RESUMEN
Klinefelter syndrome (KS) is the most prevalent genetic disorder of infertile males. This study aimed to determine in Klinefelter patients (KS) the expression levels of spermatogenic markers and testicular growth factors that might predict spermatogenesis based on conventional testicular sperm extraction (TESE). The expression levels of the pre-meiotic (OCT4, CD9, GFR-α1, α-6-INTEGRIN, SALL4, C-KIT), meiotic (CREM-1), and post-meiotic (protamine) markers, as well as the colony stimulating factor-1 (CSF-1) were examined in testicular biopsies with and without mature sperm of KS and normal karyotype of azoospermic patients (AZO) with complete spermatogenesis. In the biopsies of AZO, the expression levels (fold of expression compared to the PPI of the same sample) of OCT4 were 9.68± 7.93, CREM 42.78± 28.22, CSF-1 3.07 ± 3.19, and protamine 78498.12 ± 73214.40. Biopsies from KS included 7 with sperm and 17 without sperm. Among the biopsies with sperm, the expression levels of OCT4 were 7.27± 9.29, CREM 3.13± 7.89, CSF-1 35.5 ± 48.01, and protamine 902.97 ± 2365.92. In 14 biopsies without sperm, we found low expression levels of OCT4, CREM and CSF-1, and no expression of protamine. However, in three of the biopsies without sperm that highly expressed OCT4 and CSF-1, the expression levels of CREM-1 and protamine were high. These results may be used for further consulting with patients considering repeating conventional TESE or micro TESE and cryopreservation for possible future in-vitro spermatogenesis.
Asunto(s)
Azoospermia , Modulador del Elemento de Respuesta al AMP Cíclico , Síndrome de Klinefelter , Factor Estimulante de Colonias de Macrófagos , Factor 3 de Transcripción de Unión a Octámeros , Adulto , Azoospermia/patología , Biopsia , Modulador del Elemento de Respuesta al AMP Cíclico/genética , Humanos , Integrinas , Síndrome de Klinefelter/genética , Factor Estimulante de Colonias de Macrófagos/genética , Masculino , Factor 3 de Transcripción de Unión a Octámeros/genética , Protaminas , Semen , Recuperación de la Esperma , Espermatogénesis/genética , Espermatozoides/patología , Testículo/patologíaRESUMEN
OBJECTIVE: To investigate the prevalence of testicular microlithiasis and its association with sperm retrieval rates and histopathology in men with non-obstructive azoospermia. METHODS: A total of 120 men underwent scrotal ultrasonography prior to microsurgical testicular sperm extraction. Sperm retrieval rate, testicular histopathology, testicular size, reproductive hormones, karyotyping, Y chromosome microdeletion analyses, and presence of varicoceles and hydroceles were compared between men with and without testicular microlithiasis. RESULTS: The total sperm retrieval rate was 40%. Ten men with normal spermatogenesis were excluded. The remaining 110 men with non-obstructive azoospermia were analyzed and testicular microlithiasis was detected in 16 of them (14.5%). The sperm retrieval rate in that subgroup was only 6.2% (1/16) as opposed to 39.4% (37/94) in men with non-obstructive azoospermia and no evidence of microlithiasis (P = 0.009). The mean right and left testicular diameters were significantly lower in the microlithiasis group (P = 0.04). On multivariate logistic regression analysis, the presence of mictolithiasis (odds ratio 7.4, 95% confidence interval 2.3, 12.2; P = 0.01) was the only independent predictor of unsuccessful sperm retrieval. The 15 patients with microlithiasis and without successful sperm extraction were diagnosed by histopathology as having Sertoli cells only. The 16th patient with successful sperm retrieval had a histopathology of mixed atrophy and was diagnosed with Klinefelter syndrome. CONCLUSION: The presence of testicular microlithiasis is associated with low sperm retrieval rates among our cohort of men with non-obstructive azoospermia undergoing scrotal ultrasonography prior to microsurgical testicular sperm extraction. Larger, prospective studies should be conducted to confirm these findings.
