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.     

Localization of mouse vasa homolog protein in chromatoid body and related nuage structures of mammalian spermatogenic cells during spermatogenesis

The localization of vasa homolog protein in the spermatogenic cells of mice, rats, and guinea pigs was studied by immunofluorescence and electron microscopies with the antibody against mouse vasa homolog (MVH) protein. By immunofluorescence microscopy, four types of granular staining patterns were identified: (1) fine particles observed in diplotene and meiotic cells, (2) small granules associated with a mitochondrial marker and appearing in pachytene spermatocytes after stage V, (3) strands lacking the mitochondrial marker in late spermatocytes, and (4) large irregularly shaped granules in round spermatids. Immunoelectron microscopy defined the ultrastructural profiles of these MVH protein-positive granules: the first type consisted of small dense particles, the second had intermitochondrial cement (IMC), the third type, consisting of strands, had loose aggregates of either material dissociated from IMC or 70–90-nm particles, and the fourth had typical chromatoid bodies (CBs). The results suggest that MVH proteins function in these components of nuage. MVH protein-positive structures other than CBs disappeared during meiosis and CB appeared first in early spermatids. The results suggest that the formation of nuage is discontinued between spermatocytes and spermatids. The formation of nuage in spermatocytes and of CB in spermatids is discussed.     

Spermatogenesis in XY, XYSxra and XOSxra mice: a quantitative analysis of spermatogenesis throughout puberty

Adult XYSxra mice exhibit varying degrees of spermatogenic deficiency but are usually fertile, while XOSxra mice have severe spermatogenic failure and are always sterile. The present quantitative spermatogenic analysis documents when these anomalies first appear during puberty. The results demonstrate that in XYSxra mice there was increased degeneration of pachytene spermatocytes and, to a lesser extent, meiotic metaphase stages. On average, there were only one-half the number of spermatids compared with the XY controls. The defect in XOSxra mice appeared a little later, with an almost complete arrest and degeneration during the meiotic metaphases, so that the number of spermatids produced was only 3% of the control value. These results are discussed in relation to an hypothesis that links sex chromosome univalence during meiotic prophase with spermatogenic failure.     

Immunohistochemical study of calmodulin in developing mouse testis

The purpose of this study was to determine the localization of calmodulin in the developing mouse testis by the indirect immunoperoxidase method. In addition, the amount of calmodulin in pachytene spermatocytes, spermatids, and residual bodies isolated from the mouse testis and epididymal spermatozoa was quantitated by the adenylate cyclase activation assay and by enzyme immunoassay. The relative levels of calmodulin in the developing mouse testis and in the isolated testicular germ cells were confirmed by western transfer staining. The level of immunoreactive calmodulin was very low in the testes from immature animals. In testes from the mature mouse, calmodulin was found to be localized in spermatocytes and spermatids, but was not found in spermatogonia, Sertoli cells, and interstitial cells. By contrast, immunochemical staining of tubulin was extremely intense in Sertoli cells. Biochemical determinations also showed that pachytene spermatocytes, round spermatids, spermatozoa, and residual bodies contained 14.9 micrograms, 15.8 micrograms, 2.3 micrograms and 5.2 micrograms of calmodulin per mg of protein, respectively. Both the immunochemical and the biochemical studies revealed that levels of calmodulin were high in the spermatocytes and in the round spermatids, as compared to the level in spermatozoa. This fact strongly suggests that the large amount of calmodulin in mammalian testes may be associated primarily with meiotic divisions and/or spermatogenesis.     

Purification of DNA ligases from mouse testis and their behavior during meiosis

Two types of DNA ligase, I and II, have been purified approximately 4,000-fold from mouse testes and 500-fold from nuclei of mouse spermatocytes. DNA ligase I and II consisted of single polypeptides with molecular weights of 95,000 and 65,000, respectively, according to the estimation by SDS-polyacrylamide gel electrophoresis and the AMP-binding assay. Ligase activities were higher in premeiotic spermatogonia and spermatocytes than those in liver and bone marrow cells. Moreover, DNA ligase II showed rapid increase during meiotic prophase and a decrease in round spermatids. Since this behavior of DNA ligase II is consistent with that of m-rec and DNA polymerase beta, both of which have been shown to be involved in DNA recombination in meiotic cells, DNA ligase II might be an enzyme which works at the final step of meiotic recombination reaction.     

