A single-nucleus transcriptomic atlas of primate testicular aging reveals exhaustion of the spermatogonial stem cell reservoir and loss of Sertoli cell homeostasis

The testis is pivotal for male reproduction, and its progressive functional decline in aging is associated with infertility. However, the regulatory mechanism underlying primate testicular aging remains largely elusive. Here, we resolve the aging-related cellular and molecular alterations of primate testicular aging by establishing a single-nucleus transcriptomic atlas. Gene-expression patterns along the spermatogenesis trajectory revealed molecular programs associated with attrition of spermatogonial stem cell reservoir, disturbed meiosis and impaired spermiogenesis along the sequential continuum. Remarkably, Sertoli cell was identified as the cell type most susceptible to aging, given its deeply perturbed age-associated transcriptional profiles. Concomitantly, downregulation of the transcription factor Wilms' Tumor 1 (WT1), essential for Sertoli cell homeostasis, was associated with accelerated cellular senescence, disrupted tight junctions, and a compromised cell identity signature, which altogether may help create a hostile microenvironment for spermatogenesis. Collectively, our study depicts in-depth transcriptomic traits of non-human primate (NHP) testicular aging at single-cell resolution, providing potential diagnostic biomarkers and targets for therapeutic interventions against testicular aging and age-related male reproductive diseases.

Loss-of-function CFTR p.G970D missense mutation might cause congenital bilateral absence of the vas deferens and be associated with impaired spermatogenesis / 亚洲男科学杂志(英文版)

Congenital bilateral absence of the vas deferens (CBAVD) is observed in 1%-2% of males presenting with infertility and is clearly associated with cystic fibrosis transmembrane conductance regulator (CFTR) mutations. CFTR is one of the most well-known genes related to male fertility. The frequency of CFTR mutations or impaired CFTR expression is increased in men with nonobstructive azoospermia (NOA). CFTR mutations are highly polymorphic and have established ethnic specificity. Compared with F508Del in Caucasians, the p.G970D mutation is reported to be the most frequent CFTR mutation in Chinese patients with cystic fibrosis. However, whether p.G970D participates in male infertility remains unknown. Herein, a loss-of-function CFTR p.G970D missense mutation was identified in a patient with CBAVD and NOA. Subsequent retrospective analysis of 122 Chinese patients with CBAVD showed that the mutation is a common pathogenic mutation (4.1%, 5/122), excluding polymorphic sites. Furthermore, we generated model cell lines derived from mouse testes harboring the homozygous Cftr p.G965D mutation equivalent to the CFTR variant in patients. The Cftr p.G965D mutation may be lethal in spermatogonial stem cells and spermatogonia and affect the proliferation of spermatocytes and Sertoli cells. In spermatocyte GC-2(spd)ts (GC2) Cftr p.G965D cells, RNA splicing variants were detected and CFTR expression decreased, which may contribute to the phenotypes associated with impaired spermatogenesis. Thus, this study indicated that the CFTR p.G970D missense mutation might be a pathogenic mutation for CBAVD in Chinese males and associated with impaired spermatogenesis by affecting the proliferation of germ cells.

Characterization of the protein expression and localization of hnRNP family members during murine spermatogenesis / 亚洲男科学杂志(英文版)

Mammalian testis exhibits remarkably high transcriptome complexity, and spermatogenesis undergoes two periods of transcriptional cessation. These make the RNA-binding proteins (RBPs) the utmost importance during male germ cell development. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a large family of RBPs implicated in many steps of RNA processing; however, their roles in spermatogenesis are largely unknown. Here, we investigated the expression pattern of 12 hnRNP family members in mouse testes and found that most detected members are highly expressed in the testis. Furthermore, we found that most of the detected hnRNP proteins (hnRNPD, hnRNPK, hnRNPQ, hnRNPU, and hnRNPUL1) display the highest signals in the nuclei of pachytene spermatocytes, round spermatids, and Sertoli cells, whereas hnRNPE1 exclusively concentrates in the manchette of elongating spermatids. The expression of these hnRNP proteins showed both similarities and specificity, suggesting their diverse roles in spermatogenesis.

