Review: Sperm-oocyte interactions and their implications for bull fertility, with emphasis on the ubiquitin-proteasome system

Abstract

Fertilization is an intricate cascade of events that irreversibly alter the participating male and female gamete and ultimately lead to the union of paternal and maternal genomes in the zygote. Fertilization starts with sperm capacitation within the oviductal sperm reservoir, followed by gamete recognition, sperm-zona pellucida interactions and sperm-oolemma adhesion and fusion, followed by sperm incorporation, oocyte activation, pronuclear development and embryo cleavage. At fertilization, bull spermatozoon loses its acrosome and plasma membrane components and contributes chromosomes, centriole, perinuclear theca proteins and regulatory RNAs to the zygote. While also incorporated in oocyte cytoplasm, structures of the sperm tail, including mitochondrial sheath, axoneme, fibrous sheath and outer dense fibers are degraded and recycled. The ability of some of these sperm contributed components to give rise to functional zygotic structures and properly induce embryonic development may vary between bulls, bearing on their reproductive performance, and on the fitness, health, fertility and production traits of their offspring. Proper functioning, recycling and remodeling of gamete structures at fertilization is aided by the ubiquitin-proteasome system (UPS), the universal substrate-specific protein recycling pathway present in bovine and other mammalian oocytes and spermatozoa. This review is focused on the aspects of UPS relevant to bovine fertilization and bull fertility.

Keywords: acrosome; artificial insemination; fertilization; sperm capacitation; zygote.

Figure 1

(Colour online) Protein ubiquitination and degradation. Unconjugated monoubiquitin (U; 1) binds covalently to its substrate (S) protein, catalyzed by ubiquitin-activating and -conjugating enzymes (E1, E2, E3) and fueled by ATP, to form a multi-ubiquitin chain (2), which is recognized by the 26S proteasome (3). The multi-ubiquitin chain is removed by the 19S proteasomal regulatory complex to be disassembled by deubiquitinating enzymes (DUB) and re-enter the pool of monoubiquitin available for conjugation (4). The substrate is degraded in the 20S proteasomal core (5). Proteasomal degradation has been implicated in sperm–zona pellucida binding, acrosomal exocytosis and sperm–zona penetration. Proteasomal proteolysis also regulates cell cycle progression, and pronuclear development and apposition in the zygote. During autophagy, which has been implicated in the regulation of early embryo development and post-fertilization sperm mitophagy, polybiquitinated substrate molecules are aggregated by autophagy-associated proteins (ATG) to form an aggresome (6). Aggresomes are recognized and engulfed by the autophagophore that encloses them to form an autophagosome (7), which upon fusion with a lysososome (L) becomes an autolysosome (8) capable of degrading the entire aggresome, or an ubiquitinated organelle such as sperm mitochondrion. The ubiquitin–proteasome system-regulated post-fertilization sperm mitophagy mediates clonal, maternal inheritance of mammalian mitochondrial genes. Monoubiquitination (9) is reversible and serves regulatory purposes relevant to gametogenesis, fertilization and early development, such as histone modification to establish the epigenetic histone code, or plasma membrane receptor internalization to change cell responsiveness to specific external stimuli/ligands.

Figure 2

(Colour online) Progression of sperm incorporation, sperm nucleus decondensation and sperm aster formation during bovine fertilization in vitro. In all panels, sperm aster microtubules were labeled with monoclonal antitubulin antibody E7 (red), sperm mitochondria were pre-labeled with vital stain MitoTracker Green FM (green) before IVF and DNA in the sperm nucleus (blue) was counterstained with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine. Left columns show parfocal bright-field images acquired with differential interference contrast optics (DIC). The samples were examined and photographed under a Nikon Eclipse 800 epifluorescence microscope (Nikon Instruments Inc., Melville, NY, USA) with Cool Snap camera (Roper Scientific, Tucson, AZ, USA) and MetaMorph software (Universal Imaging Corp., Downingtown, PA, USA). Images were edited and contrast balanced by Adobe Photoshop CS5 (Adobe Systems Inc., San Jose, CA, USA).

Figure 3

(Colour online) Components of ubiquitin–proteasome system in bovine gametes and embryos. (a) Bull spermatozoa (arrows) with abnormal phenotypes are ubiquitinated on their surface (green) and lack the WBP2NL/PAWP protein (red) present at varied levels in the post-acrosomal sheaths of surrounding morphologically normal spermatozoa. (b) Aggresome-binding ProteoStat probe (red) binds exclusively to mitochondrial sheaths of normal spermatozoa but detects aggresomes in the deformed sperm head of an abnormal spermatozoon (arrow). (c) Proteasomes (red) detected in bull sperm head acrosome and sperm tail connecting piece (arrows) by polyclonal antibody recognizing the 20S proteasomal core subunit PSMB10 (PW8150, Enzo Lifesciences, Ann Arbor, MI, USA). (d,e) Proteasomes (red) in the apposing (d), and apposed (e) maternal and paternal pronuclei of bovine zygotes, detected by a polyclonal antibody recognizing multiple 20S proteasomal core subunits (PW8155, Enzo Lifesciences). Inset in (e) shows the same zygote from which the differential interference contrast layer has been subtracted and red fluorescence brightened to reveal cytoplasmic proteasome labeling. Bovine oocytes were fertilized by spermatozoa pre-labeled with MitoTracker Green FM (green) to detect sperm tail mitochondrial sheaths (arrows). (f) Detection of deubiquitinases ubiquitin C-terminal hydrolase L1 (UCHL1) (green) and UCHL3 (red) in the bovine oocyte cortex and meiotic spindle, respectively. DNA in all panels was counterstained with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine. The samples were examined and photographed under a Nikon Eclipse 800 epifluorescence microscope with Cool Snap camera and MetaMorph software. Images were edited and contrast balanced by Adobe Photoshop CS5.