Testicular biopsy specimens from 16 fertile and 10 subfertile men with normal male karyotype were studied quantitatively to provide histological and cytogenetic data for a basis of reference in assessing abnormalities of spermatogenesis. Histological studies included estimation of the proportion and activity of germinal epithelium and an assessment of tubular morphology. In cytogenetic preparations, counts were made of cells at different stages of meiosis. Studies of cells at diakinesis included chiasma counts and percentage of cells with dissociated sex chromosomes. One fertile and six subfertile men showed decreased germinal activity; the six subfertile men also had decreased MII/MI ratios. Other findings were similar in the two groups.
Failure of homologous synapsis during meiotic prophase triggers transcriptional repression. Asynapsis of the X and Y chromosomes and their consequent silencing is essential for spermatogenesis. However, asynapsis of portions of autosomes in heterozygous translocation carriers may be detrimental for meiotic progression. In fact, a wide range of phenotypic outcomes from meiotic arrest to normal spermatogenesis have been described and the causes of such a variation remain elusive. To better understand the consequences of asynapsis in male carriers of Robertsonian translocations, we focused on the dynamics of recruitment of markers of asynapsis and meiotic silencing at unsynapsed autosomal trivalents in the spermatocytes of Robertsonian translocation carrier mice. Here we report that the enrichment of breast cancer 1 (BRCA1) and histone γH2AX at unsynapsed trivalents declines during the pachytene stage of meiosis and differs from that observed in the sex body. Furthermore, histone variant H3.3S31, which associates with the sex chromosomes in metaphase I/anaphase I spermatocytes, localizes to autosomes in 12% and 31% of nuclei from carriers of one and three translocations, respectively. These data suggest that the proportion of spermatocytes with markers of meiotic silencing of unsynapsed chromatin (MSUC) at trivalents depends on both, the stage of meiosis and the number of translocations. This may explain some of the variability in phenotypic outcomes associated with Robertsonian translocations. In addition our data suggest that the dynamics of response to asynapsis in Robertsonian translocations differs from the response to sex chromosomal asynapsis in the male germ line.
47,XYY syndrome is a sex chromosomal abnormality observed in humans, with a prevalence of 0.1% of male births. Sex chromosome anomalies are more frequently associated with male infertility.
We present here four cases of infertile men with azoospermia or severe oligozoospermia attending a genetic and fertility clinic. Chromosomal analysis of the peripheral blood lymphocytes demonstrated the constitutional karyotype of 47, XYY. Using fluorescence in situ hybridization (FISH) the presence of extra Y chromosome was confirmed, supporting the cytogenetic finding.
The 47,XYY syndrome is relatively uncommon and can be missed clinically because of its variable clinical presentations. Accurate diagnosis of this constitutional karyotype provides a valuable aid in the counselling and early management of the patients who undertake fertility evaluation.
Spermatogenesis uses mitotic and meiotic cell cycles coordinated with growth and differentiation programs to generate functional sperm. Our analysis of a Drosophila mutant has revealed that asunder (asun), which encodes a conserved protein, is an essential regulator of spermatogenesis. asun spermatocytes arrest during prophase of meiosis I. Strikingly, arrested spermatocytes contain free centrosomes that fail to stably associate with the nucleus. Spermatocytes that overcome arrest exhibit severe defects in meiotic spindle assembly, chromosome segregation, and cytokinesis. Furthermore, the centriole-derived basal body is detached from the nucleus in asun postmeiotic spermatids, resulting in abnormalities later in spermatogenesis. We find that asun spermatocytes and spermatids exhibit drastic reduction of perinuclear dynein–dynactin, a microtubule motor complex. We propose a model in which asun coordinates spermatogenesis by promoting dynein–dynactin recruitment to the nuclear surface, a poorly understood process required for nucleus–centrosome coupling at M phase entry and fidelity of meiotic divisions.
Purpose: Our objective was to apply ooplasmic round spermatid nuclear injections for the treatment of nonobstructive azoospermia.