Flow cytometric analysis of the effects of 0.4 MeV fission neutrons on mouse spermatogenesis

(C57Bl/Cne X C3H/Cne)F1 male mice were irradiated with single acute doses of 0.4 MeV neutrons ranging from 0.05 to 2 Gy, and testis cell suspensions were prepared for cytometric analysis of the DNA content 2-70 days after irradiation. Various cell subpopulations could be identified in the control histogram including mature and immature spermatids, diploid spermatogonia and spermatocytes, tetraploid cells and cells in the S-phase. Variations in the relative proportions of different cell types were detected at each dose and time, reflecting lethal damage induced on specific spermatogenetic stages. The reduction of the number of elongated spermatids 28 days after irradiation was shown to be a particularly sensitive parameter for the cytometrical assessment of the radiosensitivity of differentiating gonia. A D0 value of 0.13 Gy was calculated and compared with data obtained after X-irradiation, using the same experimental protocol. In the latter case a biphasic curve was obtained over the dose range from 0.25 to 10 Gy, possibly reflecting the existence of some cell population heterogeneity. RBE values were estimated at different neutron doses relative to the radiosensitive component of the X-ray curve, and ranged from 3.3 to 4, in agreement with data in the literature. Genotoxic effects were monitored 7 days after irradiation by a dose-dependent increase of the coefficient of variation (CV) values of the round spermatid peak, reflecting the induction of numerical and structural chromosome aberrations, and 14 or 21 days after irradiation by the detection of diploid elongated spermatids, probably arising from a radiation-induced complete failure of the first or second meiotic division.     

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 relative levels of translin-associated factor X (TRAX) and testis brain RNA-binding protein determine their nucleocytoplasmic distribution in male germ cells

Testis brain RNA-binding protein (TB-RBP), the mouse orthologue of human translin, is an RNA and single-stranded DNA-binding protein abundant in testis and brain. Translin-associated factor X (TRAX) was identified as a protein that interacts with TB-RBP and is dependent upon TB-RBP for stabilization. Using immunohistochemistry to investigate the subcellular locations of TB-RBP and TRAX during spermatogenesis, both proteins localize in nuclei in meiotic pachytene spermatocytes and in the cytoplasm of subsequent meiotic and post-meiotic cells. An identical subcellular distribution is seen in female germ cells. Western blot analysis of germ cell protein extracts reveals an increased ratio of TRAX to TB-RBP in meiotic pachytene spermatocytes compared with the post-meiotic round and elongated spermatids. Using COS-1 cells and mouse embryonic fibroblasts derived from TB-RBP null mice as model systems to examine the shuttling of TB-RBP and TRAX, we demonstrate that TRAX contains a functional nuclear localization signal and TB-RBP contains a functional nuclear export signal. Coexpression of both proteins in COS-1 cells and TB-RBP-deficient mouse embryonic fibroblasts reveals that the ratio of TRAX to TB-RBP determines their subcellular locations, i.e. increased TRAX to TB-RBP ratios lead to nuclear localizations, whereas TRAX remains in the cytoplasm when TB-RBP levels are elevated. These subcellular distributions require interaction between TB-RBP and TRAX. We propose that the subcellular locations of TB-RBP and TRAX in male germ cells are modulated by the relative ratios of TRAX and TB-RBP.     

