Biomedical subjects

Eileen A McLaughlin

Publications and source records attributed to Eileen A McLaughlin.

17 recordsLinked to original sources

PubMed2006

Cis-unsaturated fatty acids stimulate reactive oxygen species generation and lipid peroxidation in human spermatozoa.

CONTEXT: Defective sperm function is the largest defined cause of human infertility; however, the etiology of this condition is poorly understood. Although oxidative stress is acknowledged as a key contributor to this pathology, there are also data indicating that defective human spermatozoa contain abnormally high amounts of cis-unsaturated fatty acids. This study investigated whether a causative relationship exists between these two attributes of impaired semen quality. OBJECTIVE: The objective of this study was to determine whether polyunsaturated fatty acids can induce oxidative stress in human spermatozoa. METHOD: Dihydroethidium and SYTOX Green were used in conjunction with flow cytometry and HPLC to investigate reactive oxygen species (ROS) generation by human spermatozoa after fatty acid exposure. RESULTS: Arachidonic acid (AA) induced a time- and dose-dependent increase in ROS generation by human spermatozoa that led to the promotion of peroxidative damage and a loss of sperm motility. This effect could not be blocked with inhibitors of the cyclooxygenase or lipoxygenase pathways of AA metabolism, rotenone, protein kinase C antagonists, or known inhibitors of plasma membrane redox systems. However, ROS generation could be triggered with other cis-unsaturated fatty acids including linoleic and docosahexaenoic acids. Saturated fatty acids, methyl esters of unsaturated fatty acids, or other amphiphiles were all ineffective. However in a cell-free system, AA could trigger a redox signal via mechanisms that were profoundly disrupted by diphenylene iodonium, a flavoprotein inhibitor. CONCLUSIONS: The presence of excess unsaturated fatty acids in defective human spermatozoa may precipitate the oxidative stress encountered in male infertility.

Arachidonic Acid↗

PubMed2006

Primordial follicle activation and follicular development in the juvenile rabbit ovary.

Of all the stages of mammalian folliculogenesis, the primordial to primary follicle transition is the least understood. In order to gain new insights into this process, we have conducted a comprehensive morphological, morphometric and molecular study of ovarian organisation and early follicle development in the rabbit. The structure of ovaries collected from rabbits aged from 2-12 weeks (a period encompassing primordial follicle formation, activation and the first wave of folliculogenesis in this species) has been analysed by light microscopy and the follicles present have been measured and scored for their developmental stage. To establish useful molecular markers of activation, we have further classified follicles according to their expression of the proliferative marker, proliferating cell nuclear antigen, and the zona pellucida protein, ZPB. The activation of primordial follicles is initiated immediately following their formation in the rabbit ovary and is characterised by oocyte growth, granulosa cell morphogenesis and increased granulosa cell mitosis. Enhanced ZPB protein expression at the oolemma is also associated with follicle activation and development. Few primordial follicles in the juvenile rabbit ovary are lost by atresia, as assessed by the TUNEL assay. The appearance of apoptotic granulosa cells is however coincident with the development of antral follicles. This study thus describes the temporal and spatial regulation of early follicular development in the post-natal rabbit ovary and, for the first time, shows that the primordial to primary transition in the juvenile rabbit is a highly ordered process occurring within quantifiable parameters.

Animals↗

PubMed2006

KIT/KIT ligand in mammalian oogenesis and folliculogenesis: roles in rabbit and murine ovarian follicle activation and oocyte growth.

