Meiotic expression of the cyclin H/Cdk7 complex in male germ cells of the mouse.

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.

Two distinct forms of the 64,000 Mr protein of the cleavage stimulation factor are expressed in mouse male germ cells.

Polyadenylation in male germ cells differs from that in somatic cells. Many germ cell mRNAs do not contain the canonical AAUAAA in their 3' ends but are efficiently polyadenylated. To determine whether the 64,000 Mr protein of the cleavage stimulation factor (CstF-64) is altered in male germ cells, we examined its expression in mouse testis. In addition to the 64,000 Mr form, we found a related approximately 70,000 Mr protein that is abundant in testis, at low levels in brain, and undetectable in all other tissues examined. Expression of the approximately 70,000 Mr CstF-64 was limited to meiotic spermatocytes and postmeiotic spermatids in testis. In contrast, the 64,000 Mr form was absent from spermatocytes, suggesting that the testis-specific CstF-64 might control expression of meiosis-specific genes. To determine why the 64,000 Mr CstF-64 is not expressed in spermatocytes, we mapped its chromosomal location to the X chromosome in both mouse and human. CstF-64 may, therefore, be absent in spermatocytes because the X chromosome is inactivated during male meiosis. By extension, the testis-specific CstF-64 may be expressed from an autosomal homolog of the X chromosomal gene.

Regulation of meiosis during mammalian spermatogenesis: the A-type cyclins and their associated cyclin-dependent kinases are differentially expressed in the germ-cell lineage.

To begin to examine the function of the A-type cyclins during meiosis in the male, we have examined the developmental and cellular distribution of the cyclin A1 and cyclin A2 proteins, as well as their candidate cyclin-dependent kinase partners, Cdk1 and Cdk2, in the spermatogenic lineage. Immunohistochemical localization revealed that cyclin A1 is present only in male germ cells just prior to or during the first, but not the second, meiotic division. By contrast, cyclin A2 was expressed in spermatogonia and was most abundant in preleptotene spermatocytes, cells which will enter the meiotic pathway. Immunohistochemical detection of Cdk1 was most apparent in early pachytene spermatocytes, while staining intensity diminished in diplotene and meiotically dividing spermatocytes, the cells in which cyclin A1 expression was strongest. Cdk2 was highly expressed in all spermatocytes. Notably, in cells undergoing the meiotic reduction divisions, Cdk2 appeared to localize specifically to the chromatin. This was not the case for spermatogonia undergoing mitotic divisions. In the testis, cyclin A1 has been shown to bind both Cdk1 and Cdk2 but we show here that cyclin A2 binds only Cdk2. These results indicate that the A-type cyclins and their associated kinases have different functions in the initiation and passage of male germ cells through meiosis.

The developmentally restricted pattern of expression in the male germ line of a murine cyclin A, cyclin A2, suggests roles in both mitotic and meiotic cell cycles.

We have isolated cDNAs encoding a murine cyclin A, designated cyclinA2, and have examined its in vivo expression at the level of both mRNA and protein, with particular focus on the male germ line. Cyclin A2 is expressed in embryos and in a variety of adult tissues, including the testis. In the testis, however, a striking cellular specificity of expression was observed. At both the DNA and protein levels, the predominant sites of cyclin A2 expression were in the germ line stem cells, the spermatogonia, and in highest levels in preleptotene spermatocytes, cells in which premeiotic DNA synthesis occurs. The concurrent localization of cyclin A2 mRNA and protein further suggested that cyclin A2 is regulated at the level of transcription in these cells. The observed cellular specificity of cyclin A2 expression is consistent with its function during mitosis in the stem cell stage of this lineage, while the restricted meiotic stage localization suggests function in G1/S or S but not in the meiotic divisions per se.

A distinct cyclin A is expressed in germ cells in the mouse.

In this paper, the existence of two A-type cyclins in the mouse is demonstrated. In the adult mouse, the expression of cyclin A1, which has greatest sequence identity with Xenopus cyclin A1, is restricted to germ cells. In contrast cyclin A2, which has greatest sequence identity with human cyclin A and Xenopus cyclin A2, is expressed in all tissues analysed. In order to explore the function of cyclin A1 in germ cells, its expression during the meiotic cell cycle and its associated kinase subunits have been characterised in the testis. The levels of cyclin A1 mRNA rise dramatically in late pachytene spermatocytes and become undetectable soon after completion of the meiotic divisions; thus its expression is cell cycle regulated. In lysates of germ cells from adult testes, cyclin A1 is present in p13suc1 precipitates, and cyclin A1 immunoprecipitates possess histone H1 kinase activity. Three kinase partners of cyclin A1 were identified: p34cdc2, a polypeptide of 39 x 10(3) M(r) that is related to p33cdk2 and, in lesser quantities, p33cdk2. Cyclin A1 was also detected in oocytes; in metaphase I and metaphase II oocytes, a proportion of the cyclin A1 colocalises with the spindle, possibly suggestive of a functional interaction. These data indicate that mammalian germ cells contain cyclin A1-dependent kinases that either act as a substitute for, or in addition to, the cyclin A2-dependent kinases characterised in somatic tissues.

Stimulation of human sperm capacitation by purified lipid transfer protein.