Ovulation and fertilization of primary and secondary oocytes in LT/Sv strain mice

In this study, the chromosome constitution of both unfertilized oocytes and fertilized eggs isolated from the oviducts of LT/Sv strain mice were analyzed. Air-dried chromosome preparations from unfertilized oocytes revealed that about one-third of those examined were ovulated as primary oocytes. These were arrested at metaphase of the first meiotic division and exhibited the characteristic “tetrad” chromosome configuration. The remaining two-thirds of the unfertilized oocytes were ovulated at metaphase of the second meiotic division. The fertilized eggs were isolated from the oviducts of LT/Sv females previously mated to (C57BL × CBA) F1 hybrid males. Analysis of the fertilized eggs at metaphase of their first cleavage mitosis revealed that about one-third of the eggs examined were digynic triploids, whereas the remaining two-thirds had the normal diploid chromsome constitution. In the triploids, the 40 female chromosomes present (mouse, n = 20) were derived from a single diploid pronucleus formed after the extrusion of a first polar body, and following the monospermic fertilization of primary oocytes. The female pronuclear-derived chromosomes invariably exhibited “homologous pairing,” and these were associated at their centromeres. The ovulation, penetration, and subsequent fertilization of primary oocytes is an extremely unusual phenomenon in mammals and only appears to occur on a regular basis in LT/Sv mice. The premature “cytoplasmic maturation” of these oocytes is of interest, as they clearly have the same developmental capacity as secondary oocytes. The significance of these observations in relation to folliculogenesis and litter size in LT/Sv mice is discussed.     

How is the flagellar length of mature sperm determined? III. Comparison of initial growth rate of flagella in spermatids from Cynops and Xenopus in the presence and absence of cycloheximide.

Previous studies on flagellar growth in round spermatids from Cynops and Xenopus in vitro have shown that the period and rate of flagellar growth are greater in Cynops than in Xenopus. The present study shows, however, that during the initial phase of flagellar growth (for the first 12 h following the second meiotic division), the growth rate is very similar in both Cynops and Xenopus (0.5-0.6 microns/h at 22 degrees C). The difference in the growth rate between Cynops and Xenopus was observed beyond 12 h following the second meiotic division. When round spermatids in both species were inoculated with 10 microM cycloheximide, flagella grew at the same rate as in the absence of cycloheximide for the first 12 h following the second meiotic division. Beyond 12 h, however, cycloheximide suppressed flagellar growth in round spermatids in both species. These results indicate that the initial flagellar growth in round spermatids is provided for by flagellar protein pools which were present just after the second meiotic division; the growth beyond 12 h in round spermatids is contributed by newly synthesized flagellar proteins.     

Sexual characteristics and spermatogenesis in males of the parthenogenetic gecko Lepidodactylus lugubris (Reptilia, Gekkonidae)

Obligately parthenogenetic lizards usually are all-female populations of hybrids producing diploid oocytes by premeiotic endomitosis and quasi-normal meiosis. In an all-female strain of the gekkonid lizard Lepidodactylus lugubris several phenotypic males arose spontaneously. The sexual characteristics of these males were studied using light and electron microscopy and compared with normal males of the bisexual genus Lygodactylus. Emphasis was layed on morphology of seminiferous tubules, occurrence of spermatogenic stages and ultrastructure of spermatozoa. The phenotypic males possessed preanal pores filled with secretions and a sexual nephric segment which were exactly the same as in normal, reproductively active males. In the testes, density and morphology of non-spermatogenic cell types, the Leydig and Sertoli cells, indicate a normal production of testicular testosterone and a normal function of the blood-testis barrier, respectively. Both in the normal and the phenotypic males, all meiotic cell types of spermatogenesis can be recognised in the seminiferous tubules and are apparently identical, indicating a normal meiosis without impairment in the phenotypic males. In contrast, the differentiation process of spermatids is markedly disturbed in the phenotypic males of L. lugubris. In the normal male, spermiogenesis results in mature spermatids and spermatozoa with small elongated nuclei, an acrosomal complex, and a flagellar tail possessing one axoneme. Spermatozoa fill both the lumen of most seminiferous tubules and the lumina of ductus epididymidis and ductus deferens. In the phenotypic male, spermiogenesis results in seemingly normal spermatids and in spermatozoa with large, non-elongated, deformed nuclei and/or irregular tails possessing more than one axoneme. Both the lumen of most seminiferous tubules and the lumina of the ductus epididymidis and the ductus deferens contain relatively few spermatozoa. We suggest that the phenotypic males inherited the ability for a premeiotic endomitosis from their all-female ancestral lineage. While in females this leads to quasi-normal meiosis and diploid oocytes capable of development, the small nuclei of the spermatozoa are unable to contain a diploid set of chromosomes. Because of the high amount of deformed spermatozoa and possibly uncontrolled loss of genetic material in structurally normal, but aneuploid spermatozoa we conclude that these otherwise perfect males are infertile, thus constituting another example of gametic sterility.     