FOXP4 promotes proliferation of human spermatogonial stem cells / 亚洲男科学杂志(英文版)

Continuous self-renewal and differentiation of spermatogonial stem cells (SSCs) is vital for maintenance of adult spermatogenesis. Although several spermatogonial stem cell regulators have been extensively investigated in rodents, regulatory mechanisms of human SSC self-renewal and differentiation have not been fully established. We analyzed single-cell sequencing data from the human testis and found that forkhead box P4 (FOXP4) expression gradually increased with development of SSCs. Further analysis of its expression patterns in human testicular tissues revealed that FOXP4 specifically marks a subset of spermatogonia with stem cell potential. Conditional inactivation of FOXP4 in human SSC lines suppressed SSC proliferation and significantly activated apoptosis. FOXP4 expressions were markedly suppressed in tissues with dysregulated spermatogenesis. These findings imply that FOXP4 is involved in human SSC proliferation, which will help elucidate on the mechanisms controlling the fate decisions in human SSCs.

Genetic pathogenesis of acephalic spermatozoa syndrome: past, present, and future / 亚洲男科学杂志(英文版)

Acephalic spermatozoa syndrome (ASS) is one of the most severe spermatogenic failures of all infertility in men. The cognition of ASS has experienced a tortuous process. Over the past years, with the in-depth understanding of spermatogenesis and the emergence of new genetic research technologies, the unraveling of the genetic causes of spermatogenic failure has become highly active. From these advances, we established a genetic background and made significant progress in the discovery of the genetic causes of ASS. It is important to identify pathogenic genes and mutations in ASS to determine the biological reasons for the occurrence of the disease as well as provide genetic diagnosis and treatment strategies for patients with this syndrome. In this review, we enumerate various technological developments, which have made a positive contribution to the discovery of candidate genes for ASS from the past to the present. Simultaneously, we summarize the known genetic etiology of this phenotype and the clinical outcomes of treatments in the present. Furthermore, we propose perspectives for further study and application of genetic diagnosis and assisted reproductive treatment in the future.

Biallelic mutations in spermatogenesis and centriole-associated 1 like (SPATC1L) cause acephalic spermatozoa syndrome and male infertility / 亚洲男科学杂志(英文版)

Acephalic spermatozoa syndrome is a rare type of teratozoospermia that severely impairs the reproductive ability of male patients, and genetic defects have been recognized as the main cause of acephalic spermatozoa syndrome. Spermatogenesis and centriole-associated 1 like (SPATC1L) is indispensable for maintaining the integrity of sperm head-to-tail connections in mice, but its roles in human sperm and early embryonic development remain largely unknown. Herein, we conducted whole-exome sequencing (WES) of 22 infertile men with acephalic spermatozoa syndrome. An in silico analysis of the candidate variants was conducted, and WES data analysis was performed using another cohort consisting of 34 patients with acephalic spermatozoa syndrome and 25 control subjects with proven fertility. We identified biallelic mutations in SPATC1L (c.910C>T:p.Arg304Cys and c.994G>T:p.Glu332X) from a patient whose sperm displayed complete acephalia. Both SPATC1L variants are rare and deleterious. SPATC1L is mainly expressed at the head-tail junction of elongating spermatids. Plasmids containing pathogenic variants decreased the level of SPATC1L in vitro. Moreover, none of the patient's four attempts at intracytoplasmic sperm injection (ICSI) resulted in a transplantable embryo, which suggests that SPATC1L defects might affect early embryonic development. In conclusion, this study provides the first identification of SPATC1L as a novel gene for human acephalic spermatozoa syndrome. Furthermore, WES might be applied for patients with acephalic spermatozoa syndrome who exhibit reiterative ICSI failures.

