Abstract
In non-rodent mammalian species, including humans, the oocyte and sperm both contribute centrosomal components that are most important for successful fertilization. Centrosome pathologies in sperm and the oocyte can be causes for infertility which may be overcome by assisted reproductive technologies based on proper diagnosis of specific centrosomal pathologies. However, we do not yet fully understand the cell and molecular mechanisms underlying centrosome functions in germ cells and in the developing embryo, which calls for directed specific investigations to identify centrosome-related pathologies that include components in sperm, egg, or centrosome regulation within the fertilized oocyte. The present review highlights cellular and molecular aspects of centrosomes and centrosome–nuclear interactions focused on nuclear mitotic apparatus protein during fertilization and proposes future directions in expanding therapeutic approaches related to centrosome pathologies that may play a role in still unexplained causes of infertility.
Introduction
The discovery of centrosomes is credited to Theodore Boveri who brilliantly utilized sea urchin eggs to show the importance of centrosomes for fertilization and cell division (Boveri 1901). He further showed that fertilization with two sperm (dispermy) resulted in fertilization and division abnormalities with multipolar mitoses as hallmarks that he implicated to play a role in cancer cell division. These landmark observations served as foundation for many of our current studies on centrosomes in reproduction and cancer and generated an enormous resurgence in centrosome research and in centrosome pathologies underlying disease (reviewed in Badano et al. (2005)). The current review is focused on the role of centrosomes in non-rodent mammalian fertilization and on centrosome abnormalities that play a role in male and female factor infertility. Infertility is a major health problem in reproduction and affects about 10–15% of couples who now have the option to seek out assisted reproductive technologies (ART) as treatment to overcome infertility problems. Currently, in the Western world 1% of all babies are produced through ART (Giritharan et al. 2007), and it can be expected that this trend will increase as many women postpone having children until advanced stages in life. The percent of women undergoing IVF is already higher in Australia (2.7%) and in Denmark (3.9%) and the number may also be higher in other countries, as an increasing number of women suffer from low quality oocytes, decreasing the chances for fertilization success. However, ARTs are not yet perfected and it oftentimes takes multiple attempts before success is achieved which currently ranges between 70 and 80% in the most successful IVF clinics. To optimize the conditions for likely success it is important to understand the cell and molecular aspects that play a role in fertilization, cell division, and subsequent embryo development.
Successful fertilization significantly depends on oocyte quality that is acquired during a complex process of oocyte maturation (reviewed in Fan et al. (2003, 2009), Voronina & Wessel (2003), Brunet & Maro (2005), Liang et al. (2007), Ai et al. (2008a, 2008b, 2009), Jones (2008), Swain & Pool (2008), Gosden & Lee (2010) and Schatten & Sun (2011b)) as well as on sperm quality that includes the capacity to activate the oocyte and contribute components to the fertilized egg that are essential for cell division and subsequent embryo development. In most mammalian systems including humans the oocyte is fertilized at metaphase of meiosis II (MII) and completes meiosis when activated by sperm. The MII stage is generally described as the stage in which the oocyte is arrested before fertilization takes place; however, it is important to emphasize that this arrest is achieved by highly active molecular dynamics that are critical for maintaining MII spindle integrity and preventing spindle deterioration (reviewed in Schatten & Sun (2011b)) which may be compromised in aging oocytes or during IVF procedures (reviewed in Miao et al. (2009b)).
While progress has been made to determine oocyte quality with non-invasive methods (reviewed by Wang & Sun (2007)) we still do not yet have reliable indicators to adequately assess oocyte quality (reviewed in Gosden & Lee (2010)) and we also do not yet have adequate methods to assess sperm quality (reviewed in Talevi & Gualtieri (2004)). However, MII spindle integrity is one of the key indicators of oocyte quality and depends on centrosome integrity; live-cell imaging using PolScope microscopy based on polarized light optics (Wang et al. 2001) is frequently used for the evaluation of the MII spindle although this method does not allow for sufficient sensitivity to determine molecular defects including centrosomal abnormalities at the spindle poles. The assessment of MII spindle integrity is especially important, as this is the structure that contains the maternal genetic material and provides the molecular machinery that separates chromosomes to precisely extrude half of the chromosomes into the second polar body (PBII) while the other set remains in the oocyte to form the female pronucleus (reviewed in Miao et al. (2009b)).
The sperm, on the other hand, contributes the centriole–centrosome complex, an essential component in all non-rodent mammalian systems studied so far including humans. This complex had been reduced during spermatogenesis but it has retained the functional proximal centriole that is critical for sperm aster formation, for the zygote aster, and for the bipolar mitotic apparatus that forms after centriole duplication during the pronuclear stage (reviewed in Manandhar et al. (2005) and Schatten & Sun (2009b); see schematic diagram in Fig. 1). The sperm also contains critical centrosomal proteins surrounding the sperm centriole that attract additional centrosomal proteins from the oocyte's stockpile that had been retained within the oocyte after completing oogenesis.
Schematic diagram of centriole complex in sperm and duplication after fertilization. The sperm's centriole complex primarily consisting of a prominent proximal centriole surrounded by sparse centrosomal material and a distal centriole that becomes degraded along with the sperm tail after fertilization (reviewed in Schatten & Sun (2009a, 2009b)). γ-Tubulin, pericentrin, and centrin are among the centrosomal proteins that have been identified around the proximal sperm centriole. Duplication of centrioles takes place at the pronuclear stage followed by separation of the duplicated centriole–centrosome complex and migration around the zygote nucleus to the opposite poles to form the centers of the mitotic spindle poles.
