A genetic strategy for differential screening of meiotic germ-cell cDNA libraries

The goals of this work were to create germ-cell-stage-specific cDNA libraries from mouse spermatogenic cells and to employ a novel two-step genetic screen to identify gene sequences present during the critical meiotic stage of spermatogenesis. Highly enriched germ-cell fractions were prepared from adult and juvenile mouse testes, and purity of these fractions was extensively analyzed by light and electron microscopy. Standard techniques were used to prepare cDNA libraries from populations of mixed leptotene and zygotene (L/Z) spermatocytes, pachytene (P) spermatocytes, and round spermatids. These libraries were analyzed with respect to representation of sequences from ubiquitously expressed genes, and from genes expressed at specific germ-cell stages as well as from genes expressed in testicular somatic cells. For the first step of the screening procedure, testicular cDNA was prepared from mutant mice carrying the T(X;11)38H chromosomal translocation that causes spermatogenic arrest at early meiotic prophase. This mixed cDNA probe was used to screen the libraries from L/Z and P spermatocytes to detect sequences that failed to hybridize. The clones identified were characterized for ability to hybridize to various germ-cell-specific cDNAs to verify that they represented sequences present in normal spermatogenic meiotic cells. These clones were then subjected to a second screening with another mutant probe; this time the cDNA probe was from testes of sterile mice bearing the T(X;16)16H chromosomal translocation that causes spermatogenic arrest at late meiotic prophase. This screen identified 27 clones that were not represented in testicular cDNA from T38-bearing mice or from T16-bearing mice. These clones may represent sequences essential for normal completion of the genetic events of meiosis during spermatogenesis. Likewise, the secondary screen identified 19 clones that were not represented in testicular cDNA from T38-bearing mice but were represented in testicular cDNA of T16-bearing mice. These clones are thus gene sequences present in spermatogenic cells during the time from early meiotic prophase to mid-to-late prophase. This strategy represents the first use of genetic aberrations in differential screening to identify genes expressed at specific times during mammalian spermatogenesis. © 1996 Wiley-Liss, Inc.     

Developmental regulation of calmodulin, actin, and tubulin RNAs during rat testis differentiation

Postnatal testis differentiation involves transition through neonatal, pre-meiotic, meiotic, haploid, and mature stages. We have examined the qualitative and quantitative changes in rat testis RNAs that specifically hybridize to cDNAs encoding the cytoskeletal proteins, calmodulin, beta-actin, alpha- and beta-tubulin at ages corresponding to each of these developmental periods. We compared the species and relative levels of specific RNAs from testes of animals engaged in normal spermatogenesis with RNA from germ cell-depleted, Sertoli cell-enriched (SCE) testis. Distinct developmental patterns of expression of the specific RNAs were found with each of the cDNAs in the two animal models. A 2.2 kb (kilobase) actin RNA and a 2.7 kb beta-tubulin RNA are maximal at 5-10 days of age, suggesting these RNAs are required by somatic and germ cells in the postnatal phase prior to puberty. Between 19 and 29 days, when pachytene spermatocytes appear in significant numbers, there is a slight increase in the 2.2-kb actin RNA, but a 4- to 10-fold increase in RNAs hybridizing to cDNAs for calmodulin, alpha- and beta-tubulin. These changes are much less pronounced in the SCE testis than in the normal testis, indicating increases in these RNAs are related to germinal cell maturation. The germ cell-related increase in 1.8-kb beta-tubulin RNA appears to reflect a developmental "switch" in the gene from which the RNA is derived. This hypothesis is based on the observation that the ratio of hybridization of a chicken brain beta-tubulin cDNA versus a rat spleen beta-tubulin cDNA to the 1.8-kb RNA band increases more than 40-fold between 5 and 29 days of age in normal testis, but is constant in SCE testis. These data suggest that a specific beta-tubulin gene is activated in maturing germ cells. Analogously, a 2.1-kb alpha-tubulin RNA is found only in maturing normal testis and increases as spermatids are produced. A 2.0-kb beta-tubulin RNA, not found in normal testes, is maximal in maturing SCE testes, suggesting this RNA is of somatic cell origin. All of the RNA species studied, except the 2.0-kb beta-tubulin RNA, decrease between 5 and 19 days in SCE testes, as Sertoli cell mitotic activity wanes, indicating that their levels may be regulated by the developmental signals that influence mitosis.     