Asunto(s)
Azoospermia , Enfermedades Testiculares , Azoospermia/diagnóstico por imagen , Azoospermia/epidemiología , Cálculos , Humanos , Masculino , Estudios Prospectivos , Estudios Retrospectivos , Recuperación de la Esperma , Enfermedades Testiculares/diagnóstico por imagen , Enfermedades Testiculares/epidemiología , Testículo/diagnóstico por imagenRESUMEN
STUDY QUESTION: Are there genetic variants that can be used for the clinical evaluation of azoospermic men? SUMMARY ANSWER: A novel homozygous frame-shift mutation in the MEIOB gene was identified in three azoospermic patients from two different families. WHAT IS KNOWN ALREADY: Up to 1% of all men have complete absence of sperm in the semen, a condition known as azoospermia. There are very few tools for determining the etiology of azoospermia and the likelihood of sperm cells in the testis. The MEIOB gene codes for a single-strand DNA binding protein required for DNA double-strand breaks repair during meiosis. MEIOB appears to be exclusively expressed in human and mouse testis, and MeioB knockout mice are azoospermic due to meiotic arrest. STUDY DESIGN, SIZE, DURATION: Two brothers with non-obstructive azoospermia (NOA) underwent whole-exome sequencing followed by comprehensive bioinformatics analyses. Candidate variations were further screened in infertile and fertile men, as well as in public and local reference databases. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study included 159 infertile and 77 fertile men. The exomes of two Arab men were completely sequenced. In addition, 213 other men of the same Arab ethnicity (136 infertile and 77 fertile men) underwent restriction fragment length polymorphism (RFLP) screening, as did 21 NOA men, of other ethnicities, with testicular impairment of spermatocyte arrest. All of the infertile men underwent Y-chromosome microdeletion and CFTR gene mutation assessments. Comprehensive bioinformatics analyses were designed to uncover candidate mutations associated with azoospermia. MAIN RESULTS AND THE ROLE OF CHANCE: A novel homozygous frame-shift mutation in the MEIOB gene was identified in two brothers of Arab ethnicity. This frame-shift is predicted to result in a truncated MEIOB protein, which lacks the conserved C-terminal DNA binding domain. RFLP screening of the mutation in 157 infertile men, including 112 NOA patients of Arab ethnicity, identified an additional unrelated NOA patient with the same homozygous mutation and a similar testicular impairment. This mutation was not found in available public databases (n > 160 000), nor in the 77 proven fertile men, nor in our database of local Israeli population variations derived from exome and genome sequencing data (n = 500). LIMITATIONS, REASONS FOR CAUTION: We have thus far screened for only two specific MEIOB probable pathogenic mutations in a relatively small local cohort. Therefore, the relative incidence of MEIOB mutations in azoospermia should be further assessed in larger and diverse cohorts in order to determine the efficiency of MEIOB sequence screening for clinical evaluations. WIDER IMPLICATIONS OF THE FINDINGS: The relatively high incidence of likely NOA-causing mutations in MEIOB that was found in our cohort supports the idea that a complete screening of this gene might be beneficial for clinical evaluation of NOA patients. STUDY FUNDING/COMPETING INTEREST(S): This research was supported in part by a grant to EA from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC grant agreement (616088). There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.
Asunto(s)
Azoospermia/genética , Proteínas de Unión al ADN/genética , Meiosis/genética , Mutación , Testículo/metabolismo , Adulto , Árabes/genética , Azoospermia/diagnóstico , Azoospermia/etnología , Azoospermia/patología , Estudios de Cohortes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Linaje , Hermanos , Secuenciación del ExomaRESUMEN
PURPOSE: Up to 1% of all men experience azoospermia, a condition of complete absence of sperm in the semen. The mechanisms and genes involved in spermatogenesis are mainly studied in model organisms, and their relevance to humans is unclear because human genetic studies are very scarce. Our objective was to uncover novel human mutations and genes causing azoospermia due to testicular meiotic maturation arrest. METHODS: Affected and unaffected siblings from three families were subjected to whole-exome or whole-genome sequencing, followed by comprehensive bioinformatics analyses to identify mutations suspected to cause azoospermia. These likely mutations were further screened in azoospermic and normozoospermic men and in men proven to be fertile, as well as in a reference database of local populations. RESULTS: We identified three novel likely causative mutations of azoospermia in three genes: MEIOB, TEX14, and DNAH6. These genes are associated with different meiotic processes: meiotic crossovers, daughter cell abscission, and possibly rapid prophase movements. CONCLUSION: The genes and pathways we identified are fundamental for delineating common causes of azoospermia originating in mutations affecting diverse meiotic processes and have great potential for accelerating approaches to diagnose, treat, and prevent infertility.Genet Med advance online publication 16 February 2017.
