Cryptorchid Rats Research Articles

Heat stress induced piRNA alterations in pachytene spermatocytes and round spermatids

BackgroundSpermatogenesis is a temperature-sensitive process, and elevation in temperature hampers this process quickly and significantly. We studied the molecular effects of testicular heating on piRNAs and gene expression in rat testicular germ cells.MethodsWe generated a cryptorchid rat model by displacing the testis from the scrotal sac (34 °C) to the abdominal area (37 °C) and sacrificed animals after 1 day, 3 days, and 5 days. Pachytene spermatocytes and round spermatids were purified using elutriation centrifugation and percoll gradient methods. We performed transcriptome sequencing in pachytene spermatocytes and round spermatids to identify differentially expressed piRNAs and their probable targets, i.e., TE transcripts and mRNAs.ResultsAs a result of heat stress, we observed significant upregulation of piRNAs and TE transcripts in testicular germ cells. In addition to this, piRNA biogenesis machinery and heat shock proteins (Hsp70 and Hsp90 family members) were upregulated. mRNAs have also been proposed as targets for piRNAs; therefore, we shortlisted certain piRNA-mRNA pairs with an inverse relationship of expression. We observed that in testicular heat stress, the heat shock proteins go hand-in-hand with the upregulation of piRNA biogenesis machinery. The dysregulation of piRNAs in heat-stressed germ cells, increased ping-pong activity, and disturbed expression of piRNA target transcripts suggest a connection between piRNAs, mRNAs, and TE transcripts.ConclusionsIn heat stress, piRNAs, piRNA machinery, and heat shock proteins are activated to deal with low levels of stress, which is followed by a rescue approach in prolonged stressaccompained by high TE activity to allow genetic mutations, perhaps for survival and adaptability.

Safety Study of an Original Mesenchymal Stromal Cell Secretome-Based Medicinal Product for Spermatogenesis Restoration

SCIENTIFIC RELEVANCE. Currently, there are no effective and safe medicinal products for idiopathic male infertility. Previous studies in two animal models of infertility (short-term cryptorchidism in rats and doxorubicin-induced testicular injury in mice) have shown the effectiveness of an originator medicinal product based on the mesenchymal stromal cell (MSC) secretome.AIM. The aim of the study was to evaluate the toxicity profile of the MSC secretome-based medicinal product in rats after local intratesticular or intramuscular administration.MATERIALS AND METHODS. The MSC secretome is a combination of factors secreted by MSCs in low-glucose Dulbecco’s modified Eagle’s medium (DMEM-LG) for MSC conditioning. In the single-dose toxicity study, the MSC secretome-based medicinal product was injected under the testicular tunica albuginea of male Wistar rats (15 per group) at doses of 15 and 25 relative units (RU) per animal, which are 1.5 and 2.5 times higher than the therapeutic dose (10 RU). In the repeat-dose toxicity study, male Wistar rats (10 per group) received intramuscular thigh injections of the medicinal product on days 1, 6, and 12 at doses of 15 and 25 RU per animal. The local tolerance study involved histopathological examination of the testes and thighs at the injection site. All studies included control groups of intact animals and animals similarly injected with blank DMEM-LG. The early follow-up period was 14 days, and the late follow-up period was 42 days.RESULTS. The rats showed no changes in the general condition after single and repeated doses of the MSC secretome-based medicinal product. Single subtunical doses induced moderate irritation; its signs included pathological changes in individual seminiferous tubules: epithelial atrophy (70% of the animals on day 14; 55% at late follow-up) and sperm stasis (70% of the animals). Similar changes were observed in the blank DMEM-LG group (up to 80% of the animals). There were no pathological changes in the tissues after repeated injections. A transient increase in alkaline phosphatase activity was detected in animals after their third intramuscular injection at a dose of 25 RU; the other biochemical parameters were normal in all study groups.CONCLUSIONS. The MSC secretome-based medicinal product has a favourable safety profile following both intratesticular and intramuscular administration, as it does not cause any permanent changes in the studied organs and tissues.

Stereological study of testes following experimentally-induced unilateral cryptorchidism in rats.

