Intrauterine Insemination Rubina Merchant, Gautam N Allahbadia
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Molecular Regulation of Key Events that Play a Role in Successful Mammalian FertilizationCHAPTER 1

Heide Schatten,
Qing-Yuan Sun
Understanding the molecular events that are critical for the fertilization process has contributed greatly to overcome infertility problems through assisted reproductive technologies (ART); it has allowed us to improve conditions for oocyte maturation and sperm capacitation as important steps towards successful spermatozoon incorporation and accurate cytoskeletal formation within the activated oocyte. The molecular mechanisms that govern sperm aster formation by centrosomes to move male and female pronuclei into close apposition are now being recognized as an important criterion for developmental potential, whereas centrosome dysfunctions have been recognized as causes for cases of male factor infertility. The present chapter is structured into two sections to address (1) the communication process of sperm and oocyte, leading to spermatozoon incorporation and, (2) subsequent intracellular events within the fertilized zygote to complete the fertilization process. We hope that it will contribute to further improve ART, taking into account the various levels in the sequence of the complex molecular program during mammalian fertilization.
 
Introduction
The process of fertilization presents the beginning of a complex cascade of molecular signaling that leads to the successful union of one haploid sperm cell and one haploid egg cell, equipped with molecular tools that allow development into a zygote cell followed by cell divisions and cellular differentiation into morula and blastocyst stages with subsequent development into an offspring. The molecular precision of the program required for fertilization to be successful has been the topic of numerous investigations and was first discovered in non-mammalian species that use external fertilization, and allowed us to study the molecular events during the fertilization process in excellent detail. This knowledge, gained through basic research, could later be utilized and expanded into clinical applications of in vitro (external) fertilization (IVF) in humans.2
We are now at a point where we have the ability to prevent fertilization through contraceptives on one hand and on the other hand, to aid the fertilization process when male or female factor infertility problems are encountered. Understanding the molecular events, that are critical for the fertilization process, has contributed greatly to the overcoming of infertility problems through ART that accounts for 1% of all babies in the Western world,1 which is even higher in Australia, with in vitro fertilization (IVF) accounting for 2.7% (1/37) of all born babies (Australian Bureau of Statistics. ‘Births’, 3301.0, 2007). To consider new approaches to overcome infertility problems and to develop new clinical strategies, the present chapter is structured into two sections to address 1) the communication process of the sperm and egg leading to sperm incorporation and, 2) subsequent intracellular events within the fertilized zygote to complete the fertilization process. We hope that it will contribute to further improvement in ART, taking into account the various levels in the sequence of the complex molecular program during mammalian fertilization. To keep this chapter concise, references related to review articles are included in which, specific topics have been addressed in detail and include pre-fertilization events, such as oocyte maturation,2 sperm maturation,3 centrosome biology;2,4–6 mitochondrial considerations,7–10 and others.
 
Clinical Discussion
 
Molecular communication between sperm and egg leading to sperm incorporation
The mechanisms that govern fertilization require maturation of the sperm and egg into fertilization-competent germ cells. During oocyte maturation, the immature oocyte needs to undergo several cellular and molecular remodeling steps including cytoskeletal remodeling, that result in fertilization-competent MII oocytes. The molecular events important for successful oocyte maturation have recently been reviewed in detail2 but will not be addressed in the present chapter. Sperm also need to undergo preparation for fertilization and must mature through biochemical differentiation and morphological and functional changes, that take place as they travel through the epididymis after release from the seminiferous epithelium in the testis. To acquire fertilization competency, sperm need to undergo a process referred to as capacitation, which takes about 5 to 6 hours in humans and occurs in the female reproductive tract; it depends on bicarbonate and other uterine effectors that involve cAMP as the intracellular second messenger. Reorganizations of membrane components during the process of capacitation are important for subsequent sperm binding, penetration through the egg coat and subsequent fusion with the egg's plasma membrane. The capacitation process primarily alters the lipid and glycoprotein composition of the sperm plasma membrane. It further increases sperm metabolism and motility and causes hyperpolarization of the membrane. For review articles on these sperm-related topics, the reader may refer to studies by Evan and Florman3 and Yanagimachi.11 The mature fertilization-competent sperm is highly-polarized and primarily contains a nucleus with highly-compacted DNA,12 the acrosome (the secretory 3granule in the apical region), the centriole complex with a proximal and a distal centriole surrounded by a sparse amount of centrosomal proteins located in the connecting piece, the sperm tail with the well-known microtubule organization of 9 outer doublet microtubules and 2 inner single microtubules (9 + 2), sheath proteins, and mitochondria located in the mid-piece that provide energy for sperm motility.
