Rat Testis Cells Research Articles

Is genotoxicity a suitable biomarker for monitoring anabolic-androgenic steroids exposure in vivo? A systematic review and meta-analysis.

Steroids stand for a class of hormones (natural and synthetic) known to be helpful for a number of disorders. Despite the aforementioned beneficial effects of using these hormones, anabolic-androgenic steroids (AAS) are also widely abused in a non-therapeutic manner for muscle-building and strength-increasing properties that may lead to genotoxicity in different tissues. The present study aims to understand whether genotoxicity may be a suitable biomarker for AAS exposure in vivo in both experimental animal and human studies. All studies published in PubMed/Medline, Scopus, and Web of Science electronic databases that presented data on DNA damage caused by AAS were analyzed. A total of 15 articles were included in this study, and after thoroughly reviewing the studies, a total of 8 articles were classified as Strong, 6 were classified as Moderate, and only 1 was classified as Weak, totaling 14 studies being considered either Strong or Moderate. This classification makes it possible to consider the present findings as reliable. The meta-analysis data revealed a statistically significant difference in Wistar rat testis cells with AAS compared to control for tail length and % tail DNA (p < 0.001), so that the selected articles were considered homogeneous and the I2 of 0% indicated low heterogeneity. In summary, genotoxicity can be considered a suitable biomarker for monitoring AAS exposure as a result of DNA breakage and oxidative DNA damage.

Nano-quercetin mitigates triazophos-induced testicular toxicity in rats by suppressing oxidative stress and apoptosis

The present study was designed to evaluate the testicular toxicity of triazophos in rats and to check the ameliorative effect of nano-quercetin against triazophos-induced toxicity. Nano-quercetin was synthesized from quercetin and characterized. Male Wistar rats were divided into seven groups. The control group received olive oil as a vehicle orally. The high-dose triazophos group and the low-dose triazophos group received 1/10th LD50 of triazophos (7.6 mg/kg) and 1/20th LD50 of triazophos (3.8 mg/kg), respectively. Two groups of animals were dosed with quercetin and nano-quercetin, both at 50 mg/kg body weight orally. The final two groups received high-dose triazophos with co-administration of quercetin and nano-quercetin, respectively.Triazophos disrupted the male endocrine axis by reducing the levels of steroidogenic enzymes 3-β-HSD and 17-β-HSD in testicular cells, further reducing FSH and testosterone. Also, triazophos increased the reactive oxygen species, induced lipid peroxidation, decreased the mitochondrial membrane potential, and elevated the number of apoptotic cells in rat testes. Nano-quercetin ameliorated the testicular oxidative stress and apoptotic and endocrine parameters more efficiently than quercetin. Besides, nano-quercetin alleviated the histopathological and biochemical alterations of triazophos. It is concluded that nano-quercetin has higher anti-oxidant efficacy than quercetin in protecting rats against triazophos-induced testicular toxicity.

The SLC9C2 Gene Product (Na+/H+ Exchanger Isoform 11; NHE11) Is a Testis-Specific Protein Localized to the Head of Mature Mammalian Sperm.

Na+/H+ exchangers (NHEs) are a family of ion transporters that regulate the pH of various cell compartments across an array of cell types. In eukaryotes, NHEs are encoded by the SLC9 gene family comprising 13 genes. SLC9C2, which encodes the NHE11 protein, is the only one of the SLC9 genes that is essentially uncharacterized. Here, we show that SLC9C2 exhibits testis/sperm-restricted expression in rats and humans, akin to its paralog SLC9C1 (NHE10). Similar to NHE10, NHE11 is predicted to contain an NHE domain, a voltage sensing domain, and finally an intracellular cyclic nucleotide binding domain. An immunofluorescence analysis of testis sections reveals that NHE11 localizes with developing acrosomal granules in spermiogenic cells in both rat and human testes. Most interestingly, NHE11 localizes to the sperm head, likely the plasma membrane overlaying the acrosome, in mature sperm from rats and humans. Therefore, NHE11 is the only known NHE to localize to the acrosomal region of the head in mature sperm cells. The physiological role of NHE11 has yet to be demonstrated but its predicted functional domains and unique localization suggests that it could modulate intracellular pH of the sperm head in response to changes in membrane potential and cyclic nucleotide concentrations that are a result of sperm capacitation events. If NHE11 is shown to be important for male fertility, it will be an attractive target for male contraceptive drugs due to its exclusive testis/sperm-specific expression.

