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1.  Comparative studies of spermatogenesis in fertile and subfertile men. 
Journal of Clinical Pathology  1981;34(2):145-150.
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.
PMCID: PMC1146440  PMID: 7229093
2.  Chromosome aberrations and spermatogenic disorders in mice with Robertsonian translocation (11; 13) 
Objective: To determine the diagnostic features of Robertsonian (Rob) translocation (11; 13) in mice and the mechanisms underlying the effect on spermatogenesis and reproductive decline. Methods: A Rob translocation (11; 13) mouse model was established by cross-breeding, and confirmed by chromosome analysis. Chromosome aberrations and translocation patterns were identified in mice with Rob translocation (11; 13) by fluorescence in situ hybridization (FISH). Spermatogenic disorders were investigated at different stages of spermatogenesis. Immunofluorescent analysis was performed on sections of testis and epididymis specimens during spermatogenic meiosis. The weight of the testes and reproductive decline were recorded. Results: The crossed Rob translocation (11; 13) mouse has 39 chromosomes, including a fusion chromosome (included chromosomes 11 and 13) using dual color FISH. There was no difference in the distribution pattern of SYCP3 and γH2AX in spermatocytes between Rob translocation and wild-type mice; however, round haploid spermatids presented characteristic morphologic changes of apoptosis and the number of haploid spermatids was decreased. Furthermore, the immature germ cells were released into the epididymis and the number of mature sperm was reduced. Conclusions: Chromosome aberrations and spermatogenic disorders may result from apoptosis of round haploid spermatids and a reduced number of mature sperm in Rob translocation (11; 13) mice. Abnormal sperm and reduced number of sperm may be one of the main reasons for reproductive decline and male infertility in Rob translocation (11; 13) mice.
PMCID: PMC4270519  PMID: 25550810
Infertility; spermatogenesis; Robertsonian translocation; mouse
3.  Isochromosome Yp and jumping translocation of Yq resulting in five cell lines in an infertile male: a case report and review of the literature 
Background
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.
Case presentation
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.
Conclusions
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.
doi:10.1186/1755-8166-6-36
PMCID: PMC3848363  PMID: 24020961
Isochromosome Yp; Jumping translocation Yq
4.  Dynamics of Response to Asynapsis and Meiotic Silencing in Spermatocytes from Robertsonian Translocation Carriers 
PLoS ONE  2013;8(9):e75970.
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.
doi:10.1371/journal.pone.0075970
PMCID: PMC3774740  PMID: 24066189
5.  Effect of FSH on testicular morphology and spermatogenesis in gonadotrophin-deficient hypogonadal mice lacking androgen receptors 
Reproduction (Cambridge, England)  2010;139(1):177-184.
FSH and androgen act to stimulate and maintain spermatogenesis. FSH acts directly on the Sertoli cells to stimulate germ cell number and acts indirectly to increase androgen production by the Leydig cells. In order to differentiate between the direct effects of FSH on spermatogenesis and those mediated indirectly through androgen action, we have crossed hypogonadal (hpg) mice, which lack gonadotrophins, with mice lacking androgen receptors (AR) either ubiquitously (ARKO) or specifically on the Sertoli cells (SCARKO). These hpg.ARKO and hpg.SCARKO mice were treated with recombinant FSH for 7 days and testicular morphology and cell numbers were assessed. In untreated hpg and hpg.SCARKO mice, germ cell development was limited and did not progress beyond the pachytene stage. In hpg.ARKO mice, testes were smaller with fewer Sertoli cells and germ cells compared to hpg mice. Treatment with FSH had no effect on Sertoli cell number but significantly increased germ cell numbers in all groups. In hpg mice, FSH increased the numbers of spermatogonia and spermatocytes, and induced round spermatid formation. In hpg.SCARKO and hpg.ARKO mice, in contrast, only spermatogonial and spermatocyte numbers were increased with no formation of spermatids. Leydig cell numbers were increased by FSH in hpg and hpg.SCARKO mice but not in hpg.ARKO mice. Results show that in rodents 1) FSH acts to stimulate spermatogenesis through an increase in spermatogonial number and subsequent entry of these cells into meiosis, 2) FSH has no direct effect on the completion of meiosis and 3) FSH effects on Leydig cell number are mediated through interstitial ARs.
doi:10.1530/REP-09-0377
PMCID: PMC2791495  PMID: 19846485
6.  Essential Roles of the Chromatin Remodeling Factor Brg1 in Spermatogenesis in Mice1  
Biology of Reproduction  2012;86(6):186.