In rodent ovaries Kit ligand (KITL) and its receptor KIT have diverse roles, including the promotion of primordial follicle activation, oocyte growth, and follicle survival. Studies were undertaken to determine whether KITL and KIT carry out similar activities in rabbits. KitlandKitmRNA and protein were localized to oocytes and granulosa cells, respectively, in the rabbit ovary. Ovarian cortical explants from juvenile rabbits and neonatal mouse ovaries were subsequently cultured with recombinant mouse KITL and/or KITL neutralizing antibody. Indices of follicle growth initiation were compared with controls and between treatment groups for each species. Recombinant mouse KITL had no stimulatory effect on primordial follicle recruitment in cultured rabbit ovarian explants. However, the mean diameter of oocytes from primordial, early primary, primary, and growing primary follicles increased significantly in recombinant mouse KITL-treated explants compared with untreated tissues. In contrast, recombinant mouse KITL promoted both primordial follicle activation and an increase in the diameter of oocytes from primordial and early primary follicles in the mouse, and these effects were inhibited by coculture with KITL-neutralizing antibody. Recombinant mouse KITL had no effect on follicle survival for either species. These data demonstrate that KITL promotes the growth of rabbit and mouse oocytes and stimulates primordial follicle activation in the mouse but not in the rabbit. We propose that the physiologic roles of KITL and KIT may differ between species, and this has important implications for the design of in vitro culture systems for folliculogenesis in mammals, including the human.

Animals↗

PubMed2006

The RNA-binding protein Musashi is required intrinsically to maintain stem cell identity.

A key goal of regenerative medicine is an understanding of the genetic factors that define the properties of stem cells. However, stem cell research in mammalian tissue has been hampered by a paucity of stem cell-specific markers. Although increasing evidence suggests that members of the Musashi (Msi) family of RNA-binding proteins play important functions in progenitor cells, it remains unclear whether there is a stem cell-autonomous requirement for Msi because of an inability to distinguish stem cells from early-lineage cells in mammalian tissues. Here, using the Drosophila testis as a model system for the study of stem cell regulation, we show specific evidence for a cell-autonomous requirement for Msi family proteins in regulating stem cell differentiation, leading to the identification of an RNA-binding protein required for spermatogonial stem cell maintenance. We found that loss of Msi function disrupts the balance between germ-line stem cell renewal and differentiation, resulting in the premature differentiation of germ-line stem cells. Moreover, we found that, although Msi is expressed in both somatic and germ cells, Msi function is required intrinsically in stem cells for maintenance of stem cell identity. We also discovered a requirement for Msi function in male meiosis, revealing that Msi has distinct roles at different stages of germ cell differentiation. We describe the complementary expression patterns of the murine Msi paralogues Msi1 and Msi2 during spermatogenesis, which support the idea of distinct, evolutionarily conserved roles of Msi.

Animals↗

PubMed2006

CXCR4/SDF1 interaction inhibits the primordial to primary follicle transition in the neonatal mouse ovary.

The molecular mechanisms behind the entry of the primordial follicle into the growing follicle pool remain poorly understood. To investigate this process further, a microarray-based comparison was undertaken between 2-day postpartum mouse ovaries consisting of primordial follicles/naked oocytes only and those with both primordial follicles and newly activated follicles (7-day postpartum). Gene candidates identified included the chemoattractive cytokine stromal derived factor-1 (SDF1) and its receptor CXCR4. SDF1 and CXCR4 have been implicated in a variety of physiological processes including the migration of embryonic germ cells to the gonads. SDF1-alpha expression increased with the developmental stage of the follicle. Embryonic expression was found to be dichotomous post-germ cell migration, with low expression in the female. Immunohistochemical studies nonetheless indicate that the autocrine pattern of expression ligand and receptor begins during embryonic life. Addition of recombinant SDF1-alpha to neonatal mouse ovaries in vitro resulted in significantly higher follicle densities than for control ovaries. TUNEL analysis indicated no detectable difference in populations of apoptotic cells of treated or control ovaries. Treated ovaries also contained a significantly lower percentage of activated follicles as determined by measurement of oocyte diameter and morphological analysis. Treatment of cultured ovaries with an inhibitor of SDF1-alpha, AMD3100, ablated the effect of SDF1-alpha. By retaining follicles in an unactivated state, SDF1/CXCR4 signaling may play an important role in maintaining the size and longevity of the primordial follicle pool.

Animals↗

PubMed2006

Definitive evidence for the nonmitochondrial production of superoxide anion by human spermatozoa.