DDB1- and CUL4-associated factor 8 plays a critical role in spermatogenesis / 医学前沿

Cullin-RING E3 ubiquitin ligase (CRL)-4 is a member of the large CRL family in eukaryotes. It plays important roles in a wide range of cellular processes, organismal development, and physiological and pathological conditions. DDB1- and CUL4-associated factor 8 (DCAF8) is a WD40 repeat-containing protein, which serves as a substrate receptor for CRL4. The physiological role of DCAF8 is unknown. In this study, we constructed Dcaf8 knockout mice. Homozygous mice were viable with no noticeable abnormalities. However, the fertility of Dcaf8-deficient male mice was markedly impaired, consistent with the high expression of DCAF8 in adult mouse testis. Sperm movement characteristics, including progressive motility, path velocity, progressive velocity, and track speed, were significantly lower in Dcaf8 knockout mice than in wild-type (WT) mice. However, the total motility was similar between WT and Dcaf8 knockout sperm. More than 40% of spermatids in Dcaf8 knockout mice showed pronounced morphological abnormalities with typical bent head malformation. The acrosome and nucleus of Dcaf8 knockout sperm looked similar to those of WT sperm. In vitro tests showed that the fertilization rate of Dcaf8 knockout mice was significantly reduced. The results demonstrated that DCAF8 plays a critical role in spermatogenesis, and DCAF8 is a key component of CRL4 function in the reproductive system.

Rescue of male infertility through correcting a genetic mutation causing meiotic arrest in spermatogonial stem cells / 亚洲男科学杂志(英文版)

Azoospermia patients who carry a monogenetic mutation that causes meiotic arrest may have their biological child through genetic correction in spermatogonial stem cells (SSCs). However, such therapy for infertility has not been experimentally investigated yet. In this study, a mouse model with an X-linked testis-expressed 11 (TEX11) mutation (Tex11

Non-coding RNAs and chromatin: key epigenetic factors from spermatogenesis to transgenerational inheritance

Cellular fate and gene expression patterns are modulated by different epigenetic factors including non-coding RNAs (ncRNAs) and chromatin organization. Both factors are dynamic throughout male germ cell differentiation on the seminiferous tubule, despite the transcriptional inactivation in the last stages of spermatogenesis. Sperm maturation during the caput-to-cauda transit on the epididymis involves changes in chromatin organization and the soma-to-germ line transference of ncRNAs that are essential to obtain a functional sperm for fertilization and embryo development. Here, the male environment (diseases, drugs, mental stress) is crucial to modulate these epigenetic factors throughout sperm maturation, affecting the corresponding offspring. Paternal transgenerational inheritance has been directly related to sperm epigenetic changes, most of them associated with variations in the ncRNA content and chromatin marks. Our aim is to give an overview about how epigenetics, focused on ncRNAs and chromatin, is pivotal to understand spermatogenesis and sperm maturation, and how the male environment impacts the sperm epigenome modulating the offspring gene expression pattern.

The role of tyrosine phosphatase Shp2 in spermatogonial differentiation and spermatocyte meiosis / 亚洲男科学杂志(英文版)