ABSTRACT
Mammalian spermatogenesis is a complex process that involves spatiotemporal regulation of gene expression and meiotic recombination, both of which require the modulation of chromatin structure. Proteins important for chromatin regulation during spermatogenesis remain poorly understood. Here we addressed the role of BRG1, the catalytic subunit of the mammalian Swi/Snf-like BAF chromatin-remodeling complex, during spermatogenesis in mice. BRG1 expression is dynamically regulated in the male germline, being weakly detectable in spermatogonia, highly expressed in pachytene spermatocytes, and turned off in maturing round spermatids. This expression pattern overlaps that of Brm, the Brg1 homolog. While Brm knockout males are known to be fertile, germline-specific Brg1 deletion completely arrests spermatogenesis at the midpachytene stage, which is associated with spermatocyte apoptosis and apparently also with impaired homologous recombination and meiotic sex chromosome inactivation. However, Brg1 is dispensable for gammaH2AX formation during meiotic recombination, contrary to its reported role in DNA repair in somatic cells. Our study reveals the essential role of Brg1 in meiosis and underscores the differences in the mechanisms of DNA repair between germ cells and somatic cells.
Chromatin remodeling factor Brg1 controls development of primary spermatocytes during meiosis in mice.
doi:10.1095/biolreprod.111.097097
PMCID: PMC3386149  PMID: 22495890
apoptosis; chromatin; spermatogenesis
7.  Melphalan Alone or Conjugated to a Follicle Stimulating Hormone-β Peptide Kills Murine Testicular Cells in vitro and Transiently Suppresses Murine Spermatogenesis in vivo 
Theriogenology  2014;82(1):152-159.
New approaches to sterilizing male animals are needed to control captive and wild animal populations. We sought to develop a non-surgical method of permanent sterilization for male animals by administering the gonadotoxicant melphalan conjugated to peptides derived from the β-chain of follicle stimulating hormone (FSHβ). We hypothesized that conjugating melphalan to FSHβ peptides would magnify the gonadotoxic effects of melphalan while minimizing systemic toxicity. The ability of conjugates of melphalan and FSHβ peptides to kill murine testicular cells was first tested in vitro in a three-dimensional testicular cell co-culture system. In this system, melphalan caused considerable cell death as measured both by increases in LDH concentrations in the culture supernatant and direct visualization of the cultures. Of the conjugates tested, melphalan conjugated to a 20 amino acid peptide derived from human FSHβ consisting of amino acids 33-53 (FSHβ (33-53)-melphalan) was very potent, with cell cytotoxicity and LDH release roughly one-half that of melphalan. The effects of melphalan and FSHβ (33-53)-melphalan on spermatogenesis were then tested in vivo in mature C56Bl/6 male mice. Four weeks after intraperitoneal injection, all mice treated with either FSHβ (33-53)-melphalan or melphalan had ~75% reductions in testicular spermatid counts compared with control animals. Testicular histology revealed significant reduction in mature spermatids and spermatocytes in most tubules. However, 12 weeks after the injection, testicular spermatid counts and histology were similar to controls, except in one animal receiving FSHβ (33-53)-melphalan that had no apparent spermatogenesis. We conclude that melphalan and FSHβ (33-53)-melphalan are potent gonadotoxicants in male mice resulting in marked suppression of spermatogenesis 4 weeks after a single intraperitoneal injection. However, this effect is transient in most mice as spermatogenesis is similar to control animals 12 weeks after drug administration. Melphalan or FSHβ (33-53)-melphalan may be useful for the temporary control of fertility in male animals, but additional research will be needed to develop a single dose method of permanent sterilization for male animals.
