Fertilization of Oocytes by Injecting Spermatozoa, Spermatids and Spermatocytes

Home , Oocyte, Spermatocyte, Spermatozoon

Reviews of Reproduction (1996) 1, 149–152

Fertilization of oocytes by injecting spermatozoa, spermatids and spermatocytes

Jan Tesarik

Division of Reproductive Biology, University of Granada Faculty of Sciences, Campus Universitario Fuentenueva, 18071 Granada, Spain; and Laboratoire d’Eylau, 55 Rue Saint-Didier, 75116 Paris, France

The feasibility of fertilization by injecting spermatozoa into oocytes has increased signiﬁcantly the possibilities for treatment of severe male infertility. However, the rapidity of human application has raised some concern about potential health hazards for the progeny. Human pregnancies and births have also occurred with the use of immature spermatozoa and spermatids, and normal offspring have been born after the injection of secondary spermatocytes into mouse oocytes. This short review deals with the problems that may arise from the injection technique and from the use of deﬁcient or immature sperm cells for fertilization, with particu- lar attention to human applications. Tests for screening parents and follow-up of children are suggested to control the main suspected risk factors.

The first successful fertilization of a mammalian oocyte by a been reported from a few centres (Fishel et al., 1995; Tesarik et al., direct intra-ooplasmic transfer of a spermatozoon was reported 1995; Vanderzwalmen et al., 1995). These efforts led to the first in hamsters 20 years ago (Uehara and Yanagimachi, 1976). In childbirth after injection of round spermatids into human oocytes rabbits and cattle, embryos obtained with the use of this (Tesarik et al., 1995) and to the establishment of other ongoing method were transferred to recipient mothers, resulting in pregnancies (Fishel et al., 1995; Tesarik et al., 1995). Kimura and the birth of normal offspring (Iritani, 1991). The importance Yanagimachi (1995) demonstrated that normal mice can also de- of this pioneering work for subsequent human application velop from oocytes injected with secondary spermatocyte nuclei, needs to be acknowledged. However, the success rate of this showing that the problem of ploidy associated with the use of the method was very low until the early 1990s when an improve- early stages of spermatogenic cells that have not yet completed ment of the micromanipulation technique, performed on human meiosis can be resolved by the use of an appropriate technique. gametes, led to a spectacular increase in fertilization rates and first childbirths (Palermo et al., 1992). Since then, the method, Questions relating to the injection technique known as ICSI (for ‘intracytoplasmic sperm injection’), has been used throughout the world as a treatment of last chance for The injection technique brings about two biologically relevant men with extremely severe oligo- and asthenozoospermia but effects that are common to all methods using intra-ooplasmic also as a ‘more efficient’ treatment in less severe cases in which injection for fertilization, regardless of the quality and maturity the standard in vitro fertilization could also be envisaged. of the injected sperm cell. These effects are disruption of the Owing to a strong clinical demand, the human application of plasma membrane and elimination of the normal interaction ICSI has advanced animal studies significantly, and experimen- between the surfaces of both gametes preceding gamete fusion. tal work on human oocytes donated by patients for research Mammalian cells do not survive disruptions to the plasma purposes has also lagged behind clinical applications mainly membrane unless an efficient resealing mechanism is rapidly set performed on a purely empirical basis. in motion after injection. This mechanism implies an accumu- The use of a microinjection technique to transfer the sper- lation and fusion with the wounded plasma membrane of one or matozoon into the oocyte has rendered unnecessary many sys- more cytoplasmic membrane compartments at the disruption site tems required for normal fertilization in which spermatozoa and is triggered by the disruption-provoked Ca2+ influx down undergo a functional maturation during the passage of sper- an approximately 104-fold gradient between the extracellular matozoa through the male genital tract (for example the motil- and the intracellular milieu (Miyake and McNeil, 1995). ity apparatus, zona pellucida receptors, acrosome) so that ICSI The contact and interaction between the surfaces of both has become a treatment of choice for patients with obstructive gametes before fusion is believed to be involved in triggering azoospermia in whom spermatozoa can be obtained by epidi- activation of mammalian oocytes; the most popular theory ex- dymal aspiration or testicular biopsy (for example Silber, 1994). plaining this event implies the binding of as yet unidentified Moreover, animal experiments showing that viable embryos molecules on the sperm surface to receptors on the oocyte can develop from oocytes fertilized with round spermatids, the surface, leading to activation of a G-protein–phosphoinositide youngest male germ cells to have a set of haploid chomosomes messenger system (Jaffe, 1990). In normal fertilization, this (Ogura et al., 1994; Sofikitis et al., 1994), have encouraged at- mechanism appears to be complemented by another oocyte- tempts at fertilizing human oocytes by injecting spermatids activating mechanism that relies on the release from the fertil- from patients with nonobstructive azoospermia. Successful fer- izing spermatozoon after sperm–oocyte fusion of a cytosolic tilization with round and elongated spermatids in humans has factor (reviewed in Tesarik and Sousa, 1994). Both mechanisms © 1996 Journals of Reproduction and Fertility 1359-6004/96 $8.50 Downloaded from Bioscientifica.com at 09/25/2021 05:50:09AM via free access 150 J. Tesarik produce an increase in the free cytoplasmic Ca2+ concentration concern only fertility but others have a complex pathology which is important for pronuclear formation and cleavage and which may affect the duration and quality of human life. which has an oscillatory character in mammals (Swann and Ozil, 1994). The first sperm-induced Ca2+ increase has