Citation: REPRODUCTION 142, 6; 10.1530/REP-11-0261
An important aspect of sperm aster, zygote aster, and mitotic apparatus formation is the regulation of centrosomal components that become remodeled throughout the first and subsequent cell cycles for cell cycle-specific functions (reviewed in Schatten & Sun (2010, 2011a)). Dysfunctions of centrosomal components in sperm or egg will result in developmental abnormalities or dysfunctions. The following sections will discuss centrosomal components and abnormalities in sperm that may affect in vivo fertilization and may require specific in vitro procedures, summarized as ART (reviewed in Schatten & Sun (2009b)). Oocyte abnormalities include defective MII spindles and failure of centrosome regulation to form the sperm aster, zygote aster, and mitotic apparatus after fertilization.
The sperm centriole–centrosome complex and pathologies that are implicated in fertility problems
Factors that play a role in human male infertility have recently been reviewed in detail by Singh & Jaiswal (2011) who focused on chromosomal aberrations including genetic and environmental components although this review did not include centrosome pathologies. Specific sperm pathologies have been well reviewed by Chemes (2000) and Chemes & Rawe (2003) listing a large number of sperm defects with most of them related to centriole or centrosome abnormalities that are the result of genetic or environmental influences. Such defects are typically identified as abnormalities based on morphological criteria or on evaluation with molecular methods, using immunofluorescence microscopy to specific antigens or western blotting aimed at analyzing specific proteins that may be absent in sperm and manifested as pathologies. As we have become aware of such pathologies an increasing number of morphological and molecular defects in sperm have been related to centrosomal and/or centriole abnormalities (reviewed in Schatten & Sun (2009b) and references therein) that are increasingly being identified as causes for infertility. While research on sperm centrioles has yielded a wealth of new data primarily based on electron microscopy studies, research on sperm centrosomes is only at the beginning. We know relatively little about centrosomal components surrounding the sperm centriole and the requirements for centrosome growth after fertilization. We do know that the sperm centrosome complex contains γ-tubulin that quantitatively increases after fertilization by recruiting additional γ-tubulin from the ooplasm, and we also know that it contains centrin as an important component for fertilization. Insufficient γ-tubulin and centrin have both been correlated with decreased ability to fertilize (Hinduja et al. 2010) and decrease in sperm aster formation after fertilization (Navara et al. 1996). Other proteins in the mature sperm are only incompletely understood although new studies have started to focus on this topic. Recently, Goto et al. (2010) reported that speriolin is present in sperm of human and mouse oocytes and is contributed to the oocyte after fertilization; it is detected at the mitotic poles in both systems despite the fact that in the mouse, the sperm centriole is destroyed and does not participate in the fertilization process (Schatten et al. 1985a). One complexity regarding sperm centrosomal components relates to the fact that many previous studies have used immunofluorescence microscopy without extensive control experiments to possibly unmask antigens that may not be detected because of inaccessibility to the antibody. In some cases western immunoblotting was able to identify the centrosomal proteins that had not been detected by immunofluorescence microscopy alone (reviewed in Manandhar et al. (2005)). However, a large body of evidence ascribes sperm pathologies to centrosomal dysfunctions that led to the novel approaches and suggestions to introduce donor centrosomal material along with ICSI procedures when IVF was performed. Such studies have been reported for the cat in which injection of testicular sperm into the oocyte resulted in short or absent sperm asters while ejaculated sperm was able to generate larger sperm asters (Comizzoli et al. 2006). Centrosome functions were restored by replacing the testicular sperm centrosome with a centrosome from ejaculated sperm. Such studies open up new possibilities for centrosomal male-factor therapies (reviewed in Schatten & Sun (2009b)).
The selection process for sperm used in ART is not yet perfected and still mainly relies on differential centrifugation or motility criteria employed for both IVF and ICSI, while during in vivo fertilization the selection process is more extensive and only one of the ∼300 million sperm will fertilize a single egg. Before fertilizing the egg in the oviduct (Fallopian tubes) the successful sperm has to enter the uterus, swim up the Fallopian tube and penetrate the zona pellucida before triggering egg activation and subsequent fertilization responses in the egg. As the sperm tail is not necessary for the fertilization process and ICSI will overcome sperm immotility problems if this is the underlying cause for infertility, we do not yet fully understand whether other factors play a role in the selection of the successful sperm during in vivo fertilization (reviewed in Talevi & Gualtieri (2004)).
New discoveries of a nuclear matrix in sperm have opened up further potential areas for sperm abnormalities that had not been addressed previously and relate to nuclear–centrosome interactions. Evidence has emerged for nuclear matrix proteins in the sperm nucleus that play a role in chromatin organization (reviewed in Johnson et al. (2011)) and may be important for sperm nuclear decondensation abnormalities after fertilization. A strong correlation between nuclear and centrosome proteins exist. Nuclear and centrosomal proteins are precisely regulated during cell cycle progression to coordinate spindle formation and chromosome segregation. One of the proteins that strongly link nuclear to centrosome functions is the nuclear mitotic apparatus protein (NUMA). NUMA is part of the nuclear matrix that becomes clearly detectable in the decondensing sperm nucleus after fertilization when immunofluorescence detection methods are employed (reviewed in Liu et al. (2006), Sun & Schatten (2006) and Alvarez Sedó et al. (2011)). Detailed direct studies on NUMA in the sperm nucleus before fertilization and in the decondensing pronucleus after fertilization are still needed to pursue important questions on the role of the nuclear matrix in sperm (reviewed by Johnson et al. (2011)) that may account for cases of infertility in which nuclear decondensation does not take place. Such studies may require more elaborate molecular or biochemical methods as well as specifically tailored ultrastructural methods to investigate nuclear matrix components in the nucleus (Degrouard et al. 2004, reviewed in Johnson et al. (2011)).