doi:10.1016/j.theriogenology.2014.03.014
PMCID: PMC4079550  PMID: 24746827
Gonadotoxicants; Sterilization; Male Animal Contraception; Spermatogenesis
8.  THE PATHOLOGICAL CHANGES IN THE TESTES IN EPIDEMIC PNEUMONIA 
The testicular changes in pneumonia are without clinical manifestations, are non-specific, focal in character, independent of the infecting organisms or the antecedent disease, and vary in severity directly with the total length of the illness. The process is a continuous one, divisible into stages in which the following features are recognizable: (1) cessation of spermatogenesis; (2) degeneration of preformed spermatocytes, spermatids, and spermatozoa; (3) desquamation of altered cells and fragments of the same; (4) formation of giant cells in the tubule walls with subsequent liberation into the lumen; (5) disappearance of all desquamated cells and all those derived from the spermatogonia by mitosis; (6) in some instances thickening of the hyaline layer of the basement membrane. Older lesions are frequently found which continue the structural alteration of the tubules by hyalinosis and destruction of cells until they ultimately disappear. These lesions are not believed to be connected with the present illness. Edema may represent the acute injury in another form, and round cell infiltration suggests that possibly other factors than toxins may have a part in the tissue alterations. In the absence of definite evidence to the contrary, the cause is assumed to be circulating toxins, as Wolbach (12) claims for influenzal cases. The hemolytic streptococcus produced more extensive changes, both epithelial and interstitial, in primary pneumonia occurring during the measles epidemic than when pneumonia followed as a secondary infection; in the latter cases the pulmonary complications covered a relatively shorter period. Measles and epidemic influenza had little apparent effect upon the testes, except that the former caused mild inhibition of spermatogenesis; evidence regarding the latter is inconclusive. The Pfeiffer bacillus was always associated with other organisms, in primary infections and in those following measles. It occurred alone in a few cases after epidemic influenza, but the testicular lesion was not distinctive. The pneumococcus when alone in primary infections or after an epidemic disease produced a uniformly mild picture which was not intensified when associated with the influenza bacillus. Giant cells were much more frequent after influenzal pneumonia regardless of its cause and were associated with large numbers of other desquamated cells. They are formed in the walls of tubules by futile mitotic effort and incomplete protoplasmic separation, the abnormality of the process being further suggested by the early severing of cytoplasmic attachments and rapid desquamation. The series is unique in its uniformity, in the care exercised in the bacteriological examinations, and in the relative freedom from complicating factors.
PMCID: PMC2126691  PMID: 19868374
9.  Molecular and cytogenetic studies of 101 infertile men with microdeletions of Y chromosome in 1,306 infertile Korean men 
Objectives
To determine the prevalence of Y chromosome microdeletions in infertile Korean men with abnormal sperm counts and to assess the clinical features and frequency of chromosomal abnormalities in Korean patients with microdeletions.
Methods
A total of 1,306 infertile men were screened for Y chromosome microdeletions, and 101 of them had microdeletions. These 101 men were then retrospectively studied for cytogenetic evaluation, testicular biopsy and outcomes of IVF and ICSI.
Results
The overall prevalence of Y chromosome microdeletions in infertile men was 7.7 % (101/1,306). Most microdeletions were in the AZFc region (87.1 %), including deletions of AZFbc (24.7 %) and AZFabc (8.9 %). All patients with AZFa, AZFbc and AZFabc deletions had azoospermia, whereas patients with an AZFc deletion usually had low levels of sperm in the ejaculate or in the testis tissues. Chromosomal studies were performed in 99 men with microdeletions, 36 (36.4 %) of whom had chromosomal abnormalities. Among the infertile men with Y chromosome microdeletions in this study, the incidence of chromosomal abnormality was 48.6 % in the azoospermic group and 3.7 % in the oligozoospermic group. Among the 69 patients with microdeletions and available histological results, 100.0 % of the azoospermic group and 85.7 % of the oligozoospermic group had histological abnormalities. The frequency of both chromosomal abnormalities and histological abnormalities was higher in the azoospermic group compared to the oligozoospermic group. Thirty-four ICSI cycles with either testicular (n = 14) or ejaculated spermatozoa (n = 20) were performed in 23 couples with men with AZFc microdeletion. Thirteen clinical pregnancies (39.4 %) were obtained, leading to the birth of 13 babies.
Conclusions
The study results revealed a close relationship between microdeletions and spermatogenesis, although IVF outcome was not significantly affected by the presence of the AZFc microdeletion. Nevertheless, Y chromosome microdeletions have the potential risk of being transmitted from infertile fathers to their offspring by ICSI. Therefore, before using ICSI in infertile patients with severe spermatogenic defects, careful evaluations of chromosomal abnormalities and Y chromosome microdeletions screening should be performed and genetic counseling should be provided before IVF-ET.
doi:10.1007/s10815-012-9748-4
PMCID: PMC3370041  PMID: 22456825
Y chromosome microdeletion; Chromosomal abnormality; Azoospermia factor (AZF); Intracytoplasmic sperm injection (ICSI)
10.  A High Incidence of Meiotic Silencing of Unsynapsed Chromatin Is Not Associated with Substantial Pachytene Loss in Heterozygous Male Mice Carrying Multiple Simple Robertsonian Translocations 
PLoS Genetics  2009;5(8):e1000625.