The transition from spermatogonia to spermatocytes and the initiation of meiosis are key steps in spermatogenesis and are precisely regulated by a plethora of proteins. However, the underlying molecular mechanism remains largely unknown. Here, we report that Src homology domain tyrosine phosphatase 2 (Shp2; encoded by the protein tyrosine phosphatase, nonreceptor type 11 [Ptpn11] gene) is abundant in spermatogonia but markedly decreases in meiotic spermatocytes. Conditional knockout of Shp2 in spermatogonia in mice using stimulated by retinoic acid gene 8 (Stra8)-cre enhanced spermatogonial differentiation and disturbed the meiotic process. Depletion of Shp2 in spermatogonia caused many meiotic spermatocytes to die; moreover, the surviving spermatocytes reached the leptotene stage early at postnatal day 9 (PN9) and the pachytene stage at PN11-13. In preleptotene spermatocytes, Shp2 deletion disrupted the expression of meiotic genes, such as disrupted meiotic cDNA 1 (Dmc1), DNA repair recombinase rad51 (Rad51), and structural maintenance of chromosome 3 (Smc3), and these deficiencies interrupted spermatocyte meiosis. In GC-1 cells cultured in vitro, Shp2 knockdown suppressed the retinoic acid (RA)-induced phosphorylation of extracellular-regulated protein kinase (Erk) and protein kinase B (Akt/PKB) and the expression of target genes such as synaptonemal complex protein 3 (Sycp3) and Dmc1. Together, these data suggest that Shp2 plays a crucial role in spermatogenesis by governing the transition from spermatogonia to spermatocytes and by mediating meiotic progression through regulating gene transcription, thus providing a potential treatment target for male infertility.

Altered microRNA profiles of testicular biopsies from patients with nonobstructive azoospermia / 亚洲男科学杂志(英文版)

Many studies have shown that microRNAs (miRNAs) play vital roles during the spermatogenesis. However, little is known about the altered miRNA profiles of testicular tissues in nonobstructive azoospermia (NOA). Using microarray technology, the miRNA expression profiles of testicular biopsies from patients with NOA and of normal testicular tissues were determined. Bioinformatics analyses were conducted to predict the enriched biological processes and functions of identified miRNAs. The microarray data were validated by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), the results of which were then validated with a larger sample size. Correlations between the miRNA expression levels and clinical characteristics were analyzed. Receiver operating characteristic (ROC) curve analysis was used to evaluate the diagnostic ability of miRNAs for azoospermia. Hierarchical clustering showed that 129 miRNAs were significantly differentially expressed between the NOA and control groups. Bioinformatics analysis indicated that the differentially expressed miRNAs were involved in spermatogenesis, cell cycle, and mitotic prometaphase. In the subsequent qRT-PCR assays, the selected miRNA expression levels were consistent with the microarray results, and similar validated results were obtained with a larger sample size. Some clinical characteristics were significantly associated with the expression of certain miRNAs. In particular, we identified a combination of two miRNAs (miR-10b-3p and miR-34b-5p) that could serve as a predictive biomarker of azoospermia. This study provides altered miRNA profiles of testicular biopsies from NOA patients and examines the roles of miRNAs in spermatogenesis. These profiles may be useful for predicting and diagnosing the presence of testicular sperm in individuals with azoospermia.

Mechanistic target of rapamycin kinase (Mtor) is required for spermatogonial proliferation and differentiation in mice / 亚洲男科学杂志(英文版)

Spermatogonial development is a vital prerequisite for spermatogenesis and male fertility. However, the exact mechanisms underlying the behavior of spermatogonia, including spermatogonial stem cell (SSC) self-renewal and spermatogonial proliferation and differentiation, are not fully understood. Recent studies demonstrated that the mTOR complex 1 (mTORC1) signaling pathway plays a crucial role in spermatogonial development, but whether MTOR itself was also involved in any specific process of spermatogonial development remained undetermined. In this study, we specifically deleted Mtor in male germ cells of mice using Stra8-Cre and assessed its effect on the function of spermatogonia. The Mtor knockout (KO) mice exhibited an age-dependent perturbation of testicular development and progressively lost germ cells and fertility with age. These age-related phenotypes were likely caused by a delayed initiation of Mtor deletion driven by Stra8-Cre. Further examination revealed a reduction of differentiating spermatogonia in Mtor KO mice, suggesting that spermatogonial differentiation was inhibited. Spermatogonial proliferation was also impaired in Mtor KO mice, leading to a diminished spermatogonial pool and total germ cell population. Our results show that MTOR plays a pivotal role in male fertility and is required for spermatogonial proliferation and differentiation.

