when late spermatids or spermatozoa were absent (Amer et al., 1997
; Tesarik, 1998
). Since the clinical value of ROSI needs to be evaluated further, patients should be informed about the uncertain safety and lower reproductive outcomes when round spermatids are to be used for conception.
Spermatocytes
The nuclei of secondary spermatocytes have been shown to complete meiosis when injected into mouse oocytes (Kimura and Yanagimachi, 1995). By 2 h after injection of secondary spermatocytes into mature oocytes, some spermatocyte nuclei exhibit premature chromosome condensation and microtubule attachment. Subsequent to electrical activation of oocytes containing a spermatocyte, however, both oocyte and spermatocyte chromosomes resumed their meiosis to form two pronuclei and two polar bodies (one of paternal and one of maternal origin). Of 2-cell embryos transferred to foster mothers, 24% reached full term. In the one example where human secondary spermatocyte nuclei were injected into electro-activated mature oocytes, this was followed by extrusion of a female second polar body and a male pseudo polar body, by formation of two pronuclei and by embryo development, with the reported delivery of a healthy child (Sofikitis et al., 1998
).
Mouse primary spermatocytes also can complete meiosis within maturing or mature oocytes and can participate in embryogenesis although with lower efficiency than can secondary spermatocytes (Kimura et al., 1998; Sasagawa et al., 1998
). Primary spermatocyte nuclei injected into electro-activated mature oocytes transformed to MII and the extrusion of two polar bodies was observed (PbII of oocyte origin and PbI of spermatocyte origin). When a polar body of spermatocyte origin is transferred into another mature oocyte, a male pronucleus may form, and the `haploidized' primary spermatocyte can support embryo development. Similarly, when primary spermatocyte nuclei were injected into MII oocytes, the high level of MPF induced chromosome condensation and spindle formation (Sasagawa et al., 1998
). Such oocytes first extruded two polar bodies, one of oocyte origin, and another of spermatocyte origin, and upon activation by an electric pulse they resumed meiosis to form two pronuclei, and two additional polar bodies were extruded. However, only two out of 258 such embryos transferred at the 2-cell stage developed to term. The reason for this poor success rate remains to be determined. Mechanical manipulation and suboptimal media, as well as incomplete imprinting and DNA repair might account for this low success and poor embryonic development. The high incidence of chromosomal abnormality in primary spermatocyte-injected oocytes (Kimura et al., 1998
) might be another plausible cause for this. Slightly better results have been reported using immature, instead of MII mouse oocytes, for the first step of nuclear haploidization (Ogura et al., 1998
).
Though still imperfect, the use of spermatocytes might one day offer a promising treatment for those patients with spermatogenic arrest, most frequently seen at the end of prophase (the primary spermatocyte level) (Remy and Martin-Du Pan, 1993). While 50% of infertile males might present anomalies of either synapsis (chromosome pairing) at zygotene or desynapsis (precocious separation of paired homologues) at late pachytene, or anomalies of the synaptonemal complex (Lange et al., 1997
), nevertheless, spermatocytes may provide a future alternative through which to treat spermatogenic arrest, particularly if healthy cells can be selected. In fact, in-vitro culture can simultaneously allow the selection of healthy cells and transmeiotic differentiation (Tesarik et al., 1998b
), and represents an interesting alternative to direct injection of immature germ cells or their nuclei into oocytes. After the application of this technique in cases of maturation arrest at the primary spermatocyte stage (Tesarik et al., 1999a
), the first two babies born showed no numerical or structural chromosomal anomalies (Tesarik et al., 1999b
).
Genomic imprinting
In diploid cells, allelic exclusion results in one of two alleles of some genes being inactivated. This inactivation of either paternal or maternal alleles, termed `imprinting', may be exerted through an epigenetic modification of their DNA by methylation of the 5' position of selected cytosine residues (Bergman and Mostoslavsky, 1998); and it is reversible through demethylation, when the silenced gene is reactivated. Tissue-specific genes are methylated in most of the tissues in which they are not expressed, and are unmodified in their tissue of expression (Yeivin and Razin, 1993
). In contrast, the housekeeping genes, which harbour a 5' CpG island, are unmethylated in all tissues (Bird, 1986
). Methylation is also involved in the maintenance of gene repression on the inactive X chromosome in female somatic cells (McCarrey and Dilworth, 1992).
Genomic imprinting is critical for normal development, and its disruption during gametogenesis or in early development underlies certain genetic diseases (e.g. PraderWilli and Angelman syndromes), and can promote the development of malignant childhood tumours (Tycko et al., 1997). The importance of genomic imprinting in mammalian development was first recognized in 1977 (Lyon and Glenister, 1977
). Gene inactivation experiments have since confirmed that imprinted genes in the female and male regulate embryonic and placental growth respectively (Barlow, 1995
). Dramatic methylation changes have been observed during the early steps of embryo development. Most of the DNA in the early blastomeres is unmethylated, and is related to their totipotency; however, an extensive wave of de-novo methylation following implantation modifies most of the genome except the housekeeping genes (Bergman and Mostoslavsky, 1998
). Aberrant methylation may be detrimental to embryo development, and murine embryos that express low values of the maintenance methyltransferase do not develop to term (Razin and Shemer, 1995
). On the other hand, over-expression of the H19 gene in transgenic mice leads to late gestational death (Brunkow and Tilghman, 1991
), and maternal duplication in the region of the Snrpn gene (paternal imprinted allele) causes postnatal lethality in mice (Cattanach et al., 1992
). In addition, methylation of the promoter of the tumour suppressor gene can contribute to tumour formation.
While the exact timing of imprinting events in human gametogenesis is still unclear, there are some speculations based on indirect evidence (Tycko et al., 1997), and the use of immature germ cells in conjunction with ICSI may help to elucidate at which stages imprinting is incomplete. A pilot study did not reveal any differences from controls in the expression of several paternally and maternally imprinted genes in mouse embryos derived from ROSI (Shamanski et al., 1999
). Differential methylation is not seen in germ cells of either sex at an early stage of their development. In spermatogenesis, the erasure of previous imprintings occurs prior to meiosis probably in primordial germ cells or replicating gonocytes. According to DNA methyltransferase activity, re-establishment of the imprinting appears to occur to a large extent in the preleptotene, leptotene and zygotene stages (Shamanski et al., 1999
). However, the finding of residual methyltransferase activity in round spermatids has provoked the suggestion that imprinting is completed only after meiosis. Some minor elements of this might be incomplete even after spermiation, since methylation of genes, e.g. Pgk-2, ApoA1 and Oct-3/4 appeared to occur as sperm transit the epididymis (Ariel et al., 1994
).
