Construction and Preliminary Characterization of a Series of Mouse and Rat Testis cDNA Libraries

We have constructed a series of 23 cDNA libraries from mouse and rat testicular cells. These include libraries made from whole, intact adult testes; seminiferous tubule cells from adult testes; combined populations of primary spermatocytes from 18‐day‐old mouse testes; and isolated populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene spermatocytes, leptotene plus zygotene spermatocytes, juvenile pachytene spermatocytes, adult pachytene spermatocytes, round spermatids, Sertoli cells from 6‐, 8‐, 17‐, and 18–20‐day‐old mice, and peritubular cells from 18–20 day old mice, all recovered from outbred white Swiss (CD‐1) mice. We also constructed libraries from whole adult testes from five other lines of mice: C57 BI6/J, C3 HEB, BDF‐1, Balb/c, and 129 Sv. Finally, there are two libraries made from populations of Sertoli cells and peritubular cells isolated from testes of 20‐day‐old Sprague‐Dawley rats. Enzymatic dissociation, followed by gradient separation or plating/lysing techniques, was used to prepare populations of specific cell types in purities of 85–98%. cDNAs were synthesized from poly A+ mRNA primed with oligo dT and unidirectionally cloned into the lambda Uni‐Zap XR expression vector from Stratagene. Primary titers ranged from 2.1 ± 105 to 2.9 × 108 plaque‐forming units, and insert sizes averaged 1.0–1.2 kb. These libraries have been amplified once and submitted to the American Type Culture Collection (ATCC) for distribution to interested investigators. ATCC accession numbers are provided

Frequent and recent retrotransposition of orthologous genes plays a role in the evolution of sperm glycolytic enzymes

<p><b>Abstract</b></p> <p>Background</p> <p>The central metabolic pathway of glycolysis converts glucose to pyruvate, with the net production of 2 ATP and 2 NADH per glucose molecule. Each of the ten reactions in this pathway is typically catalyzed by multiple isozymes encoded by a multigene family. Several isozymes in this pathway are expressed only during spermatogenesis, and gene targeting studies indicate that they are essential for sperm function and male fertility in mouse. At least three of the novel glycolytic isozymes are encoded by retrogenes (<it>Pgk2</it>, <it>Aldoart1</it>, and <it>Aldoart2</it>). Their restricted expression profile suggests that retrotransposition may play a significant role in the evolution of sperm glycolytic enzymes.</p> <p>Results</p> <p>We conducted a comprehensive genomic analysis of glycolytic enzymes in the human and mouse genomes and identified several intronless copies for all enzymes in the pathway, except <it>Pfk</it>. Within each gene family, a single orthologous gene was typically retrotransposed frequently and independently in both species. Several retroposed sequences maintained open reading frames (ORFs) and/or provided evidence of alternatively spliced exons. We analyzed expression of sequences with ORFs and <99% sequence identity in the coding region and obtained evidence for the expression of an alternative <it>Gpi1 </it>transcript in mouse spermatogenic cells.</p> <p>Conclusions</p> <p>Our analysis detected frequent, recent, and lineage-specific retrotransposition of orthologous glycolytic enzymes in the human and mouse genomes. Retrotransposition events are associated with LINE/LTR and genomic integration is random. We found evidence for the alternative splicing of parent genes. Many retroposed sequences have maintained ORFs, suggesting a functional role for these genes.</p

Evaluation of Three Sources of Validity Evidence for a Laparoscopic Duodenal Atresia Repair Simulator

Purpose: Laparoscopic duodenal atresia (DA) repair is a relatively uncommon pediatric operation requiring advanced minimally invasive skills. Currently, there are no commercial simulators available that address surgeons' needs for refining skills associated with this procedure. The purposes of this study were (1) to create an anatomically correct, size-relevant model and (2) to evaluate the content validity of the simulator. Materials and Methods: Radiologic images were used to create an abdominal domain consistent with a full-term infant. Fetal bovine tissue was used to complete the simulator. Following Institutional Review Board exempt determination, 18 participants performed the simulated laparoscopic DA repair. Participants completed a self-report, six-domain, 24-item instrument consisting of 4-point rating scales (from 1=not realistic to 4=highly realistic). Validity evidence relevant to test content and response processes was evaluated using the many-facet Rasch model, and evidence of internal structure (inter-item consistency) was estimated using Cronbach's alpha. Results: The highest observed averages were for ?Value as a training and testing tool? (both observed averages=3.9), whereas the lowest ratings were ?Palpation of liver? (observed average=3.3) and ?Realism of skin? (observed average=3.2). The Global opinion rating was 3.2, indicating the simulator can be considered for use as is, but could be improved slightly. Inter-item consistency was high (α=0.89). Conclusions: We have successfully created a size-appropriate laparoscopic DA simulator. Participants agreed that the simulator was relevant and valuable as a learning/testing tool. Prior to implementing this simulator as a training tool, minor improvements should be made, with subsequent evaluation of additional validation evidence.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140148/1/lap.2014.0358.pd