As shown in Figure 1.1, before the sperm-egg fusion events can occur, sperm must penetrate through a layer of cumulus cells (ca. 5000 cells) that are embedded in extracellular matrix components, including hyaluronic acid surrounding the mature MII oocyte; sperm must further penetrate through the oocyte's zona pellucida, a thick extracellular matrix layer composed of at least three specific glycoproteins termed ZP1, ZP2 and ZP3 that are produced by the growing oocyte.13,14 The ZP matrix of human, monkey and pig is formed by four glycoproteins (ZP1, ZP2, ZP3, ZP4);15–17 but data on these mammalian species are sparse and only gradually emerging to provide specific details,18 whereas most of the information on the zona pellucida and its interactions with sperm comes from the mouse system.
zoom view
Fig. 1.1: The early stages of fertilization begin when a capacitated spermatozoon penetrates the cumulus oophorus containing cumulus cells that are embedded in extracellular matrix material. The spermatozoon's acrosome reaction is triggered when it contacts ZP3 of the zona pellucida. After undergoing the acrosome reaction, the spermatozoon is able to pass through the perivitelline space and fuse with the egg's plasma membrane. The egg is representative of non-rodent mammalian species in which the metaphase II spindle is oriented perpendicular to the egg surface. Egg activation follows when spermatozoon triggered cortical granule exocytosis occurs (not shown in this schematic) to modify and harden the zona pellucida into a firm block to polyspermy, preventing additional sperm from entering the egg.
4
Newer research has shown that the ubiquitin proteasome system (UPS) plays an important role in mammalian fertilization as it participates in sperm capacitation, acrosomal exocytosis, and sperm penetration through the egg's zona pellucida.19–21 In physiological fertilization, for these tasks to be accomplished, sperm employ enzymatic and mechanical strategies to move through the cumulus cell layer to reach and penetrate the zona pellucida. The zona pellucida protects the egg and serves as barrier to only allow species-specific sperm penetration. It contains signals for the sperm to undergo the acrosome reaction, which allows exocytosis of contents from the spermatozoon's large secretory granule, the acrosome. The zona pellucida has been characterized with biochemical-molecular methods22,23 and with various microscopy methods, including high-resolution field emission scanning electron microscopy.24,25 Microscopy studies showed that ZP2 and ZP3 assemble into long filaments while ZP1 cross-links the filaments into the three-dimensional network. ZP3 plays the most important role in sperm adhesion to the zona pellucida by binding to sperm receptors in the anterior head of the acrosome-intact sperm. It is critically important for species-specific binding of sperm to the zona, which may involve the interaction of ZP3 receptors with proteins on the sperm surface through O-linked oligosaccharides. As sperm bind to the zona, the acrosome reaction, a process by which contents of the spermatozoon's acrosomal vesicle are released through exocytosis, is triggered. An increase in cytosolic Ca++ is necessary for triggering the acrosome reaction. Soluble proteins (SNARE proteins; N-ethylmaleimide-sensitive factor-attachment protein receptor), present in the acrosomal region, may couple spermatozoon entry with exocytosis. The acrosome reaction is essential for fertilization to occur and to help the spermatozoon to pass through the zona pellucida. The binding of the sperm to the zona pellucida also involves proteins that bind to ZP2. Sperm-egg adhesion and fusion with the plasma membrane involves proteins of the plasma membrane and the fertilin family.3 As most of our knowledge on this aspect of fertilization comes from the mouse system, it will be important to perform similar studies on human oocytes or animal models, such as the porcine or bovine system, that employ mechanisms more similar to humans compared to the mouse. The significant differences between the mouse and non-rodent mammalian systems have been detailed for other aspects of fertilization in previous review papers.2,6 In cases of infertility, where the above mentioned molecular processes are impaired, a spermatozoon can directly be injected into the matured MII oocyte, in a process utilizing intracytoplasmic sperm injection (ICSI).26
In physiological fertilization, of the 3,000,000,000 sperm cells that are released during ejaculation, only about 200 reach the site of fertilization in the oviduct and only one spermatozoon will fuse with the egg, which requires two built-in blocks by the egg to prevent polyspermy. The first block is a fast block and utilizes rapid depolarization of the egg plasma membrane (“electric block”), which is caused by sperm fusion to the egg and prevents further sperm from fusing to the egg. This first fast block to polyspermy is reversed while the second slower block takes place and is accomplished by the egg's cortical reaction, which is triggered when the spermatozoon fuses with the egg's plasma membrane.5