The association of stromal antigen 3 (STAG3) sequence variations with spermatogenic impairment in the male Korean population / 亚洲男科学杂志(英文版)

The stromal antigen 3 (STAG3) gene, encoding a meiosis-specific cohesin component, is a strong candidate for causing male infertility, but little is known about this gene so far. We identified STAG3 in patients with nonobstructive azoospermia (NOA) and normozoospermia in the Korean population. The coding regions and their intron boundaries of STAG3 were identified in 120 Korean men with spermatogenic impairments and 245 normal controls by using direct sequencing and haplotype analysis. A total of 30 sequence variations were identified in this study. Of the total, seven were exonic variants, 18 were intronic variants, one was in the 5'-UTR, and four were in the 3'-UTR. Pathogenic variations that directly caused NOA were not identified. However, two variants, c.3669+35C>G (rs1727130) and +198A>T (rs1052482), showed significant differences in the frequency between the patient and control groups (P = 0.021, odds ratio [OR]: 1.79, 95% confidence interval [CI]: 1.098-2.918) and were tightly linked in the linkage disequilibrium (LD) block. When pmir-rs1052482A was cotransfected with miR-3162-5p, there was a substantial decrease in luciferase activity, compared with pmir-rs1052482T. This result suggests that rs1052482 was located within a binding site of miR-3162-5p in the STAG3 3'-UTR, and the minor allele, the rs1052482T polymorphism, might offset inhibition by miR-3162-5p. We are the first to identify a total of 30 single-nucleotide variations (SNVs) of STAG3 gene in the Korean population. We found that two SNVs (rs1727130 and rs1052482) located in the 3'-UTR region may be associated with the NOA phenotype. Our findings contribute to understanding male infertility with spermatogenic impairment.

Stimulated by retinoic acid gene 8 (Stra8) plays important roles in many stages of spermatogenesis / 亚洲男科学杂志(英文版)

To clarify the functions and mechanism of stimulated by retinoic acid gene 8 (Stra8) in spermatogenesis, we analyzed the testes from Stra8 knockout and wild-type mice during the first wave of spermatogenesis. Comparisons showed no significant differences in morphology and number of germ cells at 11 days postpartum, while 21 differentially expressed genes (DEGs) associated with spermatogenesis were identified. We speculate that Stra8 performs many functions in different phases of spermatogenesis, such as establishment of spermatogonial stem cells, spermatogonial proliferation and self-renewal, spermatogonial differentiation and meiosis, through direct or indirect regulation of these DEGs. We therefore established a preliminary regulatory network of Stra8 during spermatogenesis. These results will provide a theoretical basis for further research on the mechanism underlying the role of Stra8 in spermatogenesis.

Espermiogênese como ferramenta citotaxonômica para diferenciar Triatoma guazu e T. williami, espécies vetoras da doença de Chagas / Spermiogenesis as cytotaxonomic tool to differentiate Triatoma guazu and T. williami, vector species of Chagas disease

Os triatomíneos pertencem à ordem Hemiptera, subordem Heteroptera, família Reduviidae e subfamília Triatominae. Todas as 148 espécies são hematófagas estritas e potenciais vetoras do protozoário Trypanosoma cruzi, agente etiológico da doença de Chagas. Algumas espécies são extremamente semelhantes do ponto de vista morfológico, o que pode dificultar o trabalho dos programas de controle de vetores. Triatoma guazu e T. williami são consideradas como espécies irmãs e não podem ser diferenciadas por diferentes abordagens, como análises morfométricas, isoenzimáticas e cromossômicas. Assim, o presente trabalho analisou as células haplóides durante a espermiogênese, com o objetivo de auxiliar na diferenciação desses vetores. A análise das espermátides de T. guazu e T. williami permitiu diferenciá-los, pois T. williami apresentou apenas um filamento heteropicnótico nas espermátides iniciais que foi parcialmente mantido durante o alongamento celular e T. guazu apresentou dois filamentos heteropicnóticos periféricos que se uniram durante o alongamento celular, dando origem a uma espermátide alongada totalmente heteropicnótica. Assim, ressaltando a importância dessa ferramenta na diferenciação de espécies relacionadas. No entanto, sugerimos que cruzamentos híbridos experimentais devem ser realizados entre esses triatomíneos, com o intuito de confirmar o status específico desses insetos vetores da doença de Chagas.(AU)