Oocyte centrosomes and centrosomal pathologies associated with oocyte dysfunctions
As mentioned in Introduction, centrosome components in the MII oocyte are critical for successful fertilization. The most prominent staining for centrosomal proteins is detected at the poles of the MI and MII spindles (see schematic diagram in Fig. 2) and these MI and MII spindle pole centrosomes are crucially important for accurate chromosome separation. The molecular mechanisms involved in chromosome separation in MI and MII oocytes have been well reviewed previously (Ai et al. 2008a, 2008b, Jones 2008, Holt & Jones 2009) while our understanding of centrosome dynamics and specific centrosome protein functions in MI and MII oocytes remains fragmented and complicated by the fact that most of the previous studies have been performed in the mouse model that we now know employs totally different mechanisms for meiotic spindle formation compared with non-rodent mammalian systems including humans (reviewed in Schatten & Sun (2010, 2011b)).
Schematic diagram of MII spindle in oocytes before fertilization. The MII spindle is organized by acentriolar centrosomes that nucleate kinetochore and pole-to-pole microtubules for separation of chromosomes.
Citation: REPRODUCTION 142, 6; 10.1530/REP-11-0261
In meiosis I, half of the homologous chromosome set as well as one set of the spindle pole centrosomes is extruded into the first polar body (PBI). In MII, haploidy is restored in the sperm-activated oocyte when one set of chromatids is extruded in the second PBII along with one set of the spindle pole centrosomes while the female pronucleus is formed in the oocyte. However, we do not yet understand the nature and composition of the extruded centrosomes and centrosome protein quantities that may play a role in the two consecutive highly asymmetric cell divisions during polar body formations. In somatic cells, centrosomes are key determinants for the establishment of cell divisions that includes establishment of asymmetry (reviewed in Schatten & Sun (2010)). We do not yet understand the fate of specific centrosome proteins during this critical stage of haploid oocyte formation. Understanding centrosome dynamics and functions during this process is highly important, as dysfunctional centrosomes may be among the underlying causes for the formation of multiple female pronuclei that indicates centrosome dysfunctions perhaps as a result of centrosome fragmentation resulting in unequal chromosome separation and subsequent formation of multiple pronuclei. To explain causes of aneuploidy, more detail on centrosome functions and dysfunctions during oocyte meiosis is needed. This topic is important, as therapies may be in reach and centrosome dysfunctions may be rescued and repaired with several agents that interact with centrosome signaling pathways and include caffeine, dithiothreitol, and nitric oxide, as well as others that are currently being explored (reviewed in Miao et al. (2009b)).
MI and MII spindle dynamics are complex and depend on precisely regulated centrosome, microtubule, and chromosome functions and on their structural and functional interactions. The molecular mechanisms that play important roles in chromosome–microtubule interactions have been well reviewed in previous papers (Ai et al. 2008a, 2008b, 2009, Jones 2008, Holt & Jones 2009) and are not detailed here while the role of centrosomes and their interactions with microtubules to form the functional meiotic spindle will be highlighted. We know little about molecular detail and mechanisms that play a role in the regulation of centrosome proteins in the MI and MII spindles while much of our knowledge about centrosome interactions with microtubules and chromosomes comes from mitotic spindles that do contain centrioles within the centrosome complex, unlike the acentriolar centrosomes in meiotic spindles. In all mammalian systems studied so far the MII spindle centrosomes do not contain centrioles; we know that centrioles had been destroyed during oogenesis (reviewed in Manandhar et al. (2005)). However, MI and MII spindles in mammalian oocytes do contain centrosomal components comparable to those described for mitotic spindles including the centrosomal components γ-tubulin, NUMA, and pericentrin although there are some variations in centrosome composition in different non-rodent mammalian species (reviewed in Schatten & Sun (2011b)) while the mouse employs totally different mechanisms regarding centrosome and cytoskeletal organization that are not addressed in this review (Maro et al. 1985, Schatten et al. 1985a, Lee et al. 2000, Schuh & Ellenberg 2007, reviewed in Schatten & Sun 2009a, 2009b, 2010, 2011b). Three centrosomal proteins, γ-tubulin, NUMA, and pericentrin are clearly associated with MI and MII spindle poles in non-rodent mammals in which centrioles are absent but the formation of the acentriolar centrosomes has not yet been addressed sufficiently well in non-rodent mammalian systems (reviewed in Schatten & Sun (2011b)).
The centrosomes in the MI and MII spindles are important indicators of oocyte quality, as deteriorating centrosomal material indicates decrease in oocyte quality and problems for aneuploidy, decrease in developmental capacity, and developmental dysfunctions. The MI and MII spindles are highly susceptible to environmental influences (reviewed in Schatten & Sun (2009a)) including aging (Miao et al. 2009a, 2009b), cocaine (Combelles et al. 2000), bisphenol A (BPA; Can et al. 2005, Eichenlaub-Ritter et al. 2008, Pacchierotti et al. 2008), 2-methylestradiol (2-ME; Eichenlaub-Ritter et al. 2007), and perhaps numerous others including stress factors that have been implicated in causes for aneuploidy and developmental disorders (reviewed in Miao et al. (2009b)). It has generally been observed that in oocytes affected by aging or by environmental factors, centrosomes disintegrate from the MII spindle poles followed by loss of microtubule tension, loss of microtubule connections to the kinetochores, and MII spindle deterioration (reviewed in Miao et al. (2009b)). Studies are underway to determine mechanisms by which centrosome deterioration can be prevented and MII spindle integrity can be restored which includes supplementation of culture medium with caffeine as mentioned above although these studies have not yet been translated into clinical applications (reviewed in Miao et al. (2009b)).