Phase transitions and mechanisms of cryoprotection of serum-/xeno-free media based on dextran and dimethyl sulfoxide

The development of serum-/xeno-free media may help avoid the drawbacks of using serum and its components, such as probable contamination, instability of composition, or difficulty in sterilization. The objectives of this research were to investigate the use of combinations of a permeating cryoprotective agent (Me2SO) and non-permeating polymers (polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, hydroxyethyl starch, dextran) for cryopreservation of interstitial cells (ICs) of rat testis, and to propose the mechanism of cryoprotection of such compositions. In the course of this study, the best combination was 100 mg/ml dextran (M.m. 40 kDa) (Dex40) with 0.7 M Me2SO in Ham's F12. The ICs were additionally cooled and warmed to different end temperatures (−30, −50, −50 and −196 °C) to determine which temperature intervals contributed most to the IC loss. Then, the cryoprotective action of this serum-/xeno-free medium was investigated in comparison with serum or albumin-containing media by differential scanning calorimetry (DSC) and thermomechanical analysis (TMA). The results showed that the medium based on Dex40 did not decrease the amount of ice formed. However, it could undergo other phase separation and phase transformation to form glassy states. Potential cell-damaging physical processes such as eutectic crystallization/melting, recrystallization of NaCl and/or Me2SO derivatives, found in serum-containing media and taking place in specific temperature intervals, were not observed in the Dex40 based media. This was in good correlation with indicators of cell survival. Additionally, the application of Dex40 allowed using Me2SO in lower concentrations (0.7 M) than required for serum-containing media (1.4 M), which may decrease the toxicity of serum-/xeno-free media.

Role of aquaporin 9 in hyperglycaemia-induced testicular leydig cell apoptosis

Hyperglycaemia is an early symptom of diabetes mellitus (DM) and hyperglycaemia-induced apoptosis is the most widely accepted cause for diabetes induced male infertility/subfertility. Aquaporin (AQP) isoforms (AQP0-12), are water channels involved in maintaining water homeostasis and regulate fluid absorption and/or excretion; thus, play a key role in reproductive function. Further, AQPs regulate diabetic complications, apoptosis induced by hyperglycaemia/high glucose (HG) and also involved in insulin regulation. Herein, we investigated AQP 9 role in hyperglycaemia-induced testicular Leydig cell apoptosis. In-vivo and in-vitro role of AQP 9 on hyperglycaemia induced Leydig cell apoptosis was analysed in diabetic rat testis and LC-540 rat Leydig cells by Real Time-PCR, western blotting and siRNA knock-down experiments. In addition, HG effect on viability and morphological changes due to apoptosis in cells were assessed by MTT and fluorescence microscopy. In-vivo and in-vitro AQP 9 expressions were increased in diabetic testicular Leydig cells. Hyperglycaemia caused apoptosis in testicular Leydig cells by increasing Cytochrome c, Bax and decreasing Bcl-2. Furthermore, AQP 9 knock-down resulted in downregulation of pro-apoptotic proteins and in contrast, upregulated anti-apoptotic protein expressions; thereby decreased apoptosis in testicular Leydig cells under hyperglycemia. Our findings collectively suggest, AQP 9 role in hyperglycaemia-induced apoptosis in testicular Leydig cells.

Reproductive Function of Male Rats and Motor Activity of Their Offspring in Fire Emissions Modeling.