Materials: Participants were nine azoospermic men who had previously undergone diagnostic testicular biopsy. Spermatogenetic arrest was diagnosed at the round spermatid stage (n=6) or primary spermatocyte stage (n=3). A second (therapeutic) testicular biopsy was performed and round spermatid nuclei were recovered from all the participants.
Results: Forty-nine mature oocytes were successfully injected with nuclei and then cultured for 72 hr. Twenty-four embryos were transferred to nine women. No pregnancy was achieved.
Conclusions: Round spermatids can be recovered from therapeutic testicular biopsy material of men negative for round spermatids in previous routine diagnostic testicular biopsy specimens. Round spermatid nuclear injections may play a role in the treatment of nonobstructive azoospermia.
azoospermia; infertility; oocyte; round spermatid
Details are given of a balanced 21q22q Robertsonian translocation ascertained through infertility in a phenotypically normal male. Chromosome analyses on the proband and his parents showed that the translocation arose as a new mutation. The patient was oligospermic and had a high frequency of morphological abnormalities in his spermatozoa. Meiotic investigations showed a chain trivalent in all primary spermatocytes examined at diakinesis/metaphase I. The testicular histology was normal.
An amniotic fluid sample from an in vitro fertilized pregnancy was referred for cytogenetic analysis based on a Down syndrome screening risk of 1 : 21. Routine cytogenetic analysis showed a nonmosaic karyotype of 46,XX,r(21)(p11.2q22.3), with partial monosomy for chromosome 21 due to a ring chromosome replacing one of the normal homologues. Detailed ultrasound scanning for the remainder of the pregnancy did not reveal any unusual findings. Parental bloods showed that the mother was mosaic for the ring 21 with a karyotype of 46,XX,r(21)(p11.2q22.3)/46,XX and the father had an unrelated Robertsonian translocation, with a karyotype of 45,XY,rob(13;14)(q10;q10). Microarray analysis of cultured amniocytes determined the extent of the deletion of chromosome 21 material in the ring. The parents were given genetic counselling, and a phenotypically normal female baby was delivered at term. This case highlights the importance of karyotyping as an initial step in the management of couples referred for in vitro fertilization.
Abnormal children of two 47,XYY men were studied. One of these men had 2 normal daughters and a child, 45,X/46,XY, with gonadal dysgenesis. The other man had 2 normal sons and a child with Down's syndrome. The extra chromosome 21 of this child came from the mother. Another 47,XYY man had 4 normal children.
Translational control plays a crucial role during gametogenesis in organisms as different as worms and mammals. Mouse knockout models have highlighted the essential function of many RNA-binding proteins during spermatogenesis. Herein we have investigated the expression and function during mammalian male meiosis of Sam68, an RNA-binding protein implicated in several aspects of RNA metabolism. Sam68 expression and localization within the cells is stage specific: it is expressed in the nucleus of spermatogonia, it disappears at the onset of meiosis (leptotene/zygotene stages), and it accumulates again in the nucleus of pachytene spermatocytes and round spermatids. During the meiotic divisions, Sam68 translocates to the cytoplasm where it is found associated with the polysomes. Translocation correlates with serine/threonine phosphorylation and it is blocked by inhibitors of the mitogen activated protein kinases ERK1/2 and of the maturation promoting factor cyclinB-cdc2 complex. Both kinases associate with Sam68 in pachytene spermatocytes and phosphorylate the regulatory regions upstream and downstream of the Sam68 RNA-binding motif. Molecular cloning of the mRNAs associated with Sam68 in mouse spermatocytes reveals a subset of genes that might be posttranscriptionally regulated by this RNA-binding protein during spermatogenesis. We also demonstrate that Sam68 shuttles between the nucleus and the cytoplasm in secondary spermatocytes, suggesting that it may promote translation of specific RNA targets during the meiotic divisions.
Post-transcriptional modification by SUMOylation is involved in numerous cellular processes including human spermatogenesis. For human male meiosis, we previously showed that the small ubiquitin-related modifier-1 (SUMO-1) protein localizes to chromatin axes in early pachytene spermatocytes, then to kinetochores as meiosis progresses. Here, we delineate possible functional roles based on subcellular localization for SUMO-1 and SUMO-2/3.
