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.     

The whole meiotic process can occur in vitro in untransformed rat spermatogenic cells

The aim of the present study was to assess whether the whole meiotic process of spermatogenic cells is able to take place in vitro. Fragments of seminiferous tubules from 20- to 22- or 28-day-old rats were seeded in medium containing 0.2% fetal calf serum in bicameral chambers and then cultured for 4 weeks in a chemically defined medium. The differentiation of meiotic germinal cells was followed by four criteria: (i) ultramicroscopic examination of the different types of germ cells present in the cell layer throughout the culture period; (ii) determination of the changes in DNA content per nucleus of the cell population seeded with time in culture; (iii) assessment of the ability of germinal cells to transcribe genes expressed after completion of meiosis; and (iv) monitoring the fate of BrdU-labeled leptotene spermatocytes. The ultrastructural study showed that the overall organization of the cells in the culture well recalls that of the seminiferous epithelium throughout the culture period. Moreover the identification of young round spermatids 21 days after seeding suggested that these spermatids had been formed very recently in culture. Determination of DNA content per nucleus showed that a 1C cell population could be observed after several days of cultures reaching 6 to 10% of total cells. An exponential-like increase in the amounts of the mRNAs encoding for TP1 or TP2 occurred from the time when 1C cells appeared in the culture until the end of the experiment. Finally, BrdU-labeled leptotene spermatocytes differentiated into pachytene spermatocytes and then into secondary spermatocytes, and BdrU-labeled round spermatids were observed from Day 21 of culture onward. Taken together these results indicate that the whole meiotic process from leptotene spermatocyte to round spermatid can indeed occur in vitro under the present culture conditions.     

Definitive expression of c-mos in late meiotic prophase leads to phosphorylation of a 34 kda protein in cultured rat spermatocytes

To investigate the role of c-mos in rat spermatogenesis, expression of c-mos, MAP kinase kinase (MAPKK), MAP kinase (MAPK), cdc2 and protein kinase A (PKA) by spermatogenic cell culture of 14 day-old rats was examined. MAPKK and PKA expressions were constitutive, whereas the expression of MAPK and cdc2 in spermatogonia initially decreased, but later increased on meiotic maturation of spermatocytes. c-mos expression was definitive of late meiotic prophase. c-mos immunoprecipitates prepared from the c-mos-enriched fraction (pI9.0-9.6) could form complex(es) in the cultured spermatogenic cell lysates. In vitro phosphorylation of the c-mos immune complexes revealed a 34 kDa protein that was phosphorylated at serine and threonine residues as a target of the c-mos signal. Its pI value was 4.4-4.5, and cdc2 was not detected, making it different from cdc2 (p34). These results suggest that the phosphorylation of the 34 kDa protein by the c-mos signal may play a crucial role in the meiotic division of rat spermatocytes.     

Regular and irregular divisions of Lepidoptera spermatocytes as related to the speed of meiotic prophase

Lepidoptera males bear two kinds of meiotic divisions. One is regular (eupyrene) and leads to nucleate, fertilizing spermatozoa. The other (apyrene) shows metaphase I chromosomes clumping together into irregular masses which later split forming daughter cells with unbalanced sets of chromosomes which are eventually extruded from the cells; hence, the spermatids develop into anucleate spermatozoa of unknown function. The apyrene divisions are induced by a haemolymph factor which becomes functional towards pupation. Using incorporation of tritiated thymidine at the premeiotic S-phase as a marker for timing, it was found that the prophase of the apyrene spermatocyte is shorter than that of the eupyrene spermatocyte. It is proposed that meiosis-specific proteins cannot be synthesized during the shortened apyrene prophase and that this is correlated with the irregular chromosome behaviour during the subsequent metaphase-telophase of these spermatocytes.     

