TRANSGENESIS

The word ‘transgenic’ refers to an animal or plant that has been transplanted with an exogenous gene. Unlike ICSI and other manipulation techniques on human oocytes and embryos, embryo manipulation in animals, particularly in mice, involves the introduction of a foreign gene to produce either overexpression or disruption of expression of the targeted gene. Transgenic animals are generated to study gene regulation in a tissue-specific (spatial) and developmental stage-specific (temporal) manner. In livestock production, transgenic technology can be applied to develop improved strains and increased disease resistance (Wheeler and Walters, 2001). Transgenic animals can also be utilized as models for various human diseases, wherein the probable causative gene mutation or deletion can be analysed fully throughout development (Campbell, 2002; Petters and Sommer, 2000) and possible therapies can be trialled to alleviate the human disease conditions. Applications of the transgenic mouse include fate mapping of cells during development and acting as markers for unknown regions in the chromosome (Jaenish, 1988). A commonly used technique developed by Gordon et al. (1980) is direct DNA microinjection into the pronuclei of fertilized mouse zygotes. This results in a stable chromosomal integration of the foreign DNA, usually in a head-to-tail array. Since the foreign DNA is present in every cell, including the germ cells, generally integration occurs at the one-cell stage before DNA replication, although later-stage integration can also occur, creating a mosaic mouse. The number of copies integrated varies, and the transgenes are usually integrated in a single chromosomal locus.

Spermatozoa and spermatids can be found within the ejaculate as well as in aspirates and biopsies taken from the epididymis and testis. Therefore, the method of preparation employed is influenced more by the condition of the sample rather than by the maturational stage of the spermatozoa, although some approaches have been developed for the enrichment of specific stages of spermatid. The procedure and requirements for the preparation of oocytes vary only according to whether immature or mature oocytes have been aspirated.

SEMEN ANALYSIS

Technical details concerning the routine production, collection and analysis of semen are outside the remit of this book. Therefore knowledge of these aspects is assumed and, in any case, may be gleaned from several already established sources (Fleming et al., 1997; Mortimer, 1994; World Health Organization, 1999).

If no spermatozoa are observed on a Makler chamber (Sefi Medical Instruments Ltd) during semen analysis, then a drop of the ejaculate should be placed under a coverslip on a glass microscope slide as a wet preparation and examined under 200× magnification using a compound microscope with phase-contrast optics. If one or more spermatozoa are then visible, then it should be possible to obtain sufficient spermatazoa for micromanipulation following standard preparation, providing the volume of the ejaculate is adequate (i.e. at least 1 ml). Even if no sperm are visible on a wet preparation, it is still worthwhile centrifuging the ejaculate over a density gradient or in medium (1: 1, v/v) at 500 g for 10 min in order to concentrate whatever spermatozoa may be present at the bottom of the tube.

Eppendorf AG are relative newcomers to the assisted reproduction technology (ART) market. Recognizing the potential for their instruments, they modified their existing Micromanipulator-5171 (a general-purpose micromanipulator, used mainly for the injection of cultured, adherent cells) in 1995 to a version dedicated to the manipulation of cells in suspension, called the TransferMan (see Figure 4.1). The specific requirements of ART forced the evolution of this instrument into the TransferMan NK (see Figure 4.2 and section 4.3) and then the TransferMan NK2 (see Figure 4.3). To complete the micromanipulation workstation, Eppendorf provides manual microinjectors and micropipettes for both the holding and injection sides.

While other manufacturers' devices maintain some sort of mechanical linkage between the controller and the pipette tip, all Eppendorf manipulators rely on microprocessor control, and the TransferMan models are no exception. The movements of the joystick are sensed, encoded digitally, and passed through a microprocessor and on to the headstage. By this means, an intuitive control of the pipette in all three dimensions can be achieved with these instruments in either a dynamic or proportional mode.

In dynamic mode (TransferMan), the joystick is spring-loaded in all three axes so that it returns to its centre position when released. Furthermore, the position of the joystick controls the speed of the micropipette tip: the further the joystick is displaced from the centre, the faster the tip moves.