Western and immunoprecipitation analyses were conducted on proteins isolated from the testis of two normal adult fertile men. Combinatorial immunofluorescence and chromosome-specific fluorescence in situ hybridization analyses were performed on male meiocytes obtained during testicular biopsy from four patients undergoing testicular sperm extraction for assisted reproduction technologies.
The synaptonemal complex (SC) and SC proteins (SCP)-1 and SCP2, but not SCP3, are SUMOylated by SUMO-1 during the pachytene substage. Likewise, two distinct localization patterns for SUMO-1 are identified: a linear pattern co-localized with autosomal SCs and isolated SUMO-1 near the centromeric heterochromatin of chromosomes 9 and 1. In contrast to SUMO-1, which is not detectable prior to pachytene in normal tissue, SUMO-2/3 is identified as early as leptotene and zygotene and in some, but not all, pachytene cells; no linear patterns were detected. Similar to SUMO-1, SUMO-2/3 localizes in two predominant subnuclear patterns: a single, dense signal near the centromere of human chromosome 9 and small, individual foci co-localized with autosomal centromeres.
Our data suggest that SUMO-1 may be involved in maintenance and/or protection of the autosomal SC. SUMO-2/3, though expressed similarly, may function separately and independently during pachytene in men.
small ubiquitin-related modifier-1; men; meiosis; heterochromatin; chromosome
Disturbed spermatogenesis and azoospermia are reported in a man with a deleted Y chromosome. The anomalous Y chromosome appears in the karyotype as a small metacentric marker. In situ hybridisation using three different Y specific DNA probes shows that deletion at Yq11 has resulted in loss of all distal heterochromatin. The sterility of the patient indicates loss also of the azoospermia factor (AZF) located at the Yq distal euchromatic/heterochromatic interface. Microspread and air dried meiotic preparations show a severe impairment of spermatogenesis but rare cells are seen to be progressing to the late prophase stage. The testicular histology shows most of the seminiferous tubules to be completely hyalinised. The father and a fertile brother of the proband show a satellited Y chromosome (Yqs) in their karyotypes. The case appears to be the first of its kind reported in which a father with a satellited Y chromosome has produced a son carrying a different Y chromosome anomaly. The possible derivation of the one from the other is discussed.
Chromosomal abnormalities are seen in nearly 1% of live born infants. We report a 5-year-old boy with the clinical features of Down syndrome, which is the most common human aneuploidy. Cytogenetic analysis showed a mosaicism for a double aneuploidy, Down syndrome and XYY. The karyotype was 47, XY,+21/48, XYY,+21. ish XYY (DXZ1 × 1, DYZ1 × 2). Mosaic double aneuploidies are very rare and features of only one of the aneuploidies may predominate in childhood. Cytogenetic analysis is recommended even if the typical features of a recognized aneuploidy are present so that any associated abnormality may be detected. This will enable early intervention to provide the adequate supportive care and management.
Disorder of sexual development; double aneuploidy; Down syndrome with XYY; meiotic non-disjunction; mosaic
Spermatogenesis in the nematode Caenorhabditis elegans uses unusual organelles, called the fibrous body-membranous organelle (FB-MO) complexes, to prepackage and deliver macromolecules to spermatids during cytokinesis that accompanies the second meiotic division. Mutations in the spe-4 (spermatogenesis-defective) gene disrupt these organelles and prevent cytokinesis during spermatogenesis, but do not prevent completion of the meiotic nuclear divisions that normally accompany spermatid formation. We report an ultrastructural analysis of spe-4 mutant sperm where the normally close association of the FB's with the MO's and the double layered membrane surrounding the FB's are both defective. The internal membrane structure of the MO's is also disrupted in spe-4 mutant sperm. Although sperm morphogenesis in spe-4 mutants arrests prior to the formation of spermatids, meiosis can apparently be completed so that haploid nuclei reside in an arrested spermatocyte. We have cloned the spe-4 gene in order to understand its role during spermatogenesis and the molecular basis of how mutation of this gene disrupts this process. The spe-4 gene encodes an approximately 1.5-kb mRNA that is expressed during spermatogenesis, and the sequence of this gene suggests that it encodes an integral membrane protein. These data suggest that mutation of an integral membrane protein within FB-MO complexes disrupts morphogenesis and prevents formation of spermatids but does not affect completion of the meiotic nuclear divisions in C. elegans sperm.