Sperm-egg fusion triggers a cascade of calcium events, starting with a local increase of cytosolic Ca++ that results in a wave throughout the egg and may be followed by prolonged Ca++ oscillations that in turn, cause egg activation and subsequent development.3 The cortical reaction is characterized by release of cortical granule contents through exocytosis and fusion of the cortical granule membrane with the egg plasma membrane. The change in the egg plasma membrane due to the fusion with cortical granule membranes is called plasma membrane reaction. The contents of the cortical granules include specific enzymes that cause changes in the structure of the zona pellucida resulting in zona pellucida hardening (zona reaction),27 which prevents subsequent sperm from entering and therefore, presents a firm block to polyspermy. During this process, proteolytic cleavage of ZP2, as well as hydrolysis of sugar components on ZP3, takes place.
Following sperm-egg fusion and sperm penetration into the egg, several processes have to occur within the cytoplasm that will be detailed in the following section.
 
Intracellular events within the fertilized zygote to complete the fertilization process
Sperm factors that activate the egg are important for successful fertilization and for resumption of MII and second polar body emission.3,6 Following incorporation of the spermatozoon into the egg, several events need to take place for fertilization to be successful. The now fertilized egg is called zygote and contains paternal and maternal components that are both important for embryo development and provide a complementary set for the molecular machinery that drives development of the embryo.
Oocyte quality is critically important for fertilization to be successful and several criteria have been used to assess oocyte quality, which includes integrity of the MII spindle.2,6,28–30 Although it is easy to determine MII spindle integrity with immunofluorescence microscopy on fixed cells, using antibodies to microtubules or centrosomes and fluorescent dyes to detect DNA,2,6 it is still challenging to assess the MII spindle with non-invasive methods. Non-invasive methods so far, have been limited to the use of polarization optics as these are easily available in IVF clinics and useful for the assessment of the MII spindle.2 However, the MII spindle can be defective at the molecular level and undetectable with polarization microscopy. A better assessment of the MII spindle with non-invasive methods may be available if live cell imaging with green fluorescence protein (GFP) detection can be employed. Basic experimentation on suitable animal models may be needed before application to human oocytes can be considered. Experimentation using GFP imaging is ongoing in several laboratories, and our animal science groups at the University of Missouri have successfully generated a variety of transgenic pigs as disease models in which specific components had been labeled with GFP.31 In other studies, live cell imaging of cell organelles, such as mitochondria, had been used successfully to assess oocyte quality; multiphoton live cell imaging has been performed in hamster oocytes without harming the embryo that developed to full-term after implantation into a surrogate mother.7,8 Such studies have not 6yet been applied to assess human oocyte quality in IVF clinics but should be entirely feasible. New non-invasive methods to assess the MII spindle are highly important for the detection of aneuploidies and for the detection of dysfunctional molecular components of the meiotic spindle that are the cause for chromosome mis-segregation and aneuploidies. As the current rate of aneuploidies is especially high in humans,2,6 it can be expected that it will rise in future years, as the demand for IVF by women past their optimal reproductive age is expected to increase. Spindles in aging oocytes have been well characterized28,32,33 and display aberrant centrosome, microtubule, and chromosome patterns. Remarkably, such spindles, in which deterioration is observed, can be rescued by interfering with specific signal transduction cascades that play a role in spindle deterioration.28 Caffeine has been identified as one of the components that can either delay or rescue spindle deterioration.28 Preventative addition of caffeine may also be useful in IVF applications in which oocyte handling procedures may be lengthy and oocyte aging may be encountered. Although proposed by several investigators, practical applications have not yet utilized these new findings, but are worthwhile considering.