Aside from the prominent meiotic spindle pole centrosomes it is important to emphasize that the oocyte contains centrosomal proteins in the ooplasm that are not easily detectable by immunofluorescence microscopy but do become detectable under activation conditions when centrosomal aggregates are formed within the ooplasm that can be induced by pH changes, calcium changes, or by microtubule stabilizing agents including heavy water (D2O) or taxol (Schatten et al. 1992, Kim et al. 1996, reviewed in Schatten & Sun 2011b). While detailed studies on specific ooplasmic centrosomal components are still missing we do know from indirect studies in invertebrate systems that these centrosomal aggregates can undergo time-dependent changes that include aggregations of over a 100 small centrosomal foci into fewer but larger centrosomal aggregates. We also know that the sperm-derived centriole–centrosome complex when introduced in these activated oocytes exerts dominance over the ooplasmic centrosome clusters and aggregates the oocyte's centrosomal components toward the sperm's centriole–centrosome complex (Schatten et al. 1982, 1992, Schatten 1994). Such studies performed in sea urchin eggs have not yet been performed in non-rodent mammalian systems but may provide new insights into the oocyte's contribution of centrosomal components. These ooplasmic centrosomal proteins are important after fertilization to contribute to the formation of the sperm aster, the zygote aster, and the mitotic apparatus. Therefore, aside from the functions in the MI and MII spindles, centrosomal components in the MII ooplasm are very important for the egg's developmental potential. The oocyte further provides the reservoir for regulation of the sperm centrosome complex after fertilization, as will be discussed in the following section.
Post-insemination centrosome regulation
Centrosome integrity is important for successful fertilization and for all stages of subsequent embryo development. In most mammalian systems fertilization takes place in the MII oocyte in which sperm triggers oocyte activation and completion of MII resulting in the extrusion of the PBII and formation of the female pronucleus in the now haploid oocyte. As mentioned above, accurate centrosome functions are very important in this process for the accurate separation of chromosomes and prevention of aneuploidy or multinuclear formations within the oocyte.
In all non-rodent mammalian systems studied so far including humans the sperm contributes the centrioles as essential components for successful fertilization and cell division and therefore provides the precursor and most critical component for all developmental stages from fertilization to late-stage development (reviewed in Sathananthan (2009), Schatten & Sun (2009a, 2009b, 2010) and Alvarez Sedó et al. (2011)). All microtubule formations throughout development depend on sperm-derived centriolar integrity; a defective centriole–centrosome complex inherited by a human oocyte may lead to abnormal chromosome separation with subsequent genomic instability, therefore compromising embryonic development (Sathananthan 1991, 2009). Sperm centrosomal dysfunctions contribute to embryonic aneuploidy, polyploidy, and mosaicism (Munné 2006).
The sperm-derived proximal centriole in non-rodent mammalian systems serves as the seed for recruitment of critical centrosomal proteins from the oocyte to form the sperm aster, zygote aster, and mitotic apparatus during first and all subsequent cell divisions. In addition to providing centrosomal components for the formation of the sperm aster, zygote aster, and mitotic apparatus, the fertilized oocyte also serves as reservoir for sperm-derived centrosome regulation that depends on intracellular oocyte changes accomplished during oocyte activation. Sperm-triggered oocyte activation includes changes in pH and calcium that are both important for cytoskeletal and centrosome remodeling after fertilization. Both are instrumental for successful fertilization, as they directly impact accurate centrosome functions and microtubule formation that in turn are highly important for signal transduction cascades that utilize the centrosome–microtubule system for cellular communication. These functions may be impaired in cases of failed IVF, especially when ICSI is employed, as critical oocyte activation may not have taken place in these cases. This topic is important and may impact IVF success that is supported by studies in which oocyte activation failures were rescued to obtain pregnancies (Eldar-Geva et al. 2003, Chi et al. 2004, Heindryckx et al. 2005) and other studies in which successful pregnancy was achieved after egg activation with calcium ionophore (Terada et al. 2009). More basic studies are still needed regarding the program of oocyte activation in non-rodent mammalian systems to improve ART technologies to build on existing data that have shown the importance of calcium oscillations after fertilization for successful subsequent embryo development (reviewed in Swain & Pool (2008)). Similarly, detailed studies on pH in non-rodent mammalian systems are still needed to build on previous studies that have shown the importance of pH for oocyte activation in several species (Schatten et al. 1985b, 1992, Epel 1988, Ruddock et al. 2001), and consequences for centrosome–microtubule functions during subsequent embryo development.
To mature into a division-competent centrosome complex after fertilization the sperm's centriole–centrosome complex needs to recruit additional centrosomal proteins and macromolecular complexes from the oocyte which includes recruitment of additional γ-tubulin to nucleate additional microtubules and form the zygote aster for the apposition of pronuclei and union of the paternal and maternal genomes and for the formation of the mitotic apparatus that separates chromosomes equally to the dividing daughter cells.
The importance of centrosomes for reproduction had been recognized in several previous key studies using sea urchin eggs (Boveri 1901) and the mouse model (Calarco-Gillam et al. 1983). With the advent of IVF we now have turned to the porcine and bovine systems as models for humans to allow reliable and repeatable experimentation that can be extrapolated to human reproduction (reviewed in Schatten & Sun (2009a, 2009b, 2010)). For centrosome and microtubule studies, the mouse has proven to be different and is not used as model for humans. Briefly, in the mouse, the mechanisms for the formation of the meiotic and mitotic spindles and fertilization as well as drug sensitivities are totally different from all other non-rodent mammalian species including humans. For example, during fertilization, in the mouse, the sperm centriole–centrosome complex is destroyed and cytoplasmic asters contribute to the formation of meiotic and mitotic spindles (reviewed in Schatten & Sun (2011b)). In all other mammalian species, the sperm centriole–centrosome complex is crucial for fertilization and provides the dominant material for the formation of the sperm aster and subsequent mitotic spindles. For these reasons, in recent years, the porcine and bovine models have been emphasized as most suitable model for humans. The fertilization mechanisms used by these species are similar to those used in human fertilization. Both systems depend on the sperm's centriole for successful fertilization and subsequent development, such as humans. For many studies related to humans the use of animal models is important, as it allows targeted and repeatable experimentation to analyze mechanisms that play critical roles in fertilization and uncover the underlying mechanisms for male or female factor infertility. For studies aimed at determining specific male and female factor infertility causes heterologous ICSI has been employed; such studies utilize bovine or porcine oocytes that are fertilized with human sperm to assess components contributing to male and female factor infertility and can be used to determine centrosomal dysfunctions (reviewed in Schatten & Sun (2009b)).