We studied the reproductive function of albino male rats after long-term exposure to wildfire smoke and the species-specific behavior of their sexually mature offspring. After the end of exposure, the mean number of spermatogonia and the relative number of Leydig cells in rat testes decreased and the index of spermatogenesis decreased. The offspring of exposed males mated with intact females demonstrated impaired cognitive abilities and disturbances of species-specific behavior. The severity of these changes depended on time interval between the end of the inhalation exposure and mating.

Transcriptional activation and regulation of urokinase plasminogen activator inducted by LPS through MyD88 independent pathway in rat Sertoli cells.

Urokinase plasminogen activator (uPA) is a serine protease and it also demonstrates proinflammatory properties. We thus hypothesized that uPA is involved in immunity of Sertoli cells in the rat testis. The uPA gene(PLAU) promoter was cloned by RT-PCR and the transcriptional activation of core domain was screened by using Dual-Luciferase Reporter Assay System. The Sertoli cells were harvested from 20-day-old Sprague-Dawley male rats and total RNA was isolated. The uPA mRNA levels and MyD88 pathway were tested by qPCR. We successfully cloned the 1517-bp rat uPA gene and screened the core domain (-455/+40) in five different fragments of uPA promoter. The uPA expression and uPA promoter activity were upregulated in lipopolysaccharide (LPS)-stimulated Sertoli cells. Furthermore, the uPA expression was regulated through the MyD88-independent pathway by interdicting IRF3 and interferon b. uPA expression is likely induced by LPS through the core promoter domain of uPA. This finding implied that uPA played a role in the immune function of Sertoli cells in rat testis, which might provide the development of new treatments for male infertility.

Quercetin ameliorates testosterone secretion disorder by inhibiting endoplasmic reticulum stress through the miR-1306-5p/HSD17B7 axis in diabetic rats.

Testicular damage and testosterone secretion disorder are associated with diabetes mellitus. Quercetin, a common flavonoid, has antioxidant, anti-cancer, and blood sugar lowering effects. Therefore, this study aims to investigate the effect of quercetin on the reproductive system of male rats with diabetes in vivo and in vitro and elucidate its mechanism. Streptozotocin (STZ) induction was used to establish a diabetes model in 40 male Sprague Dawley rats, which were subsequently administered with 20 or 50 mg/kg of quercetin. Leydig cells of rat testes were treated by high glucose (HG) followed by 5 or 10 μM quercetin. Two doses of quercetin increased rat body weight and testicular weight, decreased blood glucose, and inhibited oxidative stress. Quantitative real-time polymerase chain reaction and Western blotting revealed that quercetin alleviated STZ-induced testicular damage and promoted testosterone synthesis. Both doses of quercetin reduced reactive oxygen species and malondialdehyde levels, and increased superoxide dismutase level in HG-treated cells. Both, in vivo and in vitro results confirmed that a high dose of quercetin was more effective. MiR-1306-5p was upregulated in testicular tissue of diabetic rats and HG-treated cells. 17β-hydroxysteroid dehydrogenase (HSD17B7) was a target of miR-1306-5p and HSD17B7 was downregulated in STZ-induced rat tissues and HG-treated cells. HSD17B7 overexpression reversed the increase of C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (Grp78) protein levels as well as eIF2α phosphorylation level and promotion of cell apoptosis caused by miR-1306-5p overexpression. Moreover, overexpression of HSD17B7 activated the Janus kinase 2/signal transducer and activator of transcription 3 axis in HG-treated cells. In conclusion, quercetin inhibits endoplasmic reticulum stress and improves testosterone secretion disorder through the miR-1306-5p/HSD17B7 axis in diabetic rats.

C-type natriuretic peptide stimulates function of the murine Sertoli cells via activation of the NPR-B/cGMP/PKG signaling pathway.

