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Open Access Publications from the University of California

Scholarly Works (11 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2013)
An optofluidic microscopy platform has been developed to perform experiments in reproductive and developmental biology. This platform consists of two separate but related microscopy systems, Robolase I and Robolase IV. Specifically, this platform allows for the following. 1. Custom microfluidic chambers can be used to manipulate sperm and egg independently with automated computer controlled flow rates and valve timings (Robolase I or Robolase IV; Chapters 4-7). 2. Optical scissors can be used to damage individual organelles within an oocyte, egg, or embryo (Robolase IV; Chapter 7). 3. Individual motile sperm can be tracked in real-time (Robolase I; Chapters 2- 3, 5-6). 4. While being tracked, velocity measurements are automatically calculated and saved (Robolase I; Chapters 2 -3, 5-6). Optical tweezers can be used to manipulate sperm and automatically calculate escape power using a power decay algorithm (Robolase I; Chapters 2-3, 5-6). 6. Labeled motile sperm can be analyzed in real-time with automated single or dual channel fluorescence imaging processing (Robolase I; Chapter 2-3). Several experiments have been conducted using this platform to study sperm motility, fertilization, and early embryogenesis with applications to infertility and animal husbandry. This dissertation will describe the development of the automated optofluidic microscopy platform as well as the experiments performed
    Cover page: Development of an Automated Optofluidic Microscopy System and its Applications in Reproductive and Developmental Biology
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2012)
    Bisphenol A [BPA] is a widely dispersed environmental chemical that is of much concern because the BPA monomer is a weak transcriptional activator of human estrogen receptor α [ERα] and ERβ in cell culture. A BPA metabolite, 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene [MBP], has transcriptional activity at nM concentrations, which is 1000-fold lower than the concentration for estrogenic activity of BPA, suggesting that MBP may be an environmental estrogen. To investigate the structural basis for the activity of MBP at nM concentrations and the lower activity of BPA for human ERα and ERβ, we constructed 3D models of human ERα and ERβ with MBP and BPA for comparison with estradiol in these ERs. These 3D models suggest that MBP, but not BPA, has key contacts with amino acids in human ERα and ERβ that are important in binding of estradiol by these receptors. Metabolism of BPA to MBP increases the spacing between two phenolic rings, resulting in contacts between MBP and ERα and ERβ that mimic those of estradiol with these ERs. Mutagenesis of residues on these ERs that contact the phenolic hydroxyls will provide a test for our 3D models. Other environmental chemicals containing two appropriately spaced phenolic rings and an aliphatic spacer instead of an estrogenic B and C ring also may bind to ERα or ERβ and interfere with normal estrogen physiology. This analysis also may be useful in designing novel chemicals for regulating the actions of human ERα and ERβ.
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2009)

      Background

      Lamprey, basal vertebrate, is an important model system for understanding early events in vertebrate evolution. Lamprey contains orthologs of the estrogen receptor [ER], progesterone receptor and corticoid receptor. A perplexing property of lamprey is that 15α-hydroxy-steroids are active steroids. For example, 15α-hydroxy-estradiol [15α-OH-E2] is the estrogen, instead of estradiol [E2]. To investigate how 15α-OH-E2 binds lamprey ER, we constructed a 3D model of the lamprey ER with E2 and 15α-OH-E2.

      Methodology

      We used the 3D structure of human ERα as a template to construct a 3D model of lamprey ER. E2 and 15α-OH-E2 were inserted into the 3D model of lamprey ER and 15α-OH-E2 was inserted into human ERα. Then the each steroid-protein complex was refined using Discover 3 from Insight II software. To determine if lamprey ER had some regions that were unique among vertebrate ERs, we used the ligand-binding domain of lamprey ER as a query for a BLAST search of GenBank.

      Principal Findings

      Our 3D model of lamprey ER with 15α-OH-E2 shows that Sδ on Met-409 can form a hydrogen bond with the 15α-hydroxyl on 15α-OH-E2. In human ERα, the corresponding residue Ile-424 has a van der Waals contact with 15α-OH-E2. BLAST analysis of GenBank indicates that among vertebrate ERs, only lamprey ER contains a methionine at this position. Thus, the contact between Sδ on Met-409 and 15α-OH-E2 is unique. Interestingly, BLAST finds that five New World monkeys and a sturgeon contain a valine instead of isoleucine.

      Significance

      In addition to shedding light on the structure of the ER in a basal vertebrate, our 3D model of lamprey ER should prove useful in virtual screening of chemical libraries to identify compounds for controlling reproduction in sea lamprey, an environmental pest in Lake Michigan.