Once in the oocyte the sperm has to be modified into a male pronucleus that includes loss of sperm tail, changes in the nuclear envelope, and chromatin decondensation (reviewed in Swain & Pool (2008)). The role of MAPK for centrosome and microtubule functions and impact on subsequent cell signaling during fertilization and embryo development has been well addressed and the roles of several kinases and phosphatases in post-insemination events have been well reviewed in detail previously (Fan & Sun 2004) and will not be included in this section. In the present review, we will focus on centrosome–nuclear interactions and on interconnected cell signaling that will affect nuclear–centrosome relationships and centrosome–microtubule functions with focus on NUMA.
NUMA is essential for successful fertilization and embryo development; NUMA dysfunctions play a role in infertility and in developmental abnormalities. However, we do not yet understand the factors by which NUMA is regulated and we also do not yet understand the specific contributions by sperm and the oocyte to the fertilized embryo. New research on the sperm nuclear matrix allows the conclusion that NUMA is in part contributed by sperm and in part by the oocyte as essential component during the fertilization process and that NUMA regulation takes place by factors in the fertilized ooplasm.
As mentioned above, in unfertilized oocytes, NUMA is localized to the poles of the MI and MII spindles in non-rodent mammalian systems including humans. While we do not yet have detailed data on mechanisms by which NUMA becomes relocalized from the MI into the MII spindle and during MII (resulting in the extrusion of some NUMA from one spindle pole into the PBII and the formation of the female pronucleus in the oocyte), it is clear that NUMA becomes localized to the female pronucleus after completion of MII. In addition, as mentioned above, after fertilization, NUMA becomes detectable by immunofluorescence microscopy in the decondensing sperm pronucleus and disperses into the ooplasm after pronuclear apposition and nuclear envelope breakdown (NEBD). Failure in NUMA functions has recently been shown in human oocytes and in sperm that failed to decondense while displaying abnormal NUMA immunofluorescence staining patterns (Schatten et al. 2011, Alvarez Sedó et al. 2011). These results indicate that misregulation of NUMA may play a role in male and in female factor infertility problems, as regulation of NUMA in the decondensing sperm pronucleus and the female pronucleus involves factors that are provided by the fertilized oocyte. However, so far, research on NUMA regulation has only been reported for somatic cells in which cyclin B plays a major role (reviewed in Sun & Schatten (2006); see schematic diagram in Fig. 3) but not yet for embryonic cells that may employ regulation mechanisms that are different from somatic cells.
Schematic diagram of NUMA translocation, phosphorylation and regulation in somatic cell cycle. In interphase, NUMA is distributed as insoluble material in the nucleus where it serves as nuclear matrix protein but it is not associated with the interphase centrosome. During mitotic prophase after nuclear envelope breakdown NUMA is released into the cytoplasm, a process that requires stimulation by CDC2/cyclin kinase. NUMA becomes translocated along microtubules to the mitotic spindle poles aided by dynein/dynactin to form a stable crescent-shaped complex around the centrosome area facing chromosomes and tethers microtubules at their minus ends to form the spindle-shaped mitotic apparatus. Similar mechanisms may be employed for NUMA to move out of the zygote nucleus after nuclear envelope breakdown and associate with centrosomes during first mitosis in reproductive cells.
Citation: REPRODUCTION 142, 6; 10.1530/REP-11-0261
In somatic cells, NUMA is an intriguing multifunctional protein with roles as nuclear matrix protein involved in DNA organization in the nucleus that becomes a significant centrosome-associated protein during mitosis (reviewed in Sun & Schatten (2006)). In embryo cells, NUMA is critically important for accurate mitosis and cell divisions throughout embryogenesis. The sperm nucleus likely contains NUMA (although not detectable by immunofluorescence methods because of the sperm's nuclear density) which becomes dispersed after fertilization in the decondensing sperm pronucleus and commences its functions in the oocyte directly after fertilization which we have shown in the porcine model (Liu et al. 2006) and recently in human oocytes (Schatten et al. 2011, Alvarez Sedó et al. 2011). NUMA moves out of the nucleus during NEBD to play a critical role in the formation of the mitotic apparatus (Alvarez Sedó et al. 2011). Therefore, NUMA directly links nuclear functions with mitotic centrosome functions. Failure in NUMA's association with the mitotic centrosome core structure has been implicated in embryonic division dysfunctions. NUMA is not associated with the interphase centrosome and it is not associated with the sperm's centriole complex.
Most of the studies on NUMA in reproductive cells employed immunofluorescence microscopy in fertilized eggs and cloned embryos (Zhong et al. 2005) and NUMA dysfunctions have been reported for several mammalian systems but none of the previous studies have addressed NUMA regulation. It has been proposed that NUMA is de novo synthesized in the oocyte based on indirect data on nuclear transfer experiments (reviewed in Schatten & Sun (2009a, 2009b, 2010, 2011b)) that displayed a delay in NUMA immunofluorescence staining patterns in the transferred somatic cell nucleus (Liu et al. 2006). However, increasing evidence suggests that this may not be accurate, as new evidence has emerged for nuclear matrix proteins in the sperm nucleus that plays a role in chromatin organization (reviewed in Johnson et al. (2011)) which provokes the idea that NUMA may be part of the nuclear matrix in the sperm nucleus that becomes detectable in the decondensing sperm nucleus after fertilization. No direct studies on NUMA in the sperm nucleus before fertilization and in the decondensing pronucleus after fertilization are presently available but new methods and approaches have made it possible to pursue this important question. The NUMA-related abnormalities that we found in human oocytes (Schatten et al. 2011, Alvarez Sedó et al. 2011) may have been part of an inability of NUMA to be regulated by the fertilized ooplasm. This idea finds support by studies that provide evidence for sperm nuclear matrix instability influencing male factor fertility (reviewed in Johnson et al. (2011)).