C-type natriuretic peptide (CNP) is an important regulator of the male reproductive process. Our previous investigations showed that CNP can significantly stimulate the mRNA expression of androgen-binding protein (Abp) and transferrin (Trf) in the rat Sertoli cells, but the pathways responsible for this process remain to be elucidated. We predict that CNP binds the natriuretic peptide receptor B (NPR-B) to regulate expression of ABP and TRF through the intracellular cyclicguanosine monophosphate (cGMP) pathway. To address this question, in this study, we first confirmed the expression and localization of CNP and NPR-B in rat testes by immunohistochemistry and western blotting. Then, ELISA and real-time PCR were performed to investigate the signaling pathway of CNP in Sertoli cells in rat testes. Our results showed that CNP was mainly localized in the germ cells and Leydig cells, and its receptor, NPR-B, was mostly expressed in the Sertoli cells and vascular endothelial cells. CNP supplementation in the Sertoli cell medium was accompanied by an increase in the amount of intracellular cGMP and in the production of Abp and Trf mRNA, whereas inhibition of PKG with KT5823 led to a decrease in the expression of Abp and Trf mRNA. Moreover, Abp and Trf mRNA were no longer elevated when we used liposome-mediated RNA interference technology to silence the NPR-B gene in a mouse Sertoli cell line (TM4). These results suggest that CNP contributes to the regulation of ABP and TRF in the Sertoli cells through the NPR-B/cGMP/PKG signaling pathways.

Effects of HIF-1α on Spermatogenesis of Varicocele Rats by Regulating VEGF/PI3K/Akt Signaling Pathway.

Hypoxia-inducible factor-1α (HIF-1α) participates in the regulation of spermatogenic function in rats with varicocele (VC), and the PI3K/Akt pathway plays an important role in it. In the present research, we applied the CRISPR/Cas9 gene editing technique to silence the HIF-1α gene of VC rat testis, to explore the effect of HIF-1α on apoptosis of spermatogenic cells in VC rats through the PI3K/Akt pathway. Sprague Dawley rats were randomly assigned to four groups, including the normal rat group (group N), VC model group (group V), VC + HIF-1α-lentivirus group (group H), and VC + luciferase-lentivirus group (group L). Apoptosis of spermatogenic cells in rat testis was tested by TUNEL Kit. The morphologic changes of seminiferous tubules were viewed by a light microscope. Expressions of VEGF, Akt, p-Akt, p70S6K, and p-p70S6K were detected by means of Western blot, immunofluorescence, or immunohistochemistry methods. One-way ANOVA was applied to analyze the diverseness between groups. Compared with group N, the distribution of germ cells was disordered, apoptosis of spermatogenic cells increased significantly, and the expression of VEGF, p-Akt, and p-p70S6K was also increased in group V. Compared with group V, the damage of seminiferous epithelium in group H was improved, and the arrangement of the seminiferous epithelium was almost orderly. Apoptosis of spermatogenic cells decreased significantly, and the expression of VEGF, p-Akt, and p-p70S6K protein was decreased (P < 0.05). There was no significant difference between group N and group H (P > 0.05).In conclusion, HIF-1α is regulated by hypoxia in rats with varicocele to regulate its downstream gene VEGF which regulates spermatogenesis, and the PI3K/Akt signaling pathway plays a regulatory role in this process.

In utero exposure to low doses of genistein and di-(2-ethylhexyl) phthalate (DEHP) alters innate immune cells in neonatal and adult rat testes.