Asunto(s)
Azoospermia/diagnóstico , Azoospermia/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Mutación , Secuencia de Aminoácidos , Biomarcadores , Biopsia , Estudios de Casos y Controles , Consanguinidad , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Dineínas/genética , Familia , Pruebas Genéticas , Genotipo , Humanos , Hibridación Fluorescente in Situ , Masculino , Linaje , Espermatozoides/metabolismoRESUMEN
BACKGROUND: The study is aimed to describe a novel strategy that increases the accuracy and reliability of PGD in patients using sperm donation by pre-selecting the donor whose haplotype does not overlap the carrier's one. METHODS: A panel of 4-9 informative polymorphic markers, flanking the mutation in carriers of autosomal dominant/X-linked disorders, was tested in DNA of sperm donors before PGD. Whenever the lengths of donors' repeats overlapped those of the women, additional donors' DNA samples were analyzed. The donor that demonstrated the minimal overlapping with the patient was selected for IVF. RESULTS: In 8 out of 17 carriers the markers of the initially chosen donors overlapped the patients' alleles and 2-8 additional sperm donors for each patient were haplotyped. The selection of additional sperm donors increased the number of informative markers and reduced misdiagnosis risk from 6.00% ± 7.48 to 0.48% ±0.68. The PGD results were confirmed and no misdiagnosis was detected. CONCLUSIONS: Our study demonstrates that pre-selecting a sperm donor whose haplotype has minimal overlapping with the female's haplotype, is critical for reducing the misdiagnosis risk and ensuring a reliable PGD. This strategy may contribute to prevent the transmission of affected IVF-PGD embryos using a simple and economical procedure. TRIAL REGISTRATION: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. DNA testing of donors was approved by the institutional Helsinki committee (registration number 319-08TLV, 2008). The present study was approved by the institutional Helsinki committee (registration number 0385-13TLV, 2013).
Asunto(s)
Enfermedades Genéticas Ligadas al Cromosoma X/genética , Haplotipos/genética , Diagnóstico Preimplantación/normas , Espermatozoides/fisiología , Espermatozoides/trasplante , Donantes de Tejidos , Femenino , Enfermedades Genéticas Ligadas al Cromosoma X/terapia , Humanos , Masculino , Diagnóstico Preimplantación/métodosRESUMEN
PURPOSE: Mature sperm cells can be found in testicular specimens extracted from azoospermic men with non-mosaic Klinefelter syndrome (KS). The present study evaluates the expression of various known molecular markers of spermatogenesis in a population of men with KS and assesses the ability of those markers to predict spermatogenesis. METHODS: Two groups of men with non-obstructive azoospermia who underwent testicular sperm-retrieval procedures were included in the study: 31 had non-mosaic KS (KS group) and 91 had normal karyotype (NK group). Each group was subdivided into mixed atrophy (containing some mature sperm cells) or Sertoli cell only syndrome according to testicular histology and cytology observations. Semi-quantitative histological morphometric analysis (interstitial hyperplasia and hyalinization, tubules with cells and abnormal thickness of the basement membrane) and expression of spermatogenetic markers (DAZ, RBM, BOLL, and CDY1) were evaluated and compared among those subgroups. RESULTS: Clear differences in the histological morphometry and spermatogenetic marker expression were noted between the KS and NK groups. There was a significant difference in the expression of spermatogenetic markers between the subgroups of the NK group (as expected), while no difference could be discerned between the two subgroups in the KS group. CONCLUSION: We conclude that molecular spermatogenetic markers have a pattern of expression in men with KS that is distinctively different from that of men with NK, and that it precludes and limits their use for predicting spermatogenesis in the former. It is suggested that this difference might be due to the specific highly abnormal histological morphometric parameters in KS specimens.