Meiosis is a complex type of cell division that involves homologous chromosome pairing, synapsis, recombination, and segregation. When any of these processes is altered, cellular checkpoints arrest meiosis progression and induce cell elimination. Meiotic impairment is particularly frequent in organisms bearing chromosomal translocations. When chromosomal translocations appear in heterozygosis, the chromosomes involved may not correctly complete synapsis, recombination, and/or segregation, thus promoting the activation of checkpoints that lead to the death of the meiocytes. In mammals and other organisms, the unsynapsed chromosomal regions are subject to a process called meiotic silencing of unsynapsed chromatin (MSUC). Different degrees of asynapsis could contribute to disturb the normal loading of MSUC proteins, interfering with autosome and sex chromosome gene expression and triggering a massive pachytene cell death. We report that in mice that are heterozygous for eight multiple simple Robertsonian translocations, most pachytene spermatocytes bear trivalents with unsynapsed regions that incorporate, in a stage-dependent manner, proteins involved in MSUC (e.g., γH2AX, ATR, ubiquitinated-H2A, SUMO-1, and XMR). These spermatocytes have a correct MSUC response and are not eliminated during pachytene and most of them proceed into diplotene. However, we found a high incidence of apoptotic spermatocytes at the metaphase stage. These results suggest that in Robertsonian heterozygous mice synapsis defects on most pachytene cells do not trigger a prophase-I checkpoint. Instead, meiotic impairment seems to mainly rely on the action of a checkpoint acting at the metaphase stage. We propose that a low stringency of the pachytene checkpoint could help to increase the chances that spermatocytes with synaptic defects will complete meiotic divisions and differentiate into viable gametes. This scenario, despite a reduction of fertility, allows the spreading of Robertsonian translocations, explaining the multitude of natural Robertsonian populations described in the mouse.
Author Summary
Cells have different mechanisms to assess the proper occurrence of cellular events. These mechanisms are called checkpoints and are involved in the surveillance of processes such as DNA replication and cell division. A checkpoint at the pachytene stage arrests meiosis when defects in the process of homologous chromosome synapsis and recombination are detected. In mammals, both transcriptional inactivation of chromosomal regions that are not correctly synapsed at pachytene and activation of sex chromosome genes that are normally silent during this stage could contribute to meiotic arrest. We found that when Robertsonian translocations appear in heterozygosis, many synapsis defects occur, and mechanisms that trigger transcriptional silencing of the unsynapsed chromatin are activated. However, meiotic prophase-I progression is not greatly compromised. This questions the ability of the meiotic checkpoints to halt meiosis progression when synapsis is not completed, allowing cells with synapsis defects to reach the first meiotic division. The fertility reduction of Robertsonian heterozygous mice seems to be mainly caused by errors detected by the metaphase-I checkpoint, when most of the spermatocytes die, rather than by synapsis defects. In an evolutionary context, a permissive pachytene checkpoint could contribute to increasing the chances of Robertsonian translocations to spread into natural populations.
doi:10.1371/journal.pgen.1000625
PMCID: PMC2726437  PMID: 19714216
11.  Mammalian E-type Cyclins Control Chromosome Pairing, Telomere Stability and CDK2 Localization in Male Meiosis 
PLoS Genetics  2014;10(2):e1004165.
Loss of function of cyclin E1 or E2, important regulators of the mitotic cell cycle, yields viable mice, but E2-deficient males display reduced fertility. To elucidate the role of E-type cyclins during spermatogenesis, we characterized their expression patterns and produced additional deletions of Ccne1 and Ccne2 alleles in the germline, revealing unexpected meiotic functions. While Ccne2 mRNA and protein are abundantly expressed in spermatocytes, Ccne1 mRNA is present but its protein is detected only at low levels. However, abundant levels of cyclin E1 protein are detected in spermatocytes deficient in cyclin E2 protein. Additional depletion of E-type cyclins in the germline resulted in increasingly enhanced spermatogenic abnormalities and corresponding decreased fertility and loss of germ cells by apoptosis. Profound meiotic defects were observed in spermatocytes, including abnormal pairing and synapsis of homologous chromosomes, heterologous chromosome associations, unrepaired double-strand DNA breaks, disruptions in telomeric structure and defects in cyclin-dependent-kinase 2 localization. These results highlight a new role for E-type cyclins as important regulators of male meiosis.