Future directions
Compared with centrosome biology in somatic cells, our understanding of centrosomes in germ cells and embryos lags far behind despite the fact that centrosomes had been discovered in germ cells. To understand and design therapies for centrosome-related infertility problems basic research is still needed in suitable animal models that include but are not limited to the following. 1) Analysis of centrosomal components contributed by sperm to the fertilized oocyte; 2) investigation of the composition and regulation of centrosomes in the unfertilized meiotic spindle and in the unfertilized ooplasm; 3) analysis of the regulation of centrosomes within the fertilized oocyte; 4) investigations on the relationships of nuclear–centrosome interactions; 5) analysis of centrosome-associated proteins; and 6) characterization of centrosome components in all stages of fertilization. These studies require cell and molecular methods as well as live-cell imaging and ultrastructural methods that are now readily available for even single cell studies. We further need to develop reliable non-invasive methods to determine oocyte quality for selection of optimal oocytes for developmental potential resulting in healthy offspring.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the review.
Funding
This review did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Acknowledgements
The authors gratefully acknowledge Donald Connor's professional help with the illustrations.
References
Ai JS, Wang Q, Li M, Shi LH, Ola SI, Xiong B, Yin S, Chen DY & Sun QY 2008a Roles of microtubules and microfilaments in spindle movements during rat oocyte meiosis. Journal of Reproduction and Development 54 391–396. doi:10.1262/jrd.20034.
Ai JS, Wang Q, Yin S, Shi LH, Xiong B, Ouyang YC, Hou Y, Chen DY, Schatten H & Sun QY 2008b Regulation of peripheral spindle movement and spindle rotation during mouse oocyte meiosis: new perspectives. Microscopy and Microanalysis 14 349–356. doi:10.1017/S1431927608080343.
Ai JS, Li M, Schatten H & Sun QY 2009 Regulatory mechanism of spindle movements during oocyte meiotic division. Asian-Australasian Journal of Animal Sciences 22 1447–1486.
Alvarez Sedó C, Schatten H, Combelles CM & Rawe VY 2011 The nuclear mitotic apparatus (NuMA) protein: localization and dynamics in human oocytes, fertilization and early embryos. Molecular Human Reproduction 17 392–398. doi:10.1093/molehr/gar009.
Badano JL, Teslovich TM & Katsanis N 2005 The centrosome in human genetic disease. Nature Reviews Genetics 6 194–207. doi:10.1038/nrg1557.
Boveri T 1901 Zellen-Studien: Über die Natur der Centrosomen, vol. 28, pp 1–220. Jena, Germany: Fisher Z Med Naturw.
Brunet S & Maro B 2005 Cytoskeleton and cell cycle control during meiotic maturation of the mouse oocyte: integrating time and space. Reproduction 130 801–811. doi:10.1530/rep.1.00364.
Calarco-Gillam PC, Siebert MC, Hubble R, Mitchison T & Kirschner M 1983 Centrosome development in early mouse embryos as defined by an autoantibody against pericentriolar material. Cell 35 621–629. doi:10.1016/0092-8674(83)90094-6.
Can A, Semiz O & Cinar O 2005 Bisphenol-A induces cell cycle delay and alters centrosome and spindle microtubular organization in oocytes during meiosis. Molecular Human Reproduction 11 389–396. doi:10.1093/molehr/gah179.
Chemes HE 2000 Phenotypes of sperm pathology: genetic and acquired forms in infertile men. Journal of Andrology 21 799–808.
Chemes HE & Rawe VY 2003 Sperm pathology: a step beyond descriptive morphology. Origin, characterization and fertility potential of abnormal sperm phenotypes in infertile men. Human Reproduction Update 9 405–428. doi:10.1093/humupd/dmg034.
Chi HJ, Koo JJ, Song SJ, Lee JY & Chang SS 2004 Successful fertilization and pregnancy after intracytoplasmic sperm injection and oocyte activation with calcium ionophore in a normozoospermic patient with extremely low fertilization rates in intracytoplasmic sperm injection cycles. Fertility and Sterility 82 475–477. doi:10.1016/j.fertnstert.2004.01.038.
Combelles CM, Carabatsos MJ, London SN, Mailhes JB & Albertini DF 2000 Centrosome-specific perturbations during in vitro maturation of mouse oocytes exposed to cocaine. Experimental Cell Research 260 116–126. doi:10.1006/excr.2000.5011.
Comizzoli P, Wildt DE & Pukazhenthi BS 2006 Poor centrosomal function of cat testicular spermatozoa impairs embryo development in vitro after intracytoplasmic sperm injection. Biology of Reproduction 75 252–260. doi:10.1095/biolreprod.106.051342.
Degrouard J, Hozak P, Heyman Y & Flechon JE 2004 Nucleoskeleton of early bovine embryos and differentiated somatic cells: an ultrastructural and immunocytochemical comparison. Histochemistry and Cell Biology 121 441–451. doi:10.1007/s00418-004-0655-3.
Eichenlaub-Ritter U, Winterscheidt U, Vogt E, Shen Y, Tinneberg HR & Sorensen R 2007 2-Methylestradiol induces spindle aberrations, chromosome segregation failure, and nondisjunction in mouse oocytes. Biology of Reproduction 76 784–793. doi:10.1095/biolreprod.106.055111.
Eichenlaub-Ritter U, Vogt E, Cukurcam S, Sun F, Pacchierotti F & Parry J 2008 Exposure of mouse oocytes to bisphenol A causes meiotic arrest but not aneuploidy. Mutation Research 651 82–92. doi:10.1016/j.mrgentox.2007.10.014.