Triatomines belong to the Hemiptera order, Heteroptera suborder, Reduviidae family and Triatominae subfamily. All the 148 species are haematophagous strict and potential vectors of the protozoan Trypanosoma cruzi, etiologic agent of Chagas disease. Some species are very similar from a point of view morphological, what can difficult the work of vector control programs. Triatoma guazu and T. williami are considered sister species and cannot be differentiated by many approaches such as morphological, isoenzyme and chromosome analysis. Thus, the present study examined the haploid cells during spermiogenesis, aiming to assist the differentiation of these vectors. The analysis of spermatids of T. guazu and T. williami allowed to differentiate them because T. williami presented only one heteropyknotic filament in the early spermatids which was partially maintained during cell elongation and T. guazu presented two peripheral heteropycnotic filaments that have joined during cell elongation, leading to a elongated spermatid fully heteropycnotic. Thus highlighting the importance of this tool in the differentiation of related species. However, we suggest that experimental hybrid crosses should be made between these triatomines, in order to confirm the specific status of these insect vectors of Chagas disease.(AU)

Sperm evolution in the family Alestidae with comparative data for the genus Chalceus (Ostariophysi: Characiformes)

Spermiogenesis and spermatozoa in six genera of the African family Alestidae plus the Neotropical genus Chalceus are described. Spermiogenesis is quite similar in all Alestidae and is identified as Type I and its variants. In Type I spermiogenesis, the flagellum of earliest spermatids lies lateral to the nucleus, and rotation of the nucleus towards the centriolar complex is observed. Nuclear rotation is complete reaching 90 degrees in Bryconalestes longipinnis, Brachypetersius altus, Brycinus imberi, B. lateralis, and Alestopetersius compressus; and is incomplete reaching 20 degrees in Micralestes acutidens and Rhabdalestes rhodesiensis. Spermatozoa morphology varies from a medial nucleus with fibrillar chromatin in the most basal genus Brycinus to a strongly eccentric nucleus with highly condensed chromatin in the more derived Rhabdalestes and Micralestes. Chalceus has a very similar spermatozoon to that found in Brycinus sharing the fibrillar aspect of the chromatin in the nucleus. This feature is so far only observed in these two genera among African and Neotropical characiform fishes.

A espermiogênese e os espermatozoides de seis gêneros que compõem a família Alestidae mais o gênero Chalceus são descritos. A espermiogênese é muito similar em todas as espécies de Alestidae analisadas e pode ser considerada como sendo o Tipo I e suas variações. Neste tipo de espermiogênese, o flagelo das espermátides iniciais dispõe-se lateral ao núcleo. A rotação do núcleo em direção ao complexo centriolar está presente. A rotação nuclear é completa, atingindo 90 graus, em Bryconalestes longipinnis, Brachypetersius altus, Brycinus imberi, B. lateralis e Alestopetersius compressus; incompleta, atingindo 20 graus, em Micralestes acutidens e Rhabdalestes rhodesiensis. A morfologia dos espermatozoides de Alestidae varia desde o núcleo medial com cromatina fibrilar no gênero Brycinus, considerado mais basal, até o núcleo muito excêntrico com cromatina altamente compactada, nos gêneros Rhabdalestes e Micralestes, considerados mais derivados dentro de Alestidae. Chalceus possui um espermatozoide muito similar a Brycinus, compartilhando entre eles o aspecto fibrilar da cromatina no núcleo. Esta característica até o momento só foi observada nestes dois gêneros dentre os Characiformes Africanos e Neotropicais.