Although humans are exposed to mixtures of endocrine disruptor chemicals, few studies have examined their toxicity on male reproduction. We previously found that fetal exposure to a mixture of the phytoestrogen genistein (GEN) and the plasticizer di(2-ethylhexyl) phthalate (DEHP) altered gene expression in adult rat testes. Our goal was to investigate the effects of fetal exposure to GEN-DEHP mixtures at two doses relevant to humans on testicular function and transcriptome in neonatal and adult rats. Pregnant SD rats were gavaged with vehicle, GEN or DEHP, alone or mixed at 0.1 and 10mg/kg/day, from gestation day 14 to birth. Fertility, steroid levels, and testis morphology were examined in neonatal and adult rats. Testicular transcriptomes were examined by gene array and functional pathway analyses. Cell-specific genes/proteins were determined by quantitative real-time PCR and immunohistochemistry. GEN-DEHP mixtures increased the rates of infertility and abnormal testes in adult rats. Gene array analysis identified more genes exclusively altered by the mixtures than individual compounds. Altered top canonical pathways included urogenital/reproductive developmental and inflammatory processes. GEN-DEHP mixtures increased innate immune cells and macrophages markers at both doses and ages, more strongly and consistently than DEHP or GEN alone. Genes exclusively increased by the mixture in adult testis related to innate immune cells and macrophages included Kitlg, Rps6ka3 (Rsk2), Nr3c1, Nqo1, Lif, Fyn, Ptprj (Dep-1), Gpr116, Pfn2, and Ptgr1. These findings demonstrate that GEN-DEHP mixtures at doses relevant to human induce adverse testicular phenotypes, concurrent with age-dependent and non-monotonic changes in testicular transcriptomes. The involvement of innate immune cells such as macrophages suggests immediate and delayed inflammatory responses which may contribute to testicular dysfunction. Moreover, these effects are complex and likely involve multiple interactions between immune and non-immune testicular cell types that will entail further studies.

Evaluation of the anti-oxidant effect of ascorbic acid on apoptosis and proliferation of germinal epithelium cells of rat testis following malathion-induced toxicity.

Objective(s):The aim of this study was to determine the protective role of ascorbic acid on apoptosis and proliferation of spermatogonia and primary spermatocyte cells after malathion administration as an organophosphate pesticide in rat testis.Materials and Methods:Thirty male Wistar rats were randomly divided into five groups of 6 rats each, including control (no intervention), sham (normal saline 0.09%), malathion (50 mg/kg), malathion plus ascorbic acid (50 mg/kg and 200 mg/kg, respectively), and ascorbic acid (200 mg/kg) groups. Malathion and ascorbic acid were administrated via intraperitoneal injection once per day and seven times per week. After 6 weeks, animals were sacrificed, and testis tissue was used for evaluation of apoptosis and proliferation of germinal epithelium cells using the TUNEL and PCNA staining techniques. Results:The results of TUNEL staining showed that the numbers of apoptotic cells in spermatogonia and primary spermatocyte cells were significantly increased in the malathion 50 mg/kg group vs control group (P<0.001). Co-administration of malathion 50 mg/kg and ascorbic acid 200 mg/kg significantly decreased the apoptotic cells in both cell types in comparison with malathion 50 mg/kg group (P<0.001). The results of PCNA staining revealed that the proliferation of these cells was significantly decreased in malathion 50 mg/kg group vs control group (P<0.001), and malathion 50 mg/kg plus ascorbic acid 200 mg/kg administration increased the proliferation of cells compared with malathion 50 mg/kg group (P<0.001).Conclusion:The results provide evidence that ascorbic acid showed preventive effects on malathion-induced toxicity in male rat testis.

Effects of Moringa oleifera L. Extract on leydig and sertoli cells induced high Temperature on Rattus norvegicus

The aim of this study was to investigate the protective effect of Moringa oleifera leaves extract on the number of leydig and sertoli cells on Rattus norvegicus due to induced high temperature. Twenty five male rat were divided into five groups, five rat for each group and administered through intragastric gavage with differents treatments for 21 days. The treatment groups were C–(NaCMC 1% 1ml), C+ (NaCMC 1% 1ml + high temperature induction), T1, T2, and T3 (100, 200, and 400mg/kg bw Moringa oleifera Lam leaves ethanol extract respectively+high temperature induction). The high temperature induction was 40°C for one hour. The observation was done by examined the histopathological changes on the numbers of Leydig and Sertoli cells. The research showed that Moringa oleifera leaves extract could protect the leydig and sertoli cells in rat testes from destructive effect of high temperature induction. Keywords: Moringa oleifera, Leydig, Sertoli, Testes

Effects of Heshouwuyin on gene expression of the insulin/IGF signalling pathway in rat testis and spermatogenic cells