Expressed sequence tag analysis of expression profiles of zebrafish testis and ovary

In the present study, two gonad cDNA libraries from zebrafish testes and ovaries were constructed and a total of 1025 expressed sequence tag (EST) clones were generated from the two libraries: 501 from the testis library and 524 from the ovary library. A total of 641 of the EST clones were identified to share significant sequence identity with known sequences in GenBank, representing at least 478 different zebrafish genes. In order to understand the molecular compositions of the two gonad organs, the expression profiles of the identified clones in these two gonad cDNA libraries were analyzed. Both gonad libraries have a higher portion of clones for nuclear proteins and a lower portion for proteins in translational machinery, cytoskeleton and mitochondria than our previously characterized whole-adult cDNA library. Most abundant cDNA clones in the two gonad libraries were identified and over 10% of ovary clones were found to encode egg membrane proteins (zona pellucida or ZP proteins). Furthermore, the testis library showed a more even distribution of cDNA clones with relatively fewer abundant clones that tend to contribute redundant clones in EST projects; thus, the testis library can supply more unique and novel cDNA sequences in a zebrafish EST project. Another aim of this study is to identify cDNA clones that can be used as molecular markers for the analysis of the gonad development in zebrafish. Eleven potential clones were selected to analyze their expression patterns by Northern blot hybridization. Most of them showed a specific or predominant expression in the expected testis or ovary tissue. At last, four of the clones were found, by section in situ hybridization, to be expressed specifically in the germ cells of the testis or ovary and thus they are suitable molecular markers for analyses of spermatogenesis and oogenesis.     

A dual-labeling method for identifying differentially expressed genes: use in the identification of cDNA clones that hybridize to RNAs whose abundance in tomato flowers is potentially regulated by gibberellins

A method for identifying cDNA clones that hybridize to differentially expressed RNAs is described. Briefly, the RNA population in which the RNAs of interest are more abundant is used as a template for the synthesis of 35S-labeled cDNAs and another RNA population in which the RNAs of interest are less abundant is used as a template for the synthesis of 32P-labeled cDNAs. The labeled cDNAs are pooled and hybridized to plaque or colony lifts constructed from a cDNA library. Clones that hybridize to RNAs that are differentially expressed are identified using differential autoradiography/fluorography to discriminate between the 32P and 35S isotopes. We have used this method to identify cDNA clones that hybridize to mRNAs that are more abundant in the flowers of wild-type tomato than in the flowers of mutants that have low endogenous levels of gibberellins.     

Analysis of cDNA sequences from mouse testis

Few mammalian proteins involved in chromosome structure and function during meiosis have been characterized. As an approach to identify such proteins, cDNA clones expressed in mouse testis were analyzed by sequencing and Northern blotting. Various cDNA library screening methods were used to obtain the clones. First, hybridization with cDNA from testis or brain allowed selection of either negative or differentially expressed plaques. Second, positive plaques were identified by screening with polyclonal antisera to prepubertal testis nuclear proteins. Most clones were selected by negative hybridization to correspond to a low abundance class of mRNAs. A PCR-based solid-phase DNA sequencing protocol was used to rapidly obtain 306 single-pass cDNA sequences totaling more than 104 kb. Comparison with nucleic acid and protein databases showed that 56% of the clones have no significant match to any previously identified sequence. Northern blots indicate that many of these novel clones are testis-enriched in their expression. Further evidence that the screening strategies were appropriate is that a high proportion of the clones which do have a match encode testisenriched or meiosis-specific genes, including the mouse homolog of a rat gene that encodes a synaptonemal complex protein.The nucleotide sequence data reported in this paper have been submitted to Genbank and have been assigned the accession numbers L26606–1.26848.     

Targeted inactivation of testicular nuclear orphan receptor 4 delays and disrupts late meiotic prophase and subsequent meiotic divisions of spermatogenesis

Testicular orphan nuclear receptor 4 (TR4) is specifically and stage-dependently expressed in late-stage pachytene spermatocytes and round spermatids. In the developing mouse testis, the highest expression of TR4 can be detected at postnatal days 16 to 21 when the first wave of spermatogenesis progresses to late meiotic prophase. Using a knockout strategy to delete TR4 in mice, we found that sperm production in TR4(-/-) mice is reduced. The comparison of testes from developing TR4(+/+) and TR4(-/-) mice shows that spermatogenesis in TR4(-/-) mice is delayed. Analysis of the first wave of spermatogenesis shows that the delay can be due to delay and disruption of spermatogenesis at the end of late meiotic prophase and subsequent meiotic divisions. Seminiferous tubule staging shows that stages X to XII, where late meiotic prophase and meiotic divisions take place, are delayed and disrupted in TR4(-/-) mice. Histological examination of testis sections from TR4(-/-) mice shows degenerated primary spermatocytes and some necrotic tubules. Testis-specific gene analyses show that the expression of sperm 1 and cyclin A1, which are genes expressed at the end of meiotic prophase, was delayed and decreased in TR4(-/-) mouse testes. Taken together, results from TR4(+/+) and TR4(-/-) mice indicate that TR4 is essential for normal spermatogenesis in mice.     