Immunoreactive sites and accumulation of somatomedin-C in rat Sertoli-spermatogenic cell co-cultures

Sertoli-spermatogenic cell co-cultures prepared from sexually immature rats (20-22 days old) and maintained in serum-free, hormone/growth factor-supplemented medium were used to determine the cell-specific localization of the growth factor somatomedin-C (SM-C). SM-C localization studies were carried out by indirect immunofluorescence using a monoclonal antibody (sm-1.2) to SM-C. In cultured rat hepatocytes, Sertoli and testicular peritubular cells, SM-C immunoreactivity was observed as a diffuse distribution of discrete immunofluorescent granules. Radio-immunoassay experiments using a rabbit antibody against human SM-C showed that testicular peritubular cells and Sertoli cells in primary culture accumulated SM-C in the medium. In spermatogenic cells co-cultured with subjacent Sertoli cells, immunoreactive SM-C was associated with pachytene spermatocytes but not with spermatogonia or early meiotic prophase spermatocytes (leptotene or zygotene). Both Sertoli cells and pachytene spermatocytes displayed binding sites for exogenously added SM-C. SM-C6 binding to spermatocytes reaching an advanced stage of meiotic prophase suggests a possible role of this growth factor in the meiotic process.     

Reporter mice express green fluorescent protein at initiation of meiosis in spermatocytes

Transgenic mice were generated using a heat shock protein 2 (Hspa2) gene promoter to express green fluorescent protein (GFP) at the beginning of meiotic prophase I in spermatocytes. Expression was confirmed in four lines by in situ fluorescence, immunohistochemistry, western blotting, and PCR assays. The expression and distribution of the GFP and HSPA2 proteins co‐localized in spermatocytes and spermatids in three lines, but GFP expression was variegated in one line (F46), being present in some clones of meiotic and post‐meiotic germ cells and not in others. Fluorescence activated cell sorting (FACS) was used to isolate purified populations of spermatocytes and spermatids. Although bisulfite sequencing revealed differences in the DNA methylation patterns in the promoter regions of the transgene of the variegated expressing GFP line, a uniformly expressing GFP reporter line, and the Hspa2 gene, these differences did not correlate with variegated expression. The Hspa2‐GFP reporter mice provide a novel tool for studies of meiosis by allowing detection of GFP in situ and in isolated spermatogenic cells. They will allow sorting of meiotic and post‐meiotic germ cells for characterization of molecular features and correlation of expression of GFP with stage‐specific spermatogenic cell proteins and developmental events. genesis 52:976–984, 2014. © 2014 Wiley Periodicals, Inc.     

Identification and immunochemical characterization of spermatogenic cell surface antigens that appear during early meiotic prophase

Three spermatogenic cell populations isolated from prepuberal mice--type B spermatogonia, preleptotene spermatocytes, and leptotene/zygotene spermatocytes--were used to elicit distinct polyclonal antisera. Surface binding specificities were determined for purified IgGs by indirect immunofluorescence and rosette assays on live cells. Binding activities were assayed both before and after absorptions with a variety of somatic and spermatogenic cells. Each of these antisera binds to surface antigens that are present on germ cells throughout spermatogenesis and are not shared by splenocytes, thymocytes, and erythrocytes. Only the antiserum raised against leptotene and zygotene spermatocytes (ALZ) recognizes a stage-specific subset of surface determinants. After appropriate absorptions, ALZ binds to the surface of early pachytene spermatocytes and germ cells at subsequent stages of differentiation, including vas deferens spermatozoa. Antigens which react with this absorbed IgG are not detected on the surface of spermatogonia or meiotic cells prior to pachynema, including leptotene and zygotene spermatocytes. The observed binding specificities may result from the synthesis of one or more surface molecules during the early meiotic stages, followed by delayed insertion into the plasma membrane during the pachytene stage of meiotic prophase. Stage-specific antigens recognized by ALZ, including both protein and probably lipid, have been localized immunochemically on nitrocellulose blots from one-dimensional SDS gels. A dithiothreitol-sensitive constituent (Mr approximately 39,000) recognized by ALZ has been identified as the major protein determinant present in early meiotic cells but absent in 8-day-old seminiferous cell suspensions containing spermatogonia and Sertoli cells. This determinant is present in populations of preleptotene, leptotene/zygotene, and early pachytene spermatocytes isolated from 17-day-old animals, an observation consistent with the hypothesis of delayed insertion into the plasma membrane.     