Author Summary
Understanding the control of meiosis is fundamental to deciphering the origin of male infertility. Although the mechanisms controlling meiosis are poorly understood, key regulators of mitosis, such as cyclins, appear to be critical. In this regard, male mice deficient for cyclin E2 exhibit subfertility and defects in spermatogenesis; however, neither the stages of germ cell differentiation affected nor the responsible mechanisms are known. We investigated how E-type cyclins control male meiosis by examining their expression in spermatogenesis and the consequences that multiple deletions of Ccne1 and Ccne2 alleles produce. Loss of Ccne2 expression increases cyclin E1 levels as a compensatory effect, but there are still meiotic defects and subfertility. Further, loss of one Ccne1 allele in the absence of cyclin E2 results in infertility as does loss of the remaining Ccne1 allele, but with even more severe meiotic abnormalities. We further found that cyclin E1 is involved in sex chromosome synapsis while E2 is involved with homologous pairing and chromosome and telomere integrity. These processes and structures were severely disrupted in absence of both cyclin E1 and E2, uncovering new roles for the E-type cyclins in regulating male meiosis.
doi:10.1371/journal.pgen.1004165
PMCID: PMC3937215  PMID: 24586195
12.  The hsp60B gene of Drosophila melanogaster is essential for the spermatid individualization process 
Cell Stress & Chaperones  2001;6(1):71-77.
The 60-kDa heat shock protein family (Hsp60) is found in prokaryotes, mitochondria, and chloroplasts. The Hsp60 proteins promote proper protein folding by preventing aggregation. In Drosophila melanogaster, the hsp60 gene is essential for a variety of developmental processes, beginning at early embryogenesis. In this study we show that an additional member of the Drosophila hsp60 gene family, hsp60B, is essential in male fertility. In males homozygous for a mutation of the hsp60B gene, developmental processes appeared normal throughout most of spermatogenesis, including spermatocyte growth, meiosis, and spermatid elongation. At these stages, mitochondria also displayed a differentiation process similar to wild-types. However, we found that the mutation disrupted a late stage of spermatogenesis, the spermatid individualization process. In this process, the individualization complex is assembled at spermatid nuclear heads, traverses along spermatid tails, and generates membranes for each of the spermatids in a cyst. Our analysis further shows that the individualization complex in sterile males displayed abnormal morphology as it was traveling along the spermatid tails. The Drosophila Hsp60 proteins are believed to be exclusively localized in the mitochondria. Our observation that the hsp60B mutation displayed no apparent defect in mitochondrial differentiation during spermatogenesis suggests that the Hsp60B protein may operate in a nonmitochondrial location.
PMCID: PMC434385  PMID: 11525246
13.  Complex N-Glycans Are Essential, but Core 1 and 2 Mucin O-Glycans, O-Fucose Glycans, and NOTCH1 Are Dispensable, for Mammalian Spermatogenesis1 
Biology of Reproduction  2012;86(6):179.
ABSTRACT
To identify roles in spermatogenesis for major subclasses of N- and O-glycans and Notch signaling, male mice carrying floxed C1galt1, Pofut1, Notch1 or Mgat1 alleles and a testis-specific Cre recombinase transgene were generated. T-synthase (C1GALT1) transfers Gal to generate core 1 and core 2 mucin O-glycans; POFUT1 transfers O-fucose to particular epidermal growth factor-like repeats and is essential for canonical Notch signaling; and MGAT1 (GlcNAcT-I) transfers GlcNAc to initiate hybrid and complex N-glycan synthesis. Cre recombinase transgenes driven by various promoters were investigated, including Stra8-iCre expressed in spermatogonia, Sycp1-Cre expressed in spermatocytes, Prm1-Cre expressed in spermatids, and AMH-Cre expressed in Sertoli cells. All Cre transgenes deleted floxed alleles, but efficiencies varied widely. Stra8-iCre was the most effective, deleting floxed Notch1 and Mgat1 alleles with 100% efficiency and floxed C1galt1 and Pofut1 alleles with ∼80% efficiency, based on transmission of deleted alleles. Removal of C1galt1, Pofut1, or Notch1 in spermatogonia had no effect on testicular weight, histology, or fertility. However, males in which the synthesis of complex N-glycans was blocked by deletion of Mgat1 in spermatogonia did not produce sperm. Spermatogonia, spermatocytes, and spermatids were generated, but most spermatids formed giant multinucleated cells or symplasts, and apoptosis was increased. Therefore, although core 1 and 2 mucin O-glycans, NOTCH1, POFUT1, O-fucose glycans, and Notch signaling are dispensable, MGAT1 and complex N-glycans are essential for spermatogenesis.