Banding studies in 25 Robertsonian translocations showed that all could be interpreted as stable dicentrics. The mechanism for their stability is likely to be the proximity of their centromeres but centromeric suppression could also have a role. In many of these dicentric translocations, discontinuous centromeric suppression, as indicated by chromatid separation at one of the centromeric regions, was observed in C-banded preparations. A further observation of undefined relation to the first was that the ratio of the two constitutive centromeric heterochromatin (CCH) regions from the component chromosomes of the translocations was variable in the same translocation type, e.g. t(13;14). It is proposed that this ratio may influence the segregation ratio. Abnormal spermatogenesis is suggested as the likely mechanism for the difference in the proportion of aneuploid offspring in the progeny of maternal and paternal heterozygotes. Neither of the t dic(21;21)s could be interpreted as isochromosomes. It is proposed that Robertsonian fusion translocations be defined as stable, dicentric, whole-arm translocations, with both centromeres in a median position and resulting in the loss of a small acentric fragment during this formation. It is suggested that they occur at high frequency between telocentric or, as in man, certain acrocentric chromosomes because of some intrinsic property of those chromosomes not possessed by metacentric chromosomes and mediated by interphase association of centromeres.
Jumping translocations are a rare type of mosaicism in which the same portion of one donor chromosome is translocated to several recipient chromosomes. Constitutional forms of jumping translocations are rare, and the 48 cases reported to date have been associated with both normal and abnormal phenotypes. Concurrence of isochromosome (i) of one arm and translocation of the other is also rare, with seven reported cases. We describe a unique case involving concurrence of i(Yp) and a jumping translocation of Yq to the telomere of chromosomes 12q and 17q, which resulted in five cell lines.
The patient, an otherwise healthy 35-year-old man, was referred for cytogenetic studies because of absolute azoospermia. He had elevated levels of follicle stimulating hormone and luteinizing hormone, consistent with abnormal spermatogenesis, and decreased levels of free testosterone and inhibin B. G-banded chromosome analysis revealed a mosaic male karyotype involving five abnormal cell lines. One of the cell lines showed loss of chromosome Y and presence of i(Yp) as the sole abnormality. Three cell lines exhibited jumping translocation: two involved 17qter, and the other involved 12qter as the recipient and Yq as the common donor chromosome. One of the cell lines with der(17) additionally showed i(Yp). The other der(17) and der(12) cell lines had a missing Y chromosome. All five cell lines were confirmed by FISH. Subtelomric FISH study demonstrated no loss of chromosome material from the recipient chromosomes at the translocation junctions.
We postulate that a postzygotic pericentromeric break of the Y chromosome led to formation of isochromosome Yp, whereas Yq formed a jumping translocation through recombination between its internal telomere repeats and telomeric repeats of recipient chromosomes. This in turn led to either pairing or an exchange at the complimentary sequences. Such translocation junctions appear to be unstable and to result in a jumping translocation. Cryptic deletion or disruption of AZF (azoospermic factor) genes at Yq11 during translocation or defective pairing of X and Y chromosomes during meiosis, with abnormal sex vesicle formation and consequent spermatogenetic arrest, might be the main cause of the azoospermia in our patient.