CONTEXT: Oxidative stress in the male germ line has been associated with poor fertility, impaired embryonic development, miscarriage, and childhood disease. Such stress is known to be associated with the peroxidation of unsaturated fatty acids in the sperm plasma membrane and oxidative DNA damage to both the nuclear and mitochondrial genomes. However, the source of the free radicals responsible for such damage is still unresolved. OBJECTIVE: The objective of this study was to chemically validate the use of dihydroethidium (DHE) as a probe for detecting the generation of superoxide anion by human spermatozoa and to examine the relationship between this activity and defective sperm function. METHOD: DHE and SYTOX green were used in conjunction with flow cytometry and HPLC to investigate superoxide generation by human spermatozoa. Cause and effect relationships were established using menadione to artificially drive superoxide production by these cells. RESULTS: HPLC, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and spectrofluorometry were used to demonstrate that human spermatozoa generate the superoxide-specific product, 2-hydroxyethidium, from DHE. Spontaneous superoxide production by human spermatozoa was found to originate from a nonmitochondrial source and was inversely correlated with sperm motility. A causative relationship between superoxide generation and sperm function was demonstrated when the pharmacological stimulation of this activity with menadione was shown to result in both severe motility loss and DNA damage. CONCLUSIONS: These studies validate a methodology for investigating the origins of oxidative stress in the male germ line and demonstrate, for the first time, the significance of superoxide generation by human spermatozoa in the etiology of this condition.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗

PubMed2006

Identification and characterization of a novel Mt-retrotransposon highly represented in the female mouse germline.

The control of primordial follicle recruitment into the growing follicle population is a major limiting process in female reproduction. In order to gain insight into the molecular processes occurring at the time of primordial follicle activation, a subtractive hybridization analysis was performed between cDNAs prepared from temporally distinct mouse neonatal ovarian tissues that differed according to the state of primordial follicle activation. One highly represented clone associated with activation was an Mt retrotransposon-like sequence designated Mtfull, which was subsequently cloned and determined to be novel and restricted in expression to the ovary. The polyadenylated 1684-bp sequence has long terminal repeats, is predicted to be noncoding, and is the predominant Mti-related sequence present in the mouse ovary. In situ hybridization further localized Mtfull expression to the oocyte and confirmed that expression is concomitant with follicle activation. Together with in silico data, we predict Mtfull plays an essential role in folliculogenesis through regulation of gene expression.

3' Untranslated Regions↗

PubMed2006

Expression of c-Kit receptor mRNA and protein in the developing, adult and irradiated rodent testis.

Germ cell proliferation, migration and survival during all stages of spermatogenesis are affected by stem cell factor signalling through the c-Kit receptor, the expression and function of which are vital for normal male reproductive function. The present study comprehensively describes the c-Kit mRNA and protein cellular expression profiles in germ cells of the postnatal and adult rodent testis, revealing their significant elevation in synthesis at the onset of spermatogenesis. Real-time PCR analysis for both mice and rats matched the cellular mRNA expression profile where examined. Localization studies in normal mouse testes indicated that both c-Kit mRNA and protein are first detectable in differentiating spermatogonia. In addition, all spermatogonia isolated from 8-day-old mice displayed detectable c-Kit mRNA, but 30-50% of these lacked protein expression. The c-Kit mRNA and protein profile in normal rat testes indicated expression in gonocytes, in addition to differentiating spermatogonia. However, in the irradiated adult rat testes, in which undifferentiated spermatogonia are the only germ cell type, mRNA was also detected in the absence of protein. This persisted at 3 days and 1 and 2 weeks following treatment with gonadotrophin-releasing hormone (GnRH) antagonist to stimulate spermatogenesis recovery. By 4 weeks of GnRH antagonist treatment, accompanying the emergence of differentiating spermatogonia, both mRNA and protein were detected. Based on these observations, we propose that c-Kit mRNA and protein synthesis are regulated separately, possibly by influences linked to testis maturation and circulating hormone levels.

Animals↗

PubMed2005

Characterization of structure and expression of the Dzip1 gene in the rat and mouse.