An essential aspect for successful fertilization relates to the sperm-derived centriole complex. A critical requirement for successful fertilization is the formation of the sperm aster that is needed to move male and female pronuclei containing the male and female genome into close apposition. Sperm aster formation depends on centrosome integrity.6,29 In the mammalian zygote, the two pronuclei do not fuse but become apposed and remain together. They are distinct from each other until the pronuclear membranes have broken down in preparation for first cell division and distribution of the combined genome to the divided daughter cells. A critically important aspect for successful fertilization and embryo development is the centriole complex that is contributed by the sperm in non-rodent mammalian embryos and provides the seed for microtubule nucleation of the sperm aster, the mitotic apparatus and all subsequent spindles formed during symmetric and asymmetric cell division as well as in somatic cells of the adult. Dysfunctions of the centriole complex can be causes for diseases, including cancer in which the centriole complex becomes mis-regulated. The role of centrosomes in mammalian fertilization and its significance for ICSI has been highlighted previously.26
The sperm's centriole complex recruits centrosome proteins from the oocyte, including γ-tubulin, that is specific for microtubule nucleation within the centrosome complex. Dysfunctions in recruitment of centrosome proteins from the oocyte to the sperm's centriole complex can result in fertility problems, developmental abnormalities and diseases in adulthood, depending on the severity of centrosomal dysfunctions.2,6 The blended centriole-centrosome complex duplicates during the pronuclear stage and during the S phases in subsequent cell divisions throughout development. Regulation by cyclins and cell cycle-specific centrosome remodeling is important for fertilization and for all stages of subsequent development. Centrosome remodeling is facilitated by cell cycle regulation proteins, as well as by proteases, to reshape requirements during the first and subsequent cell cycles.2,67
As shown in Figure 1.2, the spermatozoon contains a proximal and a distal centriole. After sperm incorporation, the distal centriole deteriorates after having served as the basal body for sperm tail functions that are not needed in the fertilized oocyte in which the sperm tail is destroyed. The proximal centriole, on the other hand, is closely associated with the sperm nucleus and serves as the microtubule organizing center (MTOC) that recruits additional centrosomal proteins, such as the microtubule nucleating protein γ-tubulin, from the oocyte for centrosome and microtubule growth into the sperm aster that is essential for the apposition of maternal and paternal pronuclei and for the formation of the zygote aster that evolves into the mitotic apparatus during first cell division.
Significant male-factor derived infertility problems are associated with defects in the basal body34–37 and can have genetic or environmental components.26 Some of these defects can be overcome by ICSI, while others are related to an inability of the sperm's proximal centriole to organize a functional sperm aster. Several causes for incomplete or deficient sperm aster formation have been identified and can be related to molecular defects in the proximal centriole, to decreased centrosomal proteins surrounding the proximal centriole, to an inability to recruit centrosomal proteins for the growth of the sperm aster, and others that are currently under investigation.6,30,38–40 Heterologous sperm insemination has been used for the analysis of male-factor-derived dysfunctions to assess whether centrosome dysfunctions are the underlying cause.
zoom view
Fig. 1.2: The sperm in non-rodent mammalian species, including humans, contains a proximal and a distal centriole (A) while the distal centriole deteriorates after spermatozoon incorporation in the egg, the proximal centriole serves as a microtubule organizing center (MTOC) to form the sperm aster, (B) and after recruiting centrosomal material from the oocyte to increase centrosome size, the zygote aster, and the mitotic apparatus in preparation for cell division. The male and female pronuclei are apposed but still separated; the maternal and paternal chromosome sets will intermingle after nuclear envelope breakdown before separating into daughter cells during the first division.