Spermatogenesis is blocked when the Mgat1 gene is conditionally deleted in spermatogonia, but proceeds normally when Notch signaling is inhibited by deletion of Notch1 or Pofut1, or mucin O-glycan synthesis is prevented by deletion of C1galt1, in these cells.
doi:10.1095/biolreprod.111.098103
PMCID: PMC3386147  PMID: 22492969
glycosyltransferases; Notch signaling; sperm maturation; spermatid; testis
14.  Meiotic Telomere Distribution and Sertoli Cell Nuclear Architecture Are Altered in Atm- and Atm-p53-Deficient Mice 
Molecular and Cellular Biology  2000;20(20):7773-7783.
The ataxia telangiectasia mutant (ATM) protein is an intrinsic part of the cell cycle machinery that surveys genomic integrity and responses to genotoxic insult. Individuals with ataxia telangiectasia as well as Atm−/− mice are predisposed to cancer and are infertile due to spermatogenesis disruption during first meiotic prophase. Atm−/− spermatocytes frequently display aberrant synapsis and clustered telomeres (bouquet topology). Here, we used telomere fluorescent in situ hybridization and immunofluorescence (IF) staining of SCP3 and testes-specific histone H1 (H1t) to spermatocytes of Atm- and Atm-p53-deficient mice and investigated whether gonadal atrophy in Atm-null mice is associated with stalling of telomere motility in meiotic prophase. SCP3-H1t IF revealed that most Atm−/− p53−/− spermatocytes degenerated during late zygotene, while a few progressed to pachytene and diplotene and some even beyond metaphase II, as indicated by the presence of a few round spermatids. In Atm−/− p53−/− meiosis, the frequency of spermatocytes I with bouquet topology was elevated 72-fold. Bouquet spermatocytes with clustered telomeres were generally void of H1t signals, while mid-late pachytene and diplotene Atm−/− p53−/− spermatocytes displayed expression of H1t and showed telomeres dispersed over the nuclear periphery. Thus, it appears that meiotic telomere movements occur independently of ATM signaling. Atm inactivation more likely leads to accumulation of spermatocytes I with bouquet topology by slowing progression through initial stages of first meiotic prophase and an ensuing arrest and demise of spermatocytes I. Sertoli cells (SECs), which contribute to faithful spermatogenesis, in the Atm mutants were found to frequently display numerous heterochromatin and telomere clusters—a nuclear topology which resembles that of immature SECs. However, Atm−/− SECs exhibited a mature vimentin and cytokeratin 8 intermediate filament expression signature. Upon IF with ATM antibodies, we observed ATM signals throughout the nuclei of human and mouse SECs, spermatocytes I, and haploid round spermatids. ATM but not H1t was absent from elongating spermatid nuclei. Thus, ATM appears to be removed from spermatid nuclei prior to the occurrence of DNA nicks which emanate as a consequence of nucleoprotamine formation.
PMCID: PMC86364  PMID: 11003672
15.  Systematic variation in mRNA 3′-processing signals during mouse spermatogenesis 
Nucleic Acids Research  2006;35(1):234-246.
Gene expression and processing during mouse male germ cell maturation (spermatogenesis) is highly specialized. Previous reports have suggested that there is a high incidence of alternative 3′-processing in male germ cell mRNAs, including reduced usage of the canonical polyadenylation signal, AAUAAA. We used EST libraries generated from mouse testicular cells to identify 3′-processing sites used at various stages of spermatogenesis (spermatogonia, spermatocytes and round spermatids) and testicular somatic Sertoli cells. We assessed differences in 3′-processing characteristics in the testicular samples, compared to control sets of widely used 3′-processing sites. Using a new method for comparison of degenerate regulatory elements between sequence samples, we identified significant changes in the use of putative 3′-processing regulatory sequence elements in all spermatogenic cell types. In addition, we observed a trend towards truncated 3′-untranslated regions (3′-UTRs), with the most significant differences apparent in round spermatids. In contrast, Sertoli cells displayed a much smaller trend towards 3′-UTR truncation and no significant difference in 3′-processing regulatory sequences. Finally, we identified a number of genes encoding mRNAs that were specifically subject to alternative 3′-processing during meiosis and postmeiotic development. Our results highlight developmental differences in polyadenylation site choice and in the elements that likely control them during spermatogenesis.
doi:10.1093/nar/gkl919
PMCID: PMC1802579  PMID: 17158511
16.  The Nuclear RNA-binding Protein Sam68 Translocates to the Cytoplasm and Associates with the Polysomes in Mouse SpermatocytesD⃞ 
Molecular Biology of the Cell  2006;17(1):14-24.