Aneuploidy in late-step spermatids of mice detected by two-chromosome fluorescence in situ hybridization

A multicolor procedure employing fluorescence in situ hybridization is described for detecting chromosomal domains and germinal aneuploidy in late-step spermatids in mice using DNA probes specific for repetitive sequences near the centromeres of chromosomes 8 and X. These probes were nick-translated with biotin- or digoxigenin-labeled nucleotides, and were detected with FITC or rhodamine. Probe and hybridization specificities were confirmed using metaphase chromosomes from spleen and bone marrow cells as well as from primary and secondary spermatocytes. Late-step spermatids, identified in testicular preparations by their hooked shape, yielded compact fluorescence domains in ～ 50% and > 99% of cells when hybridized with probes for chromosomes X and 8, respectively. In a survey of > 80,000 late-step spermatids from 8 healthy young adult C57BL/6 or B6C3F1 mice, ～ 3/10,000 spermatids had fluorescence phenotypes indicative of X-X or 8–8 hyperhaploidy. These frequencies are consistent with published frequencies of aneuploidy in meiotic metaphase II and first cleavage metaphases of the mouse, providing preliminary validation of sperm hybridization for the detection of aneuploidy. No significant animal or strain differences were observed. In addition, the hyperhaploidy frequencies for murine spermatids were indistinguishable for those for sperm from healthy men obtained by a similar hybridization procedure. These procedures for detecting aneuploid male gametes are examples of “bridging biomarkers” between human and animal studies. They have promising applications for investigations of the genetic, reproductive, and toxicological factors leading to abnormal reproductive outcomes of paternal origin. © 1995 Wiley-Liss, Inc.     

Cytological and expression studies and quantitative analysis of the temporal and stage-specific effects of follicle-stimulating hormone and testosterone during cocultures of the normal human seminiferous epithelium

In vitro culturing of normal human seminiferous epithelium remains largely unexplored. To study normal human spermatogenesis in vitro, we used a micromethod for the purification and culture of Sertoli cells, spermatogonia A, spermatocytes, and early round spermatids. Cytological quantitative data for Sertoli and premeiotic germ cell cocultures isolated from normal testicular biopsies demonstrated that cells were able to proliferate (4%), complete meiosis (6.7%), and differentiate into late round (54%), elongating (49%), and elongated (17%) spermatids at similar in vivo time delays (up to 16 days) in response to FSH + testosterone stimulation. Cells maintained normal meiotic segregation, chromosome complements, and specific gene expression profiles. Follicle-stimulating hormone + testosterone stimulated spermatogonia proliferation and Sertoli cell survival. Follicle-stimulating hormone and especially FSH + testosterone increased diploid germ cell survival during the first week, whereas only FSH + testosterone was able to inhibit cell death during the second week of culture. Follicle-stimulating hormone and especially FSH + testosterone also stimulated meiosis resumption, although this was restricted to late pachytene and secondary spermatocytes. In contrast, spermiogenesis was only stimulated by FSH + testosterone. Expression studies showed that apoptosis was induced in the nucleus of diploid cells, and in nuclear and cytoplasmic compartments of spermatids, mainly triggered by the Fas pathway. Although junctional complexes between Sertoli and premeiotic germ cells were partially reacquired, the same did not apply to spermatids, suggesting that FSH potentiated by testosterone was unable to render Sertoli cells competent to bind round spermatids.