Transplantation of spermatogonia
In 1994, Brinster and Zimmermann reported that donor male stem cells could partially repopulate sterile mouse testes when injected into seminiferous tubules, and this produced some fertile spermatozoa (Brinster and Zimmermann, 1994). A remarkable aspect of the colonization was the faithful reconstruction of the complex cellular associations of normal spermatogenesis (Dym and Clermont, 1970
; Ewing et al., 1980
; Russell et al., 1990
). Subsequently, whether stem cells of the rat could colonize mouse seminiferous tubules was investigated (Clouthier et al., 1996
). The rat cells were transplanted to the testes of immunodeficient mice, and when epididymides of eight mice were examined, three belonging to mice with the longest transplants (>110 days) contained rat spermatozoa of normal morphology (Clouthier et al., 1996
). The occurrence of rat spermatogenesis within a mouse testis suggests that stem cells of other species might be transplantable, and opens the possibility of xenogeneic spermatogenesis using human stem cells. More recently, spermatogenesis was reinitiated within the seminiferous tubules 4 weeks after autologous spermatogonial transplantation in the cynomolgus monkey (Schlatt et al., 1999
).
Xenogeneic transplantation of human spermatogonia may thus represent another choice of treatment in conjunction with ICSI as an alternative to the use of meiotic or post-meiotic germ cells. For example, in cases involving X-irradiation or chemotherapy, the hormonal deprivation used to suppress the proliferative activity and so decrease the stem cell sensitivity, is not always effective (Schlatt et al., 1999). In such cases, recovery of germ cells and their later reinfusion into seminiferous tubules may prove to be a potentially valuable technique for re-establishment of the germinal epithelium, especially in pre-pubertal patients.
Another point concerns the efficiency of human spermatogenesis. This is relatively low when compared with that of most animals, perhaps due to a longer duration of spermatogenesis, the lower density of germ cells and the lower percentage of the human testis occupied by seminiferous epithelium (Johnson et al., 1992). In addition, a significant germ cell degeneration of as much as 3645% can be detected, especially during the second meiotic division (Johnson et al., 1992
). Since the success with in-vitro culture of spermatogonia in man or in any other mammals has been limited (Tres et al., 1989
, 1991
), the in-vivo model could offer a very interesting route to understanding the initiation and development of this unique process, either in physiological or pathological conditions.
Although transplantation of human spermatogonia into the mouse testis has yet to be accomplished, the ability to do this might provide a valuable technique through which to study human spermatogenesis, to preserve fertility in patients undergoing ablative chemo- or radiotherapy, and to provide a definitive form of treatment for azoospermic patients with maturation arrest. As a final step that may find a clinical application, spermatogonia could be cryopreserved since this has been successful in the rat (Avarbock et al., 1996). For example, men likely to lose their germ cells (e.g. after undergoing chemotherapy), may have spermatogonia cryopreserved for later recolonization of the irradiated testis, with the prospect of later harvesting spermatids for use in ICSI.
So far, our attempts to transplant human male germ cells have not been successful (Reis et al., 2000). Mixed populations of human spermatogonia, spermatocytes and spermatids (isolated from testis biopsies or orchiectomy specimens) were transferred to the testes of W/Wv micea sterile strain in which only a few primitive stage germ cells can be found. A total of 23 testes of 14 W/Wv mice and 24 testes of 12 SCID mice were injected successfully using 26 testicular samples. Recipient male mice were maintained between 48 and 230 days following injection of human donor germ cells. Our preliminary results, however, failed to demonstrate any reinitiation of spermatogenesis within the mouse testis (Reis et al., 1999
). Selection of a suitable xenogeneic recipient animal may be a necessary first step for successful transplantation of human spermatogonia.
Sources of oocytes
The feasibility of cloning in animals has raised concerns about the indiscriminate application of this to man, and as consequence several countries have imposed a ban on human cloning by legislation or regulation. Nevertheless, techniques such as nuclear transfer developed from cloning research might be a solution for some of the problems we face today in reproductive medicine.
A key step in the cloning procedure is the isolation of a karyoplast and its transfer into an enucleated ooplast. Oocytes can be enucleated by micromanipulation with a glass micropipette, or by chemical treatment. Then, after insertion of the karyoplast, electrical stimulation or viral agents can promote its fusion with the enucleated recipient ooplast.
Nuclear transplantation studies using embryonic and somatic cells have shown that successful embryo reconstitution requires nuclear reprogramming determined by maturation promoting factor (MPF) (Czolowska et al., 1984; Willadsen et al., 1986; Stice and Robl, 1988
; Campbell et al., 1993
). MPF is one of the major cytoplasmic controls of the cell cycle. MPF consists of a cyclin B, a regulatory component, and p34cdc2, a catalytic subunit. During the cell cycle, the concentration of p34cdc2 remains unchanged, whereas the concentration of cyclins varies. MPF activity begins to increase in the oocyte cytoplasm just before germinal vesicle breakdown and is sustained at a high level throughout metaphase I (MI). MPF values decrease at anaphase and telophase I, but increase again as the cell enters MII only to decline rapidly upon fertilization or oocyte activation. During transplantation, all nuclei transferred into a cytoplast with high MPF activity undergo nuclear envelope breakdown and chromosome condensation. The cell cycle is restarted by a suitable artificial stimulus such as electric pulses or exposure to a medium containing ethanol or strontium (Whittingham, 1980
). In addition, the stage in the cycle of the donor nucleus and recipient cytoplasm are important factors for the normality of the `ploidy' in the reconstituted cells (Kono, 1997
). Altering the timing of oocyte activation with respect to the fusion of the donor nucleus provides a number of possible approaches to synchronizing the respective cell cycles.