In the heterologous 8insemination system, human sperm are used to fertilize bovine or porcine oocytes to assess the formation of the sperm aster. While numerous reports exist on the reliability of this assessment for centriole and centrosome function, it is not being used for routine analysis in IVF clinics, perhaps because it requires expertise and experience with different animal systems. The mouse has been excluded from these studies as the mouse uses totally different cell and molecular mechanisms for fertilization involving significant differences in the sperm and oocyte.2,6
Infertility problems can also be identified when related to cytoskeletal dysfunctions and to mitochondrial dysfunctions, including mitochondrial translocation-distribution-dynamics dysfunctions41 and metabolic dysfunctions.9,10 Cytoskeletal and mitochondrial structure-function-dynamics relationships are important indicators of successful fertilization, as transport of mitochondria along microtubules to their functional destinations takes place during specific cell cycle phases in somatic cells, and during oocyte maturation and after fertilization in germ cells. So far, the importance of proper mitochondria distribution, using non-invasive live cell imaging, has primarily been assessed in hamsters,7 nonhuman primates,8 and porcine oocytes.42,43 Such non-invasive methods could potentially be used to assess mitochondrial functions in human oocytes to select high quality oocytes for culture and assess their proper mitochondrial distribution following IVF.
As the mitochondrion of the spermatozoon is destroyed shortly after its incorporation into the egg by ubiquitination44 and/or autophagy45,46 to allow mitochondrial homogeneity and ensure that all mitochondria for subsequent development are inherited from the oocyte,41 assessment of mitochondrial quality in oocytes is critically important and will play a significant role in the developmental potential.7,8,42,43,47 Importantly, recent studies have shown that preimplantation mitochondrial metabolism heavily relies on glycolysis, a strategy frequently used by fast-dividing cells including cancer cells,9,10 which will play a role in re-defining culture conditions for culture of human oocytes in IVF clinics. These studies are ongoing and promise to reveal new strategies to assess oocyte quality and define better culture conditions for in vitro maturation and subsequent culture of embryos during IVF procedures.

Recent Advances

As mentioned in the Clinical Discussion section above, clinical advances, at the molecular level, are in progress to intervene with dysfunctions that have been implicated in infertility or in fertility disorders; however, many of the proposed interventions and improvements in current manipulation procedures have been explored in animal models and have not yet advanced to practical applications in human ART procedures, although it would be entirely feasible. Understanding the mechanisms underlying dysfunctions will most certainly allow us to find additional means of intervention to improve ART in IVF clinics. Recent advances include–
Live cell imaging of the MII spindle that is important to determine spindle abnormalities, which may include abnormalities in centrosome and 9microtubule organization. Such abnormalities are especially apparent in aging oocytes,28 and may be detected by using GFP-tubulin markers. This technology is used fairly routinely in somatic cells to detect microtubule dynamics during mitosis. GFP-related technologies have also been used successfully for animal disease models in which specific components had been labeled with GFP.31 Using non-invasive GFP-tubulin methods to assess human MII oocyte quality in IVF clinics should be entirely feasible and would allow the detection of aneuploidies and dysfunctional molecular components of the meiotic spindle that are causes for chromosome mis-segregation and aneuploidies.
Spindle defects have successfully been corrected by adding components, such as caffeine, to the culture medium to interfere with signal transduction pathways that become dysfunctional during oocyte aging.28 Such applications, applied to IVF procedures, are likely to improve IVF success rates. Some of these studies used the porcine model as the most suitable model for humans.31 Preventative addition of caffeine may also be useful in IVF applications in which oocyte handling procedures may be lengthy and oocyte aging may be encountered.
Multiphoton live cell imaging of mitochondria using mitotracker detection has been used successfully in the hamster to assess perinuclear mitochondrial distribution that has been shown to predict developmental potential.7,8,41,42 Oocytes selected for perinuclear mitochondrial distribution without harming the embryo, after successful live cell imaging in hamster oocytes, developed to full-term after fertilization and implantation into a surrogate mother.7,8 Such non-invasive methods could potentially be used to assess mitochondrial functions in human oocytes; high quality oocytes, based on mitochondrial distribution, could be selected for culture and assessed during maturation and following IVF.
