The perinuclear theca (PT) is a condensed, nonionic detergent resistant cytosolic protein layer encapsulating the sperm head nucleus. It can be divided into two regions: the subacrosomal layer, whose proteins are involved in acrosomal assembly during spermiogenesis, and the postacrosomal sheath (PAS), whose proteins are implicated in sperm–oocyte interactions during fertilization. In continuation of our proteomic analysis of the PT, we have isolated two prominent PT-derived proteins of 28 and 31 kDa from demembranated bovine sperm head fractions. These proteins were identified by mass spectrometry as isoforms of glutathione-s-transferase omega 2 (GSTO2). Immunoblots probed with anti-GSTO2 antibodies confirmed the presence of the GSTO2 isoforms in these fractions while fluorescent immunocytochemistry localized the isoforms to the PAS region of the bull, boar, and murid PT. In addition to the PAS labeling of GSTO2, the performatorium of murid spermatozoa was also labeled. Immunohistochemistry of rat testes revealed that GSTO2 was expressed in the third phase of spermatogenesis (i.e., spermiogenesis) and assembled in the PAS and perforatorial regions of late elongating spermatids. Fluorescent immunocytochemistry performed on murine testis cells co-localized GSTO2 and tubulin on the transient microtubular-manchette of elongating spermatids. These findings imply that GSTO2 is transported and deposited in the PAS region by the manchette, conforming to the pattern of assembly found with other PAS proteins. The late assembly of GSTO2 and its localization in the PAS suggests a role in regulating the oxidative and reductive state of covalently linked spermatid/sperm proteins, especially during the disassembly of the sperm accessory structures after fertilization.
In New South Wales, Australia, the Child Personal Health Record (CPHR, aka ‘The Blue Book’, which includes notes on health and development) is proclaimed as an important document with widespread use throughout the state. Despite the significance of the record, there are few published evaluations of the efficacy of the CPHR. Parental use and views of the CPHR were examined using a two-phase, mixed-method design. One hundred and twenty mothers completed an online questionnaire, which included questions on demographics, use and views of the CPHR and child care experience. Six of these mothers participated in a follow-up interview. Perceived value of the CPHR was at its highest when the child was younger and if the child was first-born. The CPHR is used by medical professionals, yet broadening its use may increase efficiency of information transfer and promote parent understanding of developmental records, especially on growth indicators such as head circumference, which are not well understood. Implications for CPHRs as an empowering tool for families are considered.
Building on our recent discovery of the zinc signature phenomenon present in boar, bull, and human spermatozoa, we have further characterized the role of zinc ions in the spermatozoa’s pathway to fertilization. In boar, the zinc signature differed between the three major boar ejaculate fractions, the initial pre-rich, the sperm-rich, and the post-sperm-rich fraction. These differences set in the sperm ejaculatory sequence establish two major sperm cohorts with marked differences in their sperm capacitation progress. On the subcellular level, we show that the capacitation-induced Zn-ion efflux allows for sperm release from oviductal glycans as analyzed with the oviductal epithelium mimicking glycan binding assay. Sperm zinc efflux also activates zinc-containing enzymes and proteases involved in sperm penetration of the zona pellucida, such as the inner acrosomal membrane matrix metalloproteinase 2 (MMP2). Both MMP2 and the 26S proteasome showed severely reduced activity in the presence of zinc ions, through studies using by gel zymography and the fluorogenic substrates, respectively. In the context of the fertilization-induced oocyte zinc spark and the ensuing oocyte-issued polyspermy-blocking zinc shield, the inhibitory effect of zinc on sperm-borne enzymes may contribute to the fast block of polyspermy. Altogether, our findings establish a new paradigm on the role of zinc ions in sperm function and pave the way for the optimization of animal semen analysis, artificial insemination (AI), and human male-factor infertility diagnostics.
The sperm-borne oocyte-activating factor (SOAF) resides in the sperm perinuclear theca (PT). A consensus has been reached that SOAF most likely resides in the postacrosomal sheath (PAS), which is the first region of the PT to solubilize upon sperm-oocyte fusion. There are two SOAF candidates under consideration: PLCZ1 and WBP2NL. A mouse gene germline ablation of the latter showed that mice remain fertile with no observable phenotype despite the fact that a competitive inhibitor of WBP2NL, derived from its PPXY motif, blocks oocyte activation when coinjected with WBP2NL or spermatozoa. This suggested that the ortholog of WBP2NL, WBP2, containing the same domain and motifs associated with WBP2NL function, might compensate for its deficiency in oocyte activation. Our objectives were to examine whether WBP2 meets the developmental criteria established for SOAF and whether it has oocyte-activating potential. Immunoblotting detected WBP2 in mice testis and sperm and immunofluorescence localized WBP2 to the PAS and perforatorium of the PT. Immunohistochemistry of the testes revealed that WBP2 reactivity was highest in round spermatids and immunofluorescence detected WBP2 in the cytoplasmic lobe of elongating spermatids and colocalized it with the microtubular manchette during PT assembly. Microinjection of the recombinant forms of WBP2 and WBP2NL into metaphase II mouse oocytes resulted in comparable rates of oocyte activation. This study shows that WBP2 shares a similar testicular developmental pattern and location with WBP2NL and a shared ability to activate the oocyte, supporting its consideration as a mouse SOAF component that can compensate for a WBP2NL.
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