Asunto(s)
Azoospermia/metabolismo , Síndrome de Klinefelter/metabolismo , Espermatogénesis/genética , Testículo/metabolismo , Adulto , Azoospermia/complicaciones , Azoospermia/patología , Biomarcadores/metabolismo , Humanos , Síndrome de Klinefelter/complicaciones , Síndrome de Klinefelter/patología , Masculino , Recuperación de la Esperma , Espermatozoides/crecimiento & desarrollo , Espermatozoides/metabolismo , Espermatozoides/patologíaRESUMEN
BACKGROUND: MicroRNAs are involved in the regulation of spermatogenesis, are detected in semen and may be useful as molecular markers for predicting residual complete spermatogenesis in azoospermic men. OBJECTIVES: To study the biomarker potential of microRNAs that are detected in semen and testicular tissue. MATERIALS AND METHODS: MicroRNA profiles were analyzed in semen fractions of normozoospermic (n = 3) and azoospermic (n = 6) men by small RNA deep sequencing. Specific microRNAs were further analyzed by reverse transcription and quantitative polymerase chain reaction in eight testicular samples and 46 semen supernatants. The semen supernatant samples included 18 normozoospermic and 28 azoospermic men with various pathologies. RESULTS: The sequenced microRNA profiles of semen supernatant fraction samples were distinct from the other fractions. Significant expression differences were observed between the semen supernatant of normozoospermic and azoospermic men. Further analysis by reverse transcription and quantitative polymerase chain reaction revealed that expression of miR-202-3p was considerably reduced (undetectable in most samples) in the azoospermic semen supernatants. The expression of miR-202-3p was significantly lower in the azoospermic specimens than in the normozoospermic specimens and a trend was observed for miR-629-5p (p = 0.03 and 0.06, respectively). Differences in expression levels in the semen supernatant were observed among the various pathologies but not to a level of significance, possibly because of the small subgroups. miRNA-370-3p was significantly higher in semen supernatant samples from azoospermic men without sperm cells in testis (p = 0.05). In testes, the three microRNAs were expressed at higher levels in the obstructive and spermatocyte maturation arrest pathologies than in mixed atrophy and Sertoli cell only. miR-202-3p was detected in all testicular samples. CONCLUSIONS: MicroRNA expression profiles in semen were distinguishable between azoospermic and normozoospermic men. The microRNA profile also diverged among azoospermic men subdivided according to their testicular pathologies. The levels of specific microRNAs in testis and in the semen supernatant were not directly correlated.
Asunto(s)
Azoospermia , MicroARNs , Humanos , Masculino , Testículo/metabolismo , Semen/metabolismo , Espermatogénesis/genética , MicroARNs/genética , MicroARNs/metabolismo , Biomarcadores/metabolismoRESUMEN
Quantitative and qualitative spermatogenic impairments are major causes of men's infertility. Although in vitro fertilization (IVF) is effective, some couples persistently fail to conceive. To identify causal variants in patients with severe male infertility factor and repeated IVF failures, we sequenced the exome of two consanguineous family members who underwent several failed IVF cycles and were diagnosed with low sperm count and motility. We identified a rare homozygous nonsense mutation in a previously uncharacterized gene, RNF212B, as the causative variant. Recurrence was identified in another unrelated, infertile patient who also faced repeated failed IVF treatments. scRNA-seq demonstrated meiosis-specific expression of RNF212B. Sequence analysis located a protein domain known to be associated with aneuploidy, which can explain multiple IVF failures. Accordingly, FISH analysis revealed a high aneuploidy rate in the patients' sperm cells and their IVF embryos. Finally, inactivation of the Drosophila orthologs significantly reduced male fertility. Given that members of the evolutionary conserved RNF212 gene family are involved in meiotic recombination and crossover maturation, our findings indicate a critical role of RNF212B in meiosis, genome stability, and in human fertility. Since recombination is completely absent in Drosophila males, our findings may indicate an additional unrelated role for the RNF212-like paralogs in spermatogenesis.
Asunto(s)
Infertilidad Masculina , Ligasas , Semen , Humanos , Masculino , Aneuploidia , Fertilización In Vitro , Infertilidad Masculina/genética , Ligasas/genética , Espermatozoides , Dominios RING FingerRESUMEN
OBJECTIVE: To examine the effect of the BNT162b, mRNA, SARS-CoV-2 virus vaccine on sperm quality. METHODS: This was a prospective cohort study conducted on sperm donors at the sperm bank of a tertiary, university affiliated medical center. All sperm donors donated sperm repeatedly and the average sperm parameters of all available samples were compared before and after receiving the SARS-CoV-2 vaccine. Each donor served as his own control. For all participants, at-least one sperm sample was received 72 days after completing the second vaccine. Main outcome measures included total sperm count, total motile count and percent of motile sperm. RESULTS: A total of 898 sperm samples from 33 sperm donors that were vaccinated with the Pfizer BNT162b, mRNA, SARS-CoV-2 virus vaccine were analyzed, 425 samples were received before the vaccine, while 473 samples were received after vaccination. Total sperm count and total motile count increased after the second vaccine compared to samples before vaccination. Percent of motile sperm did not change after vaccine. CONCLUSION: The Pfizer BNT162b, SARS-CoV-2 vaccine has no deleterious effect on sperm quality. Patients and physicians should be counseled accordingly.