been Epigenetic factors shown to occur at least 7 s after sperm–oocyte membrane In addition to the male genome, the fertilizing spermato- fusion in sea urchins (McCulloh and Chambers, 1992). A sol- zoon delivers to the future embryo a number of epigenetic fac- uble sperm protein, termed oscillin and existing as an oligomer tors that are no less essential for normal development. These with a subunit of M 33 K, capable of activating mammalian r factors comprise a cytosolic oocyte-activating factor (see above), oocytes by triggering a characteristic series of Ca2+ oscillations the microtubule organizing centre (centrosome), and a male- has been cloned and shown to have sequence identity of 53% to specific imprint on certain developmentally regulated genes. In a glucosamine-6-phosphate isomerase isolated from Escherichia the absence of the normal gamete surface interaction, the cyto- coli (Parrington et al., 1996). Experience with ICSI, in which solic sperm factor is the principal agent responsible for oocyte the normal interaction between gamete surfaces is abolished, activation after ICSI (Tesarik and Sousa, 1994). Later, during suggests that this cytosolic factor alone can ensure all of the embryo cleavage, Ca2+-releasing activity becomes associated oocyte activation events. It appears that the Ca2+ influx at with blastomere nuclei (Kono et al., 1995), where it may have the puncture site is essential for oocyte activation after ICSI other specific functions. The sperm-derived centrosome is and that it performs, by an as yet undetermined mechanism, necessary for the development of the microtubule organizing the role of the surface component of the normal oocyte acti- activity of human embryonic cells (for example Sathananthan vation system (Tesarik and Sousa, 1995). This Ca2+ influx is et al., 1991) as is the case in most mammals studied. The patern- facilitated by hydrodynamic forces provoked by the aspiration ally specific mark (imprint) on the sperm-derived allelles of of the oocyte cytoplasm just before sperm expulsion from certain genes is implied in transcriptional control and is re- the microinjection needle (Tesarik and Sousa, 1995) and is im- quired for normal development. All these factors may be de- portant for resealing the damaged plasma membrane as well as ficient in abnormal spermatozoa, although direct experimental the subsequent oocyte activation. evidence for such an association is lacking. It may be relevant that elimination of the normal gamete surface interaction leads to a slight modification of the oocyte Ca2+ response to the fertilizing spermatozoon (Tesarik and Questions relating to sperm cell immaturity Sousa, 1994). The pattern of these Ca2+ signals can be modified by the injection technique (Tesarik and Sousa, 1995). Because When considering the use of immature spermatogenic cells for the fertilization-induced Ca2+ signal may act as an epigenetic fertilization, the problem of ploidy, associated with the use of factor influencing embryo quality (Swann and Ozil, 1994), the pre-meiotic male germ cells, has long been thought insurmount- injection technique must be considered as a possible source of able. However, an elegant study by Kimura and Yanagimachi abnormalities of oocyte activation that may consequently affect (1995) has shown that this problem can be resolved by manipu- the embryonic genome by provoking cell cycle irregularities lating the time at which oocyte activation is triggered. In the (Tesarik, 1995). absence of oocyte activation, the injected cell nucleus is exposed Last but not least, the injection technique increases the risk to metaphase-promoting factor (MPF) and cytostatic factor (CF), of unintentional introduction of foreign DNA into the oocyte. which are active in the oocyte cytoplasm and maintain the Thus, all materials and media coming into contact with the chromosomes of mature mammalian oocytes in metaphase of gametes used for ICSI should be tested for the absence of DNA the second meiotic division. After being translocated to the contamination, and the precautions normally taken in a mol- oocyte periphery, the injected nucleus thus reacts to MPF and ecular biology laboratory should be taken during all steps of CF in the same way as does the oocyte nucleus and enters the manipulation. metaphase (Fig. 1). When an artificial oocyte-activating signal is subsequently delivered, MPF and CF are inactivated and both the oocyte chromosomes and the chomosomes of the injected Questions relating to sperm cell deficiency nucleus enter anaphase, which leads to the haploidization of both chromosomal sets (Fig. 1). Thus, the premature chromo- Genetic factors some condensation (PCC) occurring in the injected nucleus in It is known that gross abnormalities of sperm morphology the absence of oocyte activation is essential for achieving fertil- and function can be due to genetic factors. Microdeletions on ization with secondary spermatocytes (Kimura and Yanagimachi, the long arm of the Y chromosome have been detected in 1995). However, the same phenomenon of PCC presents a risk some patients with azoospermia and severe oligozoospermia of aneuploidization when haploid spermatid nuclei are in- (Kobayashi et al., 1994). Some of these microdeletions are as- jected. Mature spermatozoa appear to be protected from PCC sociated with spermatogenic arrest at the spermatid stage by their special chromatin arrangement preventing a rapid re- (Reijo et al., 1995). In other cases, oligo(asthenoterato)zoosper- action to MPF and CF. mia may be related to X-linked androgen receptor insensitivity Regardless of ploidy, all the questions concerning sperm cell syndrome (McPhaul et al., 1993) or to hereditary elliptocytosis deficiency raised in the previous section also apply to sperm cell (Patrizio, 1995). Some cases of the congenital absence of vas immaturity. As for genetic factors, the X-linked form of Kallman deferens are caused by cystic fibrosis mutations (Patrizio, 1995). syndrome (Prager and Braunstein, 1993) must also be taken into Without previous diagnosis, ICSI is likely to increase the risk of account when the gonadotrophin concentrations are extremely transmission of these abnormalities to progeny; some of these low and, in the absence of spermatogenesis, the injection of