        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2007)

        Background: The glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) evolved from a common ancestor. Still not completely understood is how specificity for glucocorticoids ( e. g. cortisol) and mineralocorticoids ( e. g. aldosterone) evolved in these receptors. Results: Our analysis of several vertebrate GRs and MRs in the context of 3D structures of human GR and MR indicates that with the exception of skate GR, a cartilaginous fish, there is a deletion in all GRs, at the position corresponding to Ser-949 in human MR. This deletion occurs in a loop before helix 12, which contains the activation function 2 (AF2) domain, which binds coactivator proteins and influences transcriptional activity of steroids. Unexpectedly, we find that His-950 in human MR, which is conserved in the MR in chimpanzee, orangutan and macaque, is glutamine in all teleost and land vertebrate MRs, including New World monkeys and prosimians. Conclusion: Evolution of differences in the responses of the GR and MR to corticosteroids involved deletion in the GR of a residue corresponding to Ser-949 in human MR. A mutation corresponding to His-950 in human MR may have been important in physiological changes associated with emergence of Old World monkeys from prosimians.

          Cover page: Structural analysis of the evolution of steroid specificity in the mineralocorticoid and glucocorticoid receptors
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (2011)
          The purpose of this study is to investigate how the mitochondrial membrane potential affects sperm motility using laser tweezers and a non-ratiometric fluorescent probe, DiOC6(3). A 1064nm Nd:YVO4 continuous wave laser was used to trap motile sperm at a power of 450mW in the trap spot. Using customized tracking software, the curvilinear velocity (VCL) and the escape force from the laser tweezers weremeasured. Human (Homo sapiens), dog (Canis lupis familiaris) and drill (Mandrillus leucophaeus) sperm were treated with DiOC6(3) to measure the membrane potential in the mitochondria-rich sperm midpieces. Sperm from all three species exhibited an increase in fluorescence when treated with the DiOC6(3). When a cyanide inhibitor (CCCP) of aerobic respiration was applied, sperm of all three species exhibited a reduction in fluorescence to pre-dye levels. With respect to VCL and escape force, the CCCP had no effect on dog or human sperm, suggesting a major reliance upon anaerobic respiration (glycolysis) for ATP in these two species. Based on the preliminary study on drill sperm, CCCP caused a drop in the VCL, suggesting potential reliance on both glycolysis and aerobic respiration for motility. The results demonstrate that optical trapping in combination with DiOC 6(3) is an effective way to study sperm motility and energetics. © 2011 IOP Publishing Ltd.
            Cover page: Optical tweezers and non-ratiometric fluorescent-dye-based studies of respiration in sperm mitochondriaCreative Commons 'BY' version 4.0 license
            • Article
            • Peer Reviewed
            UC Irvine Previously Published Works (2008)
            An integrated robotic laser and microscope system has been developed to automatically analyze individual sperm motility and energetics. The custom-designed optical system directs near-infrared laser light into an inverted microscope to create a single-point 3-D gradient laser trap at the focal spot of the microscope objective. A two-level computer structure is described that quantifies the sperm motility (in terms of swimming speed and swimming force) and energetics (measuring mid-piece membrane potential) using real-time tracking (done by the upper-level system) and fluorescent ratio imaging (done by the lower-level system). The communication between these two systems is achieved by a gigabit network. The custom-built image processing algorithm identifies the sperm swimming trajectory in real-time using phase contrast images, and then subsequently traps the sperm by automatically moving the microscope stage to relocate the sperm to the laser trap focal plane. Once the sperm is stably trapped (determined by the algorithm), the algorithm can also gradually reduce the laser power by rotating the polarizer in the laser path to measure the trapping power at which the sperm is capable of escaping the trap. To monitor the membrane potential of the mitochondria located in a sperm's mid-piece, the sperm is treated with a ratiometrically-encoded fluorescent probe. The proposed algorithm can relocate the sperm to the center of the ratio imaging camera and the average ratio value can be measured in real-time. The three parameters, sperm escape power, sperm swimming speed and ratio values of the mid-piece membrane potential of individual sperm can be compared with respect to time. This two-level automatic system to study individual sperm motility and energetics has not only increased experimental throughput by an order of magnitude but also has allowed us to monitor sperm energetics prior to and after exposure to the laser trap. This system should have application in both the human fertility clinic and in animal husbandry.
              Cover page: An automatic system to study sperm motility and energeticsCreative Commons 'BY' version 4.0 license
              • Article
              • Peer Reviewed
              UC Irvine Previously Published Works (2006)
