Lynn J. Romrell

Separation of mouse spermatogenic cells by sedimentation velocity: A morphological characterization☆

Abstract A method utilizing sequential enzymatic incubation in collagenase (1 mg/ml) and trypsin (2.5 mg/ml) has been developed for the dissociation of the seminiferous epithelium. A significant advantage of this method is that, following collagenase incubation and washings in an enriched Krebs-Ringer bicarbonate buffer solution, isolated seminiferous tubules are obtained which are free of interstitial cells. The “purified” seminiferous epithelium is then dissociated with trypsin. A further advantage of this dissociation technique has been a reduction in the number of symplasts (multinucleate cells) which form by the opening up of the intercellular bridges that occur between synchronously differentiating clusters of germ cells. Both the elimination of the interstitial cells and the reduction in the number of symplasts have made possible the recovery of more highly enriched germ cell fractions. The homogeneity of the cell fractions was determined by light and electron microscopy. Integrity of the isolated cells was verified by Trypan blue exclusion and measurement of oxygen consumption.

Appearance of cell surface auto- and isoantigens during spermatogenesis in the rabbit

Abstract The appearance of spermatogenic cell surface auto- and isoantigens can be precisely determined by utilizing techniques that separate spermatogenic cells. Using cytotoxic (complement dependent) auto- and iso- rabbit antirabbit whole semen sera, specific spermatogenic auto- and isoantigens were first detected following the maturation of spermatogonia into primary pachytene spermatocytes. The antisera employed were cytotoxic (complement dependent) for pachytene diplotene and primary spermatocytes and spermatids but not for type A, intermediate, or type B spermatogonia. Furthermore, Sertoli cells, endothelial cells, Leydig cells, and erythrocytes were not lysed by the antisera. These observations support the concept of a blood-testis barrier. Only after migration of spermatogonia to the luminal side of the barrier can autoantigenic molecules be synthesized and/or inserted into the plasma membrane of spermatogenic cells. Thus, the appearance of surface autoantigens offers a model system to study the synthesis of specific molecules which are inserted into the plasma membrane at a precise time during development.

Fine structure of normal spermatid differentiation in Drosophila melanogaster.

Spermiogenesis in Drosophila melanogaster has been divided into eleven developmental stages based upon the fine structure of differentiating organelles. Emphasis is placed on the relative state of development of all organelles in each stage. This information will facilitate the analysis of subtle alterations in spermatid differentiation produced by mutations in the genome or by any other experimental manipulation. New details of organelle development are reported including distinctive associations of cytoplasmic microtubules with both the endoplasmic reticulum and the nuclear envelope. The centriolar complex is postulated to be the main organizational center in spermatid differentiation.

Appearance of regional surface autoantigens during spermatogenesis: comparison of anti-testis and anti-sperm autoantisera.

Abstract Autoantisera against rabbit testes and rabbit ejaculated spermatozoa have been used to study the appearance of surface autoantigens during spermatogenesis. Two distinct subclasses of autoantigens have been identified: an early subclass which first appears on pachytene spermatocytes and a late subclass which first appears on differentiating spermatids. These spermatids are just beginning to demonstrate migration of the nucleus and overlying acrosomal cap to the cell periphery and changes in nuclear shape. Some autoantigens of the early subclass do not appear on spermatozoa, but those that do are predominantly found over the acrosomal region. Autoantigens of the late subclass are predominantly found over the postacrosomal and middle-piece regions of the spermatozoon. It is suggested that morphological constraints during spermiogenesis may be responsible for the regional localization of the two subclasses.

Localization of a single sperm membrane autoantigen (RSA-1) on spermatogenic cells and spermatozoa☆

The rabbit sperm membrane autoantigen RSA-1 is a sialoglycoprotein of 13,000 daltons which first appears on the surface of pachytene spermatocytes. Using specific antiserum to RSA-1 the antigen has been localized by immunofluorescence and immunoperoxidase staining. On testicular cells labeled at 37 degrees C, RSA-1 is seen in patches on the surfaces of pachytene spermatocytes, round spermatids, and over the acrosomal area of later spermatids and spermatozoa. Over the postacrosomal and middle-piece regions of late spermatids and spermatozoa the labeling appears uniform. The uniformity can be seen to stop abruptly at the equatorial segment-postacrosomal border. Labeling cells after fixation gives a uniform distribution of label over the surface where patches were seen at 37 degrees C. The polypeptides recognized by the antiserum used for labeling were identified by immunoadsorbent chromatography and subsequent SDS-PAGE. In testicular cells anti-RSA-1 recognizes the 13,000-dalton form and another component which migrates with the dye front. In ejaculated spermatozoa anti-RSA-1 recognizes a distinct ejaculate complex of higher-molecular-weight proteins containing an 84,000-dalton major band and five minor components.

Mission-based budgeting: removing a graveyard.

Many activities in today’s medical schools no longer have medical students’ education as their central reason for existence. Faculty are hired primarily to provide clinical service or to make discoveries, with the role of educator of secondary importance. Budgeting in medical schools has not evolved in concert with these changing roles of faculty. The cost of medical students’ education is still calculated as if all faculty were hired primarily to teach medical students and their other activities were to support this “central” mission. Most medical schools still mix revenues without regard to intent and cannot accurately determine costs because they confuse expenses with costs. At the University of Florida College of Medicine, a group of administrators, chairpersons, and faculty developed a budgeting process now called mission-based budgeting. This is a three-step process: (1) revenues are prospectively identified for each mission and then aligned with intended purposes; (2) faculty productivity, i.e., faculty effort and its quality, is measured for each of the missions; and (3) productivity is linked to the prospective budget for each mission. This process allows the institution to understand the intent of its revenues, to measure how productive its faculty are, to learn the true costs of its missions, to make wise investment decisions (subsidies), and to justify to various constituents its use of revenues. The authors describe this process, focusing particularly on methods used to develop a comprehensive database for assessment of faculty productivity in education.

