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Biomedical subjects

Gary D Smith

Publications and source records attributed to Gary D Smith.

28 records · Page 2Linked to original sources
PubMed2003

Ultra-rapid freezing of very low numbers of sperm using cryoloops.

BACKGROUND: With the availability of ICSI, men with severe oligozoospermia (<5x10(6)/ml) are able to reproduce. Current methods for cryopreservation of severe oligozoospermic samples are labour intensive and costly. The objective of this study was to evaluate whether freezing small numbers of motile sperm (approximately 100) was feasible using cryoloops. METHODS: Initial tests assessed the effect of various dilutions of cryoprotectants on pre-freezing sperm motility. Several solutions were further evaluated for their ability to cryoprotect sperm during ultra-rapid freezing. Sperm were placed on cryoloops and held in liquid nitrogen vapour for 5 min prior to freezing (ultra-rapid freezing) or directly submerged into liquid nitrogen. Using the optimal cryoprotectant and technique from these experiments, ultra-rapid and standard slow-rate freezing protocols were compared. RESULTS: Optimal sperm survival was seen when sperm in cryoloops were placed in liquid nitrogen vapour in test yolk buffer with 12% v/v glycerol versus other cryoprotectants. Using this cryoprotectant, post-thaw sperm motility is comparable between ultra-rapid and slow-rate freezing methods. CONCLUSION: Ultra-rapid freezing of very low numbers of sperm is feasible using cryoloops suspended in liquid nitrogen vapour for 5 min.

Cell Survival↗
PubMed2002

Clinical phenotype and infertility treatment in a male with hypogonadotropic hypogonadism due to mutations Ala129Asp/Arg262Gln of the gonadotropin-releasing hormone receptor.

OBJECTIVE: To characterize the genotype and phenotype of a man with idiopathic hypogonadism with infertility. DESIGN: Molecular analysis and clinical description. SETTING: Medical school laboratory and reproductive endocrine clinic.A 40-year-old male with idiopathic hypogonadotropic hypogonadism. INTERVENTION(S): Denaturing gradient gel electrophoresis analysis and DNA sequencing of the gonadotropin-releasing hormone receptor (GNRHR) gene were performed. The patient was treated with hCG and FSH. MAIN OUTCOME MEASURE(S): GNRHR mutation detection, genotype/phenotype correlation, and testicular response to exogenous gonadotropin therapy. RESULT(S): The proband demonstrated compound heterozygosity for Ala129Asp/Arg262Gln GNRHR mutations. He had a complete form of idiopathic hypogonadotropic hypogonadism, with descended testes and severe oligospermia but little response to exogenous gonadotropins. CONCLUSION(S): The phenotype of this patient differs from the one other family described with the same mutations. Exogenous gonadotropin therapy may not be as beneficial for increasing sperm concentration in older men with idiopathic hypogonadotropic hypogonadism.

Adult↗
PubMed2002

Processed total motile sperm count correlates with pregnancy outcome after intrauterine insemination.

OBJECTIVES: To determine the impact of processed total motile sperm (PTMS) count on pregnancy after partner intrauterine insemination (IUI). IUI is generally attempted before proceeding to more expensive and invasive assisted-reproductive techniques such as intracytoplasmic sperm injection. Several semen parameters have been shown to correlate with IUI outcome and may be useful when counseling couples. METHODS: Four hundred thirty-eight couples with diverse causes of infertility underwent 1114 cycles of husband IUI during a 39-month period. The clinical and semen parameters were recorded for each couple and each insemination. The parameters were compared between those couples who achieved a pregnancy and those who did not. RESULTS: The total number of pregnancies was 120, resulting in a pregnancy rate per cycle of 10.8% and a couple pregnancy rate of 27.4%. On multivariable logistic regression analysis, the PTMS count was independently associated with fertility after IUI (P = 0.0014). Moreover, the pregnancy rate was significantly lower for couples with less than 10 million PTMS (P <0.05). CONCLUSIONS: The results of this study have demonstrated that the PTMS count independently predicts success with IUI. Cycles with less than 10 million total motile sperm are significantly less likely to result in a pregnancy. If cause-specific therapy has failed, alternatives to IUI should be considered for couples when the PTMS count is less than 10 million.

Adult↗
PubMed2002

Regulation of spindle formation by active mitogen-activated protein kinase and protein phosphatase 2A during mouse oocyte meiosis.

