Genetic and histopathological analysis of spermatogenesis after short-term testicular torsion in rats.

BACKGROUND: Patients with testicular torsion (TT) may exhibit impaired spermatogenesis from reperfusion injury after detorsion surgery. Alteration in the expressions of spermatogenesis-related genes induced by TT have not been fully elucidated. METHODS: Eight-week-old Sprague-Dawley rats were grouped as follows: group 1 (sham-operated), group 2 (TT without reperfusion) and group 3 (TT with reperfusion). TT was induced by rotating the left testis 720° for 1 h. Testicular reperfusion proceeded for 24 h. Histopathological examination, oxidative stress biomarker measurements, RNA sequencing and RT-PCR were performed. RESULTS: Testicular ischemia/reperfusion injury induced marked histopathological changes. Germ cell apoptosis was significantly increased in group 3 compared with group 1 and 2 (mean apoptotic index: 26.22 vs. 0.64 and 0.56; p = 0.024, and p = 0.024, respectively). Johnsen score in group 3 was smaller than that in group 1 and 2 (mean: 8.81 vs 9.45 and 9.47 points/tubule; p = 0.001, p < 0.001, respectively). Testicular ischemia/reperfusion injury significantly upregulated the expression of genes associated with apoptosis and antioxidant enzymes and significantly downregulated the expression of genes associated with spermatogenesis. CONCLUSION: One hour of TT followed by reperfusion injury caused histopathological testicular damage. The relatively high Johnsen score indicated spermatogenesis was maintained. Genes associated with spermatogenesis were downregulated in the TT rat model. IMPACT: How ischemia/reperfusion injury in testicular torsion (TT) affects the expressions of genes associated with spermatogenesis has not been fully elucidated. This is the first study to report comprehensive gene expression profiles using next generation sequencing for an animal model of TT. Our results revealed that ischemia/reperfusion injury downregulated the expression of genes associated with spermatogenesis and sperm function in addition to histopathological damage, even though the duration of ischemia was short.

Los ARNs no codificantes largos y su vinculación con las patologías testiculares / Long non-coding RNAs and their involvement in testicular pathologies / RNAs longos não-codificantes e sua ligação com patologias testiculares

Si bien la porción del genoma destinada a la síntesis de proteínas es muy pequeña, actualmente se sabe que casi todo el genoma se expresa bajo forma de ARNs no codificantes. Entre dichos ARNs se encuentran los ARNs no codificantes largos (lncRNAs). Aunque los lncRNAs han sido muy poco estudiados, recientemente han comenzado a centrar la atención de los investigadores, al descubrirse que los mismos pueden desempeñar diversas funciones en la regulación de la expresión génica. Además, su vinculación con patologías ha comenzado a ser puesta de manifiesto. Curiosamente, la cantidad de lncRNAs presentes en el testículo es abrumadoramente mayor que en cualquier otro órgano o tejido estudiado. Los perfiles de expresión de estos lncRNAs varían significativamente a lo largo de la espermatogénesis, y algunas evidencias sugieren que al menos algunos de ellos podrían participar en el proceso de formación de células germinales masculinas. No obstante, el conocimiento sobre el tema es aún muy escaso. En este trabajo revisamos la información disponible sobre la expresión de lncRNAs en el testículo y sus posibles funciones. Asimismo, analizamos algunos ejemplos que ilustran la participación de lncRNAs en el desarrollo de patologías como la infertilidad y el cáncer testicular.

Although the portion of the genome devoted to protein synthesis is very small, it is now known that almost the entire genome is expressed as non-coding RNAs. Among them, there are long noncoding RNAs (lncRNAs). Despite that lncRNAs have been very poorly studied, they have recently started to focus the attention of researchers, as it has been found out that lncRNAs can perform diverse functions in the regulation of gene expression. Besides, their involvement in pathologies is being revealed. Intriguingly, the amount of lncRNAs in the testis is overwhelmingly higher than in any other analyzed organ or tissue. LncRNA expression profiles significantly vary along spermatogenesis, and some evidence suggests that at least some of them could participate in the formation of male germ cells. However, knowledge on the subject is still very scarce. In this work we review the available information on the expression of lncRNAs in testis and their possible roles. We also analyze some examples that illustrate the participation of lncRNAs in the development of pathologies such as infertility and testicular cancer.