              We have developed a microscope system for real-time single sperm tracking with an automated laser tweezers escape power assay. Phase contrast images of swimming sperm are digitized to the computer at video rate. The custom algorithm creates a region of interest centered about a sperm in response to a mouse click and performs all subsequent tasks autonomously. Microscope stage movement responds to feedback from video analysis of swimming sperm to center the sperm with respect to the field of view. For escape power assays, sperm are automatically relocated to the laser trap focus where they are held for a user-defined duration at fixed power, or held as laser power is gradually reduced. The sperm's position is automatically monitored to measure the laser power at which the sperm escapes the trap. Sperm are tracked for extended durations before and after laser trap experiments. Motility measurements including the curvilinear velocity and the absolute position of the sperm relative to the cell chamber are calculated and written to the hard drive at video rate. Experimental throughput is increased over 30 times compared to off-line data analysis. The efficacy of the "track and trap" algorithm is validated through examples and comparisons with the manually collected data.
                Cover page: Real‐time automated tracking and trapping system for spermCreative Commons 'BY' version 4.0 license
                • Article
                • Peer Reviewed
                UC Irvine Previously Published Works (2008)
                We combine laser tweezers with custom computer tracking software and robotics to analyze the motility [swimming speed, VCL (curvilinear velocity), and swimming force in terms of escape laser power (Pesc)] and energetics [mitochondrial membrane potential (MP)] of individual sperm. Domestic dog sperm are labeled with a cationic fluorescent probe, DiOC2(3), that reports the MP across the inner membrane of the mitochondria located in the sperm's midpiece. Individual sperm are tracked to calculate VCL. Pesc is measured by reducing the laser power after the sperm is trapped using laser tweezers until the sperm is capable of escaping the trap. The MP is measured every second over a 5-s interval during the tracking phase (sperm is swimming freely) and continuously during the trapping phase. The effect of the fluorescent probe on sperm motility is addressed. The sensitivity of the probe is measured by assessing the effects of a mitochondrial uncoupling agent (CCCP) on MP of free swimming sperm. The effects of prolonged exposed to the laser tweezers on VCL and MP are analyzed. The system's capabilities are demonstrated by measuring VCL, Pesc, and MP simultaneously for individual sperm. This combination of imaging tools is useful to quantitatively assess sperm quality and viability.
                  Cover page: Use of laser tweezers to analyze sperm motility and mitochondrial membrane potentialCreative Commons 'BY' version 4.0 license
                  • Article
                  • Peer Reviewed
                  UC Irvine Previously Published Works (2008)
                  The combination of laser tweezers, fluorescent imaging, and real-time automated tracking and trapping (RATTS) can measure sperm swimming speed and swimming force simultaneously with mitochondrial membrane potential (MMP). This approach is used to study the roles of two sources of ATP in sperm motility: oxidative phosphorylation, which occurs in the mitochondria located in the sperm midpiece and glycolysis, which occurs along the length of the sperm tail (flagellum). The relationships between (a) swimming speed and MMP and (b) swimming force and MMP are studied in dog and human sperm. The effects of glucose, oxidative phosphorylation inhibitors and glycolytic inhibitors on human sperm motility are examined. The results indicate that oxidative phosphorylation does contribute some ATP for human sperm motility, but not enough to sustain high motility. The glycolytic pathway is shown to be a primary source of energy for human sperm motility.
                    Cover page: Comparison of glycolysis and oxidative phosphorylation as energy sources for mammalian sperm motility, using the combination of fluorescence imaging, laser tweezers, and real‐time automated tracking and trappingCreative Commons 'BY' version 4.0 license
                    • Article
                    • Peer Reviewed
                    UC Irvine Previously Published Works (2006)
                    Sperm cells from a domestic dog were treated with oxacarbocyanine DiOC2(3), a ratiometrically-encoded membrane potential fluorescent probe in order to monitor the mitochondria stored in an individual sperm's midpiece. This dye normally emits a red fluorescence near 610 nm as well as a green fluorescence near 515 nm. The ratio of red to green fluorescence provides a substantially accurate and precise measurement of sperm midpiece membrane potential. A two-level computer system has been developed to quantify the motility and energetics of sperm using video rate tracking, automated laser trapping (done by the upper-level system) and fluorescent imaging (done by the lower-level system). The communication between these two systems is achieved by a networked gigabit TCP/IP cat5e crossover connection. This allows for the curvilinear velocity (VCL) and ratio of the red to green fluorescent images of individual sperm to be written to the hard drive at video rates. This two-level automatic system has increased experimental throughput over our previous single-level system (Mei et al., 2005) by an order of magnitude.
                      Cover page: A real-time single sperm tracking, laser trapping, and ratiometric fluorescent imaging systemCreative Commons 'BY' version 4.0 license
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