Mitogen-activated protein kinase (MAPK) and protein phosphatase 2A (PP2A) regulate oocyte meiosis, yet little is known regarding their mechanisms of action. This study addressed the functional importance of active MAPK and PP2A in regulating oocyte meiosis. Experiments were conducted to identify MAPK activation, PP2A activity, intracellular enzyme trafficking, and ultrastructural associations during meiosis. Questions of requisite kinase and/or phosphatase activity and chromatin condensation, microtubule polymerization, and spindle formation were addressed. At the protein level, MAPK and PP2A were present in constant amounts throughout the first meiotic division. Both MAPK and PP2A were activated following germinal vesicle breakdown (GVBD) in conjunction with metaphase I development. Immunocytochemical studies confirmed the absence of active MAPK in germinal vesicle-intact (GVI) and GVBD oocytes. At metaphase I and during the metaphase I/metaphase II transition, activated MAPK colocalized with microtubules, poles, and plates of meiotic spindles. Protein phosphatase 2A was dispersed evenly throughout the GVI oocyte cytoplasm. Throughout the metaphase I/metaphase II transition, PP2A colocalized with microtubules of meiotic spindles. Both active MAPK and PP2A associated with in vitro-polymerized microtubules, suggesting that active MAPK and PP2A locally regulate spindle formation. Inhibition of MAPK activation resulted in compromised microtubule polymerization, no spindle formation, and loosely condensed chromosomes. Treatment with okadaic acid (OA) or calyculin-A (CL-A), which inhibits oocyte cytoplasmic PP2A, caused an absence of microtubule polymerization and spindles, even though MAPK activity was increased under these treatment conditions. Thus, active MAPK is required, but is not sufficient, for normal meiotic spindle formation and chromosome condensation. In addition, the oocyte OA/CL-A-sensitive PP, presumably PP2A, is essential for microtubule polymerization and meiotic spindle formation.

Animals↗
PubMed2002

Activation of protein kinase C induces mitogen-activated protein kinase dephosphorylation and pronucleus formation in rat oocytes.

Mammalian oocytes are arrested at metaphase of the second meiotic division (MII) before fertilization. When oocytes are stimulated by spermatozoa, they exit MII stage and complete meiosis. It has been suggested that an immediate increase in intracellular free calcium concentration and inactivation of maturation promoting factor (MPF) are required for oocyte activation. However, the underlying mechanism is still unclear. In the present study, we investigated the role of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase, and their interplay in rat oocyte activation. We found that MAP kinase became dephosphorylated in correlation with pronucleus formation after fertilization. Protein kinase C activators, phorbol 12-myriatate 13-acetate (PMA) and 1,2-dioctanoyl-rac-glycerol (diC8), triggered dephosphorylation of MAP kinase and pronucleus formation in a dose-dependent and time-dependent manner. Dephosphorylation of MAP kinase was also correlated with pronucleus formation when oocytes were treated with PKC activators. Effects of PKC activators were abolished by the PKC inhibitors, calphostin C and staurosporine, as well as a protein phosphatase blocker, okadaic acid (OA). These results suggest that PKC activation may cause rat oocyte pronucleus formation via MAP kinase dephosphorylation, which is probably mediated by OA-sensitive protein phosphatases. We also provide evidence supporting the involvement of such a process in fertilization.

Animals↗
PubMedDate not supplied

Isolation of motile spermatozoa from semen samples using microfluidics.

A microfluidic device was designed with two parallel laminar flow channels where non-motile spermatozoa and debris would flow along their initial streamlines and exit one outlet, whereas motile spermatozoa had an opportunity to swim into a parallel stream and exit a separate outlet. Motile sperm samples were prepared with density gradient separation (n = 5). Sperm motility was assessed the following day after exposing aliquots to polydimethylsiloxane (PDMS) used to construct the device. There was no difference in sperm motility when compared with unexposed aliquots (P > 0.05). Unprocessed semen samples (n = 10) were placed in wider channels and sperm motility and strict morphology were assessed from sorted outlets. Sperm motility increased from 44 +/- 4.5% to 98 +/- 0.4% (P < 0.05) and morphology increased from 10 +/- 1.05% to 22 +/- 3.3% (P < 0.05) following processing. Finally, density gradient prepared samples (n = 6) containing 5 x 10(6) motile spermatozoa/ml and 50 x 10(6) round immature germ cells/ml were sorted and assessed in a similar fashion. The ratio of motile spermatozoa to round immature germ cells in the wide inlet (1:10) was significantly improved in the thin outlet (33:1) (P < 0.05). This microfluidic device provides a novel method for isolating motile, morphologically normal spermatozoa from semen samples without centrifugation. This technology may prove useful in isolating motile spermatozoa from oligozoospermic samples, even with high amounts of non-motile gamete and/or non-gamete cell contamination. A movie sequence showing streaming and sorting of spermatozoa may be purchased for viewing on the internet at www.rbmonline.com/Article/847 (free to web subscribers).

Cell Separation↗
PubMedDate not supplied

Rethinking gamete/embryo isolation and culture with microfluidics.

IVF remains one of the most exciting modern scientific developments and continues to have a tremendous impact on people's lives. Since its beginnings, scientists have studied and critically analysed the techniques in order to find ways to improve outcomes; however, little has changed with the actual technology and equipment of IVF. Semen is still processed in test tubes and fertilization and culture still occurs in culture dishes. New technological possibilities exist with the burgeoning advancement of microfluidic technology. Microfluidics is based on the behaviour of liquids in a microenvironment. Although a young field, many developments have occurred which demonstrate the potential of this technology for IVF. In this review, we briefly discuss the physical principles of microfluidics and highlight some previous utilizations of this technology, ranging from chemical analysis to cell sorting. We then present the designs and outcomes for microfluidic devices utilized thus far for each step in IVF: gamete isolation and processing, fertilization, and embryo culture. Finally, we discuss and speculate on the ultimate goal of this technology--development of a single, integrated unit for in-vitro assisted reproduction techniques.

Cell Culture Techniques↗