A transcript encoding a rat homologue of DZIP1 (DAZ-interacting protein) was isolated from testis RNA. Like human DZIP1, it contains a C(2)H(2) zinc finger domain. A predicted mouse homologue of DZIP1 was found in the GenBank database. Genome analysis indicated that while DZIP1 and mouse Dzip1 contain 22 and 20 exons, respectively, the rat sequence was intronless, confirmed by PCR on genomic DNA. This rat Dzip1 sequence is homologous to mouse Dzip1 exons 1-6 and DZIP1 exons 5-9. As this rat sequence was shorter than DZIP1 it was designated rat Dzip1S. The rat genome also contained a further predicted homologue of DZIP1 displaying conserved linkage homology with mouse Dzip1 and DZIP1. This sequence, if expressed, is the true rat homologue of DZIP1, designated rat Dzip1. Rat Dzip1S mRNA was present in all tissues examined by qualitative RT-RCR, and in situ hybridization of rat testis confirmed that expression of rat Dzip1S mRNA was confined to the spermatogenic lineage, specifically premeiotic spermatogonia.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Localization and significance of molecular chaperones, heat shock protein 1, and tumor rejection antigen gp96 in the male reproductive tract and during capacitation and acrosome reaction.

Although the molecular basis of sperm-oocyte interaction is unclear, recent studies have implicated two chaperone proteins, heat shock protein 1 (HSPD1; previously known as heat shock protein 60) and tumor rejection antigen gp96 (TRA1; previously known as endoplasmin), in the formation of a functional zona-receptor complex on the surface of mammalian spermatozoa. The current study was undertaken to investigate the expression of these chaperones during the ontogeny of male germ cells through spermatogenesis, epididymal sperm maturation, capacitation, and acrosomal exocytosis. In testicular sections, both HSPD1 and TRA1 were closely associated with the mitochondria of spermatogonia and primary spermatocytes. However, this labeling pattern disappeared from the male germ line during spermiogenesis to become undetectable in testicular spermatozoa. Subsequently, these chaperones could be detected in epididymal spermatozoa and in previously unreported "dense bodies" in the epididymal lumen. The latter appeared in the precise region of the epididymis (proximal corpus), where spermatozoa acquire the capacity to recognize and bind to the zona pellucida, implicating these structures in the functional remodeling of the sperm surface during epididymal maturation. Both HSPD1 and TRA1 were subsequently found to become coexpressed on the surface of live mouse spermatozoa following capacitation in vitro and were lost once these cells had undergone the acrosome reaction, as would be expected of cell surface molecules involved in sperm-egg interaction. These data reinforce the notion that these chaperones are intimately involved in the mechanisms by which mammalian spermatozoa both acquire and express their ability to recognize the zona pellucida.

Acrosome Reaction↗

PubMed2004

Tyrosine phosphorylation activates surface chaperones facilitating sperm-zona recognition.

Mammalian spermatozoa undergo a series of molecular and biochemical changes collectively termed capacitation prior to acquiring the ability to fertilise the oocyte. Although phosphorylation of sperm proteins on tyrosine residues has been recognised as an important component of this process, the precise relationship between the phosphorylation status of mammalian spermatozoa and their capacity for fertilisation has remained unclear. In this study we demonstrate a causal relationship between tyrosine phosphorylation in spermatozoa and sperm-zona interaction. The phosphotyrosine expression associated with sperm capacitation localised to internal flagellar structures in permeabilised cells but could also be detected on the exterior surface of the sperm head in live cells. Importantly, almost all spermatozoa bound to the zona pellucida demonstrated this pattern of phosphoprotein localisation, compared to fewer than 15% of the free-swimming population. These data suggest that tyrosine phosphorylation plays a significant role in remodelling the sperm surface, so that these cells are able to recognise the zona pellucida. Phosphoproteome analysis yielded the first evidence of molecular chaperones, endoplasmin (erp99) and heat shock protein 60 (hsp60), as targets for phosphorylation on the surface of mouse spermatozoa, whereas immunofluorescence localised these proteins to the precise region of the sperm head that participates in zona recognition. Based on these results, we propose a novel mechanism for mammalian gamete interaction whereby the activation of sperm-surface chaperones by tyrosine phosphorylation during capacitation may trigger conformational changes facilitating the formation of a functional zona pellucida receptor complex on the surface of mammalian spermatozoa.

