Adding insult to injury: effects of xenobiotic-induced preantral ovotoxicity on ovarian development and oocyte fusibility.

Mammalian females are born with a finite number of nonrenewing primordial follicles, the majority of which remain in a quiescent state for many years. Because of their nonrenewing nature, these "resting" oocytes are particularly vulnerable to xenobiotic insult, resulting in premature ovarian senescence and the formation of dysfunctional oocytes. In this study, we characterized the mechanisms of ovotoxicity for three ovotoxic agents, 4-vinylcyclohexene diepoxide (VCD), methoxychlor (MXC), and menadione (MEN), all of which target immature follicles.

Evaluation of an online tobacco cessation course for health professions students.

Objective: To implement and evaluate the impact of a semester-long, online, 1-credit elective course designed to promote tobacco cessation counseling proficiency among health professions students.

Design: Online technology was used to create an elective course devoted to tobacco cessation, modeled closely after the Rx for Change curriculum. Students from pharmacy, nursing, and other health disciplines enrolled in the course.

Elucidation of the signaling pathways that underpin capacitation-associated surface phosphotyrosine expression in mouse spermatozoa.

Recent studies from within our laboratory have demonstrated a causal relationship between capacitation-associated surface phosphotyrosine expression and the ability of mouse spermatozoa to recognize the oocyte and engage in sperm-zona pellucida interaction. In the studies described herein we have sought to investigate the signaling pathways that underpin the tyrosine phosphorylation of sperm surface protein targets and validate the physiological significance of these pathways in relation to sperm-zona pellucida adhesion. Through selective pharmacological inhibition we have demonstrated that surface phosphotyrosine expression is unlikely to be mediated by the canonical cAMP-dependent protein kinase A (PKA) signaling cascade that has been most widely studied in relation to sperm capacitation.

Glioma pathogenesis-related 1-like 1 is testis enriched, dynamically modified, and redistributed during male germ cell maturation and has a potential role in sperm-oocyte binding.

The glioma pathogenesis-related 1 (GLIPR1) family consists of three genes [GLIPR1, GLIPR1-like 1 (GLIPR1L1), and GLIPR1-like 2 (GLIPR1L2)] and forms a distinct subgroup within the cysteine-rich secretory protein (CRISP), antigen 5, and pathogenesis-related 1 (CAP) superfamily. CAP superfamily proteins are found in phyla ranging from plants to humans and, based largely on expression and limited functional studies, are hypothesized to have roles in carcinogenesis, immunity, cell adhesion, and male fertility. Specifically data from a number of systems suggests that sequences within the C-terminal CAP domain of CAP proteins have the ability to promote cell-cell adhesion.

New proteins identified in epididymal fluid from the platypus (Ornithorhynchus anatinus).

The platypus epididymal proteome is being studied because epididymal proteins are essential for male fertility in mammals and it is considered that knowledge of the epididymal proteome in an early mammal would be informative in assessing the convergence and divergence of proteins that are important in the function of the mammalian epididymis. Few of the epididymal proteins that have been identified in eutherian mammals were found in platypus caudal epididymal fluid, and the major epididymal proteins in the platypus (PXN-FBPL, SPARC and E-OR20) have never been identified in the epididymis of any other mammal.

Testicular descent, sperm maturation and capacitation. Lessons from our most distant relatives, the monotremes.

The present review examines whether monotremes may help to resolve three questions relating to sperm production in mammals: why the testes descend into a scrotum in most mammals, why spermatozoa are infertile when they leave the testes and require a period of maturation in the specific milieu provided by the epididymides, and why ejaculated spermatozoa cannot immediately fertilise an ovum until they undergo capacitation within the female reproductive tract. Comparisons of monotremes with other mammals indicate that there is a need for considerable work on monotremes. It is hypothesised that testicular descent should be related to epididymal differentiation.

The biological significance of detergent-resistant membranes in spermatozoa.

As mammalian spermatozoa ascend the female reproductive tract, they acquire the ability to fertilize an oocyte via a complex cascade of biophysical and biochemical changes collectively know as 'capacitation'. In virtually all species studied, capacitation is accompanied by dramatic remodeling of the surface architecture, in order to render spermatozoa competent to recognize the oocyte and initiate fertilization. Although the fundamental mechanisms that underpin the dynamic redistribution of sperm surface proteins are poorly understood, recent evidence indicates that this process may be facilitated, at least in part, by specialized membrane microdomains or lipid rafts.