Maternal age is viewed now as the underlying cause of the chromosomal aneuploidy seen in at least 5% of human conceptions. That age-related aneuploidy is the main reason for the poor embryo implantation observed in older women, is indicated by the high pregnancy rate obtained in aged infertile women when young donor oocytes are used. Ageing of the ooplasm has been considered to be responsible for producing an abnormal meiotic spindle (Battaglia et al., 1996) with spindle abnormalities as the source of incorrect segregation of chromosomes/chromatids at MI (Dailey et al., 1996
). One approach to correction of these oocyte defects is transplantation of the nucleus of immature oocytes into the cytoplast of a younger woman (Zhang et al., 1999
).
In a preliminary study in the mouse, germinal vesicle (GV) karyoplasts were isolated and transferred into immature recipient cytoplasts (Takeuchi et al., 1999a) (Figures 1a,b, and 2a,b
). Not only was oocyte integrity restored in >90% of cases, but 90% of these reconstituted oocytes then extruded a polar body, and when fixed for cytogenetic analysis they displayed a normal chromosomal constitution. This preliminary study suggests that nuclear transplantation in the mouse can be highly efficient and does not cause genetic damage (Takeuchi et al., 1999a
). In attempting the same procedure on human oocytes, the restoration rate at the GV stage was >80%, but the maturation rate was only 60%, an outcome that may be explained by the utilization of a less-than-optimal culture medium for in-vitro maturation (Takeuchi et al., 1998
, 1999b
). While further cytogenetic information is needed about oocytes produced by grafting an old nucleus with a younger cytoplasm, this approach appears to be the only treatment option for age-related aneuploidy. Although it needs to be refined, the technique should stimulate and inspire further research aimed at the treatment of oocyte ageing.
|
|
Both of the above approaches, nuclear transfer and ooplasmic transfer, have one main problem: older women (aged >40 years) produce too few oocytes. However, a sufficient number of oocytes might be created by a form of cloningnuclear transplantation of an older patient's somatic cell into an enucleated ooplast obtained from a younger donor (Figure 3a). The construction of viable gametes from somatic cells would benefit older women, women with premature ovarian failure, or those considered as poor responders, as well as constitute a landmark in the history of reproductive medicine.
|
It is likely that the high rate of embryonic and fetal death arising from epigenetic and centriolar errors in animal cloning (Edwards and Beard, 1998) would also apply in cases where somatic cell haploidization is used to produce a `new' egg for fertilization. Nevertheless, sperm entry and sperm function might modify this outcome, since this establishes a completely new system not found in a simple cloning situation. Recently, mouse clones were produced using metaphase nuclei derived from 4-cell-stage mouse embryos (Kwon and Kono et al., 1996), and more efficiently than with somatic cell nuclei (83% of reconstituted embryos developed into blastocysts, of which 57% resulted in live young). This suggests that the ability of cytoplasm to support embryonic development and correct genomic imprinting differs markedly between fertilized and parthenogenetic 1-cell embryos. However, for the moment, any application to man using adult nuclei should be precluded until the reason for the fetal losses are fully understood, or until their use is proven to be safe in animal experiments.
Cytoplasmic implications
Each mitochondrion contains 210 copies of mitochondrial DNA (mtDNA) in all human tissues, except platelets and oocytes which contain only one copy per mitochondrion. mtDNA is 20 times more susceptible to mutation than nuclear DNA (Kagawa and Hayashi, 1997), due to the location of the mtDNA close to the site of reactive oxidative species formation, and a lack of protective histones in mtDNA (Tritschler and Medon, 1992). Contrary to widely held notions, involving base excision and nucleotide excision repair pathways, mitochondria can efficiently repair oxidative damage to their DNA (Bohr and Dianov, 1999
). Moreover, the mutation rate is highly variable across the genome. Some regions show nucleotide substitution rates similar to those of nuclear DNA, whereas synonymous sites and small rRNAs mutate ~20 times more rapidly, and tRNAs ~100 times more rapidly than their nuclear counterparts (Pesole et al., 1999
). In most related diseases, patients' cells carry a mixture of both mutant and wild-type (normal) mtDNA. This heterogeneous state of cells is called heteroplasmy, while the homogeneous state of cells containing pure mutant or normal mtDNA is termed homoplasmy (Kagawa and Hayashi, 1997
). One factor that appears to be important for the phenotypic expression of mitochondrial diseases is the relative proportion of wild-type and mutant mtDNA (Newman et al., 1991
; Boulet et al., 1992
), and the `dose' of mutant mtDNA also has an influence on the severity of the phenotype (Marchington et al., 1998
). The level of mutant mtDNA varies in different tissues and changes with time (Poulton, 1996
), probably related to the oxidative metabolism inherent in each organ.
In contrast to nuclear DNA, mtDNA is always maternally inherited. In recent years, mtDNA has been the subject of increasing attention due both to the subtle role it may have in early development (Van Blerkom, 1989), and its place in maternal age-related reduction of embryonic competence (Gaulden, 1992
). Mitochondria are also a source of a variety of hereditary disorders. However, the mechanisms controlling the segregation and inheritance of mtDNA in mammals are controversial and poorly understood.
The transmission of mitochondrial disorders is not always uniform, even though the mitochondrial genome is maternally inherited. Mutant mtDNA can arise de novo through the large-scale rearrangement of mtDNA without any familial history, or be maternally inherited as in the cases of mtDNA point mutations. Mitochondrial diseases can also follow an autosomal dominant pattern of inheritance that causes variable deletion of mtDNA or the expression of an autosomal recessive leading to profound cytochrome oxidase deficiency. Finally, X-linked transmission is also seen as a possible inheritance mode (Poulton, 1996).