Context The Chinese herbal formula Heshouwu decoction (Heshouwuyin) has protective effects on testicular function in aging male rats, but the mechanism is unknown. Objective This study investigated whether Heshouwuyin affects the testicular function of aging rats by regulating the insulin/IGF signalling pathway. Materials and methods Sixteen-month-old male Wistar rats in the Heshouwuyin group and the natural-aging group were orally administered Heshouwuyin granules (0.056 g/kg) or equivalent normal saline for 60 d. The testicular tissue of 12-month-old male Wistar rats was removed as a young control group (n = 10). The testicular tissue and spermatogenic cells were studied. Results The immunofluorescence results revealed that the insulin receptor (INSR)- (0.056 ± 0.00548), insulin receptor substrate 1(IRS1)- (0.251 ± 0.031), IRS2 (0.230 ± 0.019)- and insulin-like growth factor 1 (IGF1)-positive cell rate (0.33 ± 0.04) in the aging group was higher than that in the young control group (0.116 ± 0.011, 0.401 ± 0.0256, 0.427 ± 0.031, 0.56 ± 0.031; p < 0.01), and the IGF-binding protein 3 (IGFBP3)-positive cell rate (0.42 ± 0.024) was lower than that (0.06 ± 0.027) in the young group (p < 0.01). The intervention of Heshouwuyin reversed the above phenomena. The qPCR and immunoblot results were consistent with those of the immunofluorescence. The same results were obtained in spermatogenic cells. Conclusions Our research shows that Heshouwuyin can regulate the insulin/IGF signalling pathway to improve testicular function, and provides an experimental basis for further clinical use.

Pengaruh Tepung Kedelai Kaya Isoflavon terhadap Testosteron Serum, Jumlah Sel Leydig dan Jumlah Sel Spermatogenik pada Tubuli Seminiferi Testis Tikus (Rattus norvegicus)

The objective of this experiment was to evaluate the effects of isoflavone-riched soybean flour with different levels of isoflavone on testosterone level, total Leydig cells, and total spermatogenic cells in the seminiferous tubules of male rats. Diet was given as isonitrogen and isocaloric with 10% of dietary protein from casein. Twenty five male of Sprague Dawley weaning rats (21 days old) were divided into five groups and treated with isoflavone-riched soybean flour by oral administration with different levels (dosages). The treatment was conducted for 2 months. The treatment of isoflavone-riched soybean flour with higher dosage of isoflavone increased the testosterone levels in the serum and the total Leydig cells in the seminiferous tubules of male rats. The optimum dosage of isoflavone was 1.5 mg/day resulted in the highest total spermatogenic cells of rat testes. The treatment isoflavone-riched soybean flour with 1.5 mg isoflavone/day on male rats resulted in the testosteron level of 2.96±0.45 ng/ml, total Leydig cells 70.22±9.34; while the number of spermatogonia, spermatocytes, early spermatids, late spermatids, and total spermatogenic cells were 48.44±4.82, 60.00±3.43, 221.56± 16.12, 164.33±17.94, and 494.33±32.94, respectively.

The effects of perfluorooctanoic acid (PFOA) on fetal and adult rat testis

Perfluorooctanoic acid (PFOA) is a widely dispersed synthetic chemical, which accumulates in living organisms and has been connected with male reproductive disorders. To monitor the effects of PFOA, fetal rat testes or seminiferous tubule segments (stage VII-VIII) of adult rats were cultured in 0–100 μg/ml PFOA for 24 h. Afterwards, cAMP, progesterone, testosterone and StAR protein levels were measured from the fetal testes culture. Measurements were combined with immunohistochemistry, immunofluorescence, TUNEL and flow cytometric analysis to monitor cell death in somatic and germ cells. This study shows that the levels of cAMP, progesterone, testosterone and expression of StAR decreased significantly in PFOA 50 and 100 μg/ml. PFOA affected cell populations significantly by decreasing the amount of diploid, proliferating, meiotic I and G2/M-phase cells in adult rat testis. However, PFOA did not affect fetal, proliferating or adult rat Sertoli cells but an increased tendency of apoptosis in fetal Leydig cells was observed.