DNA methyltransferase is developmentally expressed in replicating and non-replicating male germ cells.

Genomic methylation patterns are established during maturation of primordial germ cells and during gametogenesis. While methylation is linked to DNA replication in somatic cells, active de novo methylation and demethylation occur in post-replicative spermatocytes during meiotic prophase (1). We have examined differentiating male germ cells for alternative forms of DNA (cytosine-5)-methyltransferase (DNA MTase) and have found a 6.2 kb DNA MTase mRNA that is present in appreciable quantities only in testis; in post-replicative pachytene spermatocytes it is the predominant form of DNA MTase mRNA. The 5.2 kb DNA MTase mRNA, characteristic of all somatic cells, was detected in isolated type A and B spermatogonia and haploid round spermatids. Immunobolt analysis detected a protein in spermatogenic cells with a relative mass of 180,000-200,000, which is close to the known size of the somatic form of mammalian DNA MTase. The demonstration of the differential developmental expression of DNA MTase in male germ cells argues for a role for testicular DNA methylation events, not only during replication in premeiotic cells, but also during meiotic prophase and postmeiotic development.     

Establishment of in vitro spermatogenesis from spermatocytes in the medaka, Oryzias latipes

Spermatocytes of the teleost, Oryzias latipes , at meiotic prophase were cultured without contact with somatic cells. They began to divide, progressing through the meiotic divisions and differentiating into round spermatids within 48 h. The chromosome number in both the primary and secondary spermatocytes at metaphase was n = 24. In spermatids, a single flagellum was formed and the release of residual bodies was observed in vitro . The size and shape of the flagellum were the same as those seen in vivo . The expression of protamine mRNA was detected in round spermatids. This result suggests that gene expression, as well as morphological change, is regulated by the progression of spermatogenesis in cell culture. Furthermore, when the eggs of O. latipes were inseminated with germ cells cultured for 10 days, normal embryos developed and hatched out. These results suggest that the spermatocytes of O. latipes develop into fertile sperm in cell culture.     

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.     

Deficiency of X and Y chromosomal pairing at meiotic prophase in spermatocytes of sterile interspecific hybrids between laboratory mice (Mus domesticus) and Mus spretus

The normal association between the X and Y chromosomes at metaphase I of meiosis, as seen in air-dried light microscope preparations of mouse spermatocytes, is frequently lacking in the spermatocytes of the sterile interspecific hybrid between the laboratory mouse strains C57BL/6 and Mus spretus. The purpose of this work is to determine whether the separate X and Y chromosomes in the hybrid are asynaptic, caused by failure to pair, or desynaptic, caused by precocious dissociation. Unpaired X-Y chromosomes were observed in air-dried preparations at diakinesis, just prior to metaphase I. Furthermore, immunocytology and electron microscopy studies of surface-spread pachytene spermatocytes indicate that the X and Y chromosomes frequently fail to initiate synapsis as judged by the failure to form a synaptonemal complex between the pairing regions of the X and Y Chromosomes. Several additional chromosomal abnormalities were observed in the hybrid. These include fold-backs of the unpaired X or Y cores, associations between the autosome and sex chromosome cores, and autosomal univalents. The occurrence of abnormal autosomal and XY-autosomal associations was also correlated with cell degeneration during meiotic prophase. The primary breakdown in hybrid spermatogenesis occurs at metaphase I (MI), with the appearance of degenerated cells at late MI. In those cells, the X and Y are decondensed rather than condensed as they are in normal mouse MI spermatocytes. These results, in combination with the previous genetic analysis of spermatogenesis in hybrids and backcrosses with fertile female hybrids, suggest that the spermatogenic breakdown in the interspecific hybrid is primarily correlated with the failure of XY pairing at meiotic prophase, asynapsis, followed by the degeneration of spermatocytes at metaphase I. Secondarily, the failure of XY pairing can be accompanied by failure of autosomal pairing, which appears to involve an abnormal sex vesicle and degeneration at pachytene or diplotene.by C. Heyting     