Recent studies on preimplantation porcine embryos have shown that mitochondrial metabolism heavily relies on glycolysis,9–10 which may impact re-defining culture conditions that are still suboptimal for culture of human oocytes. The new findings on mitochondrial metabolism is likely to contribute to new strategies in defining better culture conditions for in vitro maturation and subsequent culture of oocytes during IVF procedures.
Centrosome supplementation and “repair”: An emerging strategy for therapeutic intervention in male-factor infertility cases is centrosome supplementation and “repair” in sperm that lack centrosome integrity resulting in either suboptimal, significantly impaired, or absent centrosome functions. Such experiments are ongoing in the cat38,39 and other species26 and are aimed to allow sperm aster formation for apposition of pronuclei and subsequent development that critically depends on centrosome functions.
Assessment of sperm centrosomal functions by heterologous fertilization has become a most feasible method for the analysis of male factor-derived dysfunctions to assess whether centrosome dysfunctions are the underlying cause. In the heterologous insemination system, human sperm are used to fertilize bovine or porcine oocytes to assess the formation of the sperm 10aster. Although numerous reports exist on the reliability of this assessment for centriole and centrosome function, it is not being used for routine analysis in IVF clinics, perhaps because it requires expertise and experience with different animal systems. The mouse has been excluded from these studies as the mouse uses totally different cell and molecular mechanisms for fertilization, involving significant differences in the sperm and oocyte.2,6

Conclusion

Taken together, while significant progress has been made, especially during the past decade, to overcome infertility problems, new strategies for practical applications in IVF clinics are in demand and need to take into consideration molecular defects underlying infertility problems. Specific problems may be corrected by interfering with dysfunctional signaling pathways or specific molecular dysfunctions encountered during oocyte maturation, sperm capacitation, sperm-egg attachment and fusion, centrosome and cytoskeletal dysfunctions, mitochondrial abnormalities and others detailed above. Our current knowledge gained in basic studies may, at least in part, be utilized to determine more optimal culture conditions and strategies to bypass processes that are important for physiological fertilization. The best example of bypassing molecular requirements for incorporation of the spermatozoon into the oocyte has been the design and practical utilization of ICSI48 that has proven to be highly successful in overcoming sperm motility, sperm-egg fusion and sperm incorporation defects.
 
References
  1. Giritharan G, Talbi S, Donjacour A, Di Sebastiano F, Dobson AT, Rinaudo PF. Effect of in vitro fertilization on gene expression and development of mouse preimplantation embryos. Reproduction 2007;134:63–72.
  1. Schatten H, Sun Q-Y. Centrosome dynamics during meiotic spindle formation in oocyte maturation. Mol Reprod Dev 2011a;78:757-68.
  1. Evans JP, Florman HM. The state of the union: the cell biology of fertilization. Nat Cell Biol Fertility Supplement 2002;S57-S63.
  1. Schatten H. The mammalian centrosome and its functional significance. Histochem Cell Biol 2008;129:667–86.
  1. Schatten H, Sun QY. The role of centrosomes in fertilization, cell division and establishment of asymmetry during embryo development. Semin Cell Dev Biol 2010;21:174–84.
  1. Schatten H, Sun QY. New insights into the role of centrosomes in mammalian fertilisation and implications for ART. Reproduction 2011b; 142:793-801.
  1. Squirrell JM, Wokosin DL, Bavister BD, White JG. Long-term multiphoton fluorescence imaging of mammalian embryos does not compromise viability. Nature Biotechnology 1999;17:763–7.
  1. Squirrell JM, Lane M, Bavister BD. Altering intracellular pH disrupts development and cellular organization in preimplantation hamster embryos. Biol Reprod 2001;64:1845–54.
  1. Krisher RL, Prather RS. A role for the Warburg Effect in preimplantation embryo development: metabolic modification to support rapid cell proliferation. Mol Reprod Dev 2012;79:311–20.
  1. Redel BK, Brown AN, Spate LD, Whitworth KM, Green JA, Prather RS. Glycolysis in preimplantation development is partially controlled by the Warburg Effect. Mol Reprod Dev 2012;79:262–71.