SELECTING THE MICROSCOPE

The microinjection is carried out on an inverted microscope at a magnification of 200–400×. It is essential to equip the microscope with some form of contrast optics. Originally, only differential interference contrast (DIC) optics (Nomarski) was available. One of the most widely used optics systems is now Hoffman Optics' Modulation Contrast Optics, although Carl Zeiss introduced its low-cost ‘Varel’ system in 1996. The advantage of the latter two optical systems is that they can be used with plastic dishes, whereas DIC optics are optimized for working with glass. Dishes with a glass base are now available from, for example, WillCo Wells B.V. in Amsterdam, allowing DIC to be used as originally intended.

The microscope itself should be of the inverted type. The IX-71 from Olympus, the DM IRB/E from Leica, the Axiovert 200 from Zeiss, and the TE 2000 from Nikon are all good examples. These microscopes are available with a bewildering array of accessories and options. Those options considered important would be a camera port, allowing a simple camera and monitor to be attached (useful for teaching), and a movable stage.

Essential specifications for the microscope are:

Recommended options include:

This intention of this chapter is not to provide a fully comprehensive listing of all consumables, and suppliers thereof, that may be used for micromanipulation. Rather, the emphasis is on the requirement for specific types of materials to perform different aspects of micromanipulation technique. In most instances, a given type of consumable is often provided by a number of different suppliers, the choice usually being determined by personal preference. In this respect, most of the suppliers listed are either internationally well established or those with which the authors have dealt with in the past. Therefore, although these may not be the very best available, in most instances they have at least been tried and have been proven suitable for the purpose for which they are intended.

MATERIALS REQUIRED FOR THE PREPARATION OF SPERMATOZOA

It must be appreciated that spermatozoa are living cells and, therefore, should be handled gently during preparation. This point is particularly pertinent to samples with reduced viability, such as those sometimes recovered from surgical aspiration of the epididymis or testis. Also, occasionally samples with an intrinsically short lifespan are encountered. Therefore, handling should be minimized to that sufficient to provide spermatozoa in a condition free of debris, suitable for micromanipulation. In this respect, parameters such as the buffer employed in the medium to maintain physiological pH and the temperature at which the medium is maintained are less critical, as human spermatozoa are particularly susceptible only to acidic conditions and do not exhibit ‘cold-shock’.

A SHORT HISTORY AND BACKGROUND TO IN VITRO FERTILIZATION, INTRACYTOPLASMIC SPERM INJECTION AND ASSOCIATED TECHNIQUES

On 25 July in 1978, Louise Joy Brown, the world's first baby to be born as a result of in vitro fertilization (IVF), heralded a breakthrough in the alleviation of infertility. Over a million babies have now been born as a result of IVF. However, this relatively simple technique of placing eggs into medium containing thousands of sperm is of benefit only to those patients with the ability to produce large numbers of highly motile, normally shaped spermatozoa. Cases involving sperm disorders, however, are less likely to possess an adequate number of functional spermatozoa, with normal cleavage of any fertilized eggs also being less likely to occur.

Initially, most patients with male-factor infertility were treated empirically (e.g. using anti-oestrogens, gonadotrophins, androgens and antibiotics) in an attempt to optimize their semen profile, but without any great proven success. Modifications in preparation (e.g. discontinuous density gradient centrifugation purification and metabolic stimulation with phosphodiesterase inhibitors) and insemination procedures (e.g. high insemination concentration and short-duration inseminations) improve the fertilizing potential of spermatozoa but only in those amenable to such treatment (e.g. those with more than 500 000 progressively motile sperm). Hence, those with severe male-factor infertility have had to wait until the last decade of the twentieth century, during which time the application of micromanipulation technology and surgical sperm-recovery techniques have allowed us to offer hope to these patients.

The successful development of micromanipulation techniques for ICSI and PGD has stimulated research into further potential clinical applications. Putative beneficial uses of this technology have included the use of empty ZPs for the cryopreservation of spermatozoa (Cohen et al., 1997a), the transfer of ooplasm from one oocyte to another (Cohen et al., 1997b), the transfer of a GV from one oocyte to an enucleated oocyte (Zhang et al., 1999), ploidy reduction by removal of pronuclei from polyploid zygotes (Rawlins et al., 1988), and the removal of fragments of cytoplasm from fragmented embryos (Alikani et al., 1999). Almost all of these techniques are still at an early research stage and have yet to be proven unequivocally to have beneficial clinical application. Indeed, some of these techniques have become controversial due to concern over their safety, such as the possible inheritance of defective mitochondrial DNA, which could lead to disease in later life. It is not within the scope of this book to review exhaustively all of the new applications currently being researched. However, some of these techniques merit further description by virtue of their perceived benefit to the enhancement of reproductive and therapeutic potential. Consequently, these will be discussed briefly in this chapter.