Asunto(s)
COVID-19 , Motilidad Espermática , COVID-19/prevención & control , Vacunas contra la COVID-19 , Humanos , Masculino , Estudios Prospectivos , ARN Mensajero , SARS-CoV-2 , EspermatozoidesRESUMEN
BACKGROUND: Data on who among the infertile male population may benefit from round spermatid injections (ROSI) are lacking. OBJECTIVE: To determine the probability of finding round spermatids suitable for ROSI in men with non-obstructive azoospermia (NOA) in whom no spermatozoa were retrieved at testicular sperm extraction. MATERIALS AND METHODS: Four-hundred fifty-seven consecutive men with azoospermia underwent testicular sperm extraction. Clinical examination included age, secondary sexual characteristics, testicular size, reproductive hormone estimation, karyotyping, and Y chromosome microdeletion analyses. Histologic examination was performed, and histologic classification was determined by the most advanced spermatogenetic cell identified in the combined histologic and cytologic examination. RESULTS: Of the 457 azoospermic men, 342 were diagnosed with NOA, and 148 (148/342, 43%) had mixed atrophy on histopathology and retrievable spermatozoa. No spermatozoa were found in 194/342 men with NOA (57%). Histopathology diagnosed 145/194 (75%) of them with Sertoli cell only, 45/194 (23%) with spermatocyte maturation arrest, and 4/194 (2%) with spermatid maturation arrest. CONCLUSIONS: Histopathologically identified round spermatids without spermatozoa were rare in men with NOA. Only very few of them are likely to reap the benefits of ROSI, thus presenting the need to reconsider its actual clinical value.
Asunto(s)
Azoospermia , Recuperación de la Esperma , Espermátides , Adulto , Humanos , Masculino , Estudios RetrospectivosRESUMEN
Infertility affects one in six couples, half of which are caused by a male factor. Male infertility can be caused by both, qualitative and quantitative defects, leading to Oligo- astheno-terato-zoospermia (OAT; impairment in ejaculate sperm cell concentration, motility and morphology). Azoospermia defined as complete absence of sperm cells in the ejaculation. While hundreds of genes are involved in spermatogenesis the genetic etiology of men's infertility remains incomplete.We identified a hemizygous stop gain pathogenic variation (PV) in the X-linked Germ Cell Nuclear Acidic Peptidase (GCNA), in an Azoospermic patient by exome sequencing. Assessment of the prevalence of pathogenic variations in this gene in infertile males by exome sequence data of 11 additional unrelated patients identified a probable hemizygous causative missense PV in GCNA in a severe OAT patient. Expression of GCNA in the patients' testes biopsies and the stage of spermatogonial developmental arrest were determined by immunofluorescence and immunohistochemistry. The Azoospermic patient presented spermatogenic maturation arrest with an almost complete absence of early and late primary spermatocytes and thus the complete absence of sperm. GCNA is critical for genome integrity and its loss results in genomic instability and infertility in Drosophila, C. elegans, zebrafish, and mouse. PVs in GCNA appear to be incompatible with male fertility in humans as well: A stop-gain PV caused Azoospermia and a missense PV caused severe OAT with very low fertilization rates and no pregnancy in numerous IVF treatments.
Asunto(s)
Infertilidad Masculina/genética , Mutación , Proteínas Nucleares/genética , Adulto , Humanos , Infertilidad Masculina/patología , Masculino , Proteínas Nucleares/metabolismo , Espermatozoides/metabolismo , Espermatozoides/patologíaRESUMEN
BACKGROUND: The use of quarantined cryopreserved semen is mandatory in donor insemination programs. Whether sperm cells can survive and retain their ability to fertilize after long-term storage remains a controversial issue. The objective of this study was to determine the effect of the duration of cryostorage in liquid nitrogen on the sperm cells' progressive motility concentration (PMC) in a large study group. METHODS: A total of 2525 thawed sperm specimens, packed in straws and donated by 72 sperm bank donors for intrauterine insemination (IUI), were evaluated in an assisted reproduction institute. PMC was recorded after 0.5-14.4 years of cryostorage. RESULTS: The mean (+/-SD) value of PMC of all study samples was 10.8 +/- 3.3 x 10(6)/ml after freezing/thawing and before cryostorage (T0), and 12.3 +/- 2.9 x 10(6)/ml after storage and before using the specimen for IUI (T1, P < 0.0001). Specimen storage for different lengths of time revealed that storage duration had no significant influence on the PMC of the specimens (r = -0.03, P = 0.08). The PMC of partially filled straws was lower than in full straws. Cryostorage duration made no difference in the PMC of raw and washed sperm specimens. CONCLUSION: Prolonged storage of donated sperm in liquid nitrogen had no influence on the PMC of the specimens and therefore should not alter the fertilization potency of donated sperm. The high post-storage values of the PMC compared with the pre-storage PMC values was probably an artifact of the small volume of the pre-storage sample.