Comparative analysis of activator-Esigma54 complexes formed with nucleotide-metal fluoride analogues.

Bacterial RNA polymerase (RNAP) containing the major variant sigma(54) factor forms open promoter complexes in a reaction in which specialized activator proteins hydrolyse ATP. Here we probe binding interactions between sigma(54)-RNAP (Esigma(54)) and the ATPases associated with various cellular activities (AAA+) domain of the Escherichia coli activator protein, PspF, using nucleotide-metal fluoride (BeF and AlF) analogues representing ground and transition states of ATP, which allow complexes (that are otherwise too transient with ATP) to be captured. We show that the organization and functionality of the ADP-BeF- and ADP-AlF-dependent complexes greatly overlap.

DNA damage in human spermatozoa is highly correlated with the efficiency of chromatin remodeling and the formation of 8-hydroxy-2'-deoxyguanosine, a marker of oxidative stress.

DNA damage in human spermatozoa has been associated with a range of adverse clinical outcomes, including infertility, abortion, and disease in the offspring. We have advanced a two-step hypothesis to explain this damage involving impaired chromatin remodeling during spermiogenesis followed by a free radical attack to induce DNA strand breakage. The objective of the present study was to test this hypothesis by determining whether impaired chromatin protamination is correlated with oxidative base damage and DNA fragmentation in human spermatozoa.

Functional roles of the pre-sensor I insertion sequence in an AAA+ bacterial enhancer binding protein.

Molecular machines belonging to the AAA+ superfamily of ATPases use NTP hydrolysis to remodel their versatile substrates. The presence of an insertion sequence defines the major phylogenetic pre-sensor I insertion (pre-SIi) AAA+ superclade. In the bacterial sigma(54)-dependent enhancer binding protein phage shock protein F (PspF) the pre-SIi loop adopts different conformations depending on the nucleotide-bound state.

Coupling sigma factor conformation to RNA polymerase reorganisation for DNA melting.

ATP-driven remodelling of initial RNA polymerase (RNAP) promoter complexes occurs as a major post recruitment strategy used to control gene expression. Using a model-enhancer-dependent bacterial system (sigma54-RNAP, Esigma54) and a slowly hydrolysed ATP analogue (ATPgammaS), we provide evidence for a nucleotide-dependent temporal pathway leading to DNA melting involving a small set of sigma54-DNA conformational states. We demonstrate that the ATP hydrolysis-dependent remodelling of Esigma54 occurs in at least two distinct temporal steps.

ADPase activity of recombinantly expressed thermotolerant ATPases may be caused by copurification of adenylate kinase of Escherichia coli.

Except for apyrases, ATPases generally target only the gamma-phosphate of a nucleotide. Some non-apyrase ATPases from thermophilic microorganisms are reported to hydrolyze ADP as well as ATP, which has been described as a novel property of the ATPases from extreme thermophiles. Here, we describe an apparent ADP hydrolysis by highly purified preparations of the AAA+ ATPase NtrC1 from an extremely thermophilic bacterium, Aquifex aeolicus.

Reproductive biology in egg-laying mammals.

The duck-billed platypus and short-beaked echidna are iconic species in Australia. Their morphology and physiology have puzzled scientists all over the world for more than 200 years. Recent genetic studies, particularly the platypus whole-genome sequencing project, have revealed the molecular basis of some of the extraordinary characteristics of monotremes.

Composition and significance of detergent resistant membranes in mouse spermatozoa.

Mammalian spermatozoa acquire the ability to fertilize an oocyte as they ascend the female reproductive tract. This process is characterized by a complex cascade of biophysical and biochemical changes collectively know as "capacitation." The attainment of a capacitated state is accompanied by a dramatic reorganization of the surface architecture to render spermatozoa competent to recognize the oocyte and initiate fertilization.

Effects of 4-nonylphenol and 17alpha-ethynylestradiol exposure in the Sydney rock oyster, Saccostrea glomerata: Vitellogenin induction and gonadal development.

Adult Saccostrea glomerata were exposed to environmentally relevant concentrations of 4-nonylphenol (1microg/L and 100microg/L) and 17alpha-ethynylestradiol (5ng/L and 50ng/L) in seawater over 8 weeks. Exposures were performed to assess effects on vitellogenin induction and gonadal development during reproductive conditioning. Chronic direct estrogenicity within gonadal tissue was assessed via an estrogen receptor-mediated, chemical-activated luciferase reporter gene-expression assay (ER-CALUX).