  1. Yanagimachi R. The Physiology of Reproduction 189–317 (Raven Press,  New York 1994).
  1. Johnson GD, Lalancette C, Linnemann AK, Leduc F, Boissonneault G, Krawetz, S.A. The sperm nucleus: chromatin, RNA, and the nuclear matrix. Reproduction 2011;141:21–36.
  1. Wassarman PM. Zona pellucida glycoproteins. J Biol Chem 2008;283:24285–9.
  1. Wassarman PM, Litscher ES. Mammalian fertilization: the egg's multifunctional zona pellucida. Int J Dev Biol 2008;52:665–76.
  1. Hughes DC, Barratt CL. Identification of the true human orthologue of the mouse Zp1 gene: evidence for greater complexity in the mammalian zona pellucida? Biochem Biophys Acta 1999;1447:303–6.
  1. Lefievre L, Conner SJ, Salpekar A, Olufowobi O, Ashton P, Pavlovic B, et al. Four zona pellucida glycoproteins are expressed in the human. Hum Reprod 2004;19:1580–6.
  1. Ganguly A, Sharma RK, Gupta SK. Bonnet Monkey (Macaca radiata) ovaries, like human oocytes, express four zona pellucida glycoproteins. Mol Reprod Dev 2008;75:156–66.
  1. Ganguly A, Bukovsky A, Sharma RK, Bansal P, Bhandari B, Gupta SK. In humans, zona pellucida glycoprotein-1 binds to spermatozoa and induces acrosomal exocytosis. Hum Reprod 2010;25:1643–56.
  1. Sutovsky P, Manandhar G, McCauley TC, Caamano JN, Sutovsky M, Thompson WE, Day BN Proteasomal interference prevents zona pellucida penetration and fertilization in mammals. Biol Reprod 2004;71:1625–37.
  1. Yi YJ, Zimmerman SW, Manandhar G, Odhiambo JF, Kennedy C, Jonakova V, et al. Ubiquitin-activating enzyme (UBA1) is required for sperm capacitation, acrosomal exocytosis and sperm-egg coat penetration during porcine fertilization. Int J Androl 2011;35:196–210.
  1. Zimmerman SW, Manandhar G, Yi YJ, Gupta SK, Sutovsky M, Odhiambo JF, et al. Sperm proteasomes degrade sperm receptor on the egg zona pellucida during mammalian fertilization. PLoS 2011;6(1):e17256.
  1. Pang P-C, Chiu PCN, Lee C-L, Chang LY, Panico M, Morris HR, et al. Human sperm binding is mediated by the sialyl-Lewisx oligosaccharide on the zona pellucida. Science 2011;333:1761–4.
  1. Wassarman PM. The sperm's sweet tooth. Science 2011;333:1708–9.
  1. Familiari G, Heyn R, Relucenti, M, Sathananthan H. Structural changes of the zona pellucida during fertilization and embryo development. Front Biosci 2008;13:6730–51.
  1. Familiari G, Heyn R, Petruzziello L, Relucenti M. A method to visualize the microarchitecture of glycoprotein matrices with scanning electron microscopy. In: Scanning Electron Microscopy for the “Life Sciences 2012”; edited by Heide Schatten, Cambridge University Press. 
  1. Schatten H, Sun, QY. The role of centrosomes in mammalian fertilization and its significance for ICSI. Mol Hum Reprod 2009;15:531–8.
  1. Papi M, Brunelli R, Sylla L, Parasassi T, Monaci M, Maulucci G, et al. Mechanical properties of zona pellucida hardening. European Biophys Journal 2010;9:987–92.
  1. Miao YL, Kikuchi K, Sun QY, Schatten H. Oocyte aging: cellular and molecular changes, developmental potential and reversal possibility. Hum Reprod Update 2009;15:573–85.
  1. Schatten H, Sun QY. Nuclear-centrosome relationships during fertilization, cell division, embryo development, and in somatic cell nuclear transfer (SCNT) embryos. In: The Centrosome; 2012, edited by Heide Schatten, Springer Science and Business Media, LLC. 