Isochromosome Yp; Jumping translocation Yq
During spermatogenesis in mammalian testes, junction restructuring takes place at the Sertoli-Sertoli and Sertoli-germ cell interface, which is coupled with the development, such as cell cycle progression, and translocation of the germ cell in the seminiferous epithelium. In the rat testis, the restructuring of the blood-testis barrier (BTB) formed between Sertoli cells near the basal region and the disruption of the apical ectoplasmic specialization (apical ES) between Sertoli cells and fully developed spermatids (spermatozoa) at the luminal edge of the seminiferous epithelium occur concurrently at stage VIII of the seminiferous epithelial cycle of spermatogenesis. These two processes are essential for the translocation of primary spermatocytes from the basal to the apical compartment to prepare for meiosis, and the release of spermatozoa to the lumen of the seminiferous epithelium at spermiation, respectively. Cytokines, such as TNFα and TGFβ3, are present at high level in the microenvironment of the epithelium at this stage of the epithelial cycle. Since these cytokines were shown to disrupt the BTB integrity and germ cell adhesion, it was proposed that some cytokines released from germ cells particularly primary spermatocytes and Sertoli cells, would induce the junction restructuring of the BTB and apical ES at stage VIII of the seminiferous epithelial cycle. In this review, the intricate role of cytokines and testosterone to regulate the transit of primary spermatocytes at the BTB and spermiation will be discussed. Possible regulators that mediate the cytokine-induced junction restructuring, including the gap junction and extracellular matrix, will also be discussed.
Testis; spermatogenesis; cytokines; TGF-β3; TNFα; testosterone; blood-testis barrier; primary spermatocytes; seminiferous epithelial cycle
About 30–40% of male infertility is due to unknown reasons. Genetic contributions to the disruption of spermatogenesis are suggested and amongst the genetic factors studied, Y chromosome microdeletions represent the most common one. Screening for microdeletions in AZFa, b and c region of Y chromosome showed a big variation among different studies. The purpose of this study was to investigate the prevalence of such deletions in Saudi men. A total of 257 patients with idiopathic oligo- or azoospermia were screened for Y chromosome microdeletions by 19 markers in AZF region. Ten (3.9%) patients had chromosomal rearrangements, six of them showed sex chromosome abnormalities and four patients had apparently balanced autosomal rearrengements. Eight of the remaining 247 patients (3.2%) with a normal karyotype and no known causes of impaired spermatogenesis had Y chromosome microdeletions. Among these, six patients had deletions in AZFc region, one case had a deletion in AZFb and another had both AZFa and AZFc deletions.
In conclusion, our study shows that Y chromosome microdeletions are low in our population. We also report for the first time a case with unique point deletions of AZFa and AZFc regions. The lower frequency of deletions in our study suggest that other genetic, epigenetic, nutritional and local factors may be responsible for idiopathic oligo- or azoospermia in the Saudi population.
Robertsonian translocation is one of the major chromosomal rearrangements with a prevalence rate of 0.1% of the general population and 1% of the infertile population. In this report, we present a nonhomologous Robertsonian translocation in a female patient with a history of repeated abortions.
A couple with the complaint of repeated abortions was admitted in the Institute of Genetics and Hospital for Genetic Diseases in Begumpet, Hyderabad, India for cytogenetic evaluation. Chromosomal analysis of the couple revealed an abnormal karyotype in the female partner with 45, XX, rob (14, 15) (q10; q10) chromosomal constitution, while the male partner showed normal 46, XY karyotype.
The cytogenetic analysis of couples with repeated abortions is mandatory to identify any probable chromosomal aberrations. Prenatal diagnosis should be offered to couples with repeated abortions in the case of future pregnancies.
Chromosomal Aberration; Cytogenetic analysis; Genetic counseling; Recurrent abortions; Robertsonian translocations
In most sexually reproducing organisms, the fundamental process of meiosis is implemented concurrently with two differentiation programs that occur at different rates and generate distinct cell types, sperm and oocytes. However, little is known about how the meiotic program is influenced by such contrasting developmental programs. Here we present a detailed timeline of late meiotic prophase during spermatogenesis in Caenorhabditis elegans using cytological and molecular landmarks to interrelate changes in chromosome dynamics with germ cell cellularization, spindle formation, and cell cycle transitions. This analysis expands our understanding C. elegans spermatogenesis, as it identifies multiple spermatogenesis-specific features of the meiotic program and provides a framework for comparative studies. Post-pachytene chromatin of spermatocytes is distinct from that of oocytes in both composition and morphology. Strikingly, C. elegans spermatogenesis includes a previously undescribed karyosome stage, a common but poorly understood feature of meiosis in many organisms. We find that karyosome formation, in which chromosomes form a constricted mass within an intact nuclear envelope, follows desynapsis, involves a global down-regulation of transcription, and may support the sequential activation of multiple kinases that prepare spermatocytes for meiotic divisions. In spermatocytes, the presence of centrioles alters both the relative timing of meiotic spindle assembly and its ultimate structure. These microtubule differences are accompanied by differences in kinetochores, which connect microtubules to chromosomes. The sperm-specific features of meiosis revealed here illuminate how the underlying molecular machinery required for meiosis is differentially regulated in each sex.