Asunto(s)
Criopreservación , Preservación de Semen , Motilidad Espermática , Adulto , Humanos , Técnicas In Vitro , Inseminación Artificial Heteróloga , Masculino , Cuarentena , Bancos de Esperma , Factores de Tiempo , Adulto JovenRESUMEN
Refreezing of sperm samples would provide the possibility of performing more cycles of fertility treatments. Although the effect of repeated cycles of freezing on sperm quality was studied, the effect of the length of the time interval between each freeze-thaw cycle has not been reported. Hence, we assessed the effect of incubation time on the sperm quality of thawed sperm after repeated freezing. One-hundred samples of potential sperm donations with normal sperm quality were evaluated. The fresh semen samples were analyzed and cryopreserved in liquid nitrogen until use. After thawing, the samples were divided randomly to two groups and reanalyzed for motility, vitality, and DNA fragmentation. They were incubated at room temperature and reanalyzed after either 90 min (group A) or 180 min (group B) of incubation, and once again after a repeated cycle of freezing and thawing. Our results showed that the sperm parameters of fresh samples of both groups were similar. After one freeze-thaw cycle, both groups still had comparable values. At the end of their respective incubation time periods, however, there was a significant difference in the mean values of the assessed parameters between the two groups (p < 0.01). An additional freeze-thaw cycle further exacerbated those differences, with group B undergoing an even more substantial decline (p < 0.001). Our data suggest that thawed human spermatozoa sustain a significant decline in sperm parameters in association with longer incubation time, which is further exacerbated by an additional freeze-thaw cycle.
Asunto(s)
Criopreservación , Congelación , Espermatozoides , Fragmentación del ADN , Humanos , Masculino , Motilidad EspermáticaRESUMEN
FK506-binding protein 6 (Fkbp6) is a member of a gene family containing a prolyl isomerase/FK506-binding domain and tetratricopeptide protein-protein interaction domains. Recently, the targeted inactivation of Fkbp6 in mice has been observed to result in aspermic males and the absence of normal pachytene spermatocytes. The loss of Fkbp6 results in abnormal pairing and a misalignment of the homologous chromosomes, and in non-homologous partner switches and autosynapsis of the X chromosome cores in meiotic spermatocytes. In this study, we analyzed whether human FKBP6 gene defects might be associated with human azoospermia. We performed a mutation analysis in all the coding regions of the human FKBP6 gene in 19 patients with azoospermia resulting from meiotic arrest. The expression of the human FKBP6 gene was specific to the testis, and a novel polymorphism site, 245C --> G (Y60X) could be found in exon 3. Our findings suggest that the human FKBP6 gene might be imprinted in the testis based on an analysis using two polymorphism sites.
Asunto(s)
Azoospermia/genética , Polimorfismo de Nucleótido Simple , Proteínas de Unión a Tacrolimus/genética , Testículo/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Análisis Mutacional de ADN , Femenino , Impresión Genómica , Humanos , Masculino , Ratones , Datos de Secuencia MolecularRESUMEN
The bromodomain testis-specific (BRDT) protein belongs to the bromodomain extra-terminal (BET) family of proteins. It serves as a transcriptional regulator of gene expression during spermatogenesis, and is an essential factor for the normal spermatogenesis process. In this study, we characterized mice of several age groups who lacked the Brdt gene. The testes of Brdt mutant mice aged 8 weeks exhibited complete spermatocyte maturation arrest with a significantly increased number of apoptotic cells. The weights of the testes and accessory glands as well as the testosterone levels of the mutant mice were significantly lower compared to the normal mice. The mutant mice had delayed puberty, with normal levels of testosterone and accessory gland weights at the age of 14 and 28 weeks. The testes of the mutant mice at older ages also exhibited round spermatids. The presence of the BRDT protein was identified in the mice pituitary gland. Microarray analysis of mice pituitaries showed that 28 genes were down-regulated while 26 genes were up-regulated in the absence of the Brdt gene. Our results suggest that in addition to its critical role in the spermatogenesis process, the BRDT protein is also responsible for scheduling male puberty by regulation of the pituitary-gonad axis.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Nucleares/genética , Espermatogénesis/genética , Animales , Humanos , Masculino , Ratones , Ratones Noqueados , Proteínas Nucleares/biosíntesis , Sistema Hipófiso-Suprarrenal/crecimiento & desarrollo , Sistema Hipófiso-Suprarrenal/metabolismo , Espermátides/crecimiento & desarrollo , Espermátides/metabolismo , Espermatocitos/crecimiento & desarrollo , Espermatocitos/patología , Testículo/crecimiento & desarrollo , Testículo/metabolismoRESUMEN