Animals↗

PubMed2004

Biosynthesis of the canine zona pellucida requires the integrated participation of both oocytes and granulosa cells.

In the dog, attempts to localize the expression of zona pellucida (ZP) proteins during folliculogenesis have failed to demonstrate conclusively whether any or all of the zona proteins are synthesized in the oocyte or the granulosa cells. Probing of paraformaldehyde-fixed prepubertal canine ovarian tissue sections with a panel of fluorescently conjugated lectins localized the expression of glycoproteins during folliculogenesis. We confirm that six lectins (PSA, s-WGA, ECL, GSL-II, LEL, and STL) consistently labeled the ZP and adjacent granulosa cells of the developing follicle and that canine ZP expresses beta-gal(1,4)glcNAc, beta-gal(1,3)galNac, alpha-mannose, and terminal sialic acid residues in a developmentally specific manner. Riboprobes for canine ZPA and ZPC genes were produced and used for in situ hybridization studies of mRNA expression in canine folliculogenesis. In addition, we isolated a partial cDNA transcript from total ovarian RNA for the canine ZPB gene having a high degree of sequence identity with the felid and porcine ZPB homologues. Subsequently, the ZPA gene transcripts were localized to the cytoplasm of oocytes in primordial, primary, and early secondary follicles. We then localized expression of ZPB and ZPC gene transcripts to the granulosa cells of growing follicles, but not in squamous granulosa cells of primordial follicles or oocytes. These observations indicate that in the juvenile canine ovary, the oocyte is responsible for synthesis of the ZPA protein and directing synthesis of the ZPB and ZPC proteins by the granulosa cells and that ZP gene transcription occurs in a sequential manner during folliculogenesis.

Age Factors↗

PubMed2004

Redox activity associated with the maturation and capacitation of mammalian spermatozoa.

As rat spermatozoa undergo epididymal maturation, they acquire the ability to exhibit a spontaneous burst of luminol-peroxidase-dependent chemiluminescence when released into a simple, defined culture medium. This activity was suppressed by inhibitors of plasma membrane redox systems such as diphenylene iodonium, p-chloromercuribenzenesulfonic acid, and capsaicin, but was resistant to inhibition by resiniferatoxin and rotenone. The luminol-peroxidase signal was dependent on the presence of bicarbonate, enhanced by the substitution of fructose for glucose, and severely suppressed by desferoxamine, superoxide dimutase, and catalase. Both L- and D-arginine were stimulatory, suggesting the involvement of *NO in this spontaneous chemiluminescence activity. The L-arginine-dependent, but not the D-arginine-dependent, activity was significantly suppressed by an inhibitor of nitric oxide synthase (N(G)-nitro-L-arginine methyl ester). L- and D-arginine could also stimulate redox activity observed in immature caput epididymal cells, but only after prolonged incubation. The inhibitory effects of uric acid and ascorbate suggested the chemiluminescence signal might be induced by peroxynitrite. This conclusion was supported by confocal imaging of the cells following treatment with 4-amino-5-methylamino-2',7'-difluorofluorescein. Stimulation or suppression of the redox activity detected by luminol-peroxidase led to corresponding changes in the ability of the spermatozoa to exhibit acrosomal exocytosis, indicating that this pathway is of fundamental biological significance.

4-Chloromercuribenzenesulfonate↗

PubMed2004

Identification of cytochrome P450-reductase as the enzyme responsible for NADPH-dependent lucigenin and tetrazolium salt reduction in rat epididymal sperm preparations.