The proportion of mutant mtDNA transmitted from mother to offspring is variable because of a genetic bottleneck. For example, during germ-line development in early bovine embryogenesis, the number of mitochondria increases 100-fold, from ~1000 per oogonium to ~100 000 per oocyte, while the number of mtDNA increases only ~10-fold, from 10 000 to ~100 000 (Chen et al., 1995). As result, each organelle harbours ~1 mtDNA molecule, instead of the usual 510 (Chen et al., 1995
; Robin and Wong, 1998
). Only a small number of mtDNA molecules replicate and give rise to the entire cytoplasmic genotype (~100 000 mtDNA molecules) during the late stage of oogenesis (Hauswirth and Laipis, 1985
). This single restriction/amplification event or bottleneck may be a necessary solution to the accumulation of mtDNA during the ageing process (Cortopassi et al., 1992
), an accumulation that also occurs in oocytes (Chen et al., 1995
). During and after the mitotic division of cleavage, both mutant and normal mtDNA are distributed unevenly into the daughter cells that give origin to the fetus and its germ cells, thus enabling a complete switch within one or two generations (Meirelles and Smith, 1998
).
As of now, there is no effective treatment for mitochondrial diseases. Following transfer by the cytoplasm fusion method of normal mtDNA into a cell containing mutated mtDNA (Kagawa and Hayashi, 1997), dilution of abnormal mtDNA to below the pathogenic level proved to be a potentially effective way of restoring both biochemical and morphological phenotypes of defective mitochondria. Pursuing the same logic, nuclear transplantation in human germ line cells may offer an attractive therapeutic alternative in providing normal mitochondria at least, in patients compromised by a point mutation disorder. Theoretically, reconstitution of oocytes with healthy donor cytoplasm could diminish the transmission of defective mtDNA. However, since the transplanted nuclei always carry a thin surrounding cytoplasm (Takeuchi et al., 1999a
), a variable number of mitochondria may be transferred as well. However, the first polar body might be a promising source of karyoplasts in which the mitochondria content is negligible (Tsai et al., 1999
) and therefore be a preferable route to a minimization of mtDNA transmission. After injection of the first polar body, a mature enucleated mouse oocyte was later brought to a state of syngamy by ICSI and resulted in normal offspring (Wakayama and Yanagimachi, 1998
).
Nearly all the known activities required for mtDNA replication and expression are nuclear-encoded gene products, necessitating communication between these two intracellular compartments. Several reports indicated that mitochondria located in the perinuclear vicinity are preferentially replicated or initiate replication at an earlier stage than those further away from the nuclei, and give rise to a higher ratio of karyoplast-derived mitochondrial genotypes in daughter cells (Davis and Clayton, 1996). The same observation was supported by an experiment using mouse pronuclear transplantation and later karyoplast-derived mtDNA assessment (Meirelles and Smith, 1998
). Moreover, perinuclear mitochondria from avian and amphibian oocytes replicated more actively and appeared to segregate to the somatic cells of the fetus, while another subcortical group appeared to become localized in the primordial germ cells (D'Herde et al., 1995
).
Despite evidence for preferential replication of karyoplast-derived mitochondria in animals, the question of interspecies differences must be resolved before extrapolating this to man. A recent report has suggested that the genotype of mtDNA from recipient cytoplasm may become the dominant category of mtDNA in calves resulting from nuclear transfer (Takeda et al., 1999). Out of 21 calves, 20 showed a genotype identical to that of the recipient cytoplasm mtDNA. What will be the exact contribution of the karyoplast-derived mtDNA in offspring coming from nuclear transplantation? Can nuclear transplantation using a GV karyoplast actually minimize the transmission of maternal mutant mtDNA? As yet, the unpredictability of mtDNA segregation and a complex threshold variety involved in the phenotype expression do not enable us to reach a firm conclusion.
If the transfer of cytoplasm and nuclei to oocytes becomes safe and effective, this would offer women facing IVF failure a new option through which to conceive their genetically related offspring. As a treatment to improve the developmental potential of the embryo, one could assume that cytoplasm transfer might have negligible impact on the genetic constitution of the offspring, since this involves so little mtDNA. In the case of nuclear transplantation, the biparental character of the reconstituted oocytes may have significant social, psychological and legal implications. Certainly, the significance of mitochondria transferred with a karyoplast, and the putative preferential replication of the perinuclear mtDNA, both need to be better understood to ascertain the exact contribution of the donor and recipient mitochondria in the children that result.
Conclusions
Alternative sources of gametes are not merely science fiction, but already are a concrete fact. As of now, it appears that most of the manipulations are unlikely to have any immediate major impact on assisted reproduction in man. However, while it is important to stress that the genetic normality of the offspring and the safety of the procedures tested in animal experiments must first be firmly established, the results in the latter are encouraging enough to justify further research.
First, the utilization of immature germ cells may represent the only possibility for treatment of many azoospermic men. Although the success rate of this approach is still low, reports of healthy deliveries using round spermatid injection (ROSI) provide clear evidence, in man as well as in mouse, that the morphological steps of sperm formation are not a necessary corollary for participation in syngamy. Furthermore, healthy deliveries in mice using primary and secondary spermatocytes, and in a report in man using secondary spermatocytes, indicate that even spermatogenic cell precursors can sometimes support normal development. On the other hand, the use of immature germ cells has raised concerns about the implications of incomplete genetic imprinting, and the unexplained short life span and retarded growth observed among some offspring when extremely immature germ cells (e.g. primary spermatocytes) were used in mice.
Xenogeneic testicular transplantation of human spermatogonia may one day provide a valuable, if indirect option for treatment of patients with spermatogenetic arrest. Our failure in initial attempts to repopulate mouse seminiferous tubules with human spermatogonia may point to a need for a more suitable animal model. However, autologous spermatogonial transplantation has led to reinitiation of spermatogenesis within the seminiferous tubules in the cynomolgus monkeys (Schlatt et al., 1999). Therefore, autologous transplantation of spermatogonia may offer to oncological patients the possibility to reinitiate spermatogenesis with their own cryopreserved stem cell after completion of chemotherapy or radiotherapy.
Nuclear transplantation has proven to be a highly efficient procedure in mice, in that >90% of reconstituted oocytes were able to extrude a polar body and displayed a normal chromosomal constitution. With human oocytes, however, lower maturation rates have been the rule, probably due to the suboptimal procedures currently available for human oocyte in-vitro maturation. Nonetheless, nuclear transplantation might ultimately provide an attractive treatment option for the age-related aneuploidy seen especially in poor responders and in older patients.