The current study identified for the first time calretinin expression in abnormal Sertoli cells of azoospermic men who underwent testicular biopsy for sperm recovery and application of the retrieved sperm by in vitro fertilization techniques. Testicular biopsies with various spermatogenic impairments were evaluated immunohistochemically for the expression of the calretinin calcium-binding protein and the marker for immaturity of Sertoli cells, cytokeratin-18 (CK-18). Distribution of the markers was assessed in testes demonstrating a histological phenotype of mixed atrophy, Sertoli cell-only, or normal spermatogenesis (obstructive-azoospermia) and in men carrying a deletion in the azoospermia factor region located on the Y chromosome. Calretinin-immunopositive immature Sertoli cells revealed by co-localization of both markers, calretinin and CK-18, were identified in the mixed atrophy group in seminiferous tubules demonstrating spermatogenic failure. Sertoli cells expressing both markers were rarely detected in all other groups. Leydig cells in all the assessed biopsies expressed calretinin and served as a built-in control for immunoreactivity. This pattern of calretinin-selective expression in immature Sertoli cells suggests a functional relationship between calretinin expression and the degree of Sertoli cell differentiation. Disorders of Sertoli cell differentiation as indicated by calretinin and/or CK-18 expression contribute to the multifactorial mechanisms underlying spermatogenic failure.
Asunto(s)
Oligospermia/metabolismo , Oligospermia/patología , Proteína G de Unión al Calcio S100/metabolismo , Células de Sertoli/metabolismo , Células de Sertoli/patología , Biopsia , Calbindina 2 , Diferenciación Celular , Humanos , Inmunohistoquímica , Queratinas/metabolismo , MasculinoRESUMEN
OBJECTIVE: To demonstrate the pattern(s) of spermatogonial proliferation in different spermatogenic disorders. DESIGN: Retrospective case-control study. Teaching hospital. PATIENT(S): Azoospermic men who underwent testicular biopsy for sperm recovery and preparation for intracytoplasmic sperm injection. INTERVENTION(S): Testicular biopsy evaluation by quantitative immunohistochemistry for proliferating cell nuclear antigen (PCNA). MAIN OUTCOME MEASURE(S): The expression of PCNA in spermatogonia as an index of proliferating activity in testes with focal spermatogenesis, spermatocyte maturation arrest, or normal spermatogenesis. RESULT(S): In biopsies with focal spermatogenesis (11 men), there was a statistically significant reduction of PCNA-labeled spermatogonia in seminiferous tubules showing spermatocyte arrest compared with the expression in adjacent tubules with advanced spermatogenic stage or in normal spermatogenesis (obstructive azoospermia, six men). However, PCNA expression in tubules of the group with complete maturation arrest (six men) was significantly higher compared with the same spermatogenic defect-spermatocyte arrest-within focal spermatogenesis biopsies. CONCLUSION(S): Different causes underlie the spermatogenic disorders reported in this study. In focal spermatogenesis, the disorder is associated with the presence of mitotic inactive spermatogonia. The detection of normal active spermatogonia in the spermatocyte arrest group indicates that the spermatogenic defect, which is accompanied by meiosis impairment, is not related to a malfunction of spermatogonial proliferation.
Asunto(s)
Oligospermia/patología , Oligospermia/fisiopatología , Espermatogénesis , Espermatogonias/patología , Adulto , Ciclo Celular , Humanos , Inmunohistoquímica , Masculino , Antígeno Nuclear de Célula en Proliferación/metabolismo , Túbulos Seminíferos/metabolismo , Espermatocitos/patologíaRESUMEN
OBJECTIVE: To evaluate the involvement of Sertoli cell in different spermatogenic disorders. DESIGN: Retrospective case-control study. SETTING: Teaching hospital. PATIENT(S): Azoospermic men who underwent testicular biopsy for sperm recovery in preparation for intracytoplasmic sperm injection. INTERVENTION(S): Testicular biopsy evaluation by quantitative immunohistochemistry for the immature Sertoli cell markers anti-Müllerian hormone and cytokeratin 18 (CK-18). MAIN OUTCOME MEASURE(S): Relative area of immature Sertoli cells in testes with focal spermatogenesis, spermatocyte maturation arrest, or normal spermatogenesis. RESULT(S): The relative area occupied by immature Sertoli cells, as revealed by anti-Müllerian hormone and CK-18 expression, was highest in the 11 men with focal spermatogenesis. In the group representing normal spermatogenesis (obstructive azoospermia, 6 men) and in the group characterized by spermatocyte maturation arrest (6 men), the areas occupied by anti-Müllerian hormone- and CK-18-positive cells were minimal. CONCLUSION(S): Different etiologies underlie the spermatogenic disorders reported in this study. In focal spermatogenesis with high anti-Müllerian hormone and CK-18 expression, the spermatogenic impairment is associated with the presence of immature Sertoli cells. The detection of normal mature Sertoli cells in the spermatocyte maturation arrest group indicates that the spermatogenic defect that is accompanied by an impairment of meiosis is intrinsic to the germ line without affecting Sertoli cell differentiation.