Pachytene spermatocytes can achieve meiotic process in vitro

Highly enriched pachytene spermatocytes prepared from adult rats by centrifugal elutriation were tested for their capacity to enter the meiotic process when cocultured with 20-day old rat Sertoli cells. This was traced by phase-contrast microscopy and by DNA flow cytometry. We also performed a Northern blot analysis using the mouse protamine I cDNA as a probe, the expression of which being restricted to spermatids. Our results demonstrate that pachytene spermatocytes cocultured with Sertoli cells developed into spermatids. The number of pachytene spermatocytes entering meiosis was affected neither by growth factors nor by hormones. However these later were required in long term cocultures for the maintenance of cell integrity and viability.     

Acrosin biosynthesis in meiotic and postmeiotic spermatogenic cells.

It has been widely accepted that mammalian sperm acrosin is first synthesized only in the postmeiotic stages of spermatogenic cells. In this study, we carried out Northern blot analysis of RNAs prepared from purified populations of mouse spermatogenic cells. The acrosin mRNA was obviously found in meiotic pachytene spermatocytes, and the mRNA content markedly increased in postmeiotic round spermatids. Also, the acrosin mRNA in pachytene spermatocytes was functionally associated with polysomes. These results provide evidence that acrosin biosynthesis is already started in meiotic cells and continues through the early stages of spermiogenesis.     

Formation of flagella during interphase in secondary spermatocytes from Xenopus laevis in vitro

In cell culture, single motile flagella, 1 micron in length, were observed to grow from secondary spermatocytes of Xenopus laevis within 2-3 hours after telophase I, at 22 degrees C. About 90% of the secondary spermatocytes formed flagella as observed by phase-contrast microscopy. The flagella grew up to 2-6 microns in length during interphase II, which lasted about 18 hours. The presence of the "9 + 2" microtubular structure of the flagellar axonemes of secondary spermatocytes was confirmed by electron microscopy. When chromosomal condensation began (prophase II), the flagella were resorbed into the cells and, after the second meiotic division, a flagellum was formed again by each of the round spermatids. Thus, there appears to be a close relationship between the meiotic division cycle and the formation of flagella. The possible contribution of Sertoli cells to the formation of flagella in secondary spermatocytes was examined by reducing the number of Sertoli cells to less than ten per culture. Under these conditions, flagella formed in secondary spermatocytes with very high efficiency. It is very likely that secondary spermatocytes form flagella in vivo, since the secondary spermatocytes were observed to have flagella immediately after dissociation of the testes.     

Biosynthesis of specific histones during meiotic prophase of mouse spermatogenesis

A kinetics study has demonstrated histone synthesis occurring at two distinct phases during meiotic prophase of mouse spermatogenesis. These two periods have been delineated by quantifying the synthesis of DNA and basic nuclear proteins in spermatogenic cells at discrete intervals following the intratesticular injection of [3H] thymidine and [14C] arginine, respectively. One phase of histone synthesis occurs coincident with DNA synthesis in preleptotene spermatocytes. By contrast, a second phase of histone synthesis occurs during midprophase of meiosis, independent of semiconservative DNA synthesis. The [14C] arginine incorporated into the basic nuclear proteins of pachytene spermatocytes is conserved during spermiogenesis and then subsequently discarded within the residual bodies, which are formed during late spermiogenesis. Fluorographic analyses of isotopically labeled basic nuclear proteins in pachytene spermatocytes has shown that only the somatic complement of histones are synthesized during the preleptotene period, whereas the second phase involves the synthesis of proteins H1t, H2S, and "A". In addition, several nonhistone basic nuclear proteins are synthesized concomitant with the germ cell-specific histones. Thus, the data clearly demonstrate that pachytene spermatocytes actively synthesize a number of novel chromatin-associated polypeptides.