Even more audacious is the attempt to tailor gametes by haploidization of nuclei from somatic cell sources, theoretically, at any time and at any age. Despite its possible promise, centrosome evaluation as well as genetic assessment of the `manufactured oocytes' need to be further pursued to ascertain the potential clinical value of somatic cell haploidization.
Finally, since the role played by karyoplast-derived mtDNA in the mitochondrial phenotype of the offspring is still uncertain, the ethical and social implications of karyoplast transfer have to be taken into consideration before this can become even a relatively routine practice.
Acknowledgments
We thank Queenie V.Neri for editorial assistance of the manuscript, and Richard S.LaRocco for producing the illustrations.
Notes
1 To whom correspondence should be addressed
This opinion was previously published on Webtrack, February 10, 2000
References
Amer, M., Soliman, E., El-Sadek, M. et al. (1997) Is complete spermiogenesis failure a good indication for spermatid conception? Lancet, 350, 116.
Antinori, S., Versaci, C., Dani, G. et al. (1997) Fertilization with human testicular spermatids: four successful pregnancies. Hum. Reprod., 12, 286291.[Abstract]
Ariel, M., Cedar, H. and McCarrey, J. (1994) Developmental changes in methylation of spermatogenesis-specific genes include reprogramming in the epididymis. Nature Genet., 7, 5963.[ISI][Medline]
Avarbock, M.R., Brinster, C.J. and Brinster, R.L. (1996) Reconstruction of spermatogenesis from frozen spermatogonial stem cells. Nature Med., 2, 693696.[ISI][Medline]
Aumuller, G., Fuhrmann, W. and Krause, W. (1987) Spermatogenic arrest with inhibition of acrosome and sperm tail development. Andrologia, 19, 917.[ISI][Medline]
Barlow, D.P. (1995) Gametic imprinting in mammals. Science, 270, 16101613.[Abstract]
Battaglia, D.E., Goodwin, P., Klein, N.A. et al. (1996) Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women. Hum. Reprod., 11, 22172222.[Abstract]
Bergman, Y. and Mostoslavsky, R. (1998) DNA demethylation: turning genes on. Biol. Chem., 379, 401407.[ISI][Medline]
Bird, A. (1986) CpG-rich islands and the function of DNA methylation. Nature, 321, 209213[ISI][Medline]
Bohr, V. A. and Dianov, G.L. (1999) Oxidative DNA damage processing in nuclear and mitochondrial DNA. Biochimie, 81, 155160.[ISI][Medline]
Boulet, L., Karpati, G. and Shoubridge, E.A. (1992) Distribution and threshold expression of the tRNA(lys) mutation in skeletal muscle of patients with myoclonic epilepsy and ragged-red fibers (MERRF). Am. J. Hum. Genet., 51, 11871200.[ISI][Medline]
Brinster, R.L. and Zimmermann, J.W. (1994) Spermatogenesis following male germ-cell transplantation. Proc. Natl Acad. Sci. USA, 92, 1129811302.
Brunkow, M.E. and Tilghman, S.M. (1991) Ectopic expression of the H19 gene in mice causes prenatal lethality. Genes Dev., 5, 10921101.[Abstract]
Campbell, K.H.S., Ritchie, W.A. and Wilmut, I. (1993) Disappearance of maturation promoting factor and the formation of pronuclei in electrically activated in vitro matured bovine oocytes. Theriogenology, 39,199.
Cattanach, B.M., Barr, J.A., Evans, E.P. et al. (1992) A candidate mouse model for PraderWilli syndrome which shows an absence of Snrpn expression. Nature Genet., 2, 270274.[ISI][Medline]
Chen, X., Prosser, R., Simonetti, S. et al. (1995) Rearranged mitochondrial genomes are present in human oocytes. Am. J. Hum. Genet., 57, 239247.[ISI][Medline]
Cibelli, J.B., Stice, S.L., Golueke, P.J. et al. (1998) Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science, 280, 12561258.
Clouthier, D.E., Avarbock, M.R., Maika, S.D. et al. (1996) Rat spermatogenesis in mouse testis. Nature, 381, 30.[ISI][Medline]
Cohen, J., Scott, R., Alikani, M. et al. (1998) Ooplasmic transfer in mature human oocytes. Mol. Hum. Reprod., 4, 269280.[Abstract]
Cortopassi, G.A., Shibata, D., Soong, N.W. et al. (1992) A pattern of accumulation of somatic deletion of mitochondria DNA in aging human tissues. Proc. Natl Acad. Sci. USA, 89, 73707374.[Abstract]
Czolowska, R., Modlinski, J.A. and Tarkowski, A.K. (1984) Behavior of thymocyte nuclei in nonactivated and activated mouse oocytes. J. Cell Sci., 69, 1934.[Abstract]
Dailey, T., Dale, B., Cohen, J. et al. (1996) Association between nondisjunction and maternal age in meiosis-II human oocytes. Am. J. Hum. Genet., 59, 17684.[ISI][Medline]
Davis, A.F. and Clayton, D.A. (1996) In situ localization of mitochondria DNA replication in intact mammalian cells. J. Cell Biol., 135, 883893.[Abstract]
D'Herde, K.M., Gallebaut, F., Roels, B. et al. (1995) Homology between mitochondriogenesis in avian and amphibian oocyte. Reprod. Nutr. Dev., 35, 305311.[ISI][Medline]
Dym, M. and Clermont, Y. (1970) Role of spermatogonia in the repair of the seminiferous epithelium following X-irradiation of testis. Am. J. Anat., 128, 265282.[ISI][Medline]
Edwards, R.G. and Beard, H. (1998) How identical would cloned children be? An understanding essential to the ethical debate. Hum. Reprod. Update, 4, 791811.
Ewing, L.L., Davis, J.C. and Zirkin, B.R. (1980) Regulation of testicular function: a spatial and temporal view. Int. Rev. Physiol., 22, 41115.[Medline]
Fishel, S., Green, S., Bishop, M. et al. (1995) Pregnancy after intracytoplasmic injection of spermatid. Lancet, 345, 641642.[Medline]
Fishel, S., Aslam, I. and Tesarik, J. (1996) Spermatid conception: a stage too early, or a time too soon? Hum. Reprod., 11, 13711375.