Lucigenin-dependent chemiluminescence and WST-1 reduction can be detected following addition of NADPH to many cell types, including rat epididymal sperm suspensions. Although many reports suggest that such a phenomenon is due to reactive oxygen species production, other probes-such as MCLA and luminol-that are capable of detecting reactive oxygen metabolites do not produce a chemiluminescent signal in this model system. Our aim was to purify and identify the enzyme catalyzing the NADPH-dependent lucigenin and WST-1 reduction from rat epididymal spermatozoa preparations. Here, we show the identity of this enzyme as cytochrome P450-reductase. In support of this, a homogenous preparation of this protein was capable of reducing lucigenin and WST-1 in the presence of NADPH. Moreover, COS-7 cells overexpressing cytochrome P450-reductase displayed a 3-fold increase in the aforementioned activity compared with mock-transfected cells. Immunolocalization studies and biochemical analysis suggest that the majority of the NADPH-lucigenin activity is localized to the epithelial cells present within the epididymis. These results emphasize the importance of the direct NADPH-dependent reduction of superoxide-sensitive probes by cytochrome P450-reductase even though this enzyme does not, on its own accord, produce reactive oxygen species.

Acridines↗

PubMed2004

Identification of RARhoGAP, a novel putative RhoGAP gene expressed in male germ cells.

A gene encoding a novel RhoGAP of 1146 amino acids was isolated from rat testis RNA. Analysis of this protein identified two conserved domains, a RhoGAP domain and an RA domain. Thus the gene was named RARhoGAP. The RhoGAP domain contained conserved residues critical for RhoGAP activity, suggesting this domain is involved in the down-regulation of Rho GTPases. The presence of the RA domain suggests that RARhoGAP also functions as an effector for Ras- or Ral-like GTPases. RT-PCR analysis showed the transcript was ubiquitous in extragonadal tissues; however, Northern analysis indicated highest expression was in the testis. Homologues of rat RARhoGAP were found in mouse and human and were found expressed in testis by nested RT-PCR. In situ hybridization confirmed the specific expression of RARhoGAP in differentiating male germ cells. We postulate that RARhoGAP may be involved in rearrangements of the cytoskeleton and cell signaling events that occur during spermatogenesis.

Amino Acid Sequence↗

PubMed2003

Contraceptive vaccines.

The control of human fertility would be revolutionised by the development of a safe, effective, long-acting contraceptive vaccine. The pursuit of this objective has involved the selection of appropriate targets within the reproductive process that are amenable to interference with antibodies. To date, three major targets have been researched. The zona pellucida (ZP) plays key roles in folliculogenesis, fertilisation and early development, and is comprised of powerful cell-specific antigens. The induction of infertility requires high ZP antibody titres that are difficult to maintain without inducing ovarian pathology characterised by a premature loss of primordial follicles. As a premature menopause would be a high price to pay for long-term contraception, this approach to a vaccine cannot progress until the cause of the ovarian pathology has been resolved. Sperm surface antigens represent another promising approach to contraceptive vaccine development. While there is some clinical data to support the likely efficacy of this strategy, none of the gamete-specific molecules characterised to date have fulfilled this promise. Anti-human chorionic gonadotropin (hCG) vaccines terminate pregnancy by preventing the maternal recognition of pregnancy. This vaccine has reached the stage of clinical trials, and preliminary indications are that the approach is safe and potentially effective. However, reliability may be an issue, given the observed inter-individual variability in antibody generation. The future of contraceptive vaccine development will clearly involve a continuation of the intense search for suitable targets and the development of improved immunisation procedures that exploit the latest innovations in vaccine technology.

Adjuvants, Immunologic↗

PubMed2002

Cryopreservation, screening and storage of sperm the challenges for the twenty-first century.

The advent of HIV and the serious nature of the sequelae resulted in a major reassessment of artificial insemination practices in the UK. The development of human semen cryopreservation had enormous impact on reproductive medicine and the availability of cryopreserved quarantined donor semen became a mainstay for the treatment of male infertility in the UK. The regulation and accreditation of assisted reproductive technologies and the introduction of peer-reviewed guidelines have largely standardized clinical and laboratory practice. The introduction of assisted fertilization techniques such as intracytoplasmic sperm injection, testicular sperm retrieval and improved oncology treatments have placed pressure on reproductive biologists and cryobiologists to design and use cryopreservation protocols for the optimum survival of sperm.

Cryopreservation↗