Embora a porção do genoma usada para a síntese proteica seja muito pequena, sabe-se agora que quase todo o genoma é expresso na forma de RNAs não-codificantes. Entre esses RNAs estão os longos RNAs não codificantes (lncRNAs). Embora os lncRNAs tenham sido pouco estudados, eles recentemente começaram a focar a atenção dos pesquisadores, ao descobrirem que podem desempenhar diversas funções na regulação da expressão gênica. Além disso, sua ligação com as patologias começou a ser revelada. Curiosamente, a quantidade de lncRNAs presentes nos testículos é esmagadoramente maior do que em qualquer outro órgão ou tecido estudado. Os perfis de expressão destes lncRNAs variam significativamente ao longo da espermatogênese, e algumas evidências sugerem que pelo menos alguns deles poderiam participar no processo de formação de células germinativas masculinas. No entanto, o conhecimento sobre o assunto ainda é muito escasso. Neste trabalho, revisamos as informações disponíveis sobre a expressão de lncRNAs no testículo e suas possíveis funções. Também analisamos alguns exemplos que ilustram a participação dos lncRNAs no desenvolvimento de patologias como infertilidade e câncer testicular.

Whole-Exome Sequencing Identifies Novel Heterozygous Mutation in RAF1 in Family With Neonatal Testicular Torsion.

OBJECTIVE: To investigate a genetic cause of neonatal testicular torsion in 2 siblings and paternal cryptorchidism in a Caucasian family without history of consanguinity, we performed whole exome sequencing. PATIENTS AND METHODS: Targeted exon/whole-exome sequencing was performed in 2 siblings with testicular torsion. Potentially pathogenic variants passing filter criteria were validated with Sanger sequencing of parents to confirm familial segregation. Additionally, immunofluorescence staining for Raf-1, pERK (downstream from Raf-1) and c-Kit was performed on a testicular biopsy on the preserved testicle from the proband brother and compared with testicular biopsies from fertile men. RESULTS: A potentially pathogenic variant was identified in the RAF1 gene (serine/threonine-protein kinase) in exon 7 of chromosome 3: 12645786 G > C; both brothers and father were heterozygous for the variant, while the mother was negative for this mutation. This mutation in exon 7 (chr3:) of RAF1 is predicted to be damaging as a highly conserved splicing site is disrupted. The mutation is not found in the single nucleotide polymorphism database, the 1000 Genomes Project, ExACT, or esp6500. Immunofluorescence of the testis biopsy from one of the brothers demonstrated markedly decreased expression of Raf-1 as well as pERK but similar expression of c-kit when compared with fertile controls. CONCLUSION: We identified a novel nonsynonymous mutation in RAF1 in n Caucasian family with testicular torsion and cryptorchidism. We present the first human evidence that the RAF/MEK/ERK pathway is associated with testicular descent.

Genetic analysis of the human insulin-like 3 gene in pediatric patients with testicular torsion.

PURPOSE: Testicular torsion (TT) mainly affects boys under 18 years old. To avoid orchiectomy, TT requires an immediate operative management. The etiology of TT is still controversial. Observed familiar recurrence suggests the presence of a genetic involvement. The INSL3 gene consists of two exons, and it is specifically expressed in fetal and adult Leydig cells. In transgenic mice, deletion of this gene was observed an increased testicular mobility and testicular torsion. We have hypothesized the possible involvement of the INSL3 gene as a predisposing factor of human TT. METHODS: We performed genetic analysis in 25 pediatric patients with unilateral and intravaginal TT (left, n = 13, 56%; right, n = 12, 48%). The age of the patients ranged from 1 to 16 years (median age n = 10.4 ± 5.46 years). In this study, we included two first male cousins affected by TT. Venous peripheral blood samples was obtained after parental written informed consent. RESULTS: The Thr60Ala polymorphism was detected in exon 1 of INSL3 gene and other 2 rarer variants (rs1047233 and rs1003887) were identified in the 3' untranslated region. These variants are prevalent in patients with TT instead of healthy subjects. CONCLUSIONS: Additional studies in a larger population are needed to better understand the clinical consequence of the INSL 3 variations founded. This would allow in the future to identify the patients at risk of TT to improve clinical management.