Chromosomal abnormality in men with impaired spermatogenesis

Chromosomal abnormalities and Y chromosome microdeletions are regarded as two most frequent genetic causes associated with failure of spermatogenesis in the Caucasian population. To investigate the distribution of genetic defects in the Romanian population with azoospermia or severe oligozoospermia, karyotype analysis by G-banding was carried out in 850 idiopathic infertile men and in 49 fertile men with one or more children. Screening for microdeletions in the azoospermia factor [AZF] region of Y chromosome was performed by multiplex polymerase chain reaction [PCR] on a group of 67 patients with no detectable chromosomal abnormality. The results of the two groups were compared by a two-tailed Fisher's exact test. In our study chromosomal abnormalities were observed in 12.70% and 8.16% of infertile and fertile individuals respectively. Our data suggests that infertile men with severe azoospermia have higher incidences of genetic defects than fertile men and also patients from any other group. Infertile men with normal sperm present a higher rate of polymorphic variants. It is important to know whether there is a genetic cause of male infertility before patients are subjected to intracytoplasmic sperm injection [ICSI] or testicular sperm extraction [TESE]/ICSI treatment

Comparative expression analysis of septin 14 in testes of infertile men with normal spermatogenesis and spermatogenic failure

Septins are an evolutionary conserved group of GTP-binding and filament-forming proteins that have diverse cellular roles. An increasing body of data implicates the septin family in the pathogenesis of diverse states including cancers, neurodegeneration, and male infertility. The objective of the study was to evaluate the expression pattern of Septin14 in testis tissue of men with and without spermatogenic failure. The samples retrieved accessible random between infertile men who underwent diagnostic testicular biopsy in Royan institute. 10 infertile men with obstructive azoospermia and normal spermatogenesis and 20 infertile men with non-obstructive azoospermia were recruited for real-time reverse transcription [RT]-PCR analysis of the testicular tissue. Total RNA was extracted with trizol reagent. Comparison of the mRNA level of septin14 revealed that in tissues with partial [n=10] or complete spermatogenesis [n=10], the expression of septin 14 was significantly higher than sertoli cell only tissues. The testicular tissues of men with hypospermatogenesis, maturation arrest and sertoli cell only had lower levels of septin 14 transcripts than normal men. These data indicates that Septin 14 expression level is critical for human spermatogenesis

Sperm characteristics as additional evidence of close relationship between lebiasina and piabucina (characiformes: lebiasinidae: lebiasininae)

Spermiogenesis and spermatozoa of representatives of the genera Lebiasina and Piabucina are described. Spermiogenesis is quite similar among all analyzed species of these genera and is identified as Type II. In this type of spermiogenesis, the flagellum of earliest spermatids lies lateral to the nucleus. A slight movement of the nucleus towards the centriolar complex is observed. In late spermatids, the nucleus slightly elongates towards the flagellum. The formation of two striated rootlets apparently occurs in early/intermediate spermatids. The spermatozoa of species of Lebiasina and Piabucina share (1) a drop-shaped slightly elongated nucleus that is laterally positioned relative to the flagellum; (2) a superolateral centriolar complex; (3) two striated rootlets; (4) a basolateral midpiece; (5) oblong mitochondria and (6) a few spherical vesicles. The spermatic characteristics of Lebiasina and Piabucina indicate that spermiogenesis is a homologous process among all species of both genera examined to date and can be considered, along with the spermatozoa morphology, additional evidence indicating a close relationship between Lebiasina and Piabucina.