Cryptorchidism is one of the main causes of infertility and can result in testicular cancer. This study aimed to present quantitative data on the damage caused by cryptorchidism using stereological analysis. Thirty newborn rats were randomly divided into control and experimental groups. The experimental group underwent surgery to induce unilateral cryptorchidism in the left testis, whereas the control group underwent a sham surgical procedure 18 days after birth. The testes were removed at designated time points (40, 63, and 90 days after birth) for stereological evaluation and sperm analysis. Total testicular volume, interstitial tissue volume, seminiferous tubule volume and length, and seminiferous epithelium volume and surface area were measured. Other parameters, such as sperm count, sperm morphology, and sperm tail length, were also examined. Statistically significant differences (p<0.05) were observed between the experimental and the control groups at different ages regarding the volumes of various parameters, including the surface area of the germinal layer, the length of the seminiferous tubules, sperm count, and sperm morphology. However, no significant differences were observed in the epithelial volume and the sperm tail length of the groups. Given the substantial effect of cryptorchidism on different testicular parameters, as well as the irreversible damage it causes in the testes, it is important to take this abnormality seriously to prevent these consequences.

Identification of active spermatogenesis using a multiphoton microscope.

The sperm retrieval rate of microdissection testicular sperm extraction varies from 25% to 60%. Therefore, it is necessary to establish objective selection criteria for identifying seminiferous tubules with spermatozoa. Our aim was to develop a method for identifying spermatogenesis without sectioning testicular tissues. Testicular tissues of 10-week-old normal rats were fixed with 4% paraformaldehyde. Fluorescent labeling of seminiferous tubule nuclei and F-actin was performed, and the specimens were observed without sectioning using a multiphoton microscope. Cryptorchid rats were used as a model lacking elongated spermatids. Multiphoton images were compared with images of normal seminiferous tubules. In addition, seminiferous tubules of 10-week-old normal rats were labeled by testicular interstitial injection of fluorescent probes and observed by a multiphoton microscope without fixation. Terminal deoxynucleotidyl transferase dUTP nick end labeling-stained images of normal and probe-injected testes were compared. In fixed seminiferous tubules, elongated spermatids were identified. In addition, F-actin of apical ectoplasmic specialization was observed around elongated spermatids. Furthermore, spermatogenic stages were identified by an array of nuclei or F-actin. In cryptorchid testes, elongated spermatids and F-actin of the apical ectoplasmic specialization were not observed. In testes injected with fluorescent probes, F-actin of the apical ectoplasmic specialization was observed, and spermatogenic stages were identified without fixation. There was no significant difference in the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells per seminiferous tubule between normal and probe-injected testes. Seminiferous epithelium could be observed without sectioning of tissues by fluorescent probes and a multiphoton microscope. Active spermatogenesis was observed by labeling F-actin with and without fixation. Moreover, the toxicity of fluorescent probes was limited. Our method has a potential for live imaging of testicular tissue.

The protective effect of curcumin on testicular tissue in a cryptorchid rat model

Cryptorchidism is the most common abnormality of male sexual development. For the protection of testicular functions, antioxidants have emerged as novel options. This study aimed to investigate the protective effect of curcumin (Cur), a strong antioxidant, on the Flutamide-induced cryptorchidism testicular tissue. Pregnant rats were randomly allocated to 3 groups (n=10, each): a control, a model, and a Cur-treated group (100mg/kg/d). All offspring were delivered by days 21-22 of gestation and the male rats were sacrificed at postnatal birth days (PNDs) PND60. The testicles were separated and weighed, followed by TUNEL staining to detect germ cell apoptosis, an ELISA kit to measure SOD and MDA, and Western blot analysis to evaluate the expression of Bax, Bcl-2, and PCNA. Curcumin administration ameliorated the histological appearance of the testis and greatly reduced the level of apoptosis in cryptorchidism rats' testicular cells. After curcumin treatment, the expression of proliferating cell nuclear antigen (PCNA) was restored in the testis tissues of cryptorchidism rats. Curcumin therapy reduced Bax expression while increasing Bcl-2 expression, according to the molecular study. Curcumin therapy also reduced malondialdehyde (MDA) levels and enhanced superoxide dismutase (SOD) levels in cryptorchidism rats' testis tissue. It can be concluded that curcumin administration significantly reduced the germ cell apoptosis in rats with cryptorchidism, which provides new insight for antioxidant therapy in preserving testicular functions before or after surgery in cryptorchidism.