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.
doi:10.1091/mbc.E05-06-0548
PMCID: PMC1345642  PMID: 16221888
17.  Loss-of-Function Analysis Reveals Distinct Requirements of the Translation Initiation Factors eIF4E, eIF4E-3, eIF4G and eIF4G2 in Drosophila Spermatogenesis 
PLoS ONE  2015;10(4):e0122519.
In eukaryotes, post-transcriptional regulation of gene expression has a key role in many cellular and developmental processes. Spermatogenesis involves a complex developmental program that includes changes in cell cycle dynamics and dramatic cellular remodeling. Translational control is critical for spermatogenesis in Drosophila as many mRNAs synthesized in the spermatocytes are translated only much later during spermatid differentiation. Testes-specific translation initiation factors eIF4E-3 and eIF4G2 are essential specifically for male fertility. However, details of their roles during different stages of spermatogenesis are unknown, and the role of canonical translation initiation factors in spermatogenesis remains unexplored. In this study, we addressed the functional role of eIF4E-1, eIF4E-3, eIF4G and eIF4G2 in testes development and formation of mature sperm. Using the UAS-Gal4 system and RNA interference, we systematically knocked down these four genes in different stages of germ cell development, and in the somatic cells. Our results show that eIF4E-1 function in early germ cells and the surrounding somatic cells is critical for spermatogenesis. Both eIF4E-1 and eIF4E-3 are required in spermatocytes for chromosome condensation and cytokinesis during the meiotic stages. Interestingly, we find that eIF4G knockdown did not affect male fertility while eIF4G2 has distinct functions during spermatogenesis; it is required in early germ cells for proper meiotic divisions and spermatid elongation while its abrogation in spermatocytes caused meiotic arrest. Double knockdown of eIF4G and eIF4G2 shows that these proteins act redundantly during the early stages of spermatogenesis. Taken together, our analysis reveals spatio-temporal roles of the canonical and testes-specific translation initiation factors in coordinating developmental programs during spermatogenesis.
doi:10.1371/journal.pone.0122519
PMCID: PMC4388691  PMID: 25849588
18.  Structure and inheritance of some heterozygous Robertsonian translocation in man. 
Journal of Medical Genetics  1976;13(5):381-388.
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.
Images
PMCID: PMC1013444  PMID: 1003449
19.  Spermatogenesis-Specific Features of the Meiotic Program in Caenorhabditis elegans 
PLoS Genetics  2009;5(8):e1000611.
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.
Author Summary
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.
doi:10.1371/journal.pgen.1000611
PMCID: PMC2720455  PMID: 19696886
20.  Adenine nucleotide translocase 4 deficiency leads to early meiotic arrest of murine male germ cells 
Reproduction (Cambridge, England)  2009;138(3):463-470.
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.
doi:10.1530/REP-09-0201
PMCID: PMC3727218  PMID: 19556438
21.  Lymphoid-Specific Helicase (HELLS) Is Essential for Meiotic Progression in Mouse Spermatocytes1 
Biology of Reproduction  2011;84(6):1235-1241.
Lymphoid-specific helicase (HELLS; also known as LSH) is a member of the SNF2 family of chromatin remodeling proteins. Because Hells-null mice die at birth, a phenotype in male meiosis cannot be studied in these animals. Allografting of testis tissue from Hells−/− to wild-type mice was employed to study postnatal germ cell differentiation. Testes harvested at Day 18.5 of gestation from Hells−/−, Hells+/−, and Hells+/+ mice were grafted ectopically to immunodeficient mice. Bromodeoxyuridine incorporation at 1 wk postgrafting revealed fewer dividing germ cells in grafts from Hells−/− than from Hells+/+ mice. Whereas spermatogenesis proceeded through meiosis with round spermatids in grafts from Hells heterozygote and wild-type donor testes, spermatogenesis arrested at stage IV, and midpachytene spermatocytes were the most advanced germ cell type in grafts from Hells−/− mice at 4, 6, and 8 wk after grafting. Analysis of meiotic configurations at 22 days posttransplantation revealed an increase in Hells−/− spermatocytes with abnormal chromosome synapsis. These results indicate that in the absence of HELLS, proliferation of spermatogonia is reduced and germ cell differentiation arrested at the midpachytene stage, implicating an essential role for HELLS during male meiosis. This study highlights the utility of testis tissue grafting to study spermatogenesis in animal models that cannot reach sexual maturity.