Preliminary Evaluation of a Novel Thoracoscopic Infant Lobectomy Simulator

Purpose: Thoracoscopic lobectomy in infants requires advanced minimally invasive skills. Simulation-based education has the potential to improve complex procedural skills without exposing the patient to undue risks. The study purposes were (1) to create a size-appropriate infant lobectomy simulator and (2) to evaluate validity evidence to support or refute its use in surgical education. Materials and Methods: In this Institutional Review Board-exempt study, a size-appropriate rib cage for a 3-month-old infant was created. Fetal bovine tissue completed the simulator. Thirty-three participants performed the simulated thoracoscopic lobectomy. Participants completed a self-report, 26-item instrument consisting of 25 4-point rating scales (from 1=not realistic to 4=highly realistic) and a one 4-point Global Rating Scale. Validity evidence relevant to test content and response processes was evaluated using the many-facet Rasch model, and evidence of internal structure (inter-item consistency) was estimated using Cronbach's alpha. Results: Experienced surgeons (observed average=3.6) had slightly higher overall rating than novice surgeons (observed average=3.4, P=.001). The highest combined observed averages were for the domain Physical Attributes (3.7), whereas the lowest ratings were for the domains Realism of Experience and Ability to Perform Tasks (3.4). The global rating was 2.9, consistent with ?this simulator can be considered for use in infant lobectomy training, but could be improved slightly.? Inter-item consistency for items used to evaluate the simulator's quality was high (α=0.90). Conclusions: With ratings consistent with high physical attributes and realism, we successfully created an infant lobectomy simulator, and preliminary evidence relevant to test content, response processes, and internal structure was supported. Participants rated the model as realistic, relevant to clinical practice, and valuable as a learning tool. Minor improvements were suggested prior to its full implementation as an educational and testing tool.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140149/1/lap.2014.0364.pd

Evaluation of Three Sources of Validity Evidence for a Synthetic Thoracoscopic Esophageal Atresia/Tracheoesophageal Fistula Repair Simulator

Purpose: Thoracoscopic esophageal atresia (EA)/tracheoesophageal fistula (TEF) repair is technically challenging. We have previously reported our experiences with a high-fidelity hybrid model for simulation-based educational instruction in thoracoscopic EA/TEF, including the high cost of the tissue for these models. The purposes of this study were (1) to create a low-cost synthetic tissue EA/TEF repair simulation model and (2) to evaluate the content validity of the synthetic tissue simulator. Materials and Methods: Review of the literature and computed tomography images were used to create computer-aided drawings (CAD) for a synthetic, size-appropriate EA/TEF tissue insert. The inverse of the CAD image was then printed in six different sections to create a mold that could be filled with platinum-cured silicone. The silicone EA/TEF insert was then placed in a previously described neonatal thorax and covered with synthetic skin. Following institutional review board?exempt determination, 47 participants performed some or all of a simulated thoracoscopic EA/TEF during two separate international meetings (International Pediatric Endosurgery Group [IPEG] and World Federation of Associations of Pediatric Surgeons [WOFAPS]). Participants were identified as ?experts,? having 6?50 self-reported thoracoscopic EA/TEF repairs, and ?novice,? having 0?5 self-reported thoracoscopic EA/TEF repairs. Participants completed a self-report, six-domain, 24-item instrument consisting of 23 5-point rating scales and one 4-point Global Rating Scale. Validity evidence relevant to test content and response processes was evaluated using the many-facet Rasch model, and evidence of internal structure (interitem consistency) was estimated using Cronbach's alpha. Results: A review of the participants' ratings indicates there were no overall differences across sites (IPEG versus WOFAPS, P=.84) or experience (expert versus novice, P=.17). The highest observed averages were 4.4 (Value of Simulator as a Training Tool), 4.3 (Physical Attributes?chest circumference, chest depth, and intercostal space), and 4.3 (Realism of Experience?fistula location). The lowest observed averages were 3.5 (Ability to Perform?closure of fistula), 3.7 (Ability to Perform?acquisition target trocar sites), 3.8 (Physical Attributes?landmark visualization), 3.8 (Ability to Perform?anastomosis and dissection of upper pouch), and 3.9 (Realism of Materials?skin). The Global Rating Scale was 2.9, coinciding with a response of ?this simulator can be considered for use in neonatal TEF repair training, but could be improved slightly.? Material costs for the synthetic EA/TEF inserts were less than $2 U.S. per insert. Conclusions: We have successfully created a low-cost synthetic EA/TEF tissue insert for use in a neonatal thoracoscopic EA/TEF repair simulator. Analysis of the participants' ratings of the synthetic EA/TEF simulation model indicates that it has value and can be used to train pediatric surgeons, especially those early in their learning curve, to begin to perform a thoracoscopic EA/TEF repair. Areas for model improvement were identified, and these areas will be the focus for future modifications to the synthetic EA/TEF repair simulator.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140150/1/lap.2014.0370.pd