Fishel, S., Green, S., Hunter, A., et al. (1997) Human fertilization with round and elongated spermatids. Hum. Reprod., 12, 336340.[Abstract]
Fissore, R.A., Reis, M.M. and Palermo, G.D. (1999) Sperm-induced calcium oscillation. Isolation of the Ca2+ releasing component(s) of mammalian sperm extracts: the search continues. Mol. Hum. Reprod., 5, 189192.
Gaulden, M. (1992) The enigma of Down syndrome and other trisomic conditions. Mutat. Res., 269, 6988
Hassold, T.J. and Jacobs, P.A. (1984) Trisomy in man. Ann. Rev. Genet., 18, 6997.[ISI][Medline]
Hauswirth, W. and Laipis, P. (1985) Transmission genetics of mammalian mitochondria: a molecular model and experimental evidence. In Quagliarello, E. (ed.), Achievements and Perspectives of Mitochondrial Research. Vol 2. Elsevier, Amsterdam, The Netherlands, pp. 4959.
Homa, S.T., Swann, K. (1994) A cytosolic sperm factor triggers calcium oscillations and membrane hyperpolarizations in human oocytes. Hum. Reprod., 9, 23562361.[Abstract]
In't Veld, P., Brandenburg, H., Verhoeff, A. et al. (1995) Sex chromosomal abnormalities and intracytoplasmic sperm injection. Lancet, 346, 773.[ISI][Medline]
Jarow, J.P. (1991) Clinical significance of intratesticular arterial anatomy. J. Urol., 145, 777779.[ISI][Medline]
Johnson, L., Chaturvedi, P.K. and William, J.D. (1992) Missing generation of spermatocytes and spermatids in seminiferous epithelium contribute to low efficiency of spermatogenesis in humans. Biol. Reprod., 47, 10911098.[Abstract]
Kagawa, Y. and Hayashi, J.I. (1997) Gene therapy of mitochondrial diseases using human cytoplasts. Gene Therapy, 4, 610.[ISI][Medline]
Kimura, Y. and Yanagimachi, R. (1995) Development of normal mice from oocytes injected with secondary spermatocyte nuclei. Biol. Reprod., 53, 855862.[Abstract]
Kimura, Y., Tateno, H., Handel, M.A. et al. (1998) Factors affecting meiotic and developmental competence of primary spermatocyte nuclei injected into mouse oocytes. Biol. Reprod., 59, 871877.
Kono, T. (1997) Nuclear transfer and reprogramming. Rev. Reprod., 2, 7480.
Kubelka, M. and Moor, R.M. (1997) The behavior of mitotic nuclei after transplantation to early meiotic ooplasts or mitotic cytoplasts. Zygote, 5, 219227.[ISI][Medline]
Kwon, O.Y. and Kono, T. (1996) Production of identical sextuplet mice by transferring metaphase nuclei from four-cell embryos. Proc. Natl Acad. Sci. USA, 93, 1301013013.
Lange, R., Krause, W. and Engel, W. (1997) Analyses of meiotic chromosomes in testicular biopsies of infertile patients. Hum. Reprod., 12, 21542158.[Abstract]
Liu, L., Day, Y. and Moor, R.M. (1997) Nuclear transfer in sheep embryos: the effect of cell-cycle coordination between nucleus and cytoplasm and the use of in vitro matured oocytes. Mol. Reprod. Dev., 47, 255264.[ISI][Medline]
Lyon, M.F. and Glenister, P.H. (1977) Factors affecting the observed number of young resulting from adjacent-2 disjunction in mice carrying a translocation. Genet. Res., 29, 8392.[ISI][Medline]
MacCarrey, J.R. and Dilworth, D.D. (1992) Expression of Xist in mouse germ cells correlates with x-chromosome inactivation. Nat. Genet, 2, 200203.[ISI][Medline]
Martin-Du Pan, R. and Campana, A. (1993) Physiopathology of spermatogenic arrest. Fertil. Steril., 60, 937945.[ISI][Medline]
Marchington, D.R., Macaulay, V., Hartshorne, G.M. et al. (1998) Evidence from human oocytes for a genetic bottleneck in an mtDNA disease. Am. J. Hum. Genet., 63, 769775.[ISI][Medline]
Meirelles, F. and Smith, L.C. (1998) Mitochondrial genotype segregation during preimplantation development in mouse heteroplasmic embryos. Genetics, 148, 877883.
Moomjy, M., Sills E.S., Rosenwaks, Z. et al. (1998) Implication of complete fertilization failure after intracytoplasmic sperm injection for subsequent fertilization and reproductive outcome. Hum. Reprod., 13, 22122216.[Abstract]
Newman, N.J., Lott, M.T. and Wallace, D.C. (1991) The clinical characteristic of pedigrees of Leber's hereditary optic neuropathy with the 11778 mutation. Am. J. Ophtalmol., 111, 750762.[ISI][Medline]
Ogura, A. and Yanagimachi, R. (1993) Round spermatid nuclei injected into hamster oocyte form pronuclei and participate in syngamy. Biol. Reprod., 48, 219225.[Abstract]
Ogura, A, Yanagimachi, R. and Usui, N. (1993) Behavior of hamster and mouse round spermatid nuclei incorporated into mature oocytes by electrofusion. Zygote, 1, 18.[Medline]
Ogura, A, Matsuda, J. and Yanagimachi, R. (1994) Birth of normal young after electrofusion of mouse oocytes with round spermatids. Proc. Natl Acad. Sci. USA, 91, 74607462.[Abstract]
Ogura, A., Suzuki, O., Tanemura, K. et al. (1998) Development of normal mice from metaphase I oocytes fertilized with primary spermatocytes. Proc. Natl Acad. Sci. USA, 12, 56115615.
Ogura, A., Inoue, K. and Matsuda, J. (1999) Mouse spermatid nuclei can support full term development after premature chromosome condensation within mature oocytes. Hum. Reprod., 14, 12941298.