Asunto(s)
Oligospermia/etiología , Oligospermia/fisiopatología , Células de Sertoli , Adulto , Hormona Antimülleriana , Estudios de Casos y Controles , Senescencia Celular , Glicoproteínas/metabolismo , Humanos , Inmunohistoquímica , Queratinas/metabolismo , Masculino , Estudios Retrospectivos , Células de Sertoli/metabolismo , Espermatogénesis , Hormonas Testiculares/metabolismo , Testículo/metabolismo , Testículo/patología , Testículo/fisiopatologíaRESUMEN
Deletions in the q arm of the Y chromosome result in spermatogenesis impairment. The aim of the present study was to observe the X and Y chromosome alignment in the spermatocytes of men with Y chromosome microdeletion of the azoospermia factor (AZF) region. This was performed by multicolor fluorescence in situ hybridization probes for the centromere and telomere regions. Testicular biopsies were performed in a testicular sperm extraction-intracytoplasmic sperm injection set-up in 11 azoospermic men: 8 (nonobstructive) with AZF deletions and 3 (obstructive) controls. Histological sections, cytology preparations of the testicular biopsies, and evaluation of the meiosis according to the percentage of XY and 18 bivalents formation were assessed. Spermatozoa were identified in at least one location in controls and specimens with AZFc-deleted Y chromosomes. Complete spermatocyte arrest was found in those with a deletion that included the entire AZFb region. Bivalent formation rate of chromosome 18 was high in all samples (81%-99%). In contrast, the rate of bivalent X-Y as determined by centromeric probes was lower but in the range favorable with spermatozoa findings in controls and patients with the AZFc deletion (56%-90%), but not in those with AZFb-c deletions (28%-29%). A dramatic impairment in the normal alignment of X and Y telomeres in the specimen with AZFb-c deletion was shown (29%), compared to the specimens with AZFc deletion (70%-94%). It is suggested that the absence of sperm cells in specimens with the entire AZFb and with AZFb-c deletions is accompanied by meiosis impairment, perhaps as a result of the extent of the deletion or because of the absence of genes that are involved in the X and Y chromosome alignment.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos X , Cromosomas Humanos Y , Oligospermia/genética , Biopsia , Emparejamiento Cromosómico , Humanos , Hibridación Fluorescente in Situ , Masculino , Meiosis , Oligospermia/patología , Espermatocitos/citología , Testículo/patologíaRESUMEN
Germ cell-less (GCL) protein is a nuclear envelope protein highly conserved between the mammalian and Drosophila orthologues. In Drosophila, maternal GCL protein is required to establish the germ lineage during embryonic development. In mammals, it is suggested that the GCL function is mainly in spermatogenesis and that it might be related to the ability of mouse GCL to repress transcription. Using reverse transcriptase-polymerase chain reaction analyses, we investigated the role of human GCL (HGCL) in spermatogenesis by studying its expression in the testicular tissue of 67 azoospermic men with normal karyotype and no Y-chromosome microdeletion. Their testicular biopsy specimens underwent meticulous histological and cytological analysis as well as molecular analysis with various markers of spermatogenesis (RBM1, DAZ, and CDY1). The rate of X-Y and 18 chromosome bivalent formation during meiosis was additionally assessed in 22 of these biopsy specimens and correlated to HGCL expression. Expression of HGCL was affected in parallel with the severity of testicular impairment found. Defective sperm motility was associated with the absence of HGCL. Nevertheless, the absence of HGCL expression did not influence the normal process of chromosome bivalent formation in meiosis. Our results suggest that HGCL is not essential for the chromosomal events of meiosis but might be involved in later aspects of spermatogenesis.