Centrosome Inheritance during Human Fertilization and Therapeutic Cloning Reproductive and Developmental Diseases and Disorders Caused by Centrosome Dysfunction

C. S. Navara, C. Simerly and G. Schatten

Introduction

Over a century ago, van Beneden [1] and Boveri [2] discovered that the centrosome is vital for successful fertilization and the beginning of embryonic development. In the 1925 third edition of his pioneering monograph The Cell in Development and Heredity, sadly dedicated to the memory of his dear friend Theodor Boveri, E. B. Wilson writes:

"The essential postulates of (Boveri's) theory were (1) that the central body ('centrosome') is the fertilizing element proper; (2) it is actually imported into the egg by the sperm; (3) that the cleavage centers arise directly by division of the sperm-center. As outlined by Boveri the theory took the following form: during the somatic divisions the center (centrosome) is continuously handed on by division from generation to generation of cells. This process comes to an end in the mature egg after extrusion of the second polocyte (polar body), when the eggcenter degenerates or becomes physiologically ineffective; further cell-division is thus inhibited and the occurrence of parthenogenesis is avoided. The ripe egg possesses all ofthe elements necessary for development save an active division-center (centrosome). The sperm, on the other hand, possesses such a center but lacks the protoplasmic substratum in which to operate. In this respect the egg and sperm are complementary structures; their union in syngamy thus restores to each the missing element necessary for further development (p. 155 in [3]). Accepting this it follows that the nuclei of the embryo are derived equally from the two parents; the central bodies (centrosomes) are purely of paternal origin; and to this it might be added that the general cytoplasm of the embryo seems to be almost wholly of maternal origin." (From [4] p. 440; parentheses added).

More than a century ago, Boveri and Van Beneden recognized that the sperm contributes the centrosome, the structure that organizes the mitotic spindle poles (cleavage centers). Even in our time, these observations are profound, extraordinary for their clarity and elegant simplicity; modern centrosome biologists should be humbled by the brilliance of our great-great-grand-professors. While much of the past quarter-century has witnessed significant progress in the molecular dissec tion of the centrosome, as well as discoveries from laboratory experiments and natural reproduction, Boveri's theory remains pioneering, even from the vantage of an early 21st century centrosome biologist.

Even the minor criticism of Boveri's postulate on the uniparental origin of the centrosome ("the central bodies (centrosomes) are purely of paternal origin; and to this it might be added that the general cytoplasm of the embryo seems to be almost wholly of maternal origin") can be countered by the possibility of Wilson's slight mistranslation. The term "protoplasm" was coined by Purkinje when he first discovered the germinal vesicle [5], to refer to the unique cytoplasm that is found within an unfertilized egg. The egg's cytoplasm or protoplasm, unlike other cytoplasm, is uniquely capable of supporting the development of the next generation. When Boveri wrote "The sperm, on the other hand, possesses such a center but lacks the protoplasmic substratum in which to operate", perhaps he was indeed aware that while the egg cytoplasm is capable of initiating parthenogenetic development, the sperm centrosome recruits the maternal proteins that are essential for fertilization and early development.

A century after Boveri's theory, human in vitro fertilization (IVF) was achieved [6]. Now more than 1 million IVF babies have been born. The discarded specimens from IVF clinics have provided a precious and unique research resource for centrosome biologists, and a new breed of centrosome physician-scientist is emerging. Reproductive mistakes including polyspermy (fertilization by more than one sperm) and parthenogenesis (development beginning in an activated egg without any sperm) subtly challenge aspects ofthe unipaternal centrosome inheritance theory.

Here, we focus on the centrosome during fertilization, with special attention to human reproduction and development. We also consider the centrosome during nuclear transfer which represents research frontiers for the next generation of cen-trosome biologists. For obvious ethical reasons, experimental results are obtained and/or corroborated by studying non-human primate development. This is essential, ironically, because fertilization in mouse and other rodents represents rare exceptions to Boveri's theory [7]. In this chapter, we consider:

• Centrosomes during human fertilization

• Centrosome dysfunction as causes of human infertility

• Centrosome functional assays for diagnosing male infertility

• Polyspermy in humans

• "Dispermy hypothesis" for the origins of genomic imprinted disorders

• Maternal centrosome anomalies and birth defects

• Resolving the special problem of parthenogenetic development: roles of cytoplas-mic motors and NuMA

• Centrosomes during cloning, and centrosomes in embryonic stem cells derived after nuclear transfer

• Research challenges for centrosome developmental biologists: developmental centrosomopathies

14.3 Centrosome Dysfunction as Causes of Human Infertility | 281 Centrosomes during Human Fertilization

Centrosome inheritance during human fertilization [7-10] mirrors the inheritance pathway found in most animals (reviewed in [7]). Simply stated, the human sperm contributes the proximal centriole, which recruits egg proteins including y-tubulin, centrin, pericentrin, NuMA, and microtubule motors, to the sperm centriolar complex. Within the fertilized egg, the now enlarged "sperm centrosome", which more properly should be called the "zygote centrosome", nucleates microtubules that assemble the first microtubule-based structure in the fertilized egg - the sperm aster. The sperm aster in fertilized human oocytes (Figure 14.1A) is the typical radially-arrayed monaster juxtaposed to the sperm nucleus (which is called the "male pronucleus"; Figure 14.1B, M) after the sperm chromatin has decondensed within the egg cytoplasm. As in most animal eggs, the sperm tail enters the egg and one or two punctate structures, detectable with y-tubulin imaging, are found at the center of the sperm aster exactly at the junction between the sperm axoneme and the male pronuclear surface (Figure 14.1C and D). These have been shown by Satha-nanthan et al. [11] to be the sperm centriole(s).

Additional evidence supporting the sperm contribution of the centrosome and centriolar complex in humans comes from studies on polyspermic fertilization and parthenogenesis [8, 9, 12]. As shown in Figure 14.1E, because two sperm enter the oocyte (i. e. dispermic fertilization), each paternal centrosome organizes a sperm aster at the base of the sperm head (Figure 14.1F, M). Conversely, artificial activation of oocytes (i. e. parthenogenesis), in which no contribution of the paternal centrosome is provided, leads to a random, disarrayed microtubule pattern at interphase, as is shown in this activated oocyte in which the sperm failed to penetrate (Figure 14.1G and H). Collectively, these data reinforce the observation that the centrosome is paternally inherited in humans [7, 9].

Centrosome Dysfunction as Causes of Human Infertility

The inheritance of the centrosome during human reproduction has crucial implications for the diagnosis and treatment of human infertility, especially male-factor infertility. Male infertility may be the only example in medicine in which one patient carries a disorder (e. g. defective sperm centrosome), but another (his wife or partner) undergoes the surgical procedure - and perhaps without enjoying a successful outcome.

During the past decade, several teams of investigators around the world have made seminal discoveries concerning the pattern of centrosome inheritance in human and non-human primate fertilization [8, 9, 10, 13]. For obvious reasons, these studies were all carried out in gametes discarded from human infertility clinics employing assisted reproductive technologies (ART). Because couples seek ART treatment for reasons including male-factor infertility, certain forms of

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