Ozil, J.P. and Swann, K. (1995) Stimulation of repetitive calcium transients in mouse eggs. J. Physiol., 483, 331346.[Abstract]
Palermo, G., Joris, H., Devroey, P. et al. (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet, 340, 1718.[ISI][Medline]
Palermo, G.D., Cohen, J., Alikani, M. et al. (1995a) Development and Implementation of Intracytoplasmic Sperm Injection (ICSI). Reprod. Fertil. Dev., 7, 211218.[ISI][Medline]
Palermo, G.D., Cohen, J., Alikani, M. et al. (1995b) Intracytoplasmic sperm injection: A novel treatment for all forms of male factor infertility. Fertil. Steril., 63, 12311240.[ISI][Medline]
Palermo, G.D., Cohen, J. and Rosenwaks, Z. (1996) Intracytoplasmic Sperm Injection: a powerful tool to overcome fertilization failure. Fertil. Steril., 65, 899908.[ISI][Medline]
Palermo, G.D., Avrech, O.M., Colombero, L.T. et al. (1997) Human sperm cytosolic factor triggers Ca2+ oscillations and overcomes activation failure of mammalian oocytes. Mol. Hum. Reprod., 3, 367372.[Abstract]
Palermo, G.D., Schlegel, P.N., Hariprashad, J.J. et al. (1999) Fertilization and pregnancy outcome with intracytoplasmic sperm injection for azoospermic men. Hum. Reprod., 14, 741748.
Pesole, G., Gissi, C., De Chirico, A. et al. (1999) Nucleotide substitution rate of mammalian mitochondrial genomes. J. Mol. Evol., 48, 427434.[ISI][Medline]
Poulton, J. (1996) New genetics of mitochondrial DNA diseases. Br. J. Hosp. Med., 55, 712716.[ISI][Medline]
Razin, A. and Shemer, R. (1995) DNA methylation in early development. Hum. Mol. Genet., 4, 17511755.[Abstract]
Reis, M.M., Schlegel, P.N. Takeuchi, T. et al. (1999) Xenogeneic transplantation of human spermatogonia. Gynecol. Endocrinol., 13 (Suppl. 3), 24.
Reis, M.M., Tsai, M.C., Schlegel, P.N. et al. (2000) Xenogeneic transplantation of human spermatogonia. Zygote, (in press).
Re, M., Carpini-Familari, G., Iannitelli, M. and Vicari, A. (1979) Ultrastructural characteristics of idiopathic spermatidic arrest. Arch. Androl., 2, 283289.[ISI][Medline]
Remy, C. and Martin-Du Pan, M.D. (1993) Physiopathology of spermatogenic arrest. Fertil. Steril., 60, 937945.[ISI][Medline]
Robin, E.D. and Wong, R. (1998) Mitochondrial DNA molecules and virtual number of mitochondria per cell in mammalian cells. J. Cell Physiol., 136, 507513.
Russell, L.D., Ettlin, R.A., Hikim, A.P. et al. (1990) Histological and Histopathological Evaluation of the Testis. Cache River Press, Clearwater, FL, pp. 140.
Sasagawa, I., Kuretake, S., Eppig, J.J. et al. (1998) Mouse primary spermatocytes can complete two meiotic divisions within the oocyte cytoplasm. Biol. Reprod., 58, 248254.[Abstract]
Shamanski, F.L., Kimura, Y., Lavoir, M.C. et al. (1999) Status of genomic imprinting in mouse spermatids. Hum. Reprod., 14, 10501056.
Shiels, P.G., Kind, A.J., Campbell, K. H.S. et al. (1999) Analysis of telomere lengths in cloned sheep. Nature, 399, 316317.[ISI][Medline]
Schlatt, S., Rosiepen, G., Weinbauer, G.F. et al. (1999) Germ cell transfer into rat, bovine, monkey and human testes. Hum. Reprod., 14, 144150.
Schlegel, P.N. and Su, L.M. (1997) Physiological consequences of testicular sperm extraction. Hum. Reprod., 12, 16881692.[Abstract]
Schmiady, H., Tandler-Schneider, A and Kentenich, H. (1996) Premature chromosome condensation of sperm nucleus after intracytoplasmic sperm injection. Hum. Reprod., 11, 22312245.
Silber, S.J. and Johnson, L. (1998) Are spermatid injections of any clinical value? Hum. Reprod., 13, 509523.
Silber, S.J., Nagy, Z., Devroey, P. et al. (1997) Distribution of spermatogenesis in the testicles of azoospermic men: the presence or absence of spermatids in the testes of men with germinal failure. Hum. Reprod., 12, 24222428.[Abstract]
Sousa, M., Mendonza, C., Barros, A. et al. (1996) Calcium responses of human oocytes after intracytoplasmic injection of leucocyte, spermatocyte and spermatids. Mol. Hum. Reprod., 2, 853857.[Abstract]
Sofikitis, N.V., Miyagawa, I., Agapito, E. et al. (1994) Reproductive capacity of the nucleus of the male gamete after completion of meiosis. J. Assist. Reprod. Genet., 11, 335341.[ISI][Medline]
Sofikitis, N., Mantzavino, T., Loutradis, D. et al. (1998) Ooplasmic injection of secondary spermatocytes for non-obstructive azoospermia. [Letter.] Lancet, 351, 11771178.[ISI][Medline]
Stice, S.L. and Robl, J.M. (1988) Nuclear reprogramming in nuclear transplant rabbit embryos. Biol. Reprod., 39, 657664.[Abstract]
Takeuchi, T., Ergun, B., Huang, T.H. et al. (1998) Preliminary experience of nuclear transplantation in human oocytes. Fertil. Steril., 70 (Suppl. 1), S86S87.
Takeuchi, T., Ergün, B., Huang, T.H. et al. (1999a) A reliable technique of nuclear transplantation for immature mammalian oocytes. Hum. Reprod., 14, 13121317.
Takeuchi, T., Tsai, M.C., Gong, J. et al. (1999b) Cytogenetic analysis of reconstituted human oocytes after nuclear transplantation. [Abstr. no. O-057.] Hum. Reprod., 14 (Abstract Book 1), 31.
Takeuchi, T., Tsai, M.C., Spandorfer, S.D. et al. (1999c) An alternative source of oocytes. [Abstr. no. O-013]. Hum. Reprod., 14 (Abstract Book 1), 7.
Tesarik, J. (1996) Fertilization of oocyte by injecting spermatozoa, spermatids and spermatocytes. Rev. Reprod., 1, 149152.