Differential Expression of Cyclins B1 and B2 during Medaka (Oryzias latipes) Spermatogenesis

The Cdc2-cyclin B complex (named the M-phase-promoting factor, MPF) is well known to be a key regulator of G2-M transition in both mitosis and meiosis. However, MPF may have functions other than the cell cycle regulation, since its activity is detectable in post-mitotic (or post-meiotic) non-dividing cells. Cyclin B comprises several subtypes, but their functional differences are still unknown. Despite the established function of MPF during oocyte maturation, its role during spermatogenesis, where spermatogenic cells undergo drastic morphological changes after meiosis, remains to be elucidated. To address these issues, we have isolated cDNA clones encoding cyclins B1 and B2 from medaka testis and raised polyclonal antibodies against their products. Using these as probes, we examined the expression patterns of cyclins B1 and B2 in medaka testis at both mRNA and protein levels. Cyclin B1 and B2 mRNAs were expressed in all stages of spermatogenic cells except for spermatozoa, although the expression levels varied according to the spermatogenic stages. Cyclin B1 protein was expressed only in spermatogonia and spermatocytes at prophase and metaphase with a transient disappearance at anaphase. On the other hand, cyclin B2 protein was continuously expressed throughout spermatogenesis, even in spermatogonia and spermatocytes at anaphase and in post-meiotic spermatids and spermatozoa. The difference in their expression patterns suggests that cyclins B1 and B2 have distinct roles in medaka spermatogenesis; i.e., cyclin B1 controls the meiotic cell cycle, whereas cyclin B2 is involved in process(es) other than meiosis.     

Characterization of histone H2A.X expression in testis and specific labeling of germ cells at the commitment stage of meiosis with histone H2A.X promoter-enhanced green fluorescent protein transgene

To study the complex molecular mechanisms of mammalian spermatogenesis, it would be useful to be able to isolate cells at each stage of differentiation, especially at the stage in which the cells switch from mitosis to meiosis. Currently, no useful marker proteins or gene promoters specific to this important stage are known. We report here a transgenic mouse line that under the control of the promoter for a histone variant, H2A.X, expressed an enhanced green fluorescent protein (EGFP) in cells at the stage of the mitosis-meiosis switch. Endogenous H2A.X is expressed in type A spermatogonia through meiotic prophase spermatocytes in testis and in some somatic cells. However, despite the fact that its expression was driven by the H2A.X promoter, the EGFP expressed in the transgenic mice specifically labeled only the intermediate spermatogonia stage through the meiotic prophase spermatocyte stage in transgenic mice containing the -600-base pair H2A.X promoter/EGFP construct. Type A spermatogonia and somatic cells of other organs were not labeled. This expression pattern made it possible to isolate living cells from the testis of the transgenic mice at the stage of the mitosis-meiosis switch in spermatogenesis using EGFP fluorescence.     

Two novel genes expressed in Xenopus germ line: characteristic features of putative protein structures,their gene expression profiles and their possible roles in gametogenesis and embryogenesis

We compared the secondary spermatogonia and the primary spermatocytes of Xenopus for the proteins in their microsomal fractions and identified a newly synthesized protein (94 kDa) and three other proteins (99, 85, and 72 kDa) which increased their amount after entering the meiotic phase. These four proteins were used as antigens to produce polyclonal antibody which was found to react with the four proteins as well as two other proteins (208 and 60 kDa). Immunoscreening of Xenopus testis cDNA library with this polyclonal antibody yielded two cDNA clones (Xmegs and Xtr) encoding novel proteins. Xmegs mRNA was specifically expressed in the spermatogenic cells from the mid-pachytene stage to completion of two meiotic divisions. The putative Xmegs protein contained 19 tandem repeats of 26 amino acid residues rich in proline as well as potential phosphorylation sites (i.e., serine and threonine residues). Around this repetitive area, we found five PEST sequences known as a proteolytic signal to target protein for degradation. The presence of PEST sequences was believed to allow protein levels to closely parallel mRNA abundance. These results suggested the possible role of this novel protein in the regulation of two meiotic divisions specific to the spermatogenesis in a phosphorylation- and/or dephosphorylation-dependent manner. On the other hand, Xtr mRNA was expressed in both spermatogenic and oogenic cells except for round spermatids and the later stage cells. This mRNA was also expressed in the early stage embryos and its amount was kept constant from the St. I oocyte to the gastrula stage and decreased thereafter. The putative Xtr protein contained four complete and one partial tudor-like domains that were discovered in Drosophila tudor protein which plays an important role in PGC differentiation and abdominal segmentation. The characteristic expression profile of Xtr and the protein structure similar to the Drosophila tudor protein suggested its possible role in the progression of meiosis and PGC differentiation.     

RNA interference during spermatogenesis in mice

Spermatogenesis consists of complex cellular and developmental processes, such as the mitotic proliferation of spermatogonial stem cells, meiotic division of spermatocytes, and morphogenesis of haploid spermatids. In this study, we show that RNA interference (RNAi) functions throughout spermatogenesis in mice. We first carried out in vivo DNA electroporation of the testis during the first wave of spermatogenesis to enable foreign gene expression in spermatogenic cells at different stages of differentiation. Using prepubertal testes at different ages and differentiation stage-specific promoters, reporter gene expression was predominantly observed in spermatogonia, spermatocytes, and round spermatids. This method was next applied to introduce DNA vectors that express small hairpin RNAs, and the sequence-specific reduction in the reporter gene products was confirmed at each stage of spermatogenesis. RNAi against endogenous Dmc1, which encodes a DNA recombinase that is expressed and functionally required in spermatocytes, led to the same phenotypes observed in null mutant mice. Thus, RNAi is effective in male germ cells during mitosis and meiosis as well as in haploid cells. This experimental system provides a novel tool for the rapid, first-pass assessment of the physiological functions of spermatogenic genes in vivo.