Moving a Graveyard: How One School Prepared the Way for Continuous Curriculum Renewal

From 1991 to 1996, the faculty at the University of Florida College of Medicine initiated several significant changes in its curriculum. These changes, included the introduction of early clinical experience in primary care settings; the enhancement of active learning experiences in small-group settings; production and use of computer-based interactive learning materials; increased clinical teaching in the ambulatory care training in an interdisciplinary primary care clerkship; effective course and faculty evaluation; establishment and use of an assessment center for instruction and performance-based evaluations utilizing standardized patients; creation of a medical education center as the focal point for logistics support of the teaching faculty and education data handling; creation of a faculty development program; and initiation of mission-based budgeting based on the facultys teaching effort and quality. Because the faculty were relatively conservative, it was important to identify variables that would facilitate the introduction of changes and those that might hinder it. The following factors were most important: interest and support by the dean and clearly defined delegation of authority to an associate dean; introduction of a mission-based budgeting process that allocates education funds on the basis of faculty teaching effort and its quality; a clear understanding of the empowerment of the curriculum committee; and an identification of the principles that should guide educational planning and implementation. These efforts are considered the beginning of the continuous renewal needed to respond to information networking, scientific and technological innovations, and the fundamental changes in health care delivery. As these changes have taken place, a shift toward greater institutional control of the educational program leading to the MD degree has been evident.

Evaluating evidence-based medicine skills during a performance-based examination.

Purpose To measure students’ competencies in evidence-based medicine (EBM) skills [clinical decision making using evidence from published literature (content) and in transmitting clinical information to patients (communication)] within the context of a performance-based examination (PBE). Method In 2002–03, under the direction of a Performance-Based Examination Oversight Committee, 16 EBM queries were developed for a pair of third-year PBEs. At the last station of the PBE, the standardized patient (SP) for that station asked a clinical EBM question relating to their “disease process.” Students were asked to develop an appropriate clinical question, perform a Medline search for appropriate articles, critically appraise a complete selected article, reach a conclusion to their question, and transmit the information to the SP. Each students clinical question, search terms, selected articles, and rationale were evaluated by faculty question-writers, clinical librarians, and the EBM course director using a five-point Likert scale, with 1 being inadequate performance and 5 being superior performance. The SP evaluated the communication skills using a checklist. Results Students’ performances were very good, with means of 3.7 to 4.0 in each area. Agreement between the course director and station developers was good. Seventy-five percent of the students performed adequate Medline searches. Students averaged over 93% on the performance of four communication skills. Conclusion The evaluation of EBM skills can be carried out during a performance-based examination. Results can assist in developing students’ skills and directing curricular efforts.

Impacting Faculty Teaching and Student Performance: Nine Years' Experience With the Objective Structured Clinical Examination

Background: The impetus for administering the 2nd-year Objective Structured Clinical Examination (OSCE) came from the great variability in student performance observed by 3rd-year clerkship directors. Purpose: To document the effects of the OSCE on faculty teaching, student performance, and the curriculum over 9 years of administration of the examinations to more than 1,000 second-year medical students. Method: A 20-station OSCE was administered to all medical students at the end of their 2nd year. Using predetermined criteria, clinical faculty served as evaluators in each station. A mix of 1st-, 3rd-, and 4th-year medical students were recruited to serve as simulated patients. Faculty evaluators and examinees completed a questionnaire evaluating their experience with the OSCE. Students received a report card of their performance. Small-group leaders of the Introduction to Clinical Medicine course received feedback on their groups performance on each station compared to the class mean. Summative data on class performance was reported to the curriculum committee. The academic status committee received data on students who performed unsatisfactorily. Results: Faculty and examinee ratings of the OSCE experience were very positive. Over the 9-year period, student performance improved showing less variability and significantly fewer failed stations. Conclusion: The OSCE has proven to be a technically feasible, authentic evaluation method yielding valuable information for decisions regarding student performance, faculty teaching, and curriculum planning.

Capping and ultrastructural localization of sperm surface isoantigens during spermatogenesis.

Abstract Isoantisera from female rabbits injected with rabbit whole semen have been used to study the appearance of cell surface isoantigens during spermatogenesis. Using isoantiserum IgG and adjuvant control IgG the presence of surface isoantigens on separated pachytene spermatocyte populations and populations of cells at more advanced stages of differentiation was confirmed with fluorescein-labeled goat IgG anti-rabbit IgG. The label was uniformly dispersed over the cell surface on cells labeled at 4°C but occurred in caps on cells warmed to 37°C indicating isoantigen mobility within the plane of the membrane. Residual bodies and mature spermatozoa did not show cap formation. Spermatogonia, Leydig cells, and Sertoli cells were not labeled. These observations were confirmed at the ultrastructural level with peroxidase-conjugated goat IgG anti-rabbit IgG. The percentage of the cell surface labeled was determined on cells at specific stages of spermatogenesis by stereological analysis. No significant surface labeling was observed on spermatogonia, Leydig cells, or Sertoli cells. The percentage of label bound to the surface of spermatogenic cells increased from approximately 4% in the pachytene spermatocytes to greater than 96% in the most mature testicular spermatids.