Impact of estrogenic compounds on DNA integrity in human spermatozoa: evidence for cross-linking and redox cycling activities.

A great deal of circumstantial evidence has linked DNA damage in human spermatozoa with adverse reproductive outcomes including reduced fertility and high rates of miscarriage. Although oxidative stress is thought to make a significant contribution to DNA damage in the male germ line, the factors responsible for creating this stress have not been elucidated. One group of compounds that are thought to be active in this context are the estrogens, either generated as a result of the endogenous metabolism of androgens within the male reproductive tract or gaining access to the latter as a consequence of environmental exposure.

Identification of the molecular chaperone, heat shock protein 1 (chaperonin 10), in the reproductive tract and in capacitating spermatozoa in the male mouse.

Mammalian spermatozoa must undergo epididymal maturation in the male reproductive tract and capacitation in the female tract before acquiring the ability to fertilize an oocyte. Previous studies from our laboratory have demonstrated a causal relationship between capacitation-associated surface phosphotyrosine expression and the ability of mouse spermatozoa to recognize the oocyte and engage in sperm-zona pellucida interaction. Our previous analyses of the surface phosphoproteome of capacitated murine spermatozoa identified two molecular chaperones, heat shock protein (HSP) D1 and HSP90B1, with well-characterized roles in protein folding and the assemblage of multimeric protein complexes.

Preparation of templates for DNA sequencing.

This unit contains protocols for preparing DNA suitable for use as dideoxy sequencing templates and as material for end labeling and chemical sequencing. In all protocols, the starting material contains the recombinant molecule to be sequenced. DNA from M13mp-derived phage is easily prepared and is currently the most reliable source of template for large-scale dideoxy sequencing projects.

Constructing nested deletions for use in DNA sequencing.

Nested deletions useful for dideoxy DNA sequencing are a set of deletions originating at one end of a target DNA fragment and extending various lengths along the target DNA. Each successively longer deletion brings "new" regions of the target DNA into sequencing range (about 300 bp for normal sequencing gels) of the primer site for a general discussion of nested deletions in DNA sequencing). Two protocols for generating nested subclones via enzymatic digestion are included in this unit.

Investigation of the role of SRC in capacitation-associated tyrosine phosphorylation of human spermatozoa.

The process of capacitation is a pre-requisite for mammalian spermatozoa allowing them to gain the ability to fertilize an oocyte. A fundamental part of this mechanism is a dramatic increase in the level of tyrosine phosphorylation. Implicated in this process is a unique cAMP/protein kinase A (PKA)-mediated pathway involving an intermediate PKA-activated tyrosine kinase suggested to be pp60(c-src) (SRC) in the mouse.

Regulation and action of the bacterial enhancer-binding protein AAA+ domains.

Bacterial EBPs (enhancer-binding proteins) play crucial roles in regulating cellular responses to environmental changes, in part by providing efficient control over sigma(54)-dependent gene transcription. The AAA+ (ATPase associated with various cellular activites) domain of the EBPs, when assembled into a ring, uses energy from ATP binding, hydrolysis and product release to remodel the sigma(54)-RNAP (RNA polymerase) holoenzyme so that it can transition from closed to open form at promoter DNA. The assembly, and hence activity, of these ATPases are regulated by many different signal transduction mechanisms.

Molecular and functional characterization of the rabbit epididymal secretory protein 52, REP52.

As part of a systematic study of rabbit epididymal proteins involved in sperm maturation, we have identified and characterized a novel glycoprotein (rabbit epididymal secretory protein 52 [REP52]) of 52 kDa. REP52 is synthesized and secreted in a tissue-specific manner by the mid (region 6) and distal (region 7) corpus epididymidis and associates weakly with the sperm surface overlying the principal piece of the tail. Sequencing of cloned REP52 cDNA demonstrated that this protein represents a novel member of the highly conserved fibronectin type II (FN2) module protein family.

Analysis of chaperone proteins associated with human spermatozoa during capacitation.

Mammalian spermatozoa must undergo a post-ejaculatory period of maturation, known as capacitation, before they can engage in the process of fertilization. Studies in the mouse have established that capacitation facilitates sperm-zona recognition via mechanisms that involve the appearance of tyrosine phosphorylated chaperone proteins on the sperm surface overlying the acrosome, the site of sperm-zona recognition. In this study, we examined whether a similar relationship existed between the tyrosine phosphorylation events associated with capacitation and sperm-zona interaction in human spermatozoa.