Involvement of the bax and bcl-2 system in the induction of germ cell apoptosis is correlated with the time of reperfusion after testicular ischemia in a rat model.

The objective of this study was to examine the relationship between time of reperfusion and bax/bcl-2-dependent germ cell apoptosis after testicular ischemia-reperfusion injury in the rat. In ischemic testis, bax/bcl-2 ratio did not change significantly, and the elevation of germ cell apoptosis was not marked; in the contralateral testis, germ cell apoptosis increased after 6 hours of reperfusion, achieved statistical significance after 24 hours, and decreased after 72 hours of reperfusion and was initiated by decreased bcl-2 messenger RNA levels and elevated bax/bcl-2 ratio within the first 6 hours of reperfusion.

Change and significance of the expression of c-kit and SCF following recovery from unilateral testicular torsion in rats.

PURPOSE: To investigate the change in expression levels of c-kit and SCF, and the protective effects of FSH on ischemia-reperfusion injury due to testicular torsion-detorsion. METHODS: 24 adult male SD rats were divided into three groups of 8: control group, testicular torsion group and FSH-treated group. The control group was treated with sham-operation. Animals in the testicular torsion and FSH-treated groups were subjected to unilateral 720 degrees counterclockwise testicular torsion for 2 hours and then reperfusion was allowed after detorsion. The FSH-treated group received intraperitoneal injection of FSH 15min before detorsion. Then, the rats were sacrificed and the testes were harvested. Histopathological changes were observed by light microscope, and the expression levels of c-kit, SCF in testicular tissue in the different groups were detected by Immunohistochemical assay and Quantitative Real-time RT-PCR analysis. Finally, the relative proportions of germ cells were measured by FCM. RESULTS: c-kit and SCF were positive expressed in 52.58% and 61.16% of testicular cells of control tissues, respectively. Decreases of c-kit and SCF positive cells (15.01% and 9.18%) were found in the testicular torsion group. After being treated by FSH, the number of positive cells increased (31.25% and 20.01%). Moreover, the c-kit and SCF mRNA expression was increased dramatically (P < 0.01) in response to FSH stimulation. Furthermore, the number of haploid, diploid and tetraploid cells has also increased significantly in drug-treated testes (P < 0.01). CONCLUSION: The mechanism of tissue damage in the testicular torsion model, includes changes in the expression of c-kit and SCF following torsion. Also, FSH has a protective effect on germ cells after unilateral testicular torsion, which was reflected by increased c-kit and SCF levels.

Activation of Akt/protein kinase B and extracellular signal-regulated kinase in rats with acute experimental testicular torsion.

The Akt/protein kinase B (PKB) and extracellular signal-regulated kinase (ERK) pathways are involved in cell survival. This study examined the temporal profiles and localization of Akt/PKB and ERK1/2 activation in rat testis after ischemia/reperfusion (I/R). Testicular tissue was collected from normal control rats and rats exposed to reperfusion for 6, 24, and 48 hr after ischemic injury; the tissues were analyzed via Western blotting and immunohistochemistry. Western blot analysis showed that the levels of phosphorylated Akt/PKB (pAkt/PKB) and ERK1/2 (pERK1/2) increased significantly during the first 6-24 hr of reperfusion after ischemia. However, both of these activated proteins were decreased slightly at 48 hr after reperfusion. Immunohistochemically, low levels of pAkt/PKB expression were observed in Sertoli cells from the normal control. After I/R, pAkt/PKB expression increased mainly in the adluminal portion of the Sertoli cells, as well as in spermatogenic cells. In addition, pERK1/2 expression was observed in Sertoli and Leydig cells in the normal control. After I/R, pERK1/2 expression increased in some surviving spermatogenic cells (mainly spermatocytes), as well as in the adluminal portion of Sertoli cells. These results suggest that both Akt/PKB and ERK1/2 are involved in the survival of testicular cells during the early phase of testicular I/R. These pathways may represent important targets for increasing cell survival in testicular injury, including testicular torsion.