Sperm and oocytes contribute equal but unique complements of DNA to each new life. Both types of cells arise from meiosis, a multi-step program during which chromosomes replicate, pair and recombine, then divide to generate haploid gametes. Simultaneously, each cell type also differentiates via distinct developmental programs. Spermatogenesis rapidly produces many small, motile sperm with highly protected chromatin, while oogenesis occurs at a slower rate to yield fewer large, immobile, nutrient-rich oocytes. We provide a detailed molecular analysis of key landmark events of spermatogenesis and identify spermatogenesis-specific features of meiosis in the model organism C. elegans. We find that, as in many meiotic programs, C. elegans spermatogenesis includes a chromosome aggregation or “karyosome” phase. This extended stage provides a period for chromosome and microtubule remodeling prior to the meiotic divisions. Our analysis identifies several gamete-specific features of the meiotic program that may contribute to the differential timing, pace, and mechanics of meiotic progression. Our findings provide a foundation for understanding how differentiation influences meiosis, which is an essential step in identifying universal features required for reproductive success in all organisms.
The mechanism responsible for poor reproductive outcomes in type 1 diabetic males is not well understood. In light of new evidence that the Sertoli cells of the testis secrete insulin, it is currently unclear whether diabetic subfertility is the result of deficiency of pancreatic insulin, testicular insulin, or both. In this study, the Akita mouse diabetic model, which expresses a mutant, nonfunctional form of ins2 in testes and pancreas, was used to distinguish between systemic and local effects of insulin deficiency on the process of spermatogenesis and fertility. We determined that Akita homozygous male mice are infertile and have reduced testis size and abnormal morphology. Spermatogonial germ cells are still present but are unable to mature into spermatocytes and spermatids. Exogenous insulin treatment regenerates testes and restores fertility, but this plasma insulin cannot pass through the blood-testis barrier. We conclude that insulin does not rescue fertility through direct interaction with the testis; instead, it restores function of the hypothalamic-pituitary-gonadal axis and, thus, normalizes hormone levels of luteinizing hormone and testosterone. Although we show that the Sertoli cells of the testis secrete insulin protein, this insulin does not appear to be critical for fertility.
During spermatogenesis, step 1 spermatids (round spermatids) derive from spermatocytes following meiosis I and II at stage XIV of the epithelial cycle begin a series of morphological transformation and differentiation via 19 steps in rats to form spermatozoa. This process is known as spermiogenesis, which is marked by condensation of the genetic material in the spermatid head, formation of the acrosome and elongation of the tail. Since developing spermatids are lacking the robust protein synthesis and transcriptional activity, the cellular, molecular and morphological changes associated with spermiogenesis rely on the Sertoli cell in the seminiferous epithelium via desmosome and gap junction between Sertoli cells and step 1–7 spermatids. Interestingly, a unique anchoring junction type arises at the interface of step 8 spermatid and Sertoli cell known as apical ectoplasmic specialization (apical ES). Once it appears, apical ES is the only anchoring device restricted to the interface of step 8–19 spermatids and Sertoli cells to confer spermatid polarity, adhesion, signal communication and structural support, and to provide nutritional support during spermiogenesis, replacing desmosome and gap junction. While the adhesion protein complexes that constitute the apical ES are known, the signaling protein complexes that regulate apical ES dynamics, however, remain largely unknown. Herein we report the presence of a FAK (focal adhesion kinase)-p130Cas (p130 Crk-associated substrate)-DOCK180 (Dedicator of cytokinesis 180)-RhoA (Ras homolog gene family, member A)-vinculin signaling protein complex at the apical ES, which is also an integrated component of the β1-integrin-based adhesion protein complex based on co-immunoprecipitation experiment. It was also shown that besides p-FAK-Tyr397 and p-FAK-Tyr576, β1-integrin, p130Cas, RhoA and vinculin displayed stage-specific expression in the seminiferous epithelium during the epithelial cycle with predominant localization at the apical ES as demonstrated by immunohistochemistry. Based on these findings, functional studies can now be performed to assess the role of this β1-integrin-p-FAK-p130Cas-DOCK180-RhoA-vinculin protein complex in apical ES dynamics during spermiogenesis.