Eldar-Geva T, Brooks B, Margalioth EJ, Zylber-Haran E, Gal M & Silber SJ 2003 Successful pregnancy and delivery after calcium ionophore oocyte activation in a normozoospermic patient with previous repeated failed fertilization after intracytoplasmic sperm injection. Fertility and Sterility 79 1656–1658. doi:10.1016/S0015-0282(03)00369-8.
Epel D 1988 Role of pH in fertilization. In Na+/H+ Exchange, pp 209–223. Ed. Grinstein S. Boca Raton, FL: CRC Press.
Fan HY & Sun QY 2004 Involvement of mitogen-activated protein kinase cascade during oocyte maturation and fertilization in mammals. Biology of Reproduction 70 535–547. doi:10.1095/biolreprod.103.022830.
Fan HY, Huo LJ, Meng XQ, Zhong ZS, Hou Y, Chen DY & Sun QY 2003 Involvement of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes. Biology of Reproduction 69 1552–1564. doi:10.1095/biolreprod.103.015685.
Fan HY, Liu Z, Shimada M, Sterneck E, Johnson PF, Hedrick S & Richards JS 2009 MAPK3/1(ERK1/2) in ovarian granulosa cells are essential for female fertility. Science 324 938–941. doi:10.1126/science.1171396.
Giritharan G, Talbi S, Donjacour A, Di Sebastiano F, Dobson AT & Rinaudo PF 2007 Effect of in vitro fertilization on gene expression and development of mouse preimplantation embryos. Reproduction 134 63–72. doi:10.1530/REP-06-0247.
Gosden R & Lee B 2010 Portrait of an oocyte: our obscure origin. Journal of Clinical Investigation 120 973–983. doi:10.1172/JCI41294.
Goto M, O-Brien DA & Eddy EM 2010 Speriolin is a novel human and mouse sperm centrosome protein. Human Reproduction 25 1884–1894. doi:10.1093/humrep/deq138.
Heindryckx B, Van der Elst J, De Sutter P & Dhont M 2005 Treatment option for sperm or oocyte-related fertilization failure: assisted oocyte activation following diagnostic heterologous ICSI. Human Reproduction 20 237–2241. doi:10.1093/humrep/dei029.
Hinduja I, Baliga N & Zaveri K 2010 Correlation of human sperm centrosomal proteins with fertility. Journal of Human Reproductive Sciences 3 95–101. doi:10.4103/0974-1208.69344.
Holt JE & Jones KT 2009 Control of homologous chromosome division in the mammalian oocyte. Molecular Human Reproduction 15 139–147. doi:10.1093/molehr/gap007.
Johnson GD, Lalancette C, Linnemann AK, Leduc F, Boissonneault G & Krawetz SA 2011 The sperm nucleus: chromatin, RNA, and the nuclear matrix. Reproduction 141 21–36. doi:10.1530/REP-10-0322.
Jones KT 2008 Meiosis in oocytes: predisposition to aneuploidy and its increased incidence with age. Human Reproduction Update 14 143–158. doi:10.1093/humupd/dmm043.
Kim NH, Funahashi H, Prather RS, Schatten G & Day BN 1996 Microtubule and microfilament dynamics in porcine oocytes during meiotic maturation. Molecular Reproduction and Development 43 248–255. doi:10.1002/(SICI)1098-2795(199602)43:2<248::AID-MRD14>3.0.CO;2-#.
Lee J, Miyano T & Moor RM 2000 Spindle formation and dynamics of γ-tubulin and nuclear mitotic apparatus protein distribution during meiosis in pig and mouse oocytes. Biology of Reproduction 62 1184–1192. doi:10.1095/biolreprod62.5.1184.
Liang CG, Su YQ, Fan HY, Schatten H & Sun QY 2007 Mechanisms regulating oocyte meiotic resumption: roles of mitogen-activated protein kinase. Molecular Endocrinology 21 2037–2055. doi:10.1210/me.2006-0408.
Liu ZH, Hao YH, Lai LX, Wax D, Samuel M, Schatten H & Prather RS 2006 Reprogramming of the nuclear mitotic apparatus (NuMA) protein during the first cell cycle in porcine nuclear transfer embryos, parthenogenetic embryos and in vitro fertilized embryos. Frontiers in Bioscience 11 1945–1957. doi:10.2741/1937.
Manandhar G, Schatten H & Sutovsky P 2005 Centrosome reduction during gametogenesis and its significance. Biology of Reproduction 72 2–13. doi:10.1095/biolreprod.104.031245.
Maro B, Howlett SK & Webb M 1985 Non-spindle microtubule organizing centers in metaphase II-arrested mouse oocytes. Journal of Cell Biology 101 1665–1672. doi:10.1083/jcb.101.5.1665.
Miao YL, Sun QY, Zhang X, Zhao JG, Zhao MT, Spate L, Prather RS & Schatten H 2009a Centrosome abnormalities in porcine oocytes during aging. Environmental and Molecular Mutagenesis 50 666–671. doi:10.1002/em.20506.
Miao YL, Kikuchi K, Sun QY & Schatten H 2009b Oocyte aging: cellular and molecular changes, developmental potential and reversal possibility. Human Reproduction Update 15 573–585. doi:10.1093/humupd/dmp014.
Munné S 2006 Chromosome abnormalities and their relationship to morphology and development of human embryos. Reproductive Biomedicine Online 12 234–253. doi:10.1016/S1472-6483(10)60866-8.
Navara CS, First NL & Schatten G 1996 Phenotypic variations among paternal centrosomes expressed within the zygote as disparate microtubule lengths and sperm aster organization: correlations between centrosome activity and developmental success. PNAS 93 5384–5388. doi:10.1073/pnas.93.11.5384.