Downloaded from Bioscientifica.com at 09/25/2021 05:50:09AM via free access Microinjection-assisted fertilization 151 sperm precursor cells is envisaged. As for epigenetic factors, the birth of normal progeny after intra-ooplasmic injection of secondary spermatocytes (Kimura and Yanagimachi, 1995) and (a) round spermatids (Ogura et al., 1994) in mice and of round spermatids in humans (Tesarik et al., 1995) supports the contention that genomic imprinting status at the respective stages in the MPF two species can be compatible with normal development. CF However, this limited experience does not mean that the risk of genomic imprinting abnormalities linked to the use of immature sperm cells is totally excluded. Moreover, this uncertainty has not yet been overcome even for the use of more mature stages, such as testicular and epididymal spermatozoa, because experiments conducted in mice show that reprogramming of certain sperm genes continues in the epididymis (Ariel et al., 1994). Translocation

Questions relating to human application (b) The application of any new therapy to humans is normally pre- ceded by testing its safety in animal models. It must be ad- mitted that, for the reasons explained above, ICSI has been applied with minimal previous animal experience. Animal MPF experiments showing normal development of progeny after CF fertilization with spermatids (Ogura et al., 1994; Sofikitis et al., 1994) did precede the human application; but the question of whether this preliminary work was sufficient to justify human application can be posed. Although the available data do not suggest an increased risk of health problems for ICSI babies, it must be taken into account that this application is recent and the long-term effects have yet to be evaluated. The human PCC experience with the use of spermatids for fertilization is even more limited. The risk of producing abnormal babies can be reduced by (c) appropriate examinations of both partners of an infertile couple before their inclusion into the treatment group. Many of the potential health hazards associated with the use of abnormal or immature sperm cells can now be controlled (Table 1). The examination of the male partner for Y chromosome micro- MPF deletions, X-linked androgen receptor insensitivity and Kallman CF syndrome, and of both partners for hereditary elliptocytosis can predict the risk of transmission of these diseases to progeny. The examination of both partners for the mutations associated with cystic fibrosis will reveal the cases in which both partners are carriers and in which embryos should not be transferred Activation without previous preimplantation diagnosis to avoid the clinical manifestation of this disease. Testable abnormalities of known (d)

Fig. 1. Schematic representation of the mechanism by which the oocyte can reduce the chromosomes of an injected male germ cell. (a) Mature oocyte with female chromosomes (orange) maintained in metaphase conﬁguration by the action of metaphase promoting factor (MPF) and cytostatic factor (CF), just after the injection of a male germ cell nucleus (red). The ﬁrst polar body is not represented. (b) The injected male germ cell nucleus is translocated to the oocyte MPF periphery. (c) In the absence of oocyte activation, the persisting MPF CF and CF have induced premature chromosome condensation (PCC) in the injected male nucleus, resulting in the formation of a second metaphase plate. (d) The delivery of an oocyte-activation signal leads to the elimination of the MPF and CF activities leading to the entry of both the female and the male chromosomes into anaphase followed by the extrusion of the respective polar bodies.