Fertility-enhancing potential of ethanol extract of Cuscuta chinensis seeds in a rat model of unilateral cryptorchidism

&#x0D; &#x0D; &#x0D; &#x0D; Purpose: To investigate the fertility-enhancing potential of the ethanol extract of Cuscuta chinensis seeds in a rat model of unilateral cryptorchidism (ULC), and the mechanism(s) of action.&#x0D; Methods: Healthy male Sprague Dawley rats (n = 48; mean weight = 220 ± 10 g) were randomly assigned to 4 groups (12 rats/group): control, ULC, 100 mg extract/kg and 200 mg extract/kg groups. Unilateral cryptorchidism of right testis was induced via standard method using an operating microscope. Rats in the treatment groups received 100 and 200 mg of ethanol extract of Cuscuta chinensis/kg orally once a day for 60 days. Sperm count and sperm motility were determined in seminal vesicular fluid (SVF) suspension. Oxidative stress markers and histological changes in rat testis were evaluated. The levels of caspase-3 and caspase-9 in testicular tissue were assessed by enzyme-linked immunosorbent assay (ELISA), while the protein expressions of Nrf2 and heme oxygenase 1 (HO-1) were determined using Western blotting.&#x0D; Results: Body and reproductive organ weights, sperm count, sperm motility, and activities of glutathione peroxidase (GPx), catalase and superoxide dismutase (SOD) were significantly reduced in ULC group, relative to control group, but these parameters were significantly and dose-dependently increased following extract treatment (p &lt; 0.05). Malondialdehyde (MDA), 8-hydroxy-2’-deoxyguanosine (8-OHdG), caspase-3 and caspase-9 levels were significantly higher in ULC group than in control group, but they were reduced significantly and dose-dependently after extract treatment (p &lt; 0.05). Moreover, the protein expressions of Nrf2 and HO-1 were significantly downregulated in ULC group, when compared with control group, but they were significantly and dose-dependently upregulated by the extract (p &lt; 0.05). Cross sections of testicular tissues of rats in ULC group revealed narrowed and thickened seminiferous tubules (disrupted spermatogonia) characterized by increased apoptotic bodies (increased number of necrotic Sertoli and Leydig cells). However, there were few damaged or necrotic Sertoli and Leydig cells, and complete absence of thickening of seminiferous tubules in testicular tissues of rats treated with the extract.&#x0D; Conclusion: The ethanol extract of Cuscuta chinensis seeds effectively mitigates cryptorchidism in rats via mechanisms involving the regulation of Nrf2/HO-1 signaling pathway, and inhibition of apoptosis and oxidative stress. Thus, the plant extract has potentials for further development for the management of male infertility&#x0D; &#x0D; &#x0D; &#x0D;

Retinoic acid can improve autophagy through depression of the PI3K-Akt-mTOR signaling pathway via RARα to restore spermatogenesis in cryptorchid infertile rats

Cryptorchidism-caused adult infertility is a common component of idiopathic reasons for male infertility. Retinoic acid (RA) has a vital effect on the spermatogenesis process. Here, we found that the expression of c-Kit, Stra8, and Sycp3 could be up-regulated via the activation of retinoic acid receptor α (RARα) after RA supplementation in neonatal cryptorchid infertile rats. We also demonstrated that the protein expression of PI3K, p-Akt/pan-Akt, and p-mTOR/mTOR was higher in cryptorchid than in normal testes, and could be suppressed with RA in vivo. After RA treatment in infertile cryptorchid testis in vivo, the levels of the autophagy proteins LC3 and Beclin1 increased and those of P62 decreased. Biotin tracer indicated that the permeability of blood-testis barrier (BTB) in cryptorchid rats decreased after RA administration. Additionally, after blocking the RARα with AR7 (an RARα antagonist) in testicle culture in vitro, we observed that compared with normal testes, the PI3K-Akt-mTOR signaling pathway and the autophagy pathway was increased and decreased, respectively, which were coincident with cryptorchisd testes in vivo. Additionally, the appropriate concentrations of RA treatment could depress the PI3K-Akt-mTOR signaling pathway and improve the autophagy pathway. The results confirmed that RA can rehabilitate BTB function and drive key protein levels in spermatogonial differentiation through depressing the PI3K-Akt-mTOR signaling pathway via RARα.