A espermiogênese e os espermatozoides de representantes dos gêneros Lebiasina e Piabucina são descritos. O processo de espermiogênese é muito semelhante entre todas as espécies analisadas, sendo descrita como do Tipo II. Nesta variação de espermiogênese o flagelo das espermátides iniciais localiza-se lateralmente ao núcleo. O núcleo movimenta-se ligeiramente sobre o complexo centriolar. Nas espermátides finais, o núcleo alonga-se ligeiramente em direção ao eixo flagelar. A formação de duas raízes estriadas ocorre nas espermátides iniciais/intermediárias. Os espermatozoides das espécies de Lebiasiana e Piabucina compartilham principalmente (1) o núcleo em forma de gota, lateralmente posicionado em relação ao eixo flagelar, e ligeiramente alongado em direção ao flagelo, (2) o complexo centriolar superolateral, (3) duas raízes estriadas surgindo a partir do centríolo distal, (4) peça intermediária basolateral, (5) mitocôndrias oblongas e (6) algumas vesículas esféricas. As características espermáticas de Lebiasina e Piabucina foram observadas comparativamente e permitem concluir que o processo de espermiogênese é homólogo em todas as espécies destes dois gêneros examinadas até o momento. Os espermatozoides e principalmente os dados de espermiogênese são apresentados aqui como evidências adicionais que indicam uma relação estreita entre os gêneros Lebiasina e Piabucina.

Spermatic characteristics and sperm evolution on the subfamily Stevardiinae (Ostariophysi: Characiformes: Characidae)

The monophyly and phylogenetic relationships among the members of Clade A characids (sensu Malabarba & Weitzman), later redefined and named as the Stevardiinae (sensu Mirande), have been primarily supported by traditional morphological and molecular data. Herein were examined, described and compared spermiogenesis and sperm ultrastructure of 12 species of the genera Boehlkea, Bryconacidnus, Bryconamericus, Creagrutus, Cyanocharax, Hemibrycon, Knodus, Odontostoechus, Piabina, and Rhinobrycon in order to evaluate possible phylogenetic signals and their potential use in recovering relationships of the Stevardiinae. All examined species demonstrated a nuclear rotation equal or less than 95º resulting in a lateral position of the double nuclear fossa and flagellum. In all species, sperm nuclei are slightly elongate toward the flagellum, the proximal centriole is partially inside the nuclear fossa and lies anterior and oblique to the distal centriole, and the midpiece is short and strongly asymmetric. All species analyzed herein and other species previously examined for these systems in the Stevardiinae share homologous sperm characteristics as evidenced by spermiogenesis, further supporting the monophyly of this clade. Spermatozoa of the Stevardiinae further show three morphotypes (M1, M2, M3) of arrangement of centrioles, flagellum, nucleus and midpiece, hypothesized as successively derived in a series of transformation from the most basal morphotype (M1).

A monofilia e filogenia dos membros do Clado A (sensu Malabarba & Weitzman), mais tarde redefinido e nomeado Stevardiinae (sensu Mirande), é suportada por dados morfológicos e moleculares. Aqui são examinadas, descritas e comparadas a espermiogênese e ultraestrutura do espermatozoide de 12 espécies dos gêneros Boehlkea, Bryconacidnus, Bryconamericus, Creagrutus, Cyanocharax, Hemibrycon, Knodus, Odontostoechus, Piabina e Rhinobrycon, a fim de avaliar possíveis sinais filogenéticos e seu uso potencial no estudo de relações filogenéticas em Stevardiinae. Em todas as espécies examinadas observa-se uma rotação nuclear igual ou menor que 95º, resultando em uma posição lateral da fossa nuclear dupla e do flagelo. Em todas as espécies o núcleo do espermatozoide é alongado em direção ao flagelo, o centríolo proximal é anterior e oblíquo ao centríolo distal e localiza-se parcialmente inserido na fossa nuclear, e a peça intermediária é pequena e fortemente assimétrica. Todas as espécies de Stevardiinae analisadas aqui e outras analisadas previamente compartilham características homólogas dos espermatozoides evidenciadas por sua espermiogênese, corroborando a monofilia deste clado. Os espermatozoides de Stevardiinae apresentam ainda três morfotipos (M1, M2, M3) de acordo com o arranjo dos centríolos, flagelo e peça intermediária, considerados como sucessivamente derivados em uma série de transformações a partir do morfotipo mais basal (M1).