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Table 1. Testable genetic and epigenetic abnormalities representing risk factors in microinjection-assisted fertilization

Genetic abnormalities Epigenetic abnormalities Abnormality Test application Referencea Abnormality Test application Referencea

Y-chromosome Father and male Reijo et al., 1995 Relaxation of IGF2 Fetal analysis and Rainier et al., 1993 microdeletions progeny imprinting follow-up of children X-linked androgen Father and female McPhaul et al., 1993 Relaxation of H19 Fetal analysis and Rainier et al., 1993 receptor insensitivity progeny imprinting follow-up of children Hereditary elliptocytosis Both parents and Patrizio, 1995 Relaxation of SNRPN Fetal analysis and Glenn et al., 1993 progeny imprinting follow-up of children Kallman syndrome Father and female Prager and Braunstein, 1993 Centrosomal defects Analysis of fertilization Asch et al., 1995 (X-linked) progeny failure Cystic fibrosis Both parents and Patrizio, 1995 progeny aOnly one representative article is referred to for each abnormality. imprinted genes (Table 1) should be screened for in babies re- Miyake K and McNeil PL (1995) Vesicle accumulation and exocytosis at sites sulting from the injection of immature spermatozoa (epididymal of plasma membrane disruption Journal of Cell Biology 131 1737–1745 *Ogura A, Matsuda J and Yanagimachi R (1994) Birth of normal young after and testicular) and spermatids to evaluate the risk associated electrofusion of mouse oocytes with round spermatids Proceedings of the with the use of these cells for reproduction. Centrosomal de- National Academy of Sciences USA 91 7460–7462 fects can be detected in oocytes that fail to fertilize to avoid un- *Palermo G, Joris H, Devroey P and Van Steirteghem AC (1992) Pregnancies necessary repetitions of unsuccessful treatment cycles (Table 1). after intracytoplasmic injection of a single spermatozoon into an oocyte Finally, as with any other new method, there is a certain Lancet 340 17–18 *Parrington J, Swann K, Shevchenko VI, Sesay AK and Lai FA (1996) degree of unpredictable risk that cannot be controlled by any Calcium oscillations in mammalian eggs triggered by a soluble sperm pro- of the available methods. Pretreatment medical counselling and tein Nature 379 364–368 the informed consent form should include adequate infor- Patrizio P (1995) Intracytoplasmic sperm injection (ICSI): potential genetic mation about the existence of this risk. concerns Human Reproduction 10 2520–2523 Prager O and Braunstein GD (1993) X-chromosome-linked Kallman’s syndrome: pathology at the molecular level Journal of Clinical Endocrinology References and Metabolism 76 824–826 Rainier S, Johnson LA, Dobry CJ, Ping AJ, Grundy PE and Feinberg AP Key references are identified by asterisks. (1993) Relaxation of imprinted genes in human cancer Nature 362 Ariel M, Cedar H and McCarrey J (1994) Developmental changes in methyl- 747–749 ation of spermatogenesis-specific genes include reprogramming in the epi- Reijo R, Lee T-Y, Salo P, Alagappan R, Brown LG, Rosenberg M, Rozen S, didymis Nature Genetics 7 59–63 Jaffe T, Straus D, Hovatta O, de la Chapelle A, Silber S and Page DC Asch R, Simerly C, Ord T, Ord VA and Schatten G (1995) The stages at (1995) Diverse spermatogenic defects in humans caused by Y chromosome which human fertilization arrests: microtubule and chromosome con- deletions encompassing a novel RNA-binding protein gene Nature Genetics figurations in inseminated oocytes which failed to complete fertilization 10 383–392 and development in humans Human Reproduction 10 1897–1906 Sathananthan AH, Kola I, Osborne J, Trounson A, Ng SC, Bongso A and *Fishel S, Green S, Bishop M, Thornton S, Hunter A, Fleming S and Ratnam SS (1991) Centrioles in the beginning of human development Al-Hassan S (1995) Pregnancy after intracytoplasmic injection of sper- Proceedings of the National Academy of Sciences USA 88 4806–4810 matid Lancet 345 1641–1642 Silber SJ (1994) The use of epididymal sperm in assisted reproduction. 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