testis; spermatogenesis; apical ectoplasmic specialization; adjudin; seminiferous epithelial cycle; spermiogenesis
Male fertility relies on the highly specialized process of spermatogenesis to continually renew the supply of spermatozoa necessary for reproduction. Central to this unique process is meiosis that is responsible for the production of haploid spermatozoa as well as for generating genetic diversity. During meiosis I, there is a dramatic increase in the number of mitochondria present within the developing spermatocytes, suggesting an increased necessity for ATP production and utilization. Essential for the utilization of ATP is the translocation of ADP and ATP across the inner mitochondrial membrane, which is mediated by the adenine nucleotide translocases (Ant). We recently identified and characterized a novel testis specific Ant, ANT4 (also known as SLC25A31 and Aac4). The generation of Ant4-deficient animals resulted in the severe disruption of the seminiferous epithelium with an apparent spermatocytic arrest of the germ cell population. In the present study utilizing a chromosomal spread technique, we determined that Ant4-deficiency results in an accumulation of leptotene spermatocytes, a decrease in pachytene spermatocytes, and an absence of diplotene spermatocytes, indicating early meiotic arrest. Furthermore, the chromosomes of Ant4-deficient pachytene spermatocyte occasionally demonstrated sustained γH2AX association as well as synaptonemal complex protein 1 (SYCP1)/SYCP3 dissociation beyond the sex body. Large ATP supplies from mitochondria may be critical for normal progression of spermatogenesis during early stages of meiotic prophase I, including DNA double-strand break repair and chromosomal synapsis.
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.
Human embryonic stem cell (hESC) lines derived from poor quality embryos usually have either normal or abnormal karyotypes. However, it is still unclear whether their biological characteristics are similar.
Seven new hESC lines were established using discarded embryos. Five cell lines had normal karyotype, one was with an unbalanced Robertsonian translocation and one had a triploid karyotype. Their biological characteristics, short tandem repeat loci, HLA typing, differentiation capability and imprinted gene, DNA methylation and X chromosome inactivation status were compared between different cell lines.
All seven hESC lines had similar biological characteristics regardless of karyotype (five normal and two abnormal), such as expression of stage-specific embryonic antigen (SSEA)-4, tumor-rejection antigen (TRA)-1-81 and TRA-1-60 proteins, transcription factor octamer binding protein 4 mRNA, no detectable expression of SSEA-1 protein and high levels of alkaline phosphatase activity. All cell lines were able to undergo differentiation. Imprinted gene expression and DNA methylation were also similar among these cell lines. Non-random X chromosome inactivation patterns were found in XX cell lines.
The present results suggest that hESC lines with abnormal karyotype are also useful experimental materials for cell therapy, developmental biology and genetic research.
human embryonic stem cell lines; characterization; karyotype; methylation; X-inactivation
Marriages involving partners both of whom have abnormal karyotypes are rare and are usually ascertained because of a history of infertility, repeated abortions, or the birth of a balanced translocation carrier or chromosomally abnormal offspring. Abnormalities which have been noted include sex chromosome aberrations in both parents or a sex chromosome abnormality in one parent and an autosomal abnormality in the other. Four papers have reported balanced reciprocal autosomal translocations in both parents, two couples representing a first cousin marriage. We present a case of a paternal 13;14 Robertsonian translocation and a maternal (7p;13q) reciprocal translocation in a couple with repeated fetal loss.