Pacchierotti F, Ranaldi R, Eichenlaub-Ritter U, Attia S & Adler ID 2008 Evaluation of aneugenic effects of bisphenol A in somatic and germ cells of the mouse. Mutation Research 651 64–70. doi:10.1016/j.mrgentox.2007.10.009.
Ruddock NT, Macháty Z, Cabot RA & Prather RS 2001 Porcine oocyte activation: differing roles of calcium and pH. Molecular Reproduction and Development 59 227–234. doi:10.1002/mrd.1027.
Sathananthan AH 1991 Inheritance of paternal centrioles and male infertility. Proceedings of the XIIIth World Congress of Obstetrics and Gynaecology, Singapore. Abstract no. 1629.
Sathananthan AH 2009 Human centriole: origin, & how it impacts fertilization, embryogenesis, infertility & cloning. Indian Journal of Medical Research 129 348–350.
Schatten H 1994 Dithiothreitol prevents membrane fusion but not centrosome or microtubule organization during the first cell cycles in sea urchins. Cell Motility and the Cytoskeleton 27 59–68. doi:10.1002/cm.970270107.
Schatten H & Sun QY 2009a The functional significance of centrosomes in mammalian meiosis, fertilization, development, nuclear transfer, and stem cell differentiation. Environmental and Molecular Mutagenesis 50 620–636. doi:10.1002/em.20493.
Schatten H & Sun QY 2009b The role of centrosomes in mammalian fertilization and its significance for ICSI. Molecular Human Reproduction 15 531–538. doi:10.1093/molehr/gap049.
Schatten H & Sun QY 2010 The role of centrosomes in fertilization, cell division and establishment of asymmetry during embryo development. Seminars in Cell and Developmental Biology 21 174–184. doi:10.1016/j.semcdb.2010.01.012.
Schatten H & Sun Q-Y 2011a The significant role of centrosomes in stem cell division and differentiation. Microscopy and Microanalysis 17 506–512. doi:10.1017/S1431927611000018.
Schatten H & Sun QY 2011b Centrosome dynamics during mammalian oocyte maturation with a focus on meiotic spindle formation. Molecular Reproduction and Development 78 757–768. doi:10.1002/mrd.21380.
Schatten H, Schatten G, Petzelt C & Mazia D 1982 Effects of griseofulvin on fertilization and early development in sea urchins. Independence of DNA synthesis, chromosome condensation, and cytokinesis cycles from microtubule-mediated events. European Journal of Cell Biology 27 74–87.
Schatten G, Simerly C & Schatten H 1985a Microtubule configurations during fertilization, mitosis and early development in the mouse and the requirement for egg microtubule-mediated motility during mammalian fertilization. PNAS 82 4152–4156. doi:10.1073/pnas.82.12.4152.
Schatten G, Bestor T, Balczon R, Henson J & Schatten H 1985b Intracellular pH shift leads to microtubule assembly and microtubule-mediated motility during sea urchin fertilization: correlations between elevated intracellular pH and microtubule activity and depressed intracellular pH and microtubule disassembly. European Journal of Cell Biology 36 116–127.
Schatten H, Walter M, Biessmann H & Schatten G 1992 Activation of maternal centrosomes in unfertilized sea urchin eggs. Cell Motility and the Cytoskeleton 23 61–70. doi:10.1002/cm.970230107.
Schatten H, Rawe VY & Sun Q-Y 2011 Cytoskeletal architecture of human oocytes with focus on centrosomes and their significant role in fertilization. In Practical Manual of In Vitro Fertilization: Advanced Methods and Novel Devices. Eds A Agarwal, A Varghese & ZP Nagy. New York: Humana Press.
Schuh M & Ellenberg J 2007 Self-organization of MTOCs replaces centrosome function during acentrosomal spindle assembly in live mouse oocytes. Cell 130 484–498. doi:10.1016/j.cell.2007.06.025.
Singh K & Jaiswal D 2011 Human male infertility: a complex multifactorial phenotype. Reproductive Sciences 18 418–425. doi:10.1177/1933719111398148.
Sun QY & Schatten H 2006 Multiple roles of NuMA in vertebrate cells: review of an intriguing multi-functional protein. Frontiers in Bioscience 11 1137–1146. doi:10.2741/1868.
Swain JE & Pool TB 2008 ART failure: oocyte contributions to unsuccessful fertilization. Human Reproduction Update 14 431–446. doi:10.1093/humupd/dmn025.
Talevi R & Gualtieri R 2004 In vivo versus in vitro fertilization. European Journal of Obstetrics & Gynecology and Reproductive Biology 115S S68–S71. doi:10.1016/j.ejogrb.2004.01.015.
Terada Y, Hasegawa H, Takahashi A, Ugajin T, Yaegashi N & Okamura K 2009 Successful pregnancy after oocyte activation by a calcium ionophore for a patient with recurrent intracytoplasmic sperm injection failure, with an assessment of oocyte activation and sperm centrosomal function using bovine eggs. Fertility and Sterility 91 935.e11–935.e14. doi:10.1016/j.fertnstert.2008.09.043.
Voronina E & Wessel GM 2003 The regulation of oocyte maturation. Current Topics in Development Biology 58 53–110.
Wang Q & Sun QY 2007 Evaluation of oocyte quality: morphological, cellular and molecular predictors. Reproduction, Fertility, and Development 19 1–12. doi:10.1071/RD06103.
Wang WH, Meng L, Hackett RJ, Oldenbourg R & Keefe DL 2001 The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human oocytes. Fertility and Sterility 75 348–353. doi:10.1016/S0015-0282(00)01692-7.
Zhong ZS, Zhang G, Meng XQ, Zhang YL, Chen DY, Schatten H & Sun QY 2005 Function of donor cell centrosome in intraspecies and interspecies nuclear transfer embryos. Experimental Cell Research 306 35–46. doi:10.1016/j.yexcr.2005.02.009.