In Vivo Knockdown of the IP3R1 Calcium Channel Disrupts ER and Actin Morphology, as well as TBC Bulb Maturation in the Apical Processes of Sertoli Cells in Rat Testes

Spermatogenesis is the process by which sperm cells are produced, but at one phase of this process, late stage spermatids are temporarily attached to the apex of Sertoli cells in the seminiferous epithelium. Late spermatids are anchored by large adhesion like junction complexes termed ‘ectoplasmic specializations’ (ESs) that are proposed to be disassembled by novel endocytic structures only found in mammalian Sertoli cells, called ‘tubulobulbar complexes’ (TBCs). Both ESs and TBCs are heavily actin related, with parallel actin arrays and dendritic actin networks respectively being hallmarks of their identities. The endoplasmic reticulum (ER) in the Sertoli cell apical processes surrounding the late spermatids forms an extensive and continuous network. Cisternae of this network form part of the structure of the ESs, participate in a membrane contact site with the bulbs of TBCs, and have a parallel alignment with the surrounding Sertoli cell plasma membrane. Previously, we've localized the IP3R calcium channel to the TBC bulb‐ER contacts as well as other calcium exchange machinery to various parts of the ER in the apical process. Calcium release and exchange have been implicated in processes related to both actin dynamics as well as in fusion and acidification of endocytic components such as endosomes. We've proposed that calcium exchange may be a crucial mechanism in regulating both the actin networks of ESs and TBCs, as well as in facilitating the maturation and fusion of TBC bulbs as they develop into putative endosomes. If calcium exchange from the ER is responsible for regulating the local actin networks and maturation of endocytic components in the apical process, then knocking down the primary calcium release channel of the ER should alter local actin and TBC bulb morphology. To test this, we injected the testes of Sprague Dawley rats with siRNAs against IP3R1 (ITPR1) and collected tissues at 1 day, 2 day, and 3 days post injection and processed the tissues for either western blot, immunofluorescence, or electron microscopy. ITPR1 siRNA treated tissues showed significant morphological alterations to the ER, actin, and TBC bulbs in the apical processes of late spermatids compared to controls. This data supports the hypothesis that ER calcium exchange is an integral component to actin dynamics and TBC bulb maturation in the apical processes of Sertoli cells.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant awarded to AWV; Alexander Graham Bell Scholarship (NSERC) awarded to AAThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

PERK regulates Nrf2/ARE antioxidant pathway against dibutyl phthalate-induced mitochondrial damage and apoptosis dependent of reactive oxygen species in mouse spermatocyte-derived cells

Dibutyl phthalate (DBP)-induced germ cell apoptosis contributes to male reproductive toxicity, however, the primary target organelle of DBP or the molecular events triggered by DBP to initiate germ cell apoptosis remain unclear. Our previous studies demonstrated DBP could stimulate the production of intracellular reactive oxygen species (ROS), which served as an upstream mediator of activation of endoplasmic reticulum (ER) stress in mouse spermatocyte-derived GC-2 cells. In the present study, the impacts of DBP-induced ROS generation on the mitochondria-related damage and the associations between ER stress and mitochondrial-related damage were investigated in GC-2 cells. We observed significant decreases of mitochondrial mass, mtDNA copy number, COX IV protein level, and ATP level in DBP-treated GC-2 cells in a dose-dependent manner. And DBP activated mitochondrial-related apoptosis, indicated by the elevation of cytoplasmic cytochrome C (Cyt C) and the activation of caspase-9/3 cascade. Pretreatment with antioxidant melatonin obviously attenuated DBP-induced mitochondrial damage and mitochondrial-dependent apoptosis in GC-2 cells, indicating the role of ROS in DBP-caused testicular toxicity. In response to oxidative stress, the Nrf2/ARE axis was activated in DBP-treated GC-2 cells to counteract ROS overproduction and subsequent mitochondrial damage. Further experiments showed DBP treatment increased the phosphorylated expression of ER stress-related protein PERK. GSK2606414, a specific inhibitor of PERK, partly attenuated the expression of Nrf2. And both DBP-induced mitochondrial damage in GC-2 cells and mitochondrial-dependent apoptosis of the germ cells in rat testes were further aggravated by PERK inhibition. Taken together, our data suggest that PERK regulates the Nrf2/ARE antioxidant pathway functioning as a self-defense mechanism against ROS-related mitochondrial damage induced by DBP in male germ cells.