Lymphoid-specific helicase (HELLS) plays an essential role during meiotic chromosome synapsis in the mouse testis.
doi:10.1095/biolreprod.110.085720
PMCID: PMC3099587  PMID: 21349825
chromosomes; developmental biology; HELLS; LSH; meiosis; spermatogenesis; testis; transplantation
22.  Ooplasmic round spermatid nuclear injection procedures as an experimental treatment for nonobstructive azoospermia 
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.
doi:10.1007/BF02765754
PMCID: PMC3454702  PMID: 9013313
azoospermia; infertility; oocyte; round spermatid
23.  asunder Is a Critical Regulator of Dynein–Dynactin Localization during Drosophila Spermatogenesis 
Molecular Biology of the Cell  2009;20(11):2709-2721.
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.
doi:10.1091/mbc.E08-12-1165
PMCID: PMC2688550  PMID: 19357193
24.  Basigin null mutant male mice are sterile and exhibit impaired interactions between germ cells and Sertoli cells 
Developmental biology  2013;380(2):145-156.
Basigin (BSG) is a multifunctional glycoprotein that plays an important role in male reproduction since male knockout (KO) mice are sterile. The Bsg KO testis lacks elongated spermatids and mature spermatozoa, a phenotype similar to that of alpha-mannosidase IIx (MX) KO mice. MX regulates formation of N-acetylglucosamine (GlcNAc) terminated N-glycans that participate in germ cell-Sertoli cell adhesion. Results showed that Bsg KO spermatocytes displayed normal homologous chromosome synapsis and progression through meiosis. However, only punctate expression of the round spermatid marker SP-10 in the acrosomal granule of germ cells of Bsg KO mice was detected indicating that spermatogenesis in Bsg KO mice was arrested at the early round spermatid stages. We observed a large increase in the number of germ cells undergoing apoptosis in Bsg KO testes. Using lectin blotting, we determined that GlcNAc terminated N-glycans are linked to BSG. GlcNAc terminated N-glycans were significantly reduced in Bsg KO testes. These observations indicate that BSG may act as a germ cell-Sertoli cell attachment molecule. Loss of BSG significantly reduced adhesion between GC-2 and SF7 cells. Moreover, wild type testes showed strong expression of N-cadherin (CDH2) while expression was greatly reduced in the testes of Bsg KO mice. In addition, the integrity of the blood-testis barrier (BTB) was compromised in Bsg KO testes. In conclusion, although some Bsg KO spermatogonia can undergo normal progression to the spermatocyte stage, BSG-mediated germ cell-Sertoli cell interactions appear to be necessary for integrity of the BTB and spermatocyte progression to mature spermatozoa.
doi:10.1016/j.ydbio.2013.05.023
PMCID: PMC3778672  PMID: 23727514
Basigin; BSG; N-glycans; α-Mannosidase II; Spermatogenesis; Blood-testis barrier
25.  Aromatase expression and role of estrogens in male gonad : a review 
The ability of the testis to convert irreversibly androgens into estrogens is related to the presence of a microsomal enzymatic complex named aromatase, which is composed of a specific glycoprotein, the cytochrome P450 aromatase (P450arom) and an ubiquitous reductase. The aromatase gene is unique in humans and contained 18 exons, 9 of them being translated. In the rat testis we have immunolocalized the P450arom not only in Leydig cells but also in germ cells and especially in elongated spermatids. Related to the stage of germ cell maturation, we have shown that the level of P450arom mRNA transcripts decreases, it is much more abundant in pachytene spermatocytes and round spermatids than in mature germ cells whereas the aromatase activity is 2–4 fold greater in spermatozoa when compared to the younger germ cells. Using a highly specific quantitative competitive RT-PCR method we have evidenced that several factors direct the expression of the aromatase gene in Leydig cells, Sertoli cells, pachytene spermatocytes and round spermatids, and it is obvious that promoter PII is the main one but other promoters could be concerned.
In the bank-vole testis we have observed a positive correlation between a fully developed spermatogenesis and a strong immunoreactivity for both P450arom and estrogen receptor β not only in Sertoli cells but also in pachytene spermatocytes and round spermatids. Our recent data obtained from ejaculated human spermatozoa demonstrate the presence of aromatase both in terms of mRNA and protein, and in addition, we suggest that aromatase could be involved in the acquisition of sperm motility. Indeed in men the congenital aromatase deficiency is associated with severe bone maturation problems and sterility. Together with the widespread distribution of estrogen receptors in testicular cells these data clearly show that estrogens play a physiological role in the regulation of spermatogenesis in mammals.
doi:10.1186/1477-7827-1-35
PMCID: PMC155680  PMID: 12747806

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