Rubus apetalus (Rosaceae) improves spermatozoa characteristics, antioxidant enzymes and fertility potential in unilateral cryptorchid rats

BackgroundCryptorchidism (CPT) is an important cause of male infertility. Rubus apetalus is a medicinal plant with a powerful antioxidant potential. We investigated the effects of aqueous and methanolic extracts of R. apetalus on spermatozoa parameters, antioxidant enzymes and fertility potential of rats with experimental unilateral CPT.MethodNormal (n = 15), sham-operated (n = 15) and cryptorchid rats (n = 80; distributed into 16 groups of 5 rats/group) were treated for 2, 4 or 8 weeks with either distilled water (10 ml/kg/day), vitamin E (75 mg/kg/day), aqueous or methanolic extract of R. apetalus (12 and 60 mg/kg). Sex organ weights, spermatozoa parameters, testicular proteins, sex hormones, fertility potential, morphometric characteristics of testis and oxidative stress markers were measured.ResultsCPT significantly (p < 0.05–0.001) decreased testicular and epididymal weights, spermatozoa density, spermatozoa motility, spermatozoa normality, testicular proteins, LH, FSH and testosterone concentrations. In cryptorchid rats, peri-vascular fibrosis significantly increased (p < 0.001), while diameter of the seminiferous tube, germ cell thickness, gestation index and fertility index decreased when compared to control. Additionally, CPT induced oxidative stress by increasing lipid peroxidation and by reducing superoxide dismutase and catalase activities. These alterations were corrected by R. apetalus. For instance, a significantly increase (p < 0.05–0.001) in spermatozoa motility, normality, viability and density after 2, 4 and 8 weeks of treatment was noticed. R. apetalus also increased (p < 0.05–0.001) testicular proteins, gestation index (90–100%) and fertility index (90–100%), compared to the untreated cryptorchid rats.ConclusionR. apetalus boosts fertility potential in cryptorchid rats and could be considered as a promising alternative agent for the management of infertility associated with CPT.

504 - Abnormal Claudin-11 localization and its effects on the blood-testis barrier function during puberty in a cryptorchid rat model

504 - Abnormal Claudin-11 localization and its effects on the blood-testis barrier function during puberty in a cryptorchid rat model

Disorganization of claudin-11 and dysfunction of the blood-testis barrier during puberty in a cryptorchid rat model.

Cryptorchidism is known to impair spermatogenesis. The blood-testis barrier (BTB) becomes defined in seminiferous tubules around puberty and provides a suitable environment for germ cells. Little is known about the BTB in undescended testes (UDT). To determine the role of BTB during puberty in UDT using a non-surgical cryptorchid rat model. Unilateral cryptorchid male rats were intraperitoneally injected with non-steroidal antiandrogen during intrauterine development; the testes were harvested at 4, 5, and 6weeks after birth. Testicular histology, expression levels of the BTB proteins (claudin-11, occludin, zonula occludens-1), and apoptotic cells were evaluated by immunohistochemistry, Western blotting, and TUNEL assay. The functionality of the BTB was investigated by electron microscopy using the lanthanum tracer method. The testicular histology of undescended testes 6weeks after birth showed maturation arrest at the spermatocyte level. The BTB protein distributions were altered in the UDT, with a noticeable difference in claudin-11(CLDN11) localization from 4 to 5weeks after birth between control and UDT samples. BTB protein levels were similar. More apoptotic germ cells were detected in the adluminal compartment of tubules in the UDT than in the control testes. Electron microscopy showed that the lanthanum tracer was limited to the BTB of control testes, whereas it penetrated the BTB of UDT. Here, loss of normal BTB function and impaired spermatogenesis were observed in UDT during puberty. CLDN11 is a pivotal tight junction protein belonging to the BTB. Tight junctions are considered as essential for normal spermatogenesis, and abnormal CLDN11 organization may cause UDT-associated male infertility. CLDN11 disorganization within the BTB may cause spermatogenic impairment, possibly by limiting the BTB function.

A PERSPECTIVE ON APPLICATION OF MESENCHYMAL STROMAL CELLS OR THEIR SECRETOME-BASED PRODUCTS FOR TREATING MALE INFERTILITY

The study revealed the regenerative effects of MSC and MSC secretome in a model of abdominal cryptorchidism in rats.

A magic kick for regeneration: role of mesenchymal stromal cell secretome in spermatogonial stem cell niche recovery

BackgroundInjury of stem cell niches may disturb tissue homeostasis and regeneration coordinated by specific niche components. Yet, the mechanisms of stem cell niche restoration remain poorly understood. Herein, we examined the role of mesenchymal stromal cells (MSCs) as pivotal regulators of stem cell niche recovery focusing on the effects of their secretome.MethodsThe spermatogonial stem cell (SSC) niche was selected as a model. SSC niches were injured by inducing abdominal cryptorchidism in rats. Briefly, testes of anesthetized rats were elevated into the abdominal cavity through the inguinal canal for 14 days. After descent of testes, MSC or MSC secretome treatment was applied to the animals by local subtunical injections.ResultsLocal administration of MSC or MSC secretome was sufficient to recover spermatogenesis and production of functional germ cells. The effects of MSC and their secreted components were comparable, leading to restoration of Sertoli cell pools and recovery of Leydig cell secretory functions.ConclusionOur data suggest that MSCs mimic the functions of lost supportive cells within the stem cell niche, transiently providing paracrine stimuli for target cells and triggering tissue regenerative processes after damage.

Investigation of the Protective Effects of Amlodipine on the Structure and Function of Testicular Tissue following Experimental Unilateral Cryptorchidism in Rats

Objective- Cryptorchidism, common birth defect of the male genital tract, is one of the causes of fertility problems. The elevation of tissue temperature following of cryptorchidism could induce oxidative stress which influences the cellular and tissue degeneration. Amlodipine is a third-generation of calcium channel blockers which has antioxidant activity. The aim of this study was to evaluate the protective effects of amlodipine on testicular tissue alterations in an animal model of cryptorchidism. Design- Experimental study Animals- Thirty adult male Sprague-Dawley rats weighing 200-220g Procedure- Experimental cryptorchidism was induced in adult rats. Amlodipine (10 mg/kg b.w.) was administrated orally for two and four consecutive weeks. The experimental groups consisted of non-treated cryptorchidism (n=10) and treated cryptorchidism (n=10) groups. Testicular tissue samples were collected on days 14 and 28 following of cryptorchidism form non-treated and treated groups. Histopathological and morphometrical studies with the evaluation of microscopic indices of spermatogenesis were prepared on tissue samples. Results- Tubular atrophy with germinal epithelium disarrangement was observed in cryptorchidism groups. These changes were reduced dose-dependently in treated animals. The mean of Sertoli cells was reduced significantly (p=0.025) in four weeks non-treated and the mean of germ cells lineage was reduced significantly (p<0.0001) in four weeks non-treated and two weeks treated cryptorchidism groups compared to the control group. Similarly, all microscopic indices of spermatogenesis were reduced following the induction of cryptorchidism. These alterations were reduced time-dependently in amlodipine treated groups. Conclusion and clinical relevance- The results of this study revealed that the administration of amlodipine as an antioxidant agent, time-dependently could be effective on the reduction of cellular and tissue damages of testicular tissue induced by cryptorchidism. It seems some parts of these protective effects may be done through its activity as calcium blocker which declines apoptotic processes by reduction of cytoplasmic calcium levels.

Retinoic acid: A potential therapeutic agent for cryptorchidism infertility based on investigation of flutamide-induced cryptorchid rats in vivo and in vitro

Cryptorchidism is a common disorder in children and may cause infertility in adults. The BTB is essential for maintaining the microenvironment necessary for normal spermatogenesis. This study investigated whether retinoic acid (RA) may regulate the proteins that are essential for integrity of the BTB in cryptorchidism. Female Sprague-Dawley rats were administrated flutamide during late pregnancy to induce a model of cryptorchidism in male offspring. The concentrations of RA and BTB tight and gap junction protein levels were significantly lower in untreated cryptorchid pups compared with normal pups, but almost normal in cryptorchid pups given RA. Studies in vitro corroborated these findings. The sperm quality of RA-treated model pups was better compared with the untreated model. RA treatment may have therapeutic potential to restore retinoic acid and proteins associated with integrity of the BTB in cryptorchid testis.

Experimental cryptorchidism enhances testicular susceptibility to dibutyl phthalate or acrylamide in Sprague-Dawley rats.

Cryptorchidism (CPT), the most common male congenital abnormality, is variably associated with other male reproductive tract problems. We evaluated if cryptorchid rats develop enhanced testicular susceptibility to dibutyl phthalate (DBP) or acrylamide (AA) after extended exposure. Three studies with rats were performed: (1) in utero and postnatal exposure to DBP or AA; (2) establishment of CPT and orchiopexy; and (3) in utero and postnatal exposures to DBP or AA associated with CPT/orchiopexy. Seminiferous tubules were histologically scored according to the severity of lesions: (1) Rats exposed to DBP (score 1.5) or AA (score 1.1) presented mostly preserved spermatogenesis. Some seminiferous tubules showed vacuolated germinative epithelium, germ cell apoptosis, and a Sertoli cell-only (SCO) pattern. (2) CPT (score 3.3) resulted in decreased absolute testes weights, degenerated and SCO tubules, and spermatogenesis arrest that were reversed by orchiopexy (score 1.1). (3) Exposure to DBP or AA with CPT/orchiopexy led to atrophic testes, spermatogenesis arrest, germ cell exfoliation/multinucleation, and SCO tubules (both chemicals score 2.5). Exposure to chemicals such as DBP or AA prevented the recovery of cryptorchid testes by orchiopexy. The possible role of environmental contaminants should be considered when looking for factors that modulate human testicular disorders associated with CPT.

Antioxidant Enzymes Expression of the Impaired Contralateral Testes in Unilaterally Cryptorchid Rats.

In this study, explored the pathologic mechanism of the contralateral testes impairment in unilaterally cryptorchid rats by investigating the gene expression level. Thirty male Sprague-Dawley rats were randomly and evenly divided into two groups: cryptorchid group and control group. Cryptorchidism was induced by surgical relocation. RT-PCR was then applied to examine the mRNA expression level of antioxidant enzymes in descended testes, including glutathione peroxidase (GSH-PX), copper/zinc-superoxide dismutase (Cu/Zn-SOD), and catalase (CAT). The concentration of malondialdehyde (MDA) was determined by spectrophotometry. In addition, germ-cell apoptosis was detected by a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Two weeks after the operation, the mRNA expression levels of GSH-PX and Cu/Zn-SOD in the cryptorchid group were significantly downregulated; the expression of MDA, as well as the number of apoptotic germ cells, significantly increased compared to the control group (p < 0.01). The mRNA expression of CAT did not show significant changes (p > 0.05). GSH-PX and SOD were downregulated in the testis contralateral to the undescended testis, leading to the accumulation of reactive oxygen species and germ-cell apoptosis. Our results may provide molecular explanations for the impairment of the descended testis in unilateral cryptorchidism.

Application of rat cryptorchidism model for the evaluation of mesenchymal stromal cell secretome regenerative potential

Male infertility represents a severe social and medical challenge. In recent years the progress in regenerative medicine promoted the development of novel options to overcome this medical condition. We are elaborating a promising approach to restore spermatogenesis using mesenchymal stromal cell (MSC) secretome components as a novel class of cell-free cell therapy medicinal products for regenerative medicine. However, the choice of the representative in vivo model of spermatogenesis failure to evaluate the effectiveness of regenerative drugs remains challenging. Using the rat model of bilateral abdominal cryptorchidism, we studied the contribution of MSC conditioned medium contained bioactive cell secreted products to the spermatogenesis recovery. The feasibility of this model to evaluate the drug-driven regenerative effects on spermatogenesis restoration after the injury was demonstrated. We revealed significant correlations between the extent of spermatogonial stem cell niche recovery, spermatozoa count and serum concentration of androgens as an indicator of Leydig cell function. The obtained results can be applied in preclinical studies to choose the proper criteria to appraise the specific activity of novel regenerative drugs developed for the treatment of non-obstructive spermatogenesis disorders.

Role of Ozone Therapy in Preventing Testicular Damage in an Experimental Cryptorchid Rat Model.

BackgroundCryptorchidism is the most common developmental abnormality of the male reproductive system. If left untreated, it results with infertility and testicular cancer. According to current evidence, surgery is the mainstay of treatment, and hormonal therapy approaches are still under investigation. For the protection of testicular functions, antioxidants have emerged as novel options. This study aimed to evaluate the protective properties of ozone, a strong antioxidant, on testicular tissue.Material/MethodsThirty-five male Wistar-albino rats, 1-month-old, were used for the study. Groups were formed as follows: 1) control, 2) sham surgery (cryptorchidism), 3) cryptorchidism plus ozone, 4) cryptorchidism plus human chorionic gonadotropin (hCG), and 5) ozone plus hCG. Surgical procedures were performed on all rats except the control group. All rats except the control group were used to create an experimental cryptorchidism model, and left testes of animals were surgically placed into the abdomen. After 1 month of surgery, groups 3, 4, and 5 were given corresponding treatments intraperitoneally for 4 weeks. At the end of the study period, testicular atrophy index (TAI) and testicular sperm motility (TSM) were assessed and biochemical, histopathological, and immunohistochemical tests were performed.ResultsTAI and TSM were higher in the ozone, hCG, and ozone plus hCG groups than in the sham surgery group (p=0.001). TSM in the ozone group was significantly higher than in the hCG and ozone plus hCG groups. In biochemical analyses, the parameters of oxidative stress (GPx1, MDA, CAT, GSH, SOD) indicated increased oxidative activity in cryptorchidism, which was resolved by applying ozone and hCG (p=0.001). In addition, apoptotic markers, Caspase 3 and bcl-2 were significantly decreased by applying ozone and hCG (p=0.001).ConclusionsResults of this study suggest that ozone therapy, either as a single agent or in combination with hCG, is a promising approach for protection of testicular functions.

Testicular developmental impairment caused by flutamide-induced and DEHP-induced cryptorchid rat models is mediated by excessive apoptosis and deficient autophagy

Background: Cryptorchidism is a common condition of childhood, and it is known to impair fertility potential. However, the underlying mechanisms remain unclear.Methods: This study constructed two cryptorchid rat models to investigate the roles of apoptosis and autophagy in testicular impairment induced by cryptorchidism. Pregnant rats were randomly divided into three groups. Group I: non-treated rats were used as controls. Group II: injected with drug Flutamide (Flu) 25 mg/kg/bw/d from gestation day (GD) 11–19. Group III: daily intragastric administration of 750 mg/kg/bw/d di-2-ethylhexylphosphate (DEHP) from GD 7–19. The cubs were feed normally and the testes were excised on postnatal day (PND) 30.Results: Our results demonstrated cryptorchidism models induced noticeable decreased fertility, significantly reduced sperm count, increased sperm abnormality rate, decreased testosterone and severe testicular damage in histomorphology. Intriguingly, the level of apoptosis marker FAS, Cytochrome C and caspase-3 increased in Flu-induced and DEHP-induced groups. DEHP-induced treatment simultaneously increased the number of autophagosomes and the levels of autophagy marker LC3-II and p62. Significant decrease of autophagy gene (LC3-II and p62) expression is found in Flu-induced rats testes.Conclusion: Taken together, deficient autophagy is involved in testicular spermatogenesis damage of cryptorchidism rats. And this autophagy defect is caused by deficient degradation.

PD33-09 MIR-135A IS ASSOCIATED WITH CRYPTORCHIDISM INFERTILITY THROUGH SUPPRESSION OF FOXO1 IN SPERMATOGONIAL STEM CELLS

You have accessJournal of UrologyStem Cell Research: Stem Cell Research I1 Apr 2018PD33-09 MIR-135A IS ASSOCIATED WITH CRYPTORCHIDISM INFERTILITY THROUGH SUPPRESSION OF FOXO1 IN SPERMATOGONIAL STEM CELLS Yoshinobu Moritoki, Kentaro Mizuno, Hideyuki Kamisawa, Satoshi Kurokawa, Akihiro Nakane, Tetsuji Maruyama, Yutaro Hayashi, and Takahiro Yasui Yoshinobu MoritokiYoshinobu Moritoki More articles by this author , Kentaro MizunoKentaro Mizuno More articles by this author , Hideyuki KamisawaHideyuki Kamisawa More articles by this author , Satoshi KurokawaSatoshi Kurokawa More articles by this author , Akihiro NakaneAkihiro Nakane More articles by this author , Tetsuji MaruyamaTetsuji Maruyama More articles by this author , Yutaro HayashiYutaro Hayashi More articles by this author , and Takahiro YasuiTakahiro Yasui More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1564AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Spermatogonial stem cells (SSCs) function as a life-long spermatogenesis reserve. Self-renewal and stem maintenance are foundational characteristics of SSCs. To maintain these cellular characteristics, the regulation of genome transcription is mandatory, although the basis of this mechanism remains unknown. Recently, small mRNA, called microRNA (miRNA), consisting of 18–25 nucleotide bases have been recognized as key transcription regulators. The effect involves more than one-third of all transcription regulation. We hypothesized that miRNAs function as one of the key SSC regulators and are likely associated with SSC differentiation and thus aimed to find the miRNAs expressing in SSCs and its target mRNAs. METHODS We used a cryptorchid rat testes model of SSC disturbance. The model was generated using intra-abdominal injection of flutamide (7.5 mg/rat) into pregnant Sprague-Dawley rats. Normal testes, unilateral undescended testes (UDT), and contralateral descended testes (DT) were extirpated on postnatal day 9, when two types of SSCs (gonocytes and spermatogonia) co-existed and gonocytes began differentiation into spematogonia. A microarray examining DT and UDT focused on miR-135a, which exhibited lower expression in UDT. In situ hybridization (ISH) was used for the detection of the localization of miR-135a and quantitative polymerase chain reaction (qPCR) to determine the organ specificity of the gene. We then searched the miR-135a target genes using a computer program, followed by the luciferase assay to demonstrate the suppression effect of miR-135a in vitro. RESULTS qPCR revealed that miR-135a was 8.3–21.3 times over-expressed in the testes than in other organs; ISH showed miR-135a localized in SSCs. In silico analysis identified FOXO1 as a putative target gene of miR-135a. FOXO1 has a sequence complementary to miR-135a's seed sequence at its 3' untranslated region. FOXO1 localized in SSCs, and a luciferase assay demonstrated that miR-135a directly suppressed FOXO1 expression in mouse GC-1 cells. CONCLUSIONS We demonstrated that miR-135a is associated with the maintenance of SSCs and that its putative target gene is FOXO1. Because FOXO1 is already proven necessary for maintenance of SSCs and we proved that SSCs are reduced in UDT, we hypothesized that miR-135a was lowered to facilitate the active FOXO1 expression, leading to SSC maintenance. Thus, miR-135a is associated with SSC maintenance through regulation of FOXO1 expression. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e653-e654 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Yoshinobu Moritoki More articles by this author Kentaro Mizuno More articles by this author Hideyuki Kamisawa More articles by this author Satoshi Kurokawa More articles by this author Akihiro Nakane More articles by this author Tetsuji Maruyama More articles by this author Yutaro Hayashi More articles by this author Takahiro Yasui More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...