Lipid Transfer Protein I is recognized as a key component involved in high density lipoprotein metabolism. We have been studying lipid transfer in relation to sperm capacitation, a complex series of cell surface events required for the acrosome reaction and fertilization. We have previously shown that Lipid Transfer Protein I is present and active in the female reproductive tract. In the present study, we show that purified Lipid Transfer Protein I directly stimulates human sperm capacitation, but not the acrosome reaction, in the absence of other biological effectors. These results provide strong evidence for a novel role for Lipid Transfer Protein I and reveal, for the first time, a potent activator of capacitation, prior to the acrosome reaction.

Genetic control of mitosis, meiosis and cellular differentiation during mammalian spermatogenesis.

Gametogenesis in both the male and female mammal represents a specialized and highly regulated series of cell cycle events, involving both mitosis and meiosis as well as subsequent differentiation. Recent advances in our understanding of the genetic control of the eukaryotic cell cycle have underscored the evolutionarily-conserved nature of these regulatory processes. However, most of the data have been obtained from yeast model systems and mammalian cell lines. Furthermore, most of the observations focus on regulation of mitotic cell cycles. In the present paper: (i) aspects of gametogenesis in mammals that represent unique cell-cycle control points are highlighted; (ii) current knowledge on the regulation of the germ cell cycle, in the context of what is known in yeast and other model eukaryotic systems, is summarized; and (iii) strategies that can be used to identify additional cell cycle regulating genes are outlined.

Distinct patterns of expression of the D-type cyclins during testicular development in the mouse.

The three D-type cyclins have been shown to be differentially expressed in a number of isolated cell types and cell lines, suggesting distinct roles in cell cycle regulation in particular cell lineages. The testis provides unique opportunities to study genes involved in cell cycle regulation, since it contains cells in both mitosis and meiosis as well as differentiated cells with little proliferation activity. Major transcripts of 4.2 kb, 6.8 kb, and 2.3 kb were detected in the adult mouse testis by Northern hybridization analyses for cyclin D1, cyclin D2, and cyclin D3, respectively. Additional transcripts of 1.8 and 2.7 kb were detected by Northern hybridization for cyclin D3 in the testis, but not in other tissues, and these transcripts were limited to germ cells. Northern and in situ hybridization analyses of normal and germ cell-deficient testes showed the surprising result that cyclin D1 was expressed in a pattern consistent with expression in the non-dividing Sertoli cells. Cyclin D2 levels appeared slightly enriched in germ cell-deficient testes as compared to intact testis, but in situ hybridization analysis did not reveal any distinct cellular localization. Also surprising was the observation that cyclin D3 expression was highest in the non-dividing, haploid, round spermatids. The possible roles of these cyclins in the events of spermatogenesis are discussed.

[Genetic control of cellular differentiation and proliferation during gametogenesis in mammals]. / Contrôle génétique de la différenciation et de la prolifération cellulaire pendant la gamétogénèse chez les mammifères.

Understanding the genetic program that controls the regulation of cells progressing through the mitotic and meiotic cell cycles, whether in response to growth factors, other extracellular signals, or intrinsic programs, is critical to problems in infertility and in gonadal neoplasias. In this study, we present a consideration of important stages of cell cycle control in mammalian germ cells and data illustrating the experimental approaches that can be used to determine the pattern of such genes which may be involved in these events.

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.

Purification and characterization of a human follicular fluid lipid transfer protein that stimulates human sperm capacitation.

Identification of the mechanisms responsible for sperm capacitation has been an active area of research for nearly four decades. Changes in the lipid composition of the sperm membrane is one of the biochemical events that occurs during sperm capacitation. We have been studying physiological effectors of some of these changes and have identified lipid transfer activity in fractions of human follicular fluid that stimulates sperm penetration of zona-free hamster oocytes. We report here the purification of a lipid transfer protein by sequential chromatography from human follicular fluid. This protein was purified greater than 20,000-fold for lipid transfer activity and greater than 28,000-fold for sperm penetration-inducing activity. This 64,000 molecular weight protein has a pI of approximately 5.0 and shares physicochemical characteristics with the plasma lipid transfer protein, LTP-I. Antibodies to LTP-I also recognize this protein and depletion of LTP-I from human follicular fluid by immunoaffinity chromatography renders the follicular fluid incapable of stimulating sperm penetration. We conclude that purified LTP-I is able to stimulate human sperm capacitation and that LTP-I is a molecule responsible for this stimulation in follicular fluid.

Lipid transfer activity in human follicular fluid: relation to human sperm capacitation.

A potentially important event during sperm capacitation is the loss of sperm membrane cholesterol. Although the exact mechanisms mediating this loss are not known, albumin and high density lipoprotein have been proposed as lipid acceptors. The authors propose that lipid transfer may be involved in capacitation as a specific mediator in the sequence of events leading to sperm membrane cholesterol loss. We present the first direct evidence of lipid transfer activity (LTA) in human follicular fluid (HFF). The redistribution of 14C-cholesteryl ester among human plasma lipoproteins was used as a measure of LTA (% Transfer [%T]). The HFF was fractionated by S-300 gel filtration chromatography and assayed for LTA. Three peaks of activity were consistently eluted from the column. Each peak of LTA also stimulated human sperm to penetrate zona-free hamster oocytes after short capacitating incubations. The peak with highest LTA (12.75 +/- 1.11%T) with an Mr approximately 68,000, gave the greatest stimulation (penetration index, PI: 3.34 +/- 0.96 fold increase above control, n = 4). The HFF also showed a significant dose response for both LTA and PI, whereas bovine serum albumin did not. These results demonstrate the existence of LTA in HFF and suggest that a specific lipid transfer protein may have a role in human sperm capacitation or acrosome reaction.

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.