Proof of polar ejection fom the close-binary core of the planetary nebula Abell 63

We present the first detailed kinematical analysis of the planetary nebula Abell 63, which is known to contain the eclipsing close-binary nucleus UU Sge. Abell 63 provides an important test case in investigating the role of close-binary central stars on the evolution of planetary nebulae. Longslit observations were obtained using the Manchester echelle spectrometer combined with the 2.1-m San Pedro Martir Telescope. The spectra reveal that the central bright rim of Abell 63 has a tube-like structure. A deep image shows collimated lobes extending from the nebula, which are shown to be high-velocity outflows. The kinematic ages of the nebular rim and the extended lobes are calculated to be 8400+/-500 years and 12900+/-2800 years, respectively, which suggests that the lobes were formed at an earlier stage than the nebular rim. This is consistent with expectations that disk-generated jets form immediately after the common envelope phase. A morphological-kinematical model of the central nebula is presented and the best-fit model is found to have the same inclination as the orbital plane of the central binary system; this is the first proof that a close-binary system directly affects the shaping of its nebula. A Hubble-type flow is well-established in the morphological-kinematical modelling of the observed line profiles and imagery. Two possible formation models for the elongated lobes of Abell 63 are considered (1) a low-density, pressure-driven jet excavates a cavity in the remnant AGB envelope; (2) high-density bullets form the lobes in a single ballistic ejection event.Comment: 11 pages, 8 figures, accepted by MNRAS for publicatio

Structural analyses to identify selective inhibitors of glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme

Detailed structural comparisons of sperm-specific glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS) and the somatic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) isozyme should facilitate the identification of selective GAPDHS inhibitors for contraceptive development

Spermatogenic cell-specific type 1 hexokinase is the predominant hexokinase in sperm

Hexokinase is the first enzyme in the glycolytic pathway and utilizes ATP to convert glucose to glucose-6-phosphate (G6P). We previously identified three variant transcripts of Hk1 that are expressed specifically in spermatogenic cells, have different 5′ untranslated regions, and encode a protein (HK1S, spermatogenic cell-specific type 1 hexokinase) in which the porin-binding domain (PBD) of HK1 is replaced by a novel N-terminal spermatogenic cell-specific region (SSR). However, the level of expression of the individual variant transcripts or of the other members of the hexokinase gene family (Hk2, Hk3, and Gck) in spermatogenic cells remains uncertain. We show that Hk1, Hk2, and Hk3 transcripts levels are quite low in spermatocytes and spermatids and Gck transcripts are relatively abundant in spermatids, but that GCK is not detected in spermatozoa. Using real time RT-PCR (qPCR) with primers specific for each of the three variant forms and RNA from whole testis and isolated germ cells, we found that transcripts for Hk1_v2 and Hk1_v3, but not for Hk1_v1, are relatively high in spermatids. Similar results were seen using spermatogenic cells isolated by laser-capture microdissection (LCM). Immunoblotting studies found that HK1S is abundant in sperm, and immunostaining confirmed that HK1S is located mainly in the principal piece of the sperm flagellum, where other spermatogenic cell-specific glycolytic enzymes have been found. These results strongly suggest that HK1, HK2, HK3 and GCK are unlikely to have a role in glycolysis in sperm and that HK1S encoded by Hk1_v2 and Hk1_v3 serves this role

Metabolic Substrates Exhibit Differential Effects on Functional Parameters of Mouse Sperm Capacitation

Although substantial evidence exists that sperm ATP production via glycolysis is required for mammalian sperm function and male fertility, conflicting reports involving multiple species have appeared regarding the ability of individual glycolytic or mitochondrial substrates to support the physiological changes that occur during capacitation. Several mouse models with defects in the signaling pathways required for capacitation exhibit reductions in sperm ATP levels, suggesting regulatory interactions between sperm metabolism and signal transduction cascades. To better understand these interactions, we conducted quantitative studies of mouse sperm throughout a 2-h in vitro capacitation period and compared the effects of single substrates assayed under identical conditions. Multiple glycolytic and nonglycolytic substrates maintained sperm ATP levels and comparable percentages of motility, but only glucose and mannose supported hyperactivation. These monosaccharides and fructose supported the full pattern of tyrosine phosphorylation, whereas nonglycolytic substrates supported at least partial tyrosine phosphorylation. Inhibition of glycolysis impaired motility in the presence of glucose, fructose, or pyruvate but not in the presence of hydroxybutyrate. Addition of an uncoupler of oxidative phosphorylation reduced motility with pyruvate or hydroxybutyrate as substrates but unexpectedly stimulated hyperactivation with fructose. Investigating differences between glucose and fructose in more detail, we demonstrated that hyperactivation results from the active metabolism of glucose. Differences between glucose and fructose appeared to be downstream of changes in intracellular pH, which rose to comparable levels during incubation with either substrate. Sperm redox pathways were differentially affected, with higher levels of associated metabolites and reactive oxygen species generated during incubations with fructose than during incubations with glucose