  1. Schatten H, Rawe VY, Sun QY. The sperm centrosome: its role and significance in nature and human assisted reproduction. Journal of Reproductive and Stem Cell Biotechnology 2011;2:121–7.
  1. Whyte JJ, Prather RS. Genetic modifications of pigs for medicine and agriculture. Mol Reprod Develop 2011;78:879–91.
  1. Eichenlaub-Ritter U, Stahl A, Luciani JM. The microtubular cytoskeleton and chromosomes of unfertilized human oocytes aged in vitro. Hum Genet 1988;80:259–64.
  1. Pellestor F, Anahory T, Hamamah S. Effect of maternal age on the frequency of cytogenetic abnormalities in human oocytes. Cytogenet Genome Res 2005;111:206–12.
  1. Chemes HE. Phenotypes of sperm pathology: genetic and acquired forms in infertile men. J Androl 2000;21:799–808.
  1. Chemes HE, Rawe VY. Sperm pathology: a step beyond descriptive morphology. Origin, characterization and fertility potential of abnormal sperm phenotypes in infertile men. Hum Reprod Update 2007;9:405–28.
  1. Chemes HE. Sperm centrioles and their dual role in flagellogenesis and cell cycle of the zygote structure, function and pathology. In: The Centrosome; 2012. edited by Heide Schatten, Springer Science and Business Media, LLC. 
  1. Sathananthan, A.H. Human centrosomal dynamics during gametogenesis, fertilization and embryogenesis and its impact on fertility: Ultrastructural analysis. In: The Centrosome; 2012, edited by Heide Schatten, Springer Science and Business Media, LLC. 
  1. Comizzoli P, Wildt DE, Pukazhenthi BS. Poor centrosomal function of cat testicular spermatozoa impairs embryo development in vitro after intracytoplasmic sperm injection. Biol Reprod 2006;75:252–60.
  1. Comizzoli P, Wildt DE. Centrosomal functions and dysfunctions in cat spermatozoa. In: The Centrosome; 2012, edited by Heide Schatten, Springer Science and Business Media, LLC. 
  1. Hinduja I, Zaveri K, Baliga N. Correlation of human sperm centrosomal proteins with fertility. Journal of Human Reprod Science 2010;3:95–101.
  1. Schatten H, Prather RS, Sun QY. The significance of mitochondria for embryo development in cloned farm animals. Mitochondrion 2005;5:303–21.
  1. Sun QY, Wu GM, Lai L, Park KW, Cabot R, Cheong HT, Day BN, Prather RS, Schatten H. Translocation of active mitochondria during pig oocyte maturation, fertilization and early embryo development in vitro. Reproduction 2001;122:155–63.
  1. Katayama M, Zhong Z-S, Lai L, Sutovsky P, Prather RS, Schatten H. Mitochondria distribution and microtubule organization in fertilized and cloned porcine embryos: implications for developmental potential. Dev Biol 2006;299:206–20.
  1. Sutovsky P, Moreno RD, Ramalho-Santos J, Dominko T, Simerly C, Schatten G. Ubiquitin tag for sperm mitochondria. Nature 1999;402:371–2.
  1. Adastra KL, Chi MM, Riley JK, Moley KH. A differential autophagic response to hyperglycemia in the developing murine embryo. Reproduction 2011;141:607–15.
  1. Al Rawi S, Louvet-Vallée S, Djeddi A, Sachse M, Culetto E, Hajjar C, Boyd L, Legouis R, Galy V. Postfertilization autophagy of sperm organelles prevents paternal mitochondrial DNA transmission. Science 2011;334:1144–7.
  1. Zhong Z, Hao Y, Li R, Spate L, Wax D, Sun Q-Y, Prather RS, Schatten H. Analysis of heterogeneous mitochondria distribution in somatic cell nuclear transfer porcine embryos. Microsc Microanal 2008;14:463–77.
  1. Palermo G, Joris H, Devroey P, Van Steirteghem A. Pregnancies after intracytoplasmic sperm injection of single spermatozoon into an oocyte. Lancet 1992;340:17–8.