Role of cAMP-responsive element modulator-tau (CREMtau) in ipsilateral testicular injury after unilateral testicular torsion-detorsion.

OBJECTIVE: To investigate the role of cAMP-responsive element modulator-tau (CREMtau), an essential transcription factor for spermatogenesis, in ipsilateral testicular injury after unilateral testicular torsion-detorsion. DESIGN: Controlled experimental study using rats. SETTING: Research laboratory. ANIMAL(S): Twenty adult male Sprague-Dawley rats. INTERVENTION(S): Ten rats in the control group underwent a sham operation of the left testes. Ten rats in the torsion-detorsion group received 1 hour of left testicular torsion. Orchiectomy was performed on all rats 3 months after detorsion. MAIN OUTCOME MEASURE(S): Testicular spermatogenesis was evaluated by measuring testicular weight, mean seminiferous tubular diameter, number of germ cell layers, and mean testicular biopsy score. The expressions of CREMtau mRNA and protein in testes were detected by reverse transcription-polymerase chain reaction and Western blot, respectively. RESULT(S): Unilateral testicular torsion-detorsion caused significant spermatogenic damage in the ipsilateral testes, including reductions in testicular weight, mean seminiferous tubular diameter, number of germ cell layers, and mean testicular biopsy score. In ipsilateral testes with spermatogenic damage, the expressions of CREMtau mRNA and protein were also significantly reduced. CONCLUSION(S): Reduction in testicular CREMtau expression may be one of the mechanisms responsible for impairment of spermatogenesis in ipsilateral testes after unilateral testicular torsion-detorsion.

Torsion-induced injury in rat testes does not affect mitochondrial respiration or the accumulation of mitochondrial mutations.

Male rats were subjected to 1 h testicular torsion of the spermatic cord or 1 h torsion followed by detorsion and recovery up to 4 weeks. The extent of tissue damage was evaluated by a testicular biopsy score count and mitochondrial function. Torsion for 1 h followed by detorsion induced significant morphological damage, which became more severe with longer periods of recovery. This morphological damage could not be correlated with mitochondrial damage as assessed by measuring the 4834 bp mitochondrial DNA 'common deletion' using a quantitative competitive polymerase chain reaction (PCR) assay. Mitochondrial respiratory chain activity, as measured by mitochondrial oxygen consumption using an oxygen electrode, did not vary between the treated animals and the controls. We conclude that the common mitochondrial DNA deletion and oxygen consumption are not good indicators of testicular damage induced by torsion.

Determination of testicular function after torsion by DNA flow cytometry of serial fine-needle aspirates.

OBJECTIVE: To determine the efficacy of DNA flow cytometric analysis of testicular percutaneous fine-needle aspirates in the assessment and follow-up of testicular function after torsion, and to determine the relationship between the duration of torsion and testicular injury. MATERIALS AND METHODS: Three groups of 15 adult rats underwent a 720 degrees torsion, with fixation of the mesorchial ligament, for 1, 3 or 5 h. Bilateral aspirations, performed 7, 21 and 35 days after torsion were examined by flow cytometry. Testes were harvested and evaluated histologically using Johnsen's scoring. RESULTS: Irreversible testicular injury occurred in all three groups of rats, with loss of function after 1 h and loss of viability after 3 and 5 h. The results from flow cytometry suggested significant contralateral testicular injury (P < 0.025) but this was not supported by the histological evaluation. There was a strong correlation between the testicular function assessed by flow cytometry and by Johnsen's scoring of histological specimens (r2 = 0.95). CONCLUSION: The assessment of testicular aspirates by flow cytometry allows testicular function to be followed after torsion in rats, and potentially in humans. Using DNA flow cytometry, the temporal course of the twisted testis in the adult rat was determined; contralateral testicular injury following the reversal of torsion could not be excluded.