Stuart E. Ravnik

A novel view of albumin-supported sperm capacitation: role of Lipid Transfer Protein-I * † ‡

OBJECTIVE To determine if one mechanism of albumin-mediated support of human sperm capacitation is lipid (cholesterol) transfer activity and contamination of albumin with Lipid Transfer Protein-I (LTP-I). DESIGN AND MAIN OUTCOME MEASURES Measure lipid transfer activity in various bovine and human albumin preparations, relate this activity to albumin-supported capacitation (measured by zona-free hamster oocyte sperm penetration assay) and acrosome reactions; and attempt to detect LTP-I in active albumins. Remove LTP-I from albumin which supports capacitation and reassess this support. Reconstitute capacitation support by addition of purified LTP-I. SETTING AND SUBJECTS Healthy sperm donors with normal semen analyses were recruited by the Reproductive Biology-Andrology Laboratory in a university medical center. RESULTS Albumin preparations that effectively support capacitation have high levels of lipid transfer activity and of LTP-I, a protein responsible for lipid transfer activity. Preparations with lower levels of capacitation support have less lipid transfer activity. Removal of LTP-I from supportive albumin significantly reduces the capacitation support, and this is restored by purified LTP-I. Progesterone concentrations in these preparations are negligible. CONCLUSIONS The variable abilities of albumin preparations to support in vitro sperm capacitation are largely dependent on the presence of contaminating LTP-I. The cholesterol transfer activity of this protein, which is present in human serum and follicular fluid, may be one mechanism in the process of capacitation.

Meiotic Expression of the Cyclin H/Cdk7 Complex in Male Germ Cells of the Mouse

Abstract Cell division requires that cyclin-dependent kinases (Cdks) be activated by phosphorylation. In mitotic cells, this is accomplished by the Cdk-activating-kinase (CAK), which is a complex of cyclin H and Cdk7. There are currently no data on the role of CAK in meiotic cells. Previously, we have shown that cyclin A1 is meiosis-specific and forms an active kinase with Cdk2. Because cyclin A1 is required for meiosis, and its associated kinase must be phosphorylated (activated), we propose that cyclin H/Cdk7 function to activate cyclin A1/Cdk2 in meiotic cells. Here, we show that cyclin H and Cdk7 are present during meiosis. Using reverse transcription-polymerase chain reaction and in situ hybridization, we show that the mRNAs encoding cyclin H and Cdk7 are abundant in spermatocytes. Immunohistochemistry localized cyclin H and Cdk7 to the nucleus of spermatocytes in stages IV to XII of the spermatogenic cycle, overlapping the same stages that express cyclin A1-associated kinases. Finally, immunoprecipitation and histone H1-kinase assays of cyclin H and Cdk7 from testicular extracts show that these proteins interact to form an active kinase. We conclude that cyclin H/Cdk7 complexes are present and during meiosis, form active complexes in testicular cells and are strong candidates for the activating kinase for cyclin A1-associated kinase.

Self-generating density gradients of Percoll provide a simple and rapid method that consistently enriches natural killer cells

The separation or enrichment of natural killer (NK) cells from the heterogeneous cell populations in murine spleen or bone marrow is a vital step for the study of NK cells. We report in this study a simple and rapid method for the enrichment of NK cells from B cell-depleted spleen cells, using a self-generating density gradient of polyvinyl pyrrolidone-coated silica (Percoll). Nylon wool-passed spleen cells are suspended in Percoll that is isotonic and isosmotic with mouse blood at a density of 1.087 g/ml and ultracentrifuged at 30,000 x g for 10 min. This method consistently enriches for NK cell cytotoxic activity, in spleen cells of both unstimulated and interferon-stimulated mice, as measured in the chromium release assay. There is a concomitant enrichment for cells bearing the NK marker asialo GM-1 and depletion of L3T4 or Lyt-2-bearing T cells. In contrast to discontinuous, step-wise gradients, the self-generating Percoll gradient, which relies on the intrinsic property of Percoll to form a continuous density gradient, appears to provide the cells with a physiological environment both before and during the centrifugation step.