Tesarik, J. (1998) Oocyte activation after intracytoplasmic injection of mature and immature sperm cells. Hum. Reprod., 13 (Suppl. 1), 117127.
Tesarik, J. and Mendoza, C. (1996) Spermatid injection into human oocytes. I. Laboratory techniques and special features of zygote development. Hum. Reprod., 11, 772779.[Abstract]
Tesarik, J., Sousa, M. and Testart, J. (1994) Human oocyte activation after intracytoplasmic sperm injection. Hum. Reprod., 9, 511518.[Abstract]
Tesarik, J., Mendoza, C. and Testart, J. (1995) Viable embryos from injection of round spermatids into oocytes. [Letter.] N. Engl. J. Med., 333, 525.
Tesarik, J., Rolet, F., Brami, C. et al. (1996) Spermatid injection into human oocytes. II. Clinical application in the treatment of infertility due to non-obstructive azoospermia. Hum. Reprod., 11, 780783.[Abstract]
Tesarik, J., Greco, E., Cohen-Bacrie, P. et al. (1998a) Germ cell apoptosis in men with complete and incomplete spermiogenesis failure. Mol. Hum. Reprod., 4, 757762.[Abstract]
Tesarik, J., Guido, M., Mendoza, C. et al. (1998b) Human spermatogenesis in vitro: respective effects of follicle-stimulating hormone and testosterone on meiosis, spermiogenesis, and sertoli cell apoptosis. J. Clin. Endocrinol. Metab., 83, 44674473.
Tesarik, J., Bahceci, M., Ozcan, C. et al. (1999a) Restoration of fertility by in-vitro spermatogenesis. Lancet, 353, 555556.[ISI][Medline]
Tesarik, J., Bahceci, M., Ozcan, C. et al. (1999b) In-vitro spermatogenesis. Lancet, 353, 17071708.
Tietze, C. (1957) Reproductive span and rate of reproduction among Hutterite women. Fertil. Steril., 8, 8997.[ISI]
Tres, L.L., Mesrobian, H.G. and Abdullah, M. (1989) Human Sertoli spermatogenic cell cocultures prepared from biopsies of cryptorchid testes performed during orchidopexy. J. Urol., 141, 10031009.[ISI][Medline]
Tres, L.L., Smith, F.F. and Kierszenbaum, A.L. (1991) Spermatogenesis in vitro: methodological advances and cellular functional parameters. In Negro-Vilar, A. and Perez-Palacios, G. (eds), Reproduction, Growth and Development. Vol. 71. Serono Symposia, Raven Press, New York, USA, pp. 115125.
Tritschler, H.J. and Medori, R. (1992) Mitochondrial DNA alteration as a source of human disorders. Neurology, 43, 280288.[Abstract]
Tsai, M.C., Takeuchi, T., Rosenwaks, Z. et al. (1999) The mitochondrial status of karyoplast used for nuclear transplantation. [Abstract no. O-166.] Hum. Reprod., 14 (Abstract Book 1), 92.
Tycko, B., Trasler, J. and Bestor, T. (1997) Genomic imprinting: Gametic mechanisms and somatic consequences. J. Androl., 18, 480486.
Van Blerkom, J. (1989) Developmental failure in human reproduction associated with preovulatory oogenesis and pre-implantation embryogenesis, In Van Blerkom, J. and Motta, P. (eds), Ultrastructure of Human Gametogenesis and Embryogenesis. Kluwer, Dordrecht, The Netherlands, pp. 125180.
Van Blerkom, J., Davis, P., Merriam, J. et al. (1995) Nuclear cytoplasmic dynamics of sperm penetration, pronuclear formation and microtubule organization during fertilization and early preimplantation development in the human. Hum. Reprod. Update, 1, 429461.[Abstract]
Van Uem, J.F., Acosta, A.A., Swanson, R.J. et al. (1985) Male factor evolution in in vitro fertilization: Norfolk experience. Fertil. Steril., 44, 375383.[ISI][Medline]
Vanderzwalmen, P., Lejeune, B., Nijs, M. et al. (1995) Fertilization of an oocyte microinseminated with a spermatid in an in vitro fertilization programme. Hum. Reprod., 10, 502503.[ISI][Medline]
Vanderzwalmen, P., Zech, H., Birkenfeld, A. et al. (1997) Intracytoplasmic injection of spermatids retrieved from testicular tissue: influence of testicular pathology, type of selected spermatids and oocyte activation. Hum. Reprod., 12, 12031213.[ISI][Medline]
Verheyen, G., Crabbe, E., Joris, H. et al. (1998) Simple and reliable identification of the human round spermatid by inverted phase-contrast microscopy. Hum. Reprod., 13, 15701577.[Abstract]
Vitullo, A.D. and Ozil, J.P. (1992) Repetitive calcium stimuli drive meiotic resumption and pronuclear development during mouse oocyte activation. Dev. Biol., 151, 128136.[ISI][Medline]
Wakayama, T. and Yanagimachi, R (1998) The first polar body can be used for the production of normal offspring in mice. Biol. Reprod., 59, 100104.
Wells, D.N., Misica, P.M. and Tervit, H.R. (1999) Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells. Biol. Reprod., 60, 9961005.
Willadsen, S.M. (1986) Nuclear transplantation in sheep embryos. Nature, 320, 6365.[ISI][Medline]
Whittingham, D.G. (1980) Parthenogenesis in mammals. In Whittingham, D.G. (eds), Oxford Review of Reproductive Biology. Oxford University Press, Oxford, UK, pp. 205231.
Yamanaka, K., Sofikitis, N.V., Miyagawa, I. et al. (1997) Ooplasmic round spermatid nuclear injection procedure as an experimental treatment for nonobstructive azoospermia. J. Assist. Reprod. Genet., 14, 5562.[ISI][Medline]
Yeivin, A. and Razin, A. (1993) Gene methylation patterns and expression. EXS, 64, 524568.
Zhang, J., Wang, C.W. Krey, L. et al. (1999) In vitro maturation of human preovulatory oocytes reconstructed by germinal vesicle (GV) transfer. Fertil. Steril., 71, 726731.[ISI][Medline]