CRYOPRESERVATION OF SPERMATOZOA WITHIN THE ZP

Some patients have sparingly few spermatozoa, such as those presenting with cryptozoospermia and those requiring surgical sperm recovery, particularly patients with non-obstructive azoospermia.

Established in 1964 by Mike Lee and Vince Grispo, Research Instruments developed a range of mechanical micromanipulators for the microelectronics market for electrical testing of integrated circuits. The early micromanipulators, e.g. the TCV500 produced in 1964, had three axes of movement from one lever and a range of movement reduction from 500: 1 to 100: 1. Offering a price advantage and simplicity, these manipulators were purchased by large research organizations, such as the microelectronics giant GEC.

In the 1980s, Research Instruments developed the TLO500, an early derivative of the current TDU500 model, which incorporated the use of flexural hinges, giving the important advantages of stability and relatively low maintenance needs.

Approached by Simon Fishel, Research Instruments expanded into the IVF market in the early 1980s, developing micromanipulators that could be mounted on to an inverted microscope. The TDU500 was thus developed, and the Sonic Sword was designed to improve the ease of cell-wall penetration for the up and coming SUZI technique. During this period, Research Instruments concentrated on the IVF market and developed systems that responded to embryologists' needs.

With the advent of the ICSI technique, Research Instruments continued to develop innovative features to help the novice embryologist get to grips with micromanipulation. In the early 1990s, Research Instruments introduced a home function, which allowed the user to set up the micropipettes above the Petri dish and then to lower them into the dish rapidly without incurring accidental damage.

A number of good books have been published on the scientific and technical aspects of micromanipulation. However, they are usually aimed at the experienced practitioner with limited benefit to those working in an environment that is often largely devoid of expert technical support. In contrast, the primary objective of this book is to provide an easy-to-follow, step-by-step guide to micromanipulation techniques, with an emphasis on troubleshooting the myriad difficulties inevitably encountered. Indeed, the original idea for this book arose from a chance meeting between the authors. We realized that we shared a common experience despite our diverse backgrounds. Having introduced students to the equipment and techniques involved in micromanipulation, much of our time was then spent acting on a long-distance consultancy basis once these students found themselves back in the ‘real world’ of a clinical assisted reproduction centre. In this respect, the authors have also recognized that many of these challenges are most likely common to those working within the related fields of human assisted reproduction, livestock production, endangered species preservation, and transgenic research. Therefore, an attempt has been made to direct the material at a broad readership wherever relevant.

It is important to appreciate that this is fundamentally a technical manual. Hence, the chapters are designed to be read not in any particular order but as required. Related information within the same chapter as well as within different chapters is cross-referenced wherever necessary.

‘Zona drilling’ was the term used originally to denote the technique of creating a hole in the ZP surrounding the oocyte (Gordon and Talansky, 1986). The first intuitive application of this procedure in human infertility was to provide a conduit via which spermatozoa could pass through the ZP (Cohen et al., 1988; Gordon et al., 1988). This approach to alleviating male-factor forms of infertility that were related to the inability of spermatozoa to bind to or penetrate the ZP was termed partial zona dissection (PZD). This involved physically cutting a hole or slit in the ZP using micropipettes. However, this approach was considered to be inefficient because it was suspected that partial or complete closure of the slit created might compromise sperm motility or even prevent access to the oocyte altogether. Therefore, the next logical step was to use ZD as a means of introducing spermatozoa directly into the PVS with the aid of a large microinjection pipette that could be passed through the hole thus created (Laws-King et al., 1987; Ng et al., 1988). This technique, originally termed ‘microinjection sperm transfer’ later became known as ‘subzonal insemination’. With the introduction of very fine and sharp microinjection pipettes, ZD soon became unnecessary for the SUZI technique, as it became possible to pierce the ZP directly. Osmotic manipulation of the oocyte prior to SUZI was usually applied to enlarge the PVS, so as to avoid damage to the oolemma.