Drp1 Phosphorylation Is Indispensable for Steroidogenesis in Leydig Cells.

The initial steps of steroidogenesis occur in the mitochondria. Dynamic changes in the mitochondria are associated with their fission and fusion. Therefore, understanding the cellular and molecular relationships between steroidogenesis and mitochondrial dynamics is important. The hypothesis of the current study is that mitochondrial fission and fusion are closely associated with steroid hormone synthesis in testicular Leydig cells. Steroid hormone production, induced by dibutyryl cAMP (dbcAMP) in Leydig cells, was accompanied by increased mitochondrial mass. Mitochondrial elongation increased during the dbcAMP-induced steroid production, whereas mitochondrial fragmentation was reduced. Among the mitochondrial-shaping proteins, the level of dynamin-associated protein 1 (Drp1) was altered in response to dbcAMP stimulation. The increase in Drp1 Ser 637 phosphorylation correlated with steroid hormone production in the MA-10 Leydig cells as well as in the primary adult rat Leydig cells. Drp1 was differentially expressed in the Leydig cells during testicular development. Finally, gonadotropin administration altered the status of Drp1 phosphorylation in the Leydig cells of immature rat testes. Overall, mitochondrial dynamics is directly linked to steroidogenesis, and Drp1 plays an important regulatory role during steroidogenesis. This study shows that Drp1 level is regulated by cAMP and that its phosphorylation via protein kinase A (PKA) activation plays a decisive role in mitochondrial shaping by offering an optimal environment for steroid hormone biosynthesis in Leydig cells. Therefore, it is suggested that PKA-mediated Drp1 Ser 637 phosphorylation is indispensable for steroidogenesis in the Leydig cells, and this phosphorylation results in mitochondrial elongation via the relative attenuation of mitochondrial fission during steroidogenesis.

4-Nonylphenol effects on rat testis and sertoli cells determined by spectrochemical techniques coupled with chemometric analysis

Herein, vibrational spectroscopy has been applied for qualitative identification of biomolecular alterations that occur in cells and tissues following chemical treatment. Towards this end, we combined attenuated total reflection Fourier-transform infrared (ATR-FTIR) and Raman spectroscopy to assess testicular toxicology after 4-nonylphenol (NP) exposure, an estrogenic endocrine disruptor affecting testicular function in rats and other species. Rats aged 21, 35 or 50 days received NP at intra-peritoneal doses of 0, 25, 50 or 100 mg/kg for 20 consecutive days. Primary Sertoli cells (SCs) were treated with NP at various concentrations (0, 2.5, 5, 10 or 20 μM) for 12 h. Post-exposure, testicular cells, interstitial tissue and SCs were interrogated respectively using spectrochemical techniques coupled with multivariate analysis. Distinct biomolecular segregation between the NP-exposed samples vs. control were observed based on infrared (IR) spectral regions of 3200–2800 cm−1 and 1800-900 cm−1, and the Raman spectral region of 1800–900 cm−1. For in vivo experiments, the main wavenumbers responsible for segregation varied significantly among the three age classes. The main IR and Raman band differences between NP-exposed and control groups were observed for Amide (proteins), lipids and DNA/RNA. An interesting finding was that the peptide aggregation level, Amide Ӏ-to-Amide II ratio, and phosphate-to-carbohydrate ratio were considerably reduced in ex vivo NP-exposed testicular cells or SCs in vitro. This study demonstrates that ATR-FTIR and Raman spectroscopy techniques can be applied